Methods and Means to Modify Fiber Strength in Fiber-Producing Plants

Abstract

This invention relates to the field of agriculture, more specifically to the use of molecular biology techniques to alter fiber-producing plants, particularly cotton plants, and/or accelerate breeding of such fiber-producing plants. Methods and means are provided to alter fiber qualities, such as increasing fiber strength. Methods are also provided to identify molecular markers associated with fiber strength in a population of cotton varieties and related progenitor plants.

Description

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. application Ser. No. 12/992,907, filed Nov. 16, 2010, which is a §371 U.S. National Stage of International Application No. PCT/EP09/003674 filed May 25, 2009, which claims the benefit of U.S. Provisional Application Ser. No. 61/128,938, filed May 27, 2008, the contents of which are herein incorporated by reference in their entirety.

REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0002] The official copy of the sequence listing is submitted electronically via EFS-Web as an ASCII formatted sequence listing with a file named "CS9046PCTUSSequenceListingST25.txt", created on Nov. 11, 2010, and having a size of 358 kilobytes and is filed concurrently with the specification. The sequence listing contained in this ASCII formatted document is part of the specification and is herein incorporated by reference in its entirety.

FIELD OF THE INVENTION

[0003] This invention relates to the field of agriculture, more specifically to the use of molecular biology techniques to alter fiber-producing plants, particularly cotton plants, and/or accelerate breeding of such fiber-producing plants. Methods and means are provided to alter fiber qualities, such as increasing fiber strength. Methods are also provided to identify molecular markers associated with fiber strength in a population of cotton varieties and related progenitor plants.

BACKGROUND OF THE INVENTION

[0004] Cotton provides much of the high quality fiber for the textile industry. The modification of cotton fiber characteristics to better suit the requirements of the industry is a major effort in breeding by either classical methods or by genetically altering the genome of cotton plants.

[0005] About 90% of cotton grown worldwide is Gossypium hirsutum L., whereas Gossypium barbadense accounts for about 8%. As in most flowering plants, cotton genomes are thought to have incurred one or more polyploidization events and to have evolved by the joining of divergent genomes in a common nucleus. The cotton commerce is dominated by improved forms of two "AD" allotetraploid species, Gossypium hirsutum L. and Gossypium barbadense L (both 2n=4×=52). Allotetraploid cottons are thought to have formed about 1-2 million years ago, in the New World, by hybridization between a maternal Old World "A" genome taxon resembling Gossypium herbaceum (2n=2×=26) and paternal New World "D" genome taxon resembling Gossypium raimondii or Gossypium gossypioides (both 2n=2×=26). Wild A genome diploid and AD allotetraploid Gossypium taxa produce spinnable fibers. One A genome diploid species, Gossypium arboreum (2n=2×=26), remains intensively bred and cultivated in Asia. Its close relative and possible Gossypium progenitor, the A genome diploid species G. herbaceum, also produces spinnable fiber. Although the seeds of D genome diploids are pubescent, none produce spinnable fibers. No taxa from the other recognized diploid Gossypium genomes (B, C, E, F, G and K) have been domesticated. Intense directional selection by humans has consistently produced AD allotetraploid cottons that have superior yield and/or quality characteristics compared to the A genome diploid cultivars. Selective breeding of G. hirsutum (AADD; "Upland" cotton) has emphasized maximum yield, whereas G. barbadense (AADD; "Sea Island", "Pima", or "Egyptian" cotton) is prized for its fibers of superior length, strength, and fineness (Jiang et al., 1998, Proc Natl Acad Sci USA. 95(8): 4419-4424).

[0006] A cotton fiber is a single cell that initiates from the epidermis of the outer integument of the ovules, at or just prior to anthesis. Thereafter, the fibers elongate rapidly for about 3 weeks before they switch to intensive secondary cell wall cellulose synthesis. Fiber cells interconnect only to the underlying seed coat at their basal ends and influx of solute, water and other molecules occurs through either plasmodesmata or plasma membrane. Ruan et al. 2001 (Plant Cell 13: 47-63) demonstrated a transient closure of plasmodesmata during fiber elongation. Ruan et al. 2004 (Plant Physiology 136: 4104-4113) compared the duration of plasmodesmata closure among different cotton genotypes differing in fiber length and found a positive correlation between the duration of the plasmodesmata closure and fiber length. Furthermore, microscopic evidence was presented showing callose deposition and degradation at the fiber base, correlating with the timing of plasmodesmata closure and reopening. Expression of a endo-1,3-beta-glucanase gene in the fibers, allowing to degrade callose, correlated with the reopening of the plasmodesmata at the fiber base.

[0007] WO2005/017157 describes methods and means for modulating fiber length in fiber producing plants such as cotton by altering the fiber elongation phase. The fiber elongation phase may be increased or decreased by interfering with callose deposition in plasmodesmata at the base of the fiber cells.

[0008] WO2008/083969 (claiming priority of European patent application EP 07000550) discloses isolated DNA molecules comprising a nucleotide sequence encoding cotton endo-1,3-beta-glucanases and fiber cell preferential promoter or promoter regions, as well as methods for modifying the length of a fiber of a cotton plant using these sequences or promoters. WO2008/083969 also describes that the timing of expression of the A and D subgenome specific alleles of the fiber specific endo-1,3-beta-glucanase gene in Gossypium hirsutum is different. Whereas the onset of the expression of the D subgenome specific allele correlates with the end of the rapid elongation phase (about 14 to 17 days post-anthesis, hereinafter abbreviated "DPA"), onset of the expression of the A subgenome specific allele is delayed until the beginning of the late fiber maturation phase (about 35-40 DPA) depending on growth conditions.

[0009] One fiber characteristic that is of special interest for the cotton industry is fiber strength. There is not only a high correlation between fiber strength and yarn strength, but also cotton with high fiber strength is more likely to withstand breakage during the manufacturing process.

[0010] Fiber strength is, among many other textile properties of cotton fibers (e.g., fiber wall thickness or maturity, dyeability, extensibility . . . ), described to be directly dependent on the amount and properties (e.g., degree of polymerization, crystallite size, and microfibril orientation) of cellulose (Ramey, 1986, In: Mauney J. R. and Stewart J. McD. (eds.) Cotton Physiology. The Cotton Foundation, Memphis, Tenn., pp. 351-360; Triplett, 1993, In: Cellulosics: Pulp, Fibre, and Environmental Aspects. Ellis Horwood, Chichester, UK, pp. 135-140; Hsieh, 1999, In: Basra A. S. (ed.) Cotton Fibers: Developmental Biology, Quality Improvement, and Textile Processing. The Haworth Press, New York, pp. 137-166). Advances in the past decade, particularly using the model plant Arabidopsis (Arioli et al., 1998, Science 279(5351): 717-720), have led to a great increase in the knowledge of the proteins involved in cellulose synthesis. Despite this, there is still much to learn about cellulose synthesis, especially about how it is regulated at both transcriptional and post-transcriptional levels (Taylor, 2008, New Phytologist 178 (2), 239-252).

[0011] Typical primary fiber cell walls in G. hirsutum, which are about 0.5 μM thick and contain 20-25% cellulose along with pectin, xyloglucan, and protein (Meinert and Delmer 1977, Plant Physiol 59:1088-1097), are synthesized during fiber elongation (Haigler, 2007, In: R. M. Brown, Jr. and I. M. Saxena (eds.), Cellulose: Molecular and Structural Biology, 147-168, Springer.). Primary wall deposition proceeds alone until 14-17 DPA, then a transition phase with concurrent primary and secondary wall deposition occurs between 15-24 DPA (representing deposition of the "winding layer"), followed by predominantly secondary wall synthesis until at least 40 DPA. The first period of wall thickening (12-16 DPA) is accomplished by continued synthesis in the same proportions of primary wall components (Meinert and Delmer, 1977, supra), an observation that is consistent with increasing wall birefringence while the cellulose microfibrils remain transversely oriented (Seagull, 1986, Can J Bot 64:1373-1381). The secondary wall finally attains a thickness of 3-6 μM around the whole circumference of the fiber, becoming thinner only at the fiber tip. In G. barbadense, there is an overlap between primary and secondary wall deposition within each fiber rather than in the fiber population because the overlapping period is greatly prolonged, and 90% of secondary wall deposition is complete before elongation ceases (DeLanghe, 1986, In: Mauney J. R. and Stewart J. McD. (eds.) cotton Physiology. The Cotton Foundation, Memphis, Tenn., pp. 325-350). It is thought that elongation continues exclusively at the fiber tip as secondary wall is deposited over most of the cell surface.

[0012] Maltby et al. (1979, Plant Physiol. 63, 1158-1164) describe that developing fibers of Gossypium hirsutum transiently synthesize 1,3-beta-D-glucan (callose) at the onset of secondary wall deposition followed by massive synthesis of cellulose. Meier et al. (1981, Nature 289: 821-822) describe that callose may be a probable intermediate in biosynthesis of cellulose of cotton fibers. DeLanghe (1986, supra) describes that callose may be required in cotton fiber secondary walls to provide a space for the crystallization and final orientation of cellulose microfibrils in the exoplasmic zone in the absence of typical matrix molecules.

[0013] The inventions described hereinafter in the different embodiments, examples, figures and claims provide improved methods and means for modulating fiber strength. More specifically, the present invention describes how to increase fiber strength and at the same time maintain a high fiber yield in plants. In particular, the invention describes how to increase fiber strength in cotton species selected for high yield, such as Gossypium hirsutum, by introgression of fiber strength determining genes from other cotton species selected for high fiber strength, such as Gossypium barbadense. Methods are also provided to identify molecular markers associated with fiber strength in a population of cotton varieties and related progenitor plants. The inventions described hereinafter also provide novel nucleic acid molecules encoding fiber-specific Gossypium glucanase proteins (GLUC1.1) and the proteins as such.

SUMMARY OF THE INVENTION

[0014] The inventors identified a quantitative trait locus for fiber strength on chromosome A05 of Gossypium and found that Gossypium barbadense comprises an allele of this fiber strength locus that is superior to the allele of this QTL from Gossypium hirsutum, i.e. the presence of the Gossypium barbadense fiber strength allele in a Gossypium plant results in a higher fiber strength as compared to the fiber strength of a Gossypium plant comprising the Gossypium hirsutum fiber strength allele.

[0015] Thus, in a first aspect, the present invention provides a non-naturally occurring Gossypium plant, and parts and progeny thereof, comprising at least one superior allele of a fiber strength locus on chromosome A05.

[0016] In one embodiment, the plant is a plant from an A genome diploid Gossypium species, such as Gossypium herbaceum or Gossypium arboreum, or an AD genome allotetraploid Gossypium species, such as Gossypium hirsutum and Gossypium barbadense, and the superior fiber strength allele is derived from a different A or AD genome Gossypium species.

[0017] In another embodiment, the plant is a Gossypium hirsutum, a Gossypium herbaceum or a Gossypium arboreum plant, preferably a Gossypium hirsutum plant, and the superior fiber strength allele is derived from Gossypium barbadense.

[0018] In one aspect, the Gossypium barbadense fiber strength allele is located on chromosome A05 of Gossypium barbadense between AFLP marker P5M50-M126.7 and SSR marker CIR280. In another aspect, between AFLP marker P5M50-M126.7 and SSR marker BNL3992. In still another aspect, between AFLP marker P5M50-M126.7 and SSR marker CIR401c. In yet another aspect, is the LOD peak of the Gossypium barbadense fiber strength allele located between SSR marker NAU861 or the GLUC1.1 gene and SSR marker CIR401c. In a further aspect, is the LOD peak of the Gossypium barbadense fiber strength allele located at about 0 to 5 cM, more specifically at about 4.008 cM, from SSR marker NAU861 or the GLUC1.1 gene. In still a further aspect, is the LOD peak of the Gossypium barbadense fiber strength allele is located at about 0 to 12 cM, more specifically at about 10 cM, especially at about 10.52 cM, from SSR marker CIR401c.

[0019] In another aspect, the Gossypium barbadense fiber strength allele comprises at least one Gossypium barbadense ortholog of a nucleotide sequence comprised in the genomic DNA sequence spanning the Gossypium hirsutum GLUC1.1A gene represented in SEQ ID NO: 53.

[0020] In still another aspect, the Gossypium barbadense fiber strength allele comprises a GLUC1.1 gene encoding a non-functional GLUC1.1 protein. In one aspect, the Gossypium barbadense GLUC1.1 gene is characterised by the presence of a T nucleotide at a nucleotide position corresponding to nucleotide position 712 of SEQ ID NO: 5. In a further aspect, the Gossypium barbadense GLUC1.1 gene is located at about 0 to 5 cM, more specifically at about 4 cM, from the LOD peak of the Gossypium barbadense fiber strength allele. In yet a further aspect, the Gossypium barbadense GLUC1.1 gene is located at about 0 to 2 cM, at about 0 to 1 cM, more specifically at about 0.008 cM of the NAU861 marker.

[0021] In yet another embodiment, the plant is a Gossypium hirsutum, Gossypium barbadense, a Gossypium herbaceum or a Gossypium arboreum plant, preferably a Gossypium hirsutum plant, and the superior fiber strength allele is derived from Gossypium darwinii. In one aspect, the Gossypium darwinii fiber strength allele comprises a GLUC1.1 gene encoding a non-functional GLUC1.1 protein. In another aspect, the Gossypium darwinii GLUC1.1 gene is characterised by the presence of a T nucleotide at a nucleotide position corresponding to nucleotide position 761 of SEQ ID NO: 56.

[0022] In still another embodiment, the plant is a Gossypium hirsutum, Gossypium barbadense or a Gossypium herbaceum plant, preferably a Gossypium hirsutum plant, and the superior fiber strength allele is derived from Gossypium arboreum. In one aspect, the Gossypium arboreum fiber strength allele comprises a GLUC1.1 gene encoding a non-functional GLUC1.1 protein. In another aspect, the Gossypium arboreum GLUC1.1 gene is characterised by the abscence of a C nucleotide at a nucleotide position corresponding to the nucleotide position between position 327 and 328 of SEQ ID NO: 21.

[0023] In a further embodiment, the callose content of the fibers is increased in the plant compared to the callose content of the fibers of an equivalent Gossypium plant that does not comprise the at least one superior allele of the fiber strength locus.

[0024] In yet a further embodiment, the strength of the fibers is increased in the plant compared to the strength of the fibers of an equivalent Gossypium plant that does not comprise the at least one superior allele of the fiber strength locus. In one aspect, the strength of the fibers is on average between about 5% and about 10%, preferably about 7.5%, higher. In another aspect, the strength of the fibers is on average between about 1.6 g/tex and about 3.3 g/tex, preferably about 2.5 g/tex, higher. In still another aspect, the strength of the fibers is on average between about 34.6 g/tex and about 36.3 g/tex, preferably about 35.5 g/tex.

[0025] In another embodiment, the plant is a Gossypium hirsutum plant homozygous for the Gossypium barbadense fiber strength allele.

[0026] In still another embodiment, the invention provides a fiber obtainable from the plant of any one of paragraphs 13 to 23.

[0027] In a further embodiment, the invention provides a method of identifying a Gossypium barbadense allele of a fiber strength locus on chromosome A05 in a plant, preferably a Gossypium plant, such as a Gossypium hirsitum plant, comprising the step of determining the presence of a Gossypium barbadense allele of a marker linked to the fiber strength locus in the genomic DNA of the plant selected from the group consisting of: AFLP marker P5M50-M126.7, SSR marker CIR280, SSR marker BNL3992, SSR marker CIR401c, SSR marker NAU861, a polymorphic site in an ortholog of a nucleotide sequence comprised in the genomic DNA sequence spanning a Gossypium hirsutum GLUC1.1A gene represented in SEQ ID NO: 53 of the plant; and a polymorphic site in a nucleotide sequence of a GLUC1.1A gene of the plant, such as SNP marker GLUC1.1A-SNP2 located at a nucleotide position corresponding to nucleotide position 418 to 428 in SEQ ID NO: 5, SNP marker GLUC1.1A-SNP3 located at a nucleotide position corresponding to nucleotide position 573 in SEQ ID NO: 5, SNP marker GLUC1.1A-SNP5 located at a nucleotide position corresponding to nucleotide position 712 in SEQ ID NO: 5, SNP marker GLUC1.1A-SNP6 located at a nucleotide position corresponding to nucleotide position 864 in SEQ ID NO: 5 or SNP marker GLUC1.1A-SNP8 located at a nucleotide position corresponding to nucleotide position 832 in SEQ ID NO: 5.

[0028] In a particular aspect, the Gossypium barbadense allele of AFLP marker P5M50-M126.7 is detected by amplification of a DNA fragment of about 126.7 bp with at least two primers comprising at their extreme 3' end SEQ ID NO: 43 and 44, respectively; the Gossypium barbadense allele of SSR marker CIR280 is detected by amplification of a DNA fragment of about 205 bp with at least two primers comprising at their extreme 3' end SEQ ID NO: 51 and 52, respectively; the Gossypium barbadense allele of SSR marker BNL3992 is detected by amplification of a DNA fragment of about 140 bp to about 145 bp with at least two primers comprising at their extreme 3' end SEQ ID NO: 49 and 50, respectively; the Gossypium barbadense allele of SSR marker CIR401c is detected by amplification of a DNA fragment of about 245 to about 250 bp with at least two primers comprising at their extreme 3' end SEQ ID NO: 47 and 48, respectively; the Gossypium barbadense allele of SSR marker NAU861 is detected by amplification of a DNA fragment of about 215 bp to about 220 bp with at least two primers comprising at their extreme 3' end SEQ ID NO: 45 and 46, respectively; the Gossypium barbadense allele of SNP marker GLUC1.1A-SNP2 is detected by detecting a CTCATCAAA nucleotide sequence at a position corresponding to the position of SNP marker GLUC1.1A-SNP2 or by amplification of a DNA fragment of about 143 bp with at least two primers comprising at their extreme 3' end SEQ ID NO: 37 and 38, respectively; the Gossypium barbadense allele of SNP marker GLUC1.1A-SNP3 is detected by detecting a C nucleotide at a position corresponding to the position of SNP marker GLUC1.1A-SNP3; the Gossypium barbadense allele of SNP marker GLUC1.1A-SNP5 is detected by detecting a T nucleotide at a position corresponding to the position of SNP marker GLUC1.1A-SNP5; the Gossypium barbadense allele of SNP marker GLUC1.1A-SNP6 is detected by detecting an A nucleotide at a position corresponding to the position of SNP marker GLUC1.1A-SNP6; the Gossypium barbadense allele of SNP marker GLUC1.1A-SNP8 is detected by detecting a C nucleotide at a position corresponding to the position of SNP marker GLUC1.1A-SNP8.

[0029] In a further embodiment, the invention provides a method of identifying a Gossypium darwinii allele of a fiber strength locus on chromosome A05 in a plant, preferably a Gossypium plant, such as a Gossypium hirsitum plant, comprising the step of determining the presence of a Gossypium darwinii specific polymorphic site in a nucleotide sequence of a GLUC1.1A gene in the genomic DNA of the plant corresponding to the nucleotide sequence of a GLUC1.1A gene of SEQ ID NO: 56, such as SNP marker GLUC1.1A-SNP2 located at a nucleotide position corresponding to nucleotide position 476 to 477 in SEQ ID NO: 56, SNP marker GLUC1.1A-SNP3 located at a nucleotide position corresponding to nucleotide position 622 in SEQ ID NO: 56, SNP marker GLUC1.1A-SNP5 located at a nucleotide position corresponding to nucleotide position 761 in SEQ ID NO: 56, SNP marker GLUC1.1A-SNP6 located at a nucleotide position corresponding to nucleotide position 913 in SEQ ID NO: 56 or SNP marker GLUC1.1A-SNP8 located at a nucleotide position corresponding to nucleotide position 881 in SEQ ID NO: 56.

[0030] In a particular aspect, the Gossypium darwinii allele of SNP marker GLUC1.1A-SNP2 is detected by detecting a CTCATCAAA nucleotide sequence at a position corresponding to the position of SNP marker GLUC1.1A-SNP2 or by amplification of a DNA fragment of about 143 bp with at least two primers comprising at their extreme 3' end SEQ ID NO: 37 and 38, respectively; the Gossypium darwinii allele of SNP marker GLUC1.1A-SNP3 is detected by detecting a C nucleotide at a position corresponding to the position of SNP marker GLUC1.1A-SNP3; the Gossypium darwinii allele of SNP marker GLUC1.1A-SNP5 is detected by detecting a T nucleotide at a position corresponding to the position of SNP marker GLUC1.1A-SNP5; the Gossypium darwinii allele of SNP marker GLUC1.1A-SNP6 is detected by detecting an A nucleotide at a position corresponding to the position of SNP marker GLUC1.1A-SNP6, and the Gossypium darwinii allele of SNP marker GLUC1.1A-SNP8 is detected by detecting a G nucleotide at a position corresponding to the position of SNP marker GLUC1.1A-SNP8.

[0031] In a further embodiment, the invention provides a method of identifying a Gossypium arboreum allele of a fiber strength locus on chromosome A05 in a plant, preferably a Gossypium plant, such as a Gossypium hirsitum plant, comprising the step of determining the presence of a Gossypium arboreum specific polymorphic site in a nucleotide sequence of a GLUC1.1A gene in the genomic DNA of the plant corresponding to the nucleotide sequence of a GLUC1.1A gene of SEQ ID NO: 21, such as SNP marker GLUC1.1A-SNP7 located at a nucleotide position corresponding to a nucleotide position between nucleotide position 327 and 328 in SEQ ID NO: 21. In a particular aspect, the Gossypium arboreum allele of SNP marker GLUC1.1A-SNP7 is detected by detecting the absence of a C nucleotide at a position corresponding to the position of SNP marker GLUC1.1A-SNP7.

[0032] In a further embodiment, the invention provides a method of distinguishing a Gossypium barbadense allele of a fiber strength locus on chromosome A05 from a Gossypium hirsutum allele of the fiber strength locus in a plant, preferably a Gossypium plant, such as a Gossypium hirsitum plant, comprising the step of determining the presence of Gossypium barbadense alleles and/or Gossypium hirsutum alleles of markers linked to the fiber strength locus in the genomic DNA of the plant selected from the group consisting of: AFLP marker P5M50-M126.7, SSR marker CIR280, SSR marker BNL3992, SSR marker CIR401, SSR marker NAU861; a polymorphic site in an ortholog of a nucleotide sequence comprised in the genomic DNA sequence spanning the Gossypium hirsutum GLUC1.1A gene represented in SEQ ID NO: 53 of the plant; and a polymorphic site in a nucleotide sequence of a GLUC1.1A gene in the genomic DNA of the plant, such as SNP marker GLUC1.1A-SNP2 located at a nucleotide position corresponding to nucleotide position 418 to 428 in SEQ ID NO: 5, SNP marker GLUC1.1A-SNP3 located at a nucleotide position corresponding to nucleotide position 573 in SEQ ID NO: 5, SNP marker GLUC1.1A-SNP5 located at a nucleotide position corresponding to nucleotide position 712 in SEQ ID NO: 5, SNP marker GLUC1.1A-SNP6 located at a nucleotide position corresponding to nucleotide position 864 in SEQ ID NO: 5 or SNP marker GLUC1.1A-SNP8 located at a nucleotide position corresponding to nucleotide position 832 in SEQ ID NO: 5.

[0033] In a particular aspect, the Gossypium hirsutum allele is distinguished from the Gossypium barbadense allele of AFLP marker P5M50-M126.7 by amplification of, respectively, no DNA fragment and a DNA fragment of about 126.7 bp with at least two primers comprising at their extreme 3' end SEQ ID NO: 43 and 44, respectively; the Gossypium hirsutum allele is distinguished from the Gossypium barbadense allele of SSR marker CIR280 by amplification of, respectively, no DNA fragment and a DNA fragment of about 205 bp with at least two primers comprising at their extreme 3' end SEQ ID NO: 51 and 52, respectively; the Gossypium hirsutum allele is distinguished from the Gossypium barbadense allele of SSR marker BNL3992 by amplification of, respectively, two DNA fragments, one of about 160 bp to about 165 bp and one of about 85 bp to about 90 bp, and a DNA fragment of about 140 bp to about 145 bp with at least two primers comprising at their extreme 3' end SEQ ID NO: 49 and 50, respectively; the Gossypium hirsutum allele is distinguished from the Gossypium barbadense allele of SSR marker CIR401 by amplification of, respectively, a DNA fragment of about 255 bp (CIR401b) and a DNA fragment of about 245 bp to about 250 bp (CIR401c) with at least two primers comprising at their extreme 3' end SEQ ID NO: 47 and 48, respectively; the Gossypium hirsutum allele is distinguished from the Gossypium barbadense allele of SSR marker NAU861 by amplification of, respectively, a DNA fragment of about 205 bp to about 210 bp and a DNA fragment of about 215 bp to about 220 bp with at least two primers comprising at their extreme 3' end SEQ ID NO: 45 and 46, respectively; the Gossypium hirsutum allele is distinguished from the Gossypium barbadense allele of SNP marker GLUC1.1A-SNP2 by detecting, respectively, no nucleotide or a CTCATCAAA nucleotide sequence at a position corresponding to the position of SNP marker GLUC1.1A-SNP2, or by amplification of, respectively, a DNA fragment of about 134 bp and a DNA fragment of about 143 bp with at least two primers comprising at their extreme 3' end SEQ ID NO: 37 and 38, respectively; the Gossypium hirsutum allele is distinguished from the Gossypium barbadense allele of SNP marker GLUC1.1A-SNP3 by detecting, respectively, a G or a C nucleotide at a position corresponding to the position of SNP marker GLUC1.1A-SNP3; the Gossypium hirsutum allele is distinguished from the Gossypium barbadense allele of SNP marker GLUC1.1A-SNP5 by detecting, respectively, a C or a T nucleotide at a position corresponding to the position of SNP marker GLUC1.1A-SNP5; the Gossypium hirsutum allele is distinguished from the Gossypium barbadense allele of SNP marker GLUC1.1A-SNP6 by detecting, respectively, a G or an A nucleotide at a position corresponding to the position of SNP marker GLUC1.1A-SNP6; and the Gossypium hirsutum allele is distinguished from the Gossypium barbadense allele of SNP marker GLUC1.1A-SNP8 by detecting, respectively, a G or a C nucleotide at a position corresponding to the position of SNP marker GLUC1.1A-SNP8.

[0034] In another embodiment, the invention provides a method for generating and/or selecting a non-naturally occurring Gossypium plant, and parts and progeny thereof, comprising at least one superior allele of a fiber strength locus on chromosome A05, wherein the superior fiber strength allele is derived from Gossypium barbadense, comprising the steps of crossing a plant from an A genome diploid Gossypium species, such as Gossypium herbaceum or Gossypium arboreum, or an AD genome allotetraploid Gossypium species, such as Gossypium hirsutum, with a Gossypium barbadense plant, and identifying the Gossypium barbadense fiber strength allele according to paragraph 25 or 26.

[0035] In another embodiment, the invention provides a method for generating and/or selecting a non-naturally occurring Gossypium plant, and parts and progeny thereof, comprising at least one superior allele of a fiber strength locus on chromosome A05, wherein the superior fiber strength allele is derived from Gossypium darwinii, comprising the steps of crossing a plant from an A genome diploid Gossypium species, such as Gossypium herbaceum or Gossypium arboreum, or an AD genome allotetraploid Gossypium species, such as Gossypium hirsutum or Gossypium barbadense, with a Gossypium darwinii plant, and identifying the Gossypium darwinii fiber strength allele according to paragraph 27 or 28.

[0036] In another embodiment, the invention provides a method for generating and/or selecting a non-naturally occurring Gossypium plant, and parts and progeny thereof, comprising at least one superior allele of a fiber strength locus on chromosome A05, wherein the superior fiber strength allele is derived from Gossypium arboreum, comprising the steps of crossing a plant from an A genome diploid Gossypium species, such as Gossypium herbaceum, or an AD genome allotetraploid Gossypium species, such as Gossypium hirsutum or Gossypium barbadense, with a Gossypium arboreum plant, and identifying the Gossypium arboreum fiber strength allele according to paragraph 29.

[0037] In still another embodiment, the invention provides a method for altering the callose content of a fiber in a Gossypium plant, particularly increasing the callose content of a fiber, comprising the steps of: introgressing a superior allele of the fiber strength locus on chromosome A05 in the Gossypium plant according to any one of paragraph 32 to 34, and selecting a plant with an altered callose content in its fibers, in particular an increased callose content.

[0038] In yet another embodiment, the invention provides a method for altering the properties of a fiber in a Gossypium plant, particularly increasing the strength of a fiber, comprising the steps of: introgressing a superior allele of the fiber strength locus on chromosome A05 in the Gossypium plant according to any one of paragraph 32 to 34, selecting a plant with an altered fiber strength, in particular an increased fiber strength.

[0039] In a further embodiment, the invention provides a kit for of identifying a Gossypium barbadense allele of a fiber strength locus on chromosome A05 or for distinguishing a Gossypium barbadense allele of a fiber strength locus on chromosome A05 from a Gossypium hirsutum allele of the fiber strength locus in a plant, preferably a Gossypium plant, such as a Gossypium hirsitum plant, comprising primers and/or probes for determining the presence of Gossypium barbadense alleles and/or Gossypium hirsutum alleles of markers linked to the fiber strength locus in the genomic DNA of the plant selected from the group consisting of: AFLP marker P5M50-M126.7, SSR marker CIR280, SSR marker BNL3992, SSR marker CIR401, SSR marker NAU861, a polymorphic site in an ortholog of a nucleotide sequence comprised in the genomic DNA sequence spanning the Gossypium hirsutum GLUC1.1A gene represented in SEQ ID NO: 53, and a polymorphic site in a nucleotide sequence of the GLUC1.1A gene in the genomic DNA of the plant, such as SNP marker GLUC1.1A-SNP2 located at a nucleotide position corresponding to nucleotide position 418 to 428 in SEQ ID NO: 5, SNP marker GLUC1.1A-SNP3 located at a nucleotide position corresponding to nucleotide position 573 in SEQ ID NO: 5, SNP marker GLUC1.1A-SNP5 located at a nucleotide position corresponding to nucleotide position 712 in SEQ ID NO: 5, SNP marker GLUC1.1A-SNP6 located at a nucleotide position corresponding to nucleotide position 864 in SEQ ID NO: 5 or SNP marker GLUC1.1A-SNP8 located at a nucleotide position corresponding to nucleotide position 832 in SEQ ID NO: 5.

[0040] In one aspect, the kit comprises at least two primers and/or probes selected from the group consisting of: primers comprising at their extreme 3' end SEQ ID NO: 43 and 44, respectively; primers comprising at their extreme 3' end SEQ ID NO: 51 and 52, respectively; primers comprising at their extreme 3' end SEQ ID NO: 49 and 50, respectively; primers comprising at their extreme 3' end SEQ ID NO: 47 and 48, respectively; primers comprising at their extreme 3' end SEQ ID NO: 45 and 46, respectively; primers comprising at their extreme 3' end SEQ ID NO: 37 and 38, respectively.

[0041] The inventors have further found that the properties of fibers in cotton plants can be controlled by controlling the number of endo-1,3-beta-glucanase genes/alleles that are "functionally expressed", i.e. that result in functional (biologically active) endo-1,3-beta-glucanase protein (GLUC), in fibers during the secondary cell wall synthesis phase and the maturation phase, herein commonly referred to as fiber strength building phase, of fiber development. By abolishing the functional expression of a number of endo-1,3-beta-glucanase genes/alleles that are functionally expressed in fibers during the fiber strength building phase, in particular during the maturation phase, of fiber development, such as the A-subgenome specific endo-1,3-beta-glucanase gene in G. hirsutum, while maintaining the functional expression of a number of such endo-1,3-beta-glucanase genes/alleles, such as the D-subgenome specific endo-1,3-beta-glucanase gene in G. hirsutum, it is believed that the degradation of callose can be decreased to a level allowing a higher fiber strength, while maintaining a level of callose degradation sufficient to obtain an industrially relevant fiber length.

[0042] Thus, in another aspect, the present invention provides a non-naturally occurring fiber-producing plant, and parts and progeny thereof, characterized in that the functional expression of at least one allele of at least one fiber-specific GLUC gene that is functionally expressed during the fiber strength building phase, in particular the fiber maturation phase, of fiber development is abolished. Such plants, and parts and progeny thereof, can be used for obtaining plants with modified callose content and/or modified fiber properties, in particular for obtaining fiber-producing plants with increased callose content in the fibers and/or increased fiber strength that preferably maintain an industrially relevant fiber length. As used herein, "plant part" includes anything derived from a plant of the invention, including plant parts such as cells, tissues, organs, seeds, fibers, seed fats or oils.

[0043] In one embodiment, the GLUC gene is a GLUC1.1 gene encoding a GLUC protein that has at least 90% sequence identity to SEQ ID NO: 4.

[0044] In another embodiment, the plant is a Gossypium plant, wherein the GLUC gene is a GLUC1.1A gene encoding a GLUC protein that has at least 97% sequence identity to SEQ ID NO: 4 or a GLUC1.1D gene encoding a GLUC protein that has at least 97% sequence identity to SEQ ID NO: 10, preferably the GLUC1.1A gene.

[0045] In still another embodiment, the plant is a Gossypium hirsutum plant.

[0046] In a further embodiment, the amount of functional GLUC protein is significantly reduced in fibers during the fiber strength building phase, in particular the fiber maturation phase, of fiber development in the plant compared to the amount of functional GLUC protein produced in fibers during the fiber strength building phase, in particular the fiber maturation phase, of fiber development in a plant in which the functional expression of the at least one GLUC allele is not abolished.

[0047] In still a further embodiment, the callose content is significantly increased in fibers of the plant compared to the callose content in fibers in a plant in which the functional expression of the at least one GLUC allele is not abolished.

[0048] In yet a further embodiment, the strength of the fibers is significantly increased compared to the strength of the fibers in a plant in which the functional expression of the at least one GLUC allele is not abolished. In one aspect, the strength of the fibers is on average between about 5% and about 10%, preferably about 7.5%, higher. In another aspect, the strength of the fibers is on average between about 1.6 g/tex and about 3.3 g/tex, preferably about 2.5 g/tex, higher. In still another aspect, the strength of the fibers is on average between about 34.6 g/tex and about 36.3 g/tex.

[0049] In still a further embodiment, the plant is a Gossypium hirsutum plant characterized in that the functional expression of at least two alleles of at least one fiber-specific GLUC gene is abolished.

[0050] In another embodiment, the present invention provides a fiber obtainable from the fiber-producing plant of any one of paragraphs 40 to 47.

[0051] In a further embodiment, the present invention provides a nucleic acid molecule encoding a non-functional GLUC1.1 protein having an amino acid sequence wherein at least one amino acid residue similar to the active site residues or to the glycosylation site residues of the GLUC1.1 protein of SEQ ID NO: 4 is lacking or is substituted for a non-similar amino acid residue. In one aspect, the active site residues of the GLUC1.1 protein of SEQ ID NO: 4 are selected from the group consisting of Tyr48, Glu249, Trp252, and Glu308, and wherein the glycosylation site residue of the GLUC1.1 protein of SEQ ID NO: 4 is Asn202. In another aspect, the non-functional GLUC1.1 protein comprises an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 6, SEQ ID NO: 18, SEQ ID NO: 57 or SEQ ID NO: 22. In another aspect, the nucleic acid molecule comprises a nucleic acid sequence having at least 92% sequence identity to SEQ ID NO: 3 from nucleotide 101 to 1078, wherein at least one nucleic acid residue is deleted, inserted or substituted. In yet another aspect, the nucleic acid molecule comprises a nucleotide sequence at least 92% identical to the nucleic acid sequence of SEQ ID NO: 54 from nucleotide 50 to 589. In still a further aspect, the nucleic acid molecule comprises the nucleic acid sequence of SEQ ID NO: 54 from nucleotide 50 to 589. In still another aspect, the nucleic acid molecule comprises a nucleic acid sequence having at least 92% sequence identity to SEQ ID NO: 1 from nucleotide 2410 to 3499, wherein at least one nucleic acid residue is deleted, inserted or substituted. In yet another aspect, the nucleic acid molecule comprises a nucleotide sequence at least 92% identical to the nucleic acid sequence of SEQ ID NO: 5 from nucleotide 63 to 711, SEQ ID NO: 17 from nucleotide 2 to 472, SEQ ID NO: 56 from nucleotide 112 to 760 or SEQ ID NO: 21 from nucleotide 27 to 372. In still a further aspect, the nucleic acid molecule comprises the nucleic acid sequence of SEQ ID NO: 5 from nucleotide 63 to 711, SEQ ID NO: 17 from nucleotide 2 to 472, SEQ ID NO: 56 from nucleotide 112 to 760, or SEQ ID NO: 21 from nucleotide 27 to 372.

[0052] In another embodiment, the present invention provides a non-functional GLUC1.1 protein encoded by the nucleic acid molecule of paragraph 49.

[0053] In still another embodiment, the present invention provides a method for identifying a GLUC1.1 gene encoding a non-functional GLUC1.1 protein in a plant, preferably a Gossypium plant, such as a Gossypium hirsitum plant, said GLUC1.1 gene comprising a nucleic acid sequence having at least 92% sequence identity to SEQ ID NO: 1 from nucleotide 2410 to 3499, comprising the step of identifying a polymorphic site in the nucleotide sequence of the GLUC1.1 gene in the genomic DNA of the plant that results in the production of a non-functional GLUC1.1 protein. In one aspect, the present invention provides a method for identifying a GLUC1.1 gene from Gossypium barbadense or from Gossypium darwinii comprising the step of identifying a T nucleotide at a nucleotide position corresponding to nucleotide position 3050 in SEQ ID NO: 1. In another aspect, the present invention provides a method for identifying a GLUC1.1 gene from Gossypium arboreum comprising the step of identifying a deletion of a C nucleotide at a nucleotide position corresponding to nucleotide position 2674, 2675 or 2676 in SEQ ID NO: 1.

[0054] In a further embodiment, the present invention provides a method of distinguishing a GLUC1.1 gene encoding a non-functional GLUC1.1 protein from a GLUC1.1 gene encoding a functional GLUC1.1 protein, said GLUC1.1 genes both comprising a nucleic acid sequence having at least 92% sequence identity to SEQ ID NO: 1 from nucleotide 2410 to 3499, comprising the step of identifying a polymorphic site in the nucleotide sequences of the GLUC1.1 genes. In one aspect, the present invention provides a method of distinguishing a GLUC1.1 from Gossypium barbadense, from Gossypium darwinii or from Gossypium arboreum from a GLUC1.1 gene from Gossypium hirsutum, respectively, comprising the step of identifying a polymorphic site selected from the group consisting of: polymorphic sequence marker GLUC1.1A-SNP2 located between the nucleotide at position 2765 and 2766 in SEQ ID NO: 1, SNP marker GLUC1.1A-SNP3 located at nucleotide position 2911 in SEQ ID NO: 1, SNP marker GLUC1.1A-SNP5 located at nucleotide position 3050 in SEQ ID NO: 1, SNP marker GLUC1.1A-SNP6 located at nucleotide position 3202 in SEQ ID NO: 1, SNP marker GLUC1.1A-SNP7 located at nucleotide position 2674, 2675 or 2676 in SEQ ID NO: 1 and SNP marker GLUC1.1A-SNP8 located at nucleotide position 3170 in SEQ ID NO: 1. In another aspect, polymorphic sequence marker GLUC1.1A-SNP2 from Gossypium barbadense or Gossypium darwinii and from Gossypium hirsutum, respectively, is detected by amplification of a DNA fragment of about 143 bp and about 134 bp, respectively, with primers comprising at their extreme 3' end SEQ ID NO: 37 and 38, respectively. In still another aspect, SNP marker GLUC1.1A-SNP3 from Gossypium barbadense or Gossypium darwinii and from Gossypium hirsutum, respectively, is detected by amplification of a DNA fragment of about 57 bp with primers comprising SEQ ID NO: 41 and 42 and detection of the DNA fragment with fluorescently labeled probes comprising SEQ ID NO: 39 and 40, respectively.

[0055] In a further embodiment, the present invention provides a method for generating and/or selecting a non-naturally occurring fiber-producing plant, and parts and progeny thereof, wherein the functional expression of at least one allele of at least one fiber-specific GLUC gene that is functionally expressed during the fiber strength building phase, in particular the fiber maturation phase, of fiber development is abolished, comprising the step of: mutagenizing at least one allele of the GLUC gene, or introgressing at least one allele of a non-functionally expressed ortholog of the GLUC gene or at least one allele of a mutagenized GLUC gene, or introducing a chimeric gene comprises the following operably linked DNA elements: (a) a plant expressible promoter, (b) a transcribed DNA region, which when transcribed yields an inhibitory RNA molecule capable of reducing the expression of the GLUC allele, and (c) a 3' end region comprising transcription termination and polyadenylation signals functioning in cells of the plant. In one aspect, the GLUC gene is a GLUC1.1 gene encoding a GLUC protein that has at least 90% sequence identity to SEQ ID NO: 4. In another aspect, the fiber-producing plant is a Gossypium plant, and the GLUC gene is a GLUC1.1A gene encoding a GLUC protein that has at least 97% sequence identity to SEQ ID NO: 4 or a GLUC1.1D gene encoding a GLUC protein that has at least 97% sequence identity to SEQ ID NO: 9, preferably a GLUC1.1A gene. In still another aspect, the fiber-producing plant is a Gossypium plant, and the non-functionally expressed ortholog of the GLUC gene is a GLUC1.1A gene which is derived from a Gossypium barbadense, from a Gossypium darwinii or a Gossypium arboreum plant, preferably from a Gossypium barbadense. In a further aspect, the method further comprises the step of identifying the non-functionally expressed ortholog of the GLUC gene or the mutagenized GLUC gene according to the method of paragraph 51.

[0056] In a further embodiment, the present invention provides a method for altering the callose content of a fiber in a fiber-producing plant, particularly increasing the callose content of a fiber, comprising the steps of: generating and/or selecting a non-naturally occurring fiber-producing plant, and parts and progeny thereof, wherein the functional expression of at least one allele of at least one fiber-specific GLUC gene that is functionally expressed during the fiber strength building phase, in particular the fiber maturation phase, of fiber development is abolished, according to the method of paragraph 53, and selecting a plant with an altered callose content in its fibers, in particular an increased callose content.

[0057] In a further embodiment, the present invention provides a method for altering the properties of a fiber in a fiber-producing plant, particularly increasing the strength of a fiber, comprising the steps of: generating and/or selecting a non-naturally occurring fiber-producing plant, and parts and progeny thereof, wherein the functional expression of at least one allele of at least one fiber-specific GLUC gene that is functionally expressed during the fiber strength building phase, in particular the fiber maturation phase, of fiber development is abolished, according to the method of paragraph 53, and selecting a plant with an altered fiber strength, in particular an increased fiber strength.

[0058] In another embodiment, the present invention provides a kit for identifying a GLUC1.1 gene encoding a non-functional GLUC1.1 protein in a plant, said GLUC1.1 gene comprising a nucleic acid sequence having at least 92% sequence identity to SEQ ID NO: 1 from nucleotide 2410 to 3499, comprising primers and/or probes for determining the presence of a polymorphic site in the nucleotide sequence of the GLUC1.1 gene in the genomic DNA of the plant that results in the production of a non-functional GLUC1.1 protein. In one aspect, the kit comprises primers and/or probes for determining the presence of a T nucleotide at a nucleotide position corresponding to nucleotide position 3050 in SEQ ID NO: 1 or for determining a deletion of a C nucleotide at a nucleotide position corresponding to nucleotide position 2674, 2675 or 2676 in SEQ ID NO: 1.

[0059] In still another embodiment, the present invention provides a kit for distinguishing a GLUC1.1 gene encoding a non-functional GLUC1.1 protein from a GLUC1.1 gene encoding a functional GLUC1.1 protein, said GLUC1.1 genes both comprising a nucleic acid sequence having at least 92% sequence identity to SEQ ID NO: 1 from nucleotide 2410 to 3499, comprising primers and/or probes for determining the presence of a polymorphic site in the nucleotide sequences of the GLUC1.1 genes. In one aspect, the present invention provides a kit comprising primers and/or probes for distinghuishing Gossypium barbadense, Gossypium darwinii or Gossypium arboreum specific alleles from Gossypium hirsutum specific alleles of a polymorphic site selected from the group consisting of: polymorphic sequence marker GLUC1.1A-SNP2 located between the nucleotide at position 2765 and 2766 in SEQ ID NO: 1, SNP marker GLUC1.1A-SNP3 located at nucleotide position 2911 in SEQ ID NO: 1, SNP marker GLUC1.1A-SNP5 located at nucleotide position 3050 in SEQ ID NO: 1, SNP marker GLUC1.1A-SNP6 located at nucleotide position 3202 in SEQ ID NO: 1, SNP marker GLUC1.1A-SNP7 located at nucleotide position 2674, 2675 or 2676 in SEQ ID NO: 1 and SNP marker GLUC1.1A-SNP8 located at nucleotide position 3170 in SEQ ID NO: 1. In another aspect, the kit comprises at least two primers and/or probes selected from the group consisting of: primers comprising at their extreme 3' end SEQ ID NO: 37 and 38, respectively, to identify polymorphic sequence marker GLUC1.1A-SNP2, primers comprising SEQ ID NO: 41 and 42, respectively, to identify SNP marker GLUC1.1A-SNP3, probes comprising SEQ ID NO: 39 and 40, respectively, to identify SNP marker GLUC1.1A-SNP3, primers comprising SEQ ID NO: 62 and 63, respectively, to identify SNP marker GLUC1.1A-SNP5, and probes comprising SEQ ID NO: 60 and 61, respectively, to identify SNP marker GLUC1.1A-SNP5.

BRIEF DESCRIPTION OF THE FIGURES

[0060] FIG. 1A, FIG. 1B, FIG. 1C, FIG. 1D, and FIG. 1E: Alignment of genomic and cDNA sequences of A and D subgenome-specific GLUC1.1 genes from Gossypium hirsutum (`GhGLUC1.1A-gDNA` corresponds to SEQ ID NO: 1 from nucleotide 2246 to 3753, `GhGLUC1.1A-cDNA` corresponds to SEQ ID NO: 3, `GhGLUC1.1D-gDNA` corresponds to SEQ ID NO: 7 from nucleotide 3206 to 4694, and `GhGLUC1.1D-cDNA` corresponds to SEQ ID NO: 9) and Gossypium barbadense (`GbGLUC1.1A-gDNA` corresponds to SEQ ID NO: 5, `GbGLUC1.1A-cDNA` corresponds to SEQ ID NO: 54, `GbGLUC1.1D-gDNA` corresponds to SEQ ID NO: 11, and `GbGLUC1.1D-cDNA` corresponds to SEQ ID NO: 13). The putative TATA box is indicated in bold, the putative start codons and the putative first exons are indicated in bold and in bold with an arrow, respectively, the putative intron and second exon sequences are indicated in regular with an arrow, the putative intron sequences are further indicated between `I`, the putative (premature) STOP codons are indicated in italic and underlined.

[0061] FIG. 2: Alignment of amino acid sequences of A and D subgenome-specific GLUC1.1 proteins from Gossypium hirsutum (`GhGLUC1.1A` corresponds to SEQ ID NO: 2 and 4 and `GhGLUC1.1D` corresponds to SEQ ID NO: 8 and 10) and Gossypium barbadense (`GbGLUC1.1A` corresponds to SEQ ID NO: 6 and 55 and `GbGLUC1.1D` corresponds to SEQ ID NO: 12 and 14). The putative signal peptide is indicated in italic, the putative post-translational splicing site is indicated as `><`, the GH17 signature is indicated in bold. Amino acids that are identical between at least three of the four sequences are highlighted. The dashed line indicates the protein segment that is missing in GbGLUC1.1A.

[0062] FIG. 3A and FIG. 3B: Protein model of GLUC1.1A protein of G. hirsutum (FIG. 3a; right) and G. barbadense (FIG. 3b; right) based on an X-ray structure of a barley 1,3-1,4-beta-glucanase (1aq0; FIG. 3a&b; left). The active site of 1aq0 is located in an open cleft at the bottom of the barrel defined by the C-terminal ends of the parallel intra-barrel beta-strands (Muller et al., 1998, J. Biol. Chem 273 (6): 3438-3446) and is indicated by the amino acids and their position numbers displayed in the upper left part of the protein model of 1aq0 in FIGS. 3a and b at the left. Active site residues Glu288, Glu232 and Tyr33 in 1aq0 (FIG. 3a, left) correspond to Glu308, Glu249 and Tyr48 in GhGLUC1.1A (FIG. 3a, right) and are absent in GbGLUC1.1A (FIG. 3b, right). The glycosylation site Asn190 in 1aq0 (FIG. 3A, left) corresponds to Asn 202 in GhGLUC1.1A (FIG. 3a, right) and is also absent in GbGLUC1.1A (FIG. 3b, right). FIG. 3b further shows that the threonine, histidine and glutamine amino acids at position 82, 83 and 84 of GbGLUC1.1A (FIG. 3b; right) that are not present in GhGLUC1.1A (see for example FIG. 7) are located in a distant loop which is not part of the active site and not involved in glycosylation.

[0063] FIG. 4: Box plot indicating the difference in fiber strength (as determined by measuring the breaking force of single fibers; indicated in cN on the Y-axis) between untreated fibers (`untreated`) and fibers treated with exogenous glucanase (`treated`) derived from Gossypium hirsutum cultivar FM966 grown in a greenhouse in Europe (`FM966 Astene`), in the field in the US (`FM966 Sellers`) and in the field in Australia (`FM966 Australia`), from Gossypium hirsutum cultivar Coker 312 grown in a greenhouse in Europe (`Coker 312`), from Gossypium barbadense cultivar PimaS7 grown in a greenhouse in Europe (TimaS7'), and from Gossypium barbadense cultivar PimaY5 grown in the field in Australia (TimaY5').

[0064] FIG. 5: Box plot indicating the difference in callose content (as determined by fluorescence measurements of aniline blue stained fibers; indicated as the ratio of green over blue fluorescence on the Y-axis) between untreated fibers (`untreated`) and fibers treated with exogenous glucanase (`treated`) derived from Gossypium hirsutum cultivar FM966 grown in a greenhouse in Europe (`FM966 Astene`), in the field in the US (`FM966 Sellers`) and in the field in Australia (`FM966 Australia`), from Gossypium hirsutum cultivar Coker 312 grown in a greenhouse in Europe (`Coker 312`), from Gossypium barbadense cultivar PimaS7 grown in a greenhouse in Europe (TimaS7'), and from Gossypium barbadense cultivar PimaY5 grown in the field in Australia (`PimaY5`).

[0065] FIG. 6A, FIG. 6B, FIG. 6C, FIG. 6D, FIG. 6E and FIG. 6F: Alignment of genomic DNA sequences of A and D subgenome-specific GLUC1.1 genes from Gossypium hirsutum (`GhGLUC1.1A_gDNA` corresponds to SEQ ID NO: 1 from nucleotide 2348 to 3554 and `GhGLUC1.1D_gDNA` corresponds to SEQ ID NO: 7 from nucleotide 3311 to 4496), Gossypium tomentosum (`GtGLUC1.1A_gDNA` corresponds to SEQ ID NO: 15 and `GtGLUC1.1D_gDNA` corresponds to SEQ ID NO: 25), Gossypium barbadense (`GbGLUC1.1A_gDNA` corresponds to SEQ ID NO: 5 and `GbGLUC1.1D_gDNA` corresponds to SEQ ID NO: 11), Gossypium darwinii (`GdGLUC1.1A_gDNA` corresponds to SEQ ID NO: 17 and `GdGLUC1.1D_gDNA` corresponds to SEQ ID NO: 27), Gossypium mustelinum, (`GmGLUC1.1A_gDNA` corresponds to SEQ ID NO: 19 and `GmGLUC1.1D_gDNA` corresponds to SEQ ID NO: 29), Gossypium arboreum (`GaGLUC1.1A_gDNA` corresponds to SEQ ID NO: 21), Gossypium herbaceum (`GheGLUC1.1A_gDNA` corresponds to SEQ ID NO: 23), and Gossypium raimondii (`GrGLUC1.1D_gDNA` corresponds to SEQ ID NO: 31). The positions of primers SE077 and SE078, used to generate the complete coding sequence from start to stop codon, and the positions of primers SE003 and SE002, used to generate partial coding sequences, are underlined. The putative start codons and the putative first exons are indicated in bold and in bold with an arrow, respectively, the putative intron and second exon sequences are indicated in regular with an arrow, the putative intron sequences are further indicated between `I`, the putative (premature) STOP codons are indicated in italic and underlined. Five polymorphic sites (4 single nucleotide polymorphisms (SNPs) and one extended indel) that exist between the GLUC1.1A or GLUC1.1D sequences of, e.g., G. hirsutum FM966 and G. barbadense Pima S7 or G. darwinii, are indicated with arrows and named `GLUC1.1D-SNP1` and `GLUC1.1A-SNP2, 3, 5 and 6`. Allelic variants are indicated as follows: [G. hirsutum allele/G. barbadense or G. darwinii allele]. One polymorphic site (1 SNP) that exist between the GLUC1.1A sequences of, e.g., G. hirsutum FM966 and G. arboreum is indicated with an arrow and named `GLUC1.1A-SNP7`. Allelic variants are indicated as follows: [G. hirsutum allele/G. arboreum allele]. One polymorphic site (1 SNP) that exist between the GLUC1.1A sequences of, e.g., G. barbadense Pima S7 or G. darwinii is indicated with an arrow and named `GLUC1.1A-SNP8`. Allelic variants are indicated as follows: [G. barbadense allele/G. darwinii allele].

[0066] FIG. 7A and FIG. 7B: Alignment of amino acid sequences of A and D subgenome-specific GLUC1.1 proteins from Gossypium hirsutum (GhGLUC1.1A_prot' corresponds to SEQ ID NO: 2 and 4 and GhGLUC1.1D_prot' corresponds to SEQ ID NO: 8 and 10; full-length sequences), Gossypium tomentosum (GtGLUC1.1A_prot' corresponds to SEQ ID NO: 16 and GtGLUC1.1D_prot' corresponds to SEQ ID NO: 26; partial sequences), Gossypium barbadense (GbGLUC1.1A_prot' corresponds to SEQ ID NO: 6 and 55 and GbGLUC1.1D_prot' corresponds to SEQ ID NO: 12 and 14; full-length sequences), Gossypium darwinii (GdGLUC1.1A_prot' corresponds to SEQ ID NO: 57 and GdGLUC1.1D_prot' corresponds to SEQ ID NO: 59; full-length sequences), Gossypium mustelinum, (GmGLUC1.1A_prot' corresponds to SEQ ID NO: 20 and GmGLUC1.1D_prot' corresponds to SEQ ID NO: 30; partial sequences), Gossypium arboreum (GaGLUC1.1A_prot' corresponds to SEQ ID NO: 22; full-length sequence), Gossypium herbaceum (GheGLUC1.1A_prot' corresponds to SEQ ID NO: 24; full-length sequence), and Gossypium raimondii (GrGLUC1.1D_-- prot' corresponds to SEQ ID NO: 32; partial sequences). The putative signal peptide is indicated in italic, the putative post-translational splicing site is indicated as `><`, the GH17 signature is indicated in bold. Amino acids that differ from the amino acids in the upper sequence, i.e. GhGLUC1.1A_prot, are highlighted.

[0067] FIG. 8: Expression of GLUC1.1A and GLUC1.1D in G. barbadense. DNA from a cDNA library from (developing) fibers in Gossypium barbadense was extracted and equalized. PCR fragments were amplified using oligonucleotide primers SE002 and SE003 (SEQ ID NO: 35 and 36) and digested with restriction enzyme AlwI. A PCR amplified product for GLUC1.1A yields 3 fragments (479 bp+118 bp+59 bp) while for GLUC1.1D it only yields 2 fragments (538 bp+118 bp). The 59 bp fragment is not visible. Lane 1 and 12: 1 kb size markers; lanes 2 to 9: GbGLUC1.1A and D expression at 0, 5, 10, 15, 20, 25, 30 and 40 DPA; lane 10: negative (no template; NTC); lane 11: positive control (genomic DNA from Pima S7).

[0068] FIG. 9: Schematic representation of 165250 bps DNA fragment spanning the GLUC1.1A gene of Gossypium hirsutum (SEQ ID NO: 53). Box: retrotransposon region; *: position of CIR280 homology region; arrow: DNA fragment encoding protein indicated with following abbreviations: SHMT (Serine HydroxyMethylTransferase); GrpE/HSP-70 (GrpE protein/HSP-70 cofactor); ARF17: putative Auxin Response Factor similar to At-ARF17; eIF-5-1: probable eukaryotic translation Initiation Ffactor 5-1; Avr9: putative Avr9 elicitor response protein; VPS9: similar to Vacuolar Protein Sorting-associated protein VPS9; HAT: putative Histon Acetyl Transferase gene; Gluc1.1: GLUC1.1A encoding region; MEKK1: putative Mitogen-activated protein kinase kinase kinase 1; PIP5K1: Phosphatidylinositol-4-Phosphate 5-Kinase 1.

DETAILED EMBODIMENTS

[0069] The current invention is based on the unexpected finding that the presence of the Gossypium barbadense ortholog of a fiber strength locus on chromosome A05, hereinafter called Gossypium barbadense fiber strength allele, in Gossypium hirsutum plants results in an increased strength of the fibers of the Gossypium hirsutum plants compared to the strength of the fibers of Gossypium hirsutum plants comprising the Gossypium hirsutum ortholog of the fiber strength locus.

[0070] Thus, in a first aspect, the present invention provides a non-naturally occurring Gossypium plant, and parts and progeny thereof, comprising at least one superior allele of a quantitative trait locus (QTL) for fiber strength located on chromosome A05.

[0071] As used herein, the term "non-naturally occurring" or "cultivated" when used in reference to a plant, means a plant with a genome that has been modified by man. A transgenic fiber-producing plant, for example, is a non-naturally occurring fiber-producing plant that contains an exogenous nucleic acid molecule, e.g., a chimeric gene comprising a transcribed region which when transcribed yields a biologically active RNA molecule capable of reducing the expression of a GLUC gene according to the invention and, therefore, has been genetically modified by man. In addition, a fiber-producing plant that contains, for example, a mutation in an endogenous GLUC gene (e.g. in a regulatory element or in the coding sequence) as a result of an exposure to a mutagenic agent is also considered a non-naturally occurring fiber-producing plant, since it has been genetically modified by man. Furthermore, a fiber-producing plant of a particular species, such as Gossypium hirsutum, that contains, for example, a mutation in an endogenous GLUC gene that in nature does not occur in that particular plant species, as a result of, for example, directed breeding processes, such as marker-assisted breeding and selection or introgression, with another species of that fiber-producing plant, such as Gossypium barbadense, is also considered a non-naturally occurring fiber-producing plant. In contrast, a fiber-producing plant containing only spontaneous or naturally occurring mutations, i.e. a plant that has not been genetically modified by man, is not a "non-naturally occurring plant" as defined herein and, therefore, is not encompassed within the invention. One skilled in the art understands that, while a non-naturally occurring fiber-producing plant typically has a nucleotide sequence that is altered as compared to a naturally occurring fiber-producing plant, a non-naturally occurring fiber-producing plant also can be genetically modified by man without altering its nucleotide sequence, for example, by modifying its methylation pattern.

[0072] The term "quantitative trait" refers herein to a trait, such as fiber strength, whose phenotypic characteristics vary in degree and can be attributed to the interactions between two or more genes and their environment.

[0073] As used herein, the term "locus" (loci plural) or "site" means a specific place or places on a chromosome where, for example, a gene, a genetic marker or a QTL is found.

[0074] A "quantitative trait locus (QTL)" is a stretch of DNA (such as a chromosome arm, a chromosome region, a nucleotide sequence, a gene, and the like) that is closely linked to a gene that underlies the trait in question. "QTL mapping" involves the creation of a map of the genome using genetic or molecular markers, like AFLP, RAPD, RFLP, SNP, SSR, and the like, visible polymorphisms and allozymes, and determining the degree of association of a specific region on the genome to the inheritance of the trait of interest. As the markers do not necessarily involve genes, QTL mapping results involve the degree of association of a stretch of DNA with a trait rather than pointing directly at the gene responsible for that trait. Different statistical methods are used to ascertain whether the degree of association is significant or not. A molecular marker is said to be "linked" to a gene or locus, if the marker and the gene or locus have a greater association in inheritance than would be expected from independent assortment, i.e. the marker and the locus co-segregate in a segregating population and are located on the same chromosome. "Linkage" refers to the genetic distance of the marker to the locus or gene (or two loci or two markers to each other). The closer the linkage, the smaller the likelihood of a recombination event taking place, which separates the marker from the gene or locus. Genetic distance (map distance) is calculated from recombination frequencies and is expressed in centiMorgans (cM) [Kosambi (1944), Ann. Eugenet. 12:172-175].

[0075] "Fiber strength locus" or "strength locus", as used herein, refers to a stretch of DNA on chromosome A05 of Gossypium species that is closely linked to (a) gene(s) that is(are) involved in the regulation of fiber strength. The "fiber strength locus" is a QTL said to be linked to the "(fiber strength) causal gene(s)".

[0076] A "fiber", such as a "cotton fiber", as used herein, refers to a seed trichome, more specifically a single cell of a fiber-producing plant, such as cotton, that initiates from the epidermis of the outer integument of the ovules, at or just prior to anthesis. The morphological development of cotton fibers has been well documented (Basra and Malik, 1984, Int Rev of Cytology 89: 65-113; Graves and Stewart, 1988, supra; Ramsey and Berlin, 1976, American Journal of Botany 63 (6): 868-876; Ruan and Chourey, 1998, Plant Physiology 118: 399-406; Ruan et al. 2000, Aust. J. Plant Physiol. 27:795-800; Stewart, 1975, Am. J. Bot. 62, 723-730). Cotton fibers, in particular from Gossypium hirsutum, undergo four overlapping developmental stages: fiber cell initiation, elongation, secondary cell wall biosynthesis, and maturation. Fiber cell initiation is a rapid process. White fuzzy fibers begin to develop immediately after anthesis and continue up to about 3 days post-anthesis (DPA), which is followed by fiber cell elongation (until about 10 to about 17 DPA). Depending upon growth conditions, secondary cell wall biosynthesis initiates and continues to about 25 to about 40 DPA, followed by a maturation process until about 45 to about 60 DPA. The secondary cell wall synthesis and maturation phase are herein commonly referred to as "fiber strenght building phase". Only about 25 to 30% of the epidermal cells differentiate into the commercially important lint fibers (Kim and Triplett, 2001). The majority of cells does not differentiate into fibers or develop into short fibers or fuzz. During fiber elongation and secondary wall metabolism, the fiber cells elongate rapidly, synthesize secondary wall components, and show dramatic cellular, molecular and physiological changes. Fiber elongation is coupled with rapid cell growth and expansion (Seagull, 1991. In Biosynthesis and biodegradation of cellulose (Haigler, C. H. & Weimer, P. J., eds) pp. 1432163, MarcelDekker, New York) and constant synthesis of a large amount of cell metabolites and cell wall components such as cellulose. About 95% of the dry-weight in mature cotton fibers is cellulose (Pfluger and Zambryski, 2001, Curr Biol 11: R436-R439; Ruan et al., 2001, Plant Cell 13: 47-63). Non-celluloid components are also important to fiber cell development (Hayashi and Delmer, 1988, Carbohydr. Res. 181: 273-277; Huwyler et al., 1979, Planta 146: 635-642; Meinert and Delmer, 1977, Plant Physiol 59: 1088-1097; Peng et al., 2002, Science 295: 147-150). Compared to other plant cells, cotton fibers do not contain lignin in secondary walls but have large vacuoles that are presumably related to rapid cell growth and expansion (Basra and Malik, 1984, supra; Kim and Triplett, 2001, Plant Physiology 127: 1361-1366; Mauney, 1984, supra; Ruan and Chourey, 1998, supra; Ruan et al., 2000, supra; Van 't Hof, 1999, American Journal of Botany 86: 776-779).

[0077] "Fiber strength", as used herein, can be determined by determining the strength of a bundle of fibers, i.e. "fiber bundle strength", or by determining the strength of single fibers. The higher the single fiber strength and the lower the variations of single fiber breaking elongation, the closer the bundle and yarn tensile strength would be to the sum of single fiber strength; ideally, fiber bundle tenacity would equal the total single fiber breaking tenacity had all fibers within the bundle equal breaking elongation and no slack (Liu et al., February 2005, Textile Res. J).

[0078] "Fiber bundle strength", as used herein, refers to a measure that is usually expressed in terms of grams per tex. This commercial High Volume Instruments (HVI) measure of fiber bundle strength ("HVI strength") is also called "tenacity". A tex unit is equal to the weight in grams of 1,000 meters of fiber. Therefore, the strength reported is the force in grams required to break a bundle of fibers one tex unit in size. Measurements of cotton fiber bundle strength can, for example, be made according to USDA standards. A beard of cotton is clamped in two sets of jaws, one eighth inch apart, and the force required to break the fibers is determined. Table 1 can be used as a guide in interpreting fiber strength measurements.

TABLE-US-00001 TABLE 1 Interpretation of HVI fiber strength measurements Degree of Strength HVI* Strength (grams per tex) Very Strong 31 or more Strong 29-30 Average 26-28 Intermediate 24-25 Weak 23 or less *High Volume Precision Instruments

[0079] Alternatively, the strength of fibers can be compared by determining the "single fiber strength" by performing single fiber tensile tests, for example, on a FAVIMAT Robot (Textechno) as described on the World Wide Web at textechno.com and in the Examples. Briefly, a single fiber is clamped between two fiber clamps with a continuously adjustable gauge length between 5 and 100 mm (set e.g. on 8 mm) and a draw-off clamp speed between 0.1 and 100 mm/min (set e.g. on 4 mm/min), and the force (cN) required to break the fibers ("breaking force") is determined. Average breaking forces of specific cotton varieties can be found in the Examples.

[0080] "Chromosome A05", as used herein, refers to chromosome A05 (numbering according to Wang et al., 2006, Theor Appl Genet 113(1):73-80) in an A genome diploid Gossypium plant, such as Gossypium herbaceum or Gossypium arboreum, or in an AD allotetraploid Gossypium plant, such as Gossypium hirsutum, Gossypium barbadense and Gossypium darwinii. In one embodiment, the Gossypium plant is an A genome diploid Gossypium plant comprising 13 A genome chromosome pairs, numbered A01 to A13 according to Wang et al. (2006, Theor Appl Genet 113(1):73-80), such as Gossypium herbaceum or Gossypium arboreum. In another embodiment, the Gossypium plant is an AD genome allotetraploid Gossypium plant comprising 13 A genome and 13 D genome chromosome pairs, numbered A01 to A13 and D01 to D13, respectively, according to Wang et al. (supra), such as Gossypium hirsutum, Gossypium barbadense and Gossypium darwinii.

[0081] In one embodiment, the non-naturally occurring Gossypium plant is a Gossypium hirsutum, a Gossypium herbaceum or a Gossypium arboreum plant, preferably a Gossypium hirsutum plant, and the superior allele of the fiber strength locus is derived from Gossypium barbadense.

[0082] Gossypium barbadense, in particular Gossypium barbadense cv. Pima S7, seeds are publicly available and can be obtained for example from the Cotton Collection (USDA, ARS, Crop Germplasm Research, 2765 F&B Road, College Station, Tex. 77845; on the World Wide Web at ars-grin.gov).

[0083] The term "superior allele" of the fiber strength locus refers herein to an allele of the fiber strength locus the presence of which in the genome of a fiber-producing plant results in a higher fiber strength compared to the fiber strength in such fiber-producing plant not comprising the superior allele (i.e., comprising a non-superior allele).

[0084] As used herein, the term "allele(s)" means any of one or more alternative forms of a gene or a marker at a particular locus or of a quantitative trait locus (QTL). In a diploid or allotetraploid (amphidiploid) cell of an organism, alleles of a given gene, marker or QTL are located at a specific location or locus (loci plural) on a chromosome. One allele is present on each chromosome of the pair of homologous chromosomes. As used herein, the term "homologous chromosomes" means chromosomes that contain information for the same biological features and contain the same genes or markers at the same loci and the same quantitative trait loci but possibly different alleles of those genes, markers or quantitative trait loci. Homologous chromosomes are chromosomes that pair during meiosis. "Non-homologous chromosomes", representing all the biological features of an organism, form a set, and the number of sets in a cell is called ploidy. Diploid organisms contain two sets of non-homologous chromosomes, wherein each homologous chromosome is inherited from a different parent. In allotetraploid (amphidiploid) species, like cotton, essentially two sets of diploid genomes exist, whereby the chromosomes of the two genomes are referred to as "homeologous chromosomes" (and similarly, the genes, markers and loci of the two genomes are referred to as homeologous genes, markers or loci). A diploid, or allotetraploid (amphidiploid), plant species may comprise a large number of different alleles at a particular locus.

[0085] The term "ortholog" of a gene or protein or QTL refers herein to the homologous gene or protein or QTL found in another species, which has the same function as the gene or protein or QTL, but is (usually) diverged in sequence from the time point on when the species harboring the genes or quantitative trait loci diverged (i.e. the genes or quantitative trait loci evolved from a common ancestor by speciation). Orthologs of, e.g., the Gossypium barbadense GLUC genes or fiber strength locus may thus be identified in other plant species (e.g. Gossypium arboreum, Gossypium darwinii, etc.) based on both sequence comparisons (e.g. based on percentages sequence identity over the entire sequence or over specific domains) and/or functional analysis.

[0086] In one embodiment, the superior allele of the fiber strength locus is obtainable from Gossypium barbadense, in particular Gossypium barbadense cv. PimaS7, i.e. the presence of the Gossypium barbadense fiber strength allele in a Gossypium plant, such as a Gossypium hirsutum plant, results in an increased fiber strength compared to the fiber strength in the Gossypium plant, such as the Gossypium hirsutum plant, not comprising the Gossypium barbadense allele, but, for example, the Gossypium hirsutum allele.

[0087] In still another embodiment, the Gossypium barbadense fiber strength allele is located on chromosome A05 of Gossypium barbadense between AFLP marker P5M50-M126.7 and SSR marker CIR280. In another embodiment, the Gossypium barbadense fiber strength allele is located on chromosome A05 of Gossypium barbadense between AFLP marker P5M50-M126.7 and SSR marker BNL3992. In yet another embodiment, the Gossypium barbadense allele is located on chromosome A05 of Gossypium barbadense between AFLP marker P5M50-M126.7 and SSR marker CIR401c. In a further embodiment, the LOD peak of the fiber strenght QTL allele of Gossypium barbadense is located between SSR marker NAU861 or the GLUC1.1 marker and SSR marker CIR401c, in particular at about 0 to 5 cM, more specifically at about 4 cM, especially at about 4.008 cM, from SSR marker NAU861 or the GLUC1.1 marker and at about 0 to 12 cM, more specifically at about 10 cM, especially at about 10.52 cM, from SSR marker CIR401c.

[0088] A "(genetic or molecular) marker", as used herein, refers to a polymorphic locus, i.e. a polymorphic nucleotide (a so-called single nucleotide polymorphism or SNP) or a polymorphic DNA sequence at a specific locus. A marker refers to a measurable, genetic characteristic with a fixed position in the genome, which is normally inherited in a Mendelian fashion, and which can be used for mapping of a trait of interest. For example, the fiber strength trait was mapped on chromosome A05 of Gossypium barbadense between, amongst others, markers P5M50-M126.7 and CIR280, P5M50-M126.7 and BNL3992, P5M50-M126.7 and CIR401, and linked to markers NAU861, GLUC1.1, and others, as indicated, e.g., in Table 6 in the Examples. Thus, a genetic marker may be a short DNA sequence, such as a sequence surrounding a single base-pair change, i.e. a single nucleotide polymorphism or SNP, or a long DNA sequence, such as microsatellites or Simple Sequence Repeats (SSRs). The nature of the marker is dependent on the molecular analysis used and can be detected at the DNA, RNA or protein level. Genetic mapping can be performed using molecular markers such as, but not limited to, RFLP (restriction fragment length polymorphisms; Botstein et al. (1980), Am J Hum Genet 32:314-331; Tanksley et al. (1989), Bio/Technology 7:257-263), RAPD [random amplified polymorphic DNA; Williams et al. (1990), NAR 18:6531-6535], AFLP [Amplified Fragment Length Polymorphism; Vos et al. (1995) NAR 23:4407-4414], SSRs or microsatellites [Tautz et al. (1989), NAR 17:6463-6471]. Appropriate primers or probes are dictated by the mapping method used.

[0089] The term "AFLP®" (AFLP® is a registered trademark of KeyGene N. V., Wageningen, The Netherlands), "AFLP analysis" and "AFLP marker" is used according to standard terminology [Vos et al. (1995), NAR 23:4407-4414; EP0534858; on the World Wide Web at keygene.com/keygene/techs-apps]. Briefly, AFLP analysis is a DNA fingerprinting technique which detects multiple DNA restriction fragments by means of PCR amplification. The AFLP technology usually comprises the following steps: (i) the restriction of the DNA with two restriction enzymes, preferably a hexa-cutter and a tetra-cutter, such as EcoRI, PstI and MseI; (ii) the ligation of double-stranded adapters to the ends of the restriction fragments, such as EcoRI, PstI and MseI adaptors; (iii) the amplification of a subset of the restriction fragments using two primers complementary to the adapter and restriction site sequences, and extended at their 3' ends by one to three "selective" nucleotides, i.e., the selective amplification is achieved by the use of primers that extend into the restriction fragments, amplifying only those fragments in which the primer extensions match the nucleotides flanking the restriction sites. AFLP primers thus have a specific sequence and each AFLP primer has a specific code (the primer codes and their sequences can be found at the Keygene web site: keygene.com/keygene/pdf/PRIMERCO.pdf); (iv) gel electrophoresis of the amplified restriction fragments on denaturing slab gels or cappilaries; (v) the visualization of the DNA fingerprints by means of autoradiography, phospho-imaging, or other methods. Using this method, sets of restriction fragments may be visualized by PCR without knowledge of nucleotide sequence. An AFLP marker, as used herein, is a DNA fragment of a specific size, which is generated and visualized as a band on a gel by carrying out an AFLP analysis. Each AFLP marker is designated by the primer combination used to amplify it, followed by the approximate size (in base pairs) of the amplified DNA fragment, e.g. P5M50-M126.7 refers to AFLP primer combination P05 (or Keygene code P11, which is a PstI primer with additional nucleotides AA; see Table 2) and M50 (which is a MseI primer with additional nucleotides CAT; see Table 2), the use of which in Gossypium barbadense results in an amplified DNA fragment of 126.7 bp (see Table 2). It is understood that the size of these fragments may vary slightly depending on laboratory conditions and equipment used. Every time reference is made herein to an AFLP marker by referring to a primer combination and the specific size of a fragment, it is to be understood that such size is approximate, and comprises or is intended to include the slight variations observed in different labs. Each AFLP marker represents a certain locus in the genome.

[0090] The term "SSR" refers to Simple Sequence Repeats or microsatellite [Tautz et al. (1989), NAR 17:6463-6471]. Short Simple Sequence stretches occur as highly repetitive elements in all eukaryotic genomes. Simple sequence loci usually show extensive length polymorphisms. These simple sequence length polymorphisms (SSLP) can be detected by polymerase chain reaction (PCR) analysis and be used for identity testing, population studies, linkage analysis and genome mapping. "SSR marker", as used herein, refers to markers indicated as CIRx, NAUx and BNLx (wherein x is a number) that are publicly available markers which are used to create genetic maps of different Gossypium species (see Cotton Microsatellite Database at on the World Wide Web at cottonmarker.org).

[0091] A "(genetic or molecular) marker", such as an AFLP or SSR marker, can be dominant (homozygous and heterozygous individuals are not distinguishable) or co-dominant (distinguishing homozygous and heterozygous individuals, e.g., by band intensity), as exemplified in Table 2 below. A "(genetic or molecular) marker", such as an AFLP or SSR marker, can be linked to a gene or locus in "coupling phase" or in "repulsion phase`. For example, a dominant marker linked in coupling to a gene or locus is present in individuals with the gene or locus and absent in individuals without the gene or locus, while a dominant marker linked in repulsion phase to a gene or locus is absent in individuals with the gene or locus and present in individuals without the gene or locus.

[0092] Different alleles of markers can exist in different plant species. "Gossypium barbadense or Gossypium hirsutum alleles of markers linked to the fiber strength locus", as used herein, refers to a form of a marker that is derived from and specific for Gossypium barbadense or Gossypium hirsutum, respectively. Table 2 examplifies how different alleles of different markers can be identified or distinghuished: column 1 indicates different marker loci on chromosome A05 of Gossypium barbadense and/or Gossypium hirsutum, column 2 indicates for each marker locus a specific primer pair that can be used to identify the presence or absence of the specific marker locus, column 3 indicates whether a specific marker allele of Gossypium barbadense (in particular cv. Pima S7; indicated as `Pima`) and Gossypium hirsutum (in particular cv. FM966; indicated as `FM`) generates an amplified DNA fragment and, if so, the size of the amplified DNA fragment, column 4 indicates whether the marker indicated in column 1 is a dominant or a codominant marker as defined above.

TABLE-US-00002 TABLE 2 Detection of specific Gossypium barbadense or Gossypium hirsutum alleles of markers on chromosome A05 Marker Amplified locus on fragment (in bp) Codominant/ chromosome from from dominant A05 Primer pair: FM Pima marker P5M50- P5 5' GACTGCGTACAT -- 126.7 dominant M126.7 GCAGAA 3' (SEQ ID NO: 43) M50 5' GATGAGTCCTGA GTAACAT 3' (SEQ ID NO: 44) GLUC1.1A- forward 5' TAT CCC TCT 134 143 codominant SNP2 CGA TGA GTA CGA C 3' (SEQ ID NO: 37) reverse 5'CCC AAT GAT GAT GAA CCT GAA TTG 3' (SEQ ID NO: 38) NAU861 forward 5' CCAAAACTTGTC 205-210 215-220 codominant CCATTAGC 3' (SEQ ID NO: 45) reverse 5' TTCATCTGTTGC CAGATCC 3' (SEQ ID NO: 46) CIR401c forward 5' TGGCGACTCCCT -- 245-250 dominant TTT 3' (SEQ ID NO: 47) reverse 5' AAAAGATGTTAC ACACACACAC 3' (SEQ ID NO: 48) CIR401b forward 5' TGGCGACTCCCT 255 -- dominant TTT 3' (SEQ ID NO: 47) reverse 5' AAAAGATGTTAC ACACACACAC 3' (SEQ ID NO: 48) BNL3992 forward 5' CAGAAGAGGAGG 160-165/ 140-145 codominant AGGTGGAG 3' 85-90 (SEQ ID NO: 49) reverse 5' TGCCAATGATGG AAAACTCA 3' (SEQ ID NO: 50) CIR280 forward 5' ACTGCGTTCATT -- 205 dominant ACACC 3' (SEQ ID NO: 51) reverse 5' GCTTCACCCATT CATC 3' (SEQ ID NO: 52)

[0093] As indicated above, the location of the Gossypium barbadense fiber strength allele on chromosome A05 can be determined by linked AFLP and/or SSR markers, such as AFLP marker P5M50-M126.7, and SSR markers BNL3992, CIR401b and NAU861. However, it is understood that these AFLP and SSR markers can be converted into other types of molecular markers. When referring to a specific (molecular or genetic) marker in the present invention, it is understood that the definition encompasses other types of molecular markers used to detect the genetic variation originally identified by the AFLP and SSR markers. For example, if an AFLP marker is converted into another molecular marker using known methods, this other marker is included in the definition. For example, AFLP markers can be converted into sequence-specific markers such as, but not limited to STS (sequenced-tagged-site) or SCAR (sequence-characterized-amplified-region) markers using standard technology as described in Meksem et al. [(2001), Mol Gen Genomics 265(2):207-214], Negi et al. [(2000), TAG 101:146-152], Barret et al. (1989), TAG 97:828-833], Xu et al. [(2001), Genome 44(1):63-70], Dussel et al. [(2002), TAG 105:1190-1195] or Guo et al. [(2003), TAG 103:1011-1017]. For example, Dussel et al. [(2002), TAG 105:1190-1195] converted AFLP markers linked to resistance into PCR-based sequence tagged site markers such as indel (insertion/deletion) markers and CAPS (cleaved amplified polymorphic sequence) markers.

[0094] The conversion of an AFLP marker into an STS marker, for example, generally involves the purification of the DNA fragment from the AFLP gel and the cloning and sequencing of the DNA fragment. Cloning and sequencing of AFLP fragments (bands) can be carried out using known methods [Guo et al. TAG 103:1011-1017]. Based on the marker sequence (internal) locus specific PCR primers can be developed [Paran and Michelmore (1993), TAG 85:985-993], which amplify fragments of different sizes or wherein the PCR product is cleaved with a restriction enzyme after amplification to reveal a polymorphism. As internal PCR primers often do not reveal polymorphisms related to the EcoRI, MseI or PstI (or other enzymes) restriction site differences, inverse PCR [Hartl and Ochmann (1996), In: Harwood A, editor, Methods in molecular biology vol58: basic DNA and RNA protocols, Humana Press, Totowa N.J. pp 293-301] or PCR-walking [Negi et al. (2000), TAG 101:146-152; Siebert et al, (1995), NAR 23:1087-1088] may be used to identify flanking sequences, which can then be used to generate simple, locus specific, PCR based markers. Primers can easily be designed using computer software programs such as provided by Sci-Ed (Scientific & Educational Software PO Box 72045, Durham, N.C. 27722-2045 USA). The polymorphism of the STS marker can be detected by gel electrophoresis, or can be detected using fluorometric assays, such as TaqMan® technology (Roche Diagnostics).

[0095] In another embodiment, the fiber strenght QTL allele of Gossypium barbadense comprises at least one Gossypium barbadense ortholog of a nucleotide sequence comprised in the genomic DNA sequence spanning the Gossypium hirsutum GLUC1.1A gene represented in SEQ ID NO: 53 (see FIG. 9 and the sequence listing).

[0096] In another embodiment, the fiber strenght QTL allele of Gossypium barbadense comprises at least a GLUC1.1 gene encoding a non-functional GLUC1.1 protein as further described below. In one aspect the Gossypium barbadense GLUC1.1 gene is located at about 0 to 5 cM, more specifically at about 4 cM, from the LOD peak of the fiber strenght QTL allele of Gossypium barbadense. In another aspect the Gossypium barbadense GLUC1.1 gene is located at about 0 to 2 cM, at about 0 to 1 cM, more specifically at about 0.008 cM of the NAU861 marker located in the fiber strenght QTL allele of Gossypium barbadense.

[0097] In another embodiment, the non-naturally occurring Gossypium plant is a Gossypium hirsutum, Gossypium barbadense, a Gossypium herbaceum or a Gossypium arboreum plant, preferably a Gossypium hirsutum plant, and wherein the superior fiber strength allele is derived from Gossypium darwinii. In one aspect, the fiber strenght QTL allele of Gossypium darwinii comprises at least a GLUC1.1 gene as further described below.

[0098] In still another embodiment, the non-naturally occurring Gossypium plant is a Gossypium hirsutum, Gossypium barbadense or a Gossypium herbaceum plant, preferably a Gossypium hirsutum plant, and wherein the superior fiber strength allele is derived from Gossypium arboreum. In one aspect, the fiber strenght QTL allele of Gossypium arboreum comprises at least a GLUC1.1 gene as further described below.

[0099] In a particular embodiment, the callose content of the fibers of the non-naturally occurring Gossypium plant is increased compared to the callose content of the fibers of an equivalent Gossypium plant that does not comprise the at least one superior allele of the fiber strength locus.

[0100] "Callose" refers to a plant polysaccharide that comprises glucose residues linked together through beta-1,3-linkages, and is termed a beta-glucan. It is thought to be manufactured at the cell wall by callose synthases and is degraded by beta-1,3-glucanases. The callose content of fibers can be measured by staining the fibers with aniline blue, a dye specific for 1,3-beta-glucans. Under UV, callose deposits present an intense yellow-green fluorescence. Images are analyzed and the ratio Green/Blue is used as a measure for callose. "Cellulose" is the major structural polysaccharide of higher plant cell walls. Chains of beta-1,4-linked glucosyl residues assemble soon after synthesis to form rigid, chemically resistant microfibrils. Their mechanical properties together with their orientation in the wall influence the relative expansion of cells in different directions and determine many of the final mechanical properties of mature cells and organs.

[0101] In a particular embodiment, the strength of the fibers of the non-naturally occurring Gossypium plant is increased compared to the strength of the fibers of an equivalent Gossypium plant that does not comprise the at least one superior allele of the fiber strength locus.

[0102] "Increase in fiber strength", as used herein, refers to an average strength of fibers of a specific fiber-producing plant species, such as cotton, which is significantly higher than the average strength of fibers of that specific plant species normally observed. Fiber strength is largely determined by variety. However, it may be affected by plant nutrient deficiencies and weather.

[0103] In one aspect of this embodiment, the non-naturally occurring Gossypium plant is a Gossypium hirsutum plant which is homozygous for the Gossypium barbadense fiber strength allele. In a further aspect of this embodiment, the strength of the fibers of the Gossypium plant is on average between about 5% and about 10%, more specifically about 7.5%, higher than the fiber strength of a Gossypium hirsutum plant which is homozygous for the Gossypium hirsutum fiber strength allele. In still a further aspect of this embodiment, the strength of the fibers of the Gossypium plant is on average between about 1.6 g/tex and about 3.3 g/tex, more specifically about 2.5 g/tex higher than the fiber strength of a Gossypium hirsutum plant which is homozygous for the Gossypium hirsutum fiber strength allele. In yet a further aspect of this embodiment, the strength of the fibers of the Gossypium plant is on average between about 34.6 g/tex and about 36.3 g/tex, more specifically about 35.5 g/tex, as compared to a fiber strength of on average between about 32.2 g/tex and about 33.8 g/tex, more specifically about 33.0 g/tex of a Gossypium hirsutum plant which is homozygous for the Gossypium hirsutum fiber strength allele.

[0104] A "variety" (abbreviated as var.) or "cultivar" (abbreviated as cv.) is used herein in conformity with the UPOV convention and refers to a plant grouping within a single botanical taxon of the lowest known rank, which grouping can be defined by the expression of the characteristics resulting from a given genotype or combination of genotypes, can be distinguished from any other plant grouping by the expression of at least one of the said characteristics and is considered as a unit with regard to its suitability for being propagated unchanged (stable).

[0105] As used herein, the term "heterozygous" means a genetic condition existing when two different alleles reside at a specific locus, but are positioned individually on corresponding pairs of homologous chromosomes in the cell. Conversely, as used herein, the term "homozygous" means a genetic condition existing when two identical alleles reside at a specific locus, but are positioned individually on corresponding pairs of homologous chromosomes in the cell.

[0106] A "fiber-producing plant" refers to a plant species that produces fibers as defined above, such as a cotton plant. Of the Gossypium species, the A genome diploid Gossypium species and AD genome allotetraploid Gossypium species are known to produce spinnable fiber. Botanically, there are three principal groups of cotton that are of commercial importance. The first, Gossypium hirsutum (AADD), is native to Mexico and Central America and has been developed for extensive use in the United States, accounting for more than 95% of U.S. production. This group is known in the United States as American Upland cotton, and their fibers vary in length from about 7/8 to about 1 5/16 inches (about 22-about 33 mm). Worldwide it accounts for about 90% of the cotton production. A second botanical group, G. barbadense (AADD), which accounts for about 5% of U.S. production and about 8% of the worldwide production, is of early South American origin. With fibers varying in length from about 11/4 to about 1 9/16 inches (about 32-about 40 mm), it is known in the United States as American Pima, but is also commonly referred to as Extra Long Staple (ELS) cotton. A third group, G. herbaceum (AA) and G. arboreum (AA), embraces cotton plants with fibers of shorter length, about 1/2 to about 1 inch (about 13-about 25 mm), that are native to India and Eastern Asia. None from this group is cultivated in the United States.

[0107] "Fiber length", as used herein, refers to the average length of the longer one-half of the fibers (upper half mean length). In the US, it is usually reported in 100ths or 32nds of an inch (see Table 3; 1 inch is 25.4 mm). It is measured, for example, according to United States Department of Agriculture (USDA) standards by passing a "beard" of parallel fibers through a sensing point. The beard is formed when fibers from a sample of cotton are grasped by a clamp, then combed and brushed to straighten and parallel the fibers. Fiber length is largely determined by variety, but the cotton plant's exposure to extreme temperatures, water stress, or nutrient deficiencies may shorten the length. Excessive cleaning and/or drying at the gin may also result in shorter fiber length. Fiber length affects yarn strength, yarn evenness, and the efficiency of the spinning process. The fineness of the yarn which can be successfully produced from given fibers is also influenced by the length of the fiber.

TABLE-US-00003 TABLE 3 Cotton fiber length conversion chart for American Upland and Pima cotton American Upland cotton American Pima cotton inches 32nds inches 32nds inches 32nds At least 0.79 24 1.11-1.13 36 At least 1.20 40 0.80-0.85 26 1.14-1.17 37 1.21-1.25 42 0.86-0.89 28 1.18-1.20 38 1.26-1.31 44 0.90-0.92 29 1.21-1.23 39 1.32-1.36 46 0.93-0.95 30 1.24-1.26 40 1.37-1.42 48 0.96-0.98 31 1.27-1.29 41 1.43-1.47 50 0.99-1.01 32 1.30-1.32 42 At least 1.48 52 1.02-1.04 33 1.33-1.35 43 1.05-1.07 34 At least 1.36 At least 44 1.08-1.10 35 Source: on the World Wide Web at cottoninc.com; 1 inch = 2.54 cm

[0108] An "industrially relevant fiber length", as used herein, refers to a length of fibers of a specific cotton species which is on average at least equal to or not significantly smaller than the length of fibers of that specific cotton variety normally observed. For G. hirsutum, an industrially relevant fiber length is reported to vary from about 7/8 to 1 5/16 inches (about 22-about 33 mm). For G. barbadense, an industrially relevant fiber length is reported to vary from 11/4 to 1 9/16 inches (about 32-about 40 mm). For G. herbaceum (AA) and G. arboreum (AA), an industrially relevant fiber length is reported to vary from 1/2 to 1 inch (about 13-about 25 mm).

[0109] Whenever reference to a "plant" or "plants" according to the invention is made, it is understood that also plant parts (cells, tissues or organs, seeds, fibers, severed parts such as roots, leaves, flowers, pollen, etc.), progeny of the plants which retain the distinguishing characteristics of the parents (especially the fiber properties), such as seed obtained by selfing or crossing, e.g. hybrid seed (obtained by crossing two inbred parental lines), hybrid plants and plant parts derived there from are encompassed herein, unless otherwise indicated.

[0110] The term "fiber strength allele detection assay" refers herein to an assay that indicates (directly or indirectly) the presence or absence of specific alleles of the fiber strength locus of the present invention. In one embodiment it allows one to determine whether a particular fiber strength allele is homozygous or heterozygous at the locus in any individual plant.

[0111] In another aspect of the invention, methods are provided for generating and/or selecting Gossypium plants, and parts and progeny thereof, comprising at least one superior allele of the fiber strength locus.

[0112] In one embodiment, the superior allele of the fiber strength locus is the Gossypium barbadense allele and the method comprises the step of identifying a Gossypium plant that comprises the Gossypium barbadense fiber strength allele based on the presence of Gossypium barbadense alleles of markers linked to the fiber strength locus, such as the markers linked to the Gossypium barbadense fiber strength allele indicated above and in Table 6 and 13.

[0113] In a particular aspect, the method comprises the step of determining the presence of Gossypium barbadense alleles of markers linked to the fiber strength locus in the genomic DNA of a plant selected from the group consisting of: AFLP marker P5M50-M126.7, SSR marker CIR280, SSR marker BNL3992, SSR marker CIR401c, SSR marker NAU861, a polymorphic site in a genomic DNA sequence of the plant corresponding to a genomic DNA sequence comprised in SEQ ID NO: 53, and a polymorphic site in a nucleotide sequence of a GLUC1.1A gene in the genomic DNA of the plant corresponding to the nucleotide sequence of a GLUC1.1A gene of SEQ ID NO: 5, such as the SNP markers indicated as GLUC1.1A-SNP2, 3, 5, 6 and 8 below and in Table 13.

[0114] In a further embodiment, the superior allele of the fiber strength locus is the Gossypium darwinii allele and the method comprises the step of identifying a Gossypium plant that comprises the Gossypium darwinii fiber strength allele based on the presence of Gossypium darwinii alleles of markers linked to the fiber strength locus, such as the markers linked to the Gossypium darwinii fiber strength allele indicated above and in Table 13.

[0115] In a particular aspect, the method comprises the step of determining the presence of a Gossypium darwinii allele of a polymorphic site in a nucleotide sequence of a GLUC1.1A gene in the genomic DNA of the plant corresponding to the nucleotide sequence of a GLUC1.1A gene of SEQ ID NO: 56, such as the SNP markers indicated as GLUC1.1A-SNP2, 3, 5, 6 and 8 below and in Table 13.

[0116] In a further embodiment, the superior allele of the fiber strength locus is the Gossypium arboreum allele and the method comprises the step of identifying a Gossypium plant that comprises the Gossypium arboreum fiber strength allele based on the presence of Gossypium arboreum alleles of markers linked to the fiber strength locus, such as the markers linked to the Gossypium arboreum fiber strength allele indicated above and in Table 13.

[0117] In a particular aspect, the method comprises the step of determining the presence of a Gossypium arboreum allele of a polymorphic site in a nucleotide sequence of a GLUC1.1A gene in the genomic DNA of the plant corresponding to the nucleotide sequence of a GLUC1.1A gene of SEQ ID NO: 21, such as the SNP marker indicated as GLUC1.1A-SNP7 below and in Table 13.

[0118] Markers linked to the fiber strength locus can be used for marker assisted selection (MAS) or map based cloning of the fiber strength locus. MAS involves screening plants for the presence or absence of linked markers. In particular plants are screened for the presence of markers flanking the locus or gene or linked to the locus or gene. Based on the presence/absence of the marker(s) plants are selected or discarded during the breeding program. MAS can significantly speed up breeding programs and introgression of a particular locus or gene into another genetic background, and can also reduce problems with genotype×environment interactions. MAS is also useful in combining different fiber strength loci in one plant. The presence or absence of a specific fiber strength allele, such as the Gossypium barbadense fiber strength allele, can be inferred from the presence or absence of molecular markers, such as the AFLP and SSR markers indicated above (see for example Table 2) or markers derived from them, linked to the specific allele. For example, Gossypium barbadense plants, in particular Gossypium barbadense cv. Pima S7 plants, may be crossed to Gossypium hirsutum plants and progeny plants from this cross are then screened for the presence of one or more AFLP and/or SSR markers linked to the Gossypium barbadense fiber strength allele, for example, by using the barbadense allele identification protocol.

[0119] Breeding procedures such as crossing, selfing, and backcrossing are well known in the art [see Allard R W (1960) Principles of Plant Breeding. John Wiley & Sons, New York, and Fehr W R (1987) Principles of Cultivar Development, Volume 1, Theory and Techniques, Collier Macmillan Publishers, London. ISBN 0-02-949920-8]. Superior alleles of the fiber strength locus, such as the Gossypium barbadense fiber strength allele, can be transferred into other breeding lines or varieties either by using traditional breeding methods alone or by using additionally MAS. In traditional breeding methods the increased callose content and/or increased fiber strength phenotype is assessed in the field or in controlled environment tests in order to select or discard plants comprising or lacking the superior fiber strength allele. Different crosses can be made to transfer the superior fiber strength allele, such as the Gossypium barbadense fiber strength allele, into lines of other Gossypium species or varieties, such as A genome diploid Gossypium plant lines, such as Gossypium herbaceum or Gossypium arboreum plant lines, or in AD allotetraploid Gossypium plant lines, such as Gossypium hirsutum and Gossypium barbadense plant lines, in particularly in Gossypium barbadense plant lines different from the Pima S7 variety. The breeding program may involve crossing to generate an F1 (first filial generation), followed by several generations of selfing (generating F2, F3, etc.). The breeding program may also involve backcrossing (BC) steps, whereby the offspring are backcrossed to one of the parental lines (termed the recurrent parent). Breeders select for agronomically important traits, such as high yield, high fiber quality, disease resistance, etc., and develop thereby elite breeding lines (lines with good agronomic characteristics). In addition, plants are bred to comply with fiber quality standards, such as American Pima or American Upland fiber quality.

[0120] The "barbadense or hirsutum allele identification protocol", as used herein, refers to the identification of the Gossypium barbadense and/or Gossypium hirsutum allele of the fiber strength locus comprising the steps of: extracting DNA from plant tissue such as leaf tissue or seeds and carrying out an analysis of linked markers, such as an AFLP and/or SSR analysis for one or more of the linked AFLP and/or SSR markers, using, for example, specific primer pairs to identify the barbadense or hirsutum allele, such as those indicated in Table 2. The barbadense or hirsutum allele identification protocol may be carried out on DNA obtained from individual plants or on DNA obtained from bulks (or pools). In one embodiment kits for detecting the presence of the Gossypium barbadense and/or Gossypium hirsutum fiber strength allele in Gossypium DNA are provided. Such a kit comprises, for example, primers or probes able to detect a DNA marker, such as an AFLP and/or an SSR marker, linked to the Gossypium barbadense and/or Gossypium hirsutum fiber strength allele. The kit may further comprise samples, which can be used as positive or negative controls and additional reagents for AFLP and/or SSR analysis. The samples may be tissue samples or DNA samples. As positive control may, for example, Gossypium barbadense seeds, in particular from cv. Pima S7, be included. As negative controls may, for example, Gossypium hirsutum seeds, in particular from cv. FM966, be included.

[0121] In a further aspect, methods are provided to distinguish between the presence of superior and non-superior alleles of the fiber strength locus. In one embodiment, methods are provided to distinguish between the presence of the Gossypium barbadense allele and the Gossypium hirsutum allele comprising the step of determining the presence of Gossypium barbadense and/or Gossypium hirsutum alleles of markers linked to the fiber strength locus, such as the markers linked to the fiber strength locus indicated above, for example, those indicated in Table 2 and Table 13.

[0122] Thus, in one embodiment, a method is provided for distinguishing between the presence of the Gossypium barbadense and Gossypium hirsutum fiber strength alleles by determining the presence of Gossypium barbadense and Gossypium hirsutum alleles of markers linked to the fiber strength locus in the genomic DNA of a plant selected from the group consisting of: AFLP marker P5M50-M126.7, SSR marker CIR280, SSR marker BNL3992, SSR marker CIR401, SSR marker NAU861, a polymorphic site in a genomic DNA sequence of the plant corresponding to a genomic DNA sequence comprised in SEQ ID NO: 53, and a polymorphic site in a nucleotide sequence of a GLUC1.1A gene in the genomic DNA of the plant corresponding to the nucleotide sequence of a GLUC1.1A gene of SEQ ID NO: 5, such as the SNP markers indicated as GLUC1.1A-SNP2, 3, 5, 6 and 8 below and in Table 13.

[0123] According to another aspect of the invention, methods are provided for altering the callose content of a fiber in a Gossypium plant, particularly increasing the callose content of a fiber, comprising the step of introgressing a superior allele of the cotton fiber strength locus on chromosome A05, such as the Gossypium barbadense allele, in the Gossypium plant.

[0124] According to yet another aspect of the invention, methods are provided for altering the properties of a fiber in a Gossypium plant, particularly increasing the strength of a fiber, comprising the step of introgressing a superior allele of the cotton fiber strength locus on chromosome A05, such as the Gossypium barbadense allele, in the Gossypium plant.

[0125] The current invention is further based on the unexpected finding that the functionality and the timing of expression of the GLUC1.1A gene, which was located in the support interval of the strength locus, differ between G. hirsutum and G. barbadense. It was found that, while G. hirsutum plants comprise a GLUC1.1A gene which is functionally expressed during the fiber strength buiding stage of fiber development, more particularly during the fiber maturation phase, G. barbadense plants comprise a GLUC1.1A gene which is non-functionally expressed during the fiber strength building phase. The GLUC1.1D gene on the other hand is functionally expressed during the entire fiber strength building stage in both Gossypium species. It was further found that addition of exogenous endo-1,3-beta-glucanase to fibers of Gossypium barbadense reduces the callose content and the strength of the fibers. Based on these findings, it is believed that the renown strength of the fibers of G. barbadense might be, at least in part, caused by a higher callose content in the fibers and that this higher callose content might be caused by the abscence of a functionally expressed A subgenome-specific fiber-specific endo-1,3-beta-glucanase gene. It is further believed that by abolishing the functional expression of specific alleles of GLUC genes during the fiber strength building stage in fiber-producing plants while maintaining the functional expression of specific other GLUC genes during the fiber strength building stage, it is possible to fine tune the amount and/or type of functional GLUC proteins produced during the fiber strength building stage, thus influencing the degradation of callose in the fiber which in turn influences the strength and length of the fiber produced. It is believed that the absolute and relative amount of different GLUC proteins in fibers can thus be tuned in such a way so as to attain a proper balance between fiber length and strength.

[0126] Thus, in a further aspect, the present invention provides a non-naturally occurring fiber-producing plant, and parts and progeny thereof, characterized in that the functional expression of at least one allele of at least one fiber-specific GLUC gene that is functionally expressed during the fiber strength building phase, in particular during the maturation phase of fiber development, is abolished.

[0127] The term "gene" means a DNA sequence comprising a region (transcribed region), which is transcribed into an RNA molecule (e.g. into a pre-mRNA, comprising intron sequences, which is then spliced into a mature mRNA, or directly into a mRNA without intron sequences) in a cell, operable linked to regulatory regions (e.g. a promoter). A gene (genomic DNA) may thus comprise several operably linked sequences, such as a promoter, a 5' leader sequence comprising e.g. sequences involved in translation initiation, a (protein) coding region (with introns) and a 3' non-translated sequence comprising e.g. transcription termination sites. "cDNA sequence" refers to a nucleic acid sequence comprising the 5' untranslated region, the coding region without introns and the 3' untranslated region and a polyA tail. "Endogenous gene" is used to differentiate from a "foreign gene", "transgene" or "chimeric gene", and refers to a gene from a plant of a certain plant genus, species or variety, which has not been introduced into that plant by transformation (i.e. it is not a "transgene"), but which is normally present in plants of that genus, species or variety, or which is introduced in that plant from plants of another plant genus, species or variety, in which it is normally present, by normal breeding techniques or by somatic hybridization, e.g., by protoplast fusion. Similarly, an "endogenous allele" of a gene is not introduced into a plant or plant tissue by plant transformation, but is, for example, generated by plant mutagenesis and/or selection, introgressed from another plant species by, e.g., marker-assisted selection, or obtained by screening natural populations of plants.

[0128] "Expression of a gene" or "gene expression" refers to the process wherein a DNA region, which is operably linked to appropriate regulatory regions, particularly a promoter, is transcribed into an RNA molecule. The RNA molecule is then processed further (by post-transcriptional processes) within the cell, e.g. by RNA splicing and translation initiation and translation into an amino acid chain (polypeptide), and translation termination by translation stop codons. The term "functionally expressed" is used herein to indicate that a functional, i.e. biologically active, protein is produced; the term "not functionally expressed" to indicate that a protein with significantly reduced or no functionality (biological activity) is produced or that no or a significantly reduced amount of protein is produced.

[0129] The term "fiber specific" or "fiber cell specific", with respect to the expression of a gene, refers to, for practical purposes, the highly specific, expression of a gene in fiber cells of plants, such as cotton plants. In other words, transcript levels of a DNA in tissues different of fiber cells is either below the detection limit or very low (less than about 0.2 picogram per microgram total RNA).

[0130] The term "fiber strenght building phase" commonly refers herein to the secondary cell wall synthesis and maturation phase of fiber development as defined above.

[0131] The term "GLUC gene" refers herein to a nucleic acid sequence encoding an endo-1,3-beta-glucanase (GLUC) protein.

[0132] The term "nucleic acid sequence" (or nucleic acid molecule) refers to a DNA or RNA molecule in single or double stranded form, particularly a DNA encoding a protein or protein fragment according to the invention. An "endogenous nucleic acid sequence" refers to a nucleic acid sequence within a plant cell, e.g. an endogenous (allele of a) GLUC gene present within the nuclear genome of a plant cell. An "isolated nucleic acid sequence" is used to refer to a nucleic acid sequence that is no longer in its natural environment, for example in vitro or in a recombinant bacterial or plant host cell.

[0133] The terms "protein" and "polypeptide" are used interchangeably and refer to molecules consisting of a chain of amino acids, without reference to a specific mode of action, size, 3-dimensional structure or origin. A "fragment" or "portion" of a protein may thus still be referred to as a "protein". An "isolated protein" is used to refer to a protein that is no longer in its natural environment, for example in vitro or in a recombinant bacterial or plant host cell. "Amino acids" are the principal building blocks of proteins and enzymes. They are incorporated into proteins by transfer RNA according to the genetic code while messenger RNA is being decoded by ribosomes. During and after the final assembly of a protein, the amino acid content dictates the spatial and biochemical properties of the protein or enzyme. The amino acid backbone determines the primary sequence of a protein, but the nature of the side chains determines the protein's properties. "Similar amino acids", as used herein, refers to amino acids that have similar amino acid side chains, i.e. amino acids that have polar, non-polar or practically neutral side chains. "Non-similar amino acids", as used herein, refers to amino acids that have different amino acid side chains, for example an amino acid with a polar side chain is non-similar to an amino acid with a non-polar side chain. Polar side chains usually tend to be present on the surface of a protein where they can interact with the aqueous environment found in cells ("hydrophilic" amino acids). On the other hand, "non-polar" amino acids tend to reside within the center of the protein where they can interact with similar non-polar neighbors ("hydrophobic" amino acids"). Examples of amino acids that have polar side chains are arginine, asparagine, aspartate, cysteine, glutamine, glutamate, histidine, lysine, serine, and threonine (all hydrophilic, except for cysteine which is hydrophobic). Examples of amino acids that have non-polar side chains are alanine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, and tryptophan (all hydrophobic, except for glycine which is neutral).

[0134] An "enzyme" is a protein comprising enzymatic activity, such as functional, i.e. biologically active, endo-1,3-beta-glucanase or glucan endo-1,3-beta-D-glucosidase (GLUC) proteins (EC 3.2.1.39). GLUC proteins belong to the glycosyl hydrolase family 17 (GH17) enzyme grouping and are capable of hydrolyzing 1,3-beta-D-glucosidic linkages in 1,3-beta-D-glucans, including long chain 1,3-beta-D-glucans called callose (see also on the World Wide Web at cazy.org/fam/GH17.html). The GH17 group is identified by the following amino acid recognition signature: [LIVMKS]-X-[LIVMFYWA](3)-[STAG]-E-[STACVI]-G-[WY]*-P-[STN]-X-[SAGQ], where E, such as Glu249 in GhGLUC1.1A (SEQ ID NO: 2 and 4) and similar or identical amino acids in other GLUC1.1 proteins (for example as indicated in FIG. 7), is an active site residue. The GH17 recognition signal of GLUC1.1 enzymes, as described herein, further contains a conserved tryptophan (W) residue at the position indicated with *, such as Trp252 in GhGLUC1.1A (SEQ ID NO: 2 and 4) and similar or identical amino acids in other GLUC1.1 proteins (for example as indicated in FIG. 7), which is predicted to be involved in the interaction with the glucan substrate.

[0135] In one embodiment, the fiber-specific GLUC gene that is functionally expressed during the fiber strength building phase, is a GLUC1.1 gene.

[0136] The term "GLUC1.1 gene" refers herein to a nucleic acid sequence encoding a GLUC1.1 protein. In particular, a "GLUC1.1 gene", as used herein, refers to a GLUC gene encoding a cDNA sequence with at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, 100% sequence identity to SEQ ID NO: 3 or comprises a coding sequence with at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, 100% sequence identity to the nucleotide at position 2410 to the nucleotide at position 3499 of SEQ ID NO: 1.

[0137] A "GLUC1.1 protein", as used herein, refers to a GLUC protein that has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, 1000% sequence identity to SEQ ID NO: 4.

[0138] A functional "GLUC1.1 protein", as used herein, refers to a GLUC1.1 protein that is capable of hydrolyzing 1,3-beta-D-glucosidic linkages in 1,3-beta-D-glucans, that has at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to SEQ ID NO: 4 and that comprises amino acid residues similar to the active site residues of the GLUC1.1 protein of SEQ ID NO:4. A non-functional "GLUC1.1 protein", as used herein, refers to a GLUC1.1 protein that is not capable of hydrolyzing 1,3-beta-D-glucosidic linkages in 1,3-beta-D-glucans. In particular, a non-functional GLUC1.1 protein lacks one or more amino acid residues similar to the active site residues of the GLUC1.1 protein of SEQ ID NO:4.

[0139] An "active site" or "catalytic site", as used herein, refers to a position on the three-dimensional structure of an enzyme which is involved in substrate binding, such as binding of 1,3-beta-D-glucans to GLUC enzymes, and in the biological activity of the enzyme, such as the hydrolyzation of 1,3-beta-D-glucosidic linkages in 1,3-beta-D-glucans of GLUC enzymes. "Active site (amino acid) residues", as used herein, refer to amino acid residues that are located within the active site of an enzyme and play a crucial role in substrate binding or in enzyme activity. A "glycosylation site", as used herein, refers to a position on the three-dimensional structure of an enzyme which is glycosylated, i.e. a site to which (branched) oligosaccharides bind which may function in increasing stability, such as thermostability, of the protein. "Glycosylation site (amino acid) residues", as used herein, refer to amino acid residues within the glycosylation site of an enzyme to which (branched) oligosaccharides bind. Predictions of the three-dimensional structure of the endo-1,3-beta-glucanase enzymes as described herein indicate that the active site and the glycosylation site of the barley 1,3-1,4-beta-glucanase (as described by Muller et al., 1998, J of Biol Chem 273 (6): 3438-3446; called "1aq0" in the Protein Data Bank, which is freely available on the World Wide Web at rcsb.org/pdb) are conserved, for example, in the Gossypium hirsutum GLUC1.1A and D, the Gossypium barbadense GLUC1.1D and the Gossypium herbaceum GLUC1.1A proteins as described herein, while the Gossypium barbadense GLUC1.1A protein, the Gossypium darwinii GLUC1.1A protein, and the Gossypium arboreum GLUC1.1A protein as described herein lack most conserved amino acids located within these sites these sites (see, e.g., Table 4, FIG. 3 and Examples). Active site and glycosylation residues in other GLUC1.1 proteins can be determined by aligning the amino acid sequences of the different GLUC1.1 proteins with the GLUC1.1 proteins of the present invention, such as the amino acid sequence of GhGLUC1.1A in SEQ ID NO:4, and identifying identical or similar residues in the other GLUC1.1 proteins.

TABLE-US-00004 TABLE 4 Amino acid regions and positions of active site residues and glycosylation site residues in GLUC1.1A and D proteins of the three principal groups of cotton of commercial interest GLUC protein: barley 1,3-1,4- GhGLUC1.1 GbGLUC1.1 GheGLUC1.1 GaGLUC1.1 beta-glucanase A D A D A A SEQ ID NO: 2/4 8/10 6/55 12/14 24 22 Protein size (aa) 325 337 179 337 337 78 Mature protein 311 311 165 311 311 52 aa encoded by 11 23 11 23 23 23 exon 1 aa encoded by 314 314 168 314 314 55 exon 2 Active site residue Tyr33 Tyr48 Tyr60 Tyr48 Tyr60 Tyr60 Tyr60 Glu232 Glu249 Glu261 -- Glu261 Glu261 -- Trp252 Trp264 -- Trp264 Trp264 -- Glu288 Glu308 Glu320 -- Glu320 Glu320 -- Glycosylation site residue: Asn190 Asn202 ND -- ND Asn214 -- --: not present; ND: not determined

[0140] The terms "target peptide", "transit peptide" or "signal peptide" refer to amino acid sequences which target a protein to intracellular organelles. The GLUC1.1 proteins as described herein comprise a signal peptide at their N-terminal end, such as the amino acid sequence indicated before the putative post-translational splicing site in FIGS. 2 and 7. "Mature protein" refers to a protein without the signal peptide, such as the GLUC1.1 proteins as described herein without the amino acid sequence indicated before the putative post-translational splicing site in FIGS. 2 and 7. "Precursor protein" or "preproenzyme" refers to the mature protein with its signal peptide.

[0141] In another embodiment, the fiber-producing plant is a Gossypium plant. In a particular aspect, the Gossypium GLUC1.1 allele is a GLUC1.1A or D allele.

[0142] A "GLUC1.1A gene", as used herein, refers to a GLUC1.1 gene located on the A subgenome of a Gossypium diploid or allotetraploid species ("GLUC1.1A locus") and encoding a GLUC1.1A protein. In particular, a GLUC1.1A gene encodes a cDNA sequence with at least 97%, at least 98%, at least 99% sequence identity to SEQ ID NO: 3 or comprises a coding sequence with at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the nucleotide at position 2410 to the nucleotide at position 3499 of SEQ ID NO: 1. Similarly, a "GLUC1.1D gene", as used herein, refers to a GLUC1.1 gene located on the D subgenome of a Gossypium diploid or allotetraploid species ("GLUC1.1D locus") and encoding a GLUC1.1D protein. In particular, a GLUC1.1D gene encodes a cDNA sequence with at least 97%, at least 98%, at least 99% sequence identity to SEQ ID NO: 9 or comprises a coding sequence with at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the nucleotide at position 3337 to the nucleotide at position 4444 of SEQ ID NO: 7.

[0143] A "GLUC1.1A protein", as used herein, refers to a GLUC1.1 protein encoded by a GLUC1.1 gene located on the A subgenome of a Gossypium diploid or allotetraploid species and having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to SEQ ID NO: 4. Similarly, a "GLUC1.1D protein", as used herein, refers to a GLUC protein encoded by a GLUC1.1 gene located on the D subgenome of a Gossypium diploid or allotetraploid species and having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to SEQ ID NO: 10.

[0144] In another embodiment the fiber-producing plant is a Gossypium hirsutum plant. In a particular aspect, the Gossypium hirsutum GLUC1.1 allele is a GhGLUC1.1A or a GhGLUC1.1D allele, preferably a GhGLUC1.1A allele.

[0145] As described in WO2008/083969, the GLUC1.1A and GLUC1.1D genes of Gossypium hirsutum can be distinguished by the presence of a cleaved amplified polymorphic sequence (CAPS) marker using an AlwI restriction enzyme recognition site present in the nucleotide sequence of GhGLUC1.1A that is absent in the nucleotide sequence of GhGLUC1.1D and by their timing of expression: whereas the GhGLUC1.1D is expressed during the entire fiber strength building phase (from about 14 to 17 DPA on depending on growth conditions), onset of GhGLUC1.1A is delayed until the beginning of the late fiber maturation phase (about 30-40 DPA depending on growth conditions). The GLUC1.1A and GLUC1.1D genes of Gossypium barbadense can also be distinguished by the presence of the CAPS marker using the AlwI restriction enzyme recognition site present in the nucleotide sequence of GbGLUC1.1A that is absent in the nucleotide sequence of GbGLUC1.1D. Both genes are however expressed during the entire fiber strength building phase (from about 14 to 17 DPA on depending on growth conditions). The level of expression of GbGLUC1.1A is however much lower than the level of expression of GbGLUC1.1D.

[0146] In one embodiment, the functional expression of the at least one GLUC allele is abolished by mutagenesis.

[0147] "Mutagenesis", as used herein, refers to the process in which plant cells (e.g., Gossypium seeds or other parts, such as pollen, etc.) are subjected to a technique which induces mutations in the DNA of the cells, such as contact with a mutagenic agent, such as a chemical substance (such as ethylmethylsulfonate (EMS), ethylnitrosourea (ENU), etc.) or ionizing radiation (neutrons (such as in fast neutron mutagenesis, etc.), alpha rays, gamma rays (such as that supplied by a Cobalt 60 source), X-rays, UV-radiation, etc.), or a combination of two or more of these. Thus, the desired mutagenesis of one or more GLUC alleles may be accomplished by use of chemical means such as by contact of one or more plant tissues with ethylmethylsulfonate (EMS), ethylnitrosourea, etc., by the use of physical means such as x-ray, etc, or by gamma radiation, such as that supplied by a Cobalt 60 source. While mutations created by irradiation are often large deletions or other gross lesions such as translocations or complex rearrangements, mutations created by chemical mutagens are often more discrete lesions such as point mutations. For example, EMS alkylates guanine bases, which results in base mispairing: an alkylated guanine will pair with a thymine base, resulting primarily in G/C to A/T transitions. Following mutagenesis, Gossypium plants are regenerated from the treated cells using known techniques. For instance, the resulting Gossypium seeds may be planted in accordance with conventional growing procedures and following self-pollination seed is formed on the plants. Additional seed that is formed as a result of such self-pollination in the present or a subsequent generation may be harvested and screened for the presence of mutant GLUC alleles. Several techniques are known to screen for specific mutant alleles, e.g., Deleteagene® (Delete-a-gene; Li et al., 2001, Plant J 27: 235-242) uses polymerase chain reaction (PCR) assays to screen for deletion mutants generated by fast neutron mutagenesis, TILLING (targeted induced local lesions in genomes; McCallum et al., 2000, Nat Biotechnol 18:455-457) identifies EMS-induced point mutations, etc. Additional techniques to screen for the presence of specific mutant GLUC alleles are described in the Examples below.

[0148] "Wild type" (also written "wildtype" or "wild-type"), as used herein, refers to a typical form of a plant or a gene as it most commonly occurs in nature. A "wild type plant" refers to a plant with the most common phenotype of such plant in the natural population. A "wild type allele" refers to an allele of a gene required to produce the wild-type phenotype. By contrast, a "mutant plant" refers to a plant with a different rare phenotype of such plant in the natural population or produced by human intervention, e.g. by mutagenesis, and a "mutant allele" refers to an allele of a gene required to produce the mutant phenotype.

[0149] As used herein, the term "wild type GLUC" (e.g. wild type GLUC1.1A or GLUC1.1D), means a naturally occurring GLUC allele found within plants, in particular Gossypium plants, which encodes a functional GLUC protein (e.g. a functional GLUC1.1A or GLUC1.1D, respectively). In contrast, the term "mutant GLUC" (e.g. mutant GLUC1.1A or GLUC1.1D), as used herein, refers to a GLUC allele, which does not encode a functional GLUC protein, i.e. a GLUC allele encoding a non-functional GLUC protein (e.g. a non-functional GLUC1.1A or GLUC1.1D, respectively), which, as used herein, refers to a GLUC protein having no biological activity or a significantly reduced biological activity as compared to the corresponding wild-type functional GLUC protein, or encoding no GLUC protein or a significantly reduced amount of GLUC protein. Such a "mutant GLUC allele" is a GLUC allele, which comprises one or more mutations in its nucleic acid sequence, whereby the mutation(s) preferably result in a significantly reduced (absolute or relative) amount of functional GLUC protein in the cell in vivo. As used herein, a "full knock-out GLUC1.1A allele" is a mutant GLUC1.1A allele the presence of which in homozygous state in the plant (e.g. a Gossypium hirsutum plant with two full knock-out GLUC1.1A alleles and two wild-type GLUC1.1D alleles) results in an increase of fiber strength in that plant. Mutant alleles of the GLUC protein-encoding nucleic acid sequences are designated as "gluc" (e.g. gluc1.1a or gluc1.1d, respectively) herein. Mutant alleles can be either "natural mutant" alleles, which are mutant alleles found in nature (e.g. produced spontaneously without human application of mutagens), such as the Gossypium barbadense GLUC1.1A allele, the Gossypium darwinii GLUC1.1A allele, and the Gossypium arboreum GLUC1.1A allele, or "induced mutant" alleles, which are induced by human intervention, e.g. by mutagenesis.

[0150] Thus in one aspect of the embodiment, GLUC mutant plants are provided herein, whereby the mutant alleles are selected from the GLUC1.1A and/or GLUC1.1D genes. Thus in a particular aspect, the genotype of these GLUC mutant plants can be described as: GLUC1.1A/gluc1.1a; GLUC1.1D/gluc1.1d; GLUC1.1A/gluc1.1a, GLUC1.1D/GLUC1.1D; or GLUC1.1A/GLUC1.1A, GLUC1.1D/gluc1.1d.

[0151] In a further aspect of the embodiment, homozygous GLUC mutant plants or plant parts are provided, whereby the mutant alleles are selected from the GLUC1.1A and GLUC1.1D genes. Thus in a particular aspect, homozygous GLUC mutant plants are provided herein, wherein the genotype of the plant can be described as: gluc1.1a/gluc1.1a; gluc1.1d/gluc1.1d; gluc1.1a/gluc1.1a, GLUC1.1D/GLUC1.1D or GLUC1.1A/GLUC1.1A, gluc1.1d/gluc1.1d.

[0152] In a further aspect of the invention the homozygous GLUC mutant plants or plant parts comprise a further mutant allele, wherein the mutant plants or plant parts are heterozygous for the additional mutant GLUC allele. Thus in a further particular aspect, homozygous GLUC mutant plants comprising one further mutant GLUC allele are provided herein, wherein the genotype of the plant can be described as: GLUC1.1-A/gluc1.1-a, gluc1.1-d/gluc1.1-d or gluc1.1a/gluc1.1a, GLUC1.1D/gluc1.1d.

[0153] In another embodiment, the functional expression of the at least one GLUC allele is abolished by introgression of a non-functionally expressed orthologous GLUC allele or of a mutagenized allele of the GLUC gene.

[0154] In one aspect of this embodiment, the non-functionally expressed orthologous GLUC allele can be isolated from specific cotton species, for example from Gossypium barbadense, darwinii or arboreum.

[0155] In yet another embodiment, the functional expression of the at least one allele of the GLUC gene is abolished by introduction of a chimeric gene comprises the following operably linked DNA elements:

[0156] (a) a plant expressible promoter,

[0157] (b) a transcribed DNA region, which when transcribed yields an inhibitory RNA molecule capable of reducing the expression of the GLUC allele, and

[0158] (c) a 3' end region comprising transcription termination and polyadenylation signals functioning in cells of the plant.

[0159] Several methods are available in the art to produce an inhibitory or a silencing RNA molecule, i.e. an RNA molecule which when expressed reduces the expression of a particular gene or group of genes, including the so-called "sense" or "antisense" RNA technologies.

[0160] Thus in one embodiment, the inhibitory RNA molecule encoding chimeric gene is based on the so-called antisense technology. In other words, the coding region of the chimeric gene comprises a nucleotide sequence of at least 19 or 20 consecutive nucleotides of the complement of the nucleotide sequence of the GLUC allele. Such a chimeric gene may be constructed by operably linking a DNA fragment comprising at least 19 or 20 nucleotides from the GLUC allele, isolated or identified as described elsewhere in this application, in inverse orientation to a plant expressible promoter and 3' end formation region involved in transcription termination and polyadenylation.

[0161] In another embodiment, the inhibitory RNA molecule encoding chimeric gene is based on the so-called co-suppression technology. In other words, the coding region of the chimeric gene comprises a nucleotide sequence of at least 19 or 20 consecutive nucleotides of the nucleotide sequence of the GLUC allele. Such a chimeric gene may be constructed by operably linking a DNA fragment comprising at least 19 or 20 nucleotides from the GLUC allele, in direct orientation to a plant expressible promoter and 3' end formation region involved in transcription termination and polyadenylation.

[0162] The efficiency of the above mentioned chimeric genes in reducing the expression of the GLUC allele may be further enhanced by the inclusion of a DNA element which results in the expression of aberrant, unpolyadenylated inhibitory RNA molecules or results in the retention of the inhibitory RNA molecules in the nucleus of the cells. One such DNA element suitable for that purpose is a DNA region encoding a self-splicing ribozyme, as described in WO 00/01133 (incorporated by reference). Another such DNA element suitable for that purpose is a DNA region encoding an RNA nuclear localization or retention signal, as described in WO03/076619 (incorporated by reference).

[0163] A convenient and very efficient way of downregulating the expression of a gene of interest uses so-called double-stranded RNA (dsRNA) or interfering RNA (RNAi), as described e.g. in WO99/53050 (incorporated by reference). In this technology, an RNA molecule is introduced into a plant cell, whereby the RNA molecule is capable of forming a double stranded RNA region over at least about 19 to about 21 nucleotides, and whereby one of the strands of this double stranded RNA region is about identical in nucleotide sequence to the target gene ("sense region"), whereas the other strand is about identical in nucleotide sequence to the complement of the target gene or of the sense region ("antisense region"). It is expected that for silencing of the target gene expression, the nucleotide sequence of the 19 consecutive nucleotide sequences may have one mismatch, or the sense and antisense region may differ in one nucleotide. To achieve the construction of such RNA molecules or the encoding chimeric genes, use can be made of the vector as described in WO 02/059294.

[0164] Thus, in one aspect of the embodiment, the chimeric gene comprises the following operably linked DNA elements:

[0165] (a) a plant expressible promoter, preferably a plant expressible promoter which controls transcription preferentially in the fiber cells;

[0166] (b) a transcribed DNA region, which when transcribed yields a double-stranded RNA molecule capable of reducing the expression of the GLUC allele and the RNA molecule comprising a first and second RNA region wherein

[0167] i) the first RNA region comprises a nucleotide sequence of at least 19 consecutive nucleotides having at least about 94% sequence identity to the nucleotide sequence of the GLUC allele;

[0168] ii) the second RNA region comprises a nucleotide sequence complementary to the at least 19 consecutive nucleotides of the first RNA region;

[0169] iii) the first and second RNA region are capable of base-pairing to form a double stranded RNA molecule between at least the 19 consecutive nucleotides of the first and second region; and

[0170] (c) a 3' end region comprising transcription termination and polyadenylation signals functioning in cells of the plant.

[0171] The length of the first or second RNA region (sense or antisense region) may vary from about 19 nucleotides (nt) up to a length equaling the length (in nucleotides) of the GLUC allele. The total length of the sense or antisense nucleotide sequence may thus be at least about 25 nt, or at least about 50 nt, or at least about 100 nt, or at least about 150 nt, or at least about 200 nt, or at least about 500 nt. It is expected that there is no upper limit to the total length of the sense or the antisense nucleotide sequence. However for practical reasons (such as e.g. stability of the chimeric genes) it is expected that the length of the sense or antisense nucleotide sequence should not exceed 5000 nt, particularly should not exceed 2500 nt and could be limited to about 1000 nt.

[0172] It will be appreciated that the longer the total length of the sense or antisense region, the less stringent the requirements for sequence identity between these regions and the corresponding sequence in the GLUC allele or its complement. Preferably, the nucleic acid of interest should have a sequence identity of at least about 75% with the corresponding target sequence, particularly at least about 80%, more particularly at least about 85%, quite particularly about 90%, especially about 95%, more especially about 100%, quite especially be identical to the corresponding part of the target sequence or its complement. However, it is preferred that the nucleic acid of interest always includes a sequence of about 19 consecutive nucleotides, particularly about 25 nt, more particularly about 50 nt, especially about 100 nt, quite especially about 150 nt with 100% sequence identity to the corresponding part of the target nucleic acid. Preferably, for calculating the sequence identity and designing the corresponding sense or antisense sequence, the number of gaps should be minimized, particularly for the shorter sense sequences.

[0173] For the purpose of this invention, the "sequence identity" of two related nucleotide or amino acid sequences, expressed as a percentage, refers to the number of positions in the two optimally aligned sequences which have identical residues (×100) divided by the number of positions compared. A gap, i.e., a position in an alignment where a residue is present in one sequence but not in the other, is regarded as a position with non-identical residues. The "optimal alignment" of two sequences is found by aligning the two sequences over the entire length according to the Needleman and Wunsch global alignment algorithm (Needleman and Wunsch, 1970, J Mol Biol 48(3):443-53) in The European Molecular Biology Open Software Suite (EMBOSS, Rice et al., 2000, Trends in Genetics 16(6): 276-277; see e.g. on the World Wide Web at ebi.ac.uk/emboss/align/index.html) using default settings (gap opening penalty=10 (for nucleotides)/10 (for proteins) and gap extension penalty=0.5 (for nucleotides)/0.5 (for proteins)). For nucleotides the default scoring matrix used is EDNAFULL and for proteins the default scoring matrix is EBLOSUM62.

[0174] "Substantially identical", "essentially similar", or "corresponding to", as used herein, refers to sequences, which, when optimally aligned as defined above, share at least a certain minimal percentage of sequence identity (as defined further below). "(A nucleotide or a nucleotide sequence) at a position corresponding to a position of (a nucleotide or a nucleotide sequence in a specific nucleotide sequence)", as used herein, refers to (nucleotides or nucleotide sequences) of two essentially similar sequences, which are aligned with each other in an optimal alignment of the two essentially similar sequences.

[0175] dsRNA encoding chimeric genes according to the invention may comprise an intron, such as a heterologous intron, located e.g. in the spacer sequence between the sense and antisense RNA regions in accordance with the disclosure of WO 99/53050 (incorporated herein by reference).

[0176] It is preferred for the current invention that the target specific gene sequence included in the antisense, sense or double stranded RNA molecule comprises at least one nucleotide, and preferably more which are specific for the specific GLUC allele whose expression is to be downregulated. Such specific nucleotides are indicated at least in FIG. 6 by the gray boxes.

[0177] In a preferred embodiment, the inhibitory RNA molecule is specifically adapted to downregulate the A-subgenomic allele of the GLUC1.1 gene. In another preferred embodiment, the biologically active RNA is specifically adapted to downregulate the D subgenome-specific allele of the GLUC1.1 gene.

[0178] The use of synthetic micro-RNA's to downregulate expression of a particular gene in a plant cell, provides for very high sequence specificity of the target gene, and thus allows conveniently to discriminate between closely related alleles as target genes the expression of which is to be downregulated.

[0179] Thus, in another embodiment of the invention, the inhibitory RNA or silencing RNA or biologically active RNA molecule may be a microRNA molecule, designed, synthesized and/or modulated to target and cause the cleavage of specific subgenomic alleles, preferably the A subgenomic allele of the GLUC1.1 gene in a fiber producing plant, such as a cotton plant. Various methods have been described to generate and use miRNAs for a specific target gene (including but not limited to Schwab et al. (2006, Plant Cell, 18(5):1121-1133), WO2006/044322, WO2005/047505, EP 06009836, incorporated by reference). Usually, an existing miRNA scaffold is modified in the target gene recognizing portion so that the generated miRNA now guides the RISC complex to cleave the RNA molecules transcribed from the target nucleic acid. miRNA scaffolds could be modified or synthesized such that the miRNA now comprises 21 consecutive nucleotides of one of the subgenomic alleles of the fiber selective β-1,3 endoglucanase encoding nucleotide sequence, such as the sequences represented in the Sequence listing of WO2008/083969, and allowing mismatches according to the herein below described rules.

[0180] Thus, in one embodiment, the invention provides a chimeric gene comprising the following operably linked DNA regions:

[0181] (a) a plant expressible promoter;

[0182] (b) a DNA region which upon introduction and transcription in a plant cell is processed into a miRNA, whereby the miRNA is capable of recognizing and guiding the cleavage of the mRNA of a GLUC allele of the plant but not another GLUC allele, such as the mRNA of the A subgenome specific GLUC allele but not the D subgenome specific GLUC allele; and optionally,

[0183] (c) a 3' DNA region involved in transcription termination and polyadenylation.

[0184] The mentioned DNA region processed into a miRNA may comprise a nucleotide sequence which is essentially complementary to a nucleotide sequence of at least 21 consecutive nucleotides of a GLUC allele, provided that one or more of following mismatches are allowed: a mismatch between the nucleotide at the 5' end of the miRNA and the corresponding nucleotide sequence in the RNA molecule; a mismatch between any one of the nucleotides in position 1 to position 9 of the miRNA and the corresponding nucleotide sequence in the RNA molecule; three mismatches between any one of the nucleotides in position 12 to position 21 of the miRNA and the corresponding nucleotide sequence in the RNA molecule provided that there are no more than two consecutive mismatches.

[0185] As used herein, a "miRNA" is an RNA molecule of about 20 to 22 nucleotides in length which can be loaded into a RISC complex and direct the cleavage of another RNA molecule, wherein the other RNA molecule comprises a nucleotide sequence essentially complementary to the nucleotide sequence of the miRNA molecule whereby one or more of the following mismatches may occur: a mismatch between the nucleotide at the 5' end of said miRNA and the corresponding nucleotide sequence in the target RNA molecule; a mismatch between any one of the nucleotides in position 1 to position 9 of said miRNA and the corresponding nucleotide sequence in the target RNA molecule; three mismatches between any one of the nucleotides in position 12 to position 21 of said miRNA and the corresponding nucleotide sequence in the target RNA molecule provided that there are no more than two consecutive mismatches. no mismatch is allowed at positions 10 and 11 of the miRNA (all miRNA positions are indicated starting from the 5' end of the miRNA molecule).

[0186] A miRNA is processed from a "pre-miRNA" molecule by proteins, such as DCL proteins, present in any plant cell and loaded onto a RISC complex where it can guide the cleavage of the target RNA molecules.

[0187] As used herein, a "pre-miRNA" molecule is an RNA molecule of about 100 to about 200 nucleotides, preferably about 100 to about 130 nucleotides which can adopt a secondary structure comprising a double stranded RNA stem and a single stranded RNA loop and further comprising the nucleotide sequence of the miRNA (and its complement sequence) in the double stranded RNA stem. Preferably, the miRNA and its complement are located about 10 to about 20 nucleotides from the free ends of the miRNA double stranded RNA stem. The length and sequence of the single stranded loop region are not critical and may vary considerably, e.g. between 30 and 50 nt in length. Preferably, the difference in free energy between unpaired and paired RNA structure is between -20 and -60 kcal/mole, particularly around -40 kcal/mole. The complementarity between the miRNA and the miRNA* need not be perfect and about 1 to 3 bulges of unpaired nucleotides can be tolerated. The secondary structure adopted by an RNA molecule can be predicted by computer algorithms conventional in the art such as mFOLD. The particular strand of the double stranded RNA stem from the pre-miRNA which is released by DCL activity and loaded onto the RISC complex is determined by the degree of complementarity at the 5' end, whereby the strand which at its 5' end is the least involved in hydrogen bounding between the nucleotides of the different strands of the cleaved dsRNA stem is loaded onto the RISC complex and will determine the sequence specificity of the target RNA molecule degradation. However, if empirically the miRNA molecule from a particular synthetic pre-miRNA molecule is not functional (because the "wrong" strand is loaded on the RISC complex, it will be immediately evident that this problem can be solved by exchanging the position of the miRNA molecule and its complement on the respective strands of the dsRNA stem of the pre-miRNA molecule. As is known in the art, binding between A and U involving two hydrogen bounds, or G and U involving two hydrogen bounds is less strong that between G and C involving three hydrogen bounds.

[0188] Naturally occurring miRNA molecules may be comprised within their naturally occurring pre-miRNA molecules but they can also be introduced into existing pre-miRNA molecule scaffolds by exchanging the nucleotide sequence of the miRNA molecule normally processed from such existing pre-miRNA molecule for the nucleotide sequence of another miRNA of interest. The scaffold of the pre-miRNA can also be completely synthetic. Likewise, synthetic miRNA molecules may be comprised within, and processed from, existing pre-miRNA molecule scaffolds or synthetic pre-miRNA scaffolds.

[0189] The pre-miRNA molecules (and consequently also the miRNA molecules) can be conveniently introduced into a plant cell by providing the plant cells with a gene comprising a plant-expressible promoter operably linked to a DNA region, which when transcribed yields the pre-miRNA molecule. The plant expressible promoter may be the promoter naturally associated with the pre-miRNA molecule or it may be a heterologous promoter.

[0190] Suitable miRNA and pre microRNA molecules for the specific downregulation of the expression of the GhGLUC1.1A gene are set forth in the sequence listing entries SEQ ID NO: 13, 14, 17, 18 and 19 of WO2008/083969.

[0191] Suitable miRNA and pre microRNA molecules for the specific downregulation of the expression of the GhGLUC1.1D gene are set forth in the sequence listing entries SEQ ID NO: 15, 16, 20 and 21 of WO2008/083969.

[0192] As used herein, the term "plant-expressible promoter" means a DNA sequence which is capable of controlling (initiating) transcription in a plant cell. This includes any promoter of plant origin, but also any promoter of non-plant origin which is capable of directing transcription in a plant cell, i.e., certain promoters of viral or bacterial origin such as the CaMV35S, the subterranean clover virus promoter No. 4 or No. 7, or T-DNA gene promoters and the like.

[0193] A plant-expressible promoter that controls initiation and maintenance of transcription preferentially in fiber cells is a promoter that drives transcription of the operably linked DNA region to a higher level in fiber cells and the underlying epidermis cells than in other cells or tissues of the plant. Such promoters include the promoter from cotton from a fiber-specific β-tubulin gene (as described in WO0210377), the promoter from cotton from a fiber-specific actin gene (as described in WO0210413), the promoter from a fiber specific lipid transfer protein gene from cotton (as described in U.S. Pat. No. 5,792,933), a promoter from an expansin gene from cotton (WO9830698) or a promoter from a chitinase gene in cotton (US2003106097) or the promoters of the fiber specific genes described in U.S. Pat. No. 6,259,003 or U.S. Pat. No. 6,166,294. Fiber selective promoters as described herein may also be used.

[0194] The invention also encompasses the chimeric genes herein described, as well as plants, seeds, tissues comprising these chimeric genes, and fibers produced from such plants.

[0195] Methods to transform plants are well known in the art and are of minor relevance for the current invention. Methods to transform cotton plants are also well known in the art. Agrobacterium-mediated transformation of cotton has been described e.g. in U.S. Pat. No. 5,004,863 or in U.S. Pat. No. 6,483,013 and cotton transformation by particle bombardment is reported e.g. in WO 92/15675.

[0196] The chimeric genes according to the invention may be introduced into plants in a stable manner or in a transient manner using methods well known in the art. The chimeric genes may be introduced into plants, or may be generated inside the plant cell as described e.g. in EP 1339859.

[0197] The chimeric genes may be introduced by transformation in cotton plants from which embryogenic callus can be derived, such as Coker 312, Coker310, Coker 5Acala SJ-5, GSC25110, FIBERMAX 819, Siokra 1-3, T25, GSA75, Acala SJ2, Acala SJ4, Acala SJ5, Acala SJ-C1, Acala B1644, Acala B1654-26, Acala B1654-43, Acala B3991, Acala GC356, Acala GC510, Acala GAM1, Acala C1, Acala Royale, Acala Maxxa, Acala Prema, Acala B638, Acala B1810, Acala B2724, Acala B4894, Acala B5002, non Acala "picker" Siokra, "stripper" variety FC2017, Coker 315, STONEVILLE 506, STONEVILLE 825, DP50, DP61, DP90, DP77, DES119, McN235, HBX87, HBX191, HBX107, FC 3027, CHEMBRED A1, CHEMBRED A2, CHEMBRED A3, CHEMBRED A4, CHEMBRED B1, CHEMBRED B2, CHEMBRED B3, CHEMBRED C1, CHEMBRED C2, CHEMBRED C3, CHEMBRED C4, PAYMASTER 145, HS26, HS46, SICALA, PIMA S6 ORO BLANCO PIMA, FIBERMAX FM5013, FIBERMAX FM5015, FIBERMAX FM5017, FIBERMAX FM989, FIBERMAX FM832, FIBERMAX FM966, FIBERMAX FM958, FIBERMAX FM989, FIBERMAX FM958, FIBERMAX FM832, FIBERMAX FM991, FIBERMAX FM819, FIBERMAX FM800, FIBERMAX FM960, FIBERMAX FM966, FIBERMAX FM981, FIBERMAX FM5035, FIBERMAX FM5044, FIBERMAX FM5045, FIBERMAX FM5013, FIBERMAX FM5015, FIBERMAX FM5017 or FIBERMAX FM5024 and plants with genotypes derived thereof.

[0198] "Cotton" as used herein includes Gossypium hirsutum, Gossypium barbadense, Gossypium arboreum and Gossypium herbaceum. "Cotton progenitor plants" include Gossypium arboreum, Gossypium herbaceum, Gossypium raimondii, Gossypium longicalyx and Gossypium kirkii.

[0199] The methods and means of the current invention may also be employed for other plant species such as hemp, jute, flax and woody plants, including but not limited to Pinus spp., Populus spp., Picea spp., Eucalyptus spp. etc.

[0200] The obtained transformed plant can be used in a conventional breeding scheme to produce more transformed plants with the same characteristics or to introduce the chimeric gene according to the invention in other varieties of the same or related plant species, or in hybrid plants. Seeds obtained from the transformed plants contain the chimeric genes of the invention as a stable genomic insert and are also encompassed by the invention.

[0201] In one embodiment, the amount of functional GLUC protein is significantly reduced in fibers of the fiber-producing plant during the fiber strength building phase of fiber development compared to the amount of functional GLUC protein produced during the fiber strength building phase in a plant in which the functional expression of the at least one GLUC allele is not abolished.

[0202] A "significantly reduced amount of functional GLUC protein" (e.g. functional GLUC1.1A or GLUC1.1D protein) refers to a reduction in the amount of a functional GLUC protein produced by the cell comprising a mutant GLUC allele by at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% (i.e. no functional GLUC protein is produced by the cell) as compared to the amount of the functional GLUC protein produced by the cell not comprising the mutant GLUC allele. This definition encompasses the production of a "non-functional" GLUC protein (e.g. truncated GLUC protein) having no biological activity in vivo, the reduction in the absolute amount of the functional GLUC protein (e.g. no functional GLUC protein being made due to the mutation in the GLUC gene), and/or the production of a GLUC protein with significantly reduced biological activity compared to the activity of a functional wild type GLUC protein (such as a GLUC protein in which one or more amino acid residues that are crucial for the biological activity of the encoded GLUC protein, as exemplified above and below, are substituted for another amino acid residue). The term "mutant GLUC protein", as used herein, refers to a GLUC protein encoded by a mutant GLUC nucleic acid sequence ("gluc allele") whereby the mutation results in a significantly reduced and/or no GLUC activity in vivo, compared to the activity of the GLUC protein encoded by a non-mutant, wild type GLUC sequence ("GLUC allele").

[0203] In yet a further embodiment, the fibers of the non-naturally occurring fiber-producing plant have a higher callose content compared to the callose content of the fibers of an equivalent fiber-producing plant wherein the expression of the at least one GLUC allele is not abolished.

[0204] In a particular aspect of this embodiment, the strength of the fibers of the non-naturally occurring fiber-producing plant is increased compared to the strength of the fibers of an equivalent fiber-producing plant wherein the expression of the at least one GLUC allele is not abolished.

[0205] In one aspect of this embodiment, the non-naturally occuring Gossypium plant is a Gossypium hirsutum plant which is homozygous for the Gossypium barbadense GLUC1.1A allele. In a further aspect of this embodiment, the strength of the fibers of the Gossypium plant is on average between about 5% and about 10%, more specifically about 7.5%, higher than the fiber strength of a Gossypium hirsutum plant which is homozygous for the Gossypium hirsutum GLUC1.1A allele. In still a further aspect of this embodiment, the strength of the fibers of the Gossypium plant is on average between about 1.6 g/tex and about 3.3 g/tex, more specifically about 2.5 g/tex higher than the fiber strength of a Gossypium hirsutum plant which is homozygous for the Gossypium hirsutum GLUC1.1A allele. In yet a further aspect of this embodiment, the strength of the fibers of the Gossypium plant is on average between about 34.6 g/tex and about 36.3 g/tex, more specifically about 35.5 g/tex, as compared to a fiber strength of on average between about 32.2 g/tex and about 33.8 g/tex, more specifically about 33.0 g/tex of a Gossypium hirsutum plant which is homozygous for the Gossypium hirsutum GLUC1.1A allele.

[0206] Further provided herein are nucleic acid sequences of wild type and mutant GLUC1.1 genes/alleles from Gossypium species, as well as the wild type and mutant GLUC1.1 proteins. Also provided are methods of generating and combining mutant and wild type GLUC1.1 alleles in Gossypium plants, as well as Gossypium plants and plant parts comprising specific combinations of wild type and mutant GLUC1.1 alleles in their genome, whereby these plants produce fibers with altered fiber strength and whereby the plants preferably grow normally and have a normal phenotype. The use of these plants for transferring mutant GLUC1.1 alleles to other plants is also an embodiment of the invention, as are the plant products of any of the plants described. In addition kits and methods for marker assisted selection (MAS) for combining or detecting GLUC genes and/or alleles are provided. Each of the embodiments of the invention is described in detail herein below.

[0207] Provided are both wild type (GLUC1.1) nucleic acid sequences, encoding functional GLUC1.1 proteins, and mutant (gluc1.1) nucleic acid sequences (comprising one or more mutations, preferably mutations which result in a significantly reduced biological activity of the encoded GLUC1.1 protein or in no GLUC1.1 protein being produced) of GLUC1.1 genes from Gossypium species, especially from Gossypium hirsutum and Gossypium barbadense, but also from other Gossypium species. For example, Gossypium species comprising an A and/or a D genome may comprise different alleles of GLUC1.1A or GLUC1.1D genes which can be identified and combined in a single plant according to the invention. In addition, mutagenesis methods can be used to generate mutations in wild type GLUC1.1A or GLUC1.1D alleles, thereby generating mutant alleles for use according to the invention. Because specific GLUC1.1 alleles are preferably combined in a Gossypium plant by crossing and selection, in one embodiment the GLUC1.1 and/or gluc1.1 nucleic acid sequences are provided within a Gossypium plant (i.e. endogenously).

[0208] However, isolated GLUC1.1 and gluc1.1 nucleic acid sequences (e.g. isolated from the plant by cloning or made synthetically by DNA synthesis), as well as variants thereof and fragments of any of these are also provided herein, as these can be used to determine which sequence is present endogenously in a plant or plant part, whether the sequence encodes a functional protein or a protein with significantly reduced or no functionality (e.g. by expression in a recombinant host cell and enzyme assays) and for selection and transfer of specific alleles from one Gossypium plant into another, in order to generate a plant having the desired combination of functional and mutant alleles.

[0209] Nucleic acid sequences of GLUC1.1A and/or GLUC1.1D have been isolated from Gossypium hirsutum, from Gossypium barbadense, from Gossypium tomentosum, from Gossypium darwinii, from Gossypium mustelinum, from Gossypium arboreum, from Gossypium herbaceum, and from Gossypium raimondii as depicted in the sequence listing. The wild type GLUC1.1A sequences of Gossypium hirsutum, tomentosum, mustelinum and herbaceum and wild type GLUC1.1D sequences of Gossypium hirsutum, tomentosum, barbadense, darwinii, mustelinum and raimondii are depicted, while the mutant gluc1.1a and/or gluc1.1d sequences of these sequences, and of sequences essentially similar to these, are described herein below and in the Examples, with reference to the wild type GLUC1.1A and GLUC1.1D sequences. Further, the mutant GLUC1.1A sequences of Gossypium barbadense, darwinii and arboreum are depicted, while the alternative mutant gluc1.1a sequences of these sequences, and of sequences essentially similar to these, are described herein below and in the Examples. The genomic GLUC1.1A and D protein-encoding DNA, and corresponding pre-mRNA, comprises 2 exons (numbered exons 1 and 2 starting from the 5'end) interrupted by 1 intron. In the cDNA and corresponding processed mRNA (i.e. the spliced RNA), introns are removed and exons are joined, as depicted in the sequence listing and FIGS. 1 and 6. Exon sequences are more conserved evolutionarily and are therefore less variable than intron sequences.

[0210] "GLUC1.1A nucleic acid sequences" or "GLUC1.1A variant nucleic acid sequences" according to the invention are nucleic acid sequences encoding an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 4 or nucleic acid sequences encoding a cDNA sequence with at least 97%, at least 98%, at least 99% sequence identity to SEQ ID NO: 3 or comprises a coding sequence with at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the nucleotide at position 2410 to the nucleotide at position 3499 of SEQ ID NO: 1. These nucleic acid sequences may also be referred to as being "essentially similar" or "essentially identical" or "corresponding to" the GLUC1.1A sequences provided in the sequence listing.

[0211] "GLUC1.1D nucleic acid sequences" or "GLUC1.1D variant nucleic acid sequences" according to the invention are nucleic acid sequences encoding an amino acid sequence having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% sequence identity to SEQ ID NO: 10 or nucleic acid sequences encoding a cDNA sequence with at least 97%, at least 98%, at least 99% sequence identity to SEQ ID NO: 3 or comprises a coding sequence with at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to the nucleotide at position 3337 to the nucleotide at position 4444 of SEQ ID NO: 7. These nucleic acid sequences may also be referred to as being "essentially similar" or "essentially identical" or "corresponding to" the GLUC1.1A sequences provided in the sequence listing.

[0212] Thus, the invention provides both nucleic acid sequences encoding wild type, functional GLUC1.1A and GLUC1.1D proteins, including variants and fragments thereof (as defined further below), as well as mutant nucleic acid sequences of any of these, whereby the mutation in the nucleic acid sequence preferably results in one or more amino acids being inserted, deleted or substituted in comparison to the wild type protein. Preferably the mutation(s) in the nucleic acid sequence result in one or more amino acid changes (i.e. in relation to the wild type amino acid sequence one or more amino acids are inserted, deleted and/or substituted) whereby the biological activity of the GLUC1.1 protein is significantly reduced. A significant reduction in biological activity of the mutant GLUC1.1 protein, refers to a reduction in enzymatic activity by at least 30%, at least 40%, 50% or more, at least 90% or 100% (no biological activity) compared to the activity of the wild type protein.

[0213] Both endogenous and isolated nucleic acid sequences are provided herein. Also provided are fragments of the GLUC1.1 sequences and GLUC1.1 variant nucleic acid sequences defined above, for use as primers or probes and as components of kits according to another aspect of the invention (see further below). A "fragment" of a GLUC1.1 or gluc1.1 nucleic acid sequence or variant thereof (as defined) may be of various lengths, such as at least 10, 12, 15, 18, 20, 50, 100, 200, 500, 1000 contiguous nucleotides of the GLUC1.1 or gluc1.1 sequence (or of the variant sequence).

[0214] Nucleic acid sequences of GLUC1.1A and/or GLUC1.1D have been isolated from Gossypium hirsutum, from Gossypium barbadense, from Gossypium tomentosum, from Gossypium darwinii, from Gossypium mustelinum, from Gossypium arboreum, from Gossypium herbaceum, and from Gossypium raimondii as depicted in the sequence listing. The wild type GLUC1.1A sequences of Gossypium hirsutum, tomentosum, mustelinum and herbaceum and wild type GLUC1.1D sequences of Gossypium hirsutum, tomentosum, barbadense, darwinii, mustelinum and raimondii are depicted, while the mutant gluc1.1a and/or gluc1.1d sequences of these sequences, and of sequences essentially similar to these, are described herein below and in the Examples, with reference to the wild type GLUC1.1A and GLUC1.1D sequences. Further, the mutant GLUC1.1A sequences of Gossypium barbadense, darwinii and arboreum are depicted, while the alternative mutant gluc1.1a sequences of these sequences, and of sequences essentially similar to these, are described herein below and in the Examples. The genomic GLUC1.1A and D protein-encoding DNA, and corresponding pre-mRNA, comprises 2 exons (numbered exons 1 and 2 starting from the 5'end) interrupted by 1 intron. In the cDNA and corresponding processed mRNA (i.e. the spliced RNA), introns are removed and exons are joined, as depicted in the sequence listing and FIGS. 1 and 6. Exon sequences are more conserved evolutionarily and are therefore less variable than intron sequences.

[0215] The nucleic acid sequences of GLUC1.1A and/or GLUC1.1D from Gossypium hirsutum, from Gossypium barbadense, from Gossypium tomentosum, from Gossypium darwinii, from Gossypium mustelinum, from Gossypium arboreum, from Gossypium herbaceum, and from Gossypium raimondii depicted in the sequence listing encode wild type, functional GLUC1.1 proteins from these Gossypium species. Further, the mutant GLUC1.1A sequences of Gossypium barbadense, darwinii and arboreum depicted in the sequence listing encode wild type, non-functional GLUC1.1 proteins from these Gossypium species. Thus, these sequences are endogenous to the Gossypium species from which they were isolated. Other Gossypium species, varieties, breeding lines or wild accessions may be screened for other GLUC1.1A and GLUC1.1D alleles, encoding the same GLUC1.1A and GLUC1.1D proteins or variants thereof. For example, nucleic acid hybridization techniques (e.g. Southern blot, using for example stringent hybridization conditions) or PCR-based techniques may be used to identify GLUC1.1 alleles endogenous to other Gossypium plants. To screen such plants or plant tissues for the presence of GLUC1.1 alleles, the GLUC1.1 nucleic acid sequences provided in the sequence listing, or variants or fragments of any of these, may be used. For example whole sequences or fragments may be used as probes or primers. For example specific or degenerate primers may be used to amplify nucleic acid sequences encoding GLUC1.1 proteins from the genomic DNA of the plant or plant tissue. These GLUC1.1 nucleic acid sequences may be isolated and sequenced using standard molecular biology techniques. Bioinformatics analysis may then be used to characterize the allele(s), for example in order to determine which GLUC1.1 allele the sequence corresponds to and which GLUC1.1 protein or protein variant is encoded by the sequence.

[0216] Whether a nucleic acid sequence encodes a functional GLUC1.1 protein can be analyzed by recombinant DNA techniques as known in the art, e.g. expressing the nucleic acid molecule in a host cell (e.g. a bacterium, such as E. coli) and analyzing the endo-1,3-beta-glucanase activity of the resulting protein or cells.

[0217] In addition, it is understood that GLUC1.1 nucleic acid sequences and variants thereof (or fragments of any of these) may be identified in silico, by screening nucleic acid databases for essentially similar sequences. Likewise, a nucleic acid sequence may be synthesized chemically. Fragments of nucleic acid molecules according to the invention are also provided, which are described further below. Fragments include nucleic acid sequences encoding only the mature protein, or smaller fragments comprising all or part of the exon and/or intron sequences, etc.

[0218] Nucleic acid sequences comprising one or more nucleotide deletions, insertions or substitutions relative to the wild type nucleic acid sequences are another embodiment of the invention, as are fragments of such mutant nucleic acid molecules. Such mutant nucleic acid sequences (referred to as gluc1.1 sequences) can be generated and/or identified using various known methods, as described further below. Again, such nucleic acid molecules are provided both in endogenous form and in isolated form. In one embodiment, the mutation(s) result in one or more changes (deletions, insertions and/or substitutions) in the amino acid sequence of the encoded GLUC1.1 protein (i.e. it is not a "silent mutation"). In another embodiment, the mutation(s) in the nucleic acid sequence result in a significantly reduced or completely abolished biological activity of the encoded GLUC1.1 protein relative to the wild type protein.

[0219] The nucleic acid molecules may, thus, comprise one or more mutations, such as:

[0220] (a) a "missense mutation", which is a change in the nucleic acid sequence that results in the substitution of an amino acid for another amino acid;

[0221] (b) a "nonsense mutation" or "STOP codon mutation", which is a change in the nucleic acid sequence that results in the introduction of a premature STOP codon and thus the termination of translation (resulting in a truncated protein); plant genes contain the translation stop codons "TGA" (UGA in RNA), "TAA" (UAA in RNA) and "TAG" (UAG in RNA); thus any nucleotide substitution, insertion, deletion which results in one of these codons to be in the mature mRNA being translated (in the reading frame) will terminate translation.

[0222] (c) an "insertion mutation" of one or more amino acids, due to one or more codons having been added in the coding sequence of the nucleic acid;

[0223] (d) a "deletion mutation" of one or more amino acids, due to one or more codons having been deleted in the coding sequence of the nucleic acid;

[0224] (e) a "frameshift mutation", resulting in the nucleic acid sequence being translated in a different frame downstream of the mutation. A frameshift mutation can have various causes, such as the insertion, deletion or duplication of one or more nucleotides, but also mutations which affect pre-mRNA splicing (splice site mutations) can result in frameshifts;

[0225] (f) a "splice site mutation", which alters or abolishes the correct splicing of the pre-mRNA sequence, resulting in a protein of different amino acid sequence than the wild type. For example, one or more exons may be skipped during RNA splicing, resulting in a protein lacking the amino acids encoded by the skipped exons. Alternatively, the reading frame may be altered through incorrect splicing, or one or more introns may be retained, or alternate splice donors or acceptors may be generated, or splicing may be initiated at an alternate position (e.g. within an intron), or alternate polyadenylation signals may be generated. Correct pre-mRNA splicing is a complex process, which can be affected by various mutations in the nucleotide sequence of the GLUC1.1-encoding gene. In higher eukaryotes, such as plants, the major spliceosome splices introns containing GU at the 5' splice site (donor site) and AG at the 3' splice site (acceptor site). This GU-AG rule (or GT-AG rule; see Lewin, Genes VI, Oxford University Press 1998, pp 885-920, ISBN 0198577788) is followed in about 99% of splice sites of nuclear eukaryotic genes, while introns containing other dinucleotides at the 5' and 3' splice site, such as GC-AG and AU-AC account for only about 1% and 0.1% respectively.

[0226] As already mentioned, it is desired that the mutation(s) in the nucleic acid sequence preferably result in a mutant protein comprising significantly reduced or no enzymatic activity in vivo. Basically, any mutation which results in a protein comprising at least one amino acid insertion, deletion and/or substitution relative to the wild type protein can lead to significantly reduced or no enzymatic activity. It is, however, understood that mutations in certain parts of the protein are more likely to result in a reduced function of the mutant GLUC1.1 protein, such as mutations leading to truncated proteins, whereby significant portions of the functional domains, such as the catalytic domain, are lacking.

[0227] The functional GLUC1.1 proteins of Gossypium described herein are about 325-337 amino acids in length and comprise a number of structural and functional domains. These include the following: An N-terminal plastid target peptide of about 14-26 amino acids followed by what constitutes the mature GLUC1.1 protein. The mature GLUC1.1 protein comprises active site and glycosylation amino acid residues as indicated in Table 4 above.

[0228] Thus in one embodiment, nucleic acid sequences comprising one or more of any of the types of mutations described above are provided. In another embodiment, gluc1.1 sequences comprising one or more deletion mutations, one or more stop codon (nonsense) mutations and/or one or more splice site mutations are provided. Any of the above mutant nucleic acid sequences are provided per se (in isolated form), as are plants and plant parts comprising such sequences endogenously.

[0229] A deletion mutation in a GLUC1.1 allele, as used herein, is a mutation in a GLUC1.1 allele whereby at least 1, at least 2, 3, 4, 5, 10, 20, 30, 50, 100, 200, 500, 1000 or more bases are deleted from the corresponding wild type GLUC1.1 allele, and whereby the deletion results in the mutant GLUC1.1 allele being transcribed and translated into a mutant protein which has significantly reduced or no activity in vivo. A deletion may lead to a frame-shift and/or it may introduce a premature stop codon, or may lead to one amino acid or more amino acids (e.g. large parts) of coding sequence being removed, etc. The exact underlying molecular basis by which the deletion results in a mutant protein having significantly reduced biological activity is not important. Also provided herein are plants and plant parts in which specific GLUC1.1 alleles are completely deleted, i.e. plants and plant parts lacking one or more GLUC1.1 alleles.

[0230] A nonsense mutation in a GLUC1.1 allele, as used herein, is a mutation in a GLUC1.1 allele whereby one or more translation stop codons are introduced into the coding DNA and the corresponding mRNA sequence of the corresponding wild type GLUC1.1 allele. Translation stop codons are TGA (UGA in the mRNA), TAA (UAA) and TAG (UAG). Thus, any mutation (deletion, insertion or substitution) which leads to the generation of an in-frame stop codon in the coding sequence (exon sequence) will result in termination of translation and truncation of the amino acid chain. In one embodiment, a mutant GLUC1.1 allele comprising a nonsense mutation is a GLUC1.1 allele wherein an in-frame stop codon is introduced in the GLUC1.1 codon sequence by a single nucleotide substitution, such as the mutation of CAG to TAG, TGG to TAG, TGG to TGA, or CGA to TGA. In another embodiment, a mutant GLUC1.1 allele comprising a nonsense mutation is a GLUC1.1 allele wherein an in-frame stop codon is introduced in the GLUC1.1 codon sequence by double nucleotide substitutions, such as the mutation of CAG to TAA, TGG to TAA, CGG to TAG or TGA, CGA to TAA. In yet another embodiment, a mutant GLUC1.1 allele comprising a nonsense mutation is a GLUC1.1 allele wherein an in-frame stop codon is introduced in the GLUC1.1 codon sequence by triple nucleotide substitutions, such as the mutation of CGG to TAA. The truncated protein lacks the amino acids encoded by the coding DNA downstream of the mutation (i.e. the C-terminal part of the GLUC1.1 protein) and maintains the amino acids encoded by the coding DNA upstream of the mutation (i.e. the N-terminal part of the GLUC1.1 protein). In one embodiment, the nonsense mutation is present anywhere in front of the second conserved Glu residue, the Trp residue, the first Glu residue, and/or the Tyr residue of the active site, so that at least the conserved Glu residue, the Trp residue, the first Glu residue, and/or the Tyr residue is lacking, resulting in significantly reduced activity of the truncated protein. The more truncated the mutant protein is in comparison to the wild type protein, the more likely it is that it will lack any enzymatic activity. Thus in another embodiment, a mutant GLUC1.1 allele comprising a nonsense mutation which result in a truncated protein lacking the second conserved Glu, a truncated protein lacking the second conserved Glu residue and the Trp residue, a truncated protein lacking the second conserved Glu residue, the Trp residue and the first Glu residue, a truncated protein lacking the second conserved Glu residue, the Trp residue, the first Glu residue and the Tyr residue, or a truncated protein with even less amino acids in length are provided. In yet another embodiment, the nonsense mutation results in one or more exons not being translated into protein, such as exon 1, exon 2 or exons 1 and 2.

[0231] A splice site mutation in a GLUC1.1 allele, as used herein, is a mutation in a GLUC1.1 allele whereby a mutation in the corresponding wild type functional GLUC1.1 allele results in aberrant splicing of the pre-mRNA thereby resulting in a mutant protein having significantly reduced or no activity. The mutation may be in the consensus splice site sequence. For example, Table 5 describes consensus sequences, which--if mutated--are likely to affect correct splicing. The GT-AG splice sites commonly have other conserved nucleotides, such as 2 highly conserved nucleotides on the 5'end of the intron (in the exon), often being 5'-AG-3'. On the 3'-side of the GT dinucleotide (thus in the intron) high conservation can be found for a tetranucleotide 5'-AAGT-3'. This means that 8 nucleotides can be identified as highly conserved at the donor site.

TABLE-US-00005 TABLE 5 Consensus splice site sequences 5' splice Near 3' 3' splice Intron junction splice junction type (exon{circumflex over ( )}intron) site (intron{circumflex over ( )}exon) Found in GU-AG CRN{circumflex over ( )}GU A YnAG{circumflex over ( )}N nuclear (Canonical (A/G)AGU pre-mRNA introns; about 99%) (about 1%) {circumflex over ( )}GC AG{circumflex over ( )} nuclear pre-mRNA Non- {circumflex over ( )}AU AC{circumflex over ( )} nuclear canonical pre-mRNA introns (< about 0.1%) Canonical CUPuAPy 20-50 branch sites nucleotides 5' to splice-site acceptor of nuclear pre mRNA {circumflex over ( )} depicts the splice site; R = A or G; Y = C or T; N = A, C, G or T (but often G); n = multiple nucleotides; in bold = consensus dinucleotides in the intron sequence. Pu = purine base; Py = pyrimidine base.

[0232] Splice site structure and consensus sequences are described in the art and computer programs for identifying exons and splice site sequences, such as NetPLAntgene, BDGP or Genio, est2genome, FgeneSH, and the like, are available. Comparison of the genomic sequence or pre-mRNA sequence with the translated protein can be used to determine or verify splice sites and aberrant splicing.

[0233] Any mutation (insertion, deletion and/or substitution of one or more nucleotides) which alters pre-mRNA splicing and thereby leads to a protein with significantly reduced biological activity is encompassed herein. In one embodiment, a mutant GLUC1.1 allele comprising a splice site mutation is a GLUC1.1 allele wherein altered splicing is caused by the introduction in the GLUC1.1 transcribed DNA region of one or more nucleotide substitution(s) of the consensus dinucleotides depicted in bold above. For example, GU may for example be mutated to AU in the donor splice site and/or AG may be mutated to AA in the acceptor splice site sequence. In another embodiment, a mutant GLUC1.1 allele comprising a splice site mutation is a GLUC1.1 allele wherein altered splicing is caused by the introduction in the GLUC1.1 transcribed DNA region of one or more nucleotide substitution(s) in the conserved nucleotides in the exon sequences.

[0234] Further provided are both functional GLUC1.1 amino acid sequences and non-functional GLUC1.1 amino acid sequences (comprising one or more mutations, preferably mutations which result in a significantly reduced or no biological activity of the GLUC1.1 protein) from Gossypium species, especially from Gossypium hirsutum and Gossypium barbadense, but also from other Gossypium species, such as those indicated below. In addition, mutagenesis methods can be used to generate mutations in wild type functional GLUC1.1 alleles, thereby generating mutant non-functional GLUC1.1 alleles which can encode further non-functional GLUC1.1 proteins. In one embodiment the functional and/or non-functional GLUC1.1 amino acid sequences are provided within a Gossypium plant (i.e. endogenously). However, isolated GLUC1.1 amino acid sequences (e.g. isolated from the plant or made synthetically), as well as variants thereof and fragments of any of these are also provided herein.

[0235] Amino acid sequences of GLUC1.1A and GLUC1.1D proteins have been determined from Gossypium hirsutum, from Gossypium barbadense, from Gossypium tomentosum, from Gossypium darwinii, from Gossypium mustilinum, from Gossypium arboreum, from Gossypium herbaceum, and from Gossypium raimondii as depicted in the sequence listing and FIGS. 2 and 7. The wild type functional GLUC1.1A sequences of Gossypium hirsutum, tomentosum, mustilinum and herbaceum and wild type functional GLUC1.1D sequences of Gossypium hirsutum, tomentosum, barbadense, darwinii, mustilinum and raimondii are depicted, while mutant non-functional GLUC1.1A sequences of these, and of sequences essentially similar to these, are described herein below, with reference to the wild type functional GLUC1.1A and GLUC1.1D sequences. Further, the wild type non-functional GLUC1.1A sequences of Gossypium barbadense, darwinii and arboreum are depicted, while alternative (mutant) non-functional GLUC1.1A sequences of these sequences, and of sequences essentially similar to these, are described herein below and in the Examples.

[0236] As described above, the functional GLUC1.1 proteins of Gossypium described herein are about 325-337 amino acids in length and comprise a number of structural and functional domains. The sequences of the N-terminal part of the GLUC1.1 proteins are less conserved evolutionarily than the sequences of the mature GLUC1.1 proteins. The sequences of the mature GLUC1.1 proteins are therefore less variable than the sequences of the precursor proteins.

[0237] "GLUC1.1A amino acid sequences" or "GLUC1.1A variant amino acid sequences" according to the invention are amino acid sequences having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 100% sequence identity to SEQ ID NO: 4. These amino acid sequences may also be referred to as being "essentially similar" or "essentially identical" or "corresponding to" the GLUC1.1A sequences provided in the sequence listing.

[0238] "GLUC1.1D amino acid sequences" or "GLUC1.1D variant amino acid sequences" according to the invention are amino acid sequences having at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 100% sequence identity to SEQ ID NO: 10. These amino acid sequences may also be referred to as being "essentially similar" or "essentially identical" or "corresponding to" the GLUC1.1D sequences provided in the sequence listing.

[0239] Thus, the invention provides both amino acid sequences of wild type functional and non-functional GLUC1.1A and GLUC1.1D proteins, including variants and fragments thereof (as defined further below), as well as mutant non-functional amino acid sequences of any of these, whereby the mutation in the amino acid sequence preferably results in a significant reduction in the biological activity of the GLUC1.1 protein. A significant reduction in biological activity of the (wild type or mutant) non-functional GLUC1.1 protein, refers to a reduction in enzymatic activity (i.e. in endo-1,3-beta-glucanase activity) by at least 30%, at least 40%, 50% or more, at least 90% or 100% (no biological activity) compared to the activity of the functional protein.

[0240] Both endogenous and isolated amino acid sequences are provided herein. A "fragment" of a GLUC1.1 amino acid sequence or variant thereof (as defined) may be of various lengths, such as at least 10, 12, 15, 18, 20, 50, 100, 200, 400 contiguous amino acids of the GLUC1.1 sequence (or of the variant sequence).

[0241] The amino acid sequences depicted in the sequence listing are wild type GLUC1.1 proteins from Gossypium species. Thus, these sequences are endogenous to the Gossypium plants from which they were isolated. Other Gossypium species, varieties, breeding lines or wild accessions may be screened for other (functional or non-functional) GLUC1.1 proteins with the same amino acid sequences or variants thereof, as described above.

[0242] In addition, it is understood that GLUC1.1 amino acid sequences and variants thereof (or fragments of any of these) may be identified in silico, by screening amino acid databases for essentially similar sequences. Fragments of amino acid molecules according to the invention are also provided. Fragments include amino acid sequences of the mature protein, or smaller fragments comprising all or part of the amino acid sequences, etc.

[0243] Amino acid sequences comprising one or more amino acid deletions, insertions or substitutions relative to the wild type (functional or non-functional) amino acid sequences are another embodiment of the invention, as are fragments of such mutant amino acid molecules. Such mutant amino acid sequences can be generated and/or identified using various known methods, as described above. Again, such amino acid molecules are provided both in endogenous form and in isolated form.

[0244] In one embodiment, the mutation(s) in the amino acid sequence result in a significantly reduced or completely abolished biological activity of the GLUC1.1 protein relative to the wild type protein. As described above, basically, any mutation which results in a protein comprising at least one amino acid insertion, deletion and/or substitution relative to the wild type protein can lead to significantly reduced (or no) enzymatic activity. It is, however, understood that mutations in certain parts of the protein are more likely to result in a reduced function of the mutant GLUC1.1 protein, such as mutations leading to truncated proteins, whereby significant portions of the functional domains, such as the active site or glycosylation site (see above), are lacking or mutations whereby conserved amino acid residues which have a catalytic function or which are involved in substrate specificity are substituted.

[0245] Thus in one embodiment, mutant GLUC1.1 proteins are provided comprising one or more deletion or insertion mutations, whereby the deletion(s) or insertion(s) result(s) in a mutant protein which has significantly reduced or no activity in vivo. Such mutant GLUC1.1 proteins are GLUC1.1 proteins wherein at least 1, at least 2, 3, 4, 5, 10, 20, 30, 50, 100, 200, 300, 400 or more amino acids are deleted or inserted as compared to the wild type GLUC1.1 protein, whereby the deletion(s) or insertion(s) result(s) in a mutant protein which has significantly reduced or no activity in vivo.

[0246] In another embodiment, mutant GLUC1.1 proteins are provided which are truncated whereby the truncation results in a mutant protein which has significantly reduced or no activity in vivo. Such truncated GLUC1.1 proteins are GLUC1.1 proteins which lack functional domains, such as active site residues and/or glycosylation site residues, in the C-terminal part of the corresponding wild type (mature) GLUC1.1 protein and which maintain the N-terminal part of the corresponding wild type (mature) GLUC1.1 protein. Thus in one embodiment, a truncated GLUC1.1 protein comprising the N-terminal part of the corresponding wild type (mature) GLUC1.1 protein up to but not including the conserved second Glu residue (as described above) is provided. The more truncated the mutant protein is in comparison to the wild type protein, the more likely it is that it will lack any enzymatic activity. Thus in another embodiment, a truncated GLUC1.1 protein comprising the N-terminal part of the corresponding wild type (mature) GLUC1.1 protein up to but not including the conserved Trp and/or the first Glu residue (as described above) is provided. In yet another embodiment, a truncated GLUC1.1 protein comprising the N-terminal part of the corresponding wild type (mature) GLUC1.1 protein up to but not including the conserved Tyr residue (as described above), or lacking even more amino acids, is provided.

[0247] In yet another embodiment, mutant GLUC1.1 proteins are provided comprising one or more substitution mutations, whereby the substitution(s) result(s) in a mutant protein which has significantly reduced or no activity in vivo. Such mutant GLUC1.1 proteins are GLUC1.1 proteins whereby conserved amino acid residues which have a catalytic function or which are involved in substrate binding or specificity (for example, those described above) are substituted. Thus in one embodiment, a mutant GLUC1.1 protein comprising a substitution of a conserved amino acid residue which has a catalytic function, such as the conserved first or second Glu, Trp, and/or Tyr residues, is provided. In another embodiment, a mutant GLUC1.1 protein comprising a substitution of a conserved amino acid residue involved in glycosylation, such as the conserved Asn residue, is provided.

[0248] In another aspect of the invention, methods are provided for generating mutant gluc1.1 alleles (for example induced by mutagenesis) and/or identifying mutant gluc1.1 alleles using a range of methods, which are conventional in the art, for example using PCR based methods to amplify part or all of the gluc1.1 genomic or cDNA.

[0249] The term "mutagenesis", as used herein, refers to the process in which plant cells (e.g., a plurality of Gossypium seeds or other parts, such as pollen) are subjected to a technique which induces mutations in the DNA of the cells, such as contact with a mutagenic agent, such as a chemical substance (such as ethylmethylsulfonate (EMS), ethylnitrosourea (ENU), etc.) or ionizing radiation (neutrons (such as in fast neutron mutagenesis, etc.), alpha rays, gamma rays (such as that supplied by a Cobalt 60 source), X-rays, UV-radiation, etc.), or a combination of two or more of these. Thus, the desired mutagenesis of one or more GLUC1.1 alleles may be accomplished by use of chemical means such as by contact of one or more plant tissues with ethylmethylsulfonate (EMS), ethylnitrosourea, etc., by the use of physical means such as x-ray, etc, or by gamma radiation, such as that supplied by a Cobalt 60 source.

[0250] Following mutagenesis, Gossypium plants are grown from the treated seeds, or regenerated from the treated cells using known techniques. For instance, the resulting Gossypium seeds may be planted in accordance with conventional growing procedures and following self-pollination seed is formed on the plants. Additional seed which is formed as a result of such self-pollination in the present or a subsequent generation may be harvested and screened for the presence of mutant GLUC1.1 alleles, using techniques which are conventional in the art, for example polymerase chain reaction (PCR) based techniques (amplification of the gluc1.1 alleles) or hybridization based techniques, e.g. Southern blot analysis, and/or direct sequencing of gluc1.1 alleles. To screen for the presence of point mutations (so called Single Nucleotide Polymorphisms or SNPs) in mutant GLUC1.1 alleles, SNP detection methods conventional in the art can be used, for example oligoligation-based techniques, single base extension-based techniques or techniques based on differences in restriction sites, such as TILLING.

[0251] As described above, mutagenization (spontaneous as well as induced) of a specific wild-type (functional or non-functional) GLUC1.1 allele results in the presence of one or more deleted, inserted, or substituted nucleotides (hereinafter called "mutation region") in the resulting mutant GLUC1.1 allele. The mutant GLUC1.1 allele can thus be characterized by the location and the configuration of the one or more deleted, inserted, or substituted nucleotides in the wild type GLUC1.1 allele. The site in the wild type GLUC1.1 allele where the one or more nucleotides have been inserted, deleted, or substituted, respectively, is also referred to as the "mutation region". A "5' or 3' flanking region or sequence" as used herein refers to a DNA region or sequence in the mutant (or the corresponding wild type) GLUC1.1 allele of at least 20 bp, preferably at least 50 bp, at least 750 bp, at least 1500 bp, and up to 5000 bp of DNA different from the DNA containing the one or more deleted, inserted, or substituted nucleotides, preferably DNA from the mutant (or the corresponding wild type) GLUC1.1 allele which is located either immediately upstream of and contiguous with (5' flanking region or sequence") or immediately downstream of and contiguous with (3' flanking region or sequence") the mutation region in the mutant GLUC1.1 allele (or in the corresponding wild type GLUC1.1 allele).

[0252] The tools developed to identify a specific mutant GLUC1.1 allele or the plant or plant material comprising a specific mutant GLUC1.1 allele, or products which comprise plant material comprising a specific mutant GLUC1.1 allele are based on the specific genomic characteristics of the specific mutant GLUC1.1 allele as compared to the genomic characteristics of the corresponding wild type GLUC1.1 allele, such as, a specific restriction map of the genomic region comprising the mutation region, molecular markers or the sequence of the flanking and/or mutation regions.

[0253] Once a specific mutant GLUC1.1 allele has been sequenced, primers and probes can be developed which specifically recognize a sequence within the 5' flanking, 3' flanking and/or mutation regions of the mutant GLUC1.1 allele in the nucleic acid (DNA or RNA) of a sample by way of a molecular biological technique. For instance a PCR method can be developed to identify the mutant GLUC1.1 allele in biological samples (such as samples of plants, plant material or products comprising plant material). Such a PCR is based on at least two specific "primers": one recognizing a sequence within the 5' or 3' flanking region of the mutant GLUC1.1 allele and the other recognizing a sequence within the 3' or 5' flanking region of the mutant GLUC1.1 allele, respectively; or one recognizing a sequence within the 5' or 3' flanking region of the mutant GLUC1.1 allele and the other recognizing a sequence within the mutation region of the mutant GLUC1.1 allele; or one recognizing a sequence within the 5' or 3' flanking region of the mutant GLUC1.1 allele and the other recognizing a sequence spanning the joining region between the 3' or 5' flanking region and the mutation region of the specific mutant GLUC1.1 allele (as described further below), respectively.

[0254] The primers preferably have a sequence of between 15 and 35 nucleotides which under optimized PCR conditions "specifically recognize" a sequence within the 5' or 3' flanking region, a sequence within the mutation region, or a sequence spanning the joining region between the 3' or 5' flanking and mutation regions of the specific mutant GLUC1.1 allele, so that a specific fragment ("mutant GLUC1.1 specific fragment" or discriminating amplicon) is amplified from a nucleic acid sample comprising the specific mutant GLUC1.1 allele. This means that only the targeted mutant GLUC1.1 allele, and no other sequence in the plant genome, is amplified under optimized PCR conditions.

[0255] PCR primers suitable for the invention may be the following:

[0256] oligonucleotides ranging in length from 17 nt to about 200 nt, comprising a nucleotide sequence of at least 17 consecutive nucleotides, preferably 20 consecutive nucleotides selected from the 5' flanking sequence of a specific mutant GLUC1.1 allele (i.e., for example, the sequence 5' flanking the one or more nucleotides deleted, inserted or substituted in the mutant GLUC1.1 alleles of the invention, such as the sequence 5' flanking the deletion, non-sense or splice site mutations described above or the sequence 5' flanking the potential STOP codon or splice site mutations indicated above) at their 3' end (primers recognizing 5' flanking sequences); or

[0257] oligonucleotides ranging in length from 17 nt to about 200 nt, comprising a nucleotide sequence of at least 17 consecutive nucleotides, preferably 20 consecutive nucleotides, selected from the 3' flanking sequence of a specific mutant GLUC1.1 allele (i.e., for example, the complement of the sequence 3' flanking the one or more nucleotides deleted, inserted or substituted in the mutant GLUC1.1 alleles of the invention, such as the complement of the sequence 3' flanking the deletion, non-sense or splice site mutations described above or the complement of the sequence 3' flanking the potential STOP codon or splice site mutations indicated above) at their 3' end (primers recognizing 3' flanking sequences); or

[0258] oligonucleotides ranging in length from 17 nt to about 200 nt, comprising a nucleotide sequence of at least 17 consecutive nucleotides, preferably 20 nucleotides selected from the sequence of the mutation region of a specific mutant GLUC1.1 allele (i.e., for example, the sequence of nucleotides inserted or substituted in the GLUC1.1 genes of the invention, or the complement thereof) at their 3' end (primers recognizing mutation sequences).

[0259] The primers may of course be longer than the mentioned 17 consecutive nucleotides, and may e.g. be 20, 21, 30, 35, 50, 75, 100, 150, 200 nt long or even longer. The primers may entirely consist of nucleotide sequence selected from the mentioned nucleotide sequences of flanking and mutation sequences. However, the nucleotide sequence of the primers at their 5' end (i.e. outside of the 3'-located 17 consecutive nucleotides) is less critical. Thus, the 5' sequence of the primers may consist of a nucleotide sequence selected from the flanking or mutation sequences, as appropriate, but may contain several (e.g. 1, 2, 5, 10) mismatches. The 5' sequence of the primers may even entirely consist of a nucleotide sequence unrelated to the flanking or mutation sequences, such as e.g. a nucleotide sequence representing restriction enzyme recognition sites. Such unrelated sequences or flanking DNA sequences with mismatches should preferably be not longer than 100, more preferably not longer than 50 or even 25 nucleotides.

[0260] Moreover, suitable primers may comprise or consist of a nucleotide sequence at their 3' end spanning the joining region between flanking and mutation sequences (i.e., for example, the joining region between a sequence 5' flanking one or more nucleotides deleted, inserted or substituted in the mutant GLUC1.1 alleles of the invention and the sequence of the one or more nucleotides inserted or substituted or the sequence 3' flanking the one or more nucleotides deleted, such as the joining region between a sequence 5' flanking deletion, non-sense or splice site mutations in the GLUC1.1 genes of the invention described above and the sequence of the non-sense or splice site mutations or the sequence 3' flanking the deletion mutation, or the joining region between a sequence 5' flanking a potential STOP codon or splice site mutation as indicated above and the sequence of the potential STOP codon or splice site mutation), provided the mentioned 3'-located nucleotides are not derived exclusively from either the mutation region or flanking regions.

[0261] It will also be immediately clear to the skilled artisan that properly selected PCR primer pairs should also not comprise sequences complementary to each other.

[0262] For the purpose of the invention, the "complement of a nucleotide sequence represented in SEQ ID NO: X" is the nucleotide sequence which can be derived from the represented nucleotide sequence by replacing the nucleotides through their complementary nucleotide according to Chargaff's rules (AT; GC) and reading the sequence in the 5' to 3' direction, i.e in opposite direction of the represented nucleotide sequence.

[0263] Examples of primers suitable to identify specific mutant GLUC1.1 alleles are described in the Examples.

[0264] As used herein, "the nucleotide sequence of SEQ ID No. Z from position X to position Y" indicates the nucleotide sequence including both nucleotide endpoints.

[0265] Preferably, the amplified fragment has a length of between 50 and 1000 nucleotides, such as a length between 50 and 500 nucleotides, or a length between 100 and 350 nucleotides. The specific primers may have a sequence which is between 80 and 100% identical to a sequence within the 5' or 3' flanking region, a sequence within the mutation region, or a sequence spanning the joining region between the 3' or 5' flanking and mutation regions of the specific mutant GLUC1.1 allele, provided the mismatches still allow specific identification of the specific mutant GLUC1.1 allele with these primers under optimized PCR conditions. The range of allowable mismatches however, can easily be determined experimentally and are known to a person skilled in the art.

[0266] Detection and/or identification of a "mutant GLUC1.1 specific fragment" can occur in various ways, e.g., via size estimation after gel or capillary electrophoresis or via fluorescence-based detection methods. The mutant GLUC1.1 specific fragments may also be directly sequenced. Other sequence specific methods for detection of amplified DNA fragments are also known in the art.

[0267] Standard PCR protocols are described in the art, such as in `PCR Applications Manual" (Roche Molecular Biochemicals, 2nd Edition, 1999) and other references. The optimal conditions for the PCR, including the sequence of the specific primers, is specified in a "PCR identification protocol" for each specific mutant GLUC1.1 allele. It is however understood that a number of parameters in the PCR identification protocol may need to be adjusted to specific laboratory conditions, and may be modified slightly to obtain similar results. For instance, use of a different method for preparation of DNA may require adjustment of, for instance, the amount of primers, polymerase, MgCl₂ concentration or annealing conditions used. Similarly, the selection of other primers may dictate other optimal conditions for the PCR identification protocol. These adjustments will however be apparent to a person skilled in the art, and are furthermore detailed in current PCR application manuals such as the one cited above.

[0268] Examples of PCR identification protocols to identify specific mutant GLUC1.1 alleles are described in the Examples.

[0269] Alternatively, specific primers can be used to amplify a mutant GLUC1.1 specific fragment that can be used as a "specific probe" for identifying a specific mutant GLUC1.1 allele in biological samples. Contacting nucleic acid of a biological sample, with the probe, under conditions which allow hybridization of the probe with its corresponding fragment in the nucleic acid, results in the formation of a nucleic acid/probe hybrid. The formation of this hybrid can be detected (e.g. labeling of the nucleic acid or probe), whereby the formation of this hybrid indicates the presence of the specific mutant GLUC1.1 allele. Such identification methods based on hybridization with a specific probe (either on a solid phase carrier or in solution) have been described in the art. The specific probe is preferably a sequence which, under optimized conditions, hybridizes specifically to a region within the 5' or 3' flanking region and/or within the mutation region of the specific mutant GLUC1.1 allele (hereinafter referred to as "GLUC1.1 mutation specific region"). Preferably, the specific probe comprises a sequence of between 20 and 1000 bp, 50 and 600 bp, between 100 to 500 bp, between 150 to 350 bp, which is at least 80%, preferably between 80 and 85%, more preferably between 85 and 90%, especially preferably between 90 and 95%, most preferably between 95% and 100% identical (or complementary) to the nucleotide sequence of a specific region. Preferably, the specific probe will comprise a sequence of about 15 to about 100 contiguous nucleotides identical (or complementary) to a specific region of the specific mutant GLUC1.1 allele.

[0270] Specific probes suitable for the invention may be the following:

[0271] oligonucleotides ranging in length from 20 nt to about 1000 nt, comprising a nucleotide sequence of at least 20 consecutive nucleotides selected from the 5' flanking sequence of a specific mutant GLUC1.1 allele (i.e., for example, the sequence 5' flanking the one or more nucleotides deleted, inserted or substituted in the mutant GLUC1.1 alleles of the invention, such as the sequence 5' flanking the deletion, non-sense or splice site mutations described above or the sequence 5' flanking the potential STOP codon or splice site mutations indicated above), or a sequence having at least 80% sequence identity therewith (probes recognizing 5' flanking sequences); or

[0272] oligonucleotides ranging in length from 20 nt to about 1000 nt, comprising a nucleotide sequence of at least 20 consecutive nucleotides selected from the 3' flanking sequence of a specific mutant GLUC1.1 allele (i.e., for example, the sequence 3' flanking the one or more nucleotides deleted, inserted or substituted in the mutant GLUC1.1 alleles of the invention, such as the sequence 3' flanking the deletion, non-sense or splice site mutations described above or the sequence 3' flanking the potential STOP codon or splice site mutations indicated above), or a sequence having at least 80% sequence identity therewith (probes recognizing 3' flanking sequences); or

[0273] oligonucleotides ranging in length from 20 nt to about 1000 nt, comprising a nucleotide sequence of at least 20 consecutive nucleotides selected from the mutation sequence of a specific mutant GLUC1.1 allele (i.e., for example, the sequence of nucleotides inserted or substituted in the GLUC1.1 genes of the invention, or the complement thereof), or a sequence having at least 80% sequence identity therewith (probes recognizing mutation sequences).

[0274] The probes may entirely consist of nucleotide sequence selected from the mentioned nucleotide sequences of flanking and mutation sequences. However, the nucleotide sequence of the probes at their 5' or 3' ends is less critical. Thus, the 5' or 3' sequences of the probes may consist of a nucleotide sequence selected from the flanking or mutation sequences, as appropriate, but may consist of a nucleotide sequence unrelated to the flanking or mutation sequences. Such unrelated sequences should preferably be not longer than 50, more preferably not longer than 25 or even not longer than 20 or 15 nucleotides.

[0275] Moreover, suitable probes may comprise or consist of a nucleotide sequence spanning the joining region between flanking and mutation sequences (i.e., for example, the joining region between a sequence 5' flanking one or more nucleotides deleted, inserted or substituted in the mutant GLUC1.1 alleles of the invention and the sequence of the one or more nucleotides inserted or substituted or the sequence 3' flanking the one or more nucleotides deleted, such as the joining region between a sequence 5' flanking deletion, non-sense or splice site mutations in the GLUC1.1 genes of the invention described above and the sequence of the non-sense or splice site mutations or the sequence 3' flanking the deletion mutation, or the joining region between a sequence 5' flanking a potential STOP codon or splice site mutation indicated above and the sequence of the potential STOP codon or splice site mutation), provided the mentioned nucleotide sequence is not derived exclusively from either the mutation region or flanking regions.

[0276] Examples of specific probes suitable to identify specific mutant GLUC1.1 alleles are described in the Examples.

[0277] Detection and/or identification of a "mutant GLUC1.1 specific region" hybridizing to a specific probe can occur in various ways, e.g., via size estimation after gel electrophoresis or via fluorescence-based detection methods. Other sequence specific methods for detection of a "mutant GLUC1.1 specific region" hybridizing to a specific probe are also known in the art.

[0278] Alternatively, plants or plant parts comprising one or more mutant gluc1.1 alleles can be generated and identified using other methods, such as the "Delete-a-gene®" method which uses PCR to screen for deletion mutants generated by fast neutron mutagenesis (reviewed by Li and Zhang, 2002, Funct Integr Genomics 2:254-258), by the TILLING (Targeting Induced Local Lesions IN Genomes) method which identifies EMS-induced point mutations using denaturing high-performance liquid chromatography (DHPLC) to detect base pair changes by heteroduplex analysis (McCallum et al., 2000, Nat Biotech 18:455, and McCallum et al. 2000, Plant Physiol. 123, 439-442), etc. As mentioned, TILLING uses high-throughput screening for mutations (e.g. using Cel 1 cleavage of mutant-wildtype DNA heteroduplexes and detection using a sequencing gel system). Thus, the use of TILLING to identify plants, seeds and tissues comprising one or more mutant gluc1.1 alleles in one or more tissues and methods for generating and identifying such plants is encompassed herein. Thus in one embodiment, the method according to the invention comprises the steps of mutagenizing plant seeds (e.g. EMS mutagenesis), pooling of plant individuals or DNA, PCR amplification of a region of interest, heteroduplex formation and high-throughput detection, identification of the mutant plant, sequencing of the mutant PCR product. It is understood that other mutagenesis and selection methods may equally be used to generate such mutant plants.

[0279] Instead of inducing mutations in GLUC1.1 alleles, natural (spontaneous) mutant alleles may be identified by methods known in the art. For example, ECOTILLING may be used (Henikoff et al. 2004, Plant Physiology 135(2):630-6) to screen a plurality of plants or plant parts for the presence of natural mutant gluc1.1 alleles. As for the mutagenesis techniques above, preferably Gossypium species are screened which comprise an A and/or a D genome, so that the identified gluc1.1 allele can subsequently be introduced into other Gossypium species, such as Gossypium hirsutum, by crossing (inter- or intraspecific crosses) and selection. In ECOTILLING natural polymorphisms in breeding lines or related species are screened for by the TILLING methodology described above, in which individual or pools of plants are used for PCR amplification of the gluc1.1 target, heteroduplex formation and high-throughput analysis. This can be followed up by selecting individual plants having a required mutation that can be used subsequently in a breeding program to incorporate the desired mutant allele.

[0280] The identified mutant alleles can then be sequenced and the sequence can be compared to the wild type allele to identify the mutation(s). Optionally functionality can be tested by expression in a homologous or heterologous host and testing the mutant GLUC1.1 protein for functionality in an enzyme assay. Using this approach a plurality of mutant gluc1.1 alleles (and Gossypium plants comprising one or more of these) can be identified. The desired mutant alleles can then be combined with the desired wild type alleles by crossing and selection methods as described further below. Finally a single plant comprising the desired number of mutant gluc1.1 and the desired number of wild type GLUC1.1 alleles is generated.

[0281] Oligonucleotides suitable as PCR primers or specific probes for detection of a specific mutant GLUC1.1 allele can also be used to develop methods to determine the zygosity status of the specific mutant GLUC1.1 allele.

[0282] To determine the zygosity status of a specific mutant GLUC1.1 allele, a PCR-based assay can be developed to determine the presence of a mutant and/or corresponding wild type GLUC1.1 specific allele:

[0283] To determine the zygosity status of a specific mutant GLUC1.1 allele, two primers specifically recognizing the wild-type GLUC1.1 allele can be designed in such a way that they are directed towards each other and have the mutation region located in between the primers. These primers may be primers specifically recognizing the 5' and 3' flanking sequences, respectively. This set of primers allows simultaneous diagnostic PCR amplification of the mutant, as well as of the corresponding wild type GLUC1.1 allele.

[0284] Alternatively, to determine the zygosity status of a specific mutant GLUC1.1 allele, two primers specifically recognizing the wild-type GLUC1.1 allele can be designed in such a way that they are directed towards each other and that one of them specifically recognizes the mutation region. These primers may be primers specifically recognizing the sequence of the 5' or 3' flanking region and the mutation region of the wild type GLUC1.1 allele, respectively. This set of primers, together with a third primer which specifically recognizes the sequence of the mutation region in the mutant GLUC1.1 allele, allow simultaneous diagnostic PCR amplification of the mutant GLUC1.1 gene, as well as of the wild type GLUC1.1 gene.

[0285] Alternatively, to determine the zygosity status of a specific mutant GLUC1.1 allele, two primers specifically recognizing the wild-type GLUC1.1 allele can be designed in such a way that they are directed towards each other and that one of them specifically recognizes the joining region between the 5' or 3' flanking region and the mutation region. These primers may be primers specifically recognizing the 5' or 3' flanking sequence and the joining region between the mutation region and the 3' or 5' flanking region of the wild type GLUC1.1 allele, respectively. This set of primers, together with a third primer which specifically recognizes the joining region between the mutation region and the 3' or 5' flanking region of the mutant GLUC1.1 allele, respectively, allow simultaneous diagnostic PCR amplification of the mutant GLUC1.1 gene, as well as of the wild type GLUC1.1 gene.

[0286] Alternatively, the zygosity status of a specific mutant GLUC1.1 allele can be determined by using alternative primer sets which specifically recognize mutant and wild type GLUC1.1 alleles.

[0287] If the plant is homozygous for the mutant GLUC1.1 gene or the corresponding wild type GLUC1.1 gene, the diagnostic PCR assays described above will give rise to a single PCR product typical, preferably typical in length, for either the mutant or wild type GLUC1.1 allele. If the plant is hemizygous for the mutant GLUC1.1 allele, two specific PCR products will appear, reflecting both the amplification of the mutant and the wild type GLUC1.1 allele.

[0288] Identification of the wild type and mutant GLUC1.1 specific PCR products can occur e.g. by size estimation after gel or capillary electrophoresis (e.g. for mutant GLUC1.1 alleles comprising a number of inserted or deleted nucleotides which results in a size difference between the fragments amplified from the wild type and the mutant GLUC1.1 allele, such that said fragments can be visibly separated on a gel); by evaluating the presence or absence of the two different fragments after gel or capillary electrophoresis, whereby the diagnostic PCR amplification of the mutant GLUC1.1 allele can, optionally, be performed separately from the diagnostic PCR amplification of the wild type GLUC1.1 allele; by direct sequencing of the amplified fragments; or by fluorescence-based detection methods.

[0289] Examples of primers suitable to determine the zygosity of specific mutant GLUC1.1 alleles are described in the Examples.

[0290] Alternatively, to determine the zygosity status of a specific mutant GLUC1.1 allele, a hybridization-based assay can be developed to determine the presence of a mutant and/or corresponding wild type GLUC1.1 specific allele:

[0291] To determine the zygosity status of a specific mutant GLUC1.1 allele, two specific probes recognizing the wild-type GLUC1.1 allele can be designed in such a way that each probe specifically recognizes a sequence within the GLUC1.1 wild type allele and that the mutation region is located in between the sequences recognized by the probes. These probes may be probes specifically recognizing the 5' and 3' flanking sequences, respectively. The use of one or, preferably, both of these probes allows simultaneous diagnostic hybridization of the mutant, as well as of the corresponding wild type GLUC1.1 allele.

[0292] Alternatively, to determine the zygosity status of a specific mutant GLUC1.1 allele, two specific probes recognizing the wild-type GLUC1.1 allele can be designed in such a way that one of them specifically recognizes a sequence within the GLUC1.1 wild type allele upstream or downstream of the mutation region, preferably upstream of the mutation region, and that one of them specifically recognizes the mutation region. These probes may be probes specifically recognizing the sequence of the 5' or 3' flanking region, preferably the 5' flanking region, and the mutation region of the wild type GLUC1.1 allele, respectively. The use of one or, preferably, both of these probes, optionally, together with a third probe which specifically recognizes the sequence of the mutation region in the mutant GLUC1.1 allele, allow diagnostic hybridization of the mutant and of the wild type GLUC1.1 gene.

[0293] Alternatively, to determine the zygosity status of a specific mutant GLUC1.1 allele, a specific probe recognizing the wild-type GLUC1.1 allele can be designed in such a way that the probe specifically recognizes the joining region between the 5' or 3' flanking region, preferably the 5' flanking region, and the mutation region of the wild type GLUC1.1 allele. This probe, optionally, together with a second probe which specifically recognizes the joining region between the 5' or 3' flanking region, preferably the 5' flanking region, and the mutation region of the mutant GLUC1.1 allele, allows diagnostic hybridization of the mutant and of the wild type GLUC1.1 gene.

[0294] Alternatively, the zygosity status of a specific mutant GLUC1.1 allele can be determined by using alternative sets of probes which specifically recognize mutant and wild type GLUC1.1 alleles.

[0295] If the plant is homozygous for the mutant GLUC1.1 gene or the corresponding wild type GLUC1.1 gene, the diagnostic hybridization assays described above will give rise to a single specific hybridization product, such as one or more hybridizing DNA (restriction) fragments, typical, preferably typical in length, for either the mutant or wild type GLUC1.1 allele. If the plant is hemizygous for the mutant GLUC1.1 allele, two specific hybridization products will appear, reflecting both the hybridization of the mutant and the wild type GLUC1.1 allele.

[0296] Identification of the wild type and mutant GLUC1.1 specific hybridization products can occur e.g. by size estimation after gel or capillary electrophoresis (e.g. for mutant GLUC1.1 alleles comprising a number of inserted or deleted nucleotides which results in a size difference between the hybridizing DNA (restriction) fragments from the wild type and the mutant GLUC1.1 allele, such that said fragments can be visibly separated on a gel); by evaluating the presence or absence of the two different specific hybridization products after gel or capillary electrophoresis, whereby the diagnostic hybridization of the mutant GLUC1.1 allele can, optionally, be performed separately from the diagnostic hybridization of the wild type GLUC1.1 allele; by direct sequencing of the hybridizing DNA (restriction) fragments; or by fluorescence-based detection methods.

[0297] Examples of probes suitable to determine the zygosity of specific mutant GLUC1.1 alleles are described in the Examples.

[0298] Furthermore, detection methods specific for a specific mutant GLUC1.1 allele which differ from PCR- or hybridization-based amplification methods can also be developed using the specific mutant GLUC1.1 allele specific sequence information provided herein. Such alternative detection methods include linear signal amplification detection methods based on invasive cleavage of particular nucleic acid structures, also known as Invader® technology, (as described e.g. in U.S. Pat. No. 5,985,557 "Invasive Cleavage of Nucleic Acids", U.S. Pat. No. 6,001,567 "Detection of Nucleic Acid sequences by Invader Directed Cleavage, incorporated herein by reference), RT-PCR-based detection methods, such as Taqman, or other detection methods, such as SNPlex.

[0299] In another aspect of the invention, kits are provided. A "kit" as used herein refers to a set of reagents for the purpose of performing the methods of the invention, more particularly, the identification of a specific mutant GLUC1.1 allele in biological samples or the determination of the zygosity status of plant material comprising a specific mutant GLUC1.1 allele. More particularly, a preferred embodiment of the kit of the invention comprises at least two specific primers, as described above, for identification of a specific mutant GLUC1.1 allele, or at least two or three specific primers for the determination of the zygosity status. Optionally, the kit can further comprise any other reagent described herein in the PCR identification protocol. Alternatively, according to another embodiment of this invention, the kit can comprise at least one specific probe, which specifically hybridizes with nucleic acid of biological samples to identify the presence of a specific mutant GLUC1.1 allele therein, as described above, for identification of a specific mutant GLUC1.1 allele, or at least two or three specific probes for the determination of the zygosity status. Optionally, the kit can further comprise any other reagent (such as but not limited to hybridizing buffer, label) for identification of a specific mutant GLUC1.1 allele in biological samples, using the specific probe.

[0300] The kit of the invention can be used, and its components can be specifically adjusted, for purposes of quality control (e.g., purity of seed lots), detection of the presence or absence of a specific mutant GLUC1.1 allele in plant material or material comprising or derived from plant material, such as but not limited to cotton seeds, raw cotton, cotton bales, yarn, fabric, apparel, etc.

[0301] The term "primer" as used herein encompasses any nucleic acid that is capable of priming the synthesis of a nascent nucleic acid in a template-dependent process, such as PCR. Typically, primers are oligonucleotides from 10 to 30 nucleotides, but longer sequences can be employed. Primers may be provided in double-stranded form, though the single-stranded form is preferred. Probes can be used as primers, but are designed to bind to the target DNA or RNA and need not be used in an amplification process.

[0302] The term "recognizing" as used herein when referring to specific primers, refers to the fact that the specific primers specifically hybridize to a nucleic acid sequence in a specific mutant GLUC1.1 allele under the conditions set forth in the method (such as the conditions of the PCR identification protocol), whereby the specificity is determined by the presence of positive and negative controls.

[0303] The term "hybridizing" as used herein when referring to specific probes, refers to the fact that the probe binds to a specific region in the nucleic acid sequence of a specific mutant GLUC1.1 allele under standard stringency conditions. Standard stringency conditions as used herein refers to the conditions for hybridization described herein or to the conventional hybridizing conditions as described by Sambrook et al., 1989 (Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbour Laboratory Press, NY) which for instance can comprise the following steps: 1) immobilizing plant genomic DNA fragments or BAC library DNA on a filter, 2) prehybridizing the filter for 1 to 2 hours at 65° C. in 6×SSC, 5×Denhardt's reagent, 0.5% SDS and 20 μg/ml denaturated carrier DNA, 3) adding the hybridization probe which has been labeled, 4) incubating for 16 to 24 hours, 5) washing the filter once for 30 min. at 68° C. in 6×SSC, 0.1% SDS, 6) washing the filter three times (two times for 30 min. in 30 ml and once for 10 min in 500 ml) at 68° C. in 2×SSC, 0.1% SDS, and 7) exposing the filter for 4 to 48 hours to X-ray film at -70° C.

[0304] As used in herein, a "biological sample" is a sample of a plant, plant material or product comprising plant material. The term "plant" is intended to encompass Gossypium plant tissues, at any stage of maturity, as well as any cells, tissues, or organs taken from or derived from any such plant, including without limitation, any fibers, seeds, leaves, stems, flowers, roots, single cells, gametes, cell cultures, tissue cultures or protoplasts. "Plant material", as used herein refers to material which is obtained or derived from a plant. Products comprising plant material relate to food, feed or other products, such as raw cotton, cotton bales, yarn, fabric, apparel, etc., which are produced using plant material or can be contaminated by plant material. It is understood that, in the context of the present invention, such biological samples are tested for the presence of nucleic acids specific for a specific mutant GLUC1.1 allele, implying the presence of nucleic acids in the samples. Thus the methods referred to herein for identifying a specific mutant GLUC1.1 allele in biological samples, relate to the identification in biological samples of nucleic acids which comprise the specific mutant GLUC1.1 allele.

[0305] The present invention also relates to the transfer of one or more specific mutant GLUC1.1 allele(s) in one Gossypium plant to another Gossypium plant, to the combination of specific GLUC1.1 alleles in one plant, to the plants comprising one or more specific mutant GLUC1.1 allele(s), the progeny obtained from these plants and to the plant cells, or plant material derived from these plants.

[0306] Thus, in one embodiment of the invention a method for transferring a non-functionally expressed GLUC1.1 allele from one Gossypium plant to another Gossypium plant is provided comprising the steps of:

[0307] (a) crossing a Gossypium plant comprising a non-functionally expressed GLUC1.1 allele, as described above, with a second Gossypium plant,

[0308] (b) collecting F1 hybrid seeds from the cross,

[0309] (c) optionally, backcrossing the F1 plants, derived from the F1 seeds, for one or more generations (x), collecting BCx seeds from the crosses, and identifying in every generation BCx plants, derived from the BCx seeds, comprising the non-functionally expressed GLUC1.1 allele as described above,

[0310] (d) selfing the F1 or BCx plants, derived from the F1 or BCx seeds,

[0311] (e) collecting F1 S1 or BCx S1 seeds from the selfing,

[0312] (f) identifying F1 S1 or BCx S1 plants, derived from the F1 S1 or BCx S1 seeds, comprising the non-functionally expressed GLUC1.1 allele as described above.

[0313] In another embodiment of the invention a method for combining at least two non-functionally expressed GLUC1.1 alleles in one Gossypium plant is provided comprising the steps of:

[0314] (a) transferring a non-functionally expressed GLUC1.1 allele(s) from one Gossypium plant to another Gossypium plant as described above,

[0315] (b) repeating step (a) until the desired number and/or types of non-functionally expressed GLUC1.1 alleles are combined in the second plant.

[0316] In yet another embodiment of the invention, a method is provided for altering the callose content of a fiber in a fiber producing plant, such as Gossypium plants, comprising the steps of:

[0317] (a) abolishing the functional expression of at least one allele of at least one fiber specific GLUC gene that is functionally expressed during the fiber strength building phase of fiber development,

[0318] (b) identifying a plant, which produces fibers, the callose content of which is increased as compared to the callose content of the fibers of a corresponding plant in which the functional expression of the GLUC gene is not abolished.

[0319] In still another embodiment of the invention, a method is provided for altering the properties of a fiber, particularly increasing the strength of a fiber, in a fiber producing plant, such as a Gossypium plant, comprising the steps of:

[0320] (c) abolishing the functional expression of at least one allele of at least one fiber specific GLUC gene that is functionally expressed during the fiber strength building phase of fiber development,

[0321] (d) identifying a plant, which produces fibers, the strength of which is increased as compared to the strength of fibers of a corresponding plant in which the functional expression of the GLUC gene is not abolished.

[0322] In another aspect of the invention, plant fibers with increased fiber strength are provided derived from fiber-producing plants according to the invention, especially of Gossypium hirsutum plants as provided herein, but also from other Gossypium species. For example, Gossypium species wherein the expression of at least one fiber specific GLUC gene that is functionally expressed during the fiber strength building phase of fiber development, such as a GLUC1.1A and/or GLUC1.1D gene, can be abolished, for example Gossypium tomentosum, Gossypium mustilinum, Gossypium herbaceum, or Gossypium raimondii.

[0323] Also included in the invention is the use of the fibers of this invention, for example, in the production of raw cotton, cotton bales, yarn, fabric, apparel, etc.

[0324] Other applications, such as mixing fibers with a specific callose content and/or a specific modified strength according to the invention with other fibers with a lower callose content and/or a lower fiber to increase the average callose content and/or fiber strength in, for example, cotton bales, yarn, fabric, apparel, etc; thus making it more suitable for certain applications, such as but not limited to, the production of biodiesel, stronger textile, etc., are also included in the invention.

[0325] It will be clear that whenever nucleotide sequences of RNA molecules are defined by reference to nucleotide sequence of corresponding DNA molecules, the thymine (T) in the nucleotide sequence should be replaced by uracil (U). Whether reference is made to RNA or DNA molecules will be clear from the context of the application.

[0326] It is understood that when referring to a word in the singular (e.g. plant or root), the plural is also included herein (e.g. a plurality of plants, a plurality of roots). Thus, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one".

[0327] As used herein "comprising" is to be interpreted as specifying the presence of the stated features, integers, steps or components as referred to, but does not preclude the presence or addition of one or more features, integers, steps or components, or groups thereof. Thus, e.g., a nucleic acid or protein comprising a sequence of nucleotides or amino acids, may comprise more nucleotides or amino acids than the actually cited ones, i.e., be embedded in a larger nucleic acid or protein. A chimeric gene comprising a DNA region, which is functionally or structurally defined, may comprise additional DNA regions etc. A plant comprising a certain trait may thus comprise additional traits etc.

[0328] The following non-limiting Examples describe the identification of a fiber strength locus on chromosome A05 in cotton and the characterization of a GLUC1.1 gene located in the 1-LOD support interval of the Strengt QTL. Unless stated otherwise in the Examples, all recombinant DNA techniques are carried out according to standard protocols as described in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, NY and in Volumes 1 and 2 of Ausubel et al. (1994) Current Protocols in Molecular Biology, Current Protocols, USA. Standard materials and methods for plant molecular work are described in Plant Molecular Biology Labfax (1993) by R. D. D. Croy, jointly published by BIOS Scientific Publications Ltd (UK) and Blackwell Scientific Publications, UK.

[0329] Throughout the description and Examples, reference is made to the following sequences represented in the sequence listing:

[0330] SEQ ID NO: 1: amplified genomic DNA fragment of endo-1,3-beta-glucanase gene from Gossypium hirsutum cv. Fiber Max966, A-subgenome specific

[0331] SEQ ID NO: 2: endo-1,3-beta-glucanase protein encoded by SEQ ID NO: 1

[0332] SEQ ID NO: 3: amplified cDNA fragment of endo-1,3-beta-glucanase gene from Gossypium hirsutum cv. Fiber Max966, A-subgenome specific

[0333] SEQ ID NO: 4: endo-1,3-beta-glucanase protein encoded by SEQ ID NO: 3

[0334] SEQ ID NO: 5: amplified genomic DNA fragment of endo-1,3-beta-glucanase gene from Gossypium barbadense cv. PimaS7, A-subgenome specific

[0335] SEQ ID NO: 6: endo-1,3-beta-glucanase protein encoded by SEQ ID NO: 5

[0336] SEQ ID NO: 7: amplified genomic DNA fragment of endo-1,3-beta-glucanase gene from Gossypium hirsutum cv. Fiber Max966, D-subgenome specific

[0337] SEQ ID NO: 8: endo-1,3-beta-glucanase protein encoded by SEQ ID NO: 7

[0338] SEQ ID NO: 9: amplified cDNA fragment of endo-1,3-beta-glucanase gene from Gossypium hirsutum cv. Fiber Max966, D-subgenome specific

[0339] SEQ ID NO: 10: endo-1,3-beta-glucanase protein encoded by SEQ ID NO: 9

[0340] SEQ ID NO: 11: amplified genomic DNA fragment of endo-1,3-beta-glucanase gene from Gossypium barbadense cv. PimaS7, D-subgenome specific

[0341] SEQ ID NO: 12: endo-1,3-beta-glucanase protein encoded by SEQ ID NO: 11

[0342] SEQ ID NO: 13: amplified cDNA fragment of endo-1,3-beta-glucanase gene from Gossypium barbadense cv. PimaS7, D-subgenome specific

[0343] SEQ ID NO: 14: endo-1,3-beta-glucanase protein encoded by SEQ ID NO: 13

[0344] SEQ ID NO: 15: amplified genomic DNA fragment of endo-1,3-beta-glucanase gene from Gossypium tomentosum, A-subgenome specific

[0345] SEQ ID NO: 16: endo-1,3-beta-glucanase protein encoded by SEQ ID NO: 15

[0346] SEQ ID NO: 17: amplified genomic DNA fragment of endo-1,3-beta-glucanase gene from Gossypium darwinii, A-subgenome specific

[0347] SEQ ID NO: 18: endo-1,3-beta-glucanase protein encoded by SEQ ID NO: 17

[0348] SEQ ID NO: 19: amplified genomic DNA fragment of endo-1,3-beta-glucanase gene from Gossypium mustelinum, A-subgenome specific

[0349] SEQ ID NO: 20: endo-1,3-beta-glucanase protein encoded by SEQ ID NO: 19

[0350] SEQ ID NO: 21: amplified genomic DNA fragment of endo-1,3-beta-glucanase gene from Gossypium arboreum, A-subgenome specific

[0351] SEQ ID NO: 22: endo-1,3-beta-glucanase protein encoded by SEQ ID NO: 21

[0352] SEQ ID NO: 23: amplified genomic DNA fragment of endo-1,3-beta-glucanase gene from Gossypium herbaceum, A-subgenome specific

[0353] SEQ ID NO: 24: endo-1,3-beta-glucanase protein encoded by SEQ ID NO: 23

[0354] SEQ ID NO: 25: amplified genomic DNA fragment of endo-1,3-beta-glucanase gene from Gossypium tomentosum, D-subgenome specific

[0355] SEQ ID NO: 26: endo-1,3-beta-glucanase protein encoded by SEQ ID NO: 25

[0356] SEQ ID NO: 27: amplified genomic DNA fragment of endo-1,3-beta-glucanase gene from Gossypium darwinii, D-subgenome specific

[0357] SEQ ID NO: 28: endo-1,3-beta-glucanase protein encoded by SEQ ID NO: 27

[0358] SEQ ID NO: 29: amplified genomic DNA fragment of endo-1,3-beta-glucanase gene from Gossypium mustelinum, D-subgenome specific

[0359] SEQ ID NO: 30: endo-1,3-beta-glucanase protein encoded by SEQ ID NO: 29

[0360] SEQ ID NO: 31: amplified genomic DNA fragment of endo-1,3-beta-glucanase gene from Gossypium raimondii, D-subgenome specific

[0361] SEQ ID NO: 32: endo-1,3-beta-glucanase protein encoded by SEQ ID NO: 31

[0362] SEQ ID NO: 33: forward primer SE077 for amplification of endo-1,3-beta-glucanase genomic fragment

[0363] SEQ ID NO: 34: reverse primer SE078 for amplification of endo-1,3-beta-glucanase genomic fragment

[0364] SEQ ID NO: 35: forward primer SE002 for amplification of endo-1,3-beta-glucanase genomic fragment

[0365] SEQ ID NO: 36: reverse primer SE003 for amplification of endo-1,3-beta-glucanase genomic fragment

[0366] SEQ ID NO: 37: forward primer p1.3GlucaAf for amplification of endo-1,3-beta-glucanase genomic fragment, in particular for discriminating different variants of polymorphic site GLUC1.1A-SNP2

[0367] SEQ ID NO: 38: reverse primer p1.3GlucaAr for amplification of endo-1,3-beta-glucanase genomic fragment, in particular for discriminating different variants of polymorphic site GLUC1.1A-SNP2

[0368] SEQ ID NO: 39: probe TM249-GCM1 for detecting the G. barbadense variant of polymorphic site GLUC1.1A-SNP3

[0369] SEQ ID NO: 40: probe TM249-GCV1 for detecting the G. hirsutum variant of polymorphic site GLUC1.1A-SNP3

[0370] SEQ ID NO: 41: forward primer TM249-GCF for amplification of endo-1,3-beta-glucanase genomic fragment, in particular for discriminating different variants of polymorphic site GLUC1.1A-SNP3

[0371] SEQ ID NO: 42: reverse primer TM249-GCR for amplification of endo-1,3-beta-glucanase genomic fragment, in particular for discriminating different variants of polymorphic site GLUC1.1A-SNP3

[0372] SEQ ID NO: 43: AFLP primer P5 for amplification of genomic DNA fragment corresponding to marker P5M50-M126.7, in particular for discriminating different variants of marker P5M50-M126.7

[0373] SEQ ID NO: 44: AFLP primer M50 for amplification of genomic DNA fragment corresponding to marker P5M50-M126.7, in particular for discriminating different variants of marker P5M50-M126.7

[0374] SEQ ID NO: 45: forward SSR primer for amplification of genomic DNA fragment corresponding to marker NAU861, in particular for discriminating different variants of marker NAU861

[0375] SEQ ID NO: 46: reverse SSR primer for amplification of genomic DNA fragment corresponding to marker NAU861, in particular for discriminating different variants of marker NAU861

[0376] SEQ ID NO: 47: forward SSR primer for amplification of genomic DNA fragment corresponding to marker CIR401, in particular for discriminating different variants of marker CIR401

[0377] SEQ ID NO: 48: reverse SSR primer for amplification of genomic DNA fragment corresponding to marker CIR401, in particular for discriminating different variants of marker CIR401

[0378] SEQ ID NO: 49: forward SSR primer for amplification of genomic DNA fragment corresponding to marker BNL3992, in particular for discriminating different variants of marker BNL3992

[0379] SEQ ID NO: 50: reverse SSR primer for amplification of genomic DNA fragment corresponding to marker BNL3992, in particular for discriminating different variants of marker BNL3992

[0380] SEQ ID NO: 51: forward SSR primer for amplification of genomic DNA fragment corresponding to marker CIR280, in particular for discriminating different variants of marker CIR280

[0381] SEQ ID NO: 52: reverse SSR primer for amplification of genomic DNA fragment corresponding to marker CIR280, in particular for discriminating different variants of marker CIR280

[0382] SEQ ID NO: 53: DNA sequence of a 165250 bps DNA fragment spanning the GLUC1.1A gene in G. hirsutum

[0383] SEQ ID NO: 54: amplified cDNA fragment of endo-1,3-beta-glucanase gene from Gossypium barbadense cv. PimaS7, A-subgenome specific

[0384] SEQ ID NO: 55: endo-1,3-beta-glucanase protein encoded by SEQ ID NO: 54

[0385] SEQ ID NO: 56: amplified genomic DNA fragment of endo-1,3-beta-glucanase gene from Gossypium darwinii, A-subgenome specific

[0386] SEQ ID NO: 57: endo-1,3-beta-glucanase protein encoded by SEQ ID NO: 56

[0387] SEQ ID NO: 58: amplified genomic DNA fragment of endo-1,3-beta-glucanase gene from Gossypium darwinii, D-subgenome specific

[0388] SEQ ID NO: 59: endo-1,3-beta-glucanase protein encoded by SEQ ID NO: 58

[0389] SEQ ID NO: 60: probe for detecting the G. barbadense variant of polymorphic site GLUC1.1A-SNP5

[0390] SEQ ID NO: 61: probe for detecting the G. hirsutum variant of polymorphic site GLUC1.1A-SNP5

[0391] SEQ ID NO: 62: forward primer for amplification of endo-1,3-beta-glucanase genomic fragment, in particular for discriminating different variants of polymorphic site GLUC1.1A-SNP5

[0392] SEQ ID NO: 63: reverse primer for amplification of endo-1,3-beta-glucanase genomic fragment, in particular for discriminating different variants of polymorphic site GLUC1.1A-SNP5

[0393] SEQ ID NO: 64: forward primer G1.1-SGA-F for amplification of endo-1,3-beta-glucanase genomic fragment

[0394] SEQ ID NO: 65: forward primer G1.1-f1-F1 for amplification of endo-1,3-beta-glucanase genomic fragment

EXAMPLES

Example 1

Identification and Characterization of a Quantitative Trait Locus (QTL) on Cotton Chromosome A05 Linked to Fiber Strength

1.1. QTL Discovery

[0395] Discovery of quantitative trait loci associated with cotton fiber properties was performed according to standard procedures. Briefly, parental cotton plant lines with fiber phenotypes of interest were selected, segregating populations were generated and the impact of the presence of specific chromosomal regions on measurable cotton fiber phenotypes was determined. The parental lines were Gossypium hirsutum cv. FM966 (used as female parent in the initial cross; abbreviated hereinafter as "FM"; particularly known for its high fiber yield, but lower fiber quality compared to Gossypium barbadense varieties) and Gossypium barbadense cv. PimaS7 (used as male parent in the initial cross; abbreviated hereinafter as "Pima"; particularly known for its excellent fiber quality, but lower fiber yield compared to Gossypium hirsutum varieties). Backcross populations with both parental lines were generated and evaluated in the greenhouse as well as in the field.

1.2. Evaluation of Plants Derived from a First Backcross to the Gossypium barbadense Pima S7 Parental Line ("Pima BC1F1 Population")

[0396] A QTL for fiber strength on chromosome A05 was originally detected in a BC1F1 mapping population [(FM×Pima)×Pima; recurrent parent used as male parent] of 119 individuals. The population was grown under standard growing conditions in a greenhouse. A genome-wide genetic map of about 800 markers was constructed based on amplified fragment length polymorphism PCR (AFLP-PCR or AFLP) marker data and simple sequence repeat (SSR or microsatellite) marker data from the 119 individuals using JoinMap software (map version 8 and 13; Stam, 1993, Plant J 3: 739-744). Fiber strength was measured by High-Volume Instruments (HVI) (United States Department of Agriculture, Agricultural Marketing Service) on samples from 88 of the 119 individual plants. QTL mapping was performed using MapQTL software (Van Ooijen and Maliepaard, 1996, Plant Genome IV Abstracts, World Wide Web site: intl-pag.org). Final QTL data are based on the restricted multiple QTL mapping (rMQM; Jansen, 1993, Genetics 135:205-211; Jansen and Stam, 1994, Genetics 136:1447-1455) analysis.

[0397] A clear QTL associated with fiber strength (also referred to as "Strength locus" or "Stren locus") was detected on chromosome A05. The QTL had a sharp LOD (logarithm of the odds) score peak with a maximum value of LOD 4.92 at a position of 98.61 cM from the tip of chromosome A05, with a 1-LOD support interval of 14 cM (from 91.515 cM to 105.61 cM). The 1-LOD QTL support interval was flanked by one AFLP marker, P5M50-M126.7, at 85.515 cM, and one microsatellite marker, CIR401c, at 109.13 cM. Within the QTL support interval one microsatellite marker NAU861 (94.61 cM) and a GLUC1.1 gene (94.602 cM) were located at close distance (ca 4 cM) to the position of maximum LOD value (Table 6). Primer pairs used to distinguish between the G. hirsutum and G. barbadense alleles of the markers are indicated in Table 2 above.

TABLE-US-00006 TABLE 6 Estimated position (according to JoinMap version 8 and 13) on chromosome A05 of markers linked to the fiber strength locus in the FM and Pima BC1F1 population Position (in cM as estimated with JoinMap version 8 or 13) 1-LOD support Marker locus on chromosome A05 of: interval of on chromosome FM BC1F1 map Pima BC1F1 map Strength A05 8 13 8 13 locus P5M50-M126.7 104.582 107.9 85.515 105.5 91.515 Lower limit GLUC1.1A 107.599 111.1 94.602 114.6 NAU861 106.884 110.5 94.610 114.6 98.610 LOD Peak 105.610 upper limit CIR401c -- -- 109.130 129.1 CIR401b 112.813 115.4 -- -- BNL3992 117.199 119.5 nd 132.1 CIR280 nd 124.5 -- --

As indicated above, the GLUC1.1A gene was mapped within the support interval of the Strength locus (LOD of 4.431) using SNP marker GLUC1.1A-SNP2 as indicated in Table 13 and primers p1.3GlucaAf (SEQ ID NO: 37) and p1.3GlucaAr (SEQ ID NO: 38) as described in Example 6 below. Plants homozygous for the GLUC1.1A allele of Gossypium barbadense Pima S7 (Pima GLUC1.1A allele or Gbgluc1.1A) had 9.7% higher fiber strength compared to plants heterozygous for Gbgluc1.1A (Ho/He ratio of 109.7%). The QTL explained 17.8% of the variation for fiber strength in the population. 1.3. Evaluation of Plants Derived from a First Backcross to the Gossypium hirsutum FM966 Parental Line ("FM BC1F1 Population")

[0398] QTL mapping was also performed in a complementary BC1F1 population [(FM×Pima)×FM; recurrent parent used as male parent] of 130 individuals. Fiber strength was measured on samples from 94 of the 130 individual plants. The QTL for fiber strength in the region flanked by markers P5M50-M126.7 and CIR401 was not detected in this FM BC1F1 population (max LOD=0.42, i.e. below the critical threshold value of LOD=3). However, technically, plants heterozygous for the GLUC1.1A allele of Gossypium barbadense Pima S7 of this population did show about 1 to about 2% higher fiber strength compared to plants homozygous for the GLUC1.1A allele of Gossypium hirsutum FM966 (FM GLUC1.1A allele or GhGLUC1.1A). Together with the data from the Pima BC1F1 population this suggested that the GLUC1.1A allele of Gossypium barbadense Pima S7 provides superior fiber strength.

1.4. Evaluation of Plants Derived from a Fourth Backcross to the Gossypium hirsutum FM966 Parental Line ("FM BC4F1 Population")

[0399] With the purpose of improving fiber quality in Gossypium hirsutum, in particular in Gossypium hirsutum cv. FM966, genome fragments of the Gossypium barbadense parental line were backcrossed into the FM BC1F1 population by single seed descent and without selection during 4 generations (FM BC4F1 population). The Pima region of chromosome A05 carrying the candidate Strength locus was expected to be present in a number of these introgression lines.

[0400] A total of 219 FM BC4F1 plants originating from 75 FM BC3F1 plants (average 3 sister plants per line) were grown under standard growing conditions in a greenhouse. All plants were genotyped for 450 SSR markers and the strength of fibers from all plants was measured by HVI (see above). In the region of the Strength locus, 14 and 23 FM BC4F1 plants were heterozygous for the NAU861 and the GLUC1.1A markers, respectively, versus 196 and 194 plants that were homozygous for the NAU861 marker and the GLUC1.1A allele of Gossypium hirsutum FM966.

[0401] Table 7 summarizes the impact on fiber strength of the presence of different Pima marker alleles in heterozygous state versus the equivalent FM marker alleles in homozygous state (He/Ho ratio) in FM BC1F1 and FM BC4F1 populations. Markers indicated as CIRx, NAUx, JESPRx and BNLx are publicly available markers (see Cotton Microsatellite Database on the World Wide Web at cottonmarker.org). Markers indicated as `Primer combination X and Y-amplified fragment size` are AFLP markers (Vos et al., 1995, NAR 23:4407-4414).

[0402] A similar effect on fiber strength was observed in both the FM BC1F1 and FM BC4F1 populations for the presence of the Pima GLUC1.1A allele (i.e. plants heterozygous for the Pima GLUC1.1A allele showed about 1 to about 2% higher fiber strength compared to plants homozygous for the FM GLUC1.1A allele).

TABLE-US-00007 TABLE 7 Estimated position (according to JoinMap version 8 and 13) on chromosome A05 and impact on fiber strength of different allele combinations (He versus Ho FM) for markers linked to the fiber strength locus in FM BC1F1 and FM BC4F1 populations FM BC1F1 FM BC4F1 Position (cM) Marker locus on He/Ho He/Ho vers.13 vers. 8 chromosome A05 K* (%) K* (%) 107.9 104.582 P5M50-M126.7 1.926 102.52 110.5 106.884 NAU861 1.334 102.09 2.189 102.60 111.1 107.599 GLUC1.1A 0.802 101.30 2.037 101.87 115.4 112.813 CIR401b 1.85 103.90 5.786 103.20 119.5 117.199 BNL3992 1.329 103.32 5.786 103.20 124.5 nd CIR280 nd nd nd nd

1.5. Evaluation of Plants Derived from the F2 Generation of a Fourth Backcross to the Gossypium hirsutum FM966 Parental Line ("FM BC4F2 Population")

[0403] As a next step, QTL validation in FM BC4F2 families was performed under field conditions in summer in Mississippi. FM BC4F2 plants segregate in 3 genetic classes: plants homozygous for FM marker alleles, plants homozygous for Pima marker alleles and plants heterozygous for FM and Pima marker alleles. In most cases 75-80 plants were genotyped per line and fiber samples from about 50 single plants were analyzed. This allowed testing of the effect of the FM or Pima marker alleles (and predicted linked genes) in heterozygous and homozygous condition.

The field trial included 4 FM BC4F2 families (called lines 6, 10, 20 and 94) segregating for various portions of the region of chromosome A05 carrying the Strength locus from Pima S7. Segregation was tested using 6 markers: BNL0542, BNL3995, CIR139a, NAU861, GLUC1.1A, BNL3992.

[0404] All BC4F2 plants of line 6 were homozygous for the FM allele of the markers tested. Line 94 produced only 38 FM BC4F2 plants and only 10 of those produced sufficient fiber for single plant analysis. The two remaining lines, lines 10 and 20, produced larger numbers of plants and had good marker segregation. Line 10 contained a segment of chromosome A05 of Pima carrying the Strength locus centered around the GLUC1.1 gene. The second line, line 20, contained a segment of chromosome A05 of Pima shifted to the lower end of the Strength locus support region.

[0405] In line 10 the expectation that plants homozygous for the Pima GLUC1.1A allele produce stronger fibers was confirmed. The fiber strength of plants homozygous for the Pima GLUC1.1A allele was on average 2.5 grams per tex higher than the fiber strength of plants homozygous for the FM GLUC1.1A allele (35.5 g/tex versus 33.0 g/tex or 7.5% increase in fiber strength). A similar result was observed for the two markers NAU861 and BNL3992 which are closely linked to GLUC1.1A on either side. The differences in fiber strength between homozygous FM plants, homozygous Pima plants and heterozygous plants were not significant in Anova, but they were significant in paired t-test between homozygous FM plants and the other two classes.

[0406] In line 20 the Pima alleles of markers NAU861 and BNL3992 did not provide stronger fiber. This line segregates for a lower section of the region of Pima chromosome A05, in the tail of the QTL support interval. This line also does not contain the Pima allele of the GLUC1.1A gene.

[0407] The data in Table 8 consolidate the results for line 10 in terms of "Marker Trait Performance" for fiber strength (MTP, calculated as ratio of the difference in average trait performance for two marker classes (HoFM-HoPima) and the average standard deviation for trait performance in both marker classes). It is shown that plants homozygous for the Pima allele of markers NAU861, GLUC1.1A and BNL3992 had stronger fibers than plants homozygous for the FM allele of these markers (negative MTP). However, the difference in performance was smaller than the average standard deviation (MTP value between 0 and -1).

[0408] Thus, the field trial data provide evidence in support of the idea that there is a QTL associated with fiber strength on chromosome A05, close to or coinciding with the GLUC1.1A gene, with the superior allele coming from Gossypium barbadense PimaS7.

[0409] Due to the low number of plants in the FM BC4F2 population it was not possible to fine map the QTL position. In this respect it is noted that the Pima allele of a marker (BNL3992) that was included in the introgressed Pima fragment in line 10, but resided at a position outside the original support interval on the Pima BC1F1 map also segregated with the enhanced fiber strength derived from PimaS7. This can be explained by the fact that in the original BC1 population sufficient recombinations had occurred to place this marker outside the QTL support interval, while in the (smaller) BC4F2 populations it remained linked to the QTL causal gene more frequently.

TABLE-US-00008 TABLE 8 Estimated position on chromosome A05 and impact on fiber strength (indicated as MTP) of different allele combinations (HH FM versus HH Pima) for markers linked to the Strength locus in FM BC4F2 plant lines Graphical phenotype for marker of BC4F1 plants giving rise to Position FM BC4F2 plant MTP for (cM - Marker locus on line n^o fiber strength in vers. 8) chromosome A05 6 10 20 94 line n^o 10 78.883 CIR139a* h a a a 79.911 BNL3029.A h a a a 82.969 NAU1042.A h a a a 106.884 NAU861* h h a h -.70 107.599 GLUC1.1A* h h a h -.67 112.813 CIR401c h h a h -.55 117.199 BNL3992* h h a h 136.15 BNL0542* a a h h 146.257 E43M49-M260.0 a a h h 149.542 E31M48-M188.5 a a h a 159.609 E43M53-M460.0 a a h a 161.272 CIR294.A a a h a 163.129 BNL3995* a a h a

[0410] Column 2 lists markers on chromosome A05 linked to the Strength locus. Markers indicated as CIRx, NAUx and BNLx are publicly available markers (see Cotton Microsatellite Database on the World Wide Web at cottonmarker.org). Markers indicated as `Primer combination X and Y-amplified fragment size` are AFLP markers (Vos et al., 1995, NAR 23:4407-4414). Column 1 indicates their map positions on the genetic map (in cM) of the FM BC1F1 mapping population constructed using JoinMap software map version 8. Graphical genotypes for the markers are indicated for BC4F1 plants that gave rise to BC4F2 families 6, 10, 20 and 94: a=homozygous FM966, h=heterozygous. Segregation of the `h` regions in the graphical genotypes was investigated using marker data for markers indicated with *. Average phenotypic performance for fiber strength was compared for groups of plants homozygous for FM966 markers (genotype "HH FM") and for groups of plants homozygous for Pima markers (genotype "HH Pima"). Marker Trait Performance (MTP) is expressed as ((average phenotype HH FM-average phenotype HH Pima)/0.5×(SD HH FM+SD HH Pima)). Positive MTP means performance FM is higher than performance Pima. Negative MTP means performance Pima is higher than performance FM. MTP higher than 1 and MTP lower than -1 means delta performance exceeds average standard deviation (SD). Data for fiber strength properties are based on homozygous segregates among 60 plants.

Example 2

Identification and Characterization of a Glucanase Gene Linked to the Fiber Strength Locus on Cotton Chromosome A05

2.1 Characterization of the GLUC1.1A Gene Localized in the Support Interval of the Strength Locus

[0411] As described in Example 1.2, a GLUC1.1 gene was mapped within the support interval of the predicted QTL for fiber strength on chromosome A05, suggesting that the GLUC1.1A candidate gene might be the causal gene for fiber strength. As further described in Example 1, the superior allele comes from the Pima parental line rather than from the FM parental line.

[0412] Based on the GhGLUC1.1A and D nucleotide sequences described in WO2008/083969 (SEQ ID NO: 1 and 7, respectively), 2 primers (forward primer SE077 (SEQ ID NO: 33) en reverse primer SE078 (SEQ ID NO: 34)) were designed to amplify genomic DNA fragments for G. barbadense (reaction mix and PCR conditions as described in Example 4). Two genomic DNA sequences were derived: one for GbGLUC1.1A (SEQ ID NO: 5) and one for GbGLUC1.1D (SEQ ID NO: 11).

[0413] The 2 primers (forward primer SE077 (SEQ ID NO: 33) en reverse primer SE078 (SEQ ID NO: 34)) were also used to amplify GLUC1.1A and GLUC1.1D cDNA from cDNA libraries from G. hirsutum and G. barbadense (reaction mix and PCR conditions as described in Example 4). cDNA sequences were derived for GhGLUC1.1A (SEQ ID NO: 3), for GhGLUC1.1D (SEQ ID NO: 9), and for GbGLUC1.1D (SEQ ID NO: 13). Forward primer G1.1-SGA-F (SEQ ID NO: 64) en reverse primer SE078 (SEQ ID NO: 34) were used to amplify GLUC1.1A cDNA from a cDNA libraries from G. barbadense. The cDNA sequence was derived for GbGLUC1.1A (SEQ ID NO: 54).

[0414] Alignment of genomic and cDNA sequences of A and D subgenome-specific GLUC1.1 genes from Gossypium hirsutum and Gossypium barbadense indicated that the GLUC1.1A gene from Gossypium barbadense displayed a c to t nucleotide substitution (at position 712 of SEQ ID NO: 5) that resulted in a putative premature STOP codon (cga to tga) as compared to the GLUC1.1A and D genes from Gossypium hirsutum and the GLUC1.1D gene from Gossypium barbadense (FIG. 1), that is predicted to result in the production of a truncated GLUC1.1A protein in Gossypium barbadense (FIG. 2). Compared to the Gossypium hirsutum ortholog, the Gossypium barbadense GLUC1.1A amino acid sequence lacks the GH17 signature (FIG. 2).

2.2. Characterization of the GLUC1.1A Protein from Different Gossypium sp.

[0415] Protein modeling based on an X-ray structure of a barley 1,3-1,4-beta-glucanase belonging to the GH17 family of glycosidase hydrolases (1aq0 in Protein Data Bank) (FIG. 3, left), using FUGUE® and ORCHESTRAR® technologies from Sybyl7.3, showed that the GLUC1.1A protein of G. barbadense (FIG. 3b, right) is missing the active site and substrate binding cleft (located within the area indicated by the amino acids and their position numbers, displayed in the upper left part of the protein model of 1aq0 and described in Muller et al., 1998, J Biol Chem 273: 3438-3446), which was found to be present in the GLUC1.1A and D proteins of G. hirsutum and in the GLUC1.1D protein of G. barbadense (FIG. 3a, right). The GLUC1.1A protein of G. barbadense is therefore predicted to be inactive.

2.3. Characterization of the Genomic Regions Spanning the GLUC1.1 Alleles from Different Gossypium sp.

[0416] DNA sequencing of an about 165 kb and 136 kb region spanning the GLUC1.1A (SEQ ID NO: 53) and GLUC1.1D alleles (not shown), respectively, of Gossypium hirsutum was undertaken using 454 DNA sequencing (454 Life Sciences): Firstly BAC clones with genomic DNA spanning each GhGLUC1.1 allele were identified by hybridization using part of the GLUC1.1 gene as a probe against a FM BAC library. The BAC clones were isolated, confirmed by PCR and grouped into alleles. Selected BAC clones were sequenced to define neighboring genes facilitated by bioinformatics annotation software programs and EST searches (see FIG. 9). The BAC sequence data also identified an additional molecular marker (CIR280) located on an adjacent gene (HAT) (see Table 6 and 7 for estimated position on chromosome A05 in the FM BC1 population).

Example 3

Analysis of the Biological Role of Glucanase in Fiber Strength

3.1. Determination of Link Between Inactive GbGLUC1.1A Enzyme and Fiber Strength

[0417] To determine if there is a link between the inactive GbGLUC1.1A enzyme and fiber strength, the impact of glucanase activity on fiber strength was analyzed by exogenous addition of a 1,3-beta-glucanase enzyme to fibers from G. barbadense (comprising a GLUC1.1A predicted to be inactive), as well as fibers from G. hirsutum (comprising a GLUC1.1A predicted to be active). It was expected that the strength of the G. barbadense fibers would significantly decrease, if there was indeed a link between the inactive GbGLUC1.1A enzyme and fiber strength.

[0418] Individual fibers were treated with a beta-1,3-D-glucanase from Helix pomatia (Fluka, 49103). 10 mg of fibers were incubated in 10 mM sodium acetate buffer (pH 5) and 500 μl of glucanase (1 mg/ml) was added. They were subjected to infiltration under vacuum for 10 minutes and overnight incubation at 37° C. The strength of individual cotton fibers was measured using a Favimat R device (Textechno) in a single fiber tensile test at 8 mm gauge length and a speed of 4 mm/min. The strength measure is recorded in force (cN). The results were statistically analyzed and are presented in Table 9 and FIG. 4.

TABLE-US-00009 TABLE 9 Callose content (as measured by the green/blue fluorescence ratio of aniline blue stained fibers (ratio green/blue)) and strength (as measured by the breaking force (cN)) of untreated fibers (no GLUC) and fibers treated with glucanase (GLUC) from different G. hirsutum and G. barbadense varieties Ratio Force Gossypium species Treatment green/blue (cN) G. hirsutum cv. FM966 No GLUC Mean 0.44 2.92 (greenhouse) SD 0.04 1.92 GLUC Mean 0.43 3.11 SD 0.06 1.74 G. hirsutum cv. FM966 (field US) No GLUC Mean 0.51 5.50 SD 0.09 2.70 GLUC Mean 0.55 4.45 SD 0.10 2.03 G. hirsutum cv. FM966 (field AU) No GLUC Mean 0.52 4.33 SD 0.09 1.72 GLUC Mean 0.51 3.30 SD 0.14 1.43 G. hirsutum cv. Coker312 No GLUC Mean 0.47 4.49 (greenhouse) SD 0.02 2.45 GLUC Mean 0.44 3.08 SD 0.06 1.63 G. barbadense cv. PimaS7 No GLUC Mean 0.60 5.31 (greenhouse) SD 0.05 2.26 GLUC Mean 0.49 2.76 SD 0.15 1.80 G. barbadense cv. PimaY5 No GLUC Mean 0.61 5.19 (field AU) SD 0.03 2.57 GLUC Mean 0.53 2.13 SD 0.04 1.20

[0419] A pronounced drop in strength was observed for Pima fibers treated with the glucanase and a less pronounced but still noticeable reduction in strength was observed for fibers from various G. hirsutum lines. In this respect, it is important to note that the extent of secondary cell wall formation and cellulose content contribute to fiber strength in G. hirsutum, while the stronger fibers of G. barbadense have a lower cellulose content than those of G. hirsutum. The complementation experiment thus indicated that the presence of the Gbgluc1.1A allele within the fiber strength locus contributes to the renowned strength of Pima fibers.

3.2. Determination of Link Between 1,3-Beta-D-Glucan Content and Fiber Strength

[0420] 1,3-beta-D-glucans, including long chain 1,3-beta-D-glucans called callose, are the substrate for 1,3-beta-glucanase enzymes. Aniline blue is a dye specific for 1,3-beta-glucans. This dye was used to determine if fibers treated with 1,3-beta-glucanase and displaying a reduced fiber strength also displayed a reduced level of the 1,3-beta-glucan substrate in the cotton fiber walls.

[0421] A 0.05% solution of aniline blue in 0.067M K₂HPO₄ (pH 9) was used. The fibers were incubated for 15 minutes under vacuum. Under UV, callose deposits present an intense yellow-green fluorescence. Images are analyzed and the ratio Green/Blue is used as a measure for callose. The average value of 3 images was calculated.

[0422] As indicated in Table 9 and FIG. 5, this staining technique showed that cotton fibers treated with the glucanase had a lower level of 1,3-beta-glucan and that elevated 1,3-beta-glucan levels were linked to enhanced fiber strength.

3.3. Statistical Analysis of Effect of Glucanase Treatment on Fiber Strength and Callose Content

[0423] The effect of the treatment (untreated minus treated) was statistically analyzed. The results are presented in Table 10.

TABLE-US-00010 TABLE 10 Statistical analysis of glucanase treatment (untreated minus treated) on callose content and strength of fibers from different G. hirsutum and G. barbadense varieties Callose content Fiber strength (ratio G/B) (Force) differ- p- differ- p- ence value ence value G. hirsutum cv. FM966 (greenhouse) 0.01 0.882 -0.18 0.618 G. hirsutum cv. FM966 (field US) -0.04 0.634 1.05 0.041* G. hirsutum cv. FM966 (field AU) 0.01 0.922 1.03 0.003* G. hirsutum cv. Coker312 0.03 0.415 1.41 0.002* (greenhouse) G. barbadense cv. PimaS7 0.11 0.278 2.55 0.000* (greenhouse) G. barbadense cv. PimaY5 0.08 0.121 3.07 0.000* (field AU)

[0424] The correlations between the treatment and callose content as well as fiber strength were statistically analyzed. The results are presented in Table 11 for G. hirsutum and in Table 11 for G. barbadense.

TABLE-US-00011 TABLE 11 Statistical analysis of correlations between glucanase treatment of fibers of G. hirsutum, their callose content and their strength Callose Fiber Glucanase content strength treatment (ratio G/B) (Force) Glucanase Correlation 1.00 -0.03 -0.48 treatment Sig. (2-tailed) 0.944 0.233 Callose Correlation -0.03 1.00 0.66 content Sig. (2-tailed) 0.944 0.075 (ratio G/B) Fiber Correlation -0.48 0.66 1.00 strength Sig. (2-tailed) 0.233 0.075 (Force)

TABLE-US-00012 TABLE 12 Statistical analysis of correlations between glucanase treatment of fibers of G. barbadense, their callose content and their strength Callose Fiber Glucanase content strength treatment (ratio G/B) (Force) Glucanase Correlation 1.00 -0.96 -0.99 treatment Sig. (2-tailed) 0.044* 0.013* Callose Correlation -0.96 1.00 0.90 content Sig. (2-tailed) 0.044* 0.103 (ratio G/B) Fiber Correlation -0.99 0.90 1.00 strength Sig. (2-tailed) 0.013* 0.103 (Force)

[0425] In summary, cotton fibers with a higher 1,3-beta-glucan content displayed higher fiber strength and reduction in 1,3-beta-glucan content by exogenously supplied 1,3-beta-glucanase enzyme significantly reduced fiber strength and callose content in G. barbadense, indicating that 1,3-beta-glucan or callose has a specific role in cotton fiber strength which can be modulated by enzymes such as GLUC1.1.

Example 4

Identification of GLUC1.1A Alleles in Different Cotton Species

[0426] GLUC1.1 sequences were isolated from six different Gossypium hirsutum varieties (Guazuncho; DP16; Cooker 312 (C312); Fiber Max 966 (FM966); Acala SJ2; Acala Maxxa), from five different Gossypium barbadense varieties (PimaS7; Tanguis LMW 1737-60; Tanguis C N (C.P.R.)712-60; Sea Island Tipless; VH8), from Gossypium herbacium, Gossypium tomentosum, Gossypium darwinii, Gossypium arboreum, Gossypium raimondii, Gossypium kirkii, Gossypium longicalyx, and Gossypium mustelinum

[0427] Based on the GhGLUC1.1A and D nucleotide sequences described in WO2008/083969 (SEQ ID NO: 1 and 7, respectively), primer pairs (forward primer SE077 (SEQ ID NO: 33) and G1.1-f1-F1 (SEQ ID NO: 65) en reverse primer SE078 (SEQ ID NO: 34) or forward primer SE002 (SEQ ID NO: 35) en reverse primer SE003 (SEQ ID NO: 36)) were designed to amplify full-length or partial, respectively, genomic DNA fragments. The reaction mix used contained: 20 DNA (200 ng/μl genomic DNA), 10 forward primer (10 pM), 10 reverse primer (10 pM), 4 μl 5× High Fidelity buffer, 0.2 μl Phusion enzyme (Finnzymes), 0.4 μl dNTP's (10 mM), 11.4 μl water (MilliQ). The PCR protocol used was as follows: 1 min at 98° C.; 30 times: 10 sec at 98° C. (denaturation), 30 sec at 56° C. (annealing), 1 min at 72° C. (elongation); 30 sec at 58° C.; 10 min at 72° C.; 4° C.

[0428] GLUC1.1A sequences from all G. barbadense lines tested and from Gossypium darwinii display a single nucleotide substitution (c to t at position 712 of SEQ ID NO: 5 and at position 470 of SEQ ID NO: 17 or at position 761 of SEQ ID NO: 56, respectively; see also GLUC1.1A-SNP5 in Table 13) resulting in a premature stop codon (cga to tga) in their sequences (FIG. 6; since the GLUC1.1 sequences from the different Gossypium hirsutum varieties and the different Gossypium barbadense varieties, respectively, were identical to each other, only the GLUC1.1 sequences of the FM966 and PimaS7 variety, respectively, were included in the alignment). The GLUC1.1A sequence from G. arboreum displayed a single nucleotide deletion (deletion of c nucleotide between position 327 and 328 of SEQ ID NO: 21) also resulting in a premature stop codon (tga at position 373-375 of SEQ ID NO: 21) further downstream in its sequence (FIG. 6). The premature stop codons in the GLUC1.1A sequences from G. barbadense, from Gossypium darwinii and from G. arboreum resulted in a predicted truncated GLUC1.1A protein sequence (FIG. 7; GLUC1.1A protein of 179 (SEQ ID NO: 6), of 179 (SEQ ID NO: 57), and of 78 (SEQ ID NO: 22) amino acids, respectively), while the GLUC1.1A sequences from all other Gossypium species tested did not display premature stop codons and are predicted to produce a complete GLUC1.1 protein (FIGS. 6 and 7).

[0429] As indicated above, G. barbadense is commercially recognized for its superior fiber quality, particularly for fiber strength, length and fineness. G. darwinii is the closest relative of G. barbadense and some even consider it as a variety of G. barbadense rather than a separate species. However, G. darwinii produces sparse, non-spinnable, khaki or brown fiber, usually less than 1.3 cm in length (see e.g. Wendel and Percy, 1990, Bioch. Systematics And Ecology 18 (7/8): 517-528). As the fibers from G. darwinii are not commercially used, little information is available about its commercially relevant fiber qualities, such as fiber strength.

Example 5

Genotyping of GLUC1.1 Genes in Commercial Germplasm

[0430] The genotype of GLUC1.1A and GLUC1.1D genes was determined in commercially available germplasm by determining the genotype of GLUC1.1A-SNP3, 5 and 6 and GLUC1.1D-SNP1 (as indicated in FIG. 6 and Table 13) in a total of 73 G. hirsutum varieties, one G. barbadense variety, 2 G. arboreum varieties, one G. herbaceum variety, and one G. mustilinum variety using Illumina GoldenGate SNP Genotyping and BeadArray technology as prescribed by the manufacturer. Briefly, a GoldenGate Genotyping assay uses allele-specific extension and ligation for genotype calling using a discriminatory DNA polymerase and ligase (Illumina).

TABLE-US-00013 TABLE 13 Position and genotype of GLUC1.1D-SNP1 and GLUC1.1A-SNP2, 3, 5, 6, 7 and 8 in GLUC1.1D and A genes, respectively of different Gossypium species (G.h.: G. hirsutum, G.b.: G. barbadense, G.t.: G. tomentosum; G.d.: G. darwinii; G.m.: G. mustilinum; G.a.: G. arboreum G.he.: G. herbaceum G.r.: G. raimondii) GLUC1.1A G. sp.: G.h. G. b. G. t. G. d. G. m. G. a. G. he. SEQ ID: 1 5 15 56/17 19 21 23 SNP7 2674-2676 327-329 85-87 376-378/ 85-87 327-328 327-329 between 85-87 C C C C C -- C SNP2 2765-2766 418-428 176-177 467-477/ 176-177 417-418 418-419 between 176-186 -- CTCAT -- CTCAT -- -- -- CAAA CAAA SNP3 2911 573 322 622/331 322 563 564 G C G C C C C SNP5 3050 712 461 761/470 461 702 703 C T C T C C C SNP8 3170 832 581 881/590 581 821 823 G C G G G G G SNP6 3202 864 613 913/622 613 854 855 G A G A G G G GLUC1.1D G. sp.: G.h. G. b. G. t. G. d. G. m. G. r. SEQ ID: 7 11 25 58/27 29 31 SNP1 3614 304 80 352/80 80 80 C T C T C C

[0431] The results confirmed that the genotypes of GLUC1.1A-SNP3, 5 and 6 and GLUC1.1D-SNP1 in the different analysed Gossypium species and varieties were as indicated in FIG. 6 and Table 13. In particular, genotyping of GLUC1.1A-SNP5 in the different Gossypium species and varieties indicated that all analysed Gossypium species and varieties different from G. barbadense comprise the cga codon found in GLUC1.1A of Gossypium hirsutum instead of the tga stop codon found in gluc1.1A of Gossypium barbadense Pima S7.

Example 6

Detection of GLUC1.1 Allele Encoding an Inactive GLUC1.1 Protein in Gossypium Plants and/or Transfer of GLUC1.1 Allele Encoding an Inactive GLUC1.1 Protein into Gossypium Lines Comprising a Corresponding GLUC1.1 Allele Encoding an Active GLUC1.1 Protein

[0432] A GLUC1.1 allele encoding an inactive GLUC1.1 enzyme, such as a Gbgluc1.1A allele, Gdgluc1.1A allele or Gagluc1.1A allele, is transferred into cotton lines comprising a corresponding GLUC1.1 allele encoding an active GLUC1.1 enzyme, such as Gossypium hirsutum breeding lines, by the following method:

[0433] A plant containing a GLUC1.1 allele encoding an inactive GLUC1.1 enzyme, such as a Gossypium barbadense plant, a Gossypium darwinii plant or a Gossypium arboreum plant containing a GLUC1.1A allele encoding an inactive GLUC1.1A enzyme, or a mutagenized Gossypium hirsutum plant containing a mutant GLUC1.1 allele encoding an inactive GLUC1.1 enzyme (donor plant), is crossed with a plant containing a corresponding GLUC1.1 allele encoding an active GLUC1.1 enzyme, such as a Gossypium hirsutum plant containing a GLUC1.1A allele encoding an active GLUC1.1A enzyme (recurrent parent). The following introgression scheme is used (the GLUC1.1 allele encoding an inactive GLUC1.1 enzyme is abbreviated to gluc while the GLUC1.1 allele encoding an active GLUC1.1 enzyme is depicted as GLUC):

[0434] Initial cross: gluc/gluc (donor)×GLUC/GLUC (recurrent parent)

[0435] F1 plant: GLUC/gluc

[0436] BC1 cross: GLUC/gluc (F1)×GLUC/GLUC (recurrent parent)

[0437] BC1 plants: 50% GLUC/gluc and 50% GLUC/GLUC

[0438] The 50% GLUC/gluc are selected using a specific assay (e.g. PCR, TaqMan®, Invader®, and the like; see also below) for the gluc1.1 allele.

[0439] BC2 cross: GLUC/gluc (BC1)×GLUC/GLUC (recurrent parent)

[0440] BC2 plants: 50% GLUC/gluc and 50% GLUC/GLUC

[0441] The 50% GLUC/gluc are selected using a specific assay (e.g. PCR, TaqMan®, Invader®, and the like; see also below) for the gluc1.1 allele.

[0442] Backcrossing is repeated until BC4 to BC5 (e.g. if the donor plant is a Gossypium barbadense plant and the recurrent parent is a Gossypium hirsutum plant) or until BC3 (e.g. if the donor plant and the recurrent parent are Gossypium hirsutum plants)

[0443] BC3-5 plants: 50% GLUC/gluc and 50% GLUC/GLUC

[0444] The 50% GLUC/gluc are selected using a specific assay (e.g. PCR, TaqMan®, Invader®, and the like; see also below) for the gluc1.1 allele.

[0445] To reduce the number of backcrossings (e.g. until BC2 if the donor plant and the recurrent parent are Gossypium hirsutum plants, or until BC3 to BC4 if the donor plant is a Gossypium barbadense plant and the recurrent parent is a Gossypium hirsutum plant), molecular markers can be used in each generation that are specific for the genetic background of the recurrent parent.

[0446] BC3-5 S1 cross: GLUC/gluc×GLUC/gluc

[0447] BC3-5 S1 plants: 25% GLUC/GLUC and 50% GLUC/gluc and 25% gluc/gluc

[0448] Plants containing the gluc1.1 allele are selected using molecular markers for the gluc1.1 allele. Individual BC3-5 S1 plants that are homozygous for the gluc1.1 allele (gluc/gluc) are selected using molecular markers for the gluc1.1 and GLUC1.1 alleles. These plants are then used for fiber production.

[0449] Molecular markers which can be used to detect a specific gluc1.1 or GLUC1.1 allele or to discriminate between a specific gluc1.1 and GLUC1.1 allele are, for example, single nucleotide polymorphisms (SNPs) or polymorphic nucleotide sequences:

[0450] As an example, SNPs and polymorphic nucleotide sequences which can be used to discriminate between the Gbgluc1.1A or Gdgluc1.1A allele and the GhGLUC1.1A allele and between the GbGLUC1.1D or Gdgluc1.1D allele and the GhGLUC1.1D allele or to detect their presence in DNA samples or plants, are SNPs indicated as GLUC1.1A-SNP3, 5 and 6 in FIG. 6 and Table 13 and the polymorphic nucleotide sequence indicated as GLUC1.1A-SNP2 in FIG. 6 and Table 13 and the SNP indicated as GLUC1.1D-SNP1 in FIG. 6 and Table 13, respectively.

[0451] In particular, a SNP which can be used to discriminate between the Gbgluc1.1A or Gdgluc1.1A allele that comprises a premature tga STOP codon and the corresponding GhGLUC1.1A allele that comprises a cga codon instead, is the SNP indicated as GLUC1.1A-SNP5 in FIG. 6 and Table 13.

[0452] The genotype of such SNPs and polymorphic nucleotide sequences can be determined, for example, using a PCR assay.

[0453] As an example, PCR assays were developed to determine the genotype of the SNP indicated as GLUC1.1D-SNP1 in FIG. 6 and Table 13 and of the polymorphic nucleotide sequence indicated as GLUC1.1A-SNP2 in FIG. 6 and Table 13 of plants of the BC1 populations described in Example 1 in order to map the GLUC1.1D and A genes of G. hirsutum and barbadense, respectively. More specifically, following PCR assay was developed to discriminate between the Gbgluc1.1A allele and the GhGLUC1.1A allele based on the genotype of the SNP indicated as GLUC1.1A-SNP2 in FIG. 6 and Table 13:

TABLE-US-00014 Primers: Forward: (p1.3GlucaAf-SEQ ID NO: 37) 5' TAT CCC TCT CGA TGA GTA CGA C 3' Reverse: (p1.3GlucaAr-SEQ ID NO: 38) 5'CCC AAT GAT GAT GAA CCT GAA TTG3'



[0454] Amplicon size: 134 bps for G. hirsutum and 143 bps for G. barbadense.

[0455] PCR conditions: 50 gDNA (20 ng/μl)+15 μl PCR mix (PCR mix: 2 μl 10×Taq PCR buffer, 1 μl labeled p1.3GlucaAf (100 pmol/μl), 0.2 μl p1.3GlucaAr (100 pmol/μl), 0.25 μl dNTPs (20 mM), 0.5 μl MgCl₂ (50 mM), 0.2 μl Taq polymerase, 10.85 μl MiliQ)

[0456] Labeling of forward primer: 0.1 μl 10×T4 kinase buffer, 0.2 μl p1.3GlucaAf(100 pmol/μl), 0.01 μl T4 kinase, 0.1 μl P³³γ ATP, 0.59 μl MilliQ=1 μl; 1 h at 37° C. and 10 min at 65° C.

[0457] PCR profile: 5 min at 95° C.; 35 times: 45 s at 95° C., 45 s at 58° C., 1 min at 72° C.; 10 min at 72° C.

[0458] Gel analysis: PCR fragments are separted on 4.5% denaturing acrylamide gels

[0459] Overnight exposure of gel to BIOMAX MR films

[0460] Alternatively, the genotype of such SNPs can be determined, for example, using Illumina GoldenGate SNP Genotyping as indicated in Example 5 for the SNPs indicated as GLUC1.1A-SNP3, 5 and 6 and GLUC1.1D-SNP1 in FIG. 6 and Table 13.

[0461] Alternatively, the genotype of such SNPs and polymorphic nucleotide sequences can be determined by direct sequencing by standard sequencing techniques known in the art to determine the complete GLUC1.1 nucleotide sequence present in a plant followed by analysis of the obtained sequence, e.g., by alignment with the GLUC1.1 sequences described herein (see, e.g., FIGS. 6 and 7).

[0462] Alternatively, the genotype of such SNPs and polymorphic nucleotide sequences can be determined by a Taqman assay. The TaqMan assay procedure and interpretation of the data are performed as prescribed by the manufacturer (Applied Biosystems). Briefly, a probe specific for a specific variant of a polymorphic site in a GLUC1.1 gene binds the template DNA if this specific variant is present. The probe has a fluorescent reporter or fluorophore, such as 6-carboxyfluorescein (acronym: FAM) and VIC (a proprietary dye from Applied Biosystems), attached to its 5' end and a quencher (e.g., tetramethylrhodamine, acronym: TAMRA, of dihydrocyclopyrroloindole tripeptide "minor groove binder", acronym: MGB) attached to its 3' end. The close proximity between fluorophore and quencher attached to the probe inhibits fluorescence from the fluorophore. During a PCR with two primers capable of amplifying a DNA fragment comprising the polymorphic site, the 5' to 3' exonuclease activity of the Taq polymerase degrades that proportion of the probe that has annealed to the template as DNA synthesis commences. Degradation of the probe releases the fluorophore from it and breaks the close proximity to the quencher, thus relieving the quenching effect and allowing fluorescence of the fluorophore. Hence, fluorescence detected in the real-time PCR thermal cycler is directly proportional to the fluorophore released and the amount of DNA template present in the PCR. The following discriminating Taqman probes and primers were thus developed to discriminate different variants of GLUC1.1A-SNP3 and GLUC1.1A-SNP5 (see FIG. 6 and Table 13):

TABLE-US-00015 TABLE 14a GLUC1.1A- SNP3 of Probes Gbgluc1.1A 5' FAM-AACTCGCT (SEQ ID NO: 39) CGCCTCA 3' GhGLUC1.1A 5' VIC-AACTCGCT (SEQ ID NO: 40) GGCCTCA 3' Forward primer 5' CCTGGTGCCATG (SEQ ID NO: 41) AACAACATAATG 3' reverse primer 5' CGTCGTGCCTAG (SEQ ID NO: 42) CCCAAA 3'

TABLE-US-00016 TABLE 14b GLUC1.1A- SNP5 of Probes Gbgluc1.1A 5' FAM-ATCCTGTCA (SEQ ID NO: 60) AACCAG 3' GhGLUC1.1A 5' VIC-ATCCTGTCA (SEQ ID NO: 61) AACCAG 3' Forward primer 5' GCTTTTGGAAGCG (SEQ ID NO: 62) ATATAACATCGA 3' reverse primer 5' GGCATAGGCAAAA (SEQ ID NO: 63) TAAGGGTACACA 3'

[0463] Probes specific for polymorphic sites in the Gbgluc1.1A or corresponding GhGLUC1.1A target gene, such as the probes specific for GLUC1.1A-SNP3 of Gbgluc1.1A and GhGLUC1.1A indicated as "5' FAM-AACTCGCTCGCCTCA 3" and "5' VIC-AACTCGCTGGCCTCA 3', respectively, in Table 14a, and forward and reverse primers that are capable of amplifying a fragment comprising the polymorphic site and that can thus be used in combination with them are indicated in Table 14a. Generally, each probe set consists of two probes each specific for one variant of the polymorphic site in the GLUC1.1 target gene which comprises the variant nucleotide (e.g., the underlined nucleotide in Table 14) or variant nucleotide sequence (e.g. the probe with SEQ ID NO: 39 is specific for GLUC1.1A-SNP3 of Gbgluc1.1A and the probe with SEQ ID NO: 40 is specific for GLUC1.1A-SNP3 of GhGLUC1.1A) and a set of two primers that are capable of amplifying a fragment comprising the polymorphic site (e.g. the primer with SEQ ID NO: 41 is specific for a nucleotide sequence upstream of GLUC1.1A-SNP3 and the primer with SEQ ID NO: 42 is specific for a nucleotide sequence downstream of GLUC1.1A-SNP3, such that the use of both primers results in the amplification of a DNA fragment comprising GLUC1.1A-SNP3).

[0464] Alternatively, the genotype of such SNPs and polymorphic nucleotide sequences can be determined by Invader® technology (Third Wave Agbio).

Example 7

Comparison of Expression of GLUC1.1A and GLUC1.1D During Fiber Growth and Development in Gossypium barbadense and in Gossypium hirsutum

[0465] Expression of GLUC1.1A and GLUC1.1D during fiber growth and development was analyzed for G. barbadense and compared with the expression of GLUC1.1A and GLUC1.1D during fiber growth and development of G. hirsutum as described in WO2008/083969.

[0466] DNA from a cDNA library of G. barbadense created from fiber cells and seed at 0 and 5 DPA and from fiber cells at 10, 15, 20, 25, 30 and 40 DPA was extracted, the concentration was equalized and a PCR amplification was performed using primers SE002 (SEQ ID NO: 35) and SE003 (SEQ ID NO: 36). The PCR reaction mix used contained: 1 μl template DNA (200 ng/μl), 5 μl 5× GreenGoTaq buffer, 0.75 μl SE002 (10 μM), 0.75 μl SE003 (10 μM), 0.5 μl dNTP's (20 mM), 0.25 μl GoTaq polymerase, 16.75 μl MilliQ water (total of 25 μl). The PCR conditions used were as follows: 5 min at 95° C.; 5 times: 1 min at 95° C., 1 min at 58° C., 2 min at 72° C.; 25 times: 30 s at 92° C., 30 s at 58° C., 1 min at 72° C.; 10 min at 72° C., cooldown to 4° C. The expected length of the PCR product is 655 bp. After PCR amplification, the PCR fragment is digested with AlwI digest (3 h incubation at 37° C.) using 10 μl template; 1 μl AlwI enzyme; 2 μl NEB 4 restriction buffer; 7 μl MQ water. The resulting fragments are analysed on 1.5% TAE gel stained with EtBr. The expected band sizes for the A subgenome allele specific PCR fragment are: 479 bp, 118 bp and 59 bp (not visible in FIG. 8). The expected band sizes for the D subgenome allele specific PCR fragment are: 538 bp and 118 bp.

[0467] FIG. 8, lanes 2 to 9, represent GbGLUC1.1A and D expression at 0, 5, 10, 15, 20, 25, 30 and 40 DPA. Differences in band intensities in FIG. 8 correspond to relative differences in expression. A negative (no template; NTC; FIG. 8, lane 10) and a positive control (genomic DNA from Pima S7; FIG. 8, lane 11) were included. The expression profile of the GhGLUC1.1A and D and GbGLUC1.1A and D genes can be summarized as follows:

TABLE-US-00017 Days post anthesis (DPA): 0 5 10 15 20 25 30 40 GhGLUC1.1 -- -- -- D D ND A&D A & D GbGLUC1.1 -- -- -- A & D A & D A & D A & A & D D

[0468] Thus while the expression of GLUC1.1A in G. hirsutum starts only at 30 DPA, GLUC1.1A in G. barbadense is expressed from 15 DPA on. However, as indicated above, the GbGLUC1.1A gene is predicted to encode a non-functional GLUC1.1A protein.

Sequence CWU 1

1

6516009DNAGossypium hirsutumCDS(2410)..(2443)CDS(2556)..(3496) 1cgcggcatat aattttatgt gtgtaatttg ttgggttaat tacttaaaat agtatatttt 60taattgctgt aattaatgta agataatttt tattatttga atcattgcac aaaattaaaa 120tagaataatt tatttaacaa ttcaaatata ataataatcc aaattataat tatagtattt 180ttacaatatt caatatacaa tatagtttta cttcatacaa ttaatataaa aaaatattat 240tcaaaataat aactaataaa cataattacc atatattaat tattttgata tttcgaacat 300aacgctaata aaaaatttcc taatcattat taaatcattt gtataaacta taaagaaatt 360gatatattgt aaattaaact ttattcattt tttttcttaa tactcaataa attaatcata 420ataactcata aataatatat aattaaaata atcataacat ggtagattat ataaataggg 480ggcgaatcta gggagctggc atgaccccta aaatagaatt ttctattttg acctatcaaa 540atttttaaaa ttttaaatta gtaaaggtaa atttgtactt tgacctctta aaatgataaa 600attttacttt aatcctttaa aatttacatt tttactatca taaaaattac aatttgattt 660tgcccctaaa atttttttct agcttagccc tgtatataaa tatattattt ataattttta 720tatttaaaat ataaagtttt taattataca aataattaaa atctgatatt taaaactaaa 780gtaatttctt ttttcttttt actttttttt aattgcaaca taatggttta aatatctata 840taacgtatga agtaatttga tataaatttt attttaattt attattatat aaattcattt 900agtaaaaact tttaatagaa tcaaaatttt tatttgtaaa ttcgataact tttcttatca 960agtatatttg tgagaaccaa atatttagta aaattaatat tcttatttat aaatatgata 1020aatcttataa aaaaatattt aaaatgaaaa aaattgtaca aatattataa aaaaatattt 1080aaaatgaaaa acattgtaca aaggctatat aagaagttca aaagtttctt cgaccatgta 1140ctcttataga gattatagat agattataaa actatatgta gtttctctta acttttaaat 1200aagaggataa atgtatttta atgtactcaa acttatatat ttttatattg acaataatat 1260caatatcaac ctaattaaga ttcattctaa cattaatgtt gaagattttt aataaaagaa 1320aaggttaata aattaattag aacacaaaca aacacaaatt taagtggtat gtaaggtcct 1380tgacccaaag gaaaaatttg ttacgtcgat taaattataa attaatttaa agtaaaatta 1440cattttaacc taaaaaaaga gaaaagtata tctaatttct tcgaaaatgg aaagaaaatt 1500ataaatttat ggcatttcta aaaaaattct gaattcgcta ctaaaagatg aaattataaa 1560atccgaagca ttaccagaag atggatcacc aaatcacaaa caatcaatga aaagtaatga 1620taattaattg aaagtgagca tttaattttg atagccatat acttcctgct gaatttatag 1680gttctcatta atgcaattaa attatattcg acaccttttg aatgaaataa aatgacacaa 1740gaggaaagac ggttcatcta ttttttcttt caatcgccca tcaaaatacc aaaaatgtaa 1800ctacatgcaa aaaatcaaat atgaaaaata ttcatatttt gatattttaa tatattgtgt 1860gttcaaaacg taaatgtatt gaaaaattat gatggtgttg ttgctgtatg tccataaaat 1920tcaatgtact cacatttatc aaatgtatac tttgagagaa gttattttga taatactcaa 1980gtttttttta tagatgggaa aattttttaa attatttttt gattttgatg aaatgtatat 2040ataaatttta attcgataca tataaatata tatgtaaatt ttaaatttaa atttaataat 2100atacaattaa gaaaataatt tataaatatt ttccgattaa aaataaatct ggaaagaaga 2160aatgtcaaca ctttttcatt aaatacaatt aggatgggac acgatacctt catgcattga 2220tatctcaggt ggtccaaaaa ctcggaatcc tttttgaaaa aaaacttcca gagagagtat 2280ataaatccag cagtaggcac aagaaacgag caccagttat tgactttcct ttgtaaaaaa 2340aaaaagtgct gagatcaaga aatatagtga aatatgggtc caagattttc tgggttttta 2400atctaagca atg ctg ttt tta act caa ctc ctc tct cta aca g 2443 Met Leu Phe Leu Thr Gln Leu Leu Ser Leu Thr 1 5 10 gtaaaacaaa cttctctaca gtgattttac agtaaatatg gctttgaaaa atatacaaca 2503aaacatttat cttcaatcca ttttaattac tgatctacta tatatgttgc ag at ggc 2560 Asp Gly cgt gat att ggt gtt tgc tat ggt ttg aac ggc aac aat ctt cca tct 2608Arg Asp Ile Gly Val Cys Tyr Gly Leu Asn Gly Asn Asn Leu Pro Ser 15 20 25 cca gga gat gtt att aat ctt ttc aaa act agt ggc ata aac aat atc 2656Pro Gly Asp Val Ile Asn Leu Phe Lys Thr Ser Gly Ile Asn Asn Ile 30 35 40 45 agg ctc tac cag cct tac cct gaa gtg ctc gaa gca gca agg gga tcg 2704Arg Leu Tyr Gln Pro Tyr Pro Glu Val Leu Glu Ala Ala Arg Gly Ser 50 55 60 gga ata tcc ctc tcg atg agt acg aca aac gag gac ata caa agc ctc 2752Gly Ile Ser Leu Ser Met Ser Thr Thr Asn Glu Asp Ile Gln Ser Leu 65 70 75 gca acg gat caa agt gca gcc gat gca tgg gtt aac acc aac atc gtc 2800Ala Thr Asp Gln Ser Ala Ala Asp Ala Trp Val Asn Thr Asn Ile Val 80 85 90 cct tat aag gaa gat gtt caa ttc agg ttc atc atc att ggg aat gaa 2848Pro Tyr Lys Glu Asp Val Gln Phe Arg Phe Ile Ile Ile Gly Asn Glu 95 100 105 gcc att cca gga cag tca agc tct tac att cct ggt gcc atg aac aac 2896Ala Ile Pro Gly Gln Ser Ser Ser Tyr Ile Pro Gly Ala Met Asn Asn 110 115 120 125 ata atg aac tcg ctg gcc tca ttt ggg cta ggc acg acg aag gtt acg 2944Ile Met Asn Ser Leu Ala Ser Phe Gly Leu Gly Thr Thr Lys Val Thr 130 135 140 acc gtg gtc ccg atg aat gcc cta agt acc tcg tac cct cct tca gac 2992Thr Val Val Pro Met Asn Ala Leu Ser Thr Ser Tyr Pro Pro Ser Asp 145 150 155 ggc gct ttt gga agc gat ata aca tcg atc atg act agt atc atg gcc 3040Gly Ala Phe Gly Ser Asp Ile Thr Ser Ile Met Thr Ser Ile Met Ala 160 165 170 att ctg gtt cga cag gat tcg ccc ctc ctg atc aat gtg tac cct tat 3088Ile Leu Val Arg Gln Asp Ser Pro Leu Leu Ile Asn Val Tyr Pro Tyr 175 180 185 ttt gcc tat gcc tca gac ccc act cat att tcc ctc aac tac gcc ttg 3136Phe Ala Tyr Ala Ser Asp Pro Thr His Ile Ser Leu Asn Tyr Ala Leu 190 195 200 205 ttc acc tcg acc gca ccg gtg gtg gtc gac caa ggc ttg gaa tac tac 3184Phe Thr Ser Thr Ala Pro Val Val Val Asp Gln Gly Leu Glu Tyr Tyr 210 215 220 aac ctc ttt gac ggc atg gtc gat gct ttc aat gcc gcc cta gat aag 3232Asn Leu Phe Asp Gly Met Val Asp Ala Phe Asn Ala Ala Leu Asp Lys 225 230 235 atc ggc ttc ggc caa att act ctc att gta gcc gaa act gga tgg ccg 3280Ile Gly Phe Gly Gln Ile Thr Leu Ile Val Ala Glu Thr Gly Trp Pro 240 245 250 acc gcc ggt aac gag cct tac acg agt gtc gcg aac gct caa act tat 3328Thr Ala Gly Asn Glu Pro Tyr Thr Ser Val Ala Asn Ala Gln Thr Tyr 255 260 265 aac aag aac ttg ttg aat cat gtg acg cag aaa ggg act ccg aaa aga 3376Asn Lys Asn Leu Leu Asn His Val Thr Gln Lys Gly Thr Pro Lys Arg 270 275 280 285 cct gaa tat ata atg ccg acg ttt ttc ttc gag atg ttc aac gag aac 3424Pro Glu Tyr Ile Met Pro Thr Phe Phe Phe Glu Met Phe Asn Glu Asn 290 295 300 ttg aag caa ccc aca gtt gag cag aat ttc gga ttc ttc ttc ccc aat 3472Leu Lys Gln Pro Thr Val Glu Gln Asn Phe Gly Phe Phe Phe Pro Asn 305 310 315 atg aac cct gtt tat cca ttt tgg tgaacttgaa atgttattgt tggctattta 3526Met Asn Pro Val Tyr Pro Phe Trp 320 325 aatcttttgc cagagacgct tcatatagtt tctgcatatt ttgaaagtgg aaaatcaatc 3586taaatataaa taagttttat ttgttgtttt ttaattaaat aaaattttaa atattttaaa 3646aacatcttta ttggtaatta aatattaaat aaaaagttta atattcaaat tttatcaatt 3706caaaaataaa ataaaaatat attaaattta tttttacgaa taaattgatt ttctattaat 3766gcagatttta aataatttga tataaatttt caattcaaca atagtaattt tgatcacatc 3826aaaggagaaa gggaaagatt taactttaat tggtgaccta atataacacg ttgaaaacgg 3886agttcccaat aaggcaaaat gacttgtaat gacgaaagag atgtccaagt gaaatctgct 3946ttaaagtgaa agaagcataa aaggataact aaataactca tgatctaaat tgaagttcta 4006taaaatgcaa ctttcatcta gaaacaaggt atgtcttaaa tgatgtttta tgaatttgtc 4066ttaattgggt tttatgcaat gaattcatgg atagcacatc tctaattata cgttgctggt 4126ttatatgaga gtggtgcaga agttaattgt gctttaaata cttgcttagt gtttatgaaa 4186tttgaaaagt gttatatact tataataaaa ataattcgat tcggaatcca attcagggtt 4246cgactcaata taataaaatt ttacagatat cttgaagggg atcttcttct tctctacttc 4306tcgagcagtg ttatatattt acaataaaga taactcaatt cgagatccga cctaatataa 4366taaaattcta cagacatatc aaagagggag atcttcttct tccctacatc ttgaccttct 4426tgatcaaaat gaccttcctt atatttttac atacgttgat tatatgaatc aaaagaaaga 4486taccaaaaag tttttaaaaa taaacaacgg ggttcttatg tagagatgct tatgggccgg 4546gccggactca actaaaaatt taggcacatt cattgggccc aggtcgggcc taacccaaaa 4606atgggcctaa aattttgccc aagcttgact caaataaaaa tgctaaaatt cgggcctgac 4666cccgtattaa ttttatatta ttttatataa cttttaaata tatataatat ataaaaaata 4726ctaaaaaaat taaaataaat atttcccaac taaactaaaa ttattaagaa aaataattca 4786tattagcgta taaattggaa attgaccaaa attaaaatta ttgtatagtt aatctatatt 4846aaaaggacat gtaattaaaa accattaaaa ctattataca ataaattaaa tcttcattgt 4906atacatagaa aggcattaat aattaaaaaa ctatattaag atataaacta aattcaaaat 4966tattaaaaac aagaactaaa taaaaaagca attgaaaatt acgaattaat gttaaaatca 5026aatgttaaaa tcaagggact taaataaaaa tatcccaaaa tacaaaacat tagcttcctt 5086tcccatccac gtgaatgcaa agtttacatg gtgtttccta gtgtttgtgc gactccaacc 5146ttttatttac ctcttttttt ctttatttga acaattattt gataatgatt agaattttgg 5206gattgttgct catcgtacgt gcaacactta aaatcactat gatttttcat aatttatata 5266acctatatcg ttttggaaat taattttatt ttttatatta ttttaataaa aataccatct 5326acctttttta atttatgatc cctttcatat ttaaaaattc aaattgacaa ttgtctaact 5386aaacaccgtc acactccaat aagattgtaa tttcctccat cttgatatta cactcaaaag 5446catgttgcca acaaacaaat caactagcct ttttctacca ctattcatca tcttcttaag 5506agtgtgttta tgtcatgtgc cgagatttta ggtatggtca cgttgtggct ttaaactcaa 5566atctattgcc catgagtcta agttagcctc cgatcctcac taaagagagg cttggcacac 5626tttacctagc caagtacaca aggaatagag ctattagaaa gcattaaaga gttaggagaa 5686tgtggaagtg tttttattac tcaaagctaa cttggataca aataaaggag ggagcctctc 5746ctttaggcaa gcttcttttg atctgatggt tacaattaat ctcgaatagg aggggtcaaa 5806cttctcactc agtttcatat tatctcttgg tgcttggttg gcctccgcct tgagacaact 5866ttagataaca cctagtctta acacttttag cttcacattg tacgcatcct tcattactca 5926aatgccacaa agcctcctta cttaaggctc ttggtcgctc ccactacctt cggctttaga 5986ctcatctaag atcttcccaa tcg 60092325PRTGossypium hirsutum 2Met Leu Phe Leu Thr Gln Leu Leu Ser Leu Thr Asp Gly Arg Asp Ile 1 5 10 15 Gly Val Cys Tyr Gly Leu Asn Gly Asn Asn Leu Pro Ser Pro Gly Asp 20 25 30 Val Ile Asn Leu Phe Lys Thr Ser Gly Ile Asn Asn Ile Arg Leu Tyr 35 40 45 Gln Pro Tyr Pro Glu Val Leu Glu Ala Ala Arg Gly Ser Gly Ile Ser 50 55 60 Leu Ser Met Ser Thr Thr Asn Glu Asp Ile Gln Ser Leu Ala Thr Asp 65 70 75 80 Gln Ser Ala Ala Asp Ala Trp Val Asn Thr Asn Ile Val Pro Tyr Lys 85 90 95 Glu Asp Val Gln Phe Arg Phe Ile Ile Ile Gly Asn Glu Ala Ile Pro 100 105 110 Gly Gln Ser Ser Ser Tyr Ile Pro Gly Ala Met Asn Asn Ile Met Asn 115 120 125 Ser Leu Ala Ser Phe Gly Leu Gly Thr Thr Lys Val Thr Thr Val Val 130 135 140 Pro Met Asn Ala Leu Ser Thr Ser Tyr Pro Pro Ser Asp Gly Ala Phe 145 150 155 160 Gly Ser Asp Ile Thr Ser Ile Met Thr Ser Ile Met Ala Ile Leu Val 165 170 175 Arg Gln Asp Ser Pro Leu Leu Ile Asn Val Tyr Pro Tyr Phe Ala Tyr 180 185 190 Ala Ser Asp Pro Thr His Ile Ser Leu Asn Tyr Ala Leu Phe Thr Ser 195 200 205 Thr Ala Pro Val Val Val Asp Gln Gly Leu Glu Tyr Tyr Asn Leu Phe 210 215 220 Asp Gly Met Val Asp Ala Phe Asn Ala Ala Leu Asp Lys Ile Gly Phe 225 230 235 240 Gly Gln Ile Thr Leu Ile Val Ala Glu Thr Gly Trp Pro Thr Ala Gly 245 250 255 Asn Glu Pro Tyr Thr Ser Val Ala Asn Ala Gln Thr Tyr Asn Lys Asn 260 265 270 Leu Leu Asn His Val Thr Gln Lys Gly Thr Pro Lys Arg Pro Glu Tyr 275 280 285 Ile Met Pro Thr Phe Phe Phe Glu Met Phe Asn Glu Asn Leu Lys Gln 290 295 300 Pro Thr Val Glu Gln Asn Phe Gly Phe Phe Phe Pro Asn Met Asn Pro 305 310 315 320 Val Tyr Pro Phe Trp 325 31185DNAGossypium hirsutumCDS(101)..(1078) 3gcaccagtta ttgactttcc tttgtaaaaa aaaaaagtgc tgagatcaag aaatatagtg 60aaatatgggt ccaagatttt ctgggttttt aatctaagca atg ctg ttt tta act 115 Met Leu Phe Leu Thr 1 5 caa ctc ctc tct cta aca gat ggc cgt gat att ggt gtt tgc tat ggt 163Gln Leu Leu Ser Leu Thr Asp Gly Arg Asp Ile Gly Val Cys Tyr Gly 10 15 20 ttg aac ggc aac aat ctt cca tct cca gga gat gtt att aat ctt ttc 211Leu Asn Gly Asn Asn Leu Pro Ser Pro Gly Asp Val Ile Asn Leu Phe 25 30 35 aaa act agt ggc ata aac aat atc agg ctc tac cag cct tac cct gaa 259Lys Thr Ser Gly Ile Asn Asn Ile Arg Leu Tyr Gln Pro Tyr Pro Glu 40 45 50 gtg ctc gaa gca gca agg gga tcg gga ata tcc ctc tcg atg agt acg 307Val Leu Glu Ala Ala Arg Gly Ser Gly Ile Ser Leu Ser Met Ser Thr 55 60 65 aca aac gag gac ata caa agc ctc gca acg gat caa agt gca gcc gat 355Thr Asn Glu Asp Ile Gln Ser Leu Ala Thr Asp Gln Ser Ala Ala Asp 70 75 80 85 gca tgg gtt aac acc aac atc gtc cct tat aag gaa gat gtt caa ttc 403Ala Trp Val Asn Thr Asn Ile Val Pro Tyr Lys Glu Asp Val Gln Phe 90 95 100 agg ttc atc atc att ggg aat gaa gcc att cca gga cag tca agc tct 451Arg Phe Ile Ile Ile Gly Asn Glu Ala Ile Pro Gly Gln Ser Ser Ser 105 110 115 tac att cct ggt gcc atg aac aac ata atg aac tcg ctg gcc tca ttt 499Tyr Ile Pro Gly Ala Met Asn Asn Ile Met Asn Ser Leu Ala Ser Phe 120 125 130 ggg cta ggc acg acg aag gtt acg acc gtg gtc ccg atg aat gcc cta 547Gly Leu Gly Thr Thr Lys Val Thr Thr Val Val Pro Met Asn Ala Leu 135 140 145 agt acc tcg tac cct cct tca gac ggc gct ttt gga agc gat ata aca 595Ser Thr Ser Tyr Pro Pro Ser Asp Gly Ala Phe Gly Ser Asp Ile Thr 150 155 160 165 tcg atc atg act agt atc atg gcc att ctg gtt cga cag gat tcg ccc 643Ser Ile Met Thr Ser Ile Met Ala Ile Leu Val Arg Gln Asp Ser Pro 170 175 180 ctc ctg atc aat gtg tac cct tat ttt gcc tat gcc tca gac ccc act 691Leu Leu Ile Asn Val Tyr Pro Tyr Phe Ala Tyr Ala Ser Asp Pro Thr 185 190 195 cat att tcc ctc aac tac gcc ttg ttc acc tcg acc gca ccg gtg gtg 739His Ile Ser Leu Asn Tyr Ala Leu Phe Thr Ser Thr Ala Pro Val Val 200 205 210 gtc gac caa ggc ttg gaa tac tac aac ctc ttt gac ggc atg gtc gat 787Val Asp Gln Gly Leu Glu Tyr Tyr Asn Leu Phe Asp Gly Met Val Asp 215 220 225 gct ttc aat gcc gcc cta gat aag atc ggc ttc ggc caa att act ctc 835Ala Phe Asn Ala Ala Leu Asp Lys Ile Gly Phe Gly Gln Ile Thr Leu 230 235 240 245 att gta gcc gaa act gga tgg ccg acc gcc ggt aac gag cct tac acg 883Ile Val Ala Glu Thr Gly Trp Pro Thr Ala Gly Asn Glu Pro Tyr Thr 250 255 260 agt gtc gcg aac gct caa act tat aac aag aac ttg ttg aat cat gtg 931Ser Val Ala Asn Ala Gln Thr Tyr Asn Lys Asn Leu Leu Asn His Val 265 270 275 acg cag aaa ggg act ccg aaa aga cct gaa tat ata atg ccg acg ttt 979Thr Gln Lys Gly Thr Pro Lys Arg Pro Glu Tyr Ile Met Pro Thr Phe 280 285 290 ttc ttc gag atg ttc aac gag aac ttg aag caa ccc aca gtt gag cag 1027Phe Phe Glu Met Phe Asn Glu Asn Leu Lys Gln Pro Thr Val Glu Gln 295 300 305 aat ttc gga ttc ttc ttc ccc aat atg aac cct gtt tat cca ttt tgg 1075Asn Phe Gly Phe Phe Phe Pro Asn Met Asn Pro Val Tyr Pro Phe Trp 310 315 320 325 tga acttgaaatg ttattgttgg ctatttaaat cttttgccag agacgcttca 1128tatagtttct gcatattttg aaagtggaaa aaaaaaaaaa aaaaaaaaaa aaaaaaa 11854325PRTGossypium hirsutum 4Met Leu Phe Leu Thr Gln Leu Leu Ser Leu

Thr Asp Gly Arg Asp Ile 1 5 10 15 Gly Val Cys Tyr Gly Leu Asn Gly Asn Asn Leu Pro Ser Pro Gly Asp 20 25 30 Val Ile Asn Leu Phe Lys Thr Ser Gly Ile Asn Asn Ile Arg Leu Tyr 35 40 45 Gln Pro Tyr Pro Glu Val Leu Glu Ala Ala Arg Gly Ser Gly Ile Ser 50 55 60 Leu Ser Met Ser Thr Thr Asn Glu Asp Ile Gln Ser Leu Ala Thr Asp 65 70 75 80 Gln Ser Ala Ala Asp Ala Trp Val Asn Thr Asn Ile Val Pro Tyr Lys 85 90 95 Glu Asp Val Gln Phe Arg Phe Ile Ile Ile Gly Asn Glu Ala Ile Pro 100 105 110 Gly Gln Ser Ser Ser Tyr Ile Pro Gly Ala Met Asn Asn Ile Met Asn 115 120 125 Ser Leu Ala Ser Phe Gly Leu Gly Thr Thr Lys Val Thr Thr Val Val 130 135 140 Pro Met Asn Ala Leu Ser Thr Ser Tyr Pro Pro Ser Asp Gly Ala Phe 145 150 155 160 Gly Ser Asp Ile Thr Ser Ile Met Thr Ser Ile Met Ala Ile Leu Val 165 170 175 Arg Gln Asp Ser Pro Leu Leu Ile Asn Val Tyr Pro Tyr Phe Ala Tyr 180 185 190 Ala Ser Asp Pro Thr His Ile Ser Leu Asn Tyr Ala Leu Phe Thr Ser 195 200 205 Thr Ala Pro Val Val Val Asp Gln Gly Leu Glu Tyr Tyr Asn Leu Phe 210 215 220 Asp Gly Met Val Asp Ala Phe Asn Ala Ala Leu Asp Lys Ile Gly Phe 225 230 235 240 Gly Gln Ile Thr Leu Ile Val Ala Glu Thr Gly Trp Pro Thr Ala Gly 245 250 255 Asn Glu Pro Tyr Thr Ser Val Ala Asn Ala Gln Thr Tyr Asn Lys Asn 260 265 270 Leu Leu Asn His Val Thr Gln Lys Gly Thr Pro Lys Arg Pro Glu Tyr 275 280 285 Ile Met Pro Thr Phe Phe Phe Glu Met Phe Asn Glu Asn Leu Lys Gln 290 295 300 Pro Thr Val Glu Gln Asn Phe Gly Phe Phe Phe Pro Asn Met Asn Pro 305 310 315 320 Val Tyr Pro Phe Trp 325 51185DNAGossypium barbadenseCDS(63)..(96)CDS(209)..(711) 5gctgagatca agaaatatag tgaaatatgg gtccaagatt ttctgggttt ttaatctaag 60ca atg ctg ttt tta act caa ctc ctc tct cta aca g gtaaaacaaa 106 Met Leu Phe Leu Thr Gln Leu Leu Ser Leu Thr 1 5 10 cttctctaca gtgattttac agtaaatatg gctttgaaaa atatacaaca aaacatttat 166cttcaatcca ttttaattac tgatctacta tatatgttgc ag at ggc cgt gat 219 Asp Gly Arg Asp 15 att ggt gtt tgc tat ggt ttg aac ggc aac aat ctt cca tct cca gga 267Ile Gly Val Cys Tyr Gly Leu Asn Gly Asn Asn Leu Pro Ser Pro Gly 20 25 30 gat gtt att aat ctt ttc aaa act agt ggc ata aac aat atc agg ctc 315Asp Val Ile Asn Leu Phe Lys Thr Ser Gly Ile Asn Asn Ile Arg Leu 35 40 45 tac cag cct tac cct gaa gtg ctc gaa gca gca agg gga tcg gga ata 363Tyr Gln Pro Tyr Pro Glu Val Leu Glu Ala Ala Arg Gly Ser Gly Ile 50 55 60 tcc ctc tcg atg agt acg aca aac gag gac ata caa agc ctc gca acg 411Ser Leu Ser Met Ser Thr Thr Asn Glu Asp Ile Gln Ser Leu Ala Thr 65 70 75 gat caa act cat caa agt gca gcc gat gca tgg gtt aac acc aac atc 459Asp Gln Thr His Gln Ser Ala Ala Asp Ala Trp Val Asn Thr Asn Ile 80 85 90 95 gtc cct tat aag gaa gat gtt caa ttc agg ttc atc atc att ggg aat 507Val Pro Tyr Lys Glu Asp Val Gln Phe Arg Phe Ile Ile Ile Gly Asn 100 105 110 gaa gcc att cca gga cag tca agc tct tac att cct ggt gcc atg aac 555Glu Ala Ile Pro Gly Gln Ser Ser Ser Tyr Ile Pro Gly Ala Met Asn 115 120 125 aac ata atg aac tcg ctc gcc tca ttt ggg cta ggc acg acg aag gtt 603Asn Ile Met Asn Ser Leu Ala Ser Phe Gly Leu Gly Thr Thr Lys Val 130 135 140 acg acc gtg gtc ccg atg aat gcc cta agt acc tcg tac cct cct tca 651Thr Thr Val Val Pro Met Asn Ala Leu Ser Thr Ser Tyr Pro Pro Ser 145 150 155 gac ggc gct ttt gga agc gat ata aca tcg atc atg act agt atc atg 699Asp Gly Ala Phe Gly Ser Asp Ile Thr Ser Ile Met Thr Ser Ile Met 160 165 170 175 gcc att ctg gtt tgacaggatt cgcccctcct gatcaatgtg tacccttatt 751Ala Ile Leu Valttgcctatgc ctcagacccc actcatattt ccctcaacta cgccttgttc acctcgaccg 811caccggtggt ggtcgaccaa cgcttggaat actacaacct ctttgacggc atagtcgatg 871ctttcaatgc cgccctagat aagatcggct tcggccaaat tactctcatt gtagccgaaa 931ctggatggcc gaccgccggt aacgagcctt acacgagtgt cgcgaacgct caaacttata 991acaagaactt gttgaatcat gtgacgcaga aagggactcc gaaaagacct gaatatataa 1051tgccgacgtt tttcttcgag atgttcaacg agaacttgaa gcaacccaca gttgagcaga 1111tgttcaacga gatgttcaac gagaacttga aatgttattg ttggctattt aaatcttttg 1171ccagagacgc ttca 11856179PRTGossypium barbadense 6Met Leu Phe Leu Thr Gln Leu Leu Ser Leu Thr Asp Gly Arg Asp Ile 1 5 10 15 Gly Val Cys Tyr Gly Leu Asn Gly Asn Asn Leu Pro Ser Pro Gly Asp 20 25 30 Val Ile Asn Leu Phe Lys Thr Ser Gly Ile Asn Asn Ile Arg Leu Tyr 35 40 45 Gln Pro Tyr Pro Glu Val Leu Glu Ala Ala Arg Gly Ser Gly Ile Ser 50 55 60 Leu Ser Met Ser Thr Thr Asn Glu Asp Ile Gln Ser Leu Ala Thr Asp 65 70 75 80 Gln Thr His Gln Ser Ala Ala Asp Ala Trp Val Asn Thr Asn Ile Val 85 90 95 Pro Tyr Lys Glu Asp Val Gln Phe Arg Phe Ile Ile Ile Gly Asn Glu 100 105 110 Ala Ile Pro Gly Gln Ser Ser Ser Tyr Ile Pro Gly Ala Met Asn Asn 115 120 125 Ile Met Asn Ser Leu Ala Ser Phe Gly Leu Gly Thr Thr Lys Val Thr 130 135 140 Thr Val Val Pro Met Asn Ala Leu Ser Thr Ser Tyr Pro Pro Ser Asp 145 150 155 160 Gly Ala Phe Gly Ser Asp Ile Thr Ser Ile Met Thr Ser Ile Met Ala 165 170 175 Ile Leu Val 76877DNAGossypium hirsutumCDS(3337)..(3406)CDS(3501)..(4441) 7ttcaaactta ctcgcttgca caaaaataat tttataaaag tatttaaaat ataaaatttt 60tatgttgata atatttttat atacatttta tattttaaga aataataatt ttttaggaat 120tagaaaaaaa atgtagaata atatcattga tattttaatt tttcaaaaaa ttaaaaataa 180gttcacgtag tctaatttta tctattttaa tttttatact ttcaaattga gataaatatc 240aaagaacttt tggttcaata tgcaatttga tacttaaatt ttaatttgat gtaattatta 300catgaaactt ggcttgtggt ttatacgtat acatgaaatt tttattttga ttcaattgta 360cgcatttaaa gaaatgaaaa tggttctaat tcaataatat tattagtgat ttgtgaaatt 420taaaactttt atgcattaaa ccacacaaaa tcagagttta tgtatgatat tgcacattgg 480actatagttc atgcatattt tttatatttt atccatgtca aattttgaaa tttcattctt 540aacttatatg atagcagtta aatttgttaa gtcaaactct agtattagtt atatactata 600cataacttgt agagtttagt ttaagttcac taatttgatt attttttatc tgtttatttt 660ttcaatttca agatttaagt tttaagctta acttaaacaa tagtcattaa atttattaac 720taaaatgtcc tggggttttt tgtaagtatt ataatatgtt tgccacgtga gattttggta 780aaagtagagt ttaacttaac aaatttaatg gctactactt agtaaggatt agaatttcaa 840aattaaaaaa aaaattatag aggctaaaga tgatcaaatt agaggtttaa attaagtcaa 900attaaaatag ttctggatat taactattta aattaattaa tgtcattata aaattagagg 960tctaaattat gtaaaattaa aatataaaaa ctaaatctcg aatgtgagta tagtataagg 1020atcaaaagtg attttggtca ttttctttta tttacaaata ttcttagaga tgctctttta 1080tatataatgg tttctaatgt gatatgcgcg gcatataatt ttatgtgttt aatttatttt 1140attaattatt taaataatat atttttaatt actataatta atgtaaaata ttttttatta 1200tttgaatcac tgcacaaaat taaaatatac taacttaatt aacaattcaa atataataat 1260aatctaaatt ataattaaag catttttaca atattcagta tataatatag ttttacttta 1320tataattaat acaaagaaat attattcaaa ataataacta atcaacataa ttactatata 1380tcaattattt tgatatttcg aacataatgc taataaaaaa tttcctaatc attattaaat 1440catttgtata aactataaag aaattgatat attgtaaatt aaacttttaa ctattcaatt 1500ttttcttaat agtcaataaa ttaatcataa taattcataa ttaatatata attaacataa 1560ccataacata gaatttttta ttttggccca ttaaaatttt taaaatttta aattagtaaa 1620ggaaaaatta cactttgacc ccttaaaaat gataaaattt tattttaatc ctttaaaatt 1680gacattttta ctattgtaaa aattacaatt taattttgcc cccctaaaaa atttttctag 1740cttcgccctt gtgtataaat atattaatta caatttttat atttgaatta tataaataat 1800taaattttga tatttaaaac taaagtaatc tctttttttt ttactttttt ttaattgaaa 1860cataatggtt taaatatcta tattacgtat gaagtaattt aatataaatt ttattttaat 1920ttattattat ataaattcat ttagtaaaaa cttttaatag aatcaaaatt tttatttgta 1980aattcgataa cttttcttat caagtaaatt tgttgaatta aatatttagt aaaattaata 2040tttttattta taaatatgat aaatcttata aaaaataaaa aaatatttaa aatgaaaaac 2100attgtacaaa ggctatataa gaagttcaaa agtttcttcg accctgtact ctaatagaga 2160ttatagatag attatagaac tattcatagt ttctcttaac ctttaaataa gaattttagt 2220gtactcaaac ttacatattt ttatattgat aataatgtca ataccagccg agttaagatt 2280cactcgacat taatgttgaa aatttttaat aaaagaaaat gttgataagt taattagaac 2340acaagcaagc acaaatttaa gtggtaagta aggtccttga ccctaatgga aaaattgtta 2400tgttgattaa attataaatt aatttaaggt aaaattatat tttgacctaa aaaaatgaaa 2460aaaatatatc tagtttcttc gaaaatgaaa agaaaataat aaattgatac attataaaat 2520ttatggcatt tctaaaaaaa ttctgaattt gatgaaatta taataaaaaa aaagtttaaa 2580aacatataga tttcaagaat agtgggaaaa ttatatttga acaacactga agaaatccaa 2640agcattagca gaaaatggat caccaaatca caaacaatca gtgaaaagta atgataatta 2700attgaaagtg agcatttaaa tttgatagcc atatacttcc tgctgaattt ataggttctc 2760attaatgcaa ttaaattata tttgtcactt tttgaatgaa ataaatgaca cagttcatct 2820attttttttc tttcaatcgc ccatcaaaat accgaaaatg taactacatt aaaaaagatc 2880gaaaaatatt catattttga tattttaata gattgtgtgt tcaaggcgta atgtactaaa 2940aaattatgat ggtgttgtcg ctgtatgtcc ataaaattca atgtattcgc atgtatcaaa 3000tgtaaatttt gacacaagtt attctaataa taatcaagtt atttttatac atgagataca 3060tctcaaaatt atttttatat atccgaaaaa tcataacgta cgatcaaact agaaagagga 3120agtgtcaaaa cctattcatt atatgcaaat atgatgggac acgataccct catgcattga 3180tatctcatat tgtccaaaaa ctcagaatcc tttttgaaaa aaaaaaattc cagagagagt 3240gtataaatcc agcagtgtgc acaagaaacg agcaccagtt attgacattc ctttgtaaaa 3300aaaaaaagaa gctgagatca agaaatatag tgaaat atg ggt cca aca ttt tct 3354 Met Gly Pro Thr Phe Ser 1 5 ggg ttt tta atc tca gca atg gtg ttt tta act caa ctc ctc tct cta 3402Gly Phe Leu Ile Ser Ala Met Val Phe Leu Thr Gln Leu Leu Ser Leu 10 15 20 aca g gtaaaacaaa cttctctaca gtgattttac ggtaagtatg gctttgaaaa 3456Thr atatacaaca aaacatttat actgatctac catatatgtt gcag at ggc cgt gat 3511 Asp Gly Arg Asp 25 att ggt gtt tgc tat ggt ttg aac ggc aac aat ctt cca tct cca gga 3559Ile Gly Val Cys Tyr Gly Leu Asn Gly Asn Asn Leu Pro Ser Pro Gly 30 35 40 gat gtt att aat ctt tac aaa act agt ggc ata aac aat atc agg ctc 3607Asp Val Ile Asn Leu Tyr Lys Thr Ser Gly Ile Asn Asn Ile Arg Leu 45 50 55 tac cag cct tac cct gaa gtg ctc gaa gca gca agg gga tcg gga ata 3655Tyr Gln Pro Tyr Pro Glu Val Leu Glu Ala Ala Arg Gly Ser Gly Ile 60 65 70 75 tcc ctc tcg atg ggt ccg aga aac gag gac ata caa agc ctc gca aaa 3703Ser Leu Ser Met Gly Pro Arg Asn Glu Asp Ile Gln Ser Leu Ala Lys 80 85 90 gat caa agt gca gcc gat gca tgg gtt aac acc aac atc gtc cct tat 3751Asp Gln Ser Ala Ala Asp Ala Trp Val Asn Thr Asn Ile Val Pro Tyr 95 100 105 aag gac gat gtt cag ttc aag ttg atc act att ggg aat gaa gcc att 3799Lys Asp Asp Val Gln Phe Lys Leu Ile Thr Ile Gly Asn Glu Ala Ile 110 115 120 tca gga caa tca agc tct tac att cct gat gcc atg aac aac ata atg 3847Ser Gly Gln Ser Ser Ser Tyr Ile Pro Asp Ala Met Asn Asn Ile Met 125 130 135 aac tcg ctc gcc tta ttt ggg tta ggc acg acg aag gtt acg acc gtg 3895Asn Ser Leu Ala Leu Phe Gly Leu Gly Thr Thr Lys Val Thr Thr Val 140 145 150 155 gtc ccg atg aat gcc cta agt acc tcg tac cct cct tca gac ggc gct 3943Val Pro Met Asn Ala Leu Ser Thr Ser Tyr Pro Pro Ser Asp Gly Ala 160 165 170 ttt gga agc gat ata aca tcg atc atg act agt atc atg gcc att ctg 3991Phe Gly Ser Asp Ile Thr Ser Ile Met Thr Ser Ile Met Ala Ile Leu 175 180 185 gct gta cag gat tcg ccc ctc ctg atc aat gtg tac cct tat ttt gcc 4039Ala Val Gln Asp Ser Pro Leu Leu Ile Asn Val Tyr Pro Tyr Phe Ala 190 195 200 tat gcc tca gac ccc act cat att tcc ctc gat tac gcc ttg ttc acc 4087Tyr Ala Ser Asp Pro Thr His Ile Ser Leu Asp Tyr Ala Leu Phe Thr 205 210 215 tcg acc gca ccg gtg gtg gtc gac caa ggc ttg gaa tac tac aac ctc 4135Ser Thr Ala Pro Val Val Val Asp Gln Gly Leu Glu Tyr Tyr Asn Leu 220 225 230 235 ttt gac ggc atg gtc gat gct ttc aat gcc gcc cta gat aag atc ggc 4183Phe Asp Gly Met Val Asp Ala Phe Asn Ala Ala Leu Asp Lys Ile Gly 240 245 250 ttc ggc caa att act ctc att gta gcc gaa act gga tgg ccg acc gcc 4231Phe Gly Gln Ile Thr Leu Ile Val Ala Glu Thr Gly Trp Pro Thr Ala 255 260 265 ggt aac gag cct tac acg agt gtc gcg aac gct caa act tat aac aag 4279Gly Asn Glu Pro Tyr Thr Ser Val Ala Asn Ala Gln Thr Tyr Asn Lys 270 275 280 aac ttg tta aat cat gtg acg cag aag ggg act ccg aaa aga cct gaa 4327Asn Leu Leu Asn His Val Thr Gln Lys Gly Thr Pro Lys Arg Pro Glu 285 290 295 tat ata atg ccg acg ttt ttc ttc gag atg ttc aac gag gat ttg aag 4375Tyr Ile Met Pro Thr Phe Phe Phe Glu Met Phe Asn Glu Asp Leu Lys 300 305 310 315 caa ccc aca gtt gag cag aat ttc gga ttc ttc ttc ccc aat atg aac 4423Gln Pro Thr Val Glu Gln Asn Phe Gly Phe Phe Phe Pro Asn Met Asn 320 325 330 cct gtt tat cca ttt tgg tgaagttgaa atgttgttgg ctatttaaat 4471Pro Val Tyr Pro Phe Trp 335 cttttgccag agacgcttca tatagtttct gcatattttg aaagtggaaa atcaatctaa 4531atattaataa gttttatgtg ttgtttttta attaaataaa attttaaata ttttaaaaat 4591atctttattg gtaattaaat attaaataaa aagtttaata ttcaaatttt atcaattcaa 4651aaataaaata aaaatatatt aaatttattt ttacgaataa attgattttc tattaataca 4711gattttgaat aatttgatat aaattttaaa ttcaacaata gtaattttga tcacatcaaa 4771ggagaaaggg aaagatttaa ctttaattgg tgacctaata taacacgttg aaaacggagc 4831tcccaggaag gcaaaatgac ttgtaatgac gaaagagatg tccaagtaga atctgcatta 4891aagtgaaaaa agcataaaag gataagtaaa ctcatgatct gacataaatt gaagttctat 4951aaaatgcaac tttcatctag aaacaaggta tgtcttaaat gatgttttat gaatttgtct 5011taactgggtt ttatgcaatg aattcatgga tagcacctca ctaattatac gttgctggtt 5071tatatgagag tggtgcagaa gttaattgtg ctttaaatac ttgcttagtg ttcaagaaat 5131ttgaaaagta ttatatattt ataataaaaa taattcagat ccgactcaat ctagtaaaat 5191tttacaaaca ttctaaaggg gatcttcttt tttctctact tattgatcag tgttatatac 5251ttataataaa gacaacctga tttgagatcc ggcctaatat aataaaattc tacagacatc 5311tcaagggaga gatcttcttc ttccctacat cttgaccttt ttgatcaaaa tttcctcccc 5371tctatttcca cattggttga tcatatgaat caacagaaag gtaccaaaaa gtttttaaaa 5431ataaacaaag gggttcttat gaaattcata tgatatattg ggtctaatta ttagaatcaa 5491ttttaagttt aaacaaattt aaaattcaaa actcaattcc atttttgttt gaacggaaag 5551ttactaattg ttaagaaaaa taattcatat tagcgtataa attggaaatt gaccaaaact 5611aaaattattg tatagttaat ctatattaaa aggacatgta attaaaaacc attaaaacta 5671ttatagaata aattaaatct tcattctata catacaaagt cattaataat taaaaaacta 5731tattaagata taaactatat tcaaaaaata ttaaaaacaa taactaaata aaaaaaacaa

5791ttgaaaatta cgaattaatg ttaaaatcaa gggacttaaa taaaaatatc ccaaaataca 5851aaacattagc ttcctttccc atccacgtga ttgcaaagtt tacatggtgt ttcctagtgc 5911ttgtgcgact ccaacctttt atttactttt ttcttttctt tatttgaaca attatttgat 5971aatgattaga attttgggat tgttgctcat cgtacgtgca acacttaaaa tcactatgat 6031ttttcataat ttatataacc tatatcgttt tggaaattaa tgttattatt tatattgttt 6091taataaaaat accatctacc tcttttaatt tatgatccat ttcttatttg aaaattcaaa 6151ttgacagttg tctaactaaa caccatcgca ctccaataaa attgtaattt tttctatcgt 6211gaatagtaca ctcaaaagta tgttgttaac aaacaaatca attagccttt ttctacctct 6271attcatcatc ttcttaatag cgtgtttatg tcacgtgttg agattttagt tccggtcacg 6331tgtggcctta aacccgaatt tcttacgcat gagtctaagt tagcctctga tcctcgctat 6391ggagatgctt ggcacagttt acctaggtaa gtaaacaagg aatagagcta ttagaaagca 6451tcagagagtt aggagaatgt ggaagtgttt ctattactca aagctaactt ggatacaaat 6511aaaagaggga gcctctcctt taggcaagcc tattttgatc tgacggttgc aattaatctc 6571gaataggagg ggtcgaactt ctcactcagt ttcacattat ctcttggtgc ttagttggcc 6631tccgccttga gacacattca aataacacct agtcttaaca cttttggctt cttattgtgc 6691gtatccttca ttactcaaat gccacaaagc ctcattactt aagactctcg gtcgctccca 6751ctaccttcga ctttagactc atctaagatc ttcccaatcg tagacaactt ggccttggtg 6811gggaaatctt gcaccctacg gggccttaca taagaagcaa ttaaatggct ttctctcacc 6871cacctt 68778337PRTGossypium hirsutum 8Met Gly Pro Thr Phe Ser Gly Phe Leu Ile Ser Ala Met Val Phe Leu 1 5 10 15 Thr Gln Leu Leu Ser Leu Thr Asp Gly Arg Asp Ile Gly Val Cys Tyr 20 25 30 Gly Leu Asn Gly Asn Asn Leu Pro Ser Pro Gly Asp Val Ile Asn Leu 35 40 45 Tyr Lys Thr Ser Gly Ile Asn Asn Ile Arg Leu Tyr Gln Pro Tyr Pro 50 55 60 Glu Val Leu Glu Ala Ala Arg Gly Ser Gly Ile Ser Leu Ser Met Gly 65 70 75 80 Pro Arg Asn Glu Asp Ile Gln Ser Leu Ala Lys Asp Gln Ser Ala Ala 85 90 95 Asp Ala Trp Val Asn Thr Asn Ile Val Pro Tyr Lys Asp Asp Val Gln 100 105 110 Phe Lys Leu Ile Thr Ile Gly Asn Glu Ala Ile Ser Gly Gln Ser Ser 115 120 125 Ser Tyr Ile Pro Asp Ala Met Asn Asn Ile Met Asn Ser Leu Ala Leu 130 135 140 Phe Gly Leu Gly Thr Thr Lys Val Thr Thr Val Val Pro Met Asn Ala 145 150 155 160 Leu Ser Thr Ser Tyr Pro Pro Ser Asp Gly Ala Phe Gly Ser Asp Ile 165 170 175 Thr Ser Ile Met Thr Ser Ile Met Ala Ile Leu Ala Val Gln Asp Ser 180 185 190 Pro Leu Leu Ile Asn Val Tyr Pro Tyr Phe Ala Tyr Ala Ser Asp Pro 195 200 205 Thr His Ile Ser Leu Asp Tyr Ala Leu Phe Thr Ser Thr Ala Pro Val 210 215 220 Val Val Asp Gln Gly Leu Glu Tyr Tyr Asn Leu Phe Asp Gly Met Val 225 230 235 240 Asp Ala Phe Asn Ala Ala Leu Asp Lys Ile Gly Phe Gly Gln Ile Thr 245 250 255 Leu Ile Val Ala Glu Thr Gly Trp Pro Thr Ala Gly Asn Glu Pro Tyr 260 265 270 Thr Ser Val Ala Asn Ala Gln Thr Tyr Asn Lys Asn Leu Leu Asn His 275 280 285 Val Thr Gln Lys Gly Thr Pro Lys Arg Pro Glu Tyr Ile Met Pro Thr 290 295 300 Phe Phe Phe Glu Met Phe Asn Glu Asp Leu Lys Gln Pro Thr Val Glu 305 310 315 320 Gln Asn Phe Gly Phe Phe Phe Pro Asn Met Asn Pro Val Tyr Pro Phe 325 330 335 Trp 91250DNAGossypium hirsutumCDS(66)..(1076) 9gcaccagtta ttgacattcc tttgtaaaaa aaaaaagaag ctgagatcaa gaaatatagt 60gaaat atg ggt cca aca ttt tct ggg ttt tta atc tca gca atg gtg ttt 110 Met Gly Pro Thr Phe Ser Gly Phe Leu Ile Ser Ala Met Val Phe 1 5 10 15 tta act caa ctc ctc tct cta aca gat ggc cgt gat att ggt gtt tgc 158Leu Thr Gln Leu Leu Ser Leu Thr Asp Gly Arg Asp Ile Gly Val Cys 20 25 30 tat ggt ttg aac ggc aac aat ctt cca tct cca gga gat gtt att aat 206Tyr Gly Leu Asn Gly Asn Asn Leu Pro Ser Pro Gly Asp Val Ile Asn 35 40 45 ctt tac aaa act agt ggc ata aac aat atc agg ctc tac cag cct tac 254Leu Tyr Lys Thr Ser Gly Ile Asn Asn Ile Arg Leu Tyr Gln Pro Tyr 50 55 60 cct gaa gtg ctc gaa gca gca agg gga tcg gga ata tcc ctc tcg atg 302Pro Glu Val Leu Glu Ala Ala Arg Gly Ser Gly Ile Ser Leu Ser Met 65 70 75 ggt ccg aga aac gag gac ata caa agc ctc gca aaa gat caa agt gca 350Gly Pro Arg Asn Glu Asp Ile Gln Ser Leu Ala Lys Asp Gln Ser Ala 80 85 90 95 gcc gat gca tgg gtt aac acc aac atc gtc cct tat aag gac gat gtt 398Ala Asp Ala Trp Val Asn Thr Asn Ile Val Pro Tyr Lys Asp Asp Val 100 105 110 cag ttc aag ttg atc act att ggg aat gaa gcc att tca gga caa tca 446Gln Phe Lys Leu Ile Thr Ile Gly Asn Glu Ala Ile Ser Gly Gln Ser 115 120 125 agc tct tac att cct gat gcc atg aac aac ata atg aac tcg ctc gcc 494Ser Ser Tyr Ile Pro Asp Ala Met Asn Asn Ile Met Asn Ser Leu Ala 130 135 140 tta ttt ggg tta ggc acg acg aag gtt acg acc gtg gtc ccg atg aat 542Leu Phe Gly Leu Gly Thr Thr Lys Val Thr Thr Val Val Pro Met Asn 145 150 155 gcc cta agt acc tcg tac cct cct tca gac ggc gct ttt gga agc gat 590Ala Leu Ser Thr Ser Tyr Pro Pro Ser Asp Gly Ala Phe Gly Ser Asp 160 165 170 175 ata aca tcg atc atg act agt atc atg gcc att ctg gct gta cag gat 638Ile Thr Ser Ile Met Thr Ser Ile Met Ala Ile Leu Ala Val Gln Asp 180 185 190 tcg ccc ctc ctg atc aat gtg tac cct tat ttt gcc tat gcc tca gac 686Ser Pro Leu Leu Ile Asn Val Tyr Pro Tyr Phe Ala Tyr Ala Ser Asp 195 200 205 ccc act cat att tcc ctc gat tac gcc ttg ttc acc tcg acc gca ccg 734Pro Thr His Ile Ser Leu Asp Tyr Ala Leu Phe Thr Ser Thr Ala Pro 210 215 220 gtg gtg gtc gac caa ggc ttg gaa tac tac aac ctc ttt gac ggc atg 782Val Val Val Asp Gln Gly Leu Glu Tyr Tyr Asn Leu Phe Asp Gly Met 225 230 235 gtc gat gct ttc aat gcc gcc cta gat aag atc ggc ttc ggc caa att 830Val Asp Ala Phe Asn Ala Ala Leu Asp Lys Ile Gly Phe Gly Gln Ile 240 245 250 255 act ctc att gta gcc gaa act gga tgg ccg acc gcc ggt aac gag cct 878Thr Leu Ile Val Ala Glu Thr Gly Trp Pro Thr Ala Gly Asn Glu Pro 260 265 270 tac acg agt gtc gcg aac gct caa act tat aac aag aac ttg tta aat 926Tyr Thr Ser Val Ala Asn Ala Gln Thr Tyr Asn Lys Asn Leu Leu Asn 275 280 285 cat gtg acg cag aag ggg act ccg aaa aga cct gaa tat ata atg ccg 974His Val Thr Gln Lys Gly Thr Pro Lys Arg Pro Glu Tyr Ile Met Pro 290 295 300 acg ttt ttc ttc gag atg ttc aac gag gat ttg aag caa ccc aca gtt 1022Thr Phe Phe Phe Glu Met Phe Asn Glu Asp Leu Lys Gln Pro Thr Val 305 310 315 gag cag aat ttc gga ttc ttc ttc ccc aat atg aac cct gtt tat cca 1070Glu Gln Asn Phe Gly Phe Phe Phe Pro Asn Met Asn Pro Val Tyr Pro 320 325 330 335 ttt tgg tgaagttgaa atgttgttgg ctatttaaat cttttgccag agacgcttca 1126Phe Trp tatagtttct gcatattttg aaagtggaaa atcaatctaa atattaataa gttttatgtg 1186ttgtttttta attaaataaa attttaaata ttataaaaaa aaaaaaaaaa aaaaaaaaaa 1246aaaa 125010337PRTGossypium hirsutum 10Met Gly Pro Thr Phe Ser Gly Phe Leu Ile Ser Ala Met Val Phe Leu 1 5 10 15 Thr Gln Leu Leu Ser Leu Thr Asp Gly Arg Asp Ile Gly Val Cys Tyr 20 25 30 Gly Leu Asn Gly Asn Asn Leu Pro Ser Pro Gly Asp Val Ile Asn Leu 35 40 45 Tyr Lys Thr Ser Gly Ile Asn Asn Ile Arg Leu Tyr Gln Pro Tyr Pro 50 55 60 Glu Val Leu Glu Ala Ala Arg Gly Ser Gly Ile Ser Leu Ser Met Gly 65 70 75 80 Pro Arg Asn Glu Asp Ile Gln Ser Leu Ala Lys Asp Gln Ser Ala Ala 85 90 95 Asp Ala Trp Val Asn Thr Asn Ile Val Pro Tyr Lys Asp Asp Val Gln 100 105 110 Phe Lys Leu Ile Thr Ile Gly Asn Glu Ala Ile Ser Gly Gln Ser Ser 115 120 125 Ser Tyr Ile Pro Asp Ala Met Asn Asn Ile Met Asn Ser Leu Ala Leu 130 135 140 Phe Gly Leu Gly Thr Thr Lys Val Thr Thr Val Val Pro Met Asn Ala 145 150 155 160 Leu Ser Thr Ser Tyr Pro Pro Ser Asp Gly Ala Phe Gly Ser Asp Ile 165 170 175 Thr Ser Ile Met Thr Ser Ile Met Ala Ile Leu Ala Val Gln Asp Ser 180 185 190 Pro Leu Leu Ile Asn Val Tyr Pro Tyr Phe Ala Tyr Ala Ser Asp Pro 195 200 205 Thr His Ile Ser Leu Asp Tyr Ala Leu Phe Thr Ser Thr Ala Pro Val 210 215 220 Val Val Asp Gln Gly Leu Glu Tyr Tyr Asn Leu Phe Asp Gly Met Val 225 230 235 240 Asp Ala Phe Asn Ala Ala Leu Asp Lys Ile Gly Phe Gly Gln Ile Thr 245 250 255 Leu Ile Val Ala Glu Thr Gly Trp Pro Thr Ala Gly Asn Glu Pro Tyr 260 265 270 Thr Ser Val Ala Asn Ala Gln Thr Tyr Asn Lys Asn Leu Leu Asn His 275 280 285 Val Thr Gln Lys Gly Thr Pro Lys Arg Pro Glu Tyr Ile Met Pro Thr 290 295 300 Phe Phe Phe Glu Met Phe Asn Glu Asp Leu Lys Gln Pro Thr Val Glu 305 310 315 320 Gln Asn Phe Gly Phe Phe Phe Pro Asn Met Asn Pro Val Tyr Pro Phe 325 330 335 Trp 111186DNAGossypium barbadenseCDS(27)..(96)CDS(191)..(1131) 11gctgagatca agaaatatag tgaaat atg ggt cca aca ttt tct ggg ttt tta 53 Met Gly Pro Thr Phe Ser Gly Phe Leu 1 5 atc tca gca atg gtg ttt tta act caa ctc ctc tct cta aca g 96Ile Ser Ala Met Val Phe Leu Thr Gln Leu Leu Ser Leu Thr 10 15 20 gtaaaacaaa cttctctaca gtgattttac ggtaagtatg gctttgaaaa atatacaaca 156aaacatttat actgatctac catatatgtt gcag at ggc cgt gat att ggt gtt 210 Asp Gly Arg Asp Ile Gly Val 25 30 tgc tat ggt ttg aac ggc aac aat ctt cca tct cca gga gat gtt att 258Cys Tyr Gly Leu Asn Gly Asn Asn Leu Pro Ser Pro Gly Asp Val Ile 35 40 45 aat ctt tac aaa act agt ggc ata aac aat atc agg ctc tac cag tct 306Asn Leu Tyr Lys Thr Ser Gly Ile Asn Asn Ile Arg Leu Tyr Gln Ser 50 55 60 tac cct gaa gtg ctc gaa gca gca agg gga tcg gga ata tcc ctc tcg 354Tyr Pro Glu Val Leu Glu Ala Ala Arg Gly Ser Gly Ile Ser Leu Ser 65 70 75 atg ggt ccg aga aac gag gac ata caa agc ctc gca aaa gat caa agt 402Met Gly Pro Arg Asn Glu Asp Ile Gln Ser Leu Ala Lys Asp Gln Ser 80 85 90 gca gcc gat gca tgg gtt aac acc aac atc gtc cct tat aag gac gat 450Ala Ala Asp Ala Trp Val Asn Thr Asn Ile Val Pro Tyr Lys Asp Asp 95 100 105 110 gtt cag ttc aag ttg atc act att ggg aat gaa gcc att tca gga caa 498Val Gln Phe Lys Leu Ile Thr Ile Gly Asn Glu Ala Ile Ser Gly Gln 115 120 125 tca agc tct tac att cct gat gcc atg aac aac ata atg aac tcg ctc 546Ser Ser Ser Tyr Ile Pro Asp Ala Met Asn Asn Ile Met Asn Ser Leu 130 135 140 gcc tta ttt ggg tta ggc acg acg aag gtt acg acc gtg gtc ccg atg 594Ala Leu Phe Gly Leu Gly Thr Thr Lys Val Thr Thr Val Val Pro Met 145 150 155 aat gcc cta agt acc tcg tac cct cct tca gac ggc gct ttt gga agc 642Asn Ala Leu Ser Thr Ser Tyr Pro Pro Ser Asp Gly Ala Phe Gly Ser 160 165 170 gat ata aca tcg atc atg act agt atc atg gcc att ctg gct gta cag 690Asp Ile Thr Ser Ile Met Thr Ser Ile Met Ala Ile Leu Ala Val Gln 175 180 185 190 gat tcg ccc ctc ctg atc aat gtg tac cct tat ttt gcc tat gcc tca 738Asp Ser Pro Leu Leu Ile Asn Val Tyr Pro Tyr Phe Ala Tyr Ala Ser 195 200 205 gac ccc act cat att tcc ctc gat tac gcc ttg ttc acc tcg acc gca 786Asp Pro Thr His Ile Ser Leu Asp Tyr Ala Leu Phe Thr Ser Thr Ala 210 215 220 ccg gtg gtg gtc gac caa ggc ttg gaa tac tac aac ctc ttt gac ggc 834Pro Val Val Val Asp Gln Gly Leu Glu Tyr Tyr Asn Leu Phe Asp Gly 225 230 235 atg gtc gat gct ttc aat gcc gcc cta gat aag atc ggc ttc ggc caa 882Met Val Asp Ala Phe Asn Ala Ala Leu Asp Lys Ile Gly Phe Gly Gln 240 245 250 att act ctc att gta gcc gaa act gga tgg ccg acc gcc ggt aac gag 930Ile Thr Leu Ile Val Ala Glu Thr Gly Trp Pro Thr Ala Gly Asn Glu 255 260 265 270 cct tac acg agt gtc gcg aac gct caa act tat aac aag aac ttg tta 978Pro Tyr Thr Ser Val Ala Asn Ala Gln Thr Tyr Asn Lys Asn Leu Leu 275 280 285 aat cat gtg acg cag aag ggg act ccg aaa aga cct gaa tat ata atg 1026Asn His Val Thr Gln Lys Gly Thr Pro Lys Arg Pro Glu Tyr Ile Met 290 295 300 ccg acg ttt ttc ttc gag atg ttc aac gag gat ttg aag caa ccc aca 1074Pro Thr Phe Phe Phe Glu Met Phe Asn Glu Asp Leu Lys Gln Pro Thr 305 310 315 gtt gag cag aat ttc gga ttc ttc ttc ccc aat atg aac cct gtt tat 1122Val Glu Gln Asn Phe Gly Phe Phe Phe Pro Asn Met Asn Pro Val Tyr 320 325 330 cca ttt tgg tgaagttgaa atgttgttgg ctatttaaat cttttgccag 1171Pro Phe Trp 335 agacgcttca tatag 118612337PRTGossypium barbadense 12Met Gly Pro Thr Phe Ser Gly Phe Leu Ile Ser Ala Met Val Phe Leu 1 5 10 15 Thr Gln Leu Leu Ser Leu Thr Asp Gly Arg Asp Ile Gly Val Cys Tyr 20 25 30 Gly Leu Asn Gly Asn Asn Leu Pro Ser Pro Gly Asp Val Ile Asn Leu 35 40 45 Tyr Lys Thr Ser Gly Ile Asn Asn Ile Arg Leu Tyr Gln Ser Tyr Pro 50 55 60 Glu Val Leu Glu Ala Ala Arg Gly Ser Gly Ile Ser Leu Ser Met Gly 65 70 75 80 Pro Arg Asn Glu Asp Ile Gln Ser Leu Ala Lys Asp Gln Ser Ala Ala 85 90 95 Asp Ala Trp Val Asn Thr Asn Ile Val Pro Tyr Lys Asp Asp Val Gln 100 105 110 Phe Lys Leu Ile Thr Ile Gly Asn Glu Ala Ile Ser Gly Gln Ser Ser 115 120 125 Ser Tyr Ile Pro Asp Ala Met Asn Asn Ile Met Asn Ser Leu Ala Leu 130 135 140 Phe Gly Leu Gly Thr Thr Lys Val Thr Thr Val Val Pro

Met Asn Ala 145 150 155 160 Leu Ser Thr Ser Tyr Pro Pro Ser Asp Gly Ala Phe Gly Ser Asp Ile 165 170 175 Thr Ser Ile Met Thr Ser Ile Met Ala Ile Leu Ala Val Gln Asp Ser 180 185 190 Pro Leu Leu Ile Asn Val Tyr Pro Tyr Phe Ala Tyr Ala Ser Asp Pro 195 200 205 Thr His Ile Ser Leu Asp Tyr Ala Leu Phe Thr Ser Thr Ala Pro Val 210 215 220 Val Val Asp Gln Gly Leu Glu Tyr Tyr Asn Leu Phe Asp Gly Met Val 225 230 235 240 Asp Ala Phe Asn Ala Ala Leu Asp Lys Ile Gly Phe Gly Gln Ile Thr 245 250 255 Leu Ile Val Ala Glu Thr Gly Trp Pro Thr Ala Gly Asn Glu Pro Tyr 260 265 270 Thr Ser Val Ala Asn Ala Gln Thr Tyr Asn Lys Asn Leu Leu Asn His 275 280 285 Val Thr Gln Lys Gly Thr Pro Lys Arg Pro Glu Tyr Ile Met Pro Thr 290 295 300 Phe Phe Phe Glu Met Phe Asn Glu Asp Leu Lys Gln Pro Thr Val Glu 305 310 315 320 Gln Asn Phe Gly Phe Phe Phe Pro Asn Met Asn Pro Val Tyr Pro Phe 325 330 335 Trp 131211DNAGossypium barbadenseCDS(29)..(1039) 13ttgctgagat caagaaatat agtgaaat atg ggt cca aca ttt tct ggg ttt 52 Met Gly Pro Thr Phe Ser Gly Phe 1 5 tta atc tca gca atg gtg ttt tta act caa ctc ctc tct cta aca gat 100Leu Ile Ser Ala Met Val Phe Leu Thr Gln Leu Leu Ser Leu Thr Asp 10 15 20 ggc cgt gat att ggt gtt tgc tat ggt ttg aac ggc aac aat ctt cca 148Gly Arg Asp Ile Gly Val Cys Tyr Gly Leu Asn Gly Asn Asn Leu Pro 25 30 35 40 tct cca gga gat gtt att aat ctt tac aaa act agt ggc ata aac aat 196Ser Pro Gly Asp Val Ile Asn Leu Tyr Lys Thr Ser Gly Ile Asn Asn 45 50 55 atc agg ctc tac cag tct tac cct gaa gtg ctc gaa gca gca agg gga 244Ile Arg Leu Tyr Gln Ser Tyr Pro Glu Val Leu Glu Ala Ala Arg Gly 60 65 70 tcg gga ata tcc ctc tcg atg ggt ccg aga aac gag gac ata caa agc 292Ser Gly Ile Ser Leu Ser Met Gly Pro Arg Asn Glu Asp Ile Gln Ser 75 80 85 ctc gca aaa gat caa agt gca gcc gat gca tgg gtt aac acc aac atc 340Leu Ala Lys Asp Gln Ser Ala Ala Asp Ala Trp Val Asn Thr Asn Ile 90 95 100 gtc cct tat aag gac gat gtt cag ttc aag ttg atc act att ggg aat 388Val Pro Tyr Lys Asp Asp Val Gln Phe Lys Leu Ile Thr Ile Gly Asn 105 110 115 120 gaa gcc att tca gga caa tca agc tct tac att cct gat gcc atg aac 436Glu Ala Ile Ser Gly Gln Ser Ser Ser Tyr Ile Pro Asp Ala Met Asn 125 130 135 aac ata atg aac tcg ctc gcc tta ttt ggg tta ggc acg acg aag gtt 484Asn Ile Met Asn Ser Leu Ala Leu Phe Gly Leu Gly Thr Thr Lys Val 140 145 150 acg acc gtg gtc ccg atg aat gcc cta agt acc tcg tac cct cct tca 532Thr Thr Val Val Pro Met Asn Ala Leu Ser Thr Ser Tyr Pro Pro Ser 155 160 165 gac ggc gct ttt gga agc gat ata aca tcg atc atg act agt atc atg 580Asp Gly Ala Phe Gly Ser Asp Ile Thr Ser Ile Met Thr Ser Ile Met 170 175 180 gcc att ctg gct gta cag gat tcg ccc ctc ctg atc aat gtg tac cct 628Ala Ile Leu Ala Val Gln Asp Ser Pro Leu Leu Ile Asn Val Tyr Pro 185 190 195 200 tat ttt gcc tat gcc tca gac ccc act cat att tcc ctc gat tac gcc 676Tyr Phe Ala Tyr Ala Ser Asp Pro Thr His Ile Ser Leu Asp Tyr Ala 205 210 215 ttg ttc acc tcg acc gca ccg gtg gtg gtc gac caa ggc ttg gaa tac 724Leu Phe Thr Ser Thr Ala Pro Val Val Val Asp Gln Gly Leu Glu Tyr 220 225 230 tac aac ctc ttt gac ggc atg gtc gat gct ttc aat gcc gcc cta gat 772Tyr Asn Leu Phe Asp Gly Met Val Asp Ala Phe Asn Ala Ala Leu Asp 235 240 245 aag atc ggc ttc ggc caa att act ctc att gta gcc gaa act gga tgg 820Lys Ile Gly Phe Gly Gln Ile Thr Leu Ile Val Ala Glu Thr Gly Trp 250 255 260 ccg acc gcc ggt aac gag cct tac acg agt gtc gcg aac gct caa act 868Pro Thr Ala Gly Asn Glu Pro Tyr Thr Ser Val Ala Asn Ala Gln Thr 265 270 275 280 tat aac aag aac ttg tta aat cat gtg acg cag aag ggg act ccg aaa 916Tyr Asn Lys Asn Leu Leu Asn His Val Thr Gln Lys Gly Thr Pro Lys 285 290 295 aga cct gaa tat ata atg ccg acg ttt ttc ttc gag atg ttc aac gag 964Arg Pro Glu Tyr Ile Met Pro Thr Phe Phe Phe Glu Met Phe Asn Glu 300 305 310 gat ttg aag caa ccc aca gtt gag cag aat ttc gga ttc ttc ttc ccc 1012Asp Leu Lys Gln Pro Thr Val Glu Gln Asn Phe Gly Phe Phe Phe Pro 315 320 325 aat atg aac cct gtt tat cca ttt tgg tgaagttgaa atgttgttgg 1059Asn Met Asn Pro Val Tyr Pro Phe Trp 330 335 ctatttaaat cttttgccag agacgctcca tatagtttct gcatattttg aaagtggaaa 1119gtcaatctaa atattaataa gttttgtgtt gttttttaat taaataaaat tttaaatatt 1179ttggaaaaaa aaaaaaaaaa aaaaaaaaaa aa 121114337PRTGossypium barbadense 14Met Gly Pro Thr Phe Ser Gly Phe Leu Ile Ser Ala Met Val Phe Leu 1 5 10 15 Thr Gln Leu Leu Ser Leu Thr Asp Gly Arg Asp Ile Gly Val Cys Tyr 20 25 30 Gly Leu Asn Gly Asn Asn Leu Pro Ser Pro Gly Asp Val Ile Asn Leu 35 40 45 Tyr Lys Thr Ser Gly Ile Asn Asn Ile Arg Leu Tyr Gln Ser Tyr Pro 50 55 60 Glu Val Leu Glu Ala Ala Arg Gly Ser Gly Ile Ser Leu Ser Met Gly 65 70 75 80 Pro Arg Asn Glu Asp Ile Gln Ser Leu Ala Lys Asp Gln Ser Ala Ala 85 90 95 Asp Ala Trp Val Asn Thr Asn Ile Val Pro Tyr Lys Asp Asp Val Gln 100 105 110 Phe Lys Leu Ile Thr Ile Gly Asn Glu Ala Ile Ser Gly Gln Ser Ser 115 120 125 Ser Tyr Ile Pro Asp Ala Met Asn Asn Ile Met Asn Ser Leu Ala Leu 130 135 140 Phe Gly Leu Gly Thr Thr Lys Val Thr Thr Val Val Pro Met Asn Ala 145 150 155 160 Leu Ser Thr Ser Tyr Pro Pro Ser Asp Gly Ala Phe Gly Ser Asp Ile 165 170 175 Thr Ser Ile Met Thr Ser Ile Met Ala Ile Leu Ala Val Gln Asp Ser 180 185 190 Pro Leu Leu Ile Asn Val Tyr Pro Tyr Phe Ala Tyr Ala Ser Asp Pro 195 200 205 Thr His Ile Ser Leu Asp Tyr Ala Leu Phe Thr Ser Thr Ala Pro Val 210 215 220 Val Val Asp Gln Gly Leu Glu Tyr Tyr Asn Leu Phe Asp Gly Met Val 225 230 235 240 Asp Ala Phe Asn Ala Ala Leu Asp Lys Ile Gly Phe Gly Gln Ile Thr 245 250 255 Leu Ile Val Ala Glu Thr Gly Trp Pro Thr Ala Gly Asn Glu Pro Tyr 260 265 270 Thr Ser Val Ala Asn Ala Gln Thr Tyr Asn Lys Asn Leu Leu Asn His 275 280 285 Val Thr Gln Lys Gly Thr Pro Lys Arg Pro Glu Tyr Ile Met Pro Thr 290 295 300 Phe Phe Phe Glu Met Phe Asn Glu Asp Leu Lys Gln Pro Thr Val Glu 305 310 315 320 Gln Asn Phe Gly Phe Phe Phe Pro Asn Met Asn Pro Val Tyr Pro Phe 325 330 335 Trp 15656DNAGossypium tomentosumCDS(2)..(655) 15c ggc aac aat ctt cca tct cca gga gat gtt att gat ctt ttc aaa act 49 Gly Asn Asn Leu Pro Ser Pro Gly Asp Val Ile Asp Leu Phe Lys Thr 1 5 10 15 agt ggc ata aac aat atc agg ctc tac cag cct tac cct gaa gtg ctc 97Ser Gly Ile Asn Asn Ile Arg Leu Tyr Gln Pro Tyr Pro Glu Val Leu 20 25 30 gaa gca gca agg gga tcg gga ata tcc ctc tcg atg agt acg aca aac 145Glu Ala Ala Arg Gly Ser Gly Ile Ser Leu Ser Met Ser Thr Thr Asn 35 40 45 gag gac ata caa agc ctc gca acg gat caa agt gca gcc gat gca tgg 193Glu Asp Ile Gln Ser Leu Ala Thr Asp Gln Ser Ala Ala Asp Ala Trp 50 55 60 gtt aac acc aac atc gtc cct tat aag gaa gat gtt caa ttc agg ttc 241Val Asn Thr Asn Ile Val Pro Tyr Lys Glu Asp Val Gln Phe Arg Phe 65 70 75 80 atc atc att ggg aat gaa gcc att cca gga cag tca agc tct tac att 289Ile Ile Ile Gly Asn Glu Ala Ile Pro Gly Gln Ser Ser Ser Tyr Ile 85 90 95 cct ggt gcc atg aac aac ata atg aac tcg ctg gcc tca ttt ggg cta 337Pro Gly Ala Met Asn Asn Ile Met Asn Ser Leu Ala Ser Phe Gly Leu 100 105 110 ggc acg acg aag gtt acg acc gtg gtc ccg atg aat gcc cta agt acc 385Gly Thr Thr Lys Val Thr Thr Val Val Pro Met Asn Ala Leu Ser Thr 115 120 125 tcg tac cct cct tca gac ggc gct ttt gga agc gat ata aca tcg atc 433Ser Tyr Pro Pro Ser Asp Gly Ala Phe Gly Ser Asp Ile Thr Ser Ile 130 135 140 atg act agt atc atg gcc att ctg gtt cga cag gat tcg ccc ctc ctg 481Met Thr Ser Ile Met Ala Ile Leu Val Arg Gln Asp Ser Pro Leu Leu 145 150 155 160 atc aat gtg tac cct tat ttt gcc tat gcc tca gac ccc act cat att 529Ile Asn Val Tyr Pro Tyr Phe Ala Tyr Ala Ser Asp Pro Thr His Ile 165 170 175 tcc ctc aac tac gcc ttg ttc acc tcg gcc gca ccg gtg gtg gtc gac 577Ser Leu Asn Tyr Ala Leu Phe Thr Ser Ala Ala Pro Val Val Val Asp 180 185 190 caa ggc ttg gaa tac tac aac ctc ttt gac ggc atg gtc gat gct ttc 625Gln Gly Leu Glu Tyr Tyr Asn Leu Phe Asp Gly Met Val Asp Ala Phe 195 200 205 aat gcc gcc cta gat aag atc ggc ttc ggc c 656Asn Ala Ala Leu Asp Lys Ile Gly Phe Gly 210 215 16218PRTGossypium tomentosum 16Gly Asn Asn Leu Pro Ser Pro Gly Asp Val Ile Asp Leu Phe Lys Thr 1 5 10 15 Ser Gly Ile Asn Asn Ile Arg Leu Tyr Gln Pro Tyr Pro Glu Val Leu 20 25 30 Glu Ala Ala Arg Gly Ser Gly Ile Ser Leu Ser Met Ser Thr Thr Asn 35 40 45 Glu Asp Ile Gln Ser Leu Ala Thr Asp Gln Ser Ala Ala Asp Ala Trp 50 55 60 Val Asn Thr Asn Ile Val Pro Tyr Lys Glu Asp Val Gln Phe Arg Phe 65 70 75 80 Ile Ile Ile Gly Asn Glu Ala Ile Pro Gly Gln Ser Ser Ser Tyr Ile 85 90 95 Pro Gly Ala Met Asn Asn Ile Met Asn Ser Leu Ala Ser Phe Gly Leu 100 105 110 Gly Thr Thr Lys Val Thr Thr Val Val Pro Met Asn Ala Leu Ser Thr 115 120 125 Ser Tyr Pro Pro Ser Asp Gly Ala Phe Gly Ser Asp Ile Thr Ser Ile 130 135 140 Met Thr Ser Ile Met Ala Ile Leu Val Arg Gln Asp Ser Pro Leu Leu 145 150 155 160 Ile Asn Val Tyr Pro Tyr Phe Ala Tyr Ala Ser Asp Pro Thr His Ile 165 170 175 Ser Leu Asn Tyr Ala Leu Phe Thr Ser Ala Ala Pro Val Val Val Asp 180 185 190 Gln Gly Leu Glu Tyr Tyr Asn Leu Phe Asp Gly Met Val Asp Ala Phe 195 200 205 Asn Ala Ala Leu Asp Lys Ile Gly Phe Gly 210 215 17665DNAGossypium darwiniiCDS(2)..(472) 17c ggc aac aat ctt cca tct cca gga gat gtt att aat ctt ttc aaa act 49 Gly Asn Asn Leu Pro Ser Pro Gly Asp Val Ile Asn Leu Phe Lys Thr 1 5 10 15 agt ggc ata aac aat atc agg ctc tac cag cct tac cct gaa gtg ctc 97Ser Gly Ile Asn Asn Ile Arg Leu Tyr Gln Pro Tyr Pro Glu Val Leu 20 25 30 gaa gca gca agg gga tcg gga ata tcc ctc tcg atg agt acg aca aac 145Glu Ala Ala Arg Gly Ser Gly Ile Ser Leu Ser Met Ser Thr Thr Asn 35 40 45 gag gac ata caa agc ctc gca acg gat caa act cat caa agt gca gcc 193Glu Asp Ile Gln Ser Leu Ala Thr Asp Gln Thr His Gln Ser Ala Ala 50 55 60 gat gca tgg gtt aac acc aac atc gtc cct tat aag gaa gat gtt caa 241Asp Ala Trp Val Asn Thr Asn Ile Val Pro Tyr Lys Glu Asp Val Gln 65 70 75 80 ttc agg ttc atc atc att ggg aat gaa gcc att cca gga cag tca agc 289Phe Arg Phe Ile Ile Ile Gly Asn Glu Ala Ile Pro Gly Gln Ser Ser 85 90 95 tct tac att cct ggt gcc atg aac aac ata atg aac tcg ctc gcc tca 337Ser Tyr Ile Pro Gly Ala Met Asn Asn Ile Met Asn Ser Leu Ala Ser 100 105 110 ttt ggg cta ggc acg acg aag gtt acg acc gtg gtc ccg atg aat gcc 385Phe Gly Leu Gly Thr Thr Lys Val Thr Thr Val Val Pro Met Asn Ala 115 120 125 cta agt acc tcg tac cct cct tca gac ggc gct ttt gga agc gat ata 433Leu Ser Thr Ser Tyr Pro Pro Ser Asp Gly Ala Phe Gly Ser Asp Ile 130 135 140 aca tcg atc atg act agt atc atg gcc att ctg gtt tga caggattcgc 482Thr Ser Ile Met Thr Ser Ile Met Ala Ile Leu Val 145 150 155 ccctcctgat caatgtgtac ccttattttg cctatgcctc agaccccact catatttccc 542tcaactacgc cttgttcacc tcgaccgcac cggtggtggt cgaccaaggc ttggaatact 602acaacctctt tgacggcata gtcgatgctt tcaatgccgc cctagataag atcggcttcg 662gcc 66518156PRTGossypium darwinii 18Gly Asn Asn Leu Pro Ser Pro Gly Asp Val Ile Asn Leu Phe Lys Thr 1 5 10 15 Ser Gly Ile Asn Asn Ile Arg Leu Tyr Gln Pro Tyr Pro Glu Val Leu 20 25 30 Glu Ala Ala Arg Gly Ser Gly Ile Ser Leu Ser Met Ser Thr Thr Asn 35 40 45 Glu Asp Ile Gln Ser Leu Ala Thr Asp Gln Thr His Gln Ser Ala Ala 50 55 60 Asp Ala Trp Val Asn Thr Asn Ile Val Pro Tyr Lys Glu Asp Val Gln 65 70 75 80 Phe Arg Phe Ile Ile Ile Gly Asn Glu Ala Ile Pro Gly Gln Ser Ser 85 90 95 Ser Tyr Ile Pro Gly Ala Met Asn Asn Ile Met Asn Ser Leu Ala Ser 100 105 110 Phe Gly Leu Gly Thr Thr Lys Val Thr Thr Val Val Pro Met Asn Ala 115 120 125 Leu Ser Thr Ser Tyr Pro Pro Ser Asp Gly Ala Phe Gly Ser Asp Ile 130 135 140 Thr Ser Ile Met Thr Ser Ile Met Ala Ile Leu Val 145 150 155 19656DNAGossypium mustelinumCDS(2)..(655) 19c ggc aac aat ctt cca tct cca gga gat gtt att aat ctt tac aaa act 49 Gly Asn Asn Leu Pro Ser Pro Gly Asp Val Ile Asn Leu Tyr Lys Thr 1 5 10 15 agt ggc ata aac aat atc agg ctc tac cag cct tac cct gaa gtg ctc 97Ser Gly Ile Asn Asn Ile Arg Leu Tyr Gln Pro Tyr Pro Glu Val Leu 20 25 30 gaa gca gca

agg gga tcg gga ata tcc ctc tcg atg agt acg aca aac 145Glu Ala Ala Arg Gly Ser Gly Ile Ser Leu Ser Met Ser Thr Thr Asn 35 40 45 gag gac ata caa agc ctc gca acg gat caa agt gca gcc gat gca tgg 193Glu Asp Ile Gln Ser Leu Ala Thr Asp Gln Ser Ala Ala Asp Ala Trp 50 55 60 gtt aac acc aac atc gtc cct tat aag gaa gat gtt caa ttc agg ttc 241Val Asn Thr Asn Ile Val Pro Tyr Lys Glu Asp Val Gln Phe Arg Phe 65 70 75 80 atc atc att ggg aat gaa gcc att cca gga cag tca agc tct tac att 289Ile Ile Ile Gly Asn Glu Ala Ile Pro Gly Gln Ser Ser Ser Tyr Ile 85 90 95 cct ggt gcc atg aac aac ata atg aac tcg ctc gcc tca ttt ggg cta 337Pro Gly Ala Met Asn Asn Ile Met Asn Ser Leu Ala Ser Phe Gly Leu 100 105 110 ggc acg acg aag gtt acg acc gtg gtc ccg atg aat gcc cta agt acc 385Gly Thr Thr Lys Val Thr Thr Val Val Pro Met Asn Ala Leu Ser Thr 115 120 125 tcg tac cct cct tca gac ggc gct ttt gga agc gat ata aca tcg atc 433Ser Tyr Pro Pro Ser Asp Gly Ala Phe Gly Ser Asp Ile Thr Ser Ile 130 135 140 atg act agt atc atg gcc att ctg gtt cga cag gat tcg ccc ctc ctg 481Met Thr Ser Ile Met Ala Ile Leu Val Arg Gln Asp Ser Pro Leu Leu 145 150 155 160 atc aat gtg tac cct tat ttt gcc tat gcc tca gac ccc act cat att 529Ile Asn Val Tyr Pro Tyr Phe Ala Tyr Ala Ser Asp Pro Thr His Ile 165 170 175 tcc ctc aac tac gcc ttg ttc acc tcg acc gca ccg gtg gtg gtc gac 577Ser Leu Asn Tyr Ala Leu Phe Thr Ser Thr Ala Pro Val Val Val Asp 180 185 190 caa ggc ttg gaa tac tac aac ctc ttt gac ggc atg gtc gat gct ttc 625Gln Gly Leu Glu Tyr Tyr Asn Leu Phe Asp Gly Met Val Asp Ala Phe 195 200 205 aat gcc gcc cta gat aag atc ggc ttc ggc c 656Asn Ala Ala Leu Asp Lys Ile Gly Phe Gly 210 215 20218PRTGossypium mustelinum 20Gly Asn Asn Leu Pro Ser Pro Gly Asp Val Ile Asn Leu Tyr Lys Thr 1 5 10 15 Ser Gly Ile Asn Asn Ile Arg Leu Tyr Gln Pro Tyr Pro Glu Val Leu 20 25 30 Glu Ala Ala Arg Gly Ser Gly Ile Ser Leu Ser Met Ser Thr Thr Asn 35 40 45 Glu Asp Ile Gln Ser Leu Ala Thr Asp Gln Ser Ala Ala Asp Ala Trp 50 55 60 Val Asn Thr Asn Ile Val Pro Tyr Lys Glu Asp Val Gln Phe Arg Phe 65 70 75 80 Ile Ile Ile Gly Asn Glu Ala Ile Pro Gly Gln Ser Ser Ser Tyr Ile 85 90 95 Pro Gly Ala Met Asn Asn Ile Met Asn Ser Leu Ala Ser Phe Gly Leu 100 105 110 Gly Thr Thr Lys Val Thr Thr Val Val Pro Met Asn Ala Leu Ser Thr 115 120 125 Ser Tyr Pro Pro Ser Asp Gly Ala Phe Gly Ser Asp Ile Thr Ser Ile 130 135 140 Met Thr Ser Ile Met Ala Ile Leu Val Arg Gln Asp Ser Pro Leu Leu 145 150 155 160 Ile Asn Val Tyr Pro Tyr Phe Ala Tyr Ala Ser Asp Pro Thr His Ile 165 170 175 Ser Leu Asn Tyr Ala Leu Phe Thr Ser Thr Ala Pro Val Val Val Asp 180 185 190 Gln Gly Leu Glu Tyr Tyr Asn Leu Phe Asp Gly Met Val Asp Ala Phe 195 200 205 Asn Ala Ala Leu Asp Lys Ile Gly Phe Gly 210 215 211206DNAGossypium arboreumCDS(27)..(96)CDS(209)..(372) 21gctgagatca agaaatatag tgaaat atg ggt cca aga ttt tct ggg ttt tta 53 Met Gly Pro Arg Phe Ser Gly Phe Leu 1 5 atc tca gca atg ctg ttt tta act caa ctc ctc tct cta aca g 96Ile Ser Ala Met Leu Phe Leu Thr Gln Leu Leu Ser Leu Thr 10 15 20 gtaaaacaaa cttctctaca gtgattttag agtaaatatg gctttgaaaa atatacaaca 156aaacatttat cttcaatcca ttttaattac tgatctacta tatatgttgc ag at ggc 213 Asp Gly 25 cgt gat att ggt gtt tgc tat ggt ttg aac ggc aac aat ctt cca tct 261Arg Asp Ile Gly Val Cys Tyr Gly Leu Asn Gly Asn Asn Leu Pro Ser 30 35 40 cca gga gat gtt att aat ctt tac aaa act agt ggc ata aac aat atc 309Pro Gly Asp Val Ile Asn Leu Tyr Lys Thr Ser Gly Ile Asn Asn Ile 45 50 55 agg ctc tac cag cct tac ctg aag tgc tcg aag gag caa ggg gat cgg 357Arg Leu Tyr Gln Pro Tyr Leu Lys Cys Ser Lys Glu Gln Gly Asp Arg 60 65 70 gaa tat ccc tct cga tgagtacgac aaacgaggac atacaaagcc tcgcaacgga 412Glu Tyr Pro Ser Arg 75 tcaaagtgca gccgatgcat gggttaacac caacatcgtc ccttataagg acgatgttca 472attcaggttc atcatcattg ggaatgaagc cattccagga cagtcaagct cttacattcc 532tggtgccatg aacaacataa tgaactcgct cgcctcattt gggctaggca cgacgaaggt 592tacgaccgtg gtcccgatga atgccctaag tacctcgtac cctccttcag acggcgcttt 652tggaagcgat ataacatcga tcatgactag tatcatggcc attctggttc gacaggattc 712gcccctcctg atcaatgtgt acccttattt tgcctatgcc tcagacccca ctcatatttc 772cctcaactac gccttgttca cctcgaccgc accggtggtg gtcgaccaag gcttggaata 832ctacaacctc tttgacggca tggtcgatgc tttcaatgcc gccctagata agatcggctt 892cggccaaatt actctcattg tagccgaaac tggatggccg accgccggta acgagcctta 952cacgagtgtc gcgaacgctc aaacttataa caagaacttg ttgaatcatg tgacgcagaa 1012agggactccg aaaagacctg aatatataat gccgacgttt ttcttcgaga tgttcaacga 1072gaacttgaag caacccacag ttgagcagaa tttcggattc ttcttcccca atatgaaccc 1132tgtttatcca ttttggtgaa cttgaaatgt tattgttggc tatttaaatc ttttgccaga 1192gacgcttcat atag 12062278PRTGossypium arboreum 22Met Gly Pro Arg Phe Ser Gly Phe Leu Ile Ser Ala Met Leu Phe Leu 1 5 10 15 Thr Gln Leu Leu Ser Leu Thr Asp Gly Arg Asp Ile Gly Val Cys Tyr 20 25 30 Gly Leu Asn Gly Asn Asn Leu Pro Ser Pro Gly Asp Val Ile Asn Leu 35 40 45 Tyr Lys Thr Ser Gly Ile Asn Asn Ile Arg Leu Tyr Gln Pro Tyr Leu 50 55 60 Lys Cys Ser Lys Glu Gln Gly Asp Arg Glu Tyr Pro Ser Arg 65 70 75 231207DNAGossypium herbaceumCDS(27)..(96)CDS(209)..(1149) 23gctgagatca agaaatatag tgaaat atg ggt cca aga ttt tct ggg ttt tta 53 Met Gly Pro Arg Phe Ser Gly Phe Leu 1 5 atc tca gca atg ctg ttt tta act caa ctc ctc tct cta aca g 96Ile Ser Ala Met Leu Phe Leu Thr Gln Leu Leu Ser Leu Thr 10 15 20 gtaaaacaaa cttctctaca gtgattttac agtaaatatg gctttgaaaa atatacaaca 156aaacatttat cttcaatcca ttttaattac tgatctacta tatatgttgc ag at ggc 213 Asp Gly 25 cgt gat att ggt gtt tgc tat ggt ttg aac ggc aac aat ctt cca tct 261Arg Asp Ile Gly Val Cys Tyr Gly Leu Asn Gly Asn Asn Leu Pro Ser 30 35 40 cca gga gat gct att aat ctt tac aaa act agt ggc ata aac aat atc 309Pro Gly Asp Ala Ile Asn Leu Tyr Lys Thr Ser Gly Ile Asn Asn Ile 45 50 55 agg ctc tac cag cct tac cct gaa gtg ctc gaa gca gca agg gga tcg 357Arg Leu Tyr Gln Pro Tyr Pro Glu Val Leu Glu Ala Ala Arg Gly Ser 60 65 70 gga ata tcc ctc tcg atg agt acg aca aac gag gac ata caa agc ctc 405Gly Ile Ser Leu Ser Met Ser Thr Thr Asn Glu Asp Ile Gln Ser Leu 75 80 85 gca acg gat caa agt gca gcc gat gca tgg gtt aac acc aac atc gtc 453Ala Thr Asp Gln Ser Ala Ala Asp Ala Trp Val Asn Thr Asn Ile Val 90 95 100 105 cct tat aag gac gat gtt caa ttc agg ttc atc atc att ggg aat gaa 501Pro Tyr Lys Asp Asp Val Gln Phe Arg Phe Ile Ile Ile Gly Asn Glu 110 115 120 gcc att cca gga cag tca agc tct tac att cct ggt gcc atg aac aac 549Ala Ile Pro Gly Gln Ser Ser Ser Tyr Ile Pro Gly Ala Met Asn Asn 125 130 135 ata atg aac tcg ctc gcc tca ttt ggg cta ggc acg acg aag gtt acg 597Ile Met Asn Ser Leu Ala Ser Phe Gly Leu Gly Thr Thr Lys Val Thr 140 145 150 acc gtg gtc ccg atg aat gcc cta agt acc tcg tac cct cct tca gac 645Thr Val Val Pro Met Asn Ala Leu Ser Thr Ser Tyr Pro Pro Ser Asp 155 160 165 ggc gct ttt gga agc gat ata aca tcg atc atg act agt atc atg gcc 693Gly Ala Phe Gly Ser Asp Ile Thr Ser Ile Met Thr Ser Ile Met Ala 170 175 180 185 att ctg gtt cga cag gat tcg ccc ctc ctg atc aat gtg tac cct tat 741Ile Leu Val Arg Gln Asp Ser Pro Leu Leu Ile Asn Val Tyr Pro Tyr 190 195 200 ttt gcc tat gcc tca gac ccc act cat att tcc ctc aac tac gcc ttg 789Phe Ala Tyr Ala Ser Asp Pro Thr His Ile Ser Leu Asn Tyr Ala Leu 205 210 215 ttc acc tcg acc gca ccg gtg gtg gtc gac caa ggc ttg gaa tac tac 837Phe Thr Ser Thr Ala Pro Val Val Val Asp Gln Gly Leu Glu Tyr Tyr 220 225 230 aac ctc ttt gac ggc atg gtc gat gct ttc aat gcc gcc cta gat aag 885Asn Leu Phe Asp Gly Met Val Asp Ala Phe Asn Ala Ala Leu Asp Lys 235 240 245 atc ggc ttc ggc caa att act ctc att gta gcc gaa act gga tgg ccg 933Ile Gly Phe Gly Gln Ile Thr Leu Ile Val Ala Glu Thr Gly Trp Pro 250 255 260 265 acc gcc ggt aac gag cct tac acg agt gtc gcg aac gct caa act tat 981Thr Ala Gly Asn Glu Pro Tyr Thr Ser Val Ala Asn Ala Gln Thr Tyr 270 275 280 aac aag aac ttg ttg aat cat gtg acg cag aaa ggg act ccg aaa aga 1029Asn Lys Asn Leu Leu Asn His Val Thr Gln Lys Gly Thr Pro Lys Arg 285 290 295 cct gaa tat ata atg ccg acg ttt ttc ttc gag atg ttc aac gag aac 1077Pro Glu Tyr Ile Met Pro Thr Phe Phe Phe Glu Met Phe Asn Glu Asn 300 305 310 ttg aag caa ccc aca gtt gag cag aat ttc gga ttc ttc ttc ccc aat 1125Leu Lys Gln Pro Thr Val Glu Gln Asn Phe Gly Phe Phe Phe Pro Asn 315 320 325 atg aac cct gtt tat cca ttt tgg tgagcttgaa atgttattgt tggctattta 1179Met Asn Pro Val Tyr Pro Phe Trp 330 335 aatcttttgc cagagacgct tcatatag 120724337PRTGossypium herbaceum 24Met Gly Pro Arg Phe Ser Gly Phe Leu Ile Ser Ala Met Leu Phe Leu 1 5 10 15 Thr Gln Leu Leu Ser Leu Thr Asp Gly Arg Asp Ile Gly Val Cys Tyr 20 25 30 Gly Leu Asn Gly Asn Asn Leu Pro Ser Pro Gly Asp Ala Ile Asn Leu 35 40 45 Tyr Lys Thr Ser Gly Ile Asn Asn Ile Arg Leu Tyr Gln Pro Tyr Pro 50 55 60 Glu Val Leu Glu Ala Ala Arg Gly Ser Gly Ile Ser Leu Ser Met Ser 65 70 75 80 Thr Thr Asn Glu Asp Ile Gln Ser Leu Ala Thr Asp Gln Ser Ala Ala 85 90 95 Asp Ala Trp Val Asn Thr Asn Ile Val Pro Tyr Lys Asp Asp Val Gln 100 105 110 Phe Arg Phe Ile Ile Ile Gly Asn Glu Ala Ile Pro Gly Gln Ser Ser 115 120 125 Ser Tyr Ile Pro Gly Ala Met Asn Asn Ile Met Asn Ser Leu Ala Ser 130 135 140 Phe Gly Leu Gly Thr Thr Lys Val Thr Thr Val Val Pro Met Asn Ala 145 150 155 160 Leu Ser Thr Ser Tyr Pro Pro Ser Asp Gly Ala Phe Gly Ser Asp Ile 165 170 175 Thr Ser Ile Met Thr Ser Ile Met Ala Ile Leu Val Arg Gln Asp Ser 180 185 190 Pro Leu Leu Ile Asn Val Tyr Pro Tyr Phe Ala Tyr Ala Ser Asp Pro 195 200 205 Thr His Ile Ser Leu Asn Tyr Ala Leu Phe Thr Ser Thr Ala Pro Val 210 215 220 Val Val Asp Gln Gly Leu Glu Tyr Tyr Asn Leu Phe Asp Gly Met Val 225 230 235 240 Asp Ala Phe Asn Ala Ala Leu Asp Lys Ile Gly Phe Gly Gln Ile Thr 245 250 255 Leu Ile Val Ala Glu Thr Gly Trp Pro Thr Ala Gly Asn Glu Pro Tyr 260 265 270 Thr Ser Val Ala Asn Ala Gln Thr Tyr Asn Lys Asn Leu Leu Asn His 275 280 285 Val Thr Gln Lys Gly Thr Pro Lys Arg Pro Glu Tyr Ile Met Pro Thr 290 295 300 Phe Phe Phe Glu Met Phe Asn Glu Asn Leu Lys Gln Pro Thr Val Glu 305 310 315 320 Gln Asn Phe Gly Phe Phe Phe Pro Asn Met Asn Pro Val Tyr Pro Phe 325 330 335 Trp 25656DNAGossypium tomentosumCDS(2)..(655) 25c ggc aac aat ctt cca tct cca gga gat gtt att aat ctt tac aaa act 49 Gly Asn Asn Leu Pro Ser Pro Gly Asp Val Ile Asn Leu Tyr Lys Thr 1 5 10 15 agt ggc ata aac aat atc agg ctc tac cag cct tac cct gaa gtg ctc 97Ser Gly Ile Asn Asn Ile Arg Leu Tyr Gln Pro Tyr Pro Glu Val Leu 20 25 30 gaa gca gca agg gga tcg gga ata tcc ctc tcg atg ggt ccg aga aac 145Glu Ala Ala Arg Gly Ser Gly Ile Ser Leu Ser Met Gly Pro Arg Asn 35 40 45 gag gac ata caa agc ctc gca aaa gat caa agt gca gcc gat gca tgg 193Glu Asp Ile Gln Ser Leu Ala Lys Asp Gln Ser Ala Ala Asp Ala Trp 50 55 60 gtt aac acc aac atc gtc cct tat aag gac gat gtt cag ttc aag ttg 241Val Asn Thr Asn Ile Val Pro Tyr Lys Asp Asp Val Gln Phe Lys Leu 65 70 75 80 atc act att ggg aat gaa gcc att tca gga caa tca agc tct tac att 289Ile Thr Ile Gly Asn Glu Ala Ile Ser Gly Gln Ser Ser Ser Tyr Ile 85 90 95 cct gat gcc atg aac aac ata atg aac tcg ctc gcc tta ttt ggg tta 337Pro Asp Ala Met Asn Asn Ile Met Asn Ser Leu Ala Leu Phe Gly Leu 100 105 110 ggc acg acg aag gtt acg acc gtg gtc ccg atg aat gcc cta agt acc 385Gly Thr Thr Lys Val Thr Thr Val Val Pro Met Asn Ala Leu Ser Thr 115 120 125 tcg tac cct cct tca gac ggc gct ttt gga agc gat ata aca tcg atc 433Ser Tyr Pro Pro Ser Asp Gly Ala Phe Gly Ser Asp Ile Thr Ser Ile 130 135 140 atg act agt atc atg gcc att ctg gct gta cag gat tcg ccc ctc ctg 481Met Thr Ser Ile Met Ala Ile Leu Ala Val Gln Asp Ser Pro Leu Leu 145 150 155 160 atc aat gtg tac cct tat ttt gcc tat gcc tca gac ccc act cat att 529Ile Asn Val Tyr Pro Tyr Phe Ala Tyr Ala Ser Asp Pro Thr His Ile 165 170 175 tcc ctc gat tac gcc ttg ttc acc tcg acc gca ccg gtg gtg gtc gac 577Ser Leu Asp Tyr Ala Leu Phe Thr Ser Thr Ala Pro Val Val Val Asp 180 185 190 caa ggc ttg gaa tac tac aac ctc ttt gac ggc atg gtc

gat gct ttc 625Gln Gly Leu Glu Tyr Tyr Asn Leu Phe Asp Gly Met Val Asp Ala Phe 195 200 205 aat gcc gcc cta gat aag atc ggc ttc ggc c 656Asn Ala Ala Leu Asp Lys Ile Gly Phe Gly 210 215 26218PRTGossypium tomentosum 26Gly Asn Asn Leu Pro Ser Pro Gly Asp Val Ile Asn Leu Tyr Lys Thr 1 5 10 15 Ser Gly Ile Asn Asn Ile Arg Leu Tyr Gln Pro Tyr Pro Glu Val Leu 20 25 30 Glu Ala Ala Arg Gly Ser Gly Ile Ser Leu Ser Met Gly Pro Arg Asn 35 40 45 Glu Asp Ile Gln Ser Leu Ala Lys Asp Gln Ser Ala Ala Asp Ala Trp 50 55 60 Val Asn Thr Asn Ile Val Pro Tyr Lys Asp Asp Val Gln Phe Lys Leu 65 70 75 80 Ile Thr Ile Gly Asn Glu Ala Ile Ser Gly Gln Ser Ser Ser Tyr Ile 85 90 95 Pro Asp Ala Met Asn Asn Ile Met Asn Ser Leu Ala Leu Phe Gly Leu 100 105 110 Gly Thr Thr Lys Val Thr Thr Val Val Pro Met Asn Ala Leu Ser Thr 115 120 125 Ser Tyr Pro Pro Ser Asp Gly Ala Phe Gly Ser Asp Ile Thr Ser Ile 130 135 140 Met Thr Ser Ile Met Ala Ile Leu Ala Val Gln Asp Ser Pro Leu Leu 145 150 155 160 Ile Asn Val Tyr Pro Tyr Phe Ala Tyr Ala Ser Asp Pro Thr His Ile 165 170 175 Ser Leu Asp Tyr Ala Leu Phe Thr Ser Thr Ala Pro Val Val Val Asp 180 185 190 Gln Gly Leu Glu Tyr Tyr Asn Leu Phe Asp Gly Met Val Asp Ala Phe 195 200 205 Asn Ala Ala Leu Asp Lys Ile Gly Phe Gly 210 215 27656DNAGossypium darwiniiCDS(2)..(655) 27c ggc aac aat ctt cca tct cca gga gat gtt att aat ctt tac aaa act 49 Gly Asn Asn Leu Pro Ser Pro Gly Asp Val Ile Asn Leu Tyr Lys Thr 1 5 10 15 agt ggc ata aac aat atc agg ctc tac cag tct tac cct gaa gtg ctc 97Ser Gly Ile Asn Asn Ile Arg Leu Tyr Gln Ser Tyr Pro Glu Val Leu 20 25 30 gaa gca gca agg gga tcg gga ata tcc ctc tcg atg ggt ccg aga aac 145Glu Ala Ala Arg Gly Ser Gly Ile Ser Leu Ser Met Gly Pro Arg Asn 35 40 45 gag gac ata caa agc ctc gca aaa gat caa agt gca gcc gat gca tgg 193Glu Asp Ile Gln Ser Leu Ala Lys Asp Gln Ser Ala Ala Asp Ala Trp 50 55 60 gtt aac acc aac atc gtc cct tat aag gac gat gtt cag ttc aag ttg 241Val Asn Thr Asn Ile Val Pro Tyr Lys Asp Asp Val Gln Phe Lys Leu 65 70 75 80 atc act att ggg aat gaa gcc att tca gga caa tca agc tct tac att 289Ile Thr Ile Gly Asn Glu Ala Ile Ser Gly Gln Ser Ser Ser Tyr Ile 85 90 95 cct gat gcc atg aac aac ata atg aac tcg ctc gcc tta ttt ggg tta 337Pro Asp Ala Met Asn Asn Ile Met Asn Ser Leu Ala Leu Phe Gly Leu 100 105 110 ggc acg acg aag gtt acg acc gtg gtc ccg atg aat gcc cta agt acc 385Gly Thr Thr Lys Val Thr Thr Val Val Pro Met Asn Ala Leu Ser Thr 115 120 125 tcg tac cct cct tca gac ggc gct ttt gga agc gat ata aca tcg atc 433Ser Tyr Pro Pro Ser Asp Gly Ala Phe Gly Ser Asp Ile Thr Ser Ile 130 135 140 atg act agt atc atg gcc att ctg gct gta cag gat tcg ccc ctc ctg 481Met Thr Ser Ile Met Ala Ile Leu Ala Val Gln Asp Ser Pro Leu Leu 145 150 155 160 atc aat gtg tac cct tat ttt gcc tat gcc tca gac ccc act cat att 529Ile Asn Val Tyr Pro Tyr Phe Ala Tyr Ala Ser Asp Pro Thr His Ile 165 170 175 tcc ctc gat tac gcc ttg ttc acc tcg acc gca ccg gtg gtg gtc gac 577Ser Leu Asp Tyr Ala Leu Phe Thr Ser Thr Ala Pro Val Val Val Asp 180 185 190 caa ggc ttg gaa tac tac aac ctc ttt gac ggc atg gtc gat gct ttc 625Gln Gly Leu Glu Tyr Tyr Asn Leu Phe Asp Gly Met Val Asp Ala Phe 195 200 205 aat gcc gcc cta gat aag atc ggc ttc ggc c 656Asn Ala Ala Leu Asp Lys Ile Gly Phe Gly 210 215 28218PRTGossypium darwinii 28Gly Asn Asn Leu Pro Ser Pro Gly Asp Val Ile Asn Leu Tyr Lys Thr 1 5 10 15 Ser Gly Ile Asn Asn Ile Arg Leu Tyr Gln Ser Tyr Pro Glu Val Leu 20 25 30 Glu Ala Ala Arg Gly Ser Gly Ile Ser Leu Ser Met Gly Pro Arg Asn 35 40 45 Glu Asp Ile Gln Ser Leu Ala Lys Asp Gln Ser Ala Ala Asp Ala Trp 50 55 60 Val Asn Thr Asn Ile Val Pro Tyr Lys Asp Asp Val Gln Phe Lys Leu 65 70 75 80 Ile Thr Ile Gly Asn Glu Ala Ile Ser Gly Gln Ser Ser Ser Tyr Ile 85 90 95 Pro Asp Ala Met Asn Asn Ile Met Asn Ser Leu Ala Leu Phe Gly Leu 100 105 110 Gly Thr Thr Lys Val Thr Thr Val Val Pro Met Asn Ala Leu Ser Thr 115 120 125 Ser Tyr Pro Pro Ser Asp Gly Ala Phe Gly Ser Asp Ile Thr Ser Ile 130 135 140 Met Thr Ser Ile Met Ala Ile Leu Ala Val Gln Asp Ser Pro Leu Leu 145 150 155 160 Ile Asn Val Tyr Pro Tyr Phe Ala Tyr Ala Ser Asp Pro Thr His Ile 165 170 175 Ser Leu Asp Tyr Ala Leu Phe Thr Ser Thr Ala Pro Val Val Val Asp 180 185 190 Gln Gly Leu Glu Tyr Tyr Asn Leu Phe Asp Gly Met Val Asp Ala Phe 195 200 205 Asn Ala Ala Leu Asp Lys Ile Gly Phe Gly 210 215 29656DNAGossypium mustelinumCDS(2)..(655) 29c ggc aac aat ctt cca tct cca gga gat gtt att aat ctt tac aaa act 49 Gly Asn Asn Leu Pro Ser Pro Gly Asp Val Ile Asn Leu Tyr Lys Thr 1 5 10 15 agt ggc ata aac aat atc agg ctc tac cag cct tac cct gaa gtg ctc 97Ser Gly Ile Asn Asn Ile Arg Leu Tyr Gln Pro Tyr Pro Glu Val Leu 20 25 30 gaa gca gca agg gga tcg gga ata tcc ctc tcg atg ggt ccg aga aac 145Glu Ala Ala Arg Gly Ser Gly Ile Ser Leu Ser Met Gly Pro Arg Asn 35 40 45 gag gac ata caa agc ctc gca aaa gat caa agt gca gcc gat gca tgg 193Glu Asp Ile Gln Ser Leu Ala Lys Asp Gln Ser Ala Ala Asp Ala Trp 50 55 60 gtt aac acc aac atc gtc cct tat aag gac gat gtt cag ttc aag ttg 241Val Asn Thr Asn Ile Val Pro Tyr Lys Asp Asp Val Gln Phe Lys Leu 65 70 75 80 atc act att ggg aat gaa gcc att tca gga caa tca agc tct tac att 289Ile Thr Ile Gly Asn Glu Ala Ile Ser Gly Gln Ser Ser Ser Tyr Ile 85 90 95 cct gat gcc atg aac aac ata atg aac tcg ctc gcc tta ttt ggg tta 337Pro Asp Ala Met Asn Asn Ile Met Asn Ser Leu Ala Leu Phe Gly Leu 100 105 110 ggc acg acg aag gtt acg acc gtg gtc ccg atg aat gcc cta aat acc 385Gly Thr Thr Lys Val Thr Thr Val Val Pro Met Asn Ala Leu Asn Thr 115 120 125 tcg tac cct cct tca gac ggc gct ttt gga agc gat ata aca tcg atc 433Ser Tyr Pro Pro Ser Asp Gly Ala Phe Gly Ser Asp Ile Thr Ser Ile 130 135 140 atg act agt atc atg gcc att ctg gct gta cag gat tcg ccc ctc ctg 481Met Thr Ser Ile Met Ala Ile Leu Ala Val Gln Asp Ser Pro Leu Leu 145 150 155 160 atc aat gtg tac cct tat ttt gcc tat gcc tca gac ccc act cat att 529Ile Asn Val Tyr Pro Tyr Phe Ala Tyr Ala Ser Asp Pro Thr His Ile 165 170 175 tcc ctc gat tac gcc ttg ttc acc tcg acc gca ccg gtg gtg gtc gac 577Ser Leu Asp Tyr Ala Leu Phe Thr Ser Thr Ala Pro Val Val Val Asp 180 185 190 caa ggc ttg gaa tac tac aac ctc ttt gac ggc atg gtc gat gct ttc 625Gln Gly Leu Glu Tyr Tyr Asn Leu Phe Asp Gly Met Val Asp Ala Phe 195 200 205 aat gcc gct cta gat aag atc ggc ttc ggc c 656Asn Ala Ala Leu Asp Lys Ile Gly Phe Gly 210 215 30218PRTGossypium mustelinum 30Gly Asn Asn Leu Pro Ser Pro Gly Asp Val Ile Asn Leu Tyr Lys Thr 1 5 10 15 Ser Gly Ile Asn Asn Ile Arg Leu Tyr Gln Pro Tyr Pro Glu Val Leu 20 25 30 Glu Ala Ala Arg Gly Ser Gly Ile Ser Leu Ser Met Gly Pro Arg Asn 35 40 45 Glu Asp Ile Gln Ser Leu Ala Lys Asp Gln Ser Ala Ala Asp Ala Trp 50 55 60 Val Asn Thr Asn Ile Val Pro Tyr Lys Asp Asp Val Gln Phe Lys Leu 65 70 75 80 Ile Thr Ile Gly Asn Glu Ala Ile Ser Gly Gln Ser Ser Ser Tyr Ile 85 90 95 Pro Asp Ala Met Asn Asn Ile Met Asn Ser Leu Ala Leu Phe Gly Leu 100 105 110 Gly Thr Thr Lys Val Thr Thr Val Val Pro Met Asn Ala Leu Asn Thr 115 120 125 Ser Tyr Pro Pro Ser Asp Gly Ala Phe Gly Ser Asp Ile Thr Ser Ile 130 135 140 Met Thr Ser Ile Met Ala Ile Leu Ala Val Gln Asp Ser Pro Leu Leu 145 150 155 160 Ile Asn Val Tyr Pro Tyr Phe Ala Tyr Ala Ser Asp Pro Thr His Ile 165 170 175 Ser Leu Asp Tyr Ala Leu Phe Thr Ser Thr Ala Pro Val Val Val Asp 180 185 190 Gln Gly Leu Glu Tyr Tyr Asn Leu Phe Asp Gly Met Val Asp Ala Phe 195 200 205 Asn Ala Ala Leu Asp Lys Ile Gly Phe Gly 210 215 31656DNAGossypium raimondiiCDS(2)..(655) 31c ggc aac aat ctt cca tct cca gga gat gtt att aat ctt tac aaa act 49 Gly Asn Asn Leu Pro Ser Pro Gly Asp Val Ile Asn Leu Tyr Lys Thr 1 5 10 15 agt ggc ata aac aat atc agg ctc tac cag cct tac cct gaa gtg ctc 97Ser Gly Ile Asn Asn Ile Arg Leu Tyr Gln Pro Tyr Pro Glu Val Leu 20 25 30 gaa gca gca agg gga tcg gga ata tcc ctc tcg atg ggt ccg aga aac 145Glu Ala Ala Arg Gly Ser Gly Ile Ser Leu Ser Met Gly Pro Arg Asn 35 40 45 gag gac ata caa agc ctc gca aaa gat caa agt gca gcc gat gca tgg 193Glu Asp Ile Gln Ser Leu Ala Lys Asp Gln Ser Ala Ala Asp Ala Trp 50 55 60 gtt aac acc aac atc gtc cct tat aag gac gat gtt cag ttc aaa ttg 241Val Asn Thr Asn Ile Val Pro Tyr Lys Asp Asp Val Gln Phe Lys Leu 65 70 75 80 atc act att ggg aat gaa gcc att tca gga caa tca agc tct tac att 289Ile Thr Ile Gly Asn Glu Ala Ile Ser Gly Gln Ser Ser Ser Tyr Ile 85 90 95 cct gat gcc atg aac aac ata atg aac tcg ctc gcc tca ttt ggg tta 337Pro Asp Ala Met Asn Asn Ile Met Asn Ser Leu Ala Ser Phe Gly Leu 100 105 110 ggc aca acg aag gtt acg acc gtg gtc ccg atg aat gcc cta agt acc 385Gly Thr Thr Lys Val Thr Thr Val Val Pro Met Asn Ala Leu Ser Thr 115 120 125 tcg tac cct cct tca gac ggc gct ttt gga agc gat ata aca tcg atc 433Ser Tyr Pro Pro Ser Asp Gly Ala Phe Gly Ser Asp Ile Thr Ser Ile 130 135 140 atg act agt atc atg gcc att ctg gct gta cag gat tcg ccc ctc ctg 481Met Thr Ser Ile Met Ala Ile Leu Ala Val Gln Asp Ser Pro Leu Leu 145 150 155 160 atc aat gtg tac cct tat ttt gcc tat gcc tca gac ccc act cat att 529Ile Asn Val Tyr Pro Tyr Phe Ala Tyr Ala Ser Asp Pro Thr His Ile 165 170 175 tcc ctc gat tac gcc ttg ttc acc tcg acc gca ccg gtg gtg gtc gac 577Ser Leu Asp Tyr Ala Leu Phe Thr Ser Thr Ala Pro Val Val Val Asp 180 185 190 caa ggc ttg gaa tac tac aac ctc ttt gac ggc atg gtc gat gct ttc 625Gln Gly Leu Glu Tyr Tyr Asn Leu Phe Asp Gly Met Val Asp Ala Phe 195 200 205 aat gcc gcc cta gat aag atc ggc ttc ggc c 656Asn Ala Ala Leu Asp Lys Ile Gly Phe Gly 210 215 32218PRTGossypium raimondii 32Gly Asn Asn Leu Pro Ser Pro Gly Asp Val Ile Asn Leu Tyr Lys Thr 1 5 10 15 Ser Gly Ile Asn Asn Ile Arg Leu Tyr Gln Pro Tyr Pro Glu Val Leu 20 25 30 Glu Ala Ala Arg Gly Ser Gly Ile Ser Leu Ser Met Gly Pro Arg Asn 35 40 45 Glu Asp Ile Gln Ser Leu Ala Lys Asp Gln Ser Ala Ala Asp Ala Trp 50 55 60 Val Asn Thr Asn Ile Val Pro Tyr Lys Asp Asp Val Gln Phe Lys Leu 65 70 75 80 Ile Thr Ile Gly Asn Glu Ala Ile Ser Gly Gln Ser Ser Ser Tyr Ile 85 90 95 Pro Asp Ala Met Asn Asn Ile Met Asn Ser Leu Ala Ser Phe Gly Leu 100 105 110 Gly Thr Thr Lys Val Thr Thr Val Val Pro Met Asn Ala Leu Ser Thr 115 120 125 Ser Tyr Pro Pro Ser Asp Gly Ala Phe Gly Ser Asp Ile Thr Ser Ile 130 135 140 Met Thr Ser Ile Met Ala Ile Leu Ala Val Gln Asp Ser Pro Leu Leu 145 150 155 160 Ile Asn Val Tyr Pro Tyr Phe Ala Tyr Ala Ser Asp Pro Thr His Ile 165 170 175 Ser Leu Asp Tyr Ala Leu Phe Thr Ser Thr Ala Pro Val Val Val Asp 180 185 190 Gln Gly Leu Glu Tyr Tyr Asn Leu Phe Asp Gly Met Val Asp Ala Phe 195 200 205 Asn Ala Ala Leu Asp Lys Ile Gly Phe Gly 210 215 3322DNAArtificialprimer SE077 33gctgagatca agaaatatag tg 223420DNAArtificialprimer SE078 34ctatatgaag cgtctctggc 203523DNAArtificialprimer SE002 35ggccgaagcc gatcttatct agg 233623DNAArtificialprimer SE003 36cggcaacaat cttccatctc cag 233722DNAArtificialprimer p1.3GlucaAf 37tatccctctc gatgagtacg ac 223824DNAArtificialp1.3GlucaAr 38cccaatgatg atgaacctga attg 243915DNAArtificialprobe TM249-GCM1 39aactcgctcg cctca 154015DNAArtificialprobe TM249-GCV1 40aactcgctgg cctca 154124DNAArtificialprimer TM249-GCF 41cctggtgcca tgaacaacat aatg 244218DNAArtificialprimer TM249-GCR 42cgtcgtgcct agcccaaa 184318DNAArtificialAFLP primer P5 43gactgcgtac atgcagaa 184419DNAArtificialAFLP primer M50 44gatgagtcct gagtaacat 194520DNAArtificialforward SSR primer NAU861 45ccaaaacttg tcccattagc 204619DNAArtificialreverse SSR primer NAU861 46ttcatctgtt gccagatcc 194715DNAArtificialforward SSR primer CIR401 47tggcgactcc ctttt

154822DNAArtificialreverse SSR primer CIR401 48aaaagatgtt acacacacac ac 224920DNAArtificialforward SSR primer BNL3992 49cagaagagga ggaggtggag 205020DNAArtificialreverse SSR primer BNL3992 50tgccaatgat ggaaaactca 205117DNAArtificialforward SSR primer CIR280 51actgcgttca ttacacc 175216DNAArtificialreverse SSR primer CIR280 52gcttcaccca ttcatc 1653165250DNAGossypium hirsutummisc_feature(1104)..(1379)Putative microsatellite region 53caaggaacga gttgatgaac aacgattagc ctgcatccca ggaacctctg ttccatcgaa 60gaccggttct tgttcctcct cagcaagatc ttctcttgta gtttcgttca cttcttcttg 120agctttggaa tccaaatccc catccttatt catcctgtca ccttgttcag catgtttgtc 180atctttgtct tctttattac cctcctctaa cttattaaca tcgtccttgc tgatgttcag 240actatgcatc tcctccacct ccacaggttt tccctgatct tctttgtgaa taccatcagt 300atgtccctct ggtttttcat ccatgggagg atctgttata tttttgtaac tggagttacc 360ctcttcccta atgtcagaaa aacataaata ttgtcattca acttgtgaaa acatggccct 420taaacttttt catcgaggat ctcttcccag gttcctgcaa ggacatttca tatagacaaa 480ttcaacagaa gaccaattcc actacggatg aaaaatgaat ataagactct tatacttcca 540taaacagagt catatttgaa agcagacatc catttcaaaa ctgcctaaca aacaatagga 600tctccatttt tcactctttg gctatttaaa caggaaaaat gtaacaaaaa agggaacaaa 660ataatctgtt gtatataggc tcttattcat tgattttcag aaatatgaac ataatccagt 720atattaaacc aaaacactag atgaaatcca taataagaaa ttgtttcaaa gcactagatg 780tgatccggca agtcaagccc tgaagcttta gttttcgacc attcctagtc aaaagaaccg 840gtcaacctaa atcctccatg atggaagaag tagtaatatt atgaacaatg aaagtcaaag 900aaaaaaatag cagagaaatg aatctgtgac caaaattaaa taacaaaaaa gattaaatct 960ttaagtcctt aacacttgat caagttttca tttaattacc acacaactta atctccaaaa 1020agaaataata ataataaacc ctgaaatttt atcgatagga tttttgcaag ttaatcataa 1080cgggaagtat gaaaactgaa aataagagag agagagagct cttacagatc cagagagagt 1140gaagaaaaaa cagtgaatat aaaagaaaag aaaaaaagat ctggagagag ttacagaacc 1200aaaaaagaaa gatttaagtt aatatatata tcaactttgg agttacaaat ttggtgtatt 1260aatgttttga aaggaaactt ataattgaaa attgaaaagg aaagaatagt caattaatgg 1320agggtggatg attttcagaa gcagaggcgg gcggtcggga caagcgggaa atccgtgggc 1380ctatcttttt tttgtctttt tagacttaag gaccccctta tcctatttct ttattttgga 1440cacccaatac ttttgatttc tttccaatcc atcccttatt gtttccattt tttaaaattg 1500aggaagaatt aagatgacta aattgctaaa tcgaatataa tttaattcga aggactcaag 1560ggaagccaag gtccttgctt gactctctca agtctaagct atatataaga ttagaaaact 1620aattttactt ttcgattact taactttaaa attttataaa attaaaccat ttaaaaattt 1680tcgttcaaat cactagactg ttaaaatggt cactatatag ctttctttat tcacattgct 1740tgcataattg taaactctca ttaatattct cttttacagt ttagtttttt acagaccaaa 1800tttcaagtag cttttttctt ctatatctta aattaattgt cttcttctac ttgccgaaga 1860gtattgatcc actataccaa tcatcaaatt gtagcttgaa gttcgctagc tgaacttaaa 1920aaaaaaacct taatagctca gtaacttgaa taaaaatttt cgaatagttg agtgacgtaa 1980atgagaagtt ttgaatagct caataaccat tttaaaaaat tttaaattta agtaaccaaa 2040atgtaaattt actaatagtt tagcgacagt gggtataatt tacccttcat tcaattgcgc 2100agacataata aaatttattt aatgtaatag gatcctaaat atgaaattgt acatatatta 2160aatgttttaa taaaaaattc aaatgtaaaa cactatcaat ggatttagta aactataaaa 2220tactaatact aatcgttatt caactcgttg aaaactacag cacaactttt gattttgagt 2280tgttgcgtat tttactgctt tcatgttgtg gaagctttca aggacaacga cacatatcat 2340gcaaacgttt taagttatta acacgtcttc tttggattac acgaacagat tggatttacc 2400aagcaatatg aagctgtaga ttgtaaatat cttatcaaac acgttgactg caatggctgc 2460atgacatgtt cctatccgtt ttccaggcat tgcatgtgac gattacatga aaaagcagac 2520tgcttaacat tggcccttga agaggtatcc aatgcagtaa tgttataaac gaaggagaac 2580atatacttga tttctactcg tatataattc aaaattcgta cgaaaacgaa ggaagatgca 2640cgggttcgcc caggggaaaa gttatttaac atgacaaata agagcagcac agaaaataag 2700gataagtaag acatcatctt tctttttctt ttttttaaag tttgtgagta tgaactagat 2760ttacatggtg tttacaaaga acataactac ctatatgtac ctgttgccgg agtgatattc 2820agtgctcagc acagcatgat agaaaagctt gtgacatttt ttgaagctcg gaaggaaaca 2880gcaccggcat gcctttctta atacaatagg ggcaggcaat ggcgtaactg cataacagca 2940tagcgatttt cgttttaact gttatttttt ccttgtccat cgcatatatg atacatacac 3000gctgcagtga aaagaaataa ttttgacaat tgcaactcca tgcctaagac aattaggtca 3060ccactaatat ttccgatgaa ggaccttcat ccaattcaat atctccgcat gcactaagaa 3120ccgtagcgag gatgtaatca tccacctcaa aaccttctgc ttccatccga tacatgagtt 3180gcaaagcttc tcgacatagc ccgtttcttg cataacccat gatcatggcc ttccacgaaa 3240ccaaattcct ttctggcatg ctgtcaaaaa cacgagaagc ctccgctaca aaaccgcatt 3300tcgcatacat atggatcaat gcactgccca cgaagacatt agaaaaggca ggagttttat 3360ttgcgaagga gtgaattaac ttcccttttg taacagctcc aagcttagca catgctttca 3420aagctgaaga ataggtaaaa gagttaggtt ctacaccctc ctccatcatt tctttcaaaa 3480aatcaagagc ctcagcctca tgccctacgc ttgcacagcc agaaatcatg gcagtccacg 3540agacaacatc cctcagcggc atctgttgaa ggactttgga ggcaacatcg tactccccac 3600atttacaata gaaccatact agagtgcttc ctatgtacat attcctttgg atagattttt 3660ttactatttg tgcgtggact tccttgccca taagtaaatc cacaactgaa ccacaagccc 3720taagtatgct tacgatggtc aagttattag caataatatt tcgactcttc attactcgaa 3780aaagactaat ggcatcctca ccaagaccct tcctagcata ccctgctata atagaagtcc 3840aggtaaccgt atttctacta ctcatcccgt taaacacaat cctagcatct actacctccc 3900cgcattttgc atacatgtcc acaagagaag accctaagaa aacatcattt ttgaacatct 3960tttttattat ggcaccatgt aattgtctac ctggtctcaa tgccttttgc tctccacaag 4020ccttcaaaac actgcaaacg gtgaactcat taggccaaaa accatcactt agcattctcg 4080aaaacaacga gaaagcctcc tccgcatatc cttgttggga gcaagcagtt atcatagctg 4140tccaacaaac cacatccttt tctgccatcc catgaaacac ttgaaaagcc cttgacaact 4200ccccacattg tgcataaaaa taagtaacag cactatccac aatcaagttc ctacaattcg 4260ctttcaggaa acacccatga atttgtctgc ctaactcaaa atccgcccgc ctactacaca 4320aattcatcaa acaaacaagc atcttcctgt tcccttgaac cccacatgat atcgaatccc 4380aaaacaacct caaagcttca tcatcaaaac ccaatttgga gtacccatta atcattgctg 4440tccaactgac gacatttctt tcagccatat tatcaaacac ctttcgagct tccactagct 4500tcccaaattt taaatacgaa cttatcaaat tattctcaac ataggtcact gggttcccta 4560aacgcttcaa aacaaccgca tggactctcc taacttgtct gccattgcta caagattgaa 4620gcaaagctgc cagctcatca gaaccgacat ttcgactaac caacggtcgg gttctatccc 4680taaaaccagc atctgggtct tcatggtaag ttgaaatact ggttaaatca tcgaattcgg 4740gtagaaaaca tgaatctttg gaagaaaaac aagaaaattg gggtgggttt ttggtgattc 4800ttggtctgga ctcgtttttg gaattcgaat aatgaaatga tggactttga atagtgaaaa 4860aggaaggcca ttgaaagtgc gccacttggg gtgaaatcac cgttaacgaa agcatcagct 4920atatctttgg atttcttcaa aactcccggc ggaaaacaga acccagtttc ccgaagttta 4980agctcttgtt tttgttaaat cccgaggctt tcaaaaactc catcggataa aagaccattt 5040tgccactaag ccagcaaaac gacatcgtct tgcaggttgc aacctctgca caatatcact 5100tctttgccgg tcgctaaaac cttattctag aaccccctgc caaaaacctt cttaaattgg 5160ttcctgctat aaacttacaa agttaaaaat ttaatctttt tctttccatg gaattggctt 5220gtgcaagttt tcaagtttgt tctatatttc cagtaagatt aaaatcttct aaagccacaa 5280aatttgaatc ttctttagct ttacttcctt cctgtaaaag ttcaaattca cctgggattc 5340gttgtttgtc ttcaaagttc ttaagtaagt tactcttttt tacctaataa tttgcagcta 5400aaggttatta atttagcgtt ttattcactg tatttatctc tctttcccgc gctgccttcc 5460tgcgtaaata atggttcaac ctggaattga atacgttaat aataattaag tgctatagct 5520gaagtattct atgatgatta gctttatctt ttctttttct tttttttttt tgttttaagt 5580tcttttcgtt tgaagtaaga aaattgggaa cttcttctag catgattatt tgggaaagca 5640ttgtttgtta ggagcttctt tggtgcttgc agagatgcat atattatttt taggccattt 5700gtctttaaca gatagttctc atgggcaggt tattctgcat tgggtgttaa tcgggtaagc 5760gggcagcagc ggttttctct tgcggctgtt gttggtgata aaactgcggt gccaaataat 5820tgtgatgaag agaagatttc agattcagat tctgccggtt cctcggtaat taatgatgag 5880gtgaccggag atggggaaaa tgatggtgat aaaggtaatg ttgaggggtt ggatagcggt 5940aaaatgatca gagtgtgtga caagttaatt gaggttttct tggttgacaa gcctacgcca 6000actgattgga gaagattact tgctttcagt aaggaatgga acaacatccg acctcatttc 6060tttcagcgtt gtcaggaacg agctgatgtt gaaggtgatc ctggaatgaa gcataagctt 6120cttcgacttg gaaggaaatt gaaagaggta tgttatttga ttataattat aatcttttgc 6180aatcaatatc agttttttgt atgagatatt tgaatctgga gaattgtcta gctgctcatg 6240attaattcta gattgattat aaagagagta cgaacaaggg agagaaatct acctgctttt 6300atgatgctga catgctgaac agatccatgt gcagtgaaga ttcctaggaa tcgaatttct 6360tattctcttt ggatggcatg ttagtcatat ggctttaata gttgaggcat gtctgatgca 6420tactgttctt attttcaagt tcatgctgat gtcatggtga ttcttgttat acagattgat 6480gacgatgttc aaagacacaa cgaacttctt gaagtgatca agggttcacc atctgagatt 6540agtgaaattg ttgctagacg tcgtaaagat tttacaaaag aattctttgt gcatatccat 6600actgtagcag aatcatatta tgacaatcca actgaacaaa atggtaaggc agtatgattg 6660aataatttaa ttatcaaaac cttattgata attattagta tgaatgttta gtatcgtgtg 6720ccagtttaaa ctatatgcct aacaaaataa gctgtgtggt gagttgcatg gcttgagatc 6780agaactttta gttcatatac ttgttaacca tatgccttcc cgaccttatt ccttcattta 6840tttgtttata cttgacagct ctgtcaaagc ttgggaatac ttgcttggct gctgtacaag 6900cttatgatac tgctgctgaa aacgttgagg cacttaatgc agcagagttg aaattccaag 6960atatcatcaa ttcaccctct ctagatgttg cttgccggaa gattgatagt ttggctgaga 7020aaaaccaact tgactcagca ttggtgctaa tgatcactaa agcttggtca gctgccaagg 7080aatctaacat gacgaaagat gaggtactgt tgctgacttc agggaggaaa aagccctcca 7140cctgctctat aaatcgtatg atgcgatggt caactagatg tagttcttgt tttgtgactc 7200agggtgactg cacctagaag aaccctatga tgatattctt ataaattaat ggaatgatta 7260ctaggttggg gatgaaggga aaagggatta ttcattttgg cattctagta aagagcggaa 7320ctaattttag aagtacaacc atatagtcat ttgtggggaa gccaactgat gtgttccttt 7380tgataagcat taaaattttc agtaggattg ctaatttaag catcatcagc cgatggtttt 7440agaaaaaaaa ttaactttac gataaattcc tatgttttgg agtgctgctg taagatgtct 7500taccaagtta tcatctcaat tgcaggtaaa agatatattg taccacttgt atatgactgc 7560tagaggtaat ctacagaggc tccttccaaa agagattaga attgtgaagt accttcttac 7620aattgaggat cctgaggagc gactgtgtgc cctaaacgat gccttttcac ctggagaaga 7680acttgaaggg agcgatatgg acaaccttta cacgtatgtc ttttgctctg acttgatttt 7740tatcagaatt tagtaccatc taggaattag gcttacaatg cattggctta atctttcaag 7800tcaatcgtcc agccttccgg gtcctgcttt ttatagtttc aggtgcaaat gatgagttag 7860gttggtagtt atcccaggaa aggatttgat agttactctg gtgtcctgct atttgcctaa 7920accaaacatc gtttaaattt tgtgtcatcc tcttttgcgc gtaagtttgt tccttcattt 7980aactcattgt aatgaaattt taggactccg gagaagcttc ataccatgat gagagctgtg 8040gtggatgctt ataatttcag ccatgaaggc actctcttaa gggaagctag agatttgatg 8100aatccgaaga taattgaaaa gctggaggag ttgataaaga ttgtggagaa aaacttcatg 8160tgacgtggag ctaagtcgta ctttgaatag ctttacgtat atttcttggt ccagaaatca 8220taatctttaa tctctacctt gattgagaat ctgaatatat ataggtgtga taactctaaa 8280tttcgggttg gttcatagct caagcagtaa tactgcccgt acccgaaagc atcaaagtag 8340agttacttca caacaaaaat tggcaataga agaagaatag tctttaggtg tgaatgttca 8400aacatacaag gtgccaatga tctcctgcag agattgatct tgttgtggtc ccataatttt 8460cttttctttt tcttaatatt agaacttgtt aatcagatgt tacatgaatt actggcaaat 8520ataccttgat ttcatattaa atattaattt aacaatattt caataaaatt aattgccata 8580atgcgatttc atttttttct aataaagtaa cacaaaatta taaaggttca aagttctctg 8640taaagagcat ttatgaacta aattagaagc cattaactct tgtgacttgt cccacatgtt 8700ataagtttag gtaaaaactg tggtcagctc ttaattctta agaaacttag gcttgaatta 8760ttgaagttga acaccaaaaa aaaaggtctt agaatctgta aatgattact tcttcctctt 8820ctttggtggc tgaatcgatt cttcttcatc ttcgtcttca ttttcaggct cttcatcttc 8880tacagtttct ttttcttcct catcatcatt gccttcattg tcgtcaccat caccatcgtc 8940ctcatcctcg tcttccggct catcatccac attttcacct tcttcttcct ctccgttctc 9000ttcttcccct gctccatcag cctctccacc accaatttcc ttgttggagt tgccattttg 9060gtcattggaa ttgttcccat gatcttcacc atcagaaggg tctccttctt cattgtcttc 9120tccgggtcca tcattatcaa tgtcttcatc ttcgtcttca tctccatctt cctcagcatc 9180aactatcgct tcatatctat gtccacccaa atcactttct tgcttttcac cttctgggtt 9240taccatcaac agttcaacca gattctgaaa ctattacaac aatctaagct aattatcacg 9300tatgcttgac aattgcacca tgaaaatata ttgaaaccaa caaattacaa accccacaaa 9360acgaaatact cccattgtaa aatgactaaa acgggaatta gcttttcacc catggtcatg 9420cagaacccaa cccaagtttc ttcttacatg catattaatt tatgacaaat tattgaatct 9480actacattga aatatgaaac tccacaaaca gaaagatgaa tacttctcaa tatacaataa 9540caaatagtag attagaaaat taaaaccact gaatactaat ttttaaatta attctataaa 9600gcaattgaat tcaatgtgaa gttaacattt tctatataca cgaataaaat attagtaaaa 9660aagatgtcgt ttttggcgtc aaaatgattt tagttgcttt aaatagaaat tgggagtttt 9720tttcttatat cggtactcga ctaaccaatt tttcttacat ttaatcttca acttagcaat 9780ttttcttgaa attcttttca tattagttta aaatttttgt aacttttttt actttagtct 9840ctaaatttag attacgaaaa aatttcagtt tcaataaaag attaggggtc aattttttta 9900aaaaaaatct tataaatgct agattttaat aggtcgctgt tgttatttat taataaaaaa 9960attataattt aaactttttt tataaatttt gtttataatt tttatatttt taattttttt 10020taattttaaa agggttcaat tgatttttta aaaaattgtt actaagattt ttttttatcc 10080tctggcatta ttagtttcga aacattctaa tatacttcaa ataaaagagt atggatcaaa 10140ttgaataaat gtgtaaaggt tgagggctaa atttactatt atacctaaac actaaaacaa 10200acatttaaca gtacaaaaat tttaacgggt ggattgtttt cctcactcat ctaacataca 10260atagctaatt tgctcatttt ttaatagaga cgctaaaatg caatcattat atagtacagg 10320gagttttgtg ttacttttac aaattgaaaa attactatga tagcaattag accctcacgt 10380aattataagg aattctagtt ccctatacat gtttggctca aagagtataa ttatgtcata 10440attctctata tcatgtttgg taatataaat tataattaca aatttacata attttaaatt 10500ctaaatgaaa aaagaatatt agttattatc aaaattatga ataatactta agaaaaaaaa 10560ctactataca aataacaaaa cttaaaatca aatcataata tataatatgt tattccaaga 10620aaatatttca taatacaatt attaataaaa ttaataataa taattaaaca ttctacacaa 10680atttatctaa ttactctaga atttcatatt tgttgagcta tactttgaca ctaagcatat 10740acttgttata atcaaatggt ctttgcccaa atattaataa ttaataataa atattatgca 10800attagttaaa attagtataa atattttata ataaatacta tgtaatttag ttaattattt 10860agtatatcaa aatatatgat aaattatctt ttgtcaataa atttttaaaa ttattaacaa 10920gattagaaaa ataacatata attatagata ttttttaaaa ttaaaatact attattatat 10980aaaaagtaac tttttaaact taaaaaatat aggacataat tgaaaagtta taaaataatt 11040agattatagt atggttgttt gtaattaccc gcataagtac tccaatactc cccttttctt 11100aagaattgga gtgcatgatt gaggtgttct agttacattt aatttagtaa tggttatcta 11160aacataccac acatgcgtaa ttacaaataa ttaaactcaa actgaatcac taggcaattt 11220ttcaaaggca aatctataaa taaaccctta ataaataagg attaatatat ttttaatgaa 11280agcctttatg acttacaaac catttctagc cataggttta attttgtttt agaatggaag 11340gttaggattt agctttttaa ggtttaaagt tggtatagac ttttaggatt caagatttat 11400agtttataat tttataaaaa tataaattta ttttttagat ttaatattta gtatttaaaa 11460tttaagggtt atcctagtca aggtttaaat tttatttttt aaaaatatat taataattac 11520taattataca aatttaaaat aagaaataat atagtaactc actgtccgtg agtccaatag 11580gaattgagga actgtgagca tggtactacc cattttttat tgggggaggt ttgaacgctt 11640tatttaatat ttatatatat tggtgggccg acaaggaatc aaatccccgg aactcaacat 11700aattttacgt aaaacagaat ctatgttttc tatttcagtt ccgcacattg tgcgcttcac 11760ccacaccagc agcgcaagtt agcaatcacc aaaaataaaa aataaaaata gaaacctaaa 11820aagaaccaac ccttacttga ggtggaaaag caagagcttg aaacgtagct gaggaggcga 11880gtagggtgag tacggttccc caccacgcgc acattagcaa accatggtta aacttaacca 11940tcaattcgct ataaccttct ttggtctcca tttgtacggc acaagggaag aaaaaattaa 12000acctagggac aaaaaacaat aacatcaatc aaccatacta ccgttagatc tgatacctgt 12060catctcagcc gtcagatcta cagagattgg ttcgaaatta tgtaaataag acaagagata 12120gaggatagca tattaaacag ttaacgttaa ttaaaagaaa acgacaaaaa aaaagcttca 12180tacctgagaa attctaggga gctcggaaaa attttccagg aaatgggagg aaaatgtaag 12240aaagaaaaca agacaaagtt aagagtggga aggggcggtt gaaagtatag gaaaggaaaa 12300gtttctaaaa agagaaagac acttgtttgt ttagatccaa cacttgtttt ttgctacaaa 12360aacattagtg ttttttgagt caatgtttaa gggtgatgaa acacatggga gaaaaaaaaa 12420ggagaaaaaa taccttcaaa attcaaaaca ggagaaatga acaagttttt gttcatttat 12480tactattttc tttgggtggt tcgtattttg gaattggatt ccattaatag tctattattg 12540atgccttttt catcctattt ggggctgctt cttttttttt tcttttttga gaatattgta 12600gtaatagtta agttttttat tcatttattt taagtaattt atgaagaaaa ggtaaattaa 12660ttattatata aattcattta tttgtttctc atgatggaga attgattcaa gttgtggtgg 12720ccgctttgaa gaacaagtcg aaatcctcgt gaagagctgc ggtatccatt ttaattcccc 12780gaacataaaa tgctgagctt ctccatgtga cacacaggca gcgcaaaatt acgatacttt 12840cattgctcag ttaacatagg ccggtgcaga attgaaatgt ggtacgggag ccgttgggct 12900gggcccaata gctaagttgt cggaccagaa ttatgattta cggaatgcca tcgagcgtgc 12960tttcgaacat gttttagttt ttactgctaa catagccgac aagtctgctc catactctct 13020ctttaaaact gaagtaccat atgaaataaa tgatccatcc gcattatcat attaaaattt 13080attttaataa aacaaacaag aaataagaaa cgaaatcata ctaattatca cccaaaaatg 13140gaccttaaat ccataaagtt tgatcatatc aatacagcat gagatattca aaatcactga 13200acatgattac atgcagctat gaactacaat ggccgccccc ctaagcttcc cctaaccctg 13260ctgttagact tccttattta ctccaatcta tagctttgcc tttgtaattc tgcacaagtt 13320tttaactgta aagatcaccc taccccctaa gccaataaaa attattctct tgcctttctt 13380ttctttgctt tgctcccccc ccctttcaat gataaaagtg ccccactttt cttggttatt 13440gatggaattt cagcttagaa ttcatggctt tacagccttt gctggaccat ggcaggtggg 13500caatgacagc ataactcccc aaaccataaa atcttggaca atggtccctc tccaacttag 13560accataatca atattcagac aagtgttaaa ccgtaatcaa cattaagtac aataaaaaga 13620atcaaaacta atccttatag tttaattaat gccactaatt ttttttccct tcctttaatt 13680gtgtgtacct aaaacagctg gcgaactttg aagggtattg tcaggcatga aagaacattc 13740atggaggcca tttatcacat aacagctcaa gaaaaggaca aaatccaaat aaagataata 13800taaatggaaa tgggtgtcca ttaaagtcaa tgggctgttt caatgatcta accgttccca 13860tttcatgatc tgtagaagaa tggctttgcc tgacaaaagg gtttactttt ccacaaaagg 13920gacttagttg agttggtgaa ttgcatgcat tcaacatact ttaaacatca aaacgaaaga 13980ttgcagctat actatagctg tgtatatcaa ttagttatct atcaatgcac atgattcact 14040cgaactggat taaatttgaa tcaatttatt taacaaaatg aactttgaaa attggtattc 14100tttggaatta gttcaacttg attcatagta ttattagatt tacaacatgt ggcatgctcc 14160cacttggcac atgatgagct aaggtcctta atcaaattgg caatgcaaaa aggaaagaaa 14220agaaaagaaa ttctactaaa tcaaaaccat aatcatgaaa agagagagta tgccccctaa 14280atagctttca attgattcct ttatttttat ttttatcatt ctaaagccta aggcctagaa 14340agcactagta ctagcattac attactataa atgctaagtt gatgtggttt tgaggaagat 14400agtaagttta tttatttata gaactttaaa atgctccctc

ttttagggga tgagaaggag 14460ctaaaagcta ctctatagtg ggaaggaatc ccaaagtgga gttttttatt tattaaaata 14520attaactttg gatagttgga tgaaaaggct aagggaagca aagcaatctc ttccccattt 14580tttctcacca tcaatgttag tcaaagaggg taactaggct ataggatcta tctccctaaa 14640cgaatttgag cctctattgt ccttatatat atatataaat ttcgggtcca cgttgatacg 14700ggagaatggg gagggaatag tgaaatctat atattcgtag ctttgagttg atcggattgt 14760tagatattaa tttaaatttt agttatcaat attaaattag gattttagtg tgcttttatt 14820caagattatt taattgttaa atttatttaa tgaattatat ttaatagctt ggaaattttg 14880gtgaataaaa tttcttcatg aataaagttt tttggtgacc aaggaatggt ttagcacttt 14940agcggttaga attaagaagt taataaagtg gtattttatt gttttgagat tgcgtatttg 15000tatttccatt tcgtttacca taatttttac ttcattttaa gtaaactgtt tatacatgga 15060cggtaatttc cacatgtgta gtatatttca agtttattaa tattaaaaaa aatttaacca 15120caaattgatt aactaatctt ttttttaaaa gaaaactaat acgatctttc taactttcca 15180atttagaata tggtaagcag agattaacaa atatttttta acattaaatt atttttttct 15240ttttatttac ctcataaccc aaaaattaat catgtaaaac acaattcaat cgaaacccca 15300agattttcac tttggaaact acttttttga aactcgtctt ttgaatccac cattcctttt 15360caaaaacgtt aatttttagt cgagatttct cattttagct ttcacataaa atccaaattc 15420aaaacccaat taaagtggac gaaaaatgtt ggtgtgttgg ggataaaaaa atttataaaa 15480aattaatgaa agtttccagg cgtgtatcaa tatcaatttc tttaaaattt tatttatctt 15540cacctatatt atgatgtaca aaagagttac tgataaataa acctcgagga tacaataaaa 15600ctcaatgatt gtctatgttt tacactaacg gaaacagtcc actgagcact aagcggagct 15660taataaggat atcaatttct ataaatattt tttttcttta tagaacaacc taaaaatata 15720aaagatgccg agataataga aataaatttt attttatatg ttttttgagt gtcatacaaa 15780taaattttta ctcttattta tcgaatatct catgtctctt tatagtgaca taactactca 15840aacggataac aatatattta gcaataaata taattattta atagatatct acttaaataa 15900ttacgattaa ttgtaataat taattgatat aacttttgaa tttagaagat ataaagatta 15960ataaaatagt actataagaa aagaatattc catcagccgt tggcccatgt atactcttaa 16020cctagactgg aagagaataa cttacaaaaa aaaaatcata aattctttca aagagaaaat 16080atcatgagaa ttcgtaggtt attacaaata tacaacaggg aagaatacta aatcatgaaa 16140aagaaataat aatgattaat aaaattatta aagtcctgtt tgatgaatct tatttactta 16200tttatttaaa tttaaaaatt ttaaaaaata taaatattat atcaatacat taattattaa 16260aatatatgaa acaacagtta taaacatgtt aatattggta taaatatata ctagaaattt 16320aggtgcagtt tcaattttac aagtaaaatt aagaggtttt tatgttttat gtgtatttaa 16380ttggtattta aaaaatgagt aattatatca ataagtgtta ggtgcagtaa taagacatat 16440tacacgttta aagagagatg tatgttcaaa ccttggcgat aactttgttc gaaggaggag 16500ctacaaatcc caaatataaa ccgtaaaata aaaatcgaga ataccaaaaa aagaaaaggg 16560gtaatcattt tgtccaacac ctgtgggtaa aagaaaaccc acaagctcat ttatgatggt 16620gtcaagtgta ctttttaatt attttattgt tgttttatta caccctaaaa acaaatggtt 16680tcatcctaaa cttgcttatt gaattttttt tttacttcac caattgtgta tgaaataata 16740atccttaaga aaaatttaga actaaataaa taagagacat aatgattttt taatccatgg 16800tgttaaaaaa gttccaccac tgagaatata ctaaataata atatattatt tggataacca 16860ttctatacat actagattat gtcacaccta aaaaatttta tgtaaaaaaa tatttttctt 16920ttgaaattaa ttcattaatg attataggtg gaatggtcaa taatttatat aaaaatcaca 16980ttaaatatgt gttcgattct tggatatatg tgatttttag ttgtttcatt taaagattat 17040ttaaaagaaa tcaataaata ttaggcaaaa gggtctgaaa aatccttagt aaaaaaataa 17100aagaaatcaa ttgagctctt agaaaaaaaa tgcactcaaa ccctcaaaga aaaaaaaatc 17160aattacactc ttccattaat agaatggaac catcgttaac cagattgatc attaacatga 17220catgattgat agaagcaacg agaaattgac acgtgacatt ttatgtggat aaatatgacg 17280tcaacatatc tatggtatat atacaatagc agttagtaaa tcagttgata tcaacattaa 17340caattttata ttagttgatt caactaattc aataacaaaa ataactatct tcttaacata 17400ttaactatta actatttaac tgtcataact gttaagctga tttattccac aatagttact 17460catccggttt atatgatcaa agtgttgaat aacgacttca taaatttttg ccatcccaaa 17520ttactattta ataatatttt ttggtgacag aactatttat attagtgtaa atttaaaata 17580attttacatc attctgtaac tttttataca attaatattt taataatcaa atcgttgaat 17640ttttcaagat atatgtactt gttatacata tacattttta agtcaattcg atattctatt 17700atatcaattt gaaatattgc ttgtattact atatatttac cggttgcttt ttttatacat 17760aaaacgaata attagaaatt tttaatttat gaaaaatttg gcatgcatga tatatacgta 17820aatagaacat gaaatatgat agttaaatta ttaaaatatt aatcatataa aaactataca 17880aatatttaaa aatactttaa ttttacatag gtgtaaatta acgaattcct ccccacaaaa 17940ttttatttaa aattaattat aaaattatat aaatggtaaa atatatttat taatatttat 18000gattttaata gtttcaaact taaaaaaaaa tccaaacttg accccaaaca aatatggaat 18060aaaaaaattt agatcactat tttgaaaatg tttggtggta aaaaaaaaat taaggatata 18120tttgaatgag tagtaagatt gatttagata agactaaaaa tagtcttaga gagatttatg 18180gtatcaacca cattgaatat ggcaatggtt aaattaaaat tatttgtaag atatgtgttc 18240gaatttaaat atactagtat taatgtacag agagataata aaaaaaaatc atcttaattt 18300aaaacataaa cagtaaaaga aacatttatc aaatgaataa ataattataa ttgatgtagt 18360tatatttatg tcaaatagta gtttaattaa taaataatta aaagtagcat aaaaaagtaa 18420aagtattaat aaaaatatgg ttaaggcctt ttgttgttgt tgacacttta gtgtctcgtg 18480ttcaagcttt gttgatataa ttccccctta atttataatt cgattcagta aaaaaatatt 18540ataatatatc aaataatatt tatttattta tttattactc tagtagttat taaaatattt 18600tatttaaatg aaaaattaag tgatcaatat gtttttcatc tatatcaatt ttcttataaa 18660tttaaaaagt tagttcaaaa taatattgaa ccattatttt atttaaaata attacatgaa 18720tttacttttt aaatattatt tttattagaa aaatgcaaat gatataatta taaatgaaag 18780gagaggaacc cgttgtaaaa ctaaagtggt gtttcattat tttataattt tttatacgat 18840taatatttta acaaattgac cgttaaattt atcaatatat atttaccctt catgcatgta 18900attttttgaa tcgatccaat atatttatta tattaattta aaatattatc tatattaata 18960tatatattaa cagttgaaac ttttttatat aacacgaata gttaaacatt tttatttttt 19020tctcaaaact taacaaacat gatttacatg aaaaaaacat attattaaaa tattaattgt 19080ataaaaatta taaaaattaa ttttatacca atataaatag attctcccct acataaaata 19140tttttttaaa tatgctgaaa aaccttgtat aaaaaaatcc ttttaaacat aaaaagaatt 19200aagataaaaa gtacaatcga atatcttttt aagaaaaaaa atataaaaaa gtattagtgt 19260tgcggtgagg tgactacaca ttcccttcca tgtgaaaagt ttttttacgt agcttttatt 19320acattatata ttttttgaat attaatttta ttataaaatt ttgtcattta ttttttataa 19380actatttata ttttattaat aatagttaaa taaaaagata atgcgcttca acacatttga 19440aactcatcta tactaacaac aattctaatg tcgatcaaac taaaactaaa ttaattacaa 19500tattacatga tatatatata tatatagaac gcattagaaa caatcactga tcattgtcat 19560taatttgttt agagggcatt ggaccatcca gttgtatcca tctaacatca ttaagaagaa 19620aatgaaacat tccctcccta tttattacgc catttcctgg ctatttttaa tacccctttt 19680aatttaatat ctataattct aaacttaaaa cactttttct attttcatgt cccattataa 19740tctacaacta ttatattagg caatgcatat aagtataact atctcagact tttcttttga 19800caacaataat aatatccttt gaacaacata aatacaccaa caaaaaaggg ggaaaaccat 19860gaaatatgaa aactaaaaaa gatataaaaa agaccaacat cctttgaaca agtgaacatg 19920tcccattccc caaaaaccca cgaccccaat ttcttttaat ttattaatta aaacacagcc 19980agaataaaaa gtaagagtac tggtcccacc cgcacctcct atcaacaaat taaaaattaa 20040aagacaacat tcatatcatt aagaaaatta agtatggatt gtaaagaaaa ttatgataag 20100ggaatatgta tatataaata tcatccactt cagcattcta ttgcctttga ttaacaaata 20160ggaagggttt aatcattagg gatttgtttg gtcaaatgta accacaaatt tgggtcccaa 20220aacattgtat aatccataat caatgtaaac aattccacat cttttttttc ccttttgttg 20280tttaagatcc accgtttgat gggtccttaa gctcgtgaaa gcaacggttt tgattaattt 20340aagacgcatc gaattgaaaa ttgataatat catccatttt gtatgaaatt tattgctggc 20400aggcaaagga agagtacttt gattcaagca tctggaattc taaaatgaag gaagaaataa 20460ttgcaagtta aaaaaggaga aacgtagctg aatctcagtt gttaatgcct aacttgatta 20520attctaagca aagcattttt tttaaaaaat tattctttaa attacaaaaa aaaaaaaaac 20580taacttgtca tcttctctct cattctcttt atttataaat aaaaatatat aaacttcagc 20640tacacagaat ctgggggtga aaacaaaggg aagaaatcag aagttcacga aaatttcatt 20700tctttaagaa aggcgttaag ccccatcttt tctttctctt tcttttttct tgaactgttt 20760tccagattgg taattttctt tttctttttt aaatatatat tttttcattt ctgccaatta 20820aacaatgaaa atggcatatt accaatcatc ctttcccctc tataggaaac acccatcatt 20880gctgccaaca atccttttca tttcaacctt agaaacctga gcttctaaga tattccttcc 20940ccttcctttc ctttctattt cttcttctct ccctctcttt caaccttctt ctccactttc 21000ctttgttgct ttttcaacaa tggatgcatc ttctacaagc tcagtcaatg ggttctatac 21060cttcttgact cgtggcatag atgatcttga acgtgtttat ctctctaata acttcatgtc 21120catccaattc cttcaaaggg ttctttccct tctccgatct ttccactccc agctactcct 21180cctcatccaa aaactccacc ttcccgtcgg tgataagtgg ctcgatgaat acatggatga 21240aagttcgaag ctctgggaag cttgtcatgt tatcaaatca ggcatctccg gcatcgaaaa 21300ttattactcg gctggcttta atatcatttc ttcttttgat aatcatcgcc atcttactca 21360ccagctttct agacaggtcc ttctttaaat tcccaaacct tacaacaatt tcgttatatg 21420tatattcttt ctcgagtttt tagcattgaa ttcatgatct gatatggctt tttatttttt 21480ctaaaaaact atatggaaac aggtaatccg ggcaatttcg gcgtgccgca gggaagctgt 21540gggattggaa gaagaaaaca gggcgttgat ggaaacgaga atccaaccgc tttcgttaag 21600gtttgacgag aaagtttcga tcgaatcaaa gctgaacgga ttcaatggtt tccgaggagt 21660tttatacgca atgaggaatg taagctcgtt gctcctaatg atcttgctgt acggattagt 21720ttattgtcga acggaatcca gtttcctacg aggaggatat gaagggtgtc taattttcgg 21780atcagctttc atgatctcaa cagggagatt gcagcaaaga gtggcggcag agatcaacca 21840aatgaatggg aggccgggga tattgcttta tgagttcagg agatcgaagt tggcaatgga 21900ggagctgaga ggggagctgg agcggagggg cggcggaggg gtggaggagt gggaaacgga 21960ggtagggggg ataagggaaa gggttgagaa cttgaaaggg tggtttgggg tgttgagatc 22020tggtgctgac aacattgttg tgcaacttga tgatttcttt gatgagattg ttgaagggag 22080gaagaagctt ttggactttt gcagtcatag gtagagaaga aaagaagatt aaattataaa 22140aaaaaaatgt aggatggttg aaaaaaaaag ttacaaaaat acttgataga agaaggttgt 22200tgttgtacct tttgaccctt ctcttctttt ctttttcttt tttcagaaaa aaaaagattc 22260tctttttatt tttcacttca tgcatttgtt gttgtttcta ccgcagttga aacatggaaa 22320tagtggtttg ttttccccaa aaatccagaa ggaaaatata tgcaggggaa ggggaaacag 22380agtaattcag gggtctcaac tatcttaaca atgtaaacag ttaaaataaa aaaaaataaa 22440ataatggtta ggactatttt tcatgcaggg aaggggatgg aatcttctat atatataata 22500ataactagaa agtgtctatg ttagagctac atatataaat attttgtagg ttattaatta 22560attttatatg tgtttttgaa agtgacttcc ctcttgacct ggtaatttat atatacagtt 22620gtcaatttta gagatgaaaa gagaggaaaa atcaggggta gaggatgctt taagatgata 22680tattagaggg ggagggagtg cacgagagag gttttcctgg gtagctttaa atgtttaaag 22740cacaagtaat gggttcgttg ccgctgggga tgcgtctgat ttagggtttt tcaactttgg 22800aagcaatgga gtttttaatg atgaaagctt caaagttcta atcactatca ccccctttct 22860tcctgacatg tccccatcta tgggtagcag aaagtttttg cagaacagta tgaaatcgac 22920cctgctctat gcataggcta ccgttgcctg tttcaccaac agatccagca aagccccctc 22980aattatcaac ccaaatcatt ttcttcactt gctttatttg ttatttaaaa gagaaaaaga 23040aaaggggttt ccagcttttg tttcctcttt acgcactagt tactgaaatt agacaacaat 23100tattatatat gattccaaga acagaagcta ttgcaagtaa aatgcagaaa caatttaatg 23160gcaggctata agaacaaaca tcaaaagcaa caggtagata taatgcatgt ctgtggtagt 23220atatatacac tagagatcaa acatctcccg aatatcacat attcagcttc gacgaagtag 23280cataaatatg aacttaaata aatggagctc cgcaatccac caaggaacat agtagtacta 23340tgctctgtca actaattcaa taggctgctg catataaatg taggagttgt tgccaaggtt 23400tcttcacatg tggatacatg tatagttcta taaaccatga ttgtatcaaa aatattttaa 23460taccgttttg aaagaactga ctataacttt ttacaaacaa cacacatgga agacaactga 23520ttcagcaaag tgattttgag gaaaaagaac cagaagtgta tacctatgct atataattta 23580caagagtaga atgggaaaaa catagacggc caaaaatata cttaaattac caggaaaaag 23640tgagggtatg ctagagataa aacaaaatga tgtcaagtaa tatgcaggcc atcttttata 23700actgttcaat catcgaatcc gggcattgca aactgagcag caaatgcttc aactcgattc 23760cgaagatcaa tgatatcttt gttattctga agacccttga gaaactcttt ctgcaatttt 23820ccgtgatctc tctgtacagc acttgtaatc tgagctgctc tgtaaagaaa gtcagccatt 23880gtctcaaaat cagaatctga acagcctctt gatgtcatag caggcgtacc tgagagaaaa 23940ttagggaagt taaagacatt aatggaaaaa gatgggttgt aagccaatac ttaggtgaca 24000aagaagcaca aaccaaaaac acaagatgtg ctctttttaa caccttcgta tcctcaaatg 24060atctttgatt gctataatac tctctagcta tgacacgaat tagtcacttc taaatatgtt 24120attagtgtaa taaaacatat tagtgcactt gaaagaactg attaatagta gtccataaca 24180ttattttacc aattggctaa tttcaacgac tcaaaaacaa tctgcaaaca aacctagaaa 24240atgtgcttgt ctaacaataa taaaggcacg atctactttg acctttaatg aagctctatt 24300tattcccgca accccccccc cccaaaaaaa aaagaaaaca agaatactcc ttaccaattc 24360taactcctcc aggagaaata gcaccatttt caccaaatat agcggtttta ttcagggtga 24420tgtggcacat ctcacacgct ttctcatagc atttacctgg ggaacagaat aagagaatta 24480tttatagaag gaagcgttca aggaagcata tgttatgata tgaaggaaat gcactgtgta 24540tgactaattg cgcatatagg tcatttttca tggtaataaa ttttttgaat tatcaaacag 24600gttaaactcc aaattcccat caacaaatag atgggctggg atgtagacag aagaaaaact 24660tgtagagaat atagtctaga agcaattctt ccttctctta agattctcaa gaaaataggc 24720agtgtttaaa ctgaactata aacgtttatt ttcacttctc agaatatttt atttcagttg 24780ctttattagc ttttccagtt gaatttataa aaactaacct atccatactc cttggagtaa 24840aacacagtca taagctttcc ccttttaaaa catagccaag aaatggttat caaaagaaaa 24900aaaagccttg aacaagacta gtaagattta ttacaatgaa ttgcaagttg atgttcacaa 24960ggggcttatg ataaatcaag aatctatgtc atgttctcca acaaggatga agaaaagaat 25020agaaaaaatt tattgataaa gccattaaga agtttttaag tgaaaactgg ttgcaaaaat 25080ctacaatgct tacataatca aaggaaacta catggagaaa tagcttaatt ccaaccagaa 25140acaatttaaa tatattagca tatatatgct cagcatgtaa atgcaccaaa aatctatcat 25200attgagcagg caagatcaac gaacctgtca agcctagagt agtgagatcc caaagcaaca 25260aatggttgtc agtgccccca gtaaccaact tgcattttct tctcagcaga gcagatgcta 25320atgcctgagc atttttcttc acctgttgca tatatgcttt gtattctggt gttgccactt 25380gcttcaaggt tatggcaaga gcagcaatat gattattatg aggcccccct tgtaatgatg 25440gaaaaacagc aaagtttatc ttttcctcaa aatcatactg gccactacaa tcaccactgt 25500taccgagaca catgccttgc tttcttgatt ttgcacccct cctataaaaa attatacctc 25560cccttgggcc acgtagactt ttgtgagttg ttgaagtaac aatatcacag tagtcaaatg 25620gactggaaca ttcctgcaat ggaaaaaagt gaacatgctg gtaaggggaa agaagaacac 25680aaatttgcct ttgcatgaaa ggaaatactg atttcatcaa aatgaagtaa cattacaaat 25740actgattcca ttcaacttca ataagccata agttccaaat attgctagga aatacagatt 25800aaaagggtga tatttggttt cttccaacta aagtttaacc tttctctcta aatggtaggt 25860atagtatggg gcatctttgg tcttccttaa atgcaaatgt tagcatgtat gtttaggttg 25920agttcagtag caagaactcc atgcatatcc agcagtcaaa cagtaattct aattgacaat 25980ttaccaagag caatgctact tgtaagttaa cctaagtgat accaaggaaa tggacacttt 26040tatggtttag ataactaact taactcgggc ctattaatat ccaaatgaat tgcaagacac 26100ctctactcag ggcacaaaaa tctttataat agaatcagaa catcaaacca ataattactc 26160aaaacaagaa aatttcgaac cttagctgcc acaagaccac taatttgagc catatcacac 26220atcaaaaccg ctccacacct atctgcaatc tgcctaaacc tggcatagtc ccactctcta 26280ggataggaac tcccgccaca aataagaatc ttgggccggt aatcaagcgc cttttcctcg 26340agcttatcat aatcaatata ccctgtttga ggattcaact tataaggaaa actctcaaaa 26400aatatagacg cagcagatac tttcttccca cccggcatat accatccatg actcatatgc 26460cctccagacg gcggatccaa ccccattatc ctatcgcctg gcaacaagag tccggtataa 26520acagcgaaat tagcggaagt acacgaataa ggctgcacgt taacacccca tttctcggaa 26580tcaagattaa aagctgtcaa tgcacgctcg tggcataggg tttcaatttg atctataaag 26640tggttgcccg tatagtacct ggcgccaggc atcccttcgg agtacttgtt cgttaaatgg 26700cttcccaatg cttccatcgc agctcggcac acaaagtttt cggaagcaat aagctcaatt 26760cccaagaact gcctttgttt ttccttattc atgatttcat tcaattctgg atccgcctct 26820tctagcggtt ggtttcccca tgaccggaca gcggctcgtc tttggtcaaa acttggctcg 26880gaacataaca gtttacaagg gttcgaaaca ggtttaggat ccctttgtct tttcacgcac 26940atagatcgcc ctagaatcct aatttcttca tcttcgttgt tgtggtcttt gtgcttgccg 27000ttctccaaac gaggggcacc gtcgtctttt tcctcgaaga actgtaaagg aacgggccgc 27060accgggttcg acgagcagcg aaagctggta tcgatttgaa gcgaaatcga atcgtctgca 27120atggaatttt tagcaaaacc caaaggcaaa cccgattgag cctgagataa atccattcaa 27180acccttaaaa aggcaactat taaaattaaa ataaaaaaat gaagtgtttg ggagaaactc 27240tttgagggag taggggaata acagcttacg gcccttaagg gtaagcgtaa aagcaacaaa 27300aacaatggct gcacttccta aagcaattac aacggaaata acgaagcatt gcctgattct 27360ttgaagcaaa aacagagcaa aaatttgagt aacttttttt ttttcaattc atgtacaaat 27420tggaattatt aatacatata gatataatgt tgaaagggaa aggttctaat gtagacggaa 27480tcacgactag gttcaagctt gtcagggggc aaaaaaattt gaagcccggc ccagaaccag 27540aataaaaggg ggaaaaatgg aaaaggtgtg atcttttttt cttttcaatt catatattat 27600aatgcataaa aggaaaagtt aactcatgta gactgaatca ccgctaggtt caagcttgtc 27660agaggcccaa aataatttga agcccggccc agatccagaa taaaagaggg ttcgtggatt 27720ctaatacaat gcttcttttc aacctttttt gtcgtcgtcg cgtcgtccat ggagtcgcct 27780tcatcgtcga cttccttgaa ttttctttcg tactggaatt atctcaattt tctactattc 27840cgtccggctc tcgctgtgct tttcgttctc tcttttatta ttctctgtaa ttcttatttt 27900tttcctttca aaacttatcc tttacttttt cgtaatttaa gggttttgct ggctgaaaat 27960tgtcttctct tttttttttt gcttgaattt atttataaaa attttaaatt tatagggtgg 28020ctgttggcgt ggaagttggt tctggttcat gttccactgg ttcaagaaat tttcggtctg 28080aggaaaaaac cggttaaacc aaagccgccg actcgtcgat tatcaagata ttataacagc 28140atcaactctc atagttctac ttctcaatag gtaatttatt tatttatttt taatttgttg 28200cattatgttg tgttagcatg aaaaagagat tgaatttttt agttatagct tgggtgtgtg 28260ctagtttatt aatgaaagaa cataatttga ttagagaatg ttaattaagg tgaaattgaa 28320gtgataaatt gcatcattta gcactaagaa cccttaatgt agtagctgct aagatattga 28380ttcagtgatg gtgatatgct acatcacttg aactggacct agtgagtgtt taatacgagt 28440atgcgtctag acatgagttt acttattcta aattttttgg tatattttga ggatattatt 28500tcgtactcat gtctgaatcc aaatgacttc ttattccaca ccaaaccaag gtcaggttag 28560ctcatgattt ggccttagac cttggaagat acaatttggt gaatggggtt tattgctata 28620cactattcac ctgcctttgg gatttgcatt agaaaaggaa ttttctcaaa agaaagggca 28680attaaccaat gtttaattta tgaagggaag aatcagtgtt caatattctt gtatgtgttc 28740atatgcagtt tgttcgacgt tggttagtta aaaaaatctt gtataacgat tggaattgtg 28800ttcccttgca ttagccatta ggttgttttt gatggttttc ttgctgttca ttgggttgtt 28860ttacgattct tagtttcagt atttcccttg gatactctct atctatgtta tctgaacgtt 28920ttatttaact tcaagtatgt gttagatacc tctgtggaac atattcaggc gtgttggtta 28980tatttgtcaa agtaacctgc agttcagtct gttctttctt attttatttg cctgtaattt 29040agcactttag cagtgccagt ggcatatcta aactgtatgg ttggtgttga aagcagaaaa 29100aaaaaaaaaa aaagaacttt attgatagct tgtaaaatac aatgtttata taattttcat 29160ccccaatctg agtgcaactt tgtgtggatg atagcagcca caaagctctg tttccagatg 29220gtcgaaacct aaagcagtgc tggcagcttg aaatacattg gagaagattg agatgattca 29280atttgtttca tacttttgtg gacagaaagt tgtcaaacag caaaaggtaa acttgttcat 29340tatttgatct aaatgaagga aatgacattc gagatgacta gagaatgtag tgttttgttt 29400atcacttctg taaatatatt ttcaactgtt gtaaaaaact ccattactga aaatatatga 29460tgatcattaa aagtttccat ttcttgttcc attagaattg

gagtcatgag tacttccaat 29520tatagtatag gtgtcattga aattatctta accgtgcaag tgaataataa atgatttggc 29580aaccagaaat ggccttatta tgaaagtaca ttacatatga aaattttaat ccgaatttgg 29640aagtgaatgt caaatgtaag tggccttact ataaagagat gacagaaatg taaatgtgag 29700tatgtaaatg tacatttatc ctacacaggt gtggatatat atacatctct ctccaaatta 29760ctgtacgagg agtttatgta tataaataat tttaaaattt ttaatcattg tttatatatt 29820ttttataatt cttaattaat tttagatttt tttatagttt tttaagaaat aatgaactcg 29880gtaaactatt tgccctaaat cattctagat gcctttagta attttaaaaa aactatgctt 29940ttttctgtat ttaataattt taaataatac taaacatttt tataatgttt taaaaaatat 30000atgtcccaga tgaaatgaat cagtacaatt atttgttcag gctcatcatg gatgggattt 30060tttaaataat tataaaaaaa ttatttattt ttaaattttt ttaaaatatt ttcaccttaa 30120catgaggtac atatctaatt cctccatcag ctacatcaac attcaacagt agaaatggat 30180gtaatttaaa taaaatgatc aatttgtttt ttacctaaca tataaaaatt aatttattta 30240tttttaacta aaaatgaata taattttaaa caaaattatt atttttttat ttacatacga 30300gaattaattt atccattttc taagtaaaga tggatgcgat tttaaacaaa atgacgagtt 30360taccttttaa ttttatagag attaatttac tcatttctaa gtagaaaggg taaaatataa 30420tttaactctt aatatgaatg tcttcgtgca cttttaccta atcatactca ttctttaatt 30480tcatattata taaaaaaatt gaaaaagtca aattattact taataacaac acagataaaa 30540tttgaataaa aaatattaat ttatataatt tttaataata tatatagact aatttaccca 30600ttttttaaat aaaagactat actctttcca actccattat ttgaaagatc atattcctga 30660tcgcatatgt tagacatggg ttttaacaat gtgctcaaat gtagccatga gaacgttact 30720attagggacg tccgaaggat tatggacgga agaaatattc agaatggaaa tttacaaaaa 30780tctgccggtt gagttgcatg gtgaaaaacg atggttgctt ttcagctaat aatgagttga 30840tattcaccaa aaccaaagcc catccaagct caggcagcta tcaacaaaag aaaaggaatg 30900ggaaaatatg ttgctttgag aaataaataa atctgaatat tctttagttg tacaagtgtt 30960tgcccccttt ccgagaaaag aaacattaaa taaaatgtgg cccatgaaaa ccaaaatgtg 31020ataatgttac ataggcaaaa atagcaaatg aaatatgaaa aagagaaaaa gccttcacgt 31080actaatgaga tttgtcaaga ggaccaattg tatgtagcag caatgcccat ttagatttac 31140tgatcatctc ccgaagattc atgagtgacc acagttgctt tcttactact acctggccct 31200gaagaaactt taaccatcgc tggtcttagt agccgatccc caaggaggaa tccacgacga 31260aattcttgga tgataatccc ttccttgaac tcttgcgact cttcgcgtgc tattgcttcg 31320tgtagctgta acatacaagt tggaaccaag tgaatcaata acaaaaatga ctaggaatgt 31380actgattctt gtgacaagtc actccaaagc ctatacaatt tgttcaacca aatttatgga 31440tgggtatatt tctcttttgg gctcaaaaca ggaatattca atcagatata ccccgaagaa 31500taaacttatg tcaaccagat gagatgatct aaacatgaaa actaaaccga acaggtatat 31560gcaccgagct ttgtaatctt ctgtttaaga tttagaatga tccctagtat ggacacaagc 31620tctcaatcta aagaagtatg cttttgctgc tggactagca tggcatccaa ctttggagat 31680caaaagaaga ttttcatcgt atgtacctag cagtagcaca aacttgcaat atagtttctg 31740aatggatatc actcacttag cacccccata aagtgcacag cttgaagata atcaggctag 31800ctcatagagt ttgaaactga aacacaaact gaaccaagag aatccctaaa cagacgaaac 31860taacaattaa accaaagtgt ttactagact gaaggtttgg ttcctacgct acatattcta 31920accttttgca aaaatttaag cagataggaa actaactgaa ccatgtgtgt gcctgtgggt 31980ataaatgtat acaaagtatc ctacacttgc aatatgttct ctaatgtgac tgaaccaaac 32040aaaaccctat tgaaggctag caacaaataa agtaacaaac agacattact tgtagcattt 32100aaaacttaca gagggatcaa agggctttcc aactgtaggg acaacagcca cttgcaaact 32160cctcatgatc tccacaaatt gcttgtatat accctgataa ctcatatcta tcttcttttc 32220tttctccgtt tcgggtttaa tttgttgctt ggctctctca aaactgtcaa ccatgggcaa 32280cagactctcc atcacttctc ctttagcatc agaccttgcc gtaagtctct ccttctcaga 32340tcttttccga taattatcaa aatcagcttg caaacggata tacttctctt tcccagaggt 32400tatctctgcc gacaattcca aaacttgttt ttccaatcca ttcttctcgt tttctaaaat 32460gctaattcta ctctcaattt cagatacaat cctatcatct ccattgagaa gcgcctcccg 32520gtaaactcca atcatggcct ttaaaccggg aaaatcttct ccagctgatg cttttacatt 32580ttcctgttaa aattccttaa tgaaattaat actaattaca aacaattaaa caataataag 32640catctattat cgattgatat gattcaattt tcatcttcag ccaatttttt tctactgtga 32700tataataaag gagggaaatt ctacatatcc ttctctcaat ctaatgctac aacttataat 32760agatacaaga aaagaaaata aagaacccag gaggaaggga gaaaaaaaga acttacagtg 32820cgagcagatt cctgggctga aagagaagcc ttgaaagtcc acctttgggg atagtttttg 32880cggttcagag ggaaggaaag gttagatgga gaaaacccag aaaagggttt tgaaaagcta 32940ggtctttggt aaagaaattg gaccggcgat gaagctatat gggtgtgttt tgagggtttt 33000aaagaagaag cagagaagtg agaaggaaag agagagtggt tagagaatga agcagccatc 33060tagaatggtg aacgatgaaa ctgggattag tagattgtgc aatagattgg cgtgtaaaaa 33120tttaaccaaa ttaaagcctg ggaaaagagt ccttggtggc agccactagc cagcccgccc 33180tttggccttc ttgcctttgc cggacaccgt gactgagtgg aggatttctc tggagaaagg 33240agattgaatt gaagcgattt cgcttactaa ctttctcttt gggctgtgct tatgggcctg 33300gctttgtttt ggataaagcc ccccaaaaaa cccaattgga atgctctctt ctccaatatt 33360ctcaactttt cttgttttct cagaagtgaa taatcaataa taatatcttc caaaaataat 33420aatagtagta tataatacag ttgaataaaa taatgagaaa ttggattttt ttataatttt 33480ttgttttata aataataatt aatattaata ataaaacatt ttaatttttt aagactatga 33540gtataatttt aacatactaa attaataaat tattttttaa ttaccacttc aaataattaa 33600caaatttcat tcaaacattt tttttagtat aactaacatt acaatccaac caacaagtgt 33660taaatatagt gtcaaaatac attgcactcc caagagaaga acgaacaaag tgtaaatctt 33720ggatataaca ttgtagaaag gggcaatcat gaaccttaaa tataaaccgt acaataaaca 33780taaaaagtaa aaaaaaaaag gaaaaccaat cttacaatca ataaaaattt caaaactcct 33840actaatagta taccttacca ttcaacactt cttgttgtcg tttatgcaaa cataatgttt 33900tataacttct aacaaattta acaactattt tagatttttc aggacttgaa aacttctaaa 33960gaaagaaatt tccagttctc tcatgcatct ctggattctc tttgtttttg taagaaacaa 34020aagctagatc tcattaccat ggcattcatt agaggttcat ttacttttgc attgtgtcac 34080ttaagctagt catcttttta gcttattatc tagctttata tatacttgca attctggcat 34140tggtaatcct gaaagcttat aaataagtat ttgcattgtt agacaatagg ttttaacaaa 34200ttagtataat gagttttagc attgttaaac agacatattc ggcctctagc gcatgtccca 34260ttaatcatca aaatgaaaga atatagaagt tacctccgct atcaatgatg gccacttgtc 34320ctcttcttct tcgtgctctt tgctttgcat gtgtcggact cgagtcgaga atttacatat 34380gttacgaaga aacccgacaa cgcaactacg tcaaaacaaa ccactaatct ccaattcatc 34440cgtgtggaaa gagataataa gcccttttct tgttcttgtt tttgcggaat tgaggttggt 34500aagtagatat tttagtcttg aaaaagattt cattaggcga gtaatatttt ttcaaaatag 34560taccaccttc aggtttggag ttaaaattaa aaataaagag aataaatctc aaatttgata 34620aaagaataaa attttctaaa ttcataattt caataaaatc agcctcatat tggctaaatt 34680tttaacacta ttatcgattt aaactataat tagttcctca cattaagtga acaaaaaaat 34740tgtcaaacta aactatcaac attaaatttc aattttggtg atagtcaaaa gaccaaaatc 34800aaaatttgac cataatcata ttagcaagaa aaagagccca gaaaagataa tgtaacacgg 34860aaatggcaga gtcaactaat ttgctctaac tccaattcca atctaagcat ccgtccacac 34920acacgaaaaa aattccactg tcgtttagta ccaagaagaa aaagggggaa aaaaccgaga 34980ttgtgtcaaa tatcaacatc ttttaacaca gactagaatc aaaacctaag cacgtattta 35040tatgagcaag taaacaacta gagataaaat catatcaggt gtcaacctcc tgactgcctg 35100cctgcctgcc taaaattacc tgcaaaacaa aatattttag cgaacaacat atcaatatat 35160gggtcagaca gaaaacaata gcacctcaag cccgtattga aatgcgtctg tgcgggtaag 35220tatataaagc tatgcatcag aatataacag tacgatacta ttttacacca aaattatgga 35280gctcatattc aataaatgat gtttaggaat taatatatac atgtgtgtgt gtgtgtgtgt 35340gtgtgtgtgt gtgtgtgtgt gtgtgtgtgt gcgcgtttaa caggtataac taggcttaga 35400ccaaaataga tcaccattat gcattttctt acataccctt tgattttgtt ttcttattat 35460taagccaacc ttattccaac cccagaaaaa cactcacaaa gcaaactggt tttctcaaaa 35520tcctgaacga aagtcttttt ggaccagacc ccttggagac caaggctttt gggtgtacca 35580caaaaccatg agaaattgga caaactgttt ttatgactct aagcaaagcc tgcttcaagt 35640cttcaaccat ttcctggctc aactcttcta tttcctttta atactgtcgc tcaaagaaaa 35700ttaatggcag gcaaatacat cctttggaaa tagggaaatt attagtttgg gtgcacgctc 35760attcttgaca agcagtcagc tccggccagc aacaactaaa atatttatgt attccagtgt 35820tcttttttga gaaatgtagg tacaactcta tagacgacat tggtcacaac tttttcccca 35880accttcctaa tactaaaata cacaggcaag cagatgaata tggaaaagga aaacagatca 35940tttctagaac aacaggatga aaaatagata aagctttata acatccaggc ctcatgtaag 36000catcaaatag cagtagcagc atgataaaat tgtaaaatcc aataacagaa accataccta 36060aacagttact catctgcatg atctggacaa caattcattc acaaggagaa gccttcaaaa 36120gttgaggctc gttggcattg gacatctagc ctgtcaagca gcttcgaaca gccataagtc 36180aatgaaagat ctaaggagtt cttttcactc acagcttcat catgcctttt acctccaaca 36240tcatccatgc aaccaacatc atcaaatctg cttctgacag gctctttcaa agcaattatc 36300ttgccaaaca atcgaaacga accaacacct actttgcttg agttgcaacc cccatgttca 36360ataagttccg tgccaaagga aaccatgctg ctctgactat caggtgacaa gttgtcagac 36420tgtgaactgc caatattgag ctcagtggat attctagttg gcttgggtac cacatagttg 36480ccagagaagt tatcagtgga catcactggg gtattctcac tcacatcgtt ggttaaactt 36540tgcaagtgaa aatgatattg cctggctccc tgcatgccag caggaaaaga attgtaattc 36600aacagtgatg gattcaaata ccccattgtt gaattagtta accctgacac aggaaatatt 36660tcaccctctg catcagccag ccctgaatcc tgagaaaact tcagcctctt ttccgagggg 36720aacgaggaat gaagcggtga ggaggaagaa atttgaactt gccatgggtt cactttcctt 36780gcatcctgga gaacttcagg ttcatcccaa gcaacctaat gatataaata taatgcaata 36840gtgagtcaca aaagatagag gagaaaaaaa attctataag tagcaacatt gatgaaatta 36900aaaagttaga ctcaattacc atcaaaaact ttaataaaga ttaaacaaac accatcctac 36960cacaaaccca cccccaaaat gctgccttgg aaatgtttca aaatatgaat aaacttttct 37020atcattagta ccagtcatag aacatgagta aggaaaagaa aaagagagag cgagagagat 37080taaaccaagc cttgatcata gaaccaaaag cttacctatt atatgttatg aagatagtat 37140ggcatacaaa tatcacaaga tacatctcac ctaagcatca aatttaaaac ttaggaaaca 37200tgatttgatg gggctgaagt taactcaaca tgactaaact cattttcttt gttagtcagg 37260tggctcaaaa ttgaatgaat cctttattcc tttcataaat ataaagttat agaaattcag 37320aattacccac aatcaatatc aagtcaaata tttaatgatt ttacaaatac cttgccaaaa 37380tgcaacaaac agaaaaaata agaacaccca tcttactttg agagagaaaa cactacctaa 37440caaggcacac aaaatccaaa cacaacctag aagaaaagaa aagaaactgg tacaaaatca 37500tttctgatct gaatgagatt ctaaggaaat tctacaagtc taatctgatt ggcaccaccc 37560atccttggtc aaattcctaa gaagagtaac tctaaaattt gttgactttt ttccactttc 37620tgtttcttaa gtctcattaa aatgttaata atgatttgaa gcctaatata gggcactgat 37680ttttattctt tatttttttt ttggggggtg ggggggtgat gcaaaaacag atatacaaga 37740agtaaaatgc agcctactcg tagagctgta gaacattcca acctcaattc ctaagtaaaa 37800caccataact tacgttatat aagcattttg tactctttca cttttaagtg cacttaattt 37860aaatactgtt gaaagtatag tgcataattc tggattatta ctgattaaat tgttcattga 37920aaatagatca acagtaaacc acattcaatt taaacttcat ttggatgacc ggtctaccac 37980ccgccatata aatgaagctc taatcacaat gaataaccac aattcaaaca attgagcccg 38040aagcactttt acctgaagca ttcgccaagg cgagccaatc caggggccag aatccggtac 38100agcagcagac ataactgtcc cttgaaacca agccaatcgc gaggagtcct ccgtctcaac 38160tgccatcttc actctggtcc ccgcagccca gtaagtactg attccagcct ccaccaacac 38220cgccctcgcc acgaaatcag tccaaccggc ccgaggataa tacacaacct cgaacgggaa 38280ccccctcgct gccttctccg ccgcttcagc cactgcctcc gccgtcatcc tcccccttcc 38340ttcccctttc attgctcctc catcactcgg ctctctccac ctccccgaat cccctccgcc 38400ttctcccgcc ttcattgctc gccggactcc aataaacatt ttccaattac agtccctcat 38460gaaaacaaca gaatcgccgg cgataagctt cttttgatta acgaacttgc tccatcccgt 38520agtgagcaga tgcctacgtg gcgtccctcg ataaatgtga cgaaactccc aaacaccgcc 38580gcgaacgtcg gtgacggaga gagtctgaac cggcggatca gcattgtagt cgagcggcgg 38640gaaaacagaa tcggcacaaa accgcgggac ggagaatcca ccgccgttgt tggcatcaga 38700gggcgttaaa accttagcaa atgacacgat cttattccta tcagaatcct caacttcacc 38760attcacattt agaaattgat taggaagcct agaagtttca acaggggtga gtaaaagctt 38820agcgaagacc tcatcggttt tcggatcggc aagataatga acgtcggaga taacgcaatt 38880aatgagaggc ctagacagta cgagagagga cagtttgggc gtggaaccgc aaacttgttc 38940aaggtggcct tgagggaagt aataaaccct agaattaacg gtggggatct gaacggaaga 39000gccggcacaa gctcgccaga tccttggatc aacatgacga agctccggag gactagaccg 39060tgaaggcggc attgggccgc ttagggttta ataaaacgag gagagaattc aaaagggtta 39120aaaagaaaag gggcagcgag tctaagagtg agtgatgttc gttagatttt tacttgagag 39180atttatattt ttaatttgtt tttatgtttt tatagtattt tatggtctgt ctgactctga 39240cgcagactct aagaggtgag gtaggaaaga gagagagttc cgtctcaagg tcggagaagc 39300gggcaaggac gggcagatgg gcaatcacga aacccaaatg ctgttatgtt agcgtttgtt 39360tttttccttt tccttttcgg ggagaaaaaa gggcagaata tatggtcggt tagaaattta 39420cataaccgtc tcaacgtcgt cgttgtggac gctctacctt ccaaaacgta gagtacaatc 39480gctctggaaa tgaagatgaa gtttgttctg aagtaaagta cgaaagtaac cttttgatgg 39540taagaggaag tcaaccaatc aaatggtcgt gaatacttta catttggaaa aaggtcgacg 39600gagacatatt gagtgaatcc ctgtttggtt ggggtgtgtt tgtggggggg ggggggtggg 39660gggggaggtt gtgatttgtg agagaaaaaa accgagggaa ttttgcaacg accggatttg 39720gattccctct ctctgcctag ctgtcgcaat ttacattcac taccgacatt ggaatttttt 39780taatttaatt ttctcttgtt ttttgggtcg tatttaaaca caataattac tggcaatagc 39840catgacatgg taataatctg gactctcaat ttttttacaa aagatatcaa tggtttaatc 39900tgacactgaa attttgttta aaacctttcc ctgcttaatg aaaaattgag gataatttca 39960acttaatttc cttctaatct aacaaaaata aataatatta ttaaacatga ggaagtagat 40020gctatttatc atggtatacg gatcttcttt attatagtcc ttatctatca tttcatgttt 40080ttttttatat attaaatcca acattctcca tcttttcatt ccaattgctc cccctatgcc 40140taataataga ggatggggtt tagagtacat ggtggaccaa cctattggaa gaacttttgt 40200tcccgaatta tcatattcga aaaatgtggg gtcaaactta gattaaacct ttagaagtct 40260agtccaaggc gaatttctag acatttactt aaagcacctc ctcttttgat ttttataatt 40320atttatatat ttttaattaa aattttaatt ttttatacat atttgattaa tagattataa 40380ttatatagta attttttata ttttgataat ataaattata attaaacaat taattaattt 40440taaattttaa aaaatattaa ttattataaa aattatcaat aatacttaaa atctcttaac 40500aaacagtgat tcatgaaaaa tatattctac aatatcatta ctaatataat taacaaggaa 40560cctactatat attctatagt aaagttttta actaaaaagg gaaacattca gttgtggtgt 40620agtctttttg cttgataagt ggcacacctt atgaattagc atgtggacaa tagacattaa 40680aaaggtgatt aggtcttaat ttaattagta actgtattgt tgctaatgca agaatacata 40740ggttcgaatg tgttaaaatg aattattatt ctatttaaga gttggagaag ggttatagat 40800agttataaat cttatatata ataataacct ataatgaaat taatgttaaa gaaaagtcat 40860caatttatct cccatattaa ttattcaact tttttggaaa tatatactaa ataataataa 40920ttctacaaag catttaatta taattttttt ccctatattt tagaaaagat atatttaaca 40980ctatatataa aataatttta gtaaaataac aattaccgta attagaattt atagagagcc 41040tataaataaa ttgtctgcaa taaacaacac agaataaata atattttgag ttatcgttca 41100agaaaatttt gaaggtatca ttttaaattt aaactttgat ttatattcta ttatttagtt 41160tttgtaaatt atgtataata tatattttta cattttttaa agcttaaaaa aaattctcaa 41220actttaaaaa aaaagcaatt aatttttttt ctcaattaag cacttaaact ttcaaaatat 41280atcaaaaagg ccattaaagt ttttcaaaaa aaggaattaa gcctctactc tcttttttca 41340ctcaaatggg tactttaact ttcaaaatgc attaaaaaga cttttaaaat taaaaaaata 41400agcaattaag ctcctactct tattaaaaat tagaaaaaga ttataaataa taataaataa 41460taaattttag aaaaaatatt aaattttaat aaaaatttta aattttatta aaaattagaa 41520tttttttaca aaaatcgtaa caaataaaaa attgtaaaat tatataaaaa tcacaaaaaa 41580aaaatttaac gacccctagt ttaaatcaat taaagtcatc acgtgtagca atacagcatg 41640acatatggtg aaaaatgata aaataaaaat aaaaattata gaaaaattta taaaatgttt 41700cttttggtac gataattttt tataaatttt ttacaaaatt tatatttttt tacattttgt 41760ataattttct tacgacttta taaaatttta taattgttta tattttattt ctatttttat 41820atttttaata gtttttaata gttgaatttt tttatttaaa atttaataat atttatagct 41880tttaatttta atttttaaaa tttttaataa aagcaatggt ttttttttaa aaaaaaagct 41940ttttgcacca ttaacactat ttttttgggt aacccttaag cgtattttga atcttctgtt 42000tttattgaca ataactcccc tattgtaatc aaatgtggtg tgtttttatt aagctgtacg 42060tggaagagtt ttcatcgcag tagataaaaa caaatatcga ctggttatca gaaaattgca 42120tcccataaaa gtcaggtaat agcttttcaa tttatccttt tgaattattt tcctttacat 42180tcaaattttt attaagtgtt attacaaatt acacttacaa ttttcaatat tgattttttt 42240taaataataa taaattccag gatcgaaggt tcaaattcat caaaaagaat tagatttgaa 42300ttcatacaag aaaaatgatt atgaagattt atttcatgtt tctatttgat ttatttatgt 42360aatcaatttt ccatctaatt taatatggtt ttccttattc tttattgaca tgtaaatgat 42420gccaaatttg tgtaaatttg attttttttc aattacttga ttatttgtat ttagggtaaa 42480ctatacttga tcactcaatt atgcttatgt ttttgctttg gtcacctaac tttaataaat 42540ttaacatttc tcatttacaa attctatcaa tttaatcttc aaacttccta atcaaaattt 42600taatttttaa aactgaaaca ttccacatct ataaatttat aaaatctcaa aaaaaaaaac 42660cttcttcaat taaaagccgt tttccaagtc aattcctgat actaatgagt ttatcttaaa 42720tctttatttt cttttctttt tcttttttca ttagttttct tctaaatata atattttata 42780accctttcat tgataaaatt ttggctaaag aaaatgaaaa caaaaccctt taaaaaaaaa 42840cacttcagac ttactcttat tgttatcagt atgataatta tttcaaggta caaagaaaaa 42900gtcttcaaag ttggtcattg aaattagaaa ttaaatctag agttaaaaaa catagctttg 42960aaaaatccaa tggttcaatc aataattatg taagagaaaa tatatataaa ctgattaaga 43020aattattatg tgttatattt atttttaatt attaaatcca taataagtaa tatgtatcac 43080taataatcta atttagcgtc aaatcattcg ccaagttaaa ctaaaactaa atttatgtta 43140gttagagtga aaaaaaaata tttataaaaa tataaattat ttatataaat ttattatatt 43200ttttattttt taaaattttc tattattttt aatagtgttt agttaatttc taacactgac 43260ttattaaaaa ataaacaaaa agggttatat atactaaatt gaataatttc aaccataaat 43320aaaacaattt agaggtgtga aatgctttta ttttaaaact tggaagcttt agttaggaag 43380ttttaggatc aaattgatag aatttataaa tatgaatggt taaatttatt gaattattta 43440gaattaggat caaattgaca gaataagtaa gtattgagga ctaaatatgt tattttacta 43500gttagaaaaa cacttttcgt tatcaattta ccggtgccta aattaaattt ttttaaagtt 43560aagtgatcaa aacatgaacg taagtataat taggtgacta tttatatggt ttacccttat 43620atttaatgtg ttgtataatt aaaatttatt ttccaatgcc ttgattattt gtacttaata 43680gaaattaata aaaatgttaa cttttgatat caaaataaaa taaaacttct gagataaaag 43740caaatgtaaa aatacagcaa tagatcatat catattggca tgaaactaat gcaaatttta 43800gtactataat tctaatttat taaaataata ttactttgtc aaattaattt atccatatta 43860atgcaacaat taaaaatatc atattagcat gaaatagttg cattgttatt aaagttttgt 43920cttcttattg taattcacca aaaaaataaa tatttttaat tattaaatta gacatttaat 43980atgttttacg taattatatg tttattatat aaaataagct aaataatttt ttttaaatca 44040tataagtaga agcttttatt gcttactacg ccatctacta taatcttaat taatatgtaa 44100tactcgctat aatgttaata tgagagtctt aattaatata tactacttct tttaatttaa 44160gattgaattt aaatactttt aatatttaga atcactttta aattgaattt ttttctatta 44220aaaaattatg ctgagttatt tttaaaggaa attttataat ttaaattaac tccacttttt 44280taattaacta aaaacatatt tcttataata atttaatttg acatattatt tttaaaattt 44340taaattaaaa tatcttaaat tgaagtaatt taatatgttt taattattaa atataatcaa 44400ttcatgcatc gcatggaaga aaagctagtt ttatttaatt acattaacat tccatattaa 44460tcaagccttg gttcttatgt ttatacaatc aaacgcatca gtttagtgca aattttctgg 44520aaagaaaatg agcacttttg gccgaattga gtgagatagc

aaataattaa tatgacttta 44580tccagattat ataagtgaaa tgttaacaag gaaataacga caagacagaa aatgatagtc 44640tgccgctttg gcacgtttca ctttgccgtt ttggcacgat tttgagggtt ttcttttctc 44700tttctttttc tttactgttt ttgcgttttc tgttgcagtt ttgcttctgg ttatgtggtg 44760tggagaggtt ttgatttgct atgtgagtga tggaacgtgg tattgcggat ctgaatctag 44820aggatgcaga ggatgaggct ttctctttgc cggaggaatc agaagaaaaa aattctgcgt 44880atagtttttg cttggtggga tgtttcctga cagcgtggtc cattttccag ccttgatgaa 44940tactttggca aacatttagc atccattgga aggggtacaa atatcagatc tgggggaaaa 45000acgatttatt ttcaattttt tcaatgagat agatatttct cgtgttatta catgtgctcc 45060ttggactttt aataaccacc tcttgatttt tcatcggatt cagtagaatg aagacccgat 45120gtctatccca ttggtgtatt cagattggtg ggttcaagtc catgacttgc ctccgggttt 45180ttttagagat tcaatggcgg ttttgttttg aaattttgtt ggcagatttc tagaatatga 45240tacaaagtag gttcttaatg gatataggaa tttcatgcgt atatgtgttc agattgatgt 45300gcgaaaacct ttgaaacgaa gaaagaaaat tatgattgct gagtcaaagt tctcttatgc 45360aaattttaaa tatgaaaaat taacattgtt ttgtttttta tgtggctgtc ttagacatgg 45420tgagaacttt tgtctggtga gattacgtat tggagcacaa gaaatggagt ttgggtggga 45480tctatcgttg agggctcaag cgaggaaggc tttcacgatc aacagcatct ggctaaggga 45540taatggggac agttgtcatt ttggaaagtc ttagtttgag cagcaatata gccataattc 45600aagtcaaaat caaggatgtg aattatgggg taattttaat aatatccttg ggataaattt 45660ggaaggttcc aaatctattg aagagatgaa tgaagaccaa gaagggggga aatttcgtca 45720tattgatgga aagagtgtga ggcatgggaa aattcaaatg gtgggacctg ctctaaatga 45780tgatgtttta aggaatgttc cggggcggaa tctaacatcc ctatctcgta accgtcgcca 45840gaataggtaa gaggtattac cacagacaga acatcacaga tccatacaga attacggata 45900tcacataata ttaatgcata agcaattcaa caattcatcc cttatggatg tctccaagac 45960ctgagacata cttttagaaa atgtcgggac taaaccaaac atgttcagaa ttttcagaac 46020ttaaaaaaaa attcaattct attgaagtta cacacccgtg tgatcaggcc gtgtgcctta 46080cacgggtacc agacaagccc atgaggtcca gccgtgccaa aacaaagtat acatattgac 46140ttttacacac ggcagtgtga tacttaattg gctactgact tgagcccacg gccatatgac 46200acgcccatgt gtcttaaccg tgtaatctta agaggttact gctttgcata cacggccaca 46260aggcacgccc atgttccctg cccatgtggc acaatgcagg cttggtttaa gccaacttgc 46320cacccttttt tgggtcattc ctaccagcaa tattaaacaa catttatacc aaaatattta 46380gctaaaacca tgctcaaaac atgtttcatt ataactaaat catcatcatt caataaccta 46440ttcaaatcca taccaaaaca tattaaaaat cttattacaa catgccaaaa tgctcatttc 46500ataactcact tatggcatct tctaactcat ggtaaaactt accatttcct caacttggca 46560tatttcaaaa tgaccaccac atacaaggcc atataaccat tacaagcata cacaatttag 46620catcacaaac catttaccaa aactaagcac aaacatacca tttgctagcc aactctcatg 46680gcataacata tatacatatc aaaacttaaa tacatagaca ttctatccta tacatgctat 46740acttaaaaat atttacactt tcaaaagtac caaaatgaat tcgatagtat ggtgacaatc 46800ctcgactatc cccgagcctt cagtagctaa gataactgta aaatagacaa aaatcacaca 46860gagtaagcta caaagagctt agtaagccaa atacaattgg tctaactatt aaagcatata 46920aagtacaaat caacagcaag aattcatagc catttcacag aatagttcat gagcctaact 46980atgctcattt gcttgtatac atgtcatgtt ttatttccat gatttcatac atactacgca 47040ttcattattt cacagaaaca atctttcata ttcaaaaatt tactactata cgaatgtatc 47100tatagagatt atacatttca tatattcatt cccatatgtc gtactctatc atcagatggt 47160tcagaaaaga tacagatact cataaacggg tacaatgcca acgtctcaga catggtatta 47220catgtaattc agtatcgatg cctctgtccc agacagggtc ttacatgaat tcagatacga 47280tatcgatgtc ccagacacga ttttacacaa tatttaagat cgatgacaac gttcctttaa 47340aaacatacag agcttttcaa atactaacat attatcgaaa tttactcgga attcattcat 47400caggctctca tagccgtcca atacagatta taactagtat atacaacatt caatcaattt 47460aacacgtaat tgtattttga cttacctcgt acgaatttca gatggaaacg agtcgactat 47520tcaattattt tggacttccc tcgatctaag ttcgattttc tttgttcttg atctaataca 47580atttaaattc aaccattcaa tcattcattt catgcaaaat aatccatgaa cacatattta 47640gggcacttta cattttaacc cttacatttt cacactttga caatttagtc tatttttcac 47700aaaatcacaa atatgaaaaa ttcaccaaga ctatagcttg gccgaatata catatcctcc 47760atacaagccc aaataacata tttaattcac aattcagtcc ttcaaaacct cattttcaca 47820aattagccca aatagctcta ttccataaaa aatttaaaaa caaagcatga taatctcacc 47880tatatctttc ataatccata taaaaacatt acaaagctca tataatcatc aatggcacat 47940ttcataatct tcaacagaaa cagaaattca gacatggatt ttgaagaaca agaagcaacg 48000atcacagaaa cgtaaaaatt ttcaaaaaca gacgaaaatt cataccttaa tcaaggatta 48060agccgaaacc taaaatggct ttcaacacat tcatgcaatt tgttttcttt atttcatgtt 48120aaagcacgaa ttaccattgt atccctcata aataaacata tcaattacat aaaacaaggt 48180catttatgac cactcataaa ttcaatggag taattgccac ataaggccat aataatccaa 48240agcaatgcca attaaacaca ttgaatatat ggcatgcaaa ttttatcatt tatgcgagta 48300aatcattttt cataattaag catagaaaca gacaaattaa atcacagaaa cttcgaagat 48360ataaattcac atatcataga cagagaaaat aatattaaaa tattttttca aaatcgatta 48420cgtggtctca aaactactgt tccgactagg gtctaaatca gactgttaca cggaaggggc 48480ttttgactaa aatggagttg tcaaatatgg aatgcaaatt agaggacgct caaataatta 48540atgaggaagg aaaaaaaaag acaaaggtct gattttttaa tctctaatgt ttctaatgga 48600caagattcgt tagaggtttg tggtgggtgt ttcgctcaaa atcaacaagt atcagcggct 48660gccattaggt aagccgaccc acagcaatga agttattttg ttggaatatt cgtggattgg 48720ggagtctgcg agcagttaga agacttcagc acatgctgaa aatttatcat cttcaaattg 48780tcttattcat tgagactaaa ttgaatgcta atagaatgga aagggttagg aaacggtgtg 48840gattttttaa tggtattgac gttccggctg aaggttctcg aggaggatta agtctagggt 48900ggaatgaggg acacttggtc aatttgaaga gtctctcaaa aaatcacatt gatgtggaaa 48960ttcaagatga taaaggaaaa catcgacggc tgtttacagg tttttatggg gctccagatg 49020ttagaaataa agtagagacg tgggatttac ttagacgatt ggggagaaat aattcattgc 49080cttggttggt tgggggagat tttaatgata ttctctttgc acatgaaaag caataaggta 49140taccaaggga aaaggctaaa actgaggcat ttcgtagaac gttaaaggat tgtcttttgg 49200aggacattgg tttttctagt ccctggttta cttaggaaaa aggggcgaat tttggagtgg 49260aacattaggg aaagaattga cagaggagtt gctacggata cttggcttta aacttttcca 49320acttattctc tggggcatct cccgcactct ttctcagatc attgtccttt gttctttgag 49380acgaaggttg gtaagaaggg gaaaagtctt attattcaat tttattttaa atcttggtgg 49440gtccttgagg agtcgtgtga agaagaaatc aaaaagcttt gggaagaaag ttctggatct 49500tattttaatc gcatgtcaac tcttgtaaac ggtttgaaag tttgggcagg caaaattcaa 49560gccaaacaaa ggtacgaggt gaaacggtta aatagaagac ttgaggaatt gaatggtgat 49620aagagatcag atgggacttt ggcggaactt atggaggtta aaattcactt gaatatggaa 49680atgaataagg aggagagata ttgggaacaa cgtgcaagag tgaactggtt gcgaatgggt 49740gataaaaaca cttcgttata aatgtgcctc gcagagaagg cgtactaatc gagttagtgg 49800gctttagaga attgatgggt cttttgcaac caatgagaga gagattgggg atattgctta 49860ggcatatttt ttcgacttat ttgaatctag aggagttcaa gatgtgaagc acatactctt 49920agagatcaaa tcgtgcatat cagatagtat gaatcaatgt ctgatggccc cttacacaga 49980ggctgaaatt gctgatgcat tgaaaggaat gaggcctaca aaggcttctg gttctgatgg 50040ttttccggcg attttttatc agaaattcta gcatatcatt ggtaaggata ctagtgaatt 50100ttgtttggat gtgttaaatc atggtcactc gttggatgaa ataaacagaa ctcacttggt 50160gttgattcca aaacctgcca atcctattaa tctgaaaaac tttcgcccta tcagtttgta 50220tatagtgatc tataaaatta tcgctaagtc cgttgccaat catttacaaa aggtgttgga 50280tggttgtatc gatgactctc agagcgcgtt tgttcctgga aggctcatta ctgataatgt 50340gttgttggca tacgaggtac ttcattcttt taagaacaaa agatcagagc gaaaatgttt 50400tatggcctta aaacttgata tgagtaaagt atatgataga gtgaagtggc cttttattaa 50460aggcctaatg tctaagttgg gttttgcaaa tgggtttatt gatttcgtta ttcgctgtct 50520taattttgtt caatacttta tcttaattaa tggagaagaa ggattgagct ttaggtctat 50580gaggggttta tgtcaagggg acctactaag cccttactta ttcttatttt gtagagaagg 50640tctgtcagcg ttaataagac tgacttgtca ggagggaaag atttgtagag ccaaggtgta 50700cagaacttct tcatcaatca cccacctcgc attctgtttg gggaagtttc gaataggggg 50760ataagtatgc ttcaagagat ccttagggaa tatgaggttt gcttggggta atgtgttaat 50820tttgaaaaat ccatgatttt tttcaattcg aatgtaaatg atcatgatag gaacttgggg 50880tttcaggttc ttaacgttcg gtgttcaatt gaccttgaga aatatctagg acttccaaac 50940atggtgagac gaaaaaagaa atttgctttt caatgtttga aagatatacg aaacaaagga 51000ttaccagtta gagcattagg catatttcac aaggaggtag agaggttttt ataaaagtcg 51060ttttgcaagc aattcccaca tatacgatgg cttattttct tttgctaaaa tctttgtgca 51120cggagttgga aaacataatg agctcttttt ggtggaataa aagtaatagg aagagaggca 51180tgcattggtg tgattggaag tccttaagcc cactcaagga agaaggtggg atgggttttc 51240gtgatttaaa tttttttaat attgtatttt tggccaaaca gggttggcgt ttgttacgta 51300accctaagaa atattataag gattctgatt ttttaaaatc caaattgggt aatttacctt 51360cctttaccta gcagagtttg tgggtgacaa aatgcctcct tttgaaagga ctgggttgga 51420ggattggtga tgggcagaag gtctctattt gggatgatgt gtgggttcct ggaaatgatg 51480tgcttaatgg tcagaattca acttctaatt cgaggctatt agaagtcgca gatttgattg 51540atactagtac aaggaaatgg aatgctgagt tgatttctaa taactttacg gagacggatg 51600ctgagaggat tttatgtatc cctttgtctt tgagttcaca tgaggatctt atcatctggc 51660gagatgaacc tactggagag tattcggttc gaagtggcca caaagttctc tcacatgttg 51720ggcagactca agtacatgac acttacgaac ttttttacaa gagactatga aatttagatt 51780taccctctaa aattaagatt acagtttgcc acctattgaa gatgtcaaaa gggagcaaaa 51840acaagggagc acttgttcaa aaattgtcct gttgcaaagg aaacatggga gaggttagat 51900attgtttggc ctgtctcaga agaaagtacc gaattcattg aatggttaaa aaaatttttt 51960gaatctaatt ctttgggcat gtgtaagagg tttgcgtgcg cattatgagg gatttggacg 52020tccagaaata gatttattca taagggaaaa atgcgattga ggatccaaat tgaagatttt 52080gttacaaact acctaaagga acttgatggg gtgaagcagg ttttacctga caaaagaatc 52140catacaacca gatgggttgc tccatcaggg ctatgactga ggataaattt tgacgcggct 52200ttcaatagtc aaagaaagga attgtgttct gggttagtgg ttagaaatgg aaaagaataa 52260gttatttgtt caaaaaatat cataaataat aatataccgt ctgcttttgc ggccgaggtg 52320ttggtgtgtt atcaggcact agatctggga ttccaactcg gcctgaaggg cgtggaagtt 52380gagggagact ctaggtcagt gattcacaag ctgcaagaga agaaagaaga tagatctgaa 52440attgctgtgt atattgaaga ttcaaaaaaa atgagtttga gttgcagata ttgtgctttt 52500cgctttctca atagggaagc taacatggtt gctcatctta ttgctactga agacattaag 52560aatggggaaa atacttatct gttgcagagg atttcttctg gtgctgaagc gacggtgatg 52620gacgatcgca gatggacaaa aaacgtgcag gtgacaaagg tttggcaagc tgaagaggag 52680tctggagggg gttatataag gatatctgat cgtttttttt ggtggttttt gttttgtcaa 52740aaggattgcg tataagagaa ggtttttgtt tttcggggtg atatttgttg gatttgacgg 52800gtggtaataa tgattctgat aactttccct ttcagttttg ctattttcaa gagggggttt 52860atttgttggc tacagctggg acgaagtctc tagagctccc ccgtcgattc tgctgctagt 52920cttaattgtt gttgggccgt ttttttattt tgttagtttt ggtttttttc gtttttttgg 52980accttttcat gtttggggtc tacttcggtt ctgttttgga ttttatgttt ggtcttgatt 53040tctacgaata tccggtattt tctcaagaaa aaaaaacatt ccatattgat tgggtcattc 53100cttctttaac attaaacata taatctttat tatcattaat tggtcattaa caaagtataa 53160ataattaata ataaatatta tgtaatttag ttatatttag tataaataat ttatagtaat 53220ttagtttatt atttagtagt tcaaaattat taagaaatta tctataatta tatttaaaaa 53280tctgtcctaa aattaaaata ccatctttat ataaaagtgt ttaaaaaaac ttaaatcgaa 53340catttttgga aagataatta gatttgaatt taattatttc taattaccta taagaattta 53400ggagggtcca attatattta atttaataaa atatactaat tatagtagtt atctaaatat 53460gccacaaatg tataattata aatatttata cataaatctg actttctaaa catgactaaa 53520ttttattata tgtatatata ttaaacatta aataatttct taaagaaagc attaattatt 53580tttaatagat attatagata attatttgat atatttttaa tagaatttat ggagtttcaa 53640ataagatgaa tttcgaatat aagtttatgt atgtaattaa accttaaata atgagttttt 53700taattaggta aataaggtta aaaaattatt atgtattttt aattaatagt taaaaaatta 53760aaaataatat aaataatata ttttttggta caccaataat ttgaaaataa actattttaa 53820agaaagtgaa tacataatta atattgaaca acttaaaatt ctcaaatatc tttattttat 53880gtgtggaatg aaaaacttta ctatcaatat atcaaatcaa taattttgat ataactaact 53940atccacaaaa tcttagatta ttttattggg taaactatac aattagtcac aaagttatta 54000atgtgttatt gttttaacca tcgaactaca aaaattttca atttcatcac gttatcattc 54060atttctgttt tgatcaccca ttgattaaat tactaataga aatggtaagg tgaccttttt 54120ttacttggta tgataacata tttagttctc agcatttaca catttgatca aattaaccct 54180aatttaaaat aatttaacaa agttaacctt ccatatttat aaattcaatc aatttgatcc 54240tcaaatacat cttcatcatg catctcgcct cacctcgttc cattgccttt tatattgttt 54300tttttttata aatattttgt ttacataatg ttttattcac ccatatttgt cataatctag 54360taaacatagt agaactaaag agatgtgcca aaaatatata taaaaaaggc ataatgattt 54420atttgatctt ttaactttac aagaaaaaag ttattttaac ttcttattca aattttcacc 54480tttttaaccg ttgaaattgt gtactttgat aaaccatctc aaaatggatg gaaaagttaa 54540tgtttgttaa cttagttgac tagcatacac gtggatgcca catcaacaat taattattat 54600tttcaattta aaaaattcaa aaaaatataa tgttttaaaa aaattataaa aatatttttt 54660aatttaaaaa ttaattaact gctgatgtgg catacagcta gactattatg ttagcgaagt 54720taacaaacat tgactttttt catctatttt gggtgatttg aaaaataacg taagtttaag 54780ggataaaaaa aacgaaaaat taaataaata actaaaataa tttttttatg agattagaca 54840gttaaataaa ttattattcc ttctttatac atatattggc acatttctgt agttgtatca 54900cgtttacaag attatgacgc aaatgggtga ataaaacatt atgtaaagaa tatatttata 54960aaatcattat ataaaagtca aaccgagagt attaagttca aatcctccta caataataat 55020ttataaaaat attcttgaat atctgtaaag gctgtacttg gctctcacga tgcatatatt 55080tttgatacaa atattattag ttgtaaaata cattttatat ataagtatta gcttgatata 55140tatttatggt tttgcggcta ataaaggctt atggaaagaa tttcagagat attccttaat 55200gctgagcaca taaattgtgt caggtgtata catacaaatt ttaagtctaa accattccac 55260cgaagaaaag tattgaaaga tattttgtgg aagtctacaa gagcaaccta tgtgaaggag 55320tttgagaaca caatattctt acaaatggtt ctattggatt gatactaagt agtggtcaaa 55380atctcacttc tcaaccgaaa gcatgagtga tatgatgttg aagaatgttt taataaggtg 55440ataattgtaa ctttaaatca tatatattta ctcaaaattc tcaaatttat gtgaatggtt 55500actaatagct tgtttcttgc ttgtaaatac cgtactaatt cattatatta ttttttattg 55560atgtcgtgag atttattgac ttacgctacc taaataaaca aaaatcgaat gacaagcacg 55620atgctattcc aacttttacg tgttaaaatc acatcatctt gctttgccct tcaaatgtta 55680atcatagaaa tgaattcttg cgaaatgcac ttgtacaact ttctttcacc ggcctggggt 55740tactcatttt tctgttcttt actctttttt gcactgtttt agatatatat tgatttgtat 55800ttgttgtgaa ttaaactcaa tggacgctgc ttcaatgatc atatcatatg agaagagaaa 55860aacggagcaa ggctgcaacc ttttcaagcc aagctgcaat aaacactttt atggaggccg 55920ttggtgggaa acatatccga gaatacccca aataacttgt gggacaagat tttacaaatg 55980ctcaaaacat taaggtggtt tcataattaa tgcatacatt gtctctctga catttccaaa 56040ttttgttaat ttctatctat tcaaatgtaa tgcctaaggt ctgtatccaa atacgaagat 56100ttttttgatg aatccatttt tggtggtaca ggagtttaca tatatcatgg agcactcgtt 56160gatgaaaatt gcgacttcta gcatctccac gaggattatt tagaatgtgt tgcaatttgt 56220atttcttact tcatcatgtt atgttttagc tgtcaacaac gatttcaaaa tgggattcca 56280atcttttata atcatcagta tacaattatg aaggaagagg tgaataaaat ctgattcaat 56340tcaaaaaatt aaattttaaa ttttgaatta aatagtttga gttatttgag ttaatcaagt 56400tattcggatc aatcagataa aaaattaatt tttcggttta acttgaatta tgaatatcta 56460aaaattcgaa taagaaaaaa taaaactaca tcgttataat aaatgtttag ttaaaattca 56520aaactattaa gataaaagtt aaaattacgt cgttttgata aatgtttact aaatttaaag 56580acaaaatcat tatattatgt atgtagttaa gtaatcttat acttcatcta ctagttaaat 56640aatcagttca tgtaaacaca acattgagta tgataagatt cgtcaacttg atttgactca 56700aaaaattttt actcggtcca attcaattgg aaaaaaattc aaattgagtt ctgttgctaa 56760aataagattc gtcaacttaa ctaactcgaa aattttttac tcaactcgat aaaatactca 56820cccctacaac tagatacttt cctaaaatcg actcgataaa atactcatcc agcaattaga 56880tacctttcta agaagagcac taacgatatc atgttgttga tttgtctgaa aaactacagc 56940caaaaacgac ttctcttgta aactgcgatc ttgtgaatgt ttttagatta gcaccgcatg 57000tgatgttgat attttcttgt atatattatc aaagctcaag gtttttatgg agttgaaaat 57060catatttttt gaagaacttg atatttttat ttattaaaaa caaagattga gctaaaagcc 57120ctaaacttga attacactta aaaaacagaa tgttttcaaa aaatatatat tttggacagt 57180caattttcat caaatcaaac atacctaaat gttgttttgt tcaaatttaa tacttcccat 57240tatacttgtt taatagtcag gtaaagccag gccaacccaa ataggagctt tgcttttaat 57300tcgacgaaaa caaaagaagc gcacaaaaca aagtgaattc aaatcatcgt taagcacaac 57360cagaaaaaat tccaaccccg gaaaccgaac tcaatacaga caacaaaaac catagaaagc 57420acaacaaaac agcgagcatg tacgccaggc gtagctcact attttattta aagaacacga 57480ccaagtccca cacactgaaa actatagcca cggtagagaa aattattgca ctataaatag 57540cacgtctgcc ataaaaggca caataaacga ctataccaag agcttcagaa tcactcctct 57600tctgtctcag actcggcatt ctggagccat tcgatgaagg gcttggagtt cttccatatc 57660tgagaactct tattaccacc ggctacaccc ttctgatacc attccatgat gaactcttct 57720tccaagatgt cattgtcata tagtgcttta agaaccaaag ccacctcctt agctgcctcg 57780aggtttgcct tgccacagaa agactcgata gaattgagga gcatcatctg ccatccctct 57840tctctggctg ctgctacaag gtagttcttc tttttagtta cttcctccgc aaaccccttc 57900ccaacattat gaaagagtgc agtaaaaagg gcgtccatga tttcttggga cgttccagag 57960agtgaaccca ggaaggattt aagctgagct gcagatgacc ccttcttgag gtatttcttt 58020atctcatcaa caagtttctc atgggcagtg ccaccattct cttgtgcctt aacctcacgc 58080tctggcgact tcttcagtga cttcttttct tcttcagtag aaagcattac catatcagct 58140gtaacagcac tcaactgctc ttgtatacgc tgttgagcag cctccagtga agtatccgtt 58200tgccactgca cattatcatc atcatcatca tccacctctt cgttctcatc agcctggctg 58260tgagttgggg agtgatcctc atcagaatgt ttgcttttct tcttggttac tgcacctttg 58320gcagcagtag cctttgaagt agtagtggta gaggaacctt tcttttttgc ctctttttta 58380atcttcttaa gctcttcatc agcggcctca ccctccttga gtctttcctt ctcagccctc 58440ctcattgcct tcttgtcttt tgaagacttc ttagcctcag gtgggttttt aagaatgaaa 58500gttgtaagct tatctctcat gtcaacatca gaaacaaacc cacacgcggc acatttcagg 58560gtaagcatct gtgtcttagt aataactatc tcagtttcag ggttcccaca gccataacac 58620tgaacatact tcttaatgaa gttctcaaga agccctgcaa gcttggcagt gtcatgggcc 58680ccatttacaa gagaagttcc agtcttctca tcaaacttgg attgggctcc cagttcacaa 58740ccaaaatatt ttgtggtgta agaagcaggt ctagccaagg cctttgcaat ttctaccatg 58800ttaaccacgt tagtcttgat gccatttcct ctcccttcaa tcttggttat cattttaggc 58860atcttatacc tataaaaggc atcatcactg ttcgaagcac caatattttg caaagccatc 58920ttgatcaaac tgagggagac cgttaatcag aaaggagata tcagatactg gttatcaaaa 58980tcgtggggag aaatctgtta ccaagctgcc tccttgcact tggaagatgt tgcataaaac 59040ggaaacccaa actgtgtctc tgattggcct caatatgcat gcagctaatt ccagaacagg 59100aacttctatt cttttttctg tacgtgggac agtactctcc aagcaatcca attgatgaca 59160aaaagaaaga ggcccgttca gacataaaag aagactcaca agtaacagac tccactttgg 59220caatctgcag cagagcagat taatctggca acttcctgaa agacaatgat ggagtttatt 59280caggcaaagg tcagagcaca gaatatcaat aactggcata aaaccataac atgaaaattg 59340ctacatcagc gaaacttgaa cattactagc aaaaccataa cactacaatt ttcaaaattg 59400catctatagt tatgcatgaa gaaataccca tactaaacat cgtcaacttt tgtaatccaa 59460taaagcatag catattccag tattataaca tcaaaactcg ggatatctat caaaaccaag 59520attaaattat ttcttcataa gtttctctat accaataaaa agtttagatt gtaaagaaaa 59580taaaaaatga tttattaagg actgattgaa gcaacgaaac

tagacagatc aagacttaaa 59640agaaaaacct aaatccgcag ttctaaatct cctataatac aaaaaatatc ttattagaaa 59700atactgaaaa taaggtaatt aaaacgaaaa tttaagtact acgatcttgg tattaaaaaa 59760cctaagcctt ccgtccaacg ttaaataatg aaatattaag caaatataaa tatgaaagaa 59820tatctgcatt gcgataatct aaattcgaaa aaaaaggaaa cagaaaacaa accctctggt 59880ttcgatctga tcatctaaaa taatacaaat acaaagctaa attaaacaat aagctcttac 59940aacataaaca gctcaacaac gatcgaacaa aagaaaagga aaaaaaaaat ctagctatcg 60000gtcaaaaaat gagagataca atgaacgata gacaaataaa aagaaaaaaa aaagcaaaca 60060gatcaataga aaatgaaaat aataaaaaaa tatgaaacaa aaagaactaa acctttgatg 60120agatgaggaa aatgatggta ggagataaat tgtgaatctg aggcaggaga atttggaagg 60180aaaatagata ttagggattt ttcttgtgga gaagaattga gaagctctca actttataca 60240cgccaacgaa gggaaatgtg aaacgctagg gcaaacgcag catcttgaat tttctagtat 60300tgattattct cgaataaact tattttatta ttaccatatt gcccttaatt cccttttcta 60360ttaaggattc ttttgggtag ataataaaaa attttaatta cgaaattagc cggattattt 60420agcagaataa tttatcttat aaaataaaat taggtgtcgt taatgtgtta agtggtacca 60480acttaaaaaa tatactctca tttgacaatc attccaggtg aaaaatattt ttaaatggtg 60540ttattacttt gtcgagcaac acaaaataaa tactgtacaa gtaaaaaaat tatattttaa 60600aacggtgcca cctaacaatg tggtggcacc aaaatttgta tttatatccc attcccagcc 60660taaaaaataa atcgttgttg tttatcaatt ctaatgacac atattaggat ctttcctcat 60720tataaactac ttatgcaggt tgatggaacc tgactgtggg aaatatttac aaacccttct 60780tattgcagtt gtagaagatg ataatcgaaa cgtactacta atagccatta ccattatgga 60840gagtgagaac atgtaattgt gacaattttt ttgacgaact tgcggagtca tattgttaaa 60900caagacattt acattatttt ttatcgatca aaggggttaa ttgcggtgat taggtgtttt 60960gaagttccgt ggagattgtc caagcaactg atagtaaagc gacgcatgct gtaacagccc 61020gttttcagtg aaaatggaac agtggtttcg agaccacaaa tctgagtccg gaagaaaaat 61080aattttaata ttatttgtaa caccccctac ccgtatccgt caccggaata ggatacgaag 61140cattatcagt gttacaaatt tatttatcag acattttatt tcatctagca ttcatatttg 61200ggaccaatca aaatcaaaga tattgccgcc tgaacatact taatttcctt gtatcaacgt 61260atcaaagata atcacatatt tacatgtcat gataaatatc attctcttat cgtttcttca 61320taaacataaa tcagttaatt tgttatatca atatttcatg taccatcaac tcatattcct 61380tatatcacat aattaggttt cacgaactta tctggctgaa ttgcaaaaat accaagattc 61440aagggtattt cggtaatttt ctattttcct cgatttttca accaatcttg atttaaatta 61500ataatttcat tcaatttatt aatttagaca ataaataatt cattttactc aatttggtca 61560tttttgatat atttataaaa ttgcccctaa agttttactt ttattcaatt tagtcttcga 61620gcctaaaata tgcaaagtaa ccactttaat gtaacccatg ctaactaaat attcatatat 61680attttccttc actaatatat caagaacata gaaccttata taagaaaact ctaccttaac 61740atcattttca tgcttttgat attagcttac atgagaaact ctacttaaaa tatattgaag 61800tcttaaagtt cttaccttgc cctattgatt tcaatcttta actgattttt ctctctcctc 61860cagcttctat ttcttgaatc caacttgata ttataactcc ccttagtctc cttaacattt 61920ttctcttttg gtagctatgg aaattctttt gatttctaat ggtgcgtttg tttgctagga 61980aaatatttta cggaaaatat tttcttggtt ttccagtgtt tgtttgcctg aaaacatttt 62040ccatttggaa aatgatttcc aagacacggg taaaatgtct tacgttttag ggaaattgcc 62100ttacgaattt catttctgta agacattttc cagtccctcc ttcatctagg taaaagtctc 62160tccttcttcc tttcttcatt tcttttcttt cttctgttct tcattttcta gtccatcgca 62220tatatcattt tctcaagctg ctgtttcatt tcctctcttt ctctctttct caggattatt 62280ccatccctct tcactaggtc ttggcttaag gtatggcata aagctctttt ttttcttatt 62340gttccttact tctccataga ttttatgact gaaattttat gctttccttt gtttctttag 62400ctagggaaga aacttgaatg atttagggat tcccaaactc aaatttttaa aaaagagaaa 62460taccgaaaac aagcttgaaa ttgaaatcgt tcggtttcaa aaatctctaa cctttgttct 62520tttagttgtt gataaaaata aaattatgga ggtttggttt ggtacctagg agccctaacc 62580aataacacca atcttgaaat cgagagatat gtgggtttga gaagccatag ctgagttgca 62640gcaattatga gtagtcagtg tggttggttt gagaggactc aaaggatctt gaaatggtcg 62700tcaagaaacc tagattaaag tttttgagga acttgagaga gagggagtag tgaagtaagt 62760aacctagatt ttaattactg gatatttttg tggatgaatg tacttgtttc ggttatgaat 62820gcttgtgatc tgttttgtaa ttgcctttgt atatcaaaca taaaatattt tgctcacagt 62880aatgtagact aaatcacata aaagattcaa tattttgctc acaaatctga taaactagca 62940gctagaattg gagtttcttc catgttggat tacctttttt tgaaaccatt gattacccca 63000taattacatt agcattctaa aatagaattt attccatata ttgtaatcag aaatttaaga 63060gaaagaaata agacatgata atatcttaat ccaaatttaa tggttgcatg atattcgtaa 63120catgttttaa agatttataa atgactcgtt cttaagacta acttattatc acgattaagg 63180caagtgtacc tatcgaacag tagtatagtt cagcaagacc ggattgttga acccaaagga 63240aatacgagta ctagtattta cttccttttt attatctagc ctaaaaatta agaggtttgg 63300ttatctaaac tacttactaa ctaagaatgc acagaaagaa aacttgggaa aatacttttg 63360ggaaaattcg attgattgag acaataccta aggaaaaatc tgtaacgacc caaattttaa 63420ggtcatcgaa aaattaaatt ttcgggtcat tattttcgca aaataaattc gtaaacattt 63480attagaaata tttatgaagc tagtagtgta gttgattaga ttttggttaa gtgaattagc 63540ttgaattaag gctaatttag tgaaaggact agattgaatg aagagtgaaa gtttaattgt 63600agaacaaaga aaattgaggg gactaaatag gcaattaagc ctattctaaa gaatgaggcg 63660gcaaaacata aaaatctttt atttttatgt tgtttaaatt ataaatatat tattgttatt 63720attgttattg tattacaaat taaattaatt atattattat attatgaaat aaattaagaa 63780aagacaaatg tatggtgcat atggtgacat gtgtaatact aatatacata caattgtaaa 63840atacatgtat atttatttat tatataagta tattattaaa ttatatatta gtattaagta 63900aaagatattt atataataaa tagattaaag aaagacaaat gtaataatat aggtgtatgc 63960aaatgtaaaa tagatattgg atattaaata gatattttat tatagttatt attaagttat 64020tataatatat atatatatat agtaaaagga ataaaaagaa aaagaataga aaaagaaaga 64080aaggatgaaa cgaaacagag agcaagggaa agaaagaagg aaggaagaaa gaaagaagga 64140aaaagggaaa attggatttc aaggcttgaa agttaaatag gtatgtcaat ttagccattt 64200ttacttgatt ttgatgtttt agaaacttta gaacaaggtt ttgatgaagt taagttgata 64260tattgtaaga tagttgatta taagacattg ttcttgttga acaaaaagat gaattaaggg 64320ctaaattgat agaaattcaa gttagaaatg aaataaggat tgaattgtaa agtgattcat 64380aagttttgaa tagtagggac taaattgaag aattttgaaa tcatagttta tggtgaaatt 64440agagagctga aataagtttg aagtgaaaat gaaatgaaaa tattgagtta aatatgaaaa 64500ataaaagtta gtctcggttt agggactaaa ttagaattaa ggtaaaagtt ggatagaaat 64560tgaaatattc aatgtgaaaa atttaatgat agtgtattaa taatatttaa ttaattcccg 64620tagctaatga tgtctcggaa aatctttgtt aagcgaggat aaggcaaaga caacgggatt 64680tagctcggaa actacggttt gtatttctat aaactgaact taatagttaa ttgttatgtt 64740aatattcgaa ttgcttgaga atggaaatgc taaggtaaga attataatgt tttataattt 64800attgaatttg attattaatt gttgtatcat gattgatatg tgacaagtaa ttaaagtatg 64860aaatatttga atgtgtgatt attggaaaat gaattaaaag gcatgttata ttaaaattga 64920aatatgtata ttgtattgaa attgaattgc atgtgaattg atatggaaaa gtgtattgaa 64980atgaaactga aaatttgaaa gtttactaaa atccctatta acaatatcgg gctagtcgga 65040aataattggc atgccatagg attggaagtg ttcagggata tttcgactgt gtgtcgataa 65100gacactatat gtgtcgacta ctgtgactgt ttcggattca ttctgaagag gtactctata 65160cctgactgtt actgttattg tttcagattt gttccgatga ggtactttgt gtaccgttac 65220gttactgtta ctattaccgt tacgatgtat ttcggcttca gccaatgaaa cactgtatac 65280tatccccggt gtgtgggttg gatccgtgta tccgtctagg tccaagtcat gttaataagg 65340gtaattaaaa gtattaaagg ttgactgcta cggaataatt gactgttatt gataaataat 65400tgttactgca taactgactg ctatagagtg ccggaatgtt actgtataaa accgataaat 65460tgattgatac tataaatgtt tattgatact gaatcaagga ctgaagtatg agtaaaacat 65520gcgaatggaa agtattaatg tttagatgat ttatgaattc atttgaaaag ctaatcgagt 65580taataaatga taaaaattaa gtgaattatg aagagtttat ttatgaattg aatgattaaa 65640taattatgat cgttatatga ttttatgtat atattatatt ttaagtatta gtttatagaa 65700attgtaatac cctaacccgt atccatctcc gaaacagggt tacaaagtgt taccaataca 65760tacagaacat ttacagatta atcgaaacat tactattcac tttctgagat catatatata 65820taacgacctt tatttaggcc cttgaagccc aacatgaaca ttaaaatcaa gtcggagctc 65880aactgatttc tcgtaaaatt ttccgcttaa ttaatttttt ttaaaaagtt tacttgtgaa 65940cagtacccac acgcccgtgt gattaggccg tgtggattcc acacgcctgt gtggcttggg 66000acacgcccgt gtcccttgtc cgtggagctt tctgtttatg acatcatcat caatttaggg 66060gcacacggcc acatcgcacg cccgtgtcct aaagtcttgt ttcatatacg gctgagacac 66120acggccgtgt ctctgcctat gtggtcaata tctaagctat tttccaagcc ttggtcgacc 66180ttaatctctt acacacttat acaaaatcaa aagcatataa catggtattc atttaatgat 66240taaacattct caattaaact acaaacatag catttgtatg tcatcataca tgtgtctctc 66300atactcattt taccttgtct attatggtac cacttataat tttataccat gattatcatc 66360ttaccaaata ttttcagctt aatcatcaag catatatatt taaagctaga tcatatcttt 66420ataaaatacc acatttcaga tgcgcggaat aaaatacttg ccttagacat ttcaatccaa 66480cttcataccc aacaagcatc atattgaaac tagtcacata tatacatgtc atgatatgta 66540tcattctcat actgttttct tataaacata tatcatttgt tttcctcctc ctcctctcca 66600ttccacatcc ttaatgtata taacattctt gtaagtgcaa tttcacaatt tacttattaa 66660tgcttacatc aagctgttta cacgagtcat agtcactcaa tcatttataa ttcaagctac 66720agagctcgaa attaagatcc gtaaatttcc cctgaaacta gactcacata tcattccaca 66780taaaattttt agaatttttg gtttagccaa ttagtacagt ttattcattt aattttcccc 66840tgtttcacta tcctacggtt ctgacctctc ttcactaaaa attaattata tcatagtaca 66900aatctcggat aatgttccca ttgatttcta ttgaaaatag actcattaag gattctaagc 66960atataaattt gagcctctaa ttaattttat ctaatttttg gtgattttcc aaagtcaaaa 67020caggggaacc cgaattcgtt ctaaccttgt ctcacaaaat tcattatatc tcataattta 67080caattcaatt gcttacaccg tttctctata agaaactaga ctcaataagc tttaattaca 67140tattttattc atcctctaat tcaatttata caatttatgg tgatttttca aagtcaacct 67200actgctgctg tccaaaactg ttttagttca agatgtttat taccattttt cctctaaatt 67260tcacagctca tacaattcag tccttgctca attagcccat ctattaagct aatttttctc 67320aattaacatt ttattccatc attctaaact attacacaac ctttgaaaat cataatttta 67380acacgaaacc ttaattcaca actttttcac aattaggtcc taaaatcaat ttctattcaa 67440attacttgat aaaatcatca aacaacaaaa tcaaagcttc aaattcattt tatatcatca 67500taaacagcca acacttatca attatagctt ttaattttgt tcataaaatc aaaaactaat 67560gaattaaaca cttggaccta attgtaaaag tcacaaaaac ataaaaatat caaagaaaag 67620ggcaagaatt gaactcacat atgtcaaagt atgaaaaacc agcagctttc agacctccca 67680tggcgttttt gctgaagaaa aatgatgata tctctagatt tttctaattt gtcttgtttt 67740atatgtttaa tttacaaaat ttcccatttt gcccttgttt ctccttgtct tttttgttga 67800ttttcttgcc caaccgtcca gcccatacaa tttgggtcca attgcctttt aaatccctcc 67860tttttgatca cttaaactat ttaatcacaa tttaataaat ttggcactat tttcaattta 67920gtctttttta attcattgac taaccaaacg ttaaaatttt ctaacgaaac tttaatacta 67980acttaataac actccataaa tatttataaa aatatttatg gctcggttta tgaattcgag 68040gtctcgatac ctcgttttca ccctaatttc ttgattaatt cttttaaagt cgcaaaattc 68100actaattaaa aaataattct tttaagttcg cgcttggcct ataattatta tttgttaaaa 68160tttctaaaat tactcgtcag atttagtgat ctcgaatcac tgtttccgac accactgaat 68220aatttgactg ttacagaaat aacactgagt tcctactcag cgtacaattt gtttccgtgc 68280gcaggttaag gtaaagtcag attgttgagt cagcattcca ggccgatccc gaactcaata 68340aggtaaagta tgttaattga tgataatggc atgtacctag gatgtcttaa gtgtgtcata 68400ttggattgtg attgtaatag tgaaataagt aaattgataa ttgataatga tacgtgataa 68460gtgtttaaag taagtattgg atagtacata aaatgtgttt gaaattgttt aatttaagac 68520attattaagt atatgtgtta aattatgata tgatggttta atgagtatta agtgtgttta 68580accatatttg gactaattga atggagaaat tttgaaatgc ttgttgtcta caatttgcag 68640gattggtaaa ttttaaaata caaggttcat tttgagacca cgtgatttgt cacacgggcg 68700tgtgccttgg ttgccacgac cgtgtcttaa agtcagttta gtacacgggt aggccacacg 68760gacgtgtgtc atggccgtgt ttaaaagtca gtgttgtaca cgggttaagg acacgggcgt 68820gtcccaagcc gcacgggtat gttaaattta gccacacggg cgtgtggtac tgtctaaaat 68880aagaaaattt aaaattgtac gaaaaatttt ctaagcttcc gatcgagccc cagtttgttt 68940aattattctt attaagtatt gtggacccac taaagctaca taaagaaatg tataattctg 69000tatttattct tgttttacat ataaatatac tgtattgacc ggtaatactc cgtaatcctg 69060ttccggcgac gggacgggtt tagaggtgtt acaaaatcca cctagacttc acttgttatt 69120taactttgaa tcagacgatt tattcatttg acttgatccg tagaaatccc taagttatat 69180tattatctct ctcgagacaa ataatgtcta accctaggtt gaataattga aatctttttc 69240taattaacac tctagaattg cattaactcg atttatggat tcccttatta gttttcaccc 69300taatccggca aaatcttatc accctatctc taggcgtgca atgaactctg cttaattata 69360acaaatttac tcttagacag ggtctattcc tcctctgaat aagagcttaa cttgaatcaa 69420tatcctggaa tattaaaaaa agaattaaga acacataatt aagaacaagt caaatattta 69480ccatataatt cagataataa taacaagatt cgttttaggt ttcattcccc ttaggtattt 69540aagggggttt agttcatact tatgaaagaa aacctctcag aagcataaag ataacaaaac 69600ataagaaaac ccaaaactcc tgaaggaact tgaagggaga tcttcagtct tgatgatgaa 69660tccggcttct gagatggatc aatcggcttc ccttgagtaa ttccttgctt cctactttgc 69720gtcccccttc taagtgcatc ctcaggtgtt taaataggct ttggaatgcc tatgagccct 69780caaaattggc cttttccgaa ttggactaaa cttgggctcc gcagggacac gctcgtgtac 69840gattacttaa ggctgtggtc aaggctgtta aatgggcacg agcgtttgat ccacccgtgt 69900aagtcatgct tcaatcctgc caaagggaca cggccgtggg acacgcccgt gtgagaaagg 69960ccaggccgtg ttgatttccc gagtgggttc attttctcca ttttcggccc gtttcccgct 70020ttttttactc tcctatgctc acctaagtat aaaatatgaa attaaaggat taggagcatc 70080gaattcacca attctaaaga gaaaccatcc ataaatgcgc taggcatggg ataaaaatat 70140gtataaatta tggtttatca aatgccccca cacttaagca tttgcttgtc cttaagcaaa 70200atcctcaact cacaatcaaa ataaattatt ctcactttat aatctctatc aataatatct 70260caaaataatc tatatgtact catatattga aaattcaact aaaagtacat caaagtttca 70320aacattccaa gttgagcatt ttatcatgaa aacataggtg tctcccctca tctaagtgat 70380tacctttaat caaaatatca cagagtttaa catcctcact aaagattcac tcaaatcact 70440caaggtgttt aaggacatca ataaaagcac tcattagtca atatgaaaag ttattaccat 70500aggcttgctt gaaaatcaaa tctccaccac tataaattga gttgatacat caatcaaaaa 70560ggtcttttag agggttgtaa tcgtggcttt ggttaggggt gtggtcacaa gttgaaagaa 70620gatgttagaa tcgagattga attaaaaaat tatctagcta gaaaaaataa ctagtcatca 70680gttgactacg agtgagcttc ttctcagaag atggaattta aatactgcgg ctcaataata 70740ccgaattact accaatatgt aagtatgaat gtttttttta aaaaaacaac tcaaaataca 70800aaatagaata aaacatagtt aagcaactat tccaactcaa atctcgacaa aaatagggat 70860caaattaatt taggggattt caataataat gagttatggg ttaatattag gggtaaatca 70920atgaatggtt tgttaggctc aagggggttc actaagggtt aattgtgaat gtaggctttt 70980atggagtgag tgggttaaac ctaagtgcct ttatcatttt gacatatcaa atcaaacggt 71040gtggtcttga catgcataat caagcaagtt ctagaataac aattcaatac tgacgcactc 71100ataatgaaag tgagcatgaa agaaataata gatgctctta aaggctcaag atctcacaaa 71160aattatagct ttttgatgtt caaacttgtg aatttcaact caagacaata cctaaactta 71220gggaaacaac ctaaatgttt tttaattcta caaaaatcaa cttattatgc ttgattccct 71280aatgtcctaa agtttaaaca atcaatgcat aaatacctat gttttaattc aagacatatc 71340aataaaaatc ataaattaat caaaattcat tctaatagtg gtatgagtga ttcacgtgag 71400aataagataa aattcaggga tttctaatga tgatatgaaa gaactcccca cacttaagat 71460gtacattgcc ttcaatgtac aaagatagat atattgacaa agatagatat ataatcataa 71520gatagggaga gaagtgaaat ttcctgaatg atgcatggac tccttgaatt ggagtcatgg 71580agaatgaacg gcgaaagcaa tgatgagggt ggaggaggat actctggtag tggtagaggt 71640tgggttccac aaatgctgcg ccaaaagaat attatatctc aagttggcta tggtcgtggt 71700cgagcagggc atggcagtca tggagaacct ttcccagtgg agtttcaagt tcctgagtaa 71760tagtgagatt tggagctctt tataactgtg atagaatcaa aaactttttt aggaaatata 71820taaggaagaa taattactcg taattaatta cccagaaata aaaattgtaa aataataatt 71880ataaaaccta ataaaaataa gcttaaagaa aaataaaaag tagtcttaaa ataaaataaa 71940aggaataaca gaaaataata aataaaagtt tttaaacatc ttcatcgcta gatggttcgc 72000gaggtggggg tggcaatgag atgtggaatt gctaacaaat ctgctgtaga gtagcatcaa 72060tgctatcgaa gcgccgaaaa cactactgct cgaatcaagt aaggcgctca gagatgtcaa 72120cgtatgaagc taccgcatga actggacgat gagaaggtgg tggctgagtc ggtggatcct 72180cgtaacgtgg agggacatca tcagtaatat cctctggtcc tcttcatcgg tggactgggt 72240gaggcagtat tgagaagggt atatgccacg tcatttctcg atcatcctca tacttagcat 72300gctcgagatg ccctgcgggg acatttggct gattagagtg agggagaatg attgggcttg 72360taacaccctg aaaatttcta cagtaagata ttatccttaa tatagtaaaa taaggaaata 72420aagtgataag aagaggaaaa attgagttat gtcactggga agtatattat gacatattga 72480ttaaagaagg actaaattgt aaaagtgaga aaagttttgt agcctaagag taaatactaa 72540aaatttgagg gattaaagcg taaatatgaa aagttgaagg actaatagtg cgaatatttt 72600aagggttgaa tgatctagaa accaaggaaa attgatgaat taggaccaaa ttgaataggt 72660aaagaattat gagggactaa attgtaattt taccaaatta agtgataact caagaataga 72720attttaaaag atcactaagg gcaaaatggt caattggaag agagagaaat ctagagacaa 72780tgatgatgtt ggagatattt tagataaaat aaataaataa atattagttt attaatactt 72840taaattgatt tttaaatgat atttttttat tattttatta ttttatttag tatacataag 72900gaaagaaaga tgaagaatca tcatcttttc tttcccatgc aaaccaacgt gagagaggaa 72960gaagaaaaga agttttcttt ctttacaatt tagtcctttc accaaaaatt cattattttc 73020acctaaaaat taaaagaatt tccatagcca tcaagagaga aagatagcaa ggagatgatg 73080gggagcaaga atatcaaatt ggattcaaga aatagaagct ggaggagaga gaaaaatcaa 73140gttaaagatt gaagtcaata agaaaaggta agaacatcaa gatttcaata tatttttaag 73200tttaatattg ttgaaaaagc atggaattga tgttgattca gagttttctt atatatggtc 73260ttatgttctt tgtcatgtta gtgaagagaa aataagagaa agtaatgaaa aatagcgtag 73320agaaagaaaa taagggtgtt ataaacatgg taaataatac cttgcactaa aatagtttta 73380gacaacaaca atagtctaaa tttgaaaaat caccaaaaat tgtgggaacc aaattatagg 73440ttaaataaaa tatgaaatta aatctcattg agtttagttt cttataaaat aaacggtcta 73500agaaataaaa ttgtaatttg tgagatatag taaattttgt tagataaggt cagaataatt 73560tcgggttcct ctattctgac tttggaaaat cataaaaaat tttagaaaaa taattatggg 73620cttaaattta tatgattaga attctgaatg agtctatttt caagagaaat aacgggaaca 73680tcatttcaat tctgtacaat gagataatta acttttagtt aagaagggtt ggaattgtca 73740gacaacagaa taagggtgac tttaaagaat aaactatact tattggctaa accaaaaatt 73800ctaaaaattt tatggtaaga cgatacataa gtctagtttc atggaaaatt atcagatctt 73860aatttcaagt tctgtagttc aagatataaa taatttagtg actatgacgc aaatggacag 73920ttttgaatat acatataagt aaatagtgaa attattgata ttgttatttg aagcatgtta 73980tataaattaa ggatgtggaa tggagaggag gaggaggagg aggagtaaaa tatgtatgaa 74040tactcatcta gcatggctaa tttgcatgtt ttaggctcag ggactaaatt gaataaaagt 74100aaaactttat agataatttt gtaaaaatat tagaaatgac caatttacat gaaatggatc 74160attttattat ttaaaattat aaaattgaat gaaattatta atttagctca agattgggga 74220aaaacatgtt ttaaggatta aattgaaaat tgttgaaatt atggaaaatt ctgatatttt 74280atagaattca tgggttgtta tcaatttttt tgagaataac ggctggaaat aaggattaaa 74340ttgtaagaat tttatttttt ttagcttaag gatgaaattg tcattaatta aaaagtttag 74400gggtaaaatg gtaattttgt ttagagcatc aatttaatgt attagaacat gaaataaatg 74460aaaacgacga tcaaatttct ttataaagat ctggatgact cgagaatacg agacttgaat 74520gtggaaaaga aaagatatca gattaatgaa attataaaca tgaacaagta acgaggtaag 74580ttagtgtaac ttgaattgta tttttaaatg catgaaatat tgatataatg aattacctga 74640tttatgttta tgaagaaatg gcaagagaat gatatttatc

gtgacttgta attaggcgat 74700tatctttgat acgttgatac aaggaaatta attaaattaa gacgagtaat aaattcaagt 74760acaacatatc aagaaaaata agtacgttaa gggacaatat gtttgatttt aattggttcc 74820aaatatgaac tttagatgaa ataaaatatc tgataaataa atcggtaact ccggtaatgc 74880tctgtaactc tattccgctg acggattcgg gttgggggcg ttacagggct gttgtgttga 74940ggagcccaaa gtgccgagcc agtcagtcac atagggccca atagagatga cccctctcct 75000gtgtcactcc gtctggtggc gaatggccaa ggcaataaag taggcgaggt cgatgacgtg 75060cccattcacc atactttata agaaataggc gtcgtgggtg gtgatgacgc cggtgctctc 75120tcgtctctct atcagagtgt gagccaagat ggcatgtaga tacctcaagg atggagggag 75180agccgatacc ttggagcggc tgggatcgta ggtggccaag gtagggacaa agttcctcca 75240acattttgag agggagtagt ggatgtggcg gtggagggtg ttgagttcat tgtcatccat 75300gaactcctct gtgtatagac ctagtgcaat cccgaacttc ggtacgctca actggcgtac 75360tagaccacca aggcggaact agaccgttcc aggatcgtcg aagttggtca tgacggtctg 75420aagatggaac gtcgagcaga gctccaatgt aaactcgaga tacgttggct tgacggtctc 75480aaagaagagc ccccacgggt cagttattag gagggttcgg acagcgtcag ccaattgaat 75540ctgttcgagt gcgacccagt caatgcagcg gcctacacct aggggtcggg cccgtaatat 75600ttgatataat tcctcttagg gtcccggggg aacctaaaag aacgggtgcc taatctccgt 75660ggtaggactc gaggataatg ctgctcattt tcgattcttc gaggcgggaa caacagtctt 75720cttaccacgt gatgatgaca ttatacctgc gttgaaaaat taaagtttaa ccaatacgtt 75780ccaaaaatag cacagaagca caaaactaaa tagaaaattt catgagactt acgtagtgga 75840tgaagtaaac aaaactacta aatatatcaa gttatagtat tatgggaata atgtaacaag 75900aatatgaatg aatgcatgtg ggaagcataa atttcatgaa actaggaaaa aggaaaaagt 75960agagcataat ggagtaacta aaatgacaaa atttttataa acaagcatga gtgttttaat 76020attctaacta tgaatattct ttcaaaatag ttcaatagag cagagagatc atgaaataag 76080caacatattt agagaaaata gagggaaaag agtaaacaaa cgcaaaaaag aatgacttgg 76140ggcgtcgaaa ttggtgttgt aggcggcgca cgagcgtggg aggaaggcgt gtgaagttgc 76200agcggctagg gttagaaatt tttggggagg gatatgatga atagtgaggg gtttatatag 76260attttgaggc atacggccat ggggcacgcc cgtgtgccct aatttttacc cgtatgtttc 76320gtattttttt gaatttgagc acgtctgaca ttcggcccac gcctgtgttc cttgggcgtg 76380tgggtgcaca cgaccgtgtc acatggccat atgtcgcttc attcgtttct tccacgccct 76440tgtatgaagg tccacgcccg tgttaatttg gcaggttcac tcacgggtgc tgggcacggg 76500cgtgcggtat gttcatgcta atttgacagg ttcacccacg gtttcgaggc acgggcgtgt 76560ctcacgccag tgttgttttg gaaggttcac ccacggctat gtcgcacggc cgcagcaatt 76620tatcgcttcc cgtgttagaa aaattttgcc ctgttttcac acagcctaag gcacactcgg 76680gtgcctggcc gtgtgggttt tagaaagcct gcgttccatg atttggttag tacgttagat 76740gttaaaaact aaaatttaaa taaattaata ctattagtgc tcgggttgcc tcccgagaag 76800cgcttatgta tagtctaagc tcgacttacc tctctggtat atgatcatgg tggatcaagg 76860agtttacact cctcatccct gctatcaatt ttatcaacat aaggtttaag acgagtacta 76920tttaccttaa acgtgccaaa tttgaggtga tttaactcga ccatatcgta tggaaaaatg 76980ctaattatcg taagagaggt tttttcattc ggttcagaaa tggtgattcg aagatctgct 77040gtatctagta gtactttgtc tcgaacttga agttgatttt cggaagaatt aagcttatca 77100tggcgtggtt ttggtttatc gtgttttctc ggtttatgta tccgccattc atctagctcc 77160tcgatttgta accttcgttc ttcatggatg ggttctttat tgttgcttgg cttatgtatg 77220ttcttcgaac ctatttcctg caaagaaggt tgcaccatat ggttagtttt agccgaatga 77280tttgtaaagt caccttcaat tattgatgtg ttattcaaat tacgggtttg aagggtgatt 77340gtttcatctc ctacacgaag tgtgagttca tctgtgccaa cgtcaataat tgttccggta 77400gttgctaaaa agggtcttcc taaaattaaa tggacgttgt tatcctcctc tatgtctaga 77460acaatgaaat caactgggaa tataaattta tcgattttaa tgagtacatc ttcaataata 77520cccctaggaa atctgatagt tttatctgct aattgaatgc tcatcctagt ttgtttaggt 77580ttccctagac ctagttactt aaacaatttg taaggcataa cattgatact agcccctaaa 77640tcaaccaacg cattattaac atctaagcta ccaattaaac aaggaattgt aaaattctct 77700ggatctttta gtttgttgga ccgcttattc tatagaatgg ctgagtagac cgcatctagc 77760tctacatgcg atgcttcatc taacttccgt ttatttgcta gaagctcctt taaaaacttg 77820actgtgtttg gcatctacga aaaggcttca ataaacggta agttaatatg tagtttcttt 77880aaaagtttaa ggaatttacc aaattgtttg tctgagcggt ctttctttat cacattgggg 77940tatggcacac gaggtttata ttctgtaatt actggctttg ccttattttg gcctacctca 78000tctttacctt tacttaccac catttttggc cttagttctg caactagccc ttcctaatct 78060tgaatggcaa ccgcgttgag ttgctccctt gggttagatt cagtgttgct tggtaggctg 78120ccttgtggtc gttcagaaat caacttggcg agctgtccaa tctgagtttc aagcctctgg 78180attaatgctt gttgattttt gagtgctgtc tcggtattct aaaaatgagt ttctgacact 78240gagatgaatt ttgttagctt cttctcaagg ttcggctttt tctcttgttg gtaaggtggt 78300tgttggaagc ctggagatgg tggtctctga ttcccttggc ctccccatga aaaatttggg 78360tggttcttcc aacctgcatt gtaagtattg ctataaggat tgttttgaga tcaaggattg 78420ttacccatgt aatttaactg ttcgttctcc atgttgtggc cataaggtgg gtattctgaa 78480ctgcttgatc cacctccgct cgcttcgcac tgcattactg ggtgaaccta tgaaaaacta 78540agaaaaccat caattttctt attcaagagt tctacctaaa tagagagcat ggtgaccgta 78600tcgatgttat aaatgccagc tattttcatt ggctttgtcc tcgtgacttg ccattaatag 78660ttattcagtg acatctcctc tataaattca taggagtctt catctatctt attattgatg 78720gttccgccag tagctgcgtc aaccatttgt cgagtcgaat gattcaggcc attatgaaag 78780gtttgaaatt gtagccagag tggtaaccca tggtgagggc atcttctcaa gaggtctttg 78840tatctctccc atgcattgta gagtgtttct aaatctattt gtacaaaaga agagatatca 78900ttacgtaatt tagccgtttt aaccggtgaa aaatatttta ataaaaactt ttcggtcatg 78960tgttcccaag tagtgattga ccccgtggca atgagttcaa ccactgttta gctttgtttt 79020tcaatgaaaa ggggaataac cgaaggcaaa tggcgtcatt agaaacgcca ttaattttaa 79080atgtatcaca tagttccaaa aagtttgcca agtgagcatt gggatactcg tcctacaaac 79140catcaaactg aacaaactgt tgtatcattt gaattgtgtt aggtttcagt tcaaaagtat 79200ttgcggctac agcaggtcta actatgctcg attcagttcc tgttaaagaa ggtttagcat 79260aatcatacat aatgcgcgga gtaggattct gattaacagc aatcgtagga ggtagcggat 79320gttcttggtt ttcagtcatc tccttggttg tggttgaatt attgtcctct tgctcttcct 79380ctatgtatcg agcttcgcct tatttctctt cggtttttgc aaactgtgcg atcgatctaa 79440ctgttaaaaa gtagtggttc tggccgtaaa ctagaaaaat ctgtcagaag aaaatgaatg 79500aagaattaga aaagaaagta aaaacttaaa ttgcaataaa agtaaaacgg ctaaagtaat 79560aaaaatcgag tattcctaat atcctagttc ctcggcaaca atgccaaaaa cttggttgcg 79620tgatattcgt aacaggtttt aaatatttat aaatgactcg ttcctgagac taacttatta 79680tcacgattaa gacaagtgta cctatcgaac agtagtatag tttagcaaga ccggattgtc 79740aaacccaaat gaactatgag tactagtatt tacttctttt ttattatcta gcctaaaaat 79800taagaggttt ggttatctaa actaattact aactaagaat gcacagaaag aaaacttggg 79860aaaatacttt tgggaaaatt cgattgattg agacaatacc taagaaaaaa ttcacctaga 79920cttcacttgt tatttaactc tgaatcagac gatttattca tttgacttga ttcatagaaa 79980tcctaagtta tattattatc tttctcaaga ctaacaacgt ctaaccctag gttgaataat 80040tgaaatctct ttctaattaa catcctagaa ttgcattaac tcgatctatg gattccctta 80100ttaggtttca ccctaatccg gcaaaatctt atcaccctat ctctaggcat acaatcaact 80160ccgcttaatt atgacaaatt tactcttagg cagggtctat tcctcctctg aataagagct 80220taacttgaat caatatcttg gaatatcaaa acaagaatta agaacacata attaagaaca 80280agtcaaatat ttatcataca attcagataa taataacaag atctttctta ggtttcattc 80340cccttaggta tttaaggagg tttagtccat acttatgaaa gcaaacatct caaaagcata 80400aagataacaa aacataagaa aacccaaaac tcttgaagga acttgaagcg agatctacag 80460tcttaatgat gaatccggct tttgagatgg atcaatcggc ttcccttaag taattccttg 80520cttcctaccc tgcctccctc ttctaagtgt gtcctcaggt gtttaaatag gctttggaat 80580gcctaagagc cctcaaaatt ggcctttttc gaattggact aaactagggc tcggtaggga 80640cacgccagtg tgacacgtcc gtgtgtgtaa caccccttaa ccccgaactg ttaccggaac 80700gaggttatga ggcattactg gacatatcag acaacttact aataatttgc aattaatata 80760actttcatag tataatacaa taattaagtc cctatcttga actctcgaag ttcaaacacg 80820tattaaaagt agaacgggac ttgttcgagt attccaattt tttttttata aaaatttcgg 80880cagcatttct gcttattttt acctaaacct cctgcaattt caaaccaaaa caaccaacac 80940caatatttca cattcatata actattattt atatcactaa caaaatttta acttaataac 81000tatcatagca tcattcaaaa ttgattaaaa acttcattca tttaacaact taatgttcat 81060gtatcaaaat atcatgtact ttccttacta tttcatttaa ccatctaaat tttataattc 81120atcctttact acccaaagta atatacatat tcaagtgact aaacaacacc tatgtacatg 81180ccacttttac ccaaaagaaa aatatacatc accaaatttg tgttggagtc gggattgttc 81240tggatgctga gccgggacac ttgacttcta ctaacctgca cacggaaaca accgtacgct 81300gagtatggat atactcagtg gtattactat aaatcaaatt atatcaacaa tagtaaaaac 81360ataaatatta aaatacctaa caattaatgt catatgtact aattcataaa tcaatgtatt 81420ttctcaaact taaatttata tcacttatga atatacattt catacttttc tcttattatc 81480acaattccaa tttcaattcg taattcttct catttcaatg cctcaaattc acatttcaat 81540ttttcaccct attaacgtaa ctcggacttt ggcggataca cggatccaac caaacacacc 81600aatatggaac tcagtgcctc atcggatagt tcgaagtaat agttgacacc cagtgtctca 81660tcggcctagc cgaagtaaag ttggtaccca gtacctcatc gaatctatcc gaagaaatat 81720agtgacaccc agtgtctcat cgactcgagg tcgaagtatc ccttccaatc ctatggcatg 81780ccaactatat ccgactcagc ccgactagtt aatagggtat tcaattcact ttctcaatcc 81840tatatcactt tcaattcacg attcaaatca ccaatcattt ttcaatcaat acgcttttca 81900aataattaca tattccatca ttcacttatt caattcaatt tgattcaatt tgcatcactt 81960tcattttcac tcaatattca tatcaatttc acatacttac ctcaagtctt acttaccata 82020cataacaata aaaaaattca gcaatcaata ataattaaaa ttcgaattat agtaatacac 82080accgtaactc tcccgttcct cgatgacttt ctcctttcct ttcgatgctg atgcttcaag 82140ttctttgttg gctattaaac ttccaagctt taaaatccct atttccccta tttcctttct 82200ttcctttctt tttctccttc tttctcccct gtttcgtttc ttctctgttt cttttgttat 82260gtttcttcaa ttcttttatg ttatatttta tatttaatta atttaataaa tatctatttt 82320aataacaaat actaatatta caattggata catatttaat tttacatttg tatcaataat 82380tttattacaa ttgtcattat tttatttgtt tcttaaacaa aaaatctcat aatttaataa 82440tttaattatt taattaacaa acatcttttt acttaaatta taagtaattt agtatttaaa 82500ttacaaatgt accaatacaa ttcttacaca tgtatatttt attaccatac aattgtcttc 82560tatttaattt atttataata tattatcaat taagtaatca taataattta atataaaact 82620ataataataa tcattataat atatatataa tttaatatat aaaacctaaa taaaatcttt 82680gattttattt caatatgccg cctcaattta tgtaaatggc ttaattgcca ttttgatcct 82740ttttattttc tattacttta gaattaaact tttacccttt ttcaatttag ttctttttgc 82800taattattct aaattaagct aatttcacct aattaaatct taattagaca caccactagg 82860ctcataaata tttttaataa ttatttttga acttatttca ctaagacgga ggccctataa 82920ctcacttttc cggtgcccgt gaatttcagg tcattacagt gtgtgattac ttaaagccgt 82980ggtcaaggct attaaatggg catgggcgtg tggtccactc gtgtaactcg tgcttcaatc 83040ctgccaaagg gacacggcca tgggacacgt ccgtaggaga aagtctaggt cgtattgatt 83100tcccgagtga gtccattttc tctgttttcg gcccgtttcc cgctcttttt actctcctat 83160gctcacctaa atataaaata tgaaattaaa ggattaggag catcaaattc accaattcta 83220aggagaaacc atccataaat gtgttaagca tgagataaaa atatgtataa attatggttt 83280atcatttaaa caaaataaac ataatttgtt ccatctattg ggaatgctag aatctcttca 83340ttacttactc gttcactctc tgtttcttct tcatctgttt ctacttcttt gttttcatct 83400ctaggtgccc aaccatccct tttctcttgg tttttgattt aacatagtag ttttctgttt 83460agttgctgag aaagtgaaag aaaataaagg aaactgttgg tttttgtttt tttgaaaggt 83520tcaagcttga taagcttcca aaagaatatt tgtttctcat ggtcgttctt cagctgcaaa 83580taagcaggtc taatgacttg ttcgcgaatt tgaaagtgtt gttttattaa aataagaaaa 83640aaaaagtaat tagtaagtga attaaatcaa acatgtaaat attctagttg atttctttga 83700gttttggctt ttgtcattgc aaaatcaaat cttgcgtaat atatctatca ttttagtcca 83760acctttattg ttgcttagtt tagcatcatg gctgcagttg taaaaagttt ggtctgaaaa 83820aaaagtgcat ttggaaaatg ttgctttgaa aaattatggt ttgggaattt taatctttta 83880gcattgctgt taactactac tatgaaaagt taaaatgtca attttaaaaa catgatctta 83940gaagataggc gagacaaatg atgttttttt gcatttttct ttttcaaaat catattttca 84000aacagcaaaa ccatcaaagt gcattttcct tgtttaggtc aaaaagtatt ttgtcgaaaa 84060gctttggtta ctaaaatcat tttagcaatg atttgaccat caaaaagtaa tttttgtgta 84120acatccctta cccgagaccg ttgcagagtc gagcacaagg cactactaaa cttatttgag 84180cacttaacca aattcagata atttatatca tacttttcag ataagttgtc caactgcgtc 84240atagttgcta aataattcat atctcgagtt ataaaactca aaatccaaat ctgtaaattt 84300tccccgaatt tatactcata tatatactta caaatttttt tctagaattt ttggtgaagc 84360caattagtac aatttattag ttaaaatctc ccctatttca ggatttgact actctgacct 84420ttgtgtatta cgaatcagat atctctctgt acagagcttc gataactatg ccgtttgtct 84480ctaataaaac tagactcaat aaggaatctg taaatataaa ctatgacttc taattatctt 84540tgtaaaattt atggtgaatt tccaaagtca gaacagggga tccagaaatc gctctggccc 84600tgtttcacaa aaatttaaac atctcataaa atatagctca tatacctgtt tcgcttcttc 84660catatgaaaa tagactcatc aagattcgat tccataactt attcattatt taattccatt 84720tatactattt ttagtgattt ttcaaattca aactactgct acttaccaaa aactgtttta 84780gtacaaatat tgttaactag tttataacat ctttacttca attcattcaa actctataca 84840tgccatataa atctttaaac ataaaacaaa aactaccgga attgatctga atagtgtgcc 84900ctgttgtgtt gatccgatct accaacttct ctttaattca atctacaaaa gaaattatca 84960aacacacaca agtaagctta ttgaagctta gtaagctcat aggcataaaa acacaaatca 85020tatcaaatat tgtacacaat catatatcta ttagtttaac tatttcatcc tccaaatcac 85080aatttcatta ataactcatt ggaataattt ccatatggct actcacaatt taactccctt 85140aggcccattt ctcatttact tcattgtcaa attagggaac aataagggaa ttgagtgctt 85200cattatcaca ttgccatact aaattatgga ctttcacatt gttacgcatc acacactgaa 85260gccatagcct tgccatggtc ttacatggtt cacatatcat accgaagcca tatcccagac 85320atggtcttat acggaatcac attatcacat tataccgatg ccatagccca gctatggtct 85380tatacagagt cacattatca cattgcaccg atgccatagc ccagctatgg tcttaaacgg 85440gcgcacttat cacatatttt tcgtcaattc atcagggtca cagaatagaa acactcaaat 85500ccattgttcc tactaatttg tacttttagt tacacattat ttattagtta atgcaaattg 85560atagtattca tcaacaataa aatactttaa caatcataag actttcataa caaaacattg 85620aactttgcca tatgaactta cctggactaa tttgcaaaag tcgtagaaat taagggacta 85680ttcttgaatt ttctcctttc cacgattcag ttcgttttct tgatctataa ttataaaatc 85740attccttcat tagaatccat tccaattcta tttcacttca caatttatgc tgttcaaatt 85800tcgaaattac acttttaccc caaaatttac agttttcaca atttagtccc tgctcaattc 85860acccatcaat tgaactaatt tttctcaatt aacactttat tttatcatta taaactattt 85920caaaaccttt tatattctta atttcaacag caaccttcaa ttcacaactt tttcacaatt 85980aggtcctaaa tatcatttcc tatcaaaatc acttaataaa accaccttaa tataaaatta 86040gaacttaaat ttcataataa ttcatcataa acttccctta tccatctagg gtaacttcta 86100atttcaccca taaaatcaaa aactaatgaa ttctataagt ggacctaatt gtaaaagtca 86160taaaaacata aaaattatca agaaaaagca agaattaaac tcacatgatg taaaatatga 86220aaaaccagct ttctccagac cttctatggc attttagctg ataaaatatg aagagatctc 86280tagattcttc aattttattc ttattttata tgttaaagtt ataaaatttc caattttgcc 86340cttatttccc ttatttttct gctgattttc ttgcctttgc cgtccagcct attcactttt 86400aggcttaatt tccctttaaa tcttccttct tttaacactt gagctattta atccttttag 86460caaaatttat ttttattaca atttagtcct ttttatttaa ttgactacct aatcgttaaa 86520atttctcaac caaactttaa tactagctaa atgaaacttc ataaatattt ataaaaatat 86580ttatagcttg attttcaaat tcgaggtctc gatacctcgt ttttgtcccg tttgacctaa 86640taaattcttt taattcacta atttcaccat ttcacgaatt cttctaaatt cttacttgac 86700tcataaatat taaattacta tcttgttcaa tcttatttgt cggatttagt gatctcaaat 86760caccatttcc gacaccactg aaaattaggc cgttacattt tgatagcttg aagcattact 86820aaacaagaac ttgttttcct tccaaagtgc gtcaatgaag aagcattaga gtttagagat 86880gttagtggga aattataaag gaagatttaa gagcagaatt cccaatgtac ataaaaattt 86940tgttccaatc gatgggtagt agagtattga gtgacaagtc tttttccttt ggaaacagtt 87000acccaaagag ctaatgaaat caaatttgga gagccttact ggagaagacg aatcactaaa 87060gaaaaagaat caatccttaa aagtgattaa tgcttccatg aaccaaacct tgacggatca 87120agcaaaaaag attgatcagt tgaggagtga tcttttgcag ctgaagaacg accatgagaa 87180gcaaaatatg aaacaactga agggcttgtt tttgaaacag cagcatacat tagtcagatc 87240gattttagtt ttaacatcat ctcaagctta gtatgagaac tattttgggg ggttttgata 87300cttttagaaa tagttgttat tttggtagga ttacaaatat tgcttttatt aagttggagg 87360gtcatgatta tgctgataat caactccttg aaagaatata tttgaaagaa tatataaaga 87420atgttgaagg gtttatttta attgcatgtt catgatgggt ataataagat ccaaaagatt 87480atgatttttt gttcaatcat aaataagtga tggtatatga tttctttttt tttctaagaa 87540aagaatagat aatgcatgga catctccctc taggattaaa actttagcag ccaaaaagaa 87600atgttcaatt tgtgtatata taaaaaataa ttaaaaatat agagtgacaa aaatgaatat 87660taataaccat taattacact aaaagcaatg tatcatataa aatgttattg caataaagaa 87720tatataaaga tgattacttt aagtaatagt aaatgtgttc gtagcaaatt acacttacca 87780cactctttta aggttagtta acgtatcgtt atgataataa taataatatg attgtttatg 87840tttccatagg gtgtttatta aaaaatccac agtattttac ttactattaa acaatgcctt 87900ataatatata tttcaataat cattaatcct tacataatat aatatcactg ctataaatga 87960atcaattaat attgaaaaaa gacaaataaa taataataat aaaaggaagc aaaaagagaa 88020gtgcatggaa aacatgttga actttcccgg tcagtgcaaa tacttatatc gttgttttat 88080taaaaattat aaatgaatat cagatatctt catgagctac tagtgtatca aatatcagtt 88140ttcatatatg tatgtatgca ctttagtcat ggtaacttta ttcatatatg tgatcttatg 88200aacaacttgc atcttttttg tagtgatcaa atatttgtgt ggcattattt tgtgtagatg 88260gatcgtagtc aagatcaaaa tgcaattgtc ggagtcgtgg cttcagtttt agcttttggg 88320gttctttgga ttaaaaaatt aaaaactaga aaagaaattg cttctcactc tcgtgtgaat 88380cgagattatg aaagagaaaa ttatattaat agtattttat atagtggtga ccaacattgt 88440attaatgtga taaggatgag accgattgcc ttttttaatt tgtgtgatat tcttagtagg 88500aataatttgt tacaatcaac taaatctgtg aatattatgg agcaagtagt tatattttta 88560catataattg gtcataatgt aaggtttcga gtgattagat ctagatatta tagatcaact 88620gagacaattc accgttactt tagggttgta ttgagagctt ttttgaaatt gtataaacta 88680gttattagat tacctgatga gtcaactcct agtgaaatta gaaacaatcc aaggttttat 88740ccttatttta aagattgtat tggggcaata gatggaactc atgttcgtgc atccgttcca 88800cttagcattc aaggaagatt tcgtagccgt aaagggggga cgacacaaaa tgtattggct 88860gccgttacat ttaatttgaa attttcctat gttctagctg gttgggaagg tagtgcacat 88920gactctcgta ttttaagtga tgcactttca cgcccaagag gattaagaat tccggaaggt 88980aataattatc attcaatatc aaatagttct agtaagctca taatttatta gtagtaatta 89040tgttttgtaa aattgtaggt aaatattatt ttgctgatgc tggatatggc gtccgaaatg 89100gatatattac cccatattgt ggtgttcgat atcatttaaa agagtttagt gctcaagggg 89160ctgaaaatgc aaaaaaattc tttaatcttc gacattcatc attgtgaatc actattgaac 89220gtgtttttgg gattttgaag aaacggtttc atgtattaga tgctgaacca ttttggaatt 89280ttcaaactca agtagatata gttttggctt gttgtatcat tcataatcat ataatgggag 89340ttgatcctag taatttactt aatcaatgat tatacgagga gcctgagtct aatttgataa 89400tatcaactct tacagagcga gaagaaagag aataagtaag agaatggtct gctaagagag 89460atgaaattgc acaaactatg tggactgatt atatggctag aaatattagg taggtttagg 89520gcttagggtt gttgtttcta tgttatgtat gttttagttt ttttttgtta atattggttg 89580agtaatgata ttgaaatttt agtttgttgg attgaaatta ttatgtcttg aatttgttgg 89640atattgattt ttatttcctc aaaacttcga caaacaattt ttttctcctc ttacttactt 89700cagctaatct ttgtgcatag aaagggacca aatgtaatat

atacgggtaa ggaaaaaaaa 89760gagatgaggt gttggataag ttgtcatgac cgattagtca tgtcagcagt taggtgatac 89820caacctgacg cattgtaatt aatgtgatca atcggtcacg atagcctatt taacacatcc 89880tactcttttt tttctagttt tttttaattt ccaattggtg tcgccaatat atcaggcacc 89940actttaaaaa ataaattttt taatcgtacg atatttgttg tatatttaaa gaaaagacct 90000gtattgttct gtgttgcttg acaaagtaac aacactactt aaaaaatatt tttttatcta 90060gaataattat caaatgatga ggtgtttttt tttaaattgg tgtcgcctag catattgaca 90120gcacctaatc ttattgtaaa aaacatacta ttctcgtaaa taatctacaa catgtctcat 90180tttcgtaatt attatttttt gtattatata agtaaaaaaa ccagattttt tattatttat 90240ttcaagtaaa aattttgaat atattgtttc caaacaaaag aaattgtgcc aacttcccga 90300tctaaatatg atatataaga aaattgatct ataaccttat tccctactta ataaaaagcc 90360gaaatttaaa tttcttcaca actaagcaat gaatttcctt cgtcatagtg agggtaatta 90420agtaatttca ttgttaaaag aggttaatag ataaaggaaa aaaagaaagt agagaagata 90480attatgatat ttattcctat aaaatgtaat tatttaattt aaaaaattaa atttaaaatt 90540atatcattat aatagattat ataaaatacg aaaccaacta 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tatccataca gaacataaca 93120gattgtcata tagcatatac taattcaatc acacttcaac cgtaacatgt ataattccat 93180tcatgccaat ctttcaatca tttacaacca cataatgtca tttccttttc tgtcctttgc 93240tcgaaggcta cagtaaatta aactcaattg cacggaattg atttacttac atatatagat 93300gtatttggcc tgctaacact aatttcggat caagattgct aacactagct tggtttgatg 93360ttgctaacac tagctttaga gaatctgcaa catatgttgg atctcaagcc atcaagttaa 93420ttcctgatca caactcccat ttattcatac agactgatat acttgagccg ctaactttag 93480cttcagatca aggttgctaa cactagcttg gttcgatgtt gctaacacta gctttaaaga 93540attcacaaca tatgttggat ctgaagccat caaattaatt cctaattaca gcacccattt 93600attcatacag actgatatac ttgggctact aacactagct tcatatcaat gttgctaaca 93660ctagctttaa agaatccgca acatatgcaa gatctcaagc catcgaatga cccctgatca 93720taattaaatg tgctcatcgg tcgggtcagg cctaagcata atattaacat actttatgct 93780tacctaataa gtgtggcccg actcgaaaca tgggcctaaa attttgtcca aatccatcca 93840tatttacaaa agattaaccc aagcccattt tatgcccatc catattattt tttaaatatt 93900taaaaaatta tttatattat attattttaa tatttaataa aattttatat atttttattt 93960attgaaagtt tttatatagt catcttaaca ttattttaat gtttatatta aagtagtatt 94020atatattttg tataagttta tttttttaat gtgttctaaa ttacattata tataagaata 94080acataatata aagtattatt aacttaaaaa tgggttgggt ggggccaagc tcgggcctta 94140aatcttcaag ctcgagtccg tctcatattt taaacgggcc taattttttt gcccaagcca 94200attttttagg cctaattttt ttgcccaaat cttctaaaat ttcaggtgga ccttcgagtt 94260tggacgggta acctaaccca tgaacaggtc taatcataat gcacattttc atttttggga 94320atttaatagc atgccatttg catctcaata gtttactaaa ttcacacggc ttgttatcga 94380atttctatca tttctatttt tgtaactgta tacatttttc atatttcaaa taacaatcca 94440tatacctttt aaacattata gcatcatcct gtacatataa ttcgttcata taataatttc 94500acatctagtc tattttgcat atctatacgt atttagcata tatttcatat atttcaattt 94560agtcttttag ctgccgaaaa cattatactc aacaatccaa actaaaagaa gaaaataaaa 94620tacaaacata tacatatcat aaattaataa agtaaatttc atatgaactt atcagacaaa 94680atctacaatg agcgaaaagt ccaagactaa tatgcatttt ccctttttca cgatttccta 94740tcgattgatc cgaatctcga tctatacggt aattcatttc 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tattttgatt aaaatggaga 96480tgttagcttt ttaggttaaa gtttactaca agtactgtac catattataa atttcatttg 96540aataccctac tattaaaagg aatattttaa ttcttatata tttgaaaagc taacattcta 96600gcccttgcag atttaacttt aatgttaaca gaatgacatg tttaatgatg atcgactgtg 96660atattaaatt aaattaaaaa gttaaacaat aattttaaaa atgagaagcg ataagaaact 96720gaatttgctt ttacccaaaa ttgttttcaa ttttttttca taattattgg tttaaattaa 96780ttttagttta gttcacatta ctcaccatta aaccacatca ttctatctat tttttagatc 96840aataaatttt tttaaatttt attgaaaaat taacttaatg ccaagttaac cattgttaag 96900ccatgttatt ccattaacaa taaaattagc atgtaagaac taaaatgtta acttttcaaa 96960tgtacataag ttaaaatatt attttgaata atagagggat aaaaacaaat ttttgatatg 97020atagaagact tgtagtaaac tttatgctga ttttttcctt ttaactaaat caattaattg 97080tattaattat ttgaattaat taatcaatga gattgtaaaa ataaaaggat tacattagaa 97140gaggtatcaa aattagaatt tgatccaaaa taaaagagcc aagtcgggta accaaaccgg 97200ccgtataggg tgtttggttc atggtacaag tcacgacatg acatgcggca taaatggcga 97260gggcgtgaga gcgagcccaa atcaggacag aaaaactcca aacatttaat gcggcaattc 97320ctttgttgcc taactgttct cattttctgt tatttcgtcg ctcccgctcc tgcgcgccta 97380atgtttcatc caaaattacc acaacaattc caatactctt tgttcacaat ctccacctcc 97440gcttcgctcc tcctcttctc tcgcccatga agcaacgtag ttcgacgaaa atctccctta 97500aatggatccc atttctctgc atctccttct tcatcctcgg aactgtcttc tctaacaggt 97560ctccctttct gtttgttctt tcttttatta atttatgaaa tggtgtcaat gcctttcctt 97620ttgctttttc ttttctcttt ttttgggttt gggtctttga ttgttggggt ggagttattt 97680atttatttat ttatattatt ggattttggt tacaggctat ggattccaac tatattcaac 97740gattgtgaca ctaagaaagt atgttcctat tatgttcaca atgacagttt ttttaacttt 97800agtttttggt tttcttcaac tttttctttt aaaaacaaag ctttattgtg ttttgcagaa 97860gcctgcaaca gataatgatg agaacggtga agttttgaaa acccacgaag caattgagtg 97920agtgcgaaac tgggttctta ttcttttagc taaagttcat gtttttgctt tgggaagtct 97980tcacttttca ctattaattt ctttgcaatt ttttattttt gatgagggcc taactagatc 98040tctagacaag tcatttgcaa tgctccagat taagttagct cccccaggta gttctcagaa 98100aatgaagaac tcggatgcca ccggtgctgt ctcgaccttg gctggtatcg actcgccgag 98160gaagaaagca ttcatggtca ttgggattaa cactgctttt agtagtagga gacggcgtga 98220ttccatcaga gaaacttgga tgccacaagg tcttttatag agcatttgat tcgtttcttc 98280tttgtatttc attatccgaa atgaattctt atgtttatgt ttgagcaggg gaaaagcttg 98340ttcggttgga gcgtgaaaag gggattatta tccgtttcat aatcggccat aggtaaaatc 98400atttttcagt tcctttttca agtgcatgca tatggagatc aaatcttgag cgtgaatggt 98460aatgtttaaa gtgtgatgaa aatgggagaa atgtacttgc ttcagtatga aattcaaact 98520gacgtacgtt tatatctgca tttattaact ccagtgcaac atccgacagc attttagata 98580gagccattga ttcagaggat gctcaacata aggacttcct tagactggtt agacaagtct 98640tcccgtgata atgtcgaaaa ctcttactga catattatcc ccatttcata cctttaaact 98700gttaccaacc ttctaataca ggagcatgtt gaaggatatc atgaattatc tgcaaaaaca 98760aaaacttata tttgtactgc agttgcaaat tgggatgccg agttctacgt caaggtggac 98820gatgatgtcc atgttaatct tggtgggttt gccatctcta atgttagaaa catatagaac 98880ttaacaaaag cttttgattg tttcattagt gaatttgttt atgcatattt ttttctgata 98940ggtaagctag ctgcacttct tggccgttac cgttccaagc ccatggccta tatagggtgc 99000atgaaatccg gaccggttct ttctaaaaag taattagttc tggtattttt tttttcaatg 99060ataatactac tctccaattt gctttgtttt gtcctgcaaa gtttgttata taccttttct 99120tgaattcctc tatgtcctta aggtctgtca agtaccatga accggagtac tggaaattcg 99180gagaagaggg gaacaagtac tttcgacatg caactggtca gatatatgca atttcaaagg 99240atctcgcaaa ctacgttgcc gcaaaccagt gagatcatct atttttatat gaaagtcact 99300tttttttttt tttggctttg cctaattgag gatcttggtt tcatctttag agcccttgta 99360tgtgtgtgca tttggcagtg tcaccttcat ccccttcctt ccccttggtt ttctttttgt 99420tttttaccag ttagatcatg tagcagtttc tttttactta aagcatgtca tcaaaacatt 99480gaagatcggt tattaccctg cataaattct gttatatttt atatcgtaga gctaaatggc 99540agtctcagac tgttgaattt gcattttgtt ttgatgtagt gactttgcac ctttgatcct 99600cggcactgat aatattctac cttaaaaaga caggcatggt taataacatt cagggtgatt 99660ctgactgaat tgcaggcata tattgcacaa gtatgctaat gaagatgtgt ccctcggttc 99720atggtttatc ggcctcgagg ttcagcactt gaatattaag agcatgtgct gtggtactcc 99780accaggtaaa gttcttcaag aacacaagta gtttaatcaa aggaaggtcg aaacaaggat 99840ggattcatgt gaaacgcacg caaaccataa ccataggcat tctaaattag aatgaggtgg 99900gtaaattagt gtttgcttaa tggagattct gttttggaaa tgcatggtgc agattgtgag 99960ctgaaggcaa aagcaggcaa tgcgtgtgcc gcatcatttg attggagttg cagtggaatc 100020tgcagatcag tggagaagat caaaatcgtt catcaaaggt gcggggaagg ggatgctgta 100080atttggagtg ccttatttta aaccgatata aggttttctt ttgggggagg aagggggggg 100140ggtttcgtgg ttgatcaatg gtggaggaaa cttctgcttt caatgcatat cctcgaccgt 100200ttactttcag aaatggaaaa caaaaaaaag gcggcgttga gttgttagat agtatgtatt 100260tatacacaca aaattttgtt ttttctttct catagaaatt atattcattc aagaatgagc 100320ggaattttaa ttggttctca aaagtataat actcaactca aattacaagc tatattcagt 100380ctaagcccag aaaattgtca aataatctgt tggttataag ccgaaatgga taatcccgac 100440gttttcttaa gattgcaaga ttttctagag cgtaatgcgc cagccttcct ctactgattt 100500cgttaaatcc atcaaaagtt ccctctgtct tcaattattt acttttttct tttcattata 100560gttctcatgt ttaagtgtat ataagagctt aagatagaac gtgatttgtg cttaaccaga 100620attgattaat gattgtttat ttggtaataa tcaacaattt ctttttggta atgaggctgc 100680taaaattcaa aattttggag gaactagatt ttaattgttt attgcttcga attcaagtaa 100740aattctcgac acgcaagctc ttctgtttgt aaatccgttg ccaaacagaa cttaatttca 100800ctatgagaag gatcgggatg tttaattcta gaaaatcatt tctgggtttt taaccgtcgc 100860tatattgcat cttcgtagtt ttgttctcca acaatgctcc tcatcccgcg caggagttct 100920tcaccaacat ggaagctgct gttcgagctc atccgctttg gggcggatgc tcagaggagg 100980acctccacag tgctggtgaa gtaagccatc gatacttgca tatatcatgg gatttgggtc 101040taaattactt gcttttatta tttttaagca atttggatac taagcctgtt gaaaagtcaa 101100caaaggtagg aagcaacttg ttaaaaaggt tgagcaagtt gcaccgaatg ttgaaaagtt 101160gaatgggata attacaattt tggtccctaa ttttttaggc catttgcaag ttagtccctg 101220aacctcaact ataaataggc cttttcattt ttcatttcaa ccatcccaac caatctttct 101280ctcttagnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 101340nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 101400nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 101460nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 101520nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnc ttccctggga attgaacttt 101580gtgtgatttt ttagtacaat aatttacacg cttccgaccc tattggaaca acaagtggta 101640tcaagagccg aaggttaatc gtagtatgct ttgtggttgc agtttaaact gatcttccac 101700atcagaaaag atttccttag gtatattgaa agattatgga gaaaacggtc ggtgtaggag 101760cttcaacatc gtccatatgg acaagaccga caattgcaaa tgcaagattg gccgtggaga 101820tctttgatgg cacgggccat tttggtatgt ggcaaagtga ggttctagat gccctttttc 101880agcagggtct agacattgcc attgatgaag agaaaccaga tgatgtacag gagaaagatt 101940ggaaggcgat caatcggttg gcatgtggca caattcgatc atgcctttct cgagagcaga 102000ggtatgcttt ttcaaaggag acttctgcaa ataagttgtg ggtggcactt gaagaaaaat 102060ttttgaagaa aaacagtcaa aataagctcc acttgaagaa aagactgttt cgcttcacat 102120acgtcccaag taccacaatg aatgatcaca tcactaaatt taatcagtta gtcactgatt 102180tgctgaatat ggatgagaca ttcaaagatg aagatttggc tttgatgctg ttggggtcac 102240ttcctgagga gtttgagttc ctagaaacta ctctacttca tggcaggagt gatatatctc 102300tgagcgaagt ctgtgcggcc ttatacagtt atgaacagag aaagaaggac aaacagaaaa 102360actcaatcag agatacagaa gctttagtag tccgaggtcg ttcatacact cggaagaaaa 102420ctcaaaaggg gagatcaaag tcaaagtcca gactcgtgaa agatgaatgt gctttttgtc 102480atgagaaagg ccactggaag aaaaattgtc caaagctgaa gaataaggga aaagctgctg 102540tagatgcttg tgttgcaaag catgatacta gtgactctga actatcactg gttgcatcat 102600catcgtcgtt ccattcagat gagtggatat tggattcggg ttgtacctat catatgtccc 102660ctaaccggga gtggttctct gatttagtag aactaaatgg aggagttgtt tatatgggca 102720atgacaatgc ctgtaaaact gttgggatag gttcaatcca attaaagaat aaagatggat 102780caaccagagt tctgactgat gttcggtacg tgcccagttt gaagaaaaat ctcatctcat 102840tgggagcctt ggaatccaat ggttcagttg ttactatgag agatggggtt ttgaaagtga 102900catctggcgc acttgtgata ttgaagggca tcaggaaaaa taacttgtat tactaccaag 102960gtagtacagt tattggagca gtcgctgcag cttccggtaa caaagacttg gactcaatgc 103020agttgtggca tatgaagttg ggacatgcca gcgaaaaatc cttgcaaatt ctggcaaagc 103080aaggattgct gaaaggtgca aaggcttgca aattaaaatt ttgtgagcat tgtgttctgg 103140gaaagcaaaa gagagtgaaa ttcggcactg ctatccataa tacaaaaggt attttggaat 103200atattcactc agatgtgtgg gggccttcca aaacaccttc gttgggagga aaacactact 103260ttgttacttt tgttgatgac ttttccagaa gagtttgggt gtataccatg aaaactaaag 103320atgaagtgct tggagttttt cttaaatgga aaactatgat cgaaaaccag actggcaaga 103380aaatcaagcg gcttaggacg gacaatggag gggaatataa aagtgatccg ttcttcgatg 103440tgtgccaaga gtatggtatt gttcgacact tcacagttag ggatacacca caacagaatg 103500gagtggcaga gcgtatgaat cgaacattgc tggagaaagt tcgatgtatg ttgtccaatg 103560ctgggttggg caagcaattt tgggctgagg ctgtgacata cgctggccat cttgttaatc 103620gtttgccatc atctgcatta gaaagaaaaa ctcctatgga ggtatggtct ggaaaaccgg 103680ctacagatta tgattcctta catgtgtttg gatccactgc atattaccat ttgaaggagt 103740caaagttaga tccgagggca aagaaagctc tctttatggg aatcacttct ggagtgaagg 103800gatttcgtct ttggtgctta agcacaaaga aaatgatctg tagcagagat gttacctttg 103860atgaatctgc cacattgaaa aaggtagcag ataaagatat tcaaacgagc aatactccac 103920agcaggtgga gtgtactcca aaacaggtgg agtttgagca gatggggatt tgcccagtta 103980ataagtctaa ttctccagcc acaatggagg aattagaggt tgaagagatt ctgacccaag 104040aaccactaag tacaccagaa ccagttgcag ttgcaaggcc acggagagaa attcgtaaac 104100ctgctcgatt tactgatatg gtggcctacg cccttcccgt tgttgatgat attcctatca 104160cttatcaaga agcaatgcaa agcttagaaa gtgataaatg gaaaagcgcc atggatgaag 104220aaatgcagtc tctccggaag aacaatactt gggagttggc gcaattacca aaaggtaaaa 104280gggcaatcgg atgcaagtgg gtattcgcaa agaaagatgg atctcctagc aagaaggata 104340ttcgctacaa ggcaagattg gtagctaaag gctacgctca gaaggaggga attgactaca 104400atgatgtatt ttcccctgtt gtgaagcatt cctccattag aattttgttg gccttggtag 104460cacagttgaa tttggagcta gctcaacttg atgttaagac agctttcttg catggtgagt 104520tagaagagga gatctatatg actcagcccg aaggatacac agatgctggt ggtagaaact 104580gggtttgtaa gctgaacaaa tcgctatatg gattgaagca atccccgagg cagtggtaca 104640agcgatttga tagctttatg agaaggcaga agtacacaag aagcaaatat gacaattgtg 104700tatatttgca gaagctgcat gacggatctt tcatttatct actcttgtat gttgatgata 104760tgttaatcgc ttcgaagagc caaaatgaga tagataagct

gaaggctcag ttgaatcaag 104820agttcgagat gaaagatcta ggtgaggcca agaagattct cggcatggag ataagtagag 104880atagaccgag aggcaagctc tgtttaaatc agaagcaata tctgaaaaag gtattacaat 104940gttttggtgt aaatgaaaac acaaaacatg taagtacccc acttgcttct catttgaaac 105000ttagtgctca attatctccg aaaactgaag aagaaagaga atatatggca aaagtcccat 105060atgctaatgc agttgggagt ttgatgtatg cgatggtgtg tacgaggcct gacatttcac 105120aagctgttgg agttgtgagc aggtatatgc atgatcctgg aaaaggacat tggcaagctg 105180tgaaatggat tctacggtat cttcgaaaaa ccgtagatgt tggtttaatt tttgaacagg 105240atgaagcact tggtcagttt gtagttggat atgttgattc cgactttgct ggtgatttag 105300ataaacgtcg ttcaactacg gggtatctgt ttactcttgc gaaagcccca gtgagttgga 105360agtctacctt acagtctaca gtagctgtgt ctactacaga ggcagaatat atggcagtta 105420cagaagctgt taaggaggct atttggctta atggattatt gaaagacttg ggagttgttc 105480aaagtcacat tagtctatat tgtgacagtc agagtgctat tcatttagcg aaaaatcaag 105540tctatcattc aagaaccaag catatcgacg taagatatca ctttgtgcgg gaagtctttg 105600aaaaaggaaa aattctactt cagaagattc cgacagcaga taatcccgca gatatgatga 105660ccaaggtggt aacaacaatc aagtttaatc attgtttgaa cttgattaac atcctgagaa 105720tttgagcacc tttaggtgta tggcgctcga gagcgcattt ggaggcacta caaaagatag 105780ctttatcgaa tttgaggagt tgaaggaagt atgtgaagat gtgattatcc taatcaaatc 105840ttcaaggtgg agattgttga aaagtcaaca aaggtaggaa gcaacttgtt aaaaaggttg 105900agcaagttgc accgaatgtt gaaaagttga atgggataat tgcaattttg gtccctaatt 105960ttttaggcca tttgcaagtt agtccctgaa cctcaactat aaataggcct tttcattttt 106020catttcaacc atcccaacca atctttctct cttaannnnn nnnnnnnnnn nnnnnnnnnn 106080nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 106140nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 106200nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 106260nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 106320nnnnnnnttt ccctgggaat tgaactttgt gtgatttttt agtacaataa tttacacgct 106380tccgacccta ttggaacaac aaagccgacc tttcaaaatg agacatcatg gctggtaagt 106440ggtcggattt gtgtttctgg tgatttcctt tttgctatat gcagtaaaat taaagacatt 106500gctgcatcaa tcttttcgca taaggccact gatctgtttt atgctctatt gaacatactc 106560tgatttagat tttagatatt ttacagaaac aatagcatga ccattgatgc atatatgtag 106620ccattttgct tcatgctata taatatattc ccttttggcg ggcttgatgc cttattaatt 106680ttcagttcta taagctctat atctgatgaa ggatagtctt aatgacagta aacctatttg 106740atcatgtagc ttactttgag acagtacgca gctccggcag gttgttatgg ctaatatttt 106800gttgtactga gaagttattg aacccagaca aagagaactt tgcgatctga accaaatctt 106860ctgaaataaa acctttaagt agagaagatc tgtcatcaat tactaaagtt ccatcaatct 106920caaagctgga gaatcaaggg gcaaccatgc ttctgcagga ggatcagaca aggaaagtgt 106980tcgagagtgc ccatatatgt ttgttcattt tagtgacctg aaaatcaatg atgttgagga 107040cctgaataac tataaacaac ttgtcttcaa gtatgtttgc ttttgaaagg actgggtagc 107100gtttctgcaa ctattccttc ttccagttca ccagctctag ctccagaaca tgttggaaca 107160gtgaaggaat ctgaagataa tagagtgatg gaactaaata ttggactgca aaaagatact 107220gacagggtgg atgatatccc aagttcagtc tctaaacaag aaaatagagt atccaatttc 107280ccaaggatga ggcagttgca cccccaggag acaaccatga tgacacctct gaacaatagc 107340tgactgggtt gatgttaatt tttttggaat atcatggcct ttcattgttc tggtaacctt 107400gccactagga cttttttctt ccctctttta tgcagcaaat gactactgct gcagctgcaa 107460atgaaaagac aatgtaagac caagttctta atttgaactt tgaaatgatt tggtttggaa 107520attcataatg ccctgtagat gcaagttttg ctgtaacact gtgggttcaa ctcggcaaag 107580cccttatttc agcacatata ggtacaattc tttgcttcat cctcgtacac agtatcgtgg 107640ttcgttacga atggtaggaa agctgtaaac ttggaacttc tgttttgaag taagatgtgg 107700caaccttttg gatcataaac caacattgac atgccaggtt aaacatgtag ataccttgat 107760cattattgaa ttattggatt gatttattta ttttcaaacg tttttgtttg gattgagtag 107820ctttcttgtt ccttcatacg catcatcatc ccatccccaa gattgagaga tggtgcatta 107880tgtcatgcca tagatatttt gtccaaataa aatatcgtat tatgcctgat atttcttctt 107940tttttccttg gggaagtgaa tatccgatca agaggccaca acttggaatt tgcttgatat 108000ttctttttag atcttaggct aaatgcaccc ggaatttgat tcaataatag atgcatagat 108060cttctctgaa aggcatgctc caatcacttg atggagaata cgaaggcaac tgattttccc 108120agtaagtgag tgatacaacc aaatagatat gacaaaacaa gtaatagttt atttaaaatt 108180tagacaaaaa ttttctcggt attttgacag tattttattt ataattgtat acaggttgga 108240gggtaaggag agagaagagg caacagtgac agataagcat atctttgatt ttctaggcaa 108300tctctggcaa tgcaatattg atgacactga tcaggtacta acataaaaat taaaacagtt 108360gcttatattt aatcctttct ccatcctact ctttaaatta gacttctcca tgtgcttgga 108420ccggccaacg actcaattta ttatttaaaa cataacatta ttttgtatta attttatatg 108480ttttatactt atatttaatg ctttgacatg acttggattg aagaaatcaa atcaatttga 108540ttatatgaat aatccgattt ttggacagat cccgttgttt acataaaaag catattttat 108600acttatagta ccccttgaaa aagccttcat cttcctcctt tactcctaat ctctttcttt 108660cctttattcc tttgttttct tttcagattt gttaaaagaa aagcaaagga ggggccaacg 108720cagcatggga aatagatata attgccatca gaatctacgt ttgaatcata caaagagaag 108780catctttttg ccgatgttgt gttcgaaggt atccattaat aaagatgtga tgaagcttcc 108840caaatggaaa gacaagttac cggatgacga ccctttgtct ccaaagatcg gttgcatggg 108900acaagtgaag aggaacaaca ggattgttgg cttccctgca ctcgatatca ccaccaagat 108960caacaacagc tgcaatgcta ataataatac caatgataat ggcatcaagt atttcaagct 109020caagaagttg ttttccggca aaagaaaaca agggtttgta gaacatggtg ggaaagagaa 109080cagtggttcg cgttcaatca acatcgaaaa catggatccc cctttgcctg tgatcaagag 109140agtgccgaaa caaggtgata aaggagaagg agacactctt tggcagagga gatctcgtgg 109200ggtttcattg gaaagcttac agcttctaca gattcaactt aacagacgtc gagaaccgac 109260cactgtttaa taactagggg gaacactaca aggttagata cgtgtatatg atttgttctt 109320aaaatttcca tgttttttta tgattgtggt gttgaagaga ggcatcgaat tatggtgatg 109380ctagttaaga gatgtatatg aattattcat caaagattta ggaattttta tctctcaaaa 109440ttattgatgg aaacaccatc attatatttt cttgatgatt gttatgtcaa tgtggaaatc 109500aagtgagtat gcattgtggt gttagatcaa gtgtctattt caaataaatt tcacatctgt 109560gataagctcc agttgaactt taaactaaat aaagggttta aaacttatga caaacgtaat 109620tttttcttgt attatattat atatctacta accagatcat gtcataagat aaaaaaggat 109680gggataaaaa atttaatttt gcctttcacg acaagctttc tttaagccca attttttttt 109740tgtctgcaat atttgaactc aaaattttat tcttgttttt tgatgaatga attaatttac 109800accagtctta taactagaca aacaataata aacattgacc aaatgccaaa agatatgaat 109860aaatttatgg tcggcagcac agcaaacagg atccacttta aagtttaaat aagtcaaata 109920atttcagcat tcaactccta cgagtgcaag gagtgtttag tcttcaattc agaatctagg 109980tagtctgata ggtttaggat gcctaacttc gataattgat ggtggatatt gaagtttgct 110040taaaccctaa atgatttttt tttctagaca aataatgttg ccttttgtat aagatttgat 110100aattcaccaa aaacatatac tgctttcttt cacatccatt gtcaaattaa ggggcagaca 110160aacataaaaa attaagggac aagacatgaa cgatttatat aatttaaaga cttgagtttc 110220gaatgtcaaa ccaaatctaa agggtgggta caagaaagtg aaagtctcgc agtgaatatt 110280tcaatcaatg ccatttcttc aataagttta atacctgatt tttaaaatgg taaactttgc 110340ttctttttta acgtctaaac cacacacttc gagatttcat tcagtttcag catagtattt 110400gcctgtagct agaagcggtc ataaagacta attcttgtta ttttattatc tacaaaataa 110460catctatccg cgggatatga aacatactta aacatggaaa catggtagat atcctctcta 110520taaaacctaa ataagcgtat gatgtcgcac aagagtgtca ttacgttgta tctcatggtt 110580aaataaggga atttacaggt tagcttgtga aatgatgata ggagaatcta attcaatctt 110640tttaagtttc ttattctctc ctatttttta tacaaacaca agttacaatt tcctagtaaa 110700acagctacta actaaacaaa ctggttagtg ttaaacctct tctttataca agaagctatg 110760tagcattaac tctgttcttt atatggtgtc cagattaact tgctaacatc aacctcaagc 110820ccaccacgcc cagcggcttt tagatgacga tccaacacct tgggatccgc acatataaca 110880tgctgcccct tcctgtattg aatcagttct agactctgca gagtggtcaa tatatcctcc 110940gcctttatag ctgtcatgtc actgagctcc tggacagaat aggttcacgt accaaacagt 111000aataacaatt cagtgaccat tgacccaaat ttctcagaga atggtaaata cagctattca 111060gcaataccta cttaactaaa aaacaataat aataatttat cacacaagct acataaaatc 111120tatcaattca ggaatgtttt taacatgttt tcattttttc aaaatgggaa gatgaactga 111180acctcgagtg ttattttact attctgggat gctcatttat atttcctagg cgaggatact 111240cttgctcacc attttaaaat aaaagccacc atgactacta gcaagctact caagcttgac 111300tgcaaagata cccagattag atctgttgcc cacaggacaa actgaagagg ttcaagtaat 111360ctcagaaaat tttaattttc aactccaata tcctaagaac ccattcaaag cttttctttg 111420tatttgttat ttaataggta attaagtctg tctaatcaaa tttgaatata actttatgat 111480agatgggtcc agtttagttg aacttgagcg ttgtcacaat gtaggcttga ctccagtaaa 111540ttaccctcaa atgcaggcac aaattctcat ggaaaggtta ccttaccaac tgctaaccaa 111600gtttctaaag tccaatatcg ggtttaccag gtcacacaag cagatgcaat tgcataaatc 111660acaccccccc taggacttaa gatattcagg taatgatacc ttgatggaaa tatttccttt 111720atgctttttc aggatgtcta aaagaaccct tgtccagtac cctctgtagc tcaacagccc 111780tagatcggaa agtggtcttt caggtgtgcc aactttacct tctttctttg agagttcata 111840tgctggtcca taacgatgac aaaataagca ataaagctca attaataaat aacaactagg 111900atgaaatgca aaagacaata taaagagagg catttatatt caatatctag aaggctctag 111960catctagcta tttattttaa gacataagaa gggaagtctt tttttctgaa ccattgataa 112020agtaaggcaa aaaaattata aaataaagta gcaggcacag agcttacaaa aggcaattaa 112080aaacttccca tagcctttcc tttgatatgg aggaagggtg aggatacatg ccaaattata 112140ggattcctct gaatgctttt cctgcatgat cagatgcaaa atgctaagat atttgttgtg 112200acggataaga cagtctaaaa gactaagaca cggtgcagat aacaatgaaa taataataag 112260gattggcaac attctaagca ttccaggtgt aacgactaca aaaattttta ccgaacatgc 112320tatggggttg aataagaaac ttaaagtccc aaaatacaca agagaataaa ttttactact 112380tgaaaacttc taatatgatg acaataaggt caagaaatgt tgtatgcact ccatgccacc 112440agagttggaa cattgcaact acaaaatatt agaacagcct tagacataaa aagatctgtt 112500gtaacctcca cacagtgtca aattataagc aacaaaaatg cagtagaaat taattacctt 112560ggaaaagtat ccaaccatgt ggcaaccacg atcatcacat tcacacaaaa catagaatag 112620aaacaggtca acatcgtaat aaagggtctt gtggtcaagg aacaacttcg ccaaataaca 112680aagattctgc ccataaactt tgttcttttt gccatcaacc tgtctcaaaa tatttgaaca 112740taggaagatg aatgtcaaca aaggctaaag atctgcagca ttctacaaca tgacattttg 112800tagcaggagc tagaaaaaaa gaaaaatggt gaaaattatt tctacactag taagccaaag 112860ttgaaaatta attgtctcaa cacaacattt ttcaaagtat catttaaaaa gatctcaaga 112920ttattggatt ttacatacaa agaaagatgt gtgaaaattg aatttacaaa agcaaaaaca 112980atagggtagg gtcgaagaaa gccacaacaa tatagatttt acttaactag atcaaggaaa 113040ggctatttcc tttttttctt tttgtgaact ttggcacaga aagtagtagc agtaaaagct 113100attcaagaat atacctcaaa cattgacagg gtaccacttc gatatatttc gtccccaggg 113160ggatgcttca aatcacactt cctctacaga gtagcatgaa aacagaaagt tagtattgat 113220tctgctccat ttgtaacttg aggcaatcgg attaagaggc aaaactcaaa gacaattttt 113280cactacaaaa atccaaaagc aattttttaa ggcagcaata tcctacatct gtaccacatt 113340ccatcctatc cctggcatga taaagaaagt acataacaaa ataaatgaag gcataagata 113400ggaaattaca ctagggaatg caatttaaga tgagttaaca tcaagggaaa atattactgc 113460tgcaatacct tgatcttaaa acacaatggc cataaaacca ttgataatca atcatgcata 113520ataacagata cttccagctt ggcaagcaaa ttacatatct cataattgtt ttatcttaat 113580caattaggtc aagaatgaag ggaaaaaaat gataagagga aaacctaaac ctcaacttac 113640aggtctacat ctacataatc tgcatagtac tagatgatga atagaaagat aactcttaac 113700aaaccaaggt tacacaaagg aaaaaaaaaa tgtcaagtgt cacttaacaa accaaaggtc 113760acttaagtta ctttcaacca aagcatgcag aaggaaaaca tgtggcaagc aaaaattata 113820ttcatgacaa atgaagcaaa cttgcattac aatgttcaag gttgctatgg ttcacacaag 113880gttcttacaa tgctagaaaa aaaaaactag tacatagaga acatttaaaa tactataaag 113940taatgcaacc ccaagcattg cagaggggaa aagactattg ctacactgag gcaaagctca 114000ccatatgcct ttgaagttgc tctttccttt tcatgaagtt gaggcaaaac tcacaaaagt 114060acaacttcaa cgaatcatta tattctggtg gaaaggggga gaagtaccat gtctcaattt 114120catatcttcc aagttctata gtcgcaatat ttttcacctt cgtgaattcc tcatgttcac 114180gcaagctggc agcatccagc tcctcatgac cctgaagaaa ataaaatgat agacgattac 114240aaataatata atttcacaaa agcaacatgc agaaatttca aatgatcctc tgatatttac 114300acatcaccga gcaaaaggtc tacgtttact tacaacctca acgtgtgtct catcaatctt 114360acgtttctgg tggcgtgtca tttttaagct tgctacctga gaaccccatg agcaaatttg 114420ttcaaatcag aacaccctca aatgataaac aaagagtaaa cataagtgca aggttctaca 114480ttgtaaaaga atgtttacct tatcttcgac cttttcatca acaaccgttt cgacagaatc 114540aagatcaagt tgttcaagct tcacccattc atcaagcctc ctattaacta tcatttcaat 114600atcattatta taaaaaaaat atacatcaat ttccaaaatt ttctaacata ataattctaa 114660aattatcaat tgaatcacaa aaaaagaacc tgaaataatt aaattaaagc aaaaaaccct 114720aggaagaaca caaatcttac actcggtgta atgaacgtaa tattcataat cactgggctc 114780agcggactgc agctttcggc gttcgatgac tttgacagga tgatacttgc cgtctctcca 114840gcggcacatg acgcgagtac ccacctctag ggggtgtata cccgtcctcc tctttttcgt 114900agcctcagac tcctgcgctc cgtttgagga ggccaaaggc ctctggttgt cgtcggccgg 114960agcagcggcg gcgtaggatt gtgttgaacc attctccgta atcgtcggcg tgtctatgga 115020acccatgggg tggtgtcggt ctgagagggt ttcagacttc gagtgggctc aacaatacaa 115080aagagggggt agaagaaaat atttttgggg cttcagccca tcgagttcct tctcgggtaa 115140attaatatct tcgtctttgg gtcctacaaa ctttagtcac atccacaaaa atattttata 115200aagtatttaa tatataaaat tttatattga taatattttt atataaattt tatattttaa 115260gaaataatag tttttttaaa ttataaaaaa taataataat atcattgagc attttaattt 115320ttcaaaaaat aaaaaaaaag ttcatgtagt ttaatttgat ccattttaat ttttatactt 115380tcaaattggg ataaatatca aagaactttg ggttcaatat gtaatttgat acataaattt 115440taatttgata taattatata catgaaactt gaattatggt tatacgtata cataaaactt 115500ttattttgat tcaattgtac acatttaaag aaataaaaaa ttcaattatt ttcatatcaa 115560attaatataa ttgtttgagt atgcaacatg ccaacatgaa atggttctaa ttcaataata 115620ttattagtga tttgtgaaat ttgaatcaaa ttaaactttt atgcacaaaa tcagagttta 115680tgtatgattt gcacgttgga ttaaagttca tccgcatttt ttatatttat ccttttcaat 115740ttttgaaatt tcagtcttaa ctttcatgat aacaattggg ttagttacat accatacata 115800aattgtagag tttagtttat gttcactaat ttgattattt tttatttgtt tgctttttcg 115860atttcaagat ttaagtttta agcttaactt aaacaatagt cgttaaattt attaactaaa 115920atgtacgggg tttattgtga gtattataat atgtttgccg tgtgagattt tggtaatagt 115980agaatttaac ttaacaaatt taatggctac tacttagtaa ggattagaat ttcaaaatta 116040aaaaaaaata tatagaggct aaagatgatc aatttagagg tctaaattaa atcaaattaa 116100aacaattctg catattaact atttagacta actaatgtga ttataaaatt agaggttcaa 116160attatgtaaa attaaaatat aaaaactaaa tctcgaatgt gagtataata gaaggataaa 116220aagtgatttt ggtcattttc ttttatatac aaatattttt agagatgttc ttttatatat 116280aatggtttct aatgtcatat gcgcggcata taattttatg tgtttaattt gttttattaa 116340ttacttaaat aatatatttt taattactgt aattaatgta aaataatttt tattatttga 116400atcattgcac aaaattaaaa tatactaatt tatttaacaa ttcaaatata ataataatcc 116460aaattataat tatagtattt ttacaatatt caatatacaa tatagtttta cttcatacaa 116520ttaatataaa aaaatattat tcaaaataat aactaataaa cataattacc atatattaat 116580tattttgata tttcgaacat aacgctaata aaaaatttcc taatcattat taaatcattt 116640gtataaacta taaagaaatt gatatattgt aaattaaact ttattcattt tttttcttaa 116700tactcaataa attaatcata ataactcata aataatatat aattaaaata atcataacat 116760attagattat ataaataggg ggcgaatcta gggagctggc atgaccccta aaatagaatt 116820ttctattttg acctatcaaa atttttaaaa ttttaaatta gtaaaggtaa atttgtactt 116880taacctctta aaatgataaa attttacttt aatcctttaa aatttacatt tttactatca 116940taaaaattac aatttgattt tacccctaaa atttttttct agcttagccc tgtatataaa 117000tatattattt ataattttta tatttaaaat ataaagtttt taattataca aataattaaa 117060atctgatatt taaaactaaa gtaatttctt ttttcttttt actttttttt aattgcaaca 117120taatggttta aatatctata taacgtatga agtaatttga tataaatttt attttaattt 117180attattatat aaattcattt agtaaaaact tttaatagaa tcaaaatttt tatttgtaaa 117240ttcgataact tttcttatca agtatatttg tgagaaccaa atatttagta aaattaatat 117300tcttatttat aaatatgata aatcttataa aaaaatattt aaaatgaaaa aaattgtaca 117360aatattataa aaaaatattt aaaatgaaaa acattgtaca aaggctatat aagaagttca 117420aaagtttctt cgaccatgta ctcttataga gattatagat agattataaa actatatgta 117480gtttctctta acttttaaat aagaggataa atgtatttta atgtactcaa acttatatat 117540ttttatattg acaataatat caatatcaac ctaattaaga ttcattctaa cattaatgtt 117600gaagattttt aataaaagaa aaggttaata aattaattag aacacaaaca aacacaaatt 117660taagtggtat gtaaggtcct tgacccaaag gaaaaatttg ttacgtcgat taaattataa 117720attaatttaa agtaaaatta cattttaacc taaaaaaaga gaaaagtata tctaatttct 117780tcgaaaatgg aaagaaaatt ataaatttat ggcatttcta aaaaaattct gaattcgcta 117840ctaaaagatg aaattataaa atccgaagca ttaccagaag atggatcacc aaatcacaaa 117900caatcaatga aaagtaatga taattaattg aaagtgagca tttaattttg atagccatat 117960acttcctgct gaatttatag gttctcatta atgcaattaa attatattcg acaccttttg 118020aatgaaataa aatgacacaa gaggaaagac ggttcatcta ttttttcttt caatcgccca 118080tcaaaatacc aaaaatgtaa ctacatgcaa aaaatcaaat atgaaaaata ttcatatttt 118140gatattttaa tatattgtgt gttcaaaacg taaatgtatt gaaaaattat gatggtgttg 118200ttgctgtatg tccataaaat tcaatgtact cacatttatc aaatgtatac tttgagagaa 118260gttattttga taatactcaa gtttttttta tagatgggaa aattttttaa attatttttt 118320gattttgatg aaatgtatat ataaatttta attcgataca tataaatata tatgtaaatt 118380ttaaatttaa atttaataat atacaattaa gaaaataatt tacataaata tatatcctaa 118440taaaaataaa aytagaaaga ggaaatgtca aaacctcttc attatataca attatgatgg 118500gacacgatac cctcatgcat tgatatctca tgttgtccaa aaactcggaa tcctttttga 118560aaaaaaactt ccagagagag tatataaatc cagcagtagg cacaagaaac gagcaccagt 118620tattgacttt cctttgtaaa aaaaaaagtg ctgagatcaa gaaatatagt gaaatatggg 118680tccaagattt tctgggtttt taatctaagc aatgctgttt ttaactcaac tcctctctct 118740aacaggtaaa acaaacttct ctacagtgat tttacagtaa atatggcttt gaaaaatata 118800caacaaaaca tttatcttca atccatttta attactgatc tactatatat gttgcagatg 118860gccgtgatat tggtgtttgc tatggtttga acggcaacaa tcttccatct ccaggagatg 118920ttattaatct tttcaaaact agtggcataa acaatatcag gctctaccag ccttaccctg 118980aagtgctcga agcagcaagg ggatcgggaa tatccctctc gatgagtacg acaaacgagg 119040acatacaaag cctcgcaacg gatcaaagtg cagccgatgc atgggttaac accaacatcg 119100tcccttataa ggaagatgtt caattcaggt tcatcatcat tgggaatgaa gccattccag 119160gacagtcaag ctcttacatt cctggtgcca tgaacaacat aatgaactcg ctggcctcat 119220ttgggctagg cacgacgaag gttacgaccg tggtcccgat gaatgcccta agtacctcgt 119280accctccttc agacggcgct tttggaagcg atataacatc gatcatgact agtatcatgg 119340ccattctggt tcgacaggat tcgcccctcc tgatcaatgt gtacccttat tttgcctatg 119400cctcagaccc cactcatatt tccctcaact acgccttgtt cacctcgacc gcaccggtgg 119460tggtcgacca aggcttggaa tactacaacc tctttgacgg catggtcgat gctttcaatg 119520ccgccctaga taagatcggc ttcggccaaa ttactctcat tgtagccgaa actggatggc 119580cgaccgccgg taacgagcct tacacgagtg tcgcgaacgc tcaaacttat aacaagaact 119640tgttgaatca tgtgacgcag aaagggactc cgaaaagacc tgaatatata atgccgacgt 119700ttttcttcga gatgttcaac gagaacttga agcaacccac agttgagcag aatttcggat 119760tcttcttccc caatatgaac cctgtttatc cattttggtg aacttgaaat gttattgttg 119820gctatttaaa tcttttgcca gagacgcttc atatagtttc

tgcatatttt gaaagtggaa 119880aatcaatcta aatataaata agttttattt gttgtttttt aattaaataa aattttaaat 119940attttaaaaa catctttatt ggtaattaaa tattaaataa aaagtttaat attcaaattt 120000tatcaattca aaaataaaat aaaaatatat taaatttatt tttacgaata aattgatttt 120060ctattaatgc agattttaaa taatttgata taaattttca attcaacaat agtaattttg 120120atcacatcaa aggagaaagg gaaagattta actttaattg gtgacctaat ataacacgtt 120180gaaaacggag ttcccaataa ggcaaaatga cttgtaatga cgaaagagat gtccaagtga 120240aatctgcttt aaagtgaaag aagcataaaa ggataactaa ataactcatg atctaaattg 120300aagttctata aaatgcaact ttcatctaga aacaaggtat gtcttaaatg atgttttatg 120360aatttgtctt aattgggttt tatgcaatga attcatggat agcacatctc taattatacg 120420ttgctggttt atatgagagt ggtgcagaag ttaattgtgc tttaaatact tgcttagtgt 120480tcatgaaatt tgaaaagtgt tatatactta taataaaaat aattcgattc ggaatccaat 120540tcagggttcg actcaatata ataaaatttt acagatatct tgaaggggat cttcttcttc 120600tctacttctc gagcagtgtt atatatttac aataaagata actcaattcg agatccgacc 120660taatataata aaattctaca gacatatcaa agagggagat cttcttcttc cctacatctt 120720gaccttcttg atcaaaatga ccttccttat atttttacat acgttgatta tatgaatcaa 120780aagaaagata ccaaaaagtt tttaaaaata aacaacgggg ttcttatgta gagatgctta 120840tgggccgggc cggactcaac taaaaattta ggcacattca ttgggcccag gtcgggccta 120900acccaaaaat gggcctaaaa ttttgcccaa gcttgactca aataaaaatg ctaaaattcg 120960ggcctgaccc cgtattaatt ttatattatt ttatataact tttaaatata tataatatat 121020aaaaaatact aaaaaattaa aataaatatt tcccaactaa actaaaatta ttaagaaaaa 121080taattcatat tagcgtataa attggaaatt gaccaaaatt aaaattattg tatagttaat 121140ctatattaaa aggacatgta attaaaaacc attaaaacta ttatacaata aattaaatct 121200tcattgtata catagaaagg cattaataat taaaaaacta tattaagata taaactaaat 121260tcaaaattat taaaaacaag aactaaataa aaaagcaatt gaaaattacg aattaatgtt 121320aaaatcaaat gttaaaatca agggacttaa ataaaaatat cccaaaatac aaaacattag 121380cttcctttcc catccacgtg aatgcaaagt ttacatggtg tttcctagtg tttgtgcgac 121440tccaaccttt tatttacctc tttttttctt tatttgaaca attatttgat aatgattaga 121500attttgggat tgttgctcat cgtacgtgca acacttaaaa tcactatgat tttcataatt 121560tatataacct atatcgtttt ggaaattaat tttatttttt atattatttt aataaaaata 121620ccatctacct tttttaattt atgatccctt tcatatttaa aaattcaaat tgacaattgt 121680ctaactaaac accgtcacac tccaataaga ttgtaatttc ctccatcttg atattacact 121740caaaagcatg ttgccaacaa acaaatcaac tagccttttt ctaccactat tcatcatctt 121800cttaagagtg tgtttatgtc atgtgccgag attttaggta tggtcacgtt gtggctttaa 121860actcaaatct attgcccatg agtctaagtt agcctccgat cctcactaaa gagaggcttg 121920gcacacttta cctagccaag tacacaagga atagagctat tagaaagcat taaagagtta 121980ggagaatgtg gaagtgtttt tattactcaa agctaacttg gatacaaata aaggagggag 122040cctctccttt aggcaagctt cttttgatct gatggttaca attaatctcg aataggaggg 122100gtcaaacttc tcactcagtt tcatattatc tcttggtgct tggttggcct ccgccttgag 122160acaactttag ataacaccta gtcttaacac ttttagcttc acattgtacg catccttcat 122220tactcaaatg ccacaaagcc tccttactta aggctcttgg tcgctcccac taccttcggc 122280tttagactca tctaagatct tcccaatcgc agacaacttg gccttgatga ggaaatcttg 122340caccctaagg ggccttacat aagaagcaat taagtggctt tctctcaccc acttcattta 122400cggttgcctt gaggcaccct ttatctcgtc agggcttagc tcaccttgtc tcctcattcg 122460actaatggtt gttattggct cccctactct tttccttacc acgattctta aggaattcaa 122520ctcattcact agggtaatca actaaggact ctggtactgt cataactcgc ataaggttta 122580aacacccttc gttgaactct attccatcaa gaaagctgaa taaggcctca ctctcgctca 122640tattcgacat ctaaagtatc aactccgaaa acttgtgcac atggttatac accgtatcac 122700tttgggtaag ccacctaaac ttcgatcaag cctcctaatt ggcatacttg aggtagaatt 122760gtagcttaaa ctccctttaa aaggcttcaa aagtatcaac ggttccacct tcacgccttg 122820catcatcgct cttacggtgc caccaaagta aggcaacatc aaagtaaatt gaagcagtgc 122880ttaccttgag ggcatcttcc tcaatcccga tcaaataaaa ttattgctcg atgctctaga 122940gaaagttatc cacctctttg gcattactcg tgcctttgaa ctcttttggc ttcaatacat 123000ctactcaata actcgaccac attggcatgc cgccactttt ggttgccttg acacttaaaa 123060gctcaccctt aaacttcttg atttctctac tcatcacccc caccaatgct tagagggcct 123120cattctttct agcaagctca cctaaggcat cccttatagc cccattcaac tcctccataa 123180gtttttcctt tagctcatct cgagactgcc tcatctcaga tttgatctca taaagggaat 123240ctttggtgtc ctccaccttt ataaggacat cgctcataac caacttgatt ttggccatcc 123300gggtctccat aatgttcaca aagcccattg acgaaacctt cttgcccttc atggcacctc 123360gattctcaac catgttgtcc tcttgctcac ttaacttctc gatcacatta gccatcgttc 123420caaccacaat gttagaaatg caaggtttga tatcacttgt cacgtgctaa gactttagct 123480ttggtcgctt gcagccttag actctgattt ctcactaacg agtctaacac ttataggctt 123540ggcacacatc acctaggcaa ataaacaagg aagagagcac ttagacaaca ctagagagtt 123600gggagaatat ggaagtgttt ctattacaca aagctagctt ggatactgac tggttacatg 123660cgtgcgcatt aaaacaacta atttatgaaa caatttttaa agtccaattg tactgtaagt 123720atacatgtca gttgtaatat ttatagtgtt acaatgaaat actggaatat tctaaggatc 123780gaacccaaag gaagaggcga ttgggcaata actagcatac acaatagagg ctaagtgatt 123840attgatacga ttttatatta cgacgattca taaaagataa gtttgcaaaa aagattaaat 123900attcttctct agagactaat ttgcaagaaa tgtaaactag atgaactatc tattaaatga 123960ctaatggggg ttggttggct tcatccaaca cgtgactagc tagtatttta ggaggcgaca 124020tgtagctagg gaggtcgacc cgtattatat cgtccctcgt gcccttaggc aaaggacgag 124080gatatacaca taaacatact ctccttcaag ctccaacttc atcccttcat tctctcaagc 124140cctttgctcc tctttctttt ctgcttaact cctttatcct aacctctgag cttcctaaat 124200tgggtaaatt gactctgatc tccttgccaa ttttctagta tcaacaaatt ttgcctcttt 124260tctttttcta aatatatatt tttttttaca tactaacttg ctttgataat tttttgctta 124320tatttatatc ggataagcaa catttagtct cgaaacttga taacttttcc taattttggt 124380gacgaagtgg cataatttca aaatatcata tcatcgtata gactaaaatt taaaaaaatt 124440atcaagttca ataattaagt tgaaatcttt ttccaaattc aaagaataaa tattaaataa 124500ttccatatat atatggtatg tgcggttctt ttgtctacag tactgttctt tttatgaaac 124560tataatttat tgatcaatta aatcaattaa cgctatattg attaatcttc gaataaaatc 124620tcacatggcc atttggagct tatattaaac tatgcttcca gaaattttgt agcaatcaag 124680tttggtagga catttatttt ttcttttctc tctctcatta cgctaaatat aaatttacgt 124740tatattttaa cattaatttc gagttttaat ttgattaaaa taatcgttat taatggaaat 124800gcatttaaat atataatatc tccgtattaa aaaaaattaa aattaaaatt catttttatt 124860gcatttccat ataaaagtaa ttatacgaat gaactaagtt aagttttgtt actaaaattt 124920aattttttat ggaatattat tacatgttta attcttatat ttgcttagag tttacatata 124980taaaataaaa ctttgcgtga actattataa tagttatttt tgtttgtctt atgttatatt 125040ttggtcactt atgtctaaaa tattatgttt taatcactta cgttatcgtg ttgtaacatt 125100ttagtcactg aaccactaat tatcgttaag taacggtaag gttgtaacac tcctaacccg 125160tatccgttgt cggaataagg ttatgaggta ttacttgact gaacaaaact tctataaggt 125220caaagatact taccagacat aaattatcat caatgcaaac ctatctcatt gattttccat 125280aagagctctt ataaattttc aaaatgactc actatcaaaa cataaccgaa tatctaataa 125340caaattaact actatcaagt tataactaaa acatttcaac atattagttc aattataagg 125400cttctctaaa caaaatgagc aagccatctt cgcatggcta taaagtatac aaagtcgaaa 125460tatcattcta cctatagtct atcctataca tgccttaaac catgatgata tacaatcttc 125520tcaactcaca taatgactcg atagtgtgat gatatctccg gctcttccaa ctcgagctaa 125580agtgtaaacc tataagaaat ggaaaagaga acatggagta agcttcaatg cttagtaagt 125640tttaagcaat gcaaacaatt aatttactta tagatcgatt atttcaaatt ttcaagaaat 125700cattcctaga taattgccat tttggccaag tatccatgaa cataatgcat ttttagcaaa 125760ttcacctcac ttgaatctga actcaattaa aaccaaatat tgaaatcaca aaaaagatca 125820taagaactcg aaaagcatct cattaactag ttttaaccat gtttgcaaca aaatcacaaa 125880ttcactacaa gctgtcttcc tgagcaacag tcactaaatt atttatagct ggagctaaga 125940aactccaaat caagtaccgt taattttctc taaaaatagg ctcatatatc ttccatccat 126000caaattttta gaatttttgg tttgaccaat caataccaga tttttattga agtttcccct 126060gtttcactgt ttgactaatc tgaccactct tcactacgaa tcaattttct cattatacag 126120aattcaaaat atgttatcgt ttatttcatt tgaaactaga ctcattaagg agtctaagaa 126180tataaatttt atcttataat catcattata caatttacaa taattttcta aaaacaaaat 126240aggggatttc aaagtcattt tgactctatc tcacgccact tcaaatatct cattatctac 126300aattcttttg tttacacggt ttcttttata agaaaataga ctaattaatc tttaattaca 126360taatttattt agcttctaat tcaatttcca caatttatgg tgatttttca aaatcacgct 126420actattgttg tcccaatcag atttattaca aatttactct ttcacacatt ccttgcattc 126480aaattatcta aacatgtata tcatgtcatt caagatcgaa ctcatataac ataagcatta 126540aaatgcttca ctatcagctt tagttcaatt gaaacgaata aaatacaata tcatattcac 126600atttaatttt tcataatcgt aatcacctaa aaataaaatc atatacttcc acaaaccttt 126660ccacaaggac caagtgttta tatttgaata taaacataga atcacttcac ataacttcac 126720acatttactg aatatatcac gatcacattt atagtcataa cacttattca cagatgcatc 126780actttatcta tttataattt aattcaaatc aaaatcgcat acgagtacat gatacatacc 126840tggccaactt aatatgtaat gcactttcaa tttgtcaact tagtgtagga tcttgtaatt 126900gtatactttt atcaaattca tcggcacttg gcctgctagg tataaaaccc gaaattatat 126960taccagcaca aagcctacgg gactttagct cggatacatt tccagcacga agcctgcggg 127020actttagccc agatacattt ccagcacgaa gcctgtggga ctttagcccg gatacatttc 127080caacatgaag cctgcgggac tttagcccgg atacatttcc agcacgaagc ctgcgggact 127140ttagcccgga tacatttcca gcacgaagct tgcgggactt tagcccggat tcattttcaa 127200cgtgaagcct gcaggacttt agcccgaata catttccagc acgaagcctg tgggacttta 127260gcctggatac atttctagtg tcttgcatat ttattcacat gtaaacacat ttcacataac 127320atatcacatt agcaattcaa ttgcttcatt cgaatataag cacaaaatgt acacctaccc 127380tttaactttc ggttcaataa tcatacacaa ggaacacata atcttttcac tatcccagtt 127440tcacttttaa taaccattcg gctataggcc atattcacaa attatttcac acacaacctc 127500gatcaagcag gaacaatagt cacaattcat ctattataca aatatcccat tctttgactt 127560tgtttcataa tagctattcg gtcaccacat atataccatt caattcacat tcgaatttat 127620acaaacaagt acaataaaag tgtatacata ttacacactt gcacatcatc acttaatagt 127680cattcggcca cattatatac ataaatcatc catttcatat tcggctttat agcctaaata 127740caatatactt attgcaaatc gaacttgtaa aggtcaaata attacttatc attttatata 127800atcttaagcg cgtaacaaat caaatttaat tacttaagga cttacctcgg caacgataat 127860cggaacgaga cgactaatcg accactttga tttccccccc gatccaaatc cgaattccac 127920ttttgccaat ctaattaata tcaaaattaa ctcacttatt caacatttca ttaaatttta 127980tctaaaggca cataatttgg gcattttgca ttttatcccc taacatttta catttttaca 128040atttaatccc tatttcaaaa taacacaaat tactcaaaat ttcatcaaac ccctgttagg 128100ccgaatttac cttaggtctc tagtaaccca tatcttttat ttatttcacg ttttgaccca 128160tcaatttaca aatttctaaa tttagtcctt aatacacatt tttatcaaaa aaaatcactt 128220aattaaacat gaaaatcaaa catcaaagat ttattaatca tcatcaaaca acaatttcat 128280cacataataa acaatggaaa atctcaaatt cttcattaaa tccaaaaatt aaggcatgag 128340tttactagta ctcgaagcaa cgatctcaaa aaagtaaaaa ttataaaaaa ccgagtaaaa 128400cacatacccg aaataagctt tcaaagtgcc aaatattcaa agcttccaaa ctcatctttt 128460ttcttttcac attcagctat ggagaaagat gatagcataa aaagacaaaa acacatggct 128520tttgatttat ttaattaaac ttttttttaa cattttacca ttttaccatt aaaataaatt 128580catatataca caaatgccaa accaaatatc atccactatc ttataaatgg gctatttacc 128640atttaaggcc atcatattaa aaagccaagg ccaattgaca cctttaacta atagcatgca 128700acttttacgt tttacgcgat ttagtccttt ttattaaatc aagcacacaa cgataaaatt 128760ttcgtacgaa aatttcacac atatcaattc acatacttta aacacagaaa ataatattaa 128820aatatttttt tactcggatt cgtggtcctg aaaccattgt tcttactagg gtctaaacta 128880gactgttaca aaggtgacgt ggtacgttaa attatcattt caaacaaaaa aattaaggta 128940aattatataa ttggtcctta tattttttgt tttgagtgat ctaattattt tcttttatgt 129000tattttaact ttattttttc tttaatttcc attatcttca gtttctccct tttccatatc 129060ttttaatata gtttttttta tattttttat ttgttaaaat tagtcctata tttttatttt 129120ggtacttgaa cttgacactt tttagtccaa tttaatactt gaacttaaca ctttttccta 129180atttggtacc tgaacttgat atttttttta tttggtactt catctttttt ttatacaact 129240tgatacataa acatgattgt ttttattaat ttgataccta atcttttttt attaattttg 129300acatttgaac ttaacagtta agtagtttga aatgaaagca aatcaatgtt agaatggcat 129360ttataaataa aataatactg acatggtttt taggtgggat ttctaaagta aaaaataaaa 129420ataaaaatct aaaaaaagta acaatgttct tcatcttctt cttacacttt ccaccattca 129480atcgaataca atgtctctcg tctataccaa tgaactttca caaatttcga ttaaggtaaa 129540gggcctataa attggggatt tcgtcgattt gaggttccta atggaatttg ggtttcacag 129600ttagggattt agtgggtcct aagtggaatt ggaaatgagt tagggattta aaaaaaaaaa 129660cataaaaatc atattattga aatcaaggtt tcaaatttca tgggagacct gtagtagata 129720aggagtaaag acgacgcaat ttttttagtt aatttgtata acatttaata attttatgta 129780ctaaatagaa tctaaaaata atttagacat caaattaaga aaaaagtctc tataagcgct 129840ttcgagtgtt cacaatatat gctttcagat accgctatat aattataact cgtatttact 129900caatggatac aaaacaccca tagatttgag gtactcaatg tgatcttaag cccttttcca 129960attaatatat gagtccgctt ccacatcgtt taatttcaag tttttaagtg catgtatttt 130020aaacttggga aatatgtgta atgagccctt acttgaatag attcgaaaac attaggccct 130080tatttgaatg ttttaaaacg tttggccttt attcaaacaa ctttgaaaag gttaggtctt 130140atttgagtat ttagccataa aaatatgctt aggatgagat ttgaaaccat accaattgta 130200ttagtaaaac ttaaaattta ccacttaaag ttttatttta aaatatgatt aattaatttc 130260aaatcttacg ttatattatt tttaaacata tatctgtatt ttttcacatg tgcatgcaag 130320tgataggatt tttaattttt attataaaat aattgatatt taagatttag atttatctat 130380atatatttta aaagaattat ccatatcttt acatagatat acatagtaat atattatttt 130440tataaagttt ttcttaatct ttttatttga tatatattaa acctaagaga aaagaaaagg 130500aggatttatt taaaggttca catccttgcc tatcatgcca cgtggcgttc aacggtttgc 130560ggcgtgtaaa attttgaaag attttaccat cgtcaatccg gataccaaaa gattagacgg 130620aagaaaaatt gaggtgtgaa attggagaaa attaaaaata aaatgatgag aaatgaatta 130680ccatttatcc cttcaaaaac ccaaaaaatt gacttcaaaa aaaggatccg atattcctct 130740tgaagcttca ttaaaatatt atgtattgaa ttttgttgtc agacttcaat attctcaaat 130800tgaaataata aagaaaagca acttatattg aaaacgtagc ttcctttcat taccttaaaa 130860aataatgatg tacggatcat tgagaacaga atttaactca actcgctcaa ataaaataat 130920catgaaataa aagtatttgt tgttataatt ttttttaaaa tatcaaataa ataataaaaa 130980catggttatg ttatgttgga agaagatgca gaggtggaat aatggtccca ccactccgga 131040agccaaacaa tataatcttt caaaatgaca tttctcattt tccacgtggc accgcgtata 131100ctctgattta ttatctttct cactcgctcc cgacggcgtt taccagtctt ttctatctcc 131160tctttcagct tttttctctt tctccccctc ttcgactcgc ctcttttccg ctcccatatt 131220cttctcacct gaattttccc tgaaagttgg ccaagaagat aaaaagtttc gtttcccttc 131280tgaattgata tttttggaaa ccctagctta cctcgattgt agaccttttt tttaaatgga 131340tttggctgca tatgcattac ctacctcgat tctttagtcg tagaagcgcg atgtattcga 131400agaggagccg gagtaagccg agactcgagc gccgcaatgc agcgaagcac atcgactacg 131460atgcagcgtc gttttcttcg tctctcgatg atacctcttc atcttcttct ctaatcacgc 131520gatcgctcga tttgtccgat aaaaccagct tccgtatcca aggaacagag ggagagttcg 131580accttatttg tcggaccttg ggcctttctg gtcctgaaga tttttccatt ccagccgccg 131640cttgggagtc ccgtaaaatt cgatcctcgt cggatcttct gcctcggtcc agattgaacc 131700ggctggatag tcctgaggaa gagacaggca agataatttt agaagacggc actgaagtaa 131760cagtctctga attaactgat agggttttgg cttctgcttt gaccgaagat gactcgcccg 131820agttgaagtt aaacgagtgc tgctgtgatg atagaaactt ggtcgatgtt gctacttcaa 131880ctgaattgaa gtcaaacgca tgctgggttt cgaatgttgt cgatggagga gggaattatg 131940gaattaaagg gattaggcca ccgggtttaa agccgccgcc ggtgatgaag ctaccggtag 132000tcgatagcgc ttgctcaact tgggatctgt ttagggattt cgcccccgaa gatgatagag 132060ggtgtatagt tcaggttcac ttacattcat cttccgatga agaagaagtt aaaggagaga 132120aggatagggg taatgaagaa aatgctaagg aggaggataa ttcaatgaga atgggagaga 132180ctgcagtgct ttctgagtcg tgctcgttta taacttcaaa tgatgacgat tcttcgagtt 132240ctacctcaga acctatgtca aacatttccc ctaacggtag gttcaaaaga acaattactt 132300attgggagaa aggtgagctt ctggggcgtg gatcatttgg atcagttttc gaagggattt 132360ctgagtaagt gatgaaattc tgtcccaact ttatttccag cttgattaaa tgcttatagc 132420tagttctttt taatcaagat aatttatcta tcttttgata tgcgcaaatt agtttgggaa 132480ctgccttatg cctaattgtc ataattttgt cgctttgggc ggcactcttt tagaggtcag 132540aaagcatcta gggtcaaaag ctgttcttgg tgttggttaa gcatccagag aaaggcattt 132600ttgtatttgt ttttattttc tttggtcatc ctagtttcca tcgcccattc ccttagttct 132660ttggggagca ctgtattaca tgtttcaggg ttgcatccaa agattgctag atatcattaa 132720ttccgttaat accaataaat acctaatatt tggacatctt gtgttttttc tacctcgaac 132780tttaaggcgt tgacgcattt tagccttaag gttcattagc ctttaagtgt tactttgttt 132840gctgcttgaa gttttgcgta gttgatacac tcatgtagcc atttttgtgg gcaaattatg 132900gatgattatg aaccccttcc tctatggtga gtacaggtaa agtccattat tgacttatga 132960agggttctgc ttgttgattc cggtctatgg cttccgatga tcctatgtgg ttagaaactg 133020gttaactgga gatatcctgt tctatagctg attttaagat gataaattta gttgttttct 133080ccaagctcct aatctacttt taaccttccc aatcattatg taattaactc acatcaaaag 133140gacattacgt gcctaaggcc tctcgaaccc acaacctcca ggagtctgtc agcagcatgc 133200ataccatctg agctagcact tagtcggtat gaatttagtt gttacctatt gttcaagagt 133260tgaaaaataa acctagggaa gctcattgca ctgcgacctt tgagatgtga agctaattgt 133320tgatattttg gaaagctatg gaatgacatc atcttggagt gactgaatga agtactgtta 133380gaaatacttt caaaattgca aggaaaagcc cattttatct tattagctat taattggtta 133440tctacttgtc caatagatta cgtattactt ttatttagga aagagatgtt tttcttttac 133500tcatgcttta ctaattattt atttattcat gtccttgtag cgatggattc ttttttgccg 133560tgaaggaagt ttcattgctt gatcaaggaa gtcaggggaa acaaagtatt atccaacttg 133620aacatgtaag acagattttc tcttctactt ttttaattgt tcgttttcat gaaataccca 133680tcttctactt gtttctgtct gatataattt ctttatttcc tttcgttttc aggagattgc 133740tcttttaagc cagtttgaac atgaaaacat agttcagtat tatggcacag ataaggttct 133800atttttttga cactcagctt aataggataa cctcataaat gttcttcttc ctagttgtct 133860aatttttttt cttttaattt tatggtcttt gcaggatcag tcaaaattat acatctttct 133920tgagcttgta accaaaggat cccttttaaa tctatatcag aggtatcatc tcagagattc 133980tcaagtctct gcatatacaa gacagatttt gcatggattg aagtatcttc atgaccaaaa 134040tgtggttcac aggtaagtaa agggacctta tgttgctgct taattattta tgtaccactt 134100aaaaaacttt tgtttgtttt ctctagccaa atctgagttt ttatttgtac ttcttttagg 134160tgtatttgct aacagcaaat tgcttcatat gagaaataaa ataaaaattt cttcacttta 134220acagattgaa gttttcagtc cttatctcta ttgggatctg ttgtaatgaa tcataatctg 134280tatgcatatg tcatattccc tctcggaaga cttttgaagt cgagaatatg aatcataatc 134340tgtatgcata gaagattttt tcagcatttt acttgctatt attttgtatt atttttccct 134400taaggttaga tgcctgcgca tttgttattg ttatgatatt gaaagtaaaa ttgtgttctt 134460tttctatgct ctacctaagt ttccatgcac ttagaatatt cttctctcaa ggctcattta 134520cttgtatgtt gatacaggga tatcaagtgt gcaaacatat tggtggatgc aagtgggtca 134580gtgaagcttt cagattttgg gttggcaaag gtttttatcc gaaacctaag actttaattg 134640tctttcttgt tttattatct ttaagcaggt cgaactcatg ttggtgctga tgcttgtttc 134700caggcaacca agtttaatga tgttaaatca tgcaaaggga cagcattctg gatggccccc 134760gaggtgtgct tctattcttt cctttcagaa atgataatct gtaatagctg ctgtttgatt 134820tgtgtagaat atcagtttct tttgtattgg ggatcctgct tgagatattt agcttcatag 134880tatactgaat attaggaaat gatgacatgc attcttggta

aatatttctc ctgtactaga 134940gatatacagc ttatgagaat actcaattat gaaaaggatg ataacataat tcttttcaga 135000tatttatcta tagtgtacaa aaggtgtgaa tcactctttg ctgatccatc ataattttct 135060caggcttcac tcaattggct ggatcttaac atttagttta cagtgaacac ttcttttatc 135120ttttagctca ctagtaattt cctaagtagt atttggttag acaattttca ttgacggata 135180attgctaatg ttttggtgat ggcagctttc cttttctgaa acactgctgg ccctttttta 135240aatcatacct gaacggtgtc ttcagtttct agttggatgg gtttcagtac tttttttctt 135300ctagaaacac accattcttg ttgtcttttc tacgtagtag ttatttgttt ttgtgacaca 135360gactactggt gatgattagt cctcccgaat tctgattatc agctgttgaa ttacaggttg 135420tcaataggaa gggtcaaggg tatggacttc ctgctgatat atggagcctt ggttgtactg 135480tgttggagat gttaacacgt cagattccat actattattt ggaacatgta tgtacctcgt 135540cttcctgata tgaacattag tttacttgac aattaagttt atgtaaaatc caaataaaga 135600agaaaaaagg atctggaaat ttctatgctg tcatctccaa atttcaaaaa ggcttatagg 135660gttttaaagc atgaaatttg gttcctacca tgaagctttg tccagaaagg tgtggacgaa 135720tattatattt gtttcaaata atttccttct acaagagcta ttgagttaaa tttttataat 135780ctcctttatt gtgcaataat gtcatcgttt gtgtaattca aaacagatgc aagcattgtt 135840tagaattggc agaggtgagc cacctgcagt tcctgattca ttgtcgaaag atgcacggga 135900ttttatcttg caatgcctac aagtaaatcc ggatgctcgt ccaactgctg ctaaactctt 135960gcagcatcca tttgtgaaga ggtcttttcc cacacactca ggctcagcat ctcctcatct 136020tggtcgtcgg atatgaatgt ttagccatga aactaaattg caacaagtaa tcaggtaaac 136080attttctgct gatcataaat ccgttggcaa catgctcctt tggtcaggtt tcagaagaaa 136140cattgtcctg gaaccttcat tatcacaaca tgttagctat ccatatccta agatacctga 136200atattatccg gtaacaatgg tacatttttg gtatcagttt agcttcattc agagctttgt 136260tcttgtattt gtgtgcagaa agttacacat tcacggagtc tagttattgc atgcagctcc 136320gtccttcatg aggaagaaga caagtttgcc ttctcgggct ctgatgttgc tacctttagt 136380tttgctccta gagactcaaa gagtctcaaa cctggtaggc agaaagcaat tctgaagctt 136440tggctatggt ctaatggcat tgacattaga tttaactatc catgaccatg aacccacgat 136500gaagctgtag agagctgagc tgctcttaat gttaaaatta tttgttatag ttttgtcagg 136560tctgggattt gatttagccc ttattttatg tagatttttt tttttgggga tttgggtgaa 136620acattagctg taggagaaat tatttgtata tatgtatctc attattgatg caaaaaataa 136680atactagttt gctctatgta tcgactatat ctaatattga gattacgaga attacgtcgc 136740tttatgattg ttattaccat aaataatata tttatttgca tttagacctg cagttggagg 136800tttggcttaa aaatagaggg tttggattta aggttagaaa aatgaatttg gataaaaatt 136860atgggttaga ttttaggcaa gatttttttt gggctcaagt ttgacctggc ctgaatatta 136920tattataaaa taatatatta attatatata ttaaataatt atatatattt atatttatat 136980taaattatta ttttaataat aattaaattt attaattaaa actttaaaaa acgtacccaa 137040ctaaataact taactcaaaa tataaatttt aaaatttata tttaatacaa taaaatattt 137100attatattta tttgtgtttt taatataata aaacatttat tatatttatg gtagtgtttt 137160ttaatataaa tatttttaat gtattagaaa atttttattt tagccttttt tttaagtgta 137220tttagtttat tatatttaaa aaatattttt aagtaaaaat taatctaaaa aaatcaaata 137280tgaatggatc aggttaaact tgagtttaac tttattaaat taaattaaat tattaaaaaa 137340ataaatctat tttttaaacc gactagactc aaatttaaaa ctttaattta atggactcaa 137400cctacctgcc caaccatgag tacctctatt tgcattatga ttatctaatt ttgcgatgtt 137460aattctcttt acagccagtg tactggaaat tagtataaat gggtaatggt tgattataat 137520aaaatgcacc atcatgattc ctgaactaat ttatcagact caatttattt ttatcgttta 137580taaataaaaa tcatagtgct atgaaattat ttatagaata attagaaaaa ggaaatttat 137640gattacaatc acaatctctg tattttatag ttaactgtgt aatgtttaaa agaataaaaa 137700aaaatcgata acattttatt gtaactttta tcgtataaat aaaatccgga cattgtataa 137760attttttgtt tgcttgatta agaaaagagt aaccatagat ctcaggtatt ctgtactaag 137820ccagattaca ttaaaaaaaa aaaacaaaca aacttgaaaa caatatcttc cttaaaagtt 137880tgacatcaat ctcctctcaa cgactttata aaatagacat ttggattgag ttagatttgt 137940ttaaacttga aaataggtca tccaactaat ttttatagga cataaattac atataaaata 138000acattataaa attaaaatta aaaacgactt gagccaaact caaattttta aatattaaag 138060tttgaatttg actcatattt ttaaaatatt taatttttta tgaacttatt ttttaaattt 138120aatattttta tttaaattct tacaaataag taaacttttg agtttaaaca aataattgag 138180ataaagtgga atattcttga actcatacaa gaggtgttaa aagtttaata gtttcgtata 138240tcctatatca taaacatcat catgaagaat tctcaattag tatgatataa aaacaggttg 138300attcgatgta catgatggca taattatatg actaaattga atggtgaatt tattaaaatt 138360ttaattgtat aaaaattatt aaattacaat atattcacaa tgttggtaaa tatatatata 138420caacaaatat atttattaga aatttatgca ataatcaaat aacatttgat tgaaatagta 138480aagttgaagg tttcaaattt atatatcaac cgagattcaa atttcatctt atgtgatatt 138540ttattaattt tacatagaca aaacacctta ataataatga tagtaacaac aacaacaaca 138600acaatctatt ttattttatt ttataaagga atgctcattt taataatttt tcaattgaat 138660tggtgttgat taactcatga catcgactca attaagaatt ttaagtataa tgtagatgga 138720agtaaataat tttttttaat tttgtactaa taatatatat aaataaataa taaaaactac 138780tctttcagaa attaatatat atattaagtg tgtttggttc acggaatcta aagattatct 138840ctggtaatta catcaacaac acgtaagatt acttgacaca ttactgaata tgttacatta 138900ctttatttgg tttatttagt tgaaatgtaa aattttatta tttagttgat agaatataag 138960accatttaaa aactaatttt acttaattat ctttataaat ttttctcttt tttattcttt 139020actacaaaaa gattgaagtt tttttatatt ttattgtatt gataaatgaa tgaatttctt 139080aaataaatta atttcatgtc tactttctca aataaataaa ataaatatga ggaagagtaa 139140tacatagtta gggcttatct tttgattaaa gtgacgagaa aaagaaagaa ataaaaatat 139200atttttttac taaaacatgt tattttttat gatttaaggt taaatttaat taaaataata 139260gaattgataa aattgttaag tttcttcatc taaaaataac aatagttgaa atgtttgtat 139320agacaaaaat cttaattttc ctcaactgaa ataataatat atagtaaaaa aatattatta 139380ttttattttg gtttaagtaa aatagtaaat ttttttttca aaagtgtgta attgtagaaa 139440agtttacatt acaaaataat aatttttgaa aaaagaaagc aagataatga atgattaatt 139500aagaaagagg tggtttttaa gataattgat ttaagatcat ttttgaaatt cgaataaaaa 139560aatttactca tacaaatata aatttagttg agtcaaaagt ttcttgtaga gaatataaag 139620aggatattgt aatcaatgta ggaagatttg aattcgagcg tgttgaagtt cattatcctt 139680ctctttatat attagggagg ggttatgaat agttctaaac attatatcaa aaattaaata 139740taatcaaaat ttataataaa attatttaaa aatatatata tatatgtcat aatgtgggag 139800atgaagctaa gccattgtca tcattacgca ccaggaagag tggttttgtt gactacgaca 139860agcttgctgt gcccataact aacaaaccat gttgaaggct tacctttgct ttcttttcgt 139920tttcgcatta catccatccc ctagttttta tttttccgag attgagtggt atggtcaaat 139980cctacattaa actacaagct tttatgctta ctcttcttca ttaatggtaa aggatgatat 140040tttacttttg tattatttac tgctggatct ttcgctgccc tctatttttt ttagttaaaa 140100tgtaaattta ttaccatatt aaaaattgtg attatatttt attttgattg atatttgtca 140160ttataatttg agaatatgat taaatttgac ccacaatttg atcaaatttc gccattgaat 140220tttaattttt tattgaattt tattattaat ttttaattaa attttagaaa ttaataagtt 140280attattggtt tttatggttt aaaaataaaa tattatttaa atattttatt ttaacaataa 140340gttaatttat aaaataataa aggttaaaag aataactaac attttgtagt taatgataga 140400aagacttgaa aaaatatatt caataaatta agtttacttt ttataaaata aaattatttt 140460tattaatttt taatgttgta ttaattaatt tattgtttgt aagtgttaaa ttaaaatatt 140520gaaatttgtt ttcattaaaa ttaagtagtg aagttcaata aagaatcaaa ctttaacgac 140580acaattcaat cccaaagacc ctaacaattt aggcccatac ccaaacaata tatctggcgc 140640agaagaaact actccaatcc ctcatgttat gatcaaggcc caatccttca ccaggatctt 140700gtgctgctgt gttaagggtt tcaatgttgg caaatttatt agttttcctg gaatttgaac 140760ccggtaacca atgggtcttt ttttcttttc tttttttggc tattttctcc caaagagaaa 140820agataaccat tacttgtaca aaggaatgtt aattggcatc taccttatct agaatcctaa 140880aaagtaacaa gtgtttacac ctgaattgta gtcaccttca tatcaggcat cttttctttg 140940ccaagttgat atatatatac acatatatat gatcaacatg cagtaaacca aaaggtttgc 141000aggatgtagg ataattatct atataagcat aagaatctgg cattattttc acttcaactt 141060tatgtcgaca taaaggtgcc accttgcccc aaaacagaat aactccaccg gttatttata 141120ggaagggatg agcaagggag cttaggcaaa ggtaagttaa atacccaagt atctataaat 141180ttaaatatat aaattaataa gcaccaatct atggctcaac tagggtcgtc tctgtcagca 141240gaaaccgaga cactgagcaa tgtcctaagc ctggtggagg ccttcagagc atttgattca 141300gacaacgatg gcgcaatcaa tgctgcagag ctagggggaa tcctgagttc gctggggtac 141360aacgctagcg agcaagacgt gagggccatg atgcgagaag gggacgccaa caaggacgga 141420ttactgagca tggaagagtt cctagagatg aacaccaagg acatggagct tggggagctt 141480gccaatttcc tcaggaccgc tttccaagct tttgaagtcg aaggggatga tgctttgact 141540gctgccgact tgtatgaggt tatggggaac cttggcatcg atcagctttc cttggaggat 141600tgccagagtg ttattgcctc catggatgct gatggtgatg gagctgttag cttggaggac 141660ttcagactca taattaattc cttattttag attattaaac ttaagttttc tctatatatg 141720cgccttgggt gctgcggtat ttccatgcat gggaataaag atcagtcaag aaacattaat 141780attactagca gaagtactgc atgtttgtgt ttctgttctc ttactatgaa tagaaagcga 141840atacaaagtg gtctcccatt ctctaatcaa tggaagtttt cattttaatt tctttttgaa 141900aatatatcgg gaaattgaaa tttcacattt tccttacaaa ccacaaacaa aaatttatag 141960aaaaatattc ttggcaaaag ctgagttttt aacatcttta taagaggctt gaatcccacc 142020acttaaaaat atatatatat gtgagtttct gatattattc taaatttaag taccactaaa 142080taagtagttt ttaaatttat tctaatctaa aattagatat aaagaagtga atgataaaat 142140ctaagtagca aatatttttc tcatgaaatg ggattaaaag gtttggggct gcgaaaagca 142200agacctaata atcttgctat gcttgccaaa ttaggtttgt aattattcat gaataagaaa 142260tctttaaggt gttgtgtttt tcaaaataaa tatctccata atgaatcgtt ttcaatggca 142320aaattgaagg aaaatgcctc atttacatgg agagcattgc taacaataac tagggaggtg 142380actcttaaga gttgtaaatg ggcaattggt gggatgatag cattcgtttt ttgcttgatt 142440ggtggttcgg tacagaaata ttgagtcaac aagtaatggt tcaaggggta tgtacaggcc 142500aattttgggc caccccaaaa cccaactaac ctaccctaac ctaaacagcc caatacccat 142560aagcccaact aatacatcag ccaacccaaa attcaaaccc catttacaac caaaacccaa 142620taatacaata cccaaaccca atttacaaac cctaacaacc caagcccact atctaaaaaa 142680tttcagcagc aaaccctagc caccaaagtc ttcagtcgct ctcccctctt cagcctcctc 142740cactcctctg acaccagcac cgcccctggt cactcccata ccgtctgcca ccgcatgctt 142800ctcctccact tccctgacac ctccataccc tgaaaagaca gaagcagacc aaacagaata 142860ggacaaaaaa taaataatat tttcctagtt ttgtagtcgg ctataaagtt gagaaataaa 142920catttgtaag atggggggga tttttgctat gaaaacaaag attttctttc aatattaaca 142980gattgaatac aagaaccatt cgaaaataca tatacaatca ggggctctaa caccaaatcg 143040gagaatcaaa tctaaaacct aaggtgactt tttttttctt ttttctttac tgttttgagt 143100ttgtttttta cataaaaata ctaaaaaaat atcaaaacat aaaaacatat gtattattaa 143160gcaaaaagaa aaaagaaatt ttaccttttc cggccaccgc acggcgccgg cgagcctccg 143220gtggccgtcc ggtgaccggc ccccatggcc ggagctcccc ccctcccctc tcttctttcc 143280ccgttccctc ttttctctcc ctcctcttct gttttttttt ctttcatctg tttcaaatga 143340aaaaaagaac aaaaatttgg cttatatagg ggtccaaaac gcaccgtttt ggaccccccc 143400ttttaaagta gaaaacgacg ccattttgat gcgggtcggg tcgacccgac ccgtccgacc 143460aggggatccg cgtgttttta agggaggggc tatttgcgca gttggcccct ccgctttttc 143520aacgttttat aatcaagttt ttttatattt taaattcggc cccgctgttt tgccctgatt 143580tcgttctagt ccctccgtgc tgcgctgcgt tttagtaatt gagaatattg cacttttggt 143640cctcgttgtt ttcacgcgtg tccattttag tcctttattt ctttatttct ttttaaattc 143700gccctgaaat tctgttctta ttccgattta atcctttttc gtttattttc ctttttttta 143760catattacta atattgttac tattatatta tttattttca ctattattat tttcatcatt 143820attatacata tatgtatata tttatgtaat attattaata ggcgtcccaa cattattatt 143880atgtatgtat atacattttg ataccgtgca tgtgtaacac cccttacccg agaccgtttc 143940cggagtcgag cacgaggcat tacttagctt atcttaccaa ttcggagcat aaaaactagg 144000tttgaaaatt tatttcatta ttcgcagcaa atctgtccaa tcacacagca gttactaaat 144060taattataac ttgagctaca gaactcgaaa tttaattccg taaattttcc ctgaaactat 144120actcatatat ctactcacca taaaattttt agaatttttg gttcagcaaa ttagtacagt 144180ttattagtta aagtctcccc tatttcacca cctgactgcc ctgacctcta gtcactaaaa 144240ataagttttc tcactgtagg attttcatat gaagttctta cttgtttcta cagaaaatac 144300actcattaag aaatctaagc atgtaaattt caactcataa ccatttttgt acaatttgta 144360attattttct aaactcagaa caggggactc caaaaacagt tctgacccta tcttactaaa 144420attcacatat cttaaaatat aaatttcctt tttctacacc gttatttttc catgaaaata 144480gactcaacaa gctttaattc catatattat tcaccctcta attcatttta tactatcttg 144540ggtgattttt caaattcacg tcactgtgct gtctgaattc tgtttctttg caaaatttta 144600tcctttcatg atttccatgc ataatttatc acctaatctt tcataacaac aaacaccttc 144660atccttaatc attttaataa ccatacatca tcaaatactt acacatcact cattagcaaa 144720atcatcatta caaacataca aaataactaa atccctatac atgccataac tcaaacgtgt 144780ttcgatataa aataccgagc agttgtagtt gatagtgtgg acgatctccg acttctttag 144840gatccttgaa gtagctttgc aatactataa gagaaagaga aataaaagaa gtaagcataa 144900agcttagtaa gtttactagc aaataaataa caatatttaa cttaaataat taaactcaat 144960gtctatatct ctagtttact ctttagttaa tctcatacta gttctcttac ttgtttactt 145020agaatacttg tgtgcataac ttactcaatc cttgctgcat cgttgaacat caattgatag 145080tataataagt tcttaagtct tacaacttac ctgagcttgt catttatgct ttaaactgaa 145140ctttcatgaa catgattcgt ttacaagccc gttgagctac attggaataa taaggatact 145200cgggtctctt ctgataataa catgccaaag ccatgtccca gacatggtct tacatgggat 145260gttctcgtga tggtgcccat gccatgtccc agacatggtc ttatagggga cctctcatct 145320cggtgccaac gccatgtccc agacatggtc ttacatggga cctctcgtct cggtgcccat 145380gccatgtccc agacatggtc ttacagggga cctctcatga tcttaaggat gccaatgcca 145440tgccccagac atggtcttac atgggatctc tttacccaaa tgtcatgaca ttcgtatcca 145500gtaccatcct tatgtatcaa cgggactttt aaattttaat tctctatcat ttcatgcttg 145560gatcatcatc aaataaattc ataaaataaa ttcataattg ctggaaatta acagcattaa 145620taataaatat tgaaatattg catttattta ccgtaaactt acctcggtac caattatagc 145680caaattcacc aacttagtct tcaactttat tcttcccttt gtctaacctc gagtttcgta 145740cttcttgatc taaaatagta aatttaactt atttaataat cacattcatc aaaacagccc 145800tcgactctaa ctttttcaaa attacaattt tgcccctaaa cttttacata attacatttt 145860tgccccaagg ctcggaaatt aaacttcatc tcttattctt atgttttata acattctgaa 145920catttttccc ttctatggca acatcaaatt cccactctaa catgtactta tgaacattag 145980gtatttttac cgattatgtc gttttactcg ttttcactta aaatcgctta gcaaaagttg 146040tttaacataa tttatagctt catattctat cataaaacat caaaataaac acttttcacc 146100tatgggtatt tttccaaata taaaccctag gttaaattat tgctagaata agctaaatta 146160agctaccggg atctcaaaaa cgtaaagaac attaaaaacg gggcttggga tcacttacta 146220tggagattgg aagcttgaaa accctaacta tggcttcccc ccttgctgat ttcgttcata 146280tgaagaagat gatgattttt gccatctttt tcccttttaa ttcattttaa ttactagatt 146340accaaattgc ccctaactta aaaattttct atttcactta tctcatgtcc atttttgtct 146400accaagttac caatggtata attaccatat aaggacctcc aatttaaagt ttcataacaa 146460ttggacacct ctaacatgta gaactcaact tttgcacttt ttacaattta gtccttttga 146520ctaaattgag tgcccaaacg ttgaaatttt cgaacgaaat tttcaaaaaa tcattttgtg 146580aaattgtaga ccataaaaat ataagaaaaa taaaattttt cttatcggat ttgtggttcc 146640gaaactactg ttccgataac ctcaaatttg ggccattaca gcatgtatat gattctattt 146700aattgttagt tttgtatact aaattcatac gtatatttct catgtcattt catgtatcca 146760tttttattat tatatatata ggtaataatt tttcaaaact tcgttttaat cttatgcatt 146820atttgtttcg ttccctttca taatattatt atatatattg gtatatatat gttctttagg 146880tgcatgtgtt cctcaatatt tattttatgt acatatgtaa atattatgaa tatatattta 146940acattactag ttatatattt ttatgatctt atatatcttt ctaataccat taatatttat 147000atatacatat tttacatgta tactcttaat tattttcatg tgtatattca tcgtattctc 147060tttacgttct cgtattcaat gctaacattg catttgatct tgcatgcgcg gttattattt 147120tccaatatca ttgtaatatt tgttatttgt ttcaaatatg tttatagttc ttccatttat 147180ttatgtttat ttcatatcag cttgcttcac attatttcga aaaattatcc atgttttctt 147240atttacttca ataatcaagg caatataccg atttaacatt aagtcatcga gttcgtcgct 147300atgttgggtg aacgtcaatt gactcatgtt aaagcgatat acccttctaa aaaaaatgaa 147360ataaaccaaa atttctcatt cttttaatcg gattatgact aaattttaca ttgaactctt 147420atttttggaa attaagacaa cgcgtgttta tgagatacca atttgggcgt cgcgagggtg 147480ctaatacctt cctcgcgcgt aaccgactcc cgaaccctag tttttctctg gcttttaacg 147540tagacctaaa ttcagccttc cttttgtttt aaaaaatgaa tctaataggt gtccgatcac 147600acctaggaaa aaggatcggt ggcgactccc tctttatttt aaaatcgaac ttcagtttcc 147660aaactttttc actagatcgc cacaattagc gaccccggaa ccaattttta tgtcgctaca 147720gggtagaaac acttaatgag atggtaagta tgggcatata tgctcatact ttggtgtcac 147780cttgaaactc cttctacaat gtcacccaag cctcctttga tctagttgct tagttgctgt 147840cataattctt ggaaaaatat cctcatgaag agctcgctga ataaaaataa tgcttgtgca 147900tcctttttct tattttcttt cagtatggct acgtcatcga ggtcaacata cccatctcta 147960ataagaaccc ggagatcgta ataagctttt catcatcatg ctccaaagtt cttatgtttc 148020actaatcacc caaaaacaag atcaaactta gctctgatac taaatttgtc gaggggaaaa 148080aagagagaat tcacataaga gaaaatattg atcacataag agaaaacaaa atactagcca 148140cgttcgtctg aaaaaaaaaa catggcaact acgaatgctt gtccacagta tgggagtatt 148200acaaagtcga aggatcatat tttcatagaa tgcaatgttg cccaagaaat ctggacggaa 148260ctatgcgctt tgaatatcca agcgaacttc ttttcagtta gtttcgagga atagtttagg 148320cgaaattgta agttagcaag tctgtcttat gattcaatat tccttggtgt gtacttttcg 148380ctatgatctt gtggggaatt tgaaaatgta gaaacgagtt gtatttcatg gtttgcatca 148440gcaactgaca gttgcagtct ttttgtatga gaagtcttct gcacaagata tttgcaaggc 148500agtgttaaat gatgtggcaa agatagcacg aataccgctt tgtgttcatt ggttgaaacc 148560acacctgcag gaagctatat tcttaatatg aatggggctg tgaaatcgac ttcagcttgg 148620gatttgatta aaacatgaca tgggtgacta ggttttaggt ttcatgatga agtcagcgca 148680agggataatt tgcaagcgga gatctggggt gtttgagaag gttttagaat tgcattcaat 148740tactcgtcat tgaattgatg ttatgacggt tgtgaagatt ctcccaacac cttacgcttt 148800tactcatcct ttgactacac tcttattcaa tagttgaagt ttgattaatc aaggatgggt 148860aattaaagta gagccaagag ggtaatatat gtgtgccgat tatctgacga atctcgacaa 148920agccgatgct tgtggtaagg atgctcttag gatttagttt cctctagttt tcttccttta 148980tgtataaaaa aaaatatatt gatcaaatgc tatcaaataa aaattcacac acactaaagt 149040aaagttttct taatatctaa aaaaataaac cgagttattt gcaacataaa tccttttaat 149100tttttcgcat tttctgttta ttaatttagt ttctaaagtt ttatagttat tctagattca 149160aaaacaaaac ccctatatat tttcaatcac ttatgaaaaa attgttattt ttctcctttt 149220tttttaattt ataaaatctt atttttataa aattaacata aaaattttaa taattaacat 149280ataactcaaa taattaattg aaatttttta tttttatatt taaaatttta actaataaat 149340attaaaaatt atattacgta tttttaacta agtattaaaa atttcactaa tgtatattaa 149400aaattacatt tatttttaga tatcaaatgt aaattaaagt aatatttaat taatacatgt 149460taaaaaacta tatttttgta attttaaata aataaataaa ataattttct aataaataat 149520gttcaataac ctaattaaat aaataataca tgttcatatt attttaacat tattttacta 149580atatttaata tttacataac attattcaaa attatacatt tataatcaca caaaaaaata 149640agtattgtta ttaaacattt tacaatccat aaattaacaa cactagtatt tattaatgct 149700catgtacaaa ttttactatc ataatttaat attaacatta catgtttata taatttttta 149760aattgtgaat tttttattta aaatttttaa ataatacatt atcattattt gactttgaat 149820atgttttcac aaatagaaaa caattcaaat ttgttttaaa tattaatttt ttattcttat 149880attatattaa taataataca aaagtataaa gatttcaaaa tttaaatatt tatattatat 149940tatttttaat ttcttaatca tagtttttct aaaatattac

aattttgtag caatttttta 150000agaaataaaa ataatatatt taatttatat atacaatcaa actcatacat cacacatgat 150060aagaaattag tgtgtatata tatatatata tatatataat attacaatat atttgatgca 150120taagtctcat gcaccatcgg tgaataataa catgacacat catcattaaa taaaataaaa 150180atataatgaa agacttataa ataaatacta ataactttat ttaaacataa ttaaataata 150240attaattata aataaatagt aaaaccctta aacctaaata tcataaattc gagagctatt 150300aatatttgtt tataatagtt ataattaatg tttaaataaa attattaata tttattaaca 150360agtcctccat aattttatat ttaatttaat aaaaaattat catattatta tttactaata 150420gtacatgaaa cttatacacc aacaatggat ataatatcgt tccttccata agttttttgg 150480gtttgatata aaaggctata tatagttttg gggcctattg agccgaggaa gagatttcgg 150540gccataagaa gcctacttgt gctgggtttc ttacacttct ctgttagtcg caaaatttgc 150600agatgcccaa accctaaact gtcttgatgt ttcttattta caattattat gaggatgatg 150660ggtgatgggc agtgacattt ggaatcataa taaaaaacag ttggcaagaa aattggacat 150720gtaggtccca aattttcaag tggcagtcgc tagcaaccaa tttgtatgtt tgatcaccat 150780aatttctgtc caacaccaag atcttcttcc acgagaaaaa taaattgtat ttataaaaca 150840ataatttcaa aattaaaaga cattgaaaac taactagaat gataagtctc attccatttc 150900atttcagttt acctgttgat aggtttagct actcgtttaa gtctaaaaat ctattcgaaa 150960tttagaagga tttaagcaaa aacattaggc tcgaaatatg agttcgggca aaaaatttag 151020acccttttaa gatatgagtt tgactcgggc ttgaacattc aaggtcaaag cccgtcctaa 151080ctagtaatgt tttatgttat tttattttta tatattatat aatttataac acataaaaat 151140taaatctata atagtattta taatattact accatgatgt aaacattaac aattgttaag 151200gtgcctatat atgaaatttt aataaataaa aatatataaa attattaaat gataaattaa 151260aaataacata aatatatatt tttgaaattt atatatatat atagacaggc ctaaaatggg 151320ttattaggtt agtcatttac aaatataaac gagcttaagt aaaattttag gtcaatattt 151380cgaatcttta cttgagcaag tataaagtat gttaatacca ttcgtaagcc gacttaaact 151440caatccataa acacctctaa tataatccat aaaaccagtg catagtttaa gatttgggtc 151500acctttataa tctaacaaaa ttgattcaaa ttgttattta agttagctat aaaatatcaa 151560acaatttaat taaacgataa agcaaatttc gggatgtatc aaatccttaa aagaaaaaat 151620aaaataaatt attttacaac tcaaacttaa attgattcca tccttaacac tcaacagaca 151680atagctcttt cactcttcca ctatatagct ggggatgttg acaacattgc cttccaataa 151740aatcctgttc cattgctatg tatggttgtg tttcataact ttacaatctt taaaaatcaa 151800cgcaatcatt caaacttttt ttactcaata taatatattt aaaaataaaa taaagaatta 151860tatttgttct ttttaacaca aggtctaaaa ttagatatag cctttcctaa ctcataaata 151920agaggataat acgcttcaac aaacttaaac ttttatcctc ctgcattgac gactatatcg 151980atactaatca aactaaaact caatcaaaat aaaggtgatt aaacttacta ctcatgtgga 152040agctcaaagt tgacactaaa ttgagtagta tattttggtg aaacaatttc caacagtaaa 152100cttgattaaa cacactccac tataccaaaa cagaaatgat tatttaagat gatatatttt 152160attttgatct ttccttaaaa aaaaaaaaag tgaagggagg caagtaggga actggaaaat 152220gctatcataa actatgcctt tttctgaaga tagagacaat attacggtgt ggtaccctta 152280ccctgtctat aatcttcctt tgttttcctg aatataattt ctatgtagat ttagtagatg 152340aataatggga tcttttttat ttactaattt gttaaaggga agaaaacgtg aatcatgcac 152400agctcttggt gctgccactc acgttttcta gcatttctat gatataaaat aagaacaaat 152460tgcatttaaa tataaatatt agggtagtga attcccacat atgtgctttc caataaacta 152520aagattctgc tgcctaatct caagaaattc tctcacttac atgtaaaaca aagcattagt 152580taaaatttaa taacaaaaag tagtattaga atagaataat cattcaacag ctttgttttt 152640ggaattatat ctaaatgtaa atcccaatat aagcccccga cattgcggta aatggtaagt 152700caaggctgag ccctatttct gtttaagatt agactgagct ttgaaataag ctgtttaagg 152760ttacataata gcaggcagac agtacatctg attagacttt gcctgtgtct ttgcctttcg 152820gcttcaaccc tccaaactta aattaattta tgcacttcta tatctacttc attaaaaatt 152880agatttttac tgcttcaaat tttatctaaa attaattcga gtttgtttaa aatttatata 152940tatttttaaa aattacattt tatttttatt taaaatttaa aatttatttt ttattttgat 153000caaatttctt tttgtttttg aaaattattt tgatcaacat tttaaatata aacaaattaa 153060aaaaattata tttactaaat attaaataaa aatattatac tataaatatt atgaaaattt 153120taaaaaatca acctcgctta actcacttaa atatttaaat tcgagcctag tttatgttta 153180aaactttaaa tgagcttata caaatttatt tttcaagttt aatattaatt aaaccttttc 153240taaatatctg ataaaattta aataaataac taaacccttg aataaatcta tttgagatta 153300tcaaattaaa taagaataaa aaagtttaaa tacctaaatt taaaataatt agttttttca 153360gtatttagaa tttaggatta ccaacaaaaa cttaaatggc ataatgactt gtttggccct 153420tcaactttat aaaaaagtta ttttagccat ttatttaatt ttttattttt ttaaccctta 153480aacttgtatt ttttgtcaaa tcaccctaaa atagatggaa aagttaacat tttttaactt 153540tgctaatgtg gcatactcgt ggattgccat gtggatgaca cattagcatt taattaactt 153600tttaaatttt taaaagttca aaaaatatat aataaattgt tttaaaaaac ttaaaattat 153660taaaaatagt atttttaaaa tttaaaaaaa taattaaata ttggcatgtc atctatttga 153720taatccacgt gtatgtcaca ttcgcaaagt taaaaaatat taacattttc atctattttg 153780ggatgattta acaaaaaaaa atacaatttc aatggctaca aagaacaaaa attaaataaa 153840agcctaaaat aatttttttc ataaggttag agggacaact ttgaagagtt taaatttcca 153900tgaaaagaaa tgaatgggta ggaaaagaaa aatgtgaagc agaattagca atttcaatgc 153960atccaaccaa cccacccctc cgcctaaatt actaaagtat ttctattaaa agaagaaata 154020attaagtaca ataataatgc atgtatttgg gtcatcatag ggacatataa ttagggattc 154080aagctacttt ttgttgcata tataaattaa tataaaattt taaattgggt agatgaaata 154140taaataattg gtgtataaaa aaagaagaaa ttaattgttg ggggagatca tgtccccttc 154200tagacaagcc tcatgtttgg tacgagtatg ccattttccc gacatggggt ggaaccacat 154260ttaagaaaag gaaggcaaag agcccattag tctttatcaa tctcataatg taagcttccc 154320ccttcgcatc attttagata tgtttggcta catgttttaa cttgtaatcc cacacaacac 154380tccatgttat tcttgtggat gtctctattt tccttgagcc taaccccagc atgtatctct 154440cacattttgt acttaaattt tttacccttc tatcacatat tttattcata taaatcatat 154500atattaaaat tttaataatt ttaataaaaa atttaataat ttgtttagct taaaaaaaaa 154560atcatccata agggtggtct taagtagcaa gtaggagact ttaccttttc aaaaaagaca 154620aatttttaat taaaaccttt taaaaattat aatattaaaa gttaatataa tagtaaaatt 154680atatttttca aaaatatatt gtttaagaat tctatacaca cccttgaggc cttaactttt 154740tttttcaagt cctccaaagc aaaatgcagt aatccataag ggttatcatt agatttgaga 154800tttataattg aaggatagtt tgaataaatt tgagaaaagg gttatgccta acctattaat 154860tgtataatca aatttgatta ttctatgata ggttaacgta ataacctatt tttatgttga 154920gaaattaatc atagtggtga atctgattca atttagttca atcaagattt ttggaatttt 154980caaatcaatt attataatta aatatgactc tcgatttttt atactaattt tatttttatg 155040aacttgcttt gacaaaatta actttattta atagcttttg acattgaaaa aatttcgata 155100taattttcaa atgaataaaa aataaaatta attataattt aaataaaaaa caaaattttg 155160ttttgagtat attcccaaaa tcatgtaaac attgccgttg gaatcgactt ttgtttttag 155220ttttttattt ttattttttc tttgtggagc ctaattaata gtgaagtttg aatttcataa 155280ttagggacga aaaagttgct acaactctca tcacaataaa ttgaaaagaa gaaagtaata 155340atttcaatag cacatgcacc catgtactaa acctaaatta ttgcaaaaat ttgttggtca 155400ttcattgata tttgatactt gtattcatca gtttccgccg ctatacaccc tccaccaatc 155460tctaactatt cctttgaaaa tggctctccc ttctgcccta ggtatcaacc aaactcttgg 155520aattgattta attaagttat acaaaatgtt gaaacagctt atattataag cggtacaaag 155580agtcgttaaa atagagtgaa taatgtggag agccgctaga actaatggat tgagggagtc 155640gaggccgtag ttgagcaaag tttgggtaat gagctttgtc tcttgtctgc aggttttatt 155700gaaagaaggg atatctataa aggcttgaag tgtgccattt ttctcatgat aatatcttgg 155760taaagtccac cttaagtcaa gattaaagta tgagttaaca aacttagcaa attaaaaaca 155820ttaattagat tagatttgat aggatgatta ttattgacgg tgttgcagcg atatttcatc 155880tctaaataca tttataataa cttgagatat tcaaatgaat atactaaaat ttgaatgcag 155940aaacatagta cgaagcttaa tcaatcacaa ataaataatc aaatacatag taattatttg 156000ataattgtat ggtgtagttc tttatttttc ataaacaggg ttagaatacc attaattaat 156060ttaattgaac attttaaaaa caaattcgtt aataatgtag tcgctaatta tcagtacagt 156120ggaattaatg tgacatgaat ataattgggg ttagggaggt aataaggagt tgttttcaag 156180gcatttaacc aaagggaccg tcataattaa aaacgcactt catttttgaa ggcaatatat 156240acctacttct atactatttg catgtgaaca gtcaccttcc attcttcatt attactcatt 156300aaaaacttta ctagagatta gctctccctc ttatttatat agctgctgcc cagtggatca 156360caaactagct aacctaacct aacctaacct aacctagcct tggattcagt ttcatgtatt 156420aaactcccaa ctactactac gaaattcaag ttggaaaccc aacatttcat agtcatcgta 156480cgagataatt ctttagattc tcgccgatac acagaaattg aaggacttat ttctctttga 156540tatatataca tgtagttcaa tatgaaataa attcatatat attagaggac atggcagaag 156600caagtgtgga agagttggga aacattgcct gttattttct tgtttccatg agaaggtgtt 156660gggggaacta tggttgaatt aggaaggagt caagtgggca ggcttgttga tgttcctggg 156720cgtgcatgtg catgtgcatg tgcatgcatg gagattttgc caatacatgt tgacaaacta 156780tggggttttt tgcgacagtt ttctttctac ggtgcttgct ggcaaatgca catgcacgtg 156840catttttatt atgttttttg gttttagacc tattgctttc atatttatgc gtgtggactg 156900tggacagcca atacaattta tgttaatata ttgataaaaa taatggaaat ttatctaaca 156960cccccaaaat gtaaatttat ataaattata tgattatgac aataatattc cgattattaa 157020ataaataaaa tcatgataat gtattcaatt atttatattc tttcttggta aacagatatg 157080tccaatgatt gtcttgagct aacaaggcat atttttctgg tacatgtaaa aagataaata 157140aacaaaaatt tcaaaggaat gataaaataa tataatattt agttaacttc aatttcgaat 157200ttaattcatg gattaactct acctcttaat aaggaacaat tttctaagat tgtattagca 157260atcagtcaac agatcagata tttttaaaaa aatataacag tttaatgatt ttccttcctt 157320tttcctagaa aattcaggaa caagaagcat taaaagaaaa tataaatatt aattagttgt 157380tgttgcagct ttcttagata ccaaagaggg tttaattgaa aaacttttta tttttttgaa 157440aatatttata aaaaatattt tagaatttta tcgaataata tttaaaaatc ataaaacaaa 157500ctaaagttaa tcaaaataat acctaaaatt aatataaact atatcattaa aacaagagtg 157560ttgagattat taaatcaata aaatcatgat aatatagcca atttattgtc ttcaactaac 157620aaggcatttt tctggtataa catgaagaat attaacagca tgtgaataat aaaaattcgt 157680tttactgata aaatatttat atttaaaaat ctaaaatcgt gtgagcaata attatatgct 157740gctatagtca gcaagtcaga ttataaaaaa aataataata aagcagtttt ctgaattttc 157800tttttccttt ttcctggaaa cttcagtaaa aaaaaaacat tagaagacaa gagaacaagt 157860tttagtgagt tgtagttgca gctttcttag gtaacaaaga gagtttctcg tcactttcat 157920gccatatgat gccgtatgct acaaaaactt gtttaacatt attaatttcc cttacattat 157980taaaagaaaa agtgcatttt gagttattgt cagcgttagg tttattggaa aaattacggc 158040ttttagatta ataaagcttg tagtaaatga aatgtctgcc tgccttgaca aaaaaggaca 158100actctcgaaa tggccacttt atttttaact caaaaagttg aatggggaga aattaagtgt 158160taataattac actaaattct tcctttttct attattgctg acctattcgc tatttgggta 158220taaaatctga tatataacat tttaaaatct ttaatactgc tatcattcct gaaaatcacg 158280tttttagaat atagttattt gtttaaaaat aatataattt attttaaata tgaaaaatta 158340tttttataat ttttttaatt gagttggtgt aattggctct taatataact tagtctgata 158400ttttactaac agtataggta tatataatta atagagaata aaattaaaat gtaacaaatt 158460aggccttggt tggatggtaa atttaatatt tttttaatat aattagcatg agtttaaatc 158520tcattatatg tatattttta ttattttttt aaatttaaaa aatcttaaat taccatttaa 158580taacttattt taaacacata aaggtttttt tttcttaagc gaattgatac ttgattgact 158640tgttatacca acttaatcaa aaattttatt aataatatag acaaaatgat aaacgaattg 158700ttagattaat taatttggat attgatccaa accaaacata agtatattat taaggaacgt 158760agaattctaa taaacaattt tacatttcat gacccaacag tttgctttaa aggagtttat 158820ttcaagccac tgtttgctag aaaaaatcta aaaaaaaaat gaaaatataa gtgagaatta 158880tttcttcaaa aaaccaacaa attacacaaa tataatatga catttaaata tctaaattca 158940aaacaaaaat taaaaaaata agtgggaatt attttttatt ttgagtaaag ccttatttaa 159000aattcttgta caaattcaag caatattatc ctgttattta ttgatattat attttttttt 159060aaaaaccctt aaaagcctac attttcttta ctccccactt gttctaatta aaactttcgg 159120gtgaaatcaa gcctccaatg gcaaatatat gatcttctag agggaaccta gctatagact 159180ttatttttta aaagaaaatt aaagttgtta aaaagtattt ttctttaata ttttcaaact 159240tattaaattc atggacagta cttgatttag aaaccctaat caatcaagcc gattgagctt 159300caacttagtt gatatcgtta ttgagttcaa gcacatttaa gcatattttt tttatataaa 159360tattaaataa gaattttgaa aaaaaaaacc ttaatcaaga attgttgaaa aaaaattaac 159420aaaaaaccca cttaatttgt agttacaaat atttgatgta tttaattaaa aatgctaaca 159480ttttgaatat aatttgattt gaaggcatta aatatctcat gcgtcatcat cagttaataa 159540taatataata tatttctaaa ttcaaaaatt gaaactttaa aaaacaatat ccgaaaccta 159600aggcttgaaa gaaaaataaa ataaaataat tttaatttta attatgttta aaaatttgaa 159660taaaaaactt gaaattcaaa accccaaatc tttaatttaa aataaaaaaa cttgaaactc 159720aaaatttaaa cacagaaaga aaaacaaaat aattataatt aatatttaac tatctttaat 159780acagttaata ttatttattt tcaagtcctt caattttttt ttcatttttt acaccaataa 159840tgatgtaaaa caaatattaa tcatacatta aatttgtaaa ttagatctaa aaaataaaat 159900atatactttt atattaataa atattaggtg cgatgataaa ttatattata tgttttaaaa 159960aaaagaattc gagttcaaag attaaaagtg ttgattatta ggaaaacaac cgtgaactca 160020aaaaaatatt aatttttata atttgaaaaa caaaaaaaaa atctgaataa tataaaaaac 160080aataatactc ccatatgaca accaccacca taatgcttta attcaaaatg gtcccaactg 160140gaaaacaaag aaagaaaaag ggcaggaaaa cagattaaat aaaatctttc cgtacaaaga 160200tggatcgact gactgagttt ataaaatttt gttattattt attttagtaa acactgatat 160260actttttagg cattcaattg caggagaggc aacgcccacc taccttcaac cccaaacaga 160320tctcgtctct gttaaaattt gcaggcctac gcctagctat ctcctaaacg tttctcctct 160380cacgtcttcc actgtttggt tctcgagaaa taaatcaatt tacaaattta atgccattca 160440tcttcaacta tctttacctc tttcaaccca aaattttcaa tttattgcac cgtcctactc 160500gtatcgtatg ttccccgtgt atatttcgct ctacgttttt tttttaatca ttttgtaata 160560atttggtttt cctttttata tatatatatt tatttattta gggcttgaat ttttttactt 160620catttctatg aattattttt aaatatatat atttttatgt ttattttatt gtctatattg 160680gagatttgta aatatctctt ttaacaatgt tgtctcttta aactcgcgtt cttttcttgt 160740taaaaaaata tatttaaatt atttttataa ttttaaatat aaaatatttt tacatcataa 160800aaataaaaaa ttaataaaaa ataaaattcg ctccaattcg attttagtat ctacaacttt 160860ttaatttaca tttcaaaaat aatccaaaca tcaattttca tttatttttc ttttttttca 160920aaaaccctat atatggttat gaattgagct taaaataagc cttgtagata agtaagatga 160980attcgaatgc ttttggattc ttgtaagaga aacatgaagt ttgaaggaat cagagcataa 161040gatttggaac actctctgat cttttaagtc aatctagaga ctttatcata aagaaatgaa 161100agaatagagt gaaaaaagag actcaattaa aatttaaaaa ttatttatat taattattta 161160aaaaatactt aaaatttata tttcatatac cacaatatta acaatgagtt gccgtgaatt 161220attttattat atttacacta tcgttcaagt tgaatttttt ttaataccaa aataatacta 161280acttggcatc gtagtaataa taacatgtaa aactaagaat aatttacatt taattctatt 161340attaatttta ttataattta agtttatata tttattctat atatcataat attaattact 161400taatacatta ttttaaatat ttcgattata aattaagttt atatatttac tatattataa 161460tattaatgaa attaaatatt aaacatggac attttatttt tgtaaaagca ttttttaact 161520tcaatggtaa actaaattaa tttcttgcta ttttggttct cctcgtaacc agatatgttt 161580taatatagtt tttgtattgt acaaaaaaat aatttaatgt gattaagttg ttatgtattt 161640gatattagtg taaataaata taaaactcgt ctaaatatcg attcaatttt taaaatttta 161700taatgtatgt atttatttat ttaaaaaaat tatatttttt taatatactt ttattattcg 161760aaattattga tataattgta agttagattt tagtaaatta aaaatattat atgatataat 161820aaaattgaaa atatcactaa accattattt agaaaaatat taatattatt aattatatat 161880atattactat aaaagtttga ctgagttagt gttagccgtc aatcaagtcc caactcaatc 161940ataaaaatat caaaatgtta atttttttat aaagcaataa atattatttt gagggtattt 162000gtatattttt atataaataa atgaggagta tttggtacac tcagtgtact tttttttatt 162060ctactagtag tcttaaaaaa ttgacatgtt ttttttaata tatatttttt aataatttac 162120tatatttttt aataatttaa actaccataa catctctcta attcataaat aagtgaataa 162180tacgttttag cgtattcgaa tctatctttt tataacaata cttattctaa gtgagataag 162240actttatggg cataatttta ttttatgtaa taaaaaaaaa acctaaacgg taaatagaag 162300tggcaaaggt tgaatctcat gggctagaag ggaaaggaaa acattgtttt atgaaacaaa 162360atgacatgac ggttctaatt tttccttctt tttttattgt atttgtggta ttgaggagaa 162420gaaatatata gaaaatgaat aagttggtaa ttactaaatg tagcaaaacc cgaaaacatt 162480ctttgactcg aacatccagg aataaataat gtataaatca gttgacttgt aaaataatct 162540acccagggag ggaaaatatt tgtgaaactg gaagggataa acctataacc atatttcttt 162600ttaatttatt gaccacattt ttggtttatt aaattgaatt atgaaaagag acagatcata 162660tggaaaaagg tcccacttat caccagactc gtggctttgg gtctgcgcaa tcagacagta 162720gaagctccca aaaagagaag taaggtcaaa agaacccccc aacccaacct ccctttaaat 162780gaaaagcatc actctactgt ttccttagcc cgactttgac ccttccccca ttttcaatta 162840aaataccaca cccttcccaa tatatgtctt cttgtttgtc cccccaaact tcgctttcat 162900tatcattatt atccatataa tacgcgtaga ataatttagg tactattttg agttggtttt 162960caagaggatt aaattaaaaa gaaacgacag cgcatctttc tcgccttttc atgtgattta 163020aattttaaac cccccaccct tgctctttca ctgcaaaaag aaaagcgaat gagccccccc 163080cccccatttt tactttttca tatataaata aactgatcaa ataaataaaa gggaaaagag 163140atgatgagac aatcccatgg cagtatggga cttctcactg atcctcctct ttttataatt 163200tatctcattc aatatatttt ttctttttaa gaaaaaactt ttaacaaaaa tatatctcac 163260ccaaaaaaaa aatcatgata tttcccatct ccttgctata agctgtacac tttgttattc 163320caagtttccc ctcttcacct ccctactttt taatctatct atctcttgga accatcttgt 163380tccctaaatc ttcacaaatt cacaaaattt ccacccccat gaaaatcata acgaaattga 163440ataaaataaa aacaaggaac cccaccataa tcccccaatt tctgtccccc ccccacctcc 163500tttgaatgta caggagcttg caccatcaaa atcatgatga tgatgaatca atcctcactt 163560cacccaccac catttccaac cctgctgcag tcgactgaga ttgagctatc ccaagatgtt 163620ttgctcacta gcttggtttt aagtgtgccc tgtggagtct tagctcaaag cttgtaacgg 163680gggcctgaac gatttcccct atatgggaga actgaagtgg cattttcaga ctagacacag 163740gccagatgtt ttattccaat tagccttctg atcatgctct actcttcttc aatgaaaatt 163800cttcctataa aatcccgaaa cctcttcgag tagagttttg gaccaacagc tgatattgaa 163860gaaggatcag cttgtagtga tttgtaggca tgctccaatt tcttgctgat gtcgtagtct 163920tgtaagatgt caatgatgcc aaagtataac actacttcat aaacttcgcc actatgtgaa 163980aagagaccga ctccaccctg tgtatactga tcaaagtcgc ttcttcttga cattcgcact 164040gctcttgctg gcatgtttgc tcctagccgt atcaatggtt tcctgccaaa cgaaaaatac 164100atagaaaatt atttagaatt tctgttaagt tttctttccc tacagaatca caactcgcag 164160aagaggcatc aatccacaac acataaaatt ctaactgcat actatcaaag tccatttcag 164220atccaattcc ctaaagtatc tcaaatgcgt atataatttt caccaagagg gtgatgaaca 164280aatggtatgt catatggtga cagtccaaag atcattagaa gggaaagaga ccttaaaata 164340gaactagtga tatgcaccaa accatcgggt cacattctcc accattctca tccactgagt 164400tcatgaatca tttcctagaa actagataaa tgggtaacaa aaaatcaagg caacccttga 164460ttcttatcta gtaggatcat aaagggccac aattgctaca tgccccacca aaaaaaaaat 164520ttgtcttctg aattatttcc acaaaatcac tcagacaaca caatcaacct tcaattattc 164580catcgaatat gatcaagtat caacagttga ccgagaaatt taaaatttaa gcatcatcat 164640ggaggagcct tgcctaattc cacatctaca ataaggaaat aactagaacc aaaagtatta 164700tctaaggaag agagcaaaac atactggcca gctaaaatcc gatccatgtc ctgtagctca 164760gcttcaagga atctacagcc acgcataaac ttttcattct gatatgaatc ctttttgcct 164820gcatagggaa ggcatattca aatcccgatt cttatttcaa cagatattat ccattcaaat 164880ttcatgagga atagtatggt tttctattaa cttacctgtg cgcaagagaa acggtgataa 164940ccccatttta tcgcctctat tatcatcccg aaagtgtagt ccaaccaaaa gactataatc 165000cataattctc tcagcctcca agaactcgca atctcgatca

atttgcctat caccatgtag 165060tcataggaaa ttagcaaaat acacgacact gacataactg gagtaaagta ataaaatata 165120gcttacttca taagctcttg gaaccaattc ctctggaggc gaaacacata attaagatcc 165180aggtctttaa gggtagtggt ttcatcaatt tcctcttctg gcttatcagt tgagcggcca 165240tgggaggatc 165250541065DNAGossypium barbadenseCDS(50)..(589) 54aatatagtga aatatgggtc caagattttc tgggttttta atctaagca atg ctg ttt 58 Met Leu Phe 1 tta act caa ctc ctc tct cta aca gat ggc cgt gat att ggt gtt tgc 106Leu Thr Gln Leu Leu Ser Leu Thr Asp Gly Arg Asp Ile Gly Val Cys 5 10 15 tat ggt ttg aac ggc aac aat ctt cca tct cca gga gat gtt att aat 154Tyr Gly Leu Asn Gly Asn Asn Leu Pro Ser Pro Gly Asp Val Ile Asn 20 25 30 35 ctt ttc aaa act agt ggc ata aac aat atc agg ctc tac cag cct tac 202Leu Phe Lys Thr Ser Gly Ile Asn Asn Ile Arg Leu Tyr Gln Pro Tyr 40 45 50 cct gaa gtg ctc gaa gca gca agg gga tcg gga ata tcc ctc tcg atg 250Pro Glu Val Leu Glu Ala Ala Arg Gly Ser Gly Ile Ser Leu Ser Met 55 60 65 agt acg aca aac gag gac ata caa agc ctc gca acg gat caa act cat 298Ser Thr Thr Asn Glu Asp Ile Gln Ser Leu Ala Thr Asp Gln Thr His 70 75 80 caa agt gca gcc gat gca tgg gtt aac acc aac atc gtc cct tat aag 346Gln Ser Ala Ala Asp Ala Trp Val Asn Thr Asn Ile Val Pro Tyr Lys 85 90 95 gaa gat gtt caa ttc agg ttc atc atc att ggg aat gaa gcc att cca 394Glu Asp Val Gln Phe Arg Phe Ile Ile Ile Gly Asn Glu Ala Ile Pro 100 105 110 115 gga cag tca agc tct tac att cct ggt gcc atg aac aac ata atg aac 442Gly Gln Ser Ser Ser Tyr Ile Pro Gly Ala Met Asn Asn Ile Met Asn 120 125 130 tcg ctc gcc tca ttt ggg cta ggc acg acg aag gtt acg acc gtg gtc 490Ser Leu Ala Ser Phe Gly Leu Gly Thr Thr Lys Val Thr Thr Val Val 135 140 145 ccg atg aat gcc cta agt acc tcg tac cct cct tca gac ggc gct ttt 538Pro Met Asn Ala Leu Ser Thr Ser Tyr Pro Pro Ser Asp Gly Ala Phe 150 155 160 gga agc gat ata aca tcg atc atg act agt atc atg gcc att ctg gtt 586Gly Ser Asp Ile Thr Ser Ile Met Thr Ser Ile Met Ala Ile Leu Val 165 170 175 tga caggattcgc ccctcctgat caatgtgtac ccttattttg cctatgcctc 639agaccccact catatttccc tcaactacgc cttgttcacc tcgaccgcac cggtggtggt 699cgaccaaggc ttggaatact acaacctctt tgacggcata gtcgatgctt tcaatgccgc 759cctagataag atcggcttcg gccaaattac tctcattgta gccgaaactg gatggccgac 819cgccggtaac gagccttaca cgagtgtcgc gaacgctcaa acttataaca agaacttgtt 879gaatcatgtg acgcagaaag gggctccgaa aagacctgaa tatataatgc cgacgttttt 939cttcgagatg ttcaacgaga acttgaagca acccacagta gagcagatgt tcaacgagat 999gttcaacgag aacttgaaat gttattgttg gctatttaaa tcttttgcca gagacgcttc 1059atatag 106555179PRTGossypium barbadense 55Met Leu Phe Leu Thr Gln Leu Leu Ser Leu Thr Asp Gly Arg Asp Ile 1 5 10 15 Gly Val Cys Tyr Gly Leu Asn Gly Asn Asn Leu Pro Ser Pro Gly Asp 20 25 30 Val Ile Asn Leu Phe Lys Thr Ser Gly Ile Asn Asn Ile Arg Leu Tyr 35 40 45 Gln Pro Tyr Pro Glu Val Leu Glu Ala Ala Arg Gly Ser Gly Ile Ser 50 55 60 Leu Ser Met Ser Thr Thr Asn Glu Asp Ile Gln Ser Leu Ala Thr Asp 65 70 75 80 Gln Thr His Gln Ser Ala Ala Asp Ala Trp Val Asn Thr Asn Ile Val 85 90 95 Pro Tyr Lys Glu Asp Val Gln Phe Arg Phe Ile Ile Ile Gly Asn Glu 100 105 110 Ala Ile Pro Gly Gln Ser Ser Ser Tyr Ile Pro Gly Ala Met Asn Asn 115 120 125 Ile Met Asn Ser Leu Ala Ser Phe Gly Leu Gly Thr Thr Lys Val Thr 130 135 140 Thr Val Val Pro Met Asn Ala Leu Ser Thr Ser Tyr Pro Pro Ser Asp 145 150 155 160 Gly Ala Phe Gly Ser Asp Ile Thr Ser Ile Met Thr Ser Ile Met Ala 165 170 175 Ile Leu Val 561239DNAGossypium darwiniiCDS(112)..(145)CDS(258)..(760) 56aagaaacgag caccagttat tgactttcct ttgtaaaaaa aaaaaaagtg ctgagatcaa 60gaaatatagt gaaatatggg tccaagattt tctgggtttt taatctaagc a atg ctg 117 Met Leu 1 ttt tta act caa ctc ctc tct cta aca g gtaaaacaaa cttctctaca 165Phe Leu Thr Gln Leu Leu Ser Leu Thr 5 10 gtgattttac agtaaatatg gctttgaaaa atatacaaca aaacatttat cttcaatcca 225ttttaattac tgatctacta tatatgttgc ag at ggc cgt gat att ggt gtt 277 Asp Gly Arg Asp Ile Gly Val 15 tgc tat ggt ttg aac ggc aac aat ctt cca tct cca gga gat gtt att 325Cys Tyr Gly Leu Asn Gly Asn Asn Leu Pro Ser Pro Gly Asp Val Ile 20 25 30 aat ctt ttc aaa act agt ggc ata aac aat atc agg ctc tac cag cct 373Asn Leu Phe Lys Thr Ser Gly Ile Asn Asn Ile Arg Leu Tyr Gln Pro 35 40 45 50 tac cct gaa gtg ctc gaa gca gca agg gga tcg gga ata tcc ctc tcg 421Tyr Pro Glu Val Leu Glu Ala Ala Arg Gly Ser Gly Ile Ser Leu Ser 55 60 65 atg agt acg aca aac gag gac ata caa agc ctc gca acg gat caa act 469Met Ser Thr Thr Asn Glu Asp Ile Gln Ser Leu Ala Thr Asp Gln Thr 70 75 80 cat caa agt gca gcc gat gca tgg gtt aac acc aac atc gtc cct tat 517His Gln Ser Ala Ala Asp Ala Trp Val Asn Thr Asn Ile Val Pro Tyr 85 90 95 aag gaa gat gtt caa ttc agg ttc atc atc att ggg aat gaa gcc att 565Lys Glu Asp Val Gln Phe Arg Phe Ile Ile Ile Gly Asn Glu Ala Ile 100 105 110 cca gga cag tca agc tct tac att cct ggt gcc atg aac aac ata atg 613Pro Gly Gln Ser Ser Ser Tyr Ile Pro Gly Ala Met Asn Asn Ile Met 115 120 125 130 aac tcg ctc gcc tca ttt ggg cta ggc acg acg aag gtt acg acc gtg 661Asn Ser Leu Ala Ser Phe Gly Leu Gly Thr Thr Lys Val Thr Thr Val 135 140 145 gtc ccg atg aat gcc cta agt acc tcg tac cct cct tca gac ggc gct 709Val Pro Met Asn Ala Leu Ser Thr Ser Tyr Pro Pro Ser Asp Gly Ala 150 155 160 ttt gga agc gat ata aca tcg atc atg act agt atc atg gcc att ctg 757Phe Gly Ser Asp Ile Thr Ser Ile Met Thr Ser Ile Met Ala Ile Leu 165 170 175 gtt tgacaggatt cgcccctcct gatcaatgtg tacccttatt ttgcctatgc 810Val ctcagacccc actcatattt ccctcaacta cgccttgttc acctcgaccg caccggtggt 870ggtcgaccaa ggcttggaat actacaacct ctttgacggc atagtcgatg ctttcaatgc 930cgccctagat aagatcggct tcggccaaat tactctcatt gtagccgaaa ctggatggcc 990gaccgccggt aacgagcctt acacgagtgt cgcgaacgct caaacttata acaagaactt 1050gttgaatcat gtgacgcaga aagggactcc gaaaagacct gaatatataa tgccgacgtt 1110tttcttcgag atgttcaacg agaacttgaa gcaacccaca gttgagcaga tgttcaacga 1170gatgttcaac gagaacttga aatgttattg ttggctattt aaatcttttg ccagagacgc 1230ttcatatag 123957179PRTGossypium darwinii 57Met Leu Phe Leu Thr Gln Leu Leu Ser Leu Thr Asp Gly Arg Asp Ile 1 5 10 15 Gly Val Cys Tyr Gly Leu Asn Gly Asn Asn Leu Pro Ser Pro Gly Asp 20 25 30 Val Ile Asn Leu Phe Lys Thr Ser Gly Ile Asn Asn Ile Arg Leu Tyr 35 40 45 Gln Pro Tyr Pro Glu Val Leu Glu Ala Ala Arg Gly Ser Gly Ile Ser 50 55 60 Leu Ser Met Ser Thr Thr Asn Glu Asp Ile Gln Ser Leu Ala Thr Asp 65 70 75 80 Gln Thr His Gln Ser Ala Ala Asp Ala Trp Val Asn Thr Asn Ile Val 85 90 95 Pro Tyr Lys Glu Asp Val Gln Phe Arg Phe Ile Ile Ile Gly Asn Glu 100 105 110 Ala Ile Pro Gly Gln Ser Ser Ser Tyr Ile Pro Gly Ala Met Asn Asn 115 120 125 Ile Met Asn Ser Leu Ala Ser Phe Gly Leu Gly Thr Thr Lys Val Thr 130 135 140 Thr Val Val Pro Met Asn Ala Leu Ser Thr Ser Tyr Pro Pro Ser Asp 145 150 155 160 Gly Ala Phe Gly Ser Asp Ile Thr Ser Ile Met Thr Ser Ile Met Ala 165 170 175 Ile Leu Val 581234DNAGossypium darwiniiCDS(75)..(144)CDS(239)..(1179) 58aagaaacgag caccagttat tgacattcct ttgtaaaaaa aagaagaagc tgagatcaag 60aaatatagtg aaat atg ggt cca aca ttt tct ggg ttt tta atc tca gca 110 Met Gly Pro Thr Phe Ser Gly Phe Leu Ile Ser Ala 1 5 10 atg gtg ttt tta act caa ctc ctc tct cta aca g gtaaaacaaa 154Met Val Phe Leu Thr Gln Leu Leu Ser Leu Thr 15 20 cttctctaca gtgattttac ggtaagtatg gctttgaaaa atatacaaca aaacatttat 214actgatctac catatatgtt gcag at ggc cgt gat att ggt gtt tgc tat 264 Asp Gly Arg Asp Ile Gly Val Cys Tyr 25 30 ggt ttg aac ggc aac aat ctt cca tct cca gga gat gtt att aat ctt 312Gly Leu Asn Gly Asn Asn Leu Pro Ser Pro Gly Asp Val Ile Asn Leu 35 40 45 tac aaa act agt ggc ata aac aat atc agg ctc tac cag tct tac cct 360Tyr Lys Thr Ser Gly Ile Asn Asn Ile Arg Leu Tyr Gln Ser Tyr Pro 50 55 60 gaa gtg ctc gaa gca gca agg gga tcg gga ata tcc ctc tcg atg ggt 408Glu Val Leu Glu Ala Ala Arg Gly Ser Gly Ile Ser Leu Ser Met Gly 65 70 75 80 ccg aga aac gag gac ata caa agc ctc gca aaa gat caa agt gca gcc 456Pro Arg Asn Glu Asp Ile Gln Ser Leu Ala Lys Asp Gln Ser Ala Ala 85 90 95 gat gca tgg gtt aac acc aac atc gtc cct tat aag gac gat gtt cag 504Asp Ala Trp Val Asn Thr Asn Ile Val Pro Tyr Lys Asp Asp Val Gln 100 105 110 ttc aag ttg atc act att ggg aat gaa gcc att tca gga caa tca agc 552Phe Lys Leu Ile Thr Ile Gly Asn Glu Ala Ile Ser Gly Gln Ser Ser 115 120 125 tct tac att cct gat gcc atg aac aac ata atg aac tcg ctc gcc tta 600Ser Tyr Ile Pro Asp Ala Met Asn Asn Ile Met Asn Ser Leu Ala Leu 130 135 140 ttt ggg tta ggc acg acg aag gtt acg acc gtg gtc ccg atg aat gcc 648Phe Gly Leu Gly Thr Thr Lys Val Thr Thr Val Val Pro Met Asn Ala 145 150 155 160 cta agt acc tcg tac cct cct tca gac ggc gct ttt gga agc gat ata 696Leu Ser Thr Ser Tyr Pro Pro Ser Asp Gly Ala Phe Gly Ser Asp Ile 165 170 175 aca tcg atc atg act agt atc atg gcc att ctg gct gta cag gat tcg 744Thr Ser Ile Met Thr Ser Ile Met Ala Ile Leu Ala Val Gln Asp Ser 180 185 190 ccc ctc ctg atc aat gtg tac cct tat ttt gcc tat gcc tca gac ccc 792Pro Leu Leu Ile Asn Val Tyr Pro Tyr Phe Ala Tyr Ala Ser Asp Pro 195 200 205 act cat att tcc ctc gat tac gcc ttg ttc acc tcg acc gca ccg gtg 840Thr His Ile Ser Leu Asp Tyr Ala Leu Phe Thr Ser Thr Ala Pro Val 210 215 220 gtg gtc gac caa ggc ttg gaa tac tac aac ctc ttt gac ggc atg gtc 888Val Val Asp Gln Gly Leu Glu Tyr Tyr Asn Leu Phe Asp Gly Met Val 225 230 235 240 gat gct ttc aat gcc gcc cta gat aag atc ggc ttc ggc caa att act 936Asp Ala Phe Asn Ala Ala Leu Asp Lys Ile Gly Phe Gly Gln Ile Thr 245 250 255 ctc att gta gcc gaa act gga tgg ccg acc gcc ggt aac gag cct tac 984Leu Ile Val Ala Glu Thr Gly Trp Pro Thr Ala Gly Asn Glu Pro Tyr 260 265 270 acg agt gtc gcg aac gct caa act tat aac aag aac ttg tta aat cat 1032Thr Ser Val Ala Asn Ala Gln Thr Tyr Asn Lys Asn Leu Leu Asn His 275 280 285 gtg acg cag aag ggg act ccg aaa aga cct gaa tat ata atg ccg acg 1080Val Thr Gln Lys Gly Thr Pro Lys Arg Pro Glu Tyr Ile Met Pro Thr 290 295 300 ttt ttc ttc gag atg ttc aac gag gat ttg aag caa ccc aca gtt gag 1128Phe Phe Phe Glu Met Phe Asn Glu Asp Leu Lys Gln Pro Thr Val Glu 305 310 315 320 cag aat ttc gga ttc ttc ttc ccc aat atg aac cct gtt tat cca ttt 1176Gln Asn Phe Gly Phe Phe Phe Pro Asn Met Asn Pro Val Tyr Pro Phe 325 330 335 tgg tgaagttgaa atgttgttgg ctatttaaat cttttgccag agacgcttca tatag 1234Trp 59337PRTGossypium darwinii 59Met Gly Pro Thr Phe Ser Gly Phe Leu Ile Ser Ala Met Val Phe Leu 1 5 10 15 Thr Gln Leu Leu Ser Leu Thr Asp Gly Arg Asp Ile Gly Val Cys Tyr 20 25 30 Gly Leu Asn Gly Asn Asn Leu Pro Ser Pro Gly Asp Val Ile Asn Leu 35 40 45 Tyr Lys Thr Ser Gly Ile Asn Asn Ile Arg Leu Tyr Gln Ser Tyr Pro 50 55 60 Glu Val Leu Glu Ala Ala Arg Gly Ser Gly Ile Ser Leu Ser Met Gly 65 70 75 80 Pro Arg Asn Glu Asp Ile Gln Ser Leu Ala Lys Asp Gln Ser Ala Ala 85 90 95 Asp Ala Trp Val Asn Thr Asn Ile Val Pro Tyr Lys Asp Asp Val Gln 100 105 110 Phe Lys Leu Ile Thr Ile Gly Asn Glu Ala Ile Ser Gly Gln Ser Ser 115 120 125 Ser Tyr Ile Pro Asp Ala Met Asn Asn Ile Met Asn Ser Leu Ala Leu 130 135 140 Phe Gly Leu Gly Thr Thr Lys Val Thr Thr Val Val Pro Met Asn Ala 145 150 155 160 Leu Ser Thr Ser Tyr Pro Pro Ser Asp Gly Ala Phe Gly Ser Asp Ile 165 170 175 Thr Ser Ile Met Thr Ser Ile Met Ala Ile Leu Ala Val Gln Asp Ser 180 185 190 Pro Leu Leu Ile Asn Val Tyr Pro Tyr Phe Ala Tyr Ala Ser Asp Pro 195 200 205 Thr His Ile Ser Leu Asp Tyr Ala Leu Phe Thr Ser Thr Ala Pro Val 210 215 220 Val Val Asp Gln Gly Leu Glu Tyr Tyr Asn Leu Phe Asp Gly Met Val 225 230 235 240 Asp Ala Phe Asn Ala Ala Leu Asp Lys Ile Gly Phe Gly Gln Ile Thr 245 250 255 Leu Ile Val Ala Glu Thr Gly Trp Pro Thr Ala Gly Asn Glu Pro Tyr 260 265 270 Thr Ser Val Ala Asn Ala Gln Thr Tyr Asn Lys Asn Leu Leu Asn His 275 280 285 Val Thr Gln Lys Gly Thr Pro Lys Arg Pro Glu Tyr Ile Met Pro Thr 290 295 300 Phe Phe Phe Glu Met Phe Asn Glu Asp Leu Lys Gln Pro Thr Val Glu 305 310 315 320 Gln Asn Phe Gly Phe Phe Phe Pro Asn Met Asn Pro Val Tyr Pro Phe 325 330 335 Trp 6015DNAArtificialoligonucleotide 60atcctgtcaa accag

156115DNAArtificialoligonucleotide 61atcctgtcaa accag 156225DNAArtificialoligonucleotide 62gcttttggaa gcgatataac atcga 256325DNAArtificialoligonucleotide 63ggcataggca aaataagggt acaca 256424DNAArtificialoligonucleotide 64aatatagtga aatatgggtc caag 246524DNAArtificialoligonucleotide 65aagaaacgag caccagttat tgac 24

Claims

1. A non-naturally occurring Gossypium hirsutum plant, and parts and progeny thereof, characterized in that the functional expression of at least one allele of at least one fiber-specific GLUC gene that is functionally expressed during the fiber strength building phase, in particular the fiber maturation phase, of fiber development is abolished, wherein the GLUC gene is a GLUC1.1A gene encoding a GLUC protein that has at least 97% sequence identity to SEQ ID NO: 4.

2. The plant of claim 1, wherein the amount of functional GLUC protein is significantly reduced in fibers during the fiber strength building phase, in particular the fiber maturation phase, of fiber development compared to the amount of functional GLUC protein produced in fibers during the fiber strength building phase, in particular the fiber maturation phase, of fiber development in a plant in which the functional expression of the at least one GLUC allele is not abolished.

3. The plant of claim 1, wherein the strength of the fibers is significantly increased compared to the strength of the fibers in a plant in which the functional expression of the at least one GLUC allele is not abolished.

4. The plant of claim 3, wherein the strength of the fibers is on average a. between about 5% and about 10%, preferably about 7.5%, higher; b. between about 1.6 g/tex and about 3.3 g/tex, preferably about 2.5 g/tex, higher; c. between about 34.6 g/tex and about 36.3 g/tex, preferably about 35.5 g/tex.

4. The plant of claim 3, which is characterized in that the functional expression of at least two alleles of at least one fiber-specific GLUC gene is abolished.

5. A fiber obtainable from the fiber-producing plant of claim 1.

6. A nucleic acid molecule encoding a non-functional GLUC1.1 protein having an amino acid sequence wherein at least one amino acid residue similar to the active site residues or to the glycosylation site residues of the GLUC1.1 protein of SEQ ID NO: 4 is lacking or is substituted for a non-similar amino acid residue.

7. The nucleic acid molecule of claim 6, wherein the active site residues of the GLUC1.1 protein of SEQ ID NO: 4 are selected from the group consisting of Tyr48, Glu249, Trp252, and Glu308, and wherein the glycosylation site residue of the GLUC1.1 protein of SEQ ID NO: 4 is Asn202.

8. The nucleic acid molecule of claim 6, comprising: d. a nucleic acid sequence having at least 92% sequence identity to SEQ ID NO: 3 from nucleotide 101 to 1078, wherein at least one nucleic acid residue is deleted, inserted or substituted; e. a nucleotide sequence at least 92% identical to the nucleic acid sequence of SEQ ID NO: 54 from nucleotide 50 to 589; f. the nucleic acid sequence of SEQ ID NO: 54 from nucleotide 50 to 589; g. a nucleic acid sequence having at least 92% sequence identity to SEQ ID NO: 1 from nucleotide 2410 to 3499, wherein at least one nucleic acid residue is deleted, inserted or substituted; h. a nucleotide sequence at least 92% identical to the nucleic acid sequence of SEQ ID NO: 5 from nucleotide 63 to 711; i. the nucleic acid sequence of SEQ ID NO: 5 from nucleotide 63 to 711.

9. A method for generating and/or selecting a non-naturally occurring Gossypium hirsutum plant, and parts and progeny thereof, wherein the functional expression of at least one allele of at least one fiber-specific GLUC gene that is functionally expressed during the fiber strength building phase, in particular the fiber maturation phase, of fiber development is abolished, comprising the step of: mutagenizing at least one allele of the GLUC gene, or introgressing at least one allele of a non-functionally expressed ortholog of the GLUC gene or at least one allele of a mutagenized GLUC gene, or introducing a chimeric gene comprises the following operably linked DNA elements: a. a plant expressible promoter, b. a transcribed DNA region, which when transcribed yields an inhibitory RNA molecule capable of reducing the expression of the GLUC allele, and c. a 3' end region comprising transcription termination and polyadenylation signals functioning in cells of the plant, wherein the GLUC gene is a GLUC1.1A gene encoding a GLUC1.1A protein that has at least 97% sequence identity to SEQ ID NO: 4.

10. The method of claim 9, wherein the non-functionally expressed ortholog of the GLUC1.1A gene is derived from a Gossypium barbadense,

11. A method for altering the properties of a fiber in a Gossypium hirsutum plant, particularly increasing the strength of a fiber, comprising the steps of: generating and/or selecting a non-naturally occurring fiber-producing plant, and parts and progeny thereof, wherein the functional expression of at least one allele of at least one fiber-specific GLUC1.1A gene that is functionally expressed during the fiber strength building phase, in particular the fiber maturation phase, of fiber development is abolished, according to claim 9, selecting a plant with an altered fiber strength, in particular an increased fiber strength.

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