Patent application title: Gene for resistance to plant disease
Inventors:
Otto Törjék (Einbeck, DE)
Dietrich Borchardt (Einbeck, DE)
Margaret Rekoske (Shakopee, MN, US)
Wolfgang Mechelke (Einbeck, DE)
Britta Schulz (Einbeck, DE)
Jens Christoph Lein (Gottingen, DE)
Assignees:
KWS SAAT SE & Co. KGaA
IPC8 Class: AC12N1582FI
USPC Class:
1 1
Class name:
Publication date: 2020-08-20
Patent application number: 20200263194
Abstract:
A more efficient breeding against Cercospora leaf spot disease, or the
development of new resistant lines, is enabled via the provision of the
Cercospora resistance-mediating gene according to the invention; in
particular, a dominant resistance effect in the target plant is evoked by
the property of the identified gene alone. The Cercospora
resistance-mediating gene, and embodiments of the present invention that
are described in the preceding, offer additional applications, e.g., the
use of the resistant gene allele in cis-genetic or trans-genetic
approaches, with the goal of developing new resistant cultivars.Claims:
1. A pelleted seed of a sugar beet plant comprising a nucleic acid
molecule encoding a polypeptide that is able to confer resistance to
Cercospora beticola in a sugar beet plant in which the polypeptide is
expressed, wherein the polypeptide comprises an amino acid sequence that
is at least 95% identical to the amino acid sequence according to SEQ ID
NO: 3.
2. The pelleted seed of the sugar beet plant according to claim 1, wherein the pelleted seed of the sugar beet plant is a monogerm seed.
3. The pelleted seed of the sugar beet plant according to claim 1, wherein the sugar beet plant is harvestable before bolting.
4. (canceled)
5. The pelleted seed of the sugar beet plant according to claim 1, wherein the pelleted seed has been polished before pelleting.
6. The pelleted seed of the sugar beet plant according to claim 1, wherein the pelleted seed has been technically treated, wherein the technical treatment is selected from the group consisting of: (a) dressing; (b) incrustation; and (c) coloring.
7. The pelleted seed of the sugar beet plant according to claim 1, wherein the pellet comprises at least one chemical selected from the group selected of: (a) insecticide; (b) fungicide; and (c) fertilizer.
8. The pelleted seed of the sugar beet plant according to claim 1, wherein the seed has been subjected to priming or pre-germination before or during pelleting.
9. The pelleted seed of the sugar beet plant according to claim 1, wherein the sugar beet plant is a hybrid sugar beet plant.
10. The pelleted seed of the sugar beet plant according to claim 1, wherein the resistance to Cercospora beticola is dominant.
11. A packing or a bag containing the pelleted seed of the sugar beet plant according to claim 1.
12. A mixture of a pelleting mass and a sugar beet plant seed wherein the sugar beet plant seed comprises a nucleic acid sequence which encodes a polypeptide that is able to confer resistance to Cercospora beticola in a sugar beet plant in which the polypeptide is expressed, wherein the polypeptide comprises an amino acid sequence that is at least 95% identical to the amino acid sequence according to SEQ ID NO: 3.
13. A method for producing the pelleted seed of the sugar beet plant according to claim 1 comprising the following steps: I) providing a sugar beet plant seed comprising a nucleic acid sequence which encodes a polypeptide that is able to confer resistance to Cercospora, wherein the polypeptide comprises an amino acid sequence that is at least 95% identical to the amino acid sequence according to SEQ ID NO: 3. II) embedding the sugar beet plant seed in a pelleting mass III) allowing the pelleting mass to dry or drying the pelleting mass.
Description:
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to European Patent Application No. 19157888.9, filed Feb. 18, 2019 under 35 U.S.C. .sctn. 119 and to PCT Patent Application No. PCT/EP2019/054008, filed Feb. 18, 2019 under 35 U.S.C. .sctn. 365, the contents of both of which are herein fully incorporated by reference in their entirety.
SEQUENCE LISTING
[0002] This application contains a Sequence Listing which has been submitted in ASCII format via EFS-Web and is hereby incorporated by reference herein in its entirety. The ASCII text file was created on Feb. 18, 2020, is named KWS0270US2sequence_listing_ST25.txt and is 238,124 bytes in size.
FIELD OF THE INVENTION
[0003] The present invention relates to a nucleic acid molecule which encodes a polypeptide, which nucleic acid molecule is able to confer a resistance to Cercospora--in particular, to the fungus Cercospora beticola in a plant, and, in particular, in a plant of the species Beta vulgaris in which the polypeptide is expressed--as well as to the polypeptide encoded by the nucleic acid molecule according to the invention. In particular, the nucleic acid molecule according to the invention is characterized in that the resistance effect to Cercospora that is conferred by the polypeptide is dominant. Furthermore, the invention relates to a Cercospora-resistant plant, plant cell, plant organ, plant tissue, plant part, or a seed or descendant of a plant, which comprises the nucleic acid molecule or portions thereof as an endogenous gene, as an edited gene, or as a transgene. Furthermore, the present invention also encompasses methods for increasing the resistance to Cercospora in a plant of the species Beta vulgaris, as well as methods for producing or identifying and possibly selecting a Cercospora-resistant plant. The present invention also encompasses methods for monitoring an infestation by the pathogen Cercospora beticola, as well as oligonucleotide probes and primers for hybridization with the nucleic acid molecule according to the invention.
BACKGROUND OF THE INVENTION
[0004] Cercospora leaf spot is one of the most important, globally prevalent leaf diseases of plants from the species Beta vulgaris and Spinacia oleracea. It is caused by the fungus Cercospora beticola. Plants infested by this disease typically form small, relatively round leaf spots (2-3 mm) that are light gray in the middle and are surrounded by a red-brown border. In a severe infestation, the leaf spots overlap, so that entire portions of the leaf blade dry out. Small black dots (pseudostromata) are visible within the fully formed spots, and a gray, felt-like covering (conidia bearers with conidia) forms under damp conditions--predominantly, on the leaf underside. Severely infested leaves first turn yellow, then turn brown and die. New leaf growth occurs in parallel, wherein the leaves become diseased again and die, however. At first, damage symptoms only on individual plants are visible; however, with spread of the disease, formation of persistent infestation nests often occurs. The further propagation over the entire field takes place via rain and wind.
[0005] The pathogen Cercospora beticola was first described in the second half of the 19th century, in Italy. Up to 40% crop losses may occur due to a severe infestation, which may be triggered by humid weather, early row closure, a high infection potential from previous years, or strong irrigation. These losses result from a reduced beet crop and reduced sugar content; see Holtschulte ((2000) "Cercospora beticola--worldwide distribution and incidence," pp. 5-16, in "Cercospora beticola Sacc. Biology, Agronomic Influence and Control Measures in Sugar Beet," vol. 2 (M. J. C. Asher, B. Holtschulte, M. R. Molard, F. Rosso, G. Steinrucken, R. Beckers, eds.). International Institute for Beet Research, Brussels, Belgium, 215 pp.). In order to fight back against the disease, intercropping or fungicides are often used. A chemical control of Cercospora beticola via fungicides incurs costs to the farmer and pollutes the environment. Repeated applications of fungicides additionally increase the selection pressure on fungicide-tolerant Cercospora beticola strains. This is contrary to a sustainable agricultural practice. It is worth mentioning that during the last few years stems of Cercospora beticola occurred which showed resistance against one or more fungicides; see for Trkulja, Nenad R., et al. "Molecular and experimental evidence of multi-resistance of Cercospora beticola field populations to MBC, DMI and Qol fungicides." European Journal of Plant Pathology 149.4 (2017): 895-910. The problem became such severe that the German Federal Office of Consumer Protection and Food Safety (BVL) approved the exemptional admission of copper-based fungicides for combating Cercospora. However, copper-based fungicides are generally regarded (depending on the dosage) as harmful for humans and environment. Copper is a heavy metal which may accumulate in the soil.
[0006] Indirect combat is done via the selection of cultivars with healthy leaves and cultivation of the beets with at least a 3-year crop rotation. Markedly better control of the infestation may be achieved with a combination of tolerant or resistant cultivars. Less susceptible Cercospora-tolerant beet cultivars have been offered on the market since 2000 (Steinrucken 1997, "Die Zuchtung von Cercospora-resistenten Zuckerruben." ["The breeding of Cercospora-resistant sugar beets." ], Vortrage fur Pflanzenzuchtung [Lectures on Plant Breeding], Volume 37, Lecture symposium, Mar. 4-5, 1997, Kiel). These cultivars are furnished with a quantitative resistance to Cercospora beticola. The resistance of these cultivars is based upon several genes and is quantitatively passed down, wherein the exact number of the genes that are responsible for the resistance is not known; see Weiland and Koch (2004), Sugarbeet leaf spot disease (Cercospora beticola Sacc.), The Plant Journal, 5(3), 157-166. The complex quantitative heredity was confirmed via several Quantitative Trait Loci (QTL) analyses. This method allows the mapping of polygenic inherited resistances and is a reliable technique for identifying the number and position of genetic resistance factors on the genetic linkage map of a host plant. In this way, multiple causative QTL's could be determined on each chromosome of the sugar beet.
[0007] The mappings were performed with different Cercospora-resistance donors, wherein the observed QTL effects were, for the most part, small. The maximum declared phenotypical values were at 5%.
[0008] In continuative studies, lists of differentially expressed genes have been described. In a study by Weltmeier et al., ((2011) Transcript profiles in sugar beet genotypes uncover timing and strength of defense reactions to Cercospora beticola infection, Molecular plant-microbe interactions, 24(7), 758-772), a genome-side expression profile for various genotypes of sugar beet (i.e., Cercospora-resistant, -tolerant, -susceptible, etc.) was created with the aid of a microarray-based technology during the pathogen infection in order to analyze transcriptional changes in the expression profile in connection with leaf spot. Via these analyses, the authors were in a position to create a pathogen-induced transcription profile in various tested genotypes of sugar beet and to determine potential candidate genes. However, these genes have not yet been characterized in detail. The genetic and functional background of Cercospora resistance and the identity of the resistance genes have until now been entirely unclear.
[0009] However, with the quantitative heredity of QTL, not only is the desired resistance to Cercospora beticola introduced into the plant, but, rather, often unwanted features as well, such as, for example, reduced yield, due to the inheritance of additional genes that are linked with the positive feature of Cercospora resistance. This phenomenon is also known by the term, "linkage drag." Furthermore, the enormous breeding cost that is required in order to select for multiple resistance loci without thereby reducing the yield may have negative effects on the vitality of the plants; see Weiland and Koch, 2004.
[0010] Breeding companies have offered Cercospora-tolerant cultivars on the market for more than a decade. The resistance of these cultivars is inherited via multiple resistance genes with small effect. However, a disadvantage of these cultivars consists in the cultivar development being very laborious and complicated due to the complicated heredity, and in such cultivars having a markedly poorer yield performance relative to normal cultivars, in the absence of an infestation. Among other things, this may be linked to the epigenetic interaction of some resistance genes with genes that are responsible for sugar production, which leads to reduced fitness of the plants, in the absence of the pathogen. Furthermore, Cercospora shows the tendency to overcome the tolerance of long-established cultivars. Moreover, the so far available resistance scores of non-adapted, wild genetic resources is usually not reliable and not comparable among each other as the underlying studies took place at different environment conditions, under different infestation pressure and with different pathogenic stems of Cercospora. In this regard it should be mentioned that environmental parameters like moisture, temperature, wind etc. (which tend to be unstable) have significant influence on the progress of the Cercospora disease after infection. It is common that a specific genetic resource shows high level of tolerance/resistance in one study and tends to be completely susceptible in another study. Due to the above given factors it was so far not possible to identify a dominant resistance gene having a major effect towards Cercospora although there is a strong demand for such a gene which could be easily transferred into already existing cultivars and varieties to establish resistance towards Cercospora.
[0011] The use of new breeding techniques based upon gene editing, e.g., by means of TALE nucleases or CRISPR systems, and of transgenic approaches, is not applicable on the so far available genetic material due to the complicated heredity and the multitude of the genes which are involved in the resistance development, the majority of which have not yet been identified and characterized.
[0012] For sustainable breeding against Cercospora leaf spot that is to counteract the danger of Cercospora variants that overcome resistance, it is necessary to continuously identify new resistance genes and integrate these into the gene pools of cultivated plants such as sugar beets. In particular, the aim consisted in the provision of suitable resistance genes that, after expression in the plant, on their own already produce a very large, dominant resistance effect against Cercospora beticola.
[0013] According to the invention, this aim is achieved via the embodiments characterized in the claims and in the specification.
SUMMARY OF THE INVENTION
[0014] The present invention relates to a nucleic acid molecule that is able to confer a resistance to Cercospora--in particular, to the fungus Cercospora beticola--in a plant, and, in particular, in Beta vulgaris subsp. vulgaris. The polypeptide which is encoded by the nucleic acid molecule is thereby produced in the plant. The nucleic acid molecule, after whose expression the polypeptide is produced, on its own, already produces in the plant a very large, dominant resistance effect against Cercospora beticola.
[0015] Furthermore, the invention relates to a Cercospora-resistant plant, plant cell, plant organ, plant tissue, plant part, a seed, seed stock, or descendant of a plant, which endogenously or transgenically comprises the nucleic acid molecule or portions thereof. According to a specific optional embodiment, those plants and their components that have been obtained via an essentially biological process are excluded.
[0016] Methods for increasing the resistance to Cercospora in a plant of the species Beta vulgaris, as well as methods for producing or identifying and possibly selecting a Cercospora-resistant plant, are likewise encompassed by the present invention. The present invention also encompasses methods for monitoring an infestation of the pathogen Cercospora beticola, as well as oligonucleotides as probes and primers for hybridization with the nucleic acid molecule according to the invention.
[0017] The present invention therefore relates to the embodiments that are listed in the following points and illustrated in the examples and figures.
[0018] [1] Nucleic acid molecule encoding a polypeptide that is able to confer resistance to Cercospora in a plant in which the polypeptide is expressed, characterized in that the nucleic acid molecule comprises a nucleotide sequence which is selected from
[0019] (a) a nucleotide sequence encoding a polypeptide having an amino acid sequence according to SEQ ID No. 3;
[0020] (b) a nucleotide sequence that comprises the DNA sequence according to SEQ ID No. 2;
[0021] (c) a nucleotide sequence that comprises a DNA sequence selected from the group consisting of SEQ ID No. 1 or SEQ ID No. 53;
[0022] (d) a nucleotide sequence that hybridizes to a nucleotide sequence which is complementary to the nucleotide sequence according to (a), (b), or (c), under stringent conditions;
[0023] (e) a nucleotide sequence encoding a polypeptide which, via substitution, deletion, and/or addition of one or more amino acids of the amino acid sequence, differs from a polypeptide encoded by the nucleotide sequence according to (a), (b), or (c);
[0024] (f) a nucleotide sequence encoding a polypeptide which has an amino acid sequence that is at least 70% identical to an amino acid sequence according to SEQ ID No. 3;
[0025] (g) a nucleotide sequence that is at least 70% identical to a DNA sequence according to SEQ ID No. 1 or SEQ ID No. 2;
[0026] wherein the resistance to Cercospora is preferably a resistance to Cercospora beticola, or wherein the plant is preferably a plant of the subspecies Beta vulgaris subsp. vulgaris, and is, particularly preferably, sugar beet.
[0027] [2] Nucleic acid molecule according to [1], characterized in that the resistance effect to Cercospora that is conferred by the polypeptide is dominant in the plant--preferably, wherein the polypeptide confers a resistance effect of at least one rating score, and, preferably, of more than one rating score, particularly preferably, of at least two rating scores, particularly preferably, of at least three rating scores, and, especially preferably, of at least four rating scores.
[0028] [3] Nucleic acid molecule according to [1] or [2], characterized in that the nucleic acid molecule originates from Beta vulgaris subsp. maritima.
[0029] [4] Polypeptide encoded by the nucleic acid molecule according to one of [1] through [3].
[0030] [5] Vector or expression cassette comprising the nucleic acid molecule according to one of [1] through [3], wherein the nucleic acid molecule is preferably heterologous to the vector or to the expression cassette.
[0031] [6] Cell which comprises the nucleic acid molecule according to one of [1] through [3], or the vector or the expression cassette according to [5], wherein the nucleic acid molecule or the expression cassette are preferably present as an endogene or transgene.
[0032] [7] Cercospora-resistant plant or a portion thereof, characterized in that the plant or its portion contains the nucleic acid molecule according to one of [1] through [3], endogenously or transgenically, or the vector or the expression cassette according to [5], wherein the plant which endogenously contains the nucleic acid molecule is a plant of the species Beta vulgaris--but not Beta vulgaris subsp. maritima--or of Beta vulgaris subsp. vulgaris.
[0033] [8] Plant according to [7], characterized in that the plant is a hybrid plant.
[0034] [9] Plant according to [7] or [8], characterized in that the nucleic acid molecule is present heterozygously or homozygously in the genome of the plant.
[0035] [10] Seeds or descendants of the plant according to one of [7] through [9], wherein the seed or the descendant transgenically or endogenously comprises the nucleic acid molecule according to one of [1] through [3], or the vector or the expression cassette according to [5].
[0036] [11] Method for increasing the resistance to Cercospora in a plant, including the following steps:
[0037] (i) integration of the nucleic acid molecule according to one of [1] through [3], or of the vector or of the expression cassette according to [5], by means of homology-directed repair or homologous recombination--preferably, supported by a site-directed nuclease--into the genome of at least one cell of a plant, and optional regeneration of a plant from the at least one plant cell; or
[0038] (ii) increase in the expression of the nucleic acid molecule according to one of [1] through [3] in at least one cell of the plant--preferably, via modification of the native promoter, e.g., comprising a DNA sequence according to SEQ ID No. 7, or via linking of the nucleic acid molecule according to one of [1] through [3] with a heterologous promoter that has a higher level of activity in comparison to the native promoter, e.g., comprising a DNA sequence according to SEQ ID No. 7--in particular, after Cercospora infection--and optional regeneration of a plant from the at least one plant cell; or
[0039] (iii) increase in the activity and/or stability of the polypeptide according to [4] via modification of the nucleotide sequence of the nucleic acid molecule according to one of [1] through [3] in at least one cell of the plant, and optional regeneration of a plant from the at least one plant cell; or
[0040] (iv) transformation of a plant cell with the nucleic acid molecule according to one of [1] through [3], or the vector or the expression cassette according to [5], and optional regeneration of a (transgenic) plant from the transformed plant cell;
[0041] wherein the resistance to Cercospora is preferably a resistance to Cercospora beticola, or the plant is preferably a plant of the species Beta vulgaris--preferably, Beta vulgaris subsp. vulgaris--and, in particular, is sugar beet.
[0042] [12] Method for producing a Cercospora-resistant plant according to one of [7] through [9], including the following steps:
[0043] (a) transformation of a plant cell with the nucleic acid molecule according to one of [1] through [3], or the vector or the expression cassette according to [5]; and
[0044] (b) regeneration of the transgenic plant from the transformed plant cell; or
[0045] (i) introduction of a site-directed nuclease and a repair matrix into a cell of a plant of the species Beta vulgaris, wherein the site-directed nuclease is able to generate at least one double-strand break of the DNA in the genome of the cell--preferably, upstream and/or downstream of a target region--and the repair matrix comprises the nucleic acid molecule according to one of [1] through [3];
[0046] (ii) cultivation of the cell from (i) under conditions that allow a homology-directed repair or a homologous recombination, wherein the nucleic acid molecule is incorporated from the repair matrix into the genome of the plant; and
[0047] (iii) regeneration of a plant from the cell modified in (ii).
[0048] [13] Method according to [12], characterized in that the target region comprises an allelic variant of the nucleic acid molecule according to one of [1] through [3], wherein the allelic variant encodes a polypeptide not conferring resistance or a slight resistance to Cercospora.
[0049] [14] Method according to [12] or [13], characterized in that the at least one double-strand break occurs at a position that is at most 10,000 base pairs upstream and/or downstream of the target region, or that is at most 10,000 base pairs distant from the allelic variant as defined in [13].
[0050] [15] Method according to [12] or [13], characterized in that the allelic variant of the nucleic acid molecule comprises a nucleotide sequence which is selected from
[0051] (a) a nucleotide sequence that encodes a polypeptide having an amino acid sequence according to SEQ ID No. 6;
[0052] (b) a nucleotide sequence that comprises the DNA sequence according to SEQ ID No. 5;
[0053] (c) a nucleotide sequence that comprises a DNA sequence according to SEQ ID No. 4;
[0054] (d) a nucleotide sequence that hybridizes to a nucleotide sequence which is complementary to the nucleotide sequence according to (a), (b), or (c), under stringent conditions;
[0055] (e) a nucleotide sequence that encodes a polypeptide which, via substitution, deletion, and/or addition of one or more amino acids of the amino acid sequence, differs from a polypeptide that is encoded by the nucleotide sequence according to (a), (b), or (c); or
[0056] (f) a nucleotide sequence that encodes a polypeptide which has an amino acid sequence that is at least 80% identical to an amino acid sequence according to SEQ ID No. 6.
[0057] [16] Plant, or a portion thereof, obtained or obtainable according to a method according to one of [12] through [15].
[0058] [17] Method for identifying, and optionally providing, a plant of the species Beta vulgaris that is resistant to Cercospora, characterized in that the method includes at least step (i) or (ii):
[0059] (i) detection of the presence and/or expression of the nucleic acid molecule according to one of [1] through [3], or the presence of the polypeptide according to [4], in the plant or a portion of the plant; and/or
[0060] (ii) detection of at least one marker locus in the nucleotide sequence of the nucleic acid molecule according to one of [1] through [3] or in a cosegregating region; and
[0061] (iii) possible selection of the Cercospora beticola-resistant plant.
[0062] [18] Method for identification of a nucleic acid molecule which encodes a polypeptide that is able to confer a resistance to Cercospora in a plant of the species Beta vulgaris in which the polypeptide is expressed, characterized in that the method includes the following steps:
[0063] (i) comparison of the amino acid sequence of the polypeptide according to [4] with amino acid sequences from a sequence database, or identification of allelic variants which encode the polypeptide according to [4] in genotypes of the species Beta vulgaris;
[0064] (ii) identification of the amino acid sequence, or an allelic variant, encoding an amino acid sequence, wherein the amino acid sequence is at least 80% identical to the amino acid sequence of the polypeptide according to [4];
[0065] (iii) introduction of a nucleic acid molecule, or the allelic variant, encoding the identified amino acid sequence into a plant of the species Beta vulgaris, and expression of the nucleic acid molecule in the plant; and
[0066] (iv) detection of the resistance to Cercospora.
[0067] [19] Method for farming of plants of the species Beta vulgaris, including
[0068] (i) the provision of plants according to one of [7] through [9], the planting of a pelleted seed of a sugar beet plant or of a plant of the genus Beta according to one of [26]-[39], the production of plants of the species Beta vulgaris with the aid of a method according to one of [12] through [15], or the identification and selection of plants of the genus Beta with the aid of a method according to [17], and
[0069] (ii) cultivation of the plants from (i) or descendants thereof, wherein the method counteracts an infestation of the cultivated plants with Cercospora.
[0070] [20] Oligonucleotide of at least 15, 16, 17, 18, 19, or 20--preferably, at least 21, 22, 23, 24, or 25, particularly preferably, at least 30, 35, 40, 45, or 50, and, especially preferably, at least 100, 200, 300, or 500--nucleotides in length, which oligonucleotide hybridizes with a nucleotide sequence as defined in one of [1] through [3].
[0071] [21] A pair of oligonucleotides--preferably, oligonucleotides according to [20] or a kit containing these oligonucleotides--wherein the oligonucleotides are suitable for hybridization as forward primer and reverse primer to a region in the Beta vulgaris genome that, in Beta vulgaris, has a cosegregation with the Cercospora resistance conferred by the polypeptide according to [4], or with the nucleic acid molecule according to one of [1] through [3].
[0072] [22] Use of the nucleic acid molecule according to one of [1] through [3] in the production of Cercospora-resistant plants of the subspecies Beta vulgaris subsp. vulgaris.
[0073] [23] Method for the production of an organism which comprises a mutated version according to [1] and/or a mutated version of a promoter comprising a nucleic acid sequence selected from
[0074] (a) SEQ ID NO: 7
[0075] (b) a nucleotide sequence, which hybridizes under stringent conditions with a sequence which is complementary to the sequence according to (a)
[0076] (c) a nucleotide sequence which is at least 70% identical to a sequence according to SEQ ID NO: 7
[0077] wherein the method includes the following steps:
[0078] (I) Provision of an organism or a cell comprising the nucleic acid molecule and/or the promoter
[0079] (II) Increase of the mutation rate of the organism or the cell or mutagenesis of the organism or the cell
[0080] (III) Phenotypic selection of an organism, which as a result of a mutation exhibits an altered resistance or altered resistance level towards Cercospora beticola or Genotypic selection of an organism or a cell which comprises a mutation in the nucleic acid molecule and/or the promoter wherein the mutation has been created via step (II) and optionally
[0081] (IV) Regeneration of the organism from the cell obtained via step (III).
[0082] [24] Method according to [23], wherein the organism is a plant.
[0083] [25] Method according to [24] wherein the plant is a Beta vulgaris, preferably a Beta vulgaris subsp. vulgaris, more preferably a sugar beet.
[0084] [26] A pelleted seed of a sugar beet plant or a plant of the genus Beta comprising a nucleic acid molecule according to [1].
[0085] [27] The pelleted seed according to [26], wherein the beet body is suitable as raw material for industrial sugar production.
[0086] [28] The pelleted seed according to [26] or [27], wherein the pelleted seed is a monogerm seed.
[0087] [29] The pelleted seed according to [26] to [28], wherein the sugar beet plant is harvestable before bolting.
[0088] [30] The pelleted seed according to [26] to [29], wherein the resistance to Cercospora is a resistance to Cercospora beticola.
[0089] [31] The pelleted seed according to [26] to [30], wherein the sugar beet plant is biannual.
[0090] [32] The pelleted seed according to [26] to [31], which has been technically treated, wherein the technical treatment is selected from the group consisting of:
[0091] (a) polishing;
[0092] (b) dressing;
[0093] (c) incrustation; and
[0094] (d) coloring.
[0095] [33] The pelleted seed according to [26] to [32], wherein the pellet comprises at least one chemical selected from the group selected of:
[0096] (a) insecticide;
[0097] (b) fungicide; and
[0098] (c) fertilizer.
[0099] [34] The pelleted seed according to [26] to [33], wherein the seed has been subjected to priming or pre-germination before or during pelleting.
[0100] [35] The pelleted seed according to [26] to [34], wherein the sugar beet plant is a hybrid sugar beet plant.
[0101] [36] The pelleted seed according to [26] to [35] wherein the nucleotide sequence includes at least one mutation.
[0102] [37] The pelleted seed according to [36] wherein the at least one mutation is a mutation relative to SEQ ID No. 1 or SEQ ID No 2.
[0103] [38] The pelleted seed according to [36] or [37] wherein the nucleotide sequence including the at least one mutation encodes a polypeptide which has an amino acid sequence that is at least 99% identical to an amino acid sequence according to SEQ ID No. 3.
[0104] [39] The pelleted seed according to [38] wherein the nucleotide sequence including the at least one mutation encodes a polypeptide having an amino acid sequence according to SEQ ID No. 3.
[0105] [40] A packing containing the pelleted seed according to [26] to [39] or containing seed stock comprising the nucleic acid molecule according to [1] wherein the seed stock preferable is seed stock of a plant of the genus Beta.
[0106] [41] A mixture of a pelleting mass and a sugar beet plant seed wherein the sugar beet plant seed comprises a nucleic acid sequence which encodes a polypeptide that is able to confer resistance to Cercospora, wherein the nucleotide sequence is selected from the group consisting of
[0107] (a) a nucleotide sequence encoding a polypeptide having an amino acid sequence according to SEQ ID No. 3;
[0108] (b) a nucleotide sequence that comprises the DNA sequence according to SEQ ID No. 2;
[0109] (c) a nucleotide sequence that comprises a DNA sequence selected from the group consisting of SEQ ID No. 1 or SEQ ID No. 53;
[0110] (d) a nucleotide sequence that hybridizes to a nucleotide sequence which is complementary to the nucleotide sequence according to (a), (b), or (c), under stringent conditions;
[0111] (e) a nucleotide sequence encoding a polypeptide which, via substitution, deletion, and/or addition of one or more amino acids of the amino acid sequence, differs from a polypeptide encoded by the nucleotide sequence according to (a), (b), or (c);
[0112] (f) a nucleotide sequence encoding a polypeptide which has an amino acid sequence that is at least 70% identical to an amino acid sequence according to SEQ ID No. 3;
[0113] (g) a nucleotide sequence that is at least 70% identical to a DNA sequence according to SEQ ID No. 1 or SEQ ID No. 2.
[0114] [42] A method for producing the pelleted sugar beet plant seed according to [26] to [39] comprising the following steps:
[0115] a) providing a sugar beet plant seed comprising a nucleic acid sequence which encodes a polypeptide that is able to confer resistance to Cercospora,
[0116] wherein the nucleotide sequence which is selected from the group consisting of
[0117] (i) a nucleotide sequence that encodes a polypeptide which has an amino acid sequence that is at least 95% identical to an amino acid sequence according to SEQ ID No. 3; and
[0118] (ii) a nucleotide sequence that is at least 95% identical to a DNA sequence according to SEQ ID No. 1 or SEQ ID No. 2
[0119] b) embedding the sugar beet plant seed in a pelleting mass
[0120] c) allow the pelleting mass to dry or dry the pelleting mass.
[0121] [43] The pelleted seed according to [36] to [39] wherein the nucleotide sequence including the at least one mutation is an artificial nucleotide sequence which does not occur naturally.
[0122] [44] The method according to [23] wherein during step (II) a mutagenizing chemical like EMS or mutagenizing radiation is applied.
[0123] [45] A variety or cultivar of the genus Beta comprising the nucleic acid molecule according to [1] or a pelleted seed the variety or cultivar.
[0124] First, some of the terms used in this application are explained in detail in the following:
[0125] What is understood by "rating score" in the sense of the present invention is a qualitative assessment of the resistance to a Cercospora infestation that is represented using a scale from 1 to 9 (with 1=strong resistance and 9=no resistance).
TABLE-US-00001 TABLE 1A 9-level resistance rating for Cercospora Rating score Leaf phenotype Whole plant phenotype 1 Healthy leaf Healthy leaf, whole 3 Diseased leaf, spots on Whole plant, beginning of the outer leaves disease, spots on the outer leaves 5 Diseased leaf, merging of Whole plant, advanced disease, the spots into dying areas merging of the spots into dying areas 7 Diseased leaf, large part Whole diseased plant, large of the leaf brown and dead, portions of the outer leaves only lower lamina is still alive are dying off 9 Diseased leaves, lamina and Whole diseased plant, outer petiole are dead and dried leaves have died, inner leaves with severe damage, strong new leaf growth
[0126] The genus Cercospora encompasses various species, e.g., the species Cercospora arachidicola, Cercospora ariminiensis, Cercospora asparagi, Cercospora bertoreae, Cercospora beticola, Cercospora bizzozeriana, Cercospora canescens, Cercospora carotae, Cercospora chenopodii, Cercospora cistinearum, Cercospora cladosporioides, Cercospora diazu, Cercospora dulcamarae, Cercospora erysimi, Cercospora hayii, Cercospora kikuchii, Cercospora malvacearum, Cercospora malvicola, Cercospora medicaginis, Cercospora oryzaem, Cercospora personata, Cercospora plantaginis, Cercospora ricinella, Cercospora setariae, Cercospora unamunoi, Cercospora violae, or Cercospora zeae-maydis.
[0127] In conjunction with the specification of a length of a nucleotide sequence, the term, "approximately," means a deviation by +/-200 base pairs--preferably, by +/-100 base pairs, and, particularly preferably, by +/-50 base pairs.
[0128] A "plant of the genus Beta" belongs to the amaranth family (Amaranthaceae). Numbering among these plants are plants of the species Beta macrocarpa, Beta vulgaris, Beta lomatogona, Beta macrorhiza, Beta corolliflora, Beta trigyna, and Beta nana. A plant of the species Beta vulgaris is, in particular, a plant of the subspecies Beta vulgaris subsp. vulgaris. For example, numbering among these are Beta vulgaris subsp. vulgaris var. altissima (sugar beet in a narrower sense), Beta vulgaris ssp. vulgaris var. vulgaris (chard), Beta vulgaris ssp. vulgaris var. conditiva (beetroot/red beet), Beta vulgaris ssp. vulgaris var. crassa/alba (fodder beet). It is noted that the nucleic acid according to the invention does not naturally occur in sugar beet, chard, beetroot, or fodder beet, but may be introduced into these via human action.
[0129] A "plant of the genus Spinacia" belongs to the amaranth family (Amaranthaceae). This genus especially encompasses Spinacia oleracea.
[0130] A "functional fragment" of a nucleotide sequence means a segment of a nucleotide sequence which has a functionality identical or comparable to that of the complete nucleotide sequence from which the functional fragment originates. As such, the functional fragment may possess a nucleotide sequence which is identical or homologous to the total nucleotide sequence over a length of at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94% 96%, 97%, 98%, or 99%. This also explicitly encompasses the range of 90-100%. Furthermore, a "functional fragment" of a nucleotide sequence may also mean a segment of a nucleotide sequence which modifies the functionality of the entire nucleotide sequence, e.g., in the course of post-transcriptional or transcriptional gene silencing. As such, the functional fragment of a nucleotide sequence may comprise at least 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25--preferably, at least 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, or 140, and, particularly preferably, at least 160, 180, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, or 1,000--successive nucleotides of the total nucleotide sequence. This also explicitly encompasses the range of 21 to 50 nucleotides.
[0131] A "functional part" of a protein means a segment of a protein, or a section of the amino acid sequence, that encodes the protein, wherein the segment may exert functionality identical or comparable to that of the entire protein in a plant cell. Over a length of at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 92%, 94% 96%, 97%, 98%, or 99%, a functional part of a protein has an amino acid sequence that is identical or, with consideration of conservative and semi-conservative amino acid exchanges, similar to the protein from which the functional part originates.
[0132] The term, "heterologous," means that the introduced polynucleotide originates from a cell or an organism with a different genetic background, of the same species or a different species, or is homologous to the prokaryotic or eukaryotic host cell, but is then located in a different genetic environment and thus differs from a corresponding polynucleotide that is possibly naturally present. A heterologous polynucleotide may be present in addition to a corresponding endogenous gene.
[0133] In the sense of the invention, what is understood by a "homolog" is a protein of the same phylogenetic origin; what is understood by an "analog" is a protein which exerts the same function, but has a different phylogenetic origin; what is understood by an "ortholog" is a protein from a different species that exerts the same function; and what is understood by a "paralog" is a protein that has appeared within a species due to duplication, wherein this copy either retains the same protein function, alters its expression template, but not the function, changes its protein function, or divides up the original gene function between both copies.
[0134] What is to be understood by "hybridizing" or "hybridization" is a process in which a single-stranded nucleic acid molecule binds to a nucleic acid strand that is complementary to the greatest possible extent, i.e., forms base pairs with this. Standard methods for hybridization are described in, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001. What is preferably understood by this is that at least 60%--more preferably, at least 65%, 70%, 75%, 80%, or 85%, and, particularly preferably, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%--of the bases of the nucleic acid molecule form a base pairing with the nucleic acid strand that is complementary to the greatest possible extent. The possibility of such an annealing depends upon the stringency of the hybridization conditions. The term, "stringency," relates to the hybridization conditions. High stringency is present when a base pairing is made more difficult; low stringency is present if a base pairing is made easier. For example, the stringency of the hybridization conditions depends upon the salt concentration or ionic strength and the temperature. In general, the stringency may be increased by increasing the temperature and/or decreasing the salt content. What are to be understood by "stringent hybridization conditions" are those conditions given which a hybridization predominantly occurs only between homologous nucleic acid molecules. The term, "hybridization conditions," thereby relates not only to the conditions prevailing in the actual addition of the nucleic acids, but also to the conditions prevailing in the following washing steps. For example, stringent hybridization conditions are conditions under which, predominantly, only those nucleic acid molecules hybridize that have at least 70%--preferably, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%--sequence identity. Stringent hybridization conditions are, for example: hybridization in 4.times.SSC at 65.degree. C., and subsequent repeated washing in 0.1.times.SSC at 65.degree. C. for approximately 1 hour in total. A hybridization preferably occurs under stringent conditions.
[0135] In relation to a nucleic acid in the form of a double-stranded DNA, "complementary" nucleotide sequence means that the second DNA strand complementary to the first DNA strand has the nucleotides that correspond to the bases of the first strand, in accordance with the base pairing rules. A complementary sequence is, preferably, entirely complementary to the counter-sequence, and thus preferably has the same length.
[0136] What is understood by an "isolated nucleic acid molecule" is a nucleic acid molecule extracted from its natural or original environment. The term also encompasses a synthetically-produced nucleic acid molecule. What is understood by an "isolated polypeptide" is a polypeptide extracted from its natural or original environment. The term also encompasses a synthetically-produced polypeptide.
[0137] A "molecular marker" is a nucleic acid that is polymorphic in a plant population and is used as a reference or orientation point. A marker for the detection of a recombination event should be suitable for monitoring differences or polymorphisms within a plant population. Such a marker is thus able to detect and differentiate between various allelic states (alleles). The term, "molecular marker," also relates to nucleotide sequences which are complementary or at least largely complementary or homologous to genomic sequences--for example, nucleic acids which are used as probes or primers. These differences at the DNA level are to be found as markers and are, for example, polynucleotide sequence differences, e.g., SSR's (simple sequence repeats), RFLP's (restriction fragment length polymorphisms), FLP's (fragment length polymorphisms) or SNP's (single nucleotide polymorphisms). The markers may be derived from genomic or expressed nucleic acids, e.g., spliced RNA, cDNA, or EST's, and may also relate to nucleic acids that are used as probes or primer pairs and as such are suitable for amplifying a sequence fragment using PCR-based methods. Markers that describe genetic polymorphisms (between parts of a population) may be detected using well-established methods from the prior art (An Introduction to Genetic Analysis, 7th edition, Griffiths, Miller, Suzuki, et al., 2000). For example, among these are DNA sequencing, PCR-based, sequence-specific amplification, verification of RFLP's, verification of polynucleotide polymorphisms by means of allele-specific hybridization (ASH), detection of amplified variable sequences of the plant genome, detection of a 3SR (self-sustained sequence replication), detection of SSR's, SNP's, RFLP's, or AFLP's (amplified fragment length polymorphisms). Furthermore, the methods for detection of EST's (expressed sequence tags) and SSR markers derived from EST sequences and RAPD (randomly amplified polymorphic DNA) are also known. Depending upon the context, the term, "marker," in the description may also mean a specific chromosome position in the genome of a species where a specific marker (SNP, for example) may be found.
[0138] Markers also include synthetic oligonucleotides that may be connected with one or more detection molecules, wherein the detection molecules may be used for a detection reaction or the generation of a signal within the scope of a verification method. Synthetic oligonucleotides also include labeled primers. Synthetic oligonucleotides and labeled primers are artificial compounds, do not occur in nature, and cannot be isolated from nature. The production of such compounds is explained further below.
[0139] A "promoter" is a non-translated, regulatory DNA sequence, typically upstream of a coding region, which contains the binding point for the RNA polymerase and initiates the transcription of the DNA. A promoter additionally contains other elements that act as a regulator gene for gene expression (for example, cis-regulatory elements). A "core or minimal promoter" is a promoter that has the basic elements which are needed for transcription initiation (for example, TATA box and/or initiator).
[0140] A "pathogen" means an organism that, in interactions with a plant, leads to disease symptoms in one or more organs in the plant. For example, animal, fungal, bacterial, or viral organisms or oomycetes number among these pathogens.
[0141] What is to be understood by a "pathogenic infection" is the earliest point in time at which a pathogen interacts with a plant host tissue. In this sense, "infestation" means the occurrence of contact between pathogen and host. With an anchorage of a pathogen at a host, e.g., of a fungal spore on a leaf surface of a plant, mechanisms of pathogen detection and signal relaying begin in the plant host cell. In the case of Cercospora beticola, conidia are formed in humid, warm weather and transferred to neighboring plants by rain and wind. New infections most often show individual leaf spots first at the physiologically older outer leaves. These are most often quite clearly delimited from the healthy leaf tissue by a brown ring. The brown conidia carriers of the fungus in the middle part of the spots may be observed with the aid of a magnifying glass (rating score 3). The number of these brown spots increases rapidly, wherein the sporocarps initially overlap even smaller dead areas (rating score 5). In the further course of the disease, which now also spans to the inner leaves, dying-off of the outer leaves finally occurs for the first time (rating score 7), and, then, of practically all leaves (rating score 9). Course of disease and symptom severity are strongly dependent upon the site and on the annually fluctuating weather conditions.
[0142] Plant "organs" means, for example, leaves, shoot, stem, roots, hypocotyl, vegetative buds, meristems, embryos, anthers, ovula, seeds, or fruits. "Plant parts" include, but are not limited to, the shoot or the stalk, leaves, blossoms, inflorescence, roots, fruits, and seeds, as well as the pollen. The term, "plant parts," also means an association of multiple organs, e.g., a blossom or a seed, or a part of an organ, e.g., a cross-section through the plant shoot. Plant "tissues" are, for example, callus tissue, storage tissue, meristematic tissue, leaf tissue, shoot tissue, root tissue, plant tumor tissue, or reproductive tissue, as well as the cambium, parenchyma, vascular tissue, sclerenchyma, and epidermis. However, the tissue is not limited to this listing. For example, what are to be understood by plant "cells" are, for example, isolated cells having a cell wall or aggregates thereof, or protoplasts.
[0143] "Variety" means a plant grouping within a single botanical taxon of the lowest known rank, which grouping, irrespective of whether the conditions for the grant of a breeder's right are fully met, can be--defined by the expression of the characteristics resulting from a given genotype or combination of genotypes,
[0144] distinguished from any other plant grouping by the expression of at least one of the said characteristics and
[0145] considered as a unit with regard to its suitability for being propagated unchanged.
[0146] In conjunction with the present invention, the term, "regulatory sequence," relates to a nucleotide sequence which influences the specificity and/or the expression strength, e.g., in that the regulatory sequence confers a defined tissue specificity. Such a regulatory sequence may be located upstream of the transcription initiation point of a minimal promoter, but also downstream thereof, e.g., in a transcribed, but not translated, leader sequence or within an intron.
[0147] The term, "resistance," is to be understood broadly and covers the range of the protection from a retardation up to a complete blocking of the development of the disease. One example of an important pathogen is Cercospora beticola. A resistant plant cell of the invention or resistant plant of the invention preferably achieves a resistance to Cercospora beticola. A resistance to a pathogen is to be equated to a resistance to the disease which this pathogen causes; for example, a resistance to Cercospora beticola is also a resistance to leaf spot disease. For example, an increase in the resistance can be measured via a reduced fungal biomass on the host plant; for this, the fungal DNA may be determined with the aid of quantitative PCR in comparison to the plant DNA in the infested plant tissue. An additional approach to the measurement of resistance is optical rating, wherein rating scores of 1 (not susceptible) to 9 (very susceptible) are awarded. "Transgenic plant" relates to a plant into whose genome is integrated at least one polynucleotide.
[0148] It may thereby be a heterologous polynucleotide. The polynucleotide is, preferably, stably integrated, which means that the integrated polynucleotide is stably preserved in the plant, is expressed, and also may be stably passed on to the descendants. The stable introduction of a polynucleotide into the genome of a plant also includes the integration into the genome of a plant of the preceding parental generation, wherein the polynucleotide may be stably passed on further. The term, "heterologous," means that the introduced polynucleotide originates from a cell or an organism with a different genetic background, of the same species or a different species, or is homologous to the prokaryotic or eukaryotic host cell, for example, but then is located in a different genetic environment and thus differs from a corresponding polynucleotide that is possibly naturally present. A heterologous polynucleotide may be present in addition to a corresponding endogenous gene.
[0149] "Raw material for industrial sugar production" means plant material which can be fed into a sugar production facility which is specialized in the extraction of sugar from sugar beets. Such raw material is typically the beet body (taproot) of the harvested sugar beet. To ensure the conformity with the extraction process the beet body needs to have sufficient mass, volume and a conical shape so that the raw material can be mechanically cut into shreds (beet strips). These beet strips maximize the surface area for sugar extraction and should have a low content of Sodium, Potassium and Nitrogen to allow an efficient extraction. After the extraction remaining beet pulp is pressed, dried and used as animal feed.
[0150] "Saccharose concentration" is expressed as percentage of the fresh weight of the root.
[0151] "Monogerm" means that a seed grows into exactly one plant whereas a polygerm or multigerm seed (also called "seed ball") grows into several plants.
[0152] "Bolting" is the production of a flowering stem (or stems) on a sugar beet in a natural attempt to produce seeds and reproduce. Bolting is triggered in sugar beet due to vernalization, i.e. a chilling stress which might occur e.g. during overwintering. However, commercially grown sugar beets are harvested before bolting as the bolting process and subsequent seed setting reduces the saccharose content in the beet body.
[0153] "Introgression" means that a nucleotide sequence has been transferred into the genome of a plant wherein this nucleotide sequence originates from a plant that does not belong to the same species or subspecies. This can for example mean that a nucleotide sequence deriving from a plant of the subspecies Beta vulgaris maritima has been transferred into a plant of the subspecies Beta vulgaris vulgaris.
BRIEF DESCRIPTION OF THE FIGURES
[0154] Designs and embodiments of the present invention are described by way of example with reference to the pending sequences and figures.
[0155] FIG. 1: Protein sequence alignment between the resistant protein (protein which confers Cercospora resistance in a plant) and the sensitive protein (protein which does not confer Cercospora resistance in a plant). The polymorphisms are highlighted in gray.
[0156] FIG. 2: Protein sequence alignment between the resistant protein (protein which confers Cercospora resistance in a plant) and the sensitive protein (protein which does not confer Cercospora resistance in a plant). The polymorphisms are highlighted in gray.
[0157] FIG. 3: Vector map of the vector pZFN-nptII including the LRR region.
[0158] FIG. 4: Statistical box-plot evaluation of the data generated eight days post infection during the transgenic verification of the resistance gene.
[0159] FIG. 5: Statistical box-plot evaluation of the data generated eleven days post infection during the transgenic verification of the resistance gene.
[0160] FIG. 6: Statistical box-plot evaluation of the data generated eight days post infection during the transgenic verification of the resistance gene.
[0161] FIG. 7: Statistical box-plot evaluation of the data generated fifteen days post infection during the transgenic verification of the resistance gene.
DETAILED DESCRIPTION OF THE INVENTION
[0162] The present invention relates to a nucleic acid molecule that is able to confer a resistance to Cercospora in a plant--in particular, in Beta vulgaris subsp. vulgaris--in which the polypeptide which is encoded by the nucleic acid molecule is expressed. According to a preferred embodiment of the invention, the pathogen is the fungus Cercospora beticola, which is among the most important and destructive leaf pathogens of sugar beets, beetroot, and chard, among others, and may cause crop losses of over 40%. The fungus produces the secondary metabolite cercosporin, which reacts with oxygen in the presence of light and leads to the formation of reactive oxygen species (ROS). The ROS cause massive cell damage in the leaf tissue of the infested plant that visible in the form of necroses.
[0163] The present invention is based upon the genetic fine mapping, identification, isolation, and characterization of a gene or of a gene locus that originates from the donor Beta vulgaris subsp. maritima, whose presence in a plant--in particular, in Beta vulgaris subsp. vulgaris--correlates with or is causative of the resistance of the plant concerned to Cercospora leaf spot disease. Initial material was a Beta vulgaris subsp. maritima population which was developed from 37 Beta vulgaris subsp. maritima accessions from different sources.
[0164] The nucleotide and amino acid encoding sequence of the nucleic acid molecule according to the invention is characterized by numerous polymorphisms, which differentiates the NPS-LRR gene identified according to the invention from the "sensitive" variant of the gene, i.e., the variant of the gene that does not confer resistance to Cercospora. Examples of polymorphisms are presented in FIG. 1.
[0165] The nucleic acid molecule according to the invention may be an isolated nucleic acid molecule. It is preferably DNA, and, particularly preferably, cDNA (coding DNA). The polypeptide which is encoded by the nucleic acid molecule according to the invention preferably confers a resistance to the pathogen Cercospora beticola, which causes the plant disease Cercospora leaf spot. Furthermore, the polypeptide which is encoded by the nucleic acid molecule according to the invention confers--in particular, in a plant of the genus Beta--a resistance to this pathogen. The plant is preferably a plant of the species Beta vulgaris--particularly preferably, a plant of the subspecies Beta vulgaris subsp. vulgaris; among these are, for example, the cultivars sugar beet, beetroot, fodder beet, chard, and Swiss chard.
[0166] In one embodiment of the present invention, the nucleic acid molecule according to the invention comprises a nucleotide sequence that encodes a polypeptide with an amino acid sequence according to SEQ ID No. 3 and/or the coding DNA sequence according to SEQ ID No. 2. Furthermore, the present invention provides a nucleotide sequence that comprises the DNA sequences according to SEQ ID No. 1 and SEQ ID No. 53.
[0167] The gene identified according to the invention is a resistance gene/protein of the type NBS-LRR, which is characterized by specific structural motifs. The general structure of such resistance proteins in plants has already been well-examined (Martin et al., Annual Review Plant Biology 54 (2003), 23-61). However, the principle of the structural embodiment--in particular, of what is known as the LRR domain, which applies as a potential detection domain for most unknown pathogenic effectors--is unpredictable, and the functional background of the resistance genes i.e., the genetic structure, is generally largely unknown. The identification of a Cercospora resistance-conferring gene or protein solely on the basis of the known structural motif is, consequently, impossible. Furthermore, the sequence region has turned out to be a highly repetitive region that contains, among other things, tandem repeats with very high sequence homology, which makes the development of diagnostic markers, as well as the assembly of sequence data, especially difficult.
[0168] With the aid of the setup of a population of over 4,000 dividing descendants and the development of special recombination screens, the target region was reduced, and thus ever further isolated, via analysis of informative recombinants (genotypical and phenotypical) in a series of resistance tests. This genetic mapping, as well as the creation of physical maps accompanied by WHG sequencing ("whole genome sequencing"), comparative BAC (Bac-by-Bac) sequencing, and bioinformatic analyses, led to the identification of three recombinant genotypes that confirmed the resistance gene (1 recombinant in the neighboring gene, on the one hand, and 2 recombinants in the neighboring gene, on the other). In light of particular requirements, the inventors placed the highly repetitive structure in the target region, which, among other things, contains tandem repeats with very high sequence homology, which made the marker development, and thus the identification of informative recombinants, enormously more difficult. The following steps were particularly decisive for the location of the genetic structure of the resistance gene:
[0169] development of the markers s4p0264s01, s4p2271s01, sxh0678s01, s4p4293s01, s4p4295s01, s4p4301s01 (see Table 1B).
[0170] Fine mapping coupled with intensive phenotyping. The phenotypes were verified with 90-180 descendants per plant in a greenhouse test, and with intensive statistical methods (for example, t-test, power analysis, etc.).
[0171] BAC clone identification and sequencing from BAC pools of the resistant genotype.
[0172] Sequence evaluation, as well as sequence and protein comparison between RR (i.e., resistant) and ss (i.e., sensitive) genotypes; an unambiguous assembly of the RR and ss sequence data was thereby not always possible, due to the sequence complexity.
TABLE-US-00002
[0172] TABLE 1B Marker in the target region relating to sensitive genotype, resistant genotype and consensus sequence. Position on Position on Sequences: sensitive/ genetic physical Marker resistant/consensus map [cM] map [bp] s4p0264s01 SEQ ID No. 54/SEQ ID No. 62,79590373 57208510 55/SEQ ID No. 10 s4p2271s01 SEQ ID No. 56/SEQ ID No. 62,81185523 57212240 57/SEQ ID No. 11 s4p4293s01 SEQ ID No. 58/SEQ ID No. 62,84491806 57219956 59/SEQ ID No. 12 s4p4295s01 SEQ ID No. 60/SEQ ID No. 62,85399055 57222060 61/SEQ ID No. 13 s4p4301s01 SEQ ID No. 62/SEQ ID No. 62,94635089 57243521 63/SEQ ID No. 14 sxh0678s01 SEQ ID No. 64/SEQ ID No. 62,97474964 57250119 65/SEQ ID No. 15
[0173] The compounds provided in Table 1B can be used as molecular markers according to the invention.
[0174] Analyses yielded that the LRR gene has a moderate protein homology to the Cf-2 resistance protein from the tomato (UNIPROT|Q41397_SOLPI P. Cf-2.1) (sequence identity 322/830=38%). In fact, the identified Cercospora resistance-conferring protein is the best sugar beet protein homolog to the Cf-2 tomato resistance protein. The Cf-2 resistance protein from the tomato confers a resistance to Cladosporium fulvum--a type of black mold fungus (U.S. Pat. No. 6,287,865 B1)--via interaction with the avirulence protein Avr2 from C. fulvum. This leads to the activation of the plant immune defense against the pathogen; see Dixon et al., 1996 (Dixon, Mark S., et al., "The tomato Cf-2 disease resistance locus comprises two functional genes encoding leucine-rich repeat proteins." Cell 84.3 (1996): 451-459). Due to the sequence homology between the Cf-2 gene and the identified LRR gene, it is to be assumed--but without thereby being bound to one theory--that a similar defense mechanism forming the basis of Cercospora resistance also occurs in the case of the sugar beet. However, a different mechanism is not to be precluded, due to the moderate sequence homology.
[0175] Furthermore, substitutions, deletions, insertions, additions, and/or any other change may be introduced into the nucleotide sequence according to the invention that, alone or in combinations, do in fact change the nucleotide sequence, wherein the modified nucleotide sequence may, however, perform the same function as the initial sequence. The present case deals with the coding of an amino acid sequence which confers resistance to Cercospora leaf spot disease. In a further embodiment, the invention therefore includes a nucleotide sequence that encodes a polypeptide which represents a derivative of the polypeptide which is encoded by the nucleotide sequence according to the invention, or which includes the amino acid sequence according to the invention. A derived amino acid sequence which has at least one substitution, deletion, insertion, or addition of one or more amino acids, wherein the functionality of the encoded polypeptide/protein is preserved, represents a derivative of the polypeptide. Substitutions, deletions, insertions, additions, and/or any other change, either solely or in combinations, that do in fact change the nucleotide sequence, but perform the same function as the initial sequence, may thereby be introduced into the nucleotide sequence using conventional methods that are known in the prior art, e.g., via site-directed mutagenesis, PCR-mediated mutagenesis, transposon mutagenesis, genome editing, etc.
[0176] The substitution of one amino acid by a different amino acid having the same or equivalent or similar chemical/physical properties is referred to as a "conservative substitution" or "semi-conservative substitution." Examples of physical/chemical properties of an amino acid are, for example, hydrophobia or the charge. Which amino acid substitution represents a conservative or semi-conservative substitution is known to the person skilled in the art. Moreover, general expertise allows the person skilled in the art to recognize, identify, and detect which amino acid deletions and additions are harmless to the functionality of the resistance protein, and at which positions these are possible. The person skilled in the art is aware that, in the case of the present NBS-LRR protein for modifications of the amino acid sequence (substitutions, deletions, insertion, or additions of one or more amino acids), the functionality, in particular, of the conserved domains must be preserved, and that therefore only limited preceding modifications are possible in these domains.
[0177] The invention thus includes a functional fragment of the nucleotide sequence according to the invention. The term, "fragment," thereby includes genes with a nucleotide sequence sufficiently similar to the aforementioned nucleotide sequence. The term, "sufficiently similar," means that a first nucleotide sequence or amino acid sequence has a sufficient or minimum number of identical or equivalent nucleotides or amino acid groups relative to a second nucleotide sequence or a second amino acid sequence.
[0178] With regard to the amino acid sequence, after modification via an aforementioned method, this also has a common structural domain and/or possesses common functional activity. Nucleotide sequences or amino acid sequences that have an identity of at least approximately 70%, at least approximately 75%, at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, at least approximately 99%, or at least approximately 100% with the nucleotide sequence or amino acid sequence according to the invention are defined here as being sufficiently similar. This also explicitly encompasses the range of 90% to 100%. For the functional fragments, a sufficient similarity is established if the nucleotide sequence or amino acid sequence generally has the same property as the previously-named nucleotide sequence or amino acid sequence of the present invention. Those nucleotide sequences which encode a derivative or for a derived amino acid sequence are generated either directly or indirectly (for example, via amplification or replication steps) from an initial nucleotide sequence which corresponds to the nucleotide sequence according to the invention over the entire length, or at least in part.
[0179] Accordingly, the present invention includes a nucleotide sequence that is able to hybridize, under stringent conditions, with a nucleotide sequence complementary to a nucleotide sequence according to the invention or to the nucleotide sequence that encodes the amino acid sequence according to the invention.
[0180] In a further embodiment, the nucleic acid molecule according to the invention is characterized in that, after expression in a plant, it already, on its own, confers a dominant resistance effect against a pathogen--preferably, against Cercospora beticola--or that it encodes for a polypeptide that is able to confer a dominant resistance effect against Cercospora. In a preferred embodiment, the nucleic acid molecule or the polypeptide confers a resistance effect of at least one rating score--preferably, of at least two rating scores, and, particularly preferably, of three to four rating scores. Such a gene that already, on its own, confers such a strongly pronounced resistance to Cercospora in a plant, or that encodes a polypeptide that is able to confer such a pronounced resistance, is not known from the prior art. As was already described above, in previously available varieties on the market, the Cercospora resistance is transmitted via many resistance genes having little effect, and a disadvantage of such varieties is that their development is very slow and expensive due to the complicated transmission, and that such varieties have a markedly poorer crop yield relative to normal varieties, in the absence of an infestation. Among other things, this may be linked to the epigenetic interaction of some resistance genes with genes that are responsible for sugar production, which leads to reduced fitness of the plants, in the absence of the pathogen.
[0181] The inventors could thus for the first time provide a Cercospora resistance gene that may be used for markedly simplified breeding. Via the incorporation of this gene in elite lines, it is now possible to very quickly develop very high-yield varieties with a high Cercospora resistance. Accordingly, in the framework of the present invention there are provided for the first time a sugar beet plant, a chard plant, a red beet or beetroot plant, a fodder beet plant having the resistance according to the invention against Cercospora beticola and thus being encompassed by the present invention. As the listed plants are all cultivated plants, crops or plants which are suitable for the agricultural cultivation and which have the resistance according to the invention, are part of the invention. Especially such crops are part of the invention which comprise a subterrestrial storage organ usable as food, raw material or industrial source of sugar and which comprise the resistance according to the invention are a further aspect of the present invention. The storage organ can be for example the sugar containing beet body of the sugar beet, the consumable beet body of the red beet or the feedable beet body of the fodder beet. The subterrestrial storage organ can sum up to more than 50% and for the sugar beet even to more than 70% of the total mass of the full-grown plant. Furthermore, also seeds or seeding material of these plants are part of the invention. The seeds or the seeding material can be technically treated as described further below. Part of the invention are also plants of the genus Spinacia comprising the resistance gene according to the invention. Especially plants of the species Spinacia oleracea and their varieties comprising the resistance gene according to the invention are included.
[0182] In this context, the invention also includes a nucleic acid that encodes the protein according to SEQ ID No. 3, wherein, in a specific embodiment, the naturally occurring nucleic acid according to SEQ ID No. 1 is excluded.
[0183] Furthermore, the present invention relates to a recombinant and/or heterologous DNA molecule that comprises the sequences of the nucleic acid molecule according to the invention. This DNA molecule, furthermore, preferably has a regulatory sequence. It may thereby be operatively linked with this regulatory sequence or be under the influence of this regulatory sequence. This regulatory sequence is preferably a promoter sequence and/or other sequences of transcription or translation control elements--for example, cis-elements. The regulatory sequence, which controls the expression of a gene that includes the nucleic acid molecule according to the invention, is preferably a sequence that is able to confer or modulate the expression, as a result of a pathogenic infection. This promoter is preferably able to control the expression of the DNA sequence specifically in leaves of the plant. The regulatory sequence may be heterologous to the expressing sequence. Such an approach has the advantage that the person skilled in the art may better adjust the expression rate of the expressing sequence, the tissue in which the expression occurs, and the point in time at which the expression occurs, in that he selects that regulatory sequence which is best suited to the respective use case. The heterologous DNA sequence preferably includes a nucleotide sequence which encodes a component of the plant pathogen defense (example: resistance genes (R-genes) or genes which encode enzymes involved in signal transfer, such as kinases or phosphatases, and for G-protein, or which encode a pathogenic effector (what are known as avirulence genes (avr))). The heterologous DNA sequence may be one of the DNA sequences according to the invention. The heterologous DNA sequence may also additionally encode further components of the plant pathogen defense. The heterologous DNA sequence may therefore be designed such that a polycistronic mRNA is created after its transcription.
[0184] The present invention furthermore also relates to a polypeptide which can be encoded by the nucleic acid molecule according to the invention and a functionally and/or immunologically active fragment thereof, as well as an antibody that specifically binds to the polypeptide or to its fragment. The polypeptide particularly preferably has an amino acid sequence according to SEQ ID No. 3. The recombinant production of proteins, polypeptides, and fragments is familiar to the person skilled in the art (Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001, or Wingfield, P. T., 2008, Production of Recombinant Proteins, Current Protocols in Protein Science, 52:5.0:5.0.1-5.0.4). Polyclonal or monoclonal antibodies to the protein according to the invention may be produced by the person skilled in the art according to known methods (E. Harlow et al., editor, Antibodies: A Laboratory Manual (1988)). The production of monoclonal antibodies, as well as of Fab and F(ab')2 fragments that are also useful in protein detection methods, may be performed via various conventional methods (Goding, Monoclonal Antibodies: Principles and Practice, pp. 98-118, New York: Academic Press (1983)). The antibodies may then be used for the screening of expression cDNA libraries in order to identify identical, homologous, or heterologous genes by means of immunological screening (Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989, or Ausubel et al., 1994, "Current Protocols in Molecular Biology." John Wiley & Sons), or may be used for western blot analyses. In particular, the present invention relates to antibodies that selectively detect a polypeptide encoded by the Cercospora resistance-conferring allele according to the invention, and essentially do not detect the polypeptide encoded by the correspondingly sensitive allele, i.e., that they detect less, by a factor of 2--preferably, a factor of 5, and, more preferably, a factor or 10 or more--of the polypeptide encoded by the correspondingly sensitive allele than the polypeptide encoded by the Cercospora resistance-conferring allele according to the invention.
[0185] In a preferred embodiment, the antibody according to the invention is characterized in that it is a synthetic polypeptide which does not occur in nature.
[0186] Furthermore, the antibodies according to the invention may be linked with a fluorescent dye in order to be usable in an immunohistochemical method, for example, and evoke an antibody coloration. The fluorescent dye may be fluorochrome. The antibodies according to the invention may also be present linked with other signaling molecules. Among these are, for example, biotin, radioisotopes, reporter enzymes such as alkaline phosphatase, or oligonucleotides.
[0187] An additional subject matter of the invention is vectors or expression cassettes that include the nucleic acid molecule or the recombinant DNA molecule according to the invention--possibly under control of regulatory elements and, in particular, under control of functional regulatory elements in plants, as well as negative and/or positive selection markers. The vector backbone is thereby heterologous to the nucleic acid molecule according to the invention, which means that such a vector does not occur in nature and cannot be isolated from nature. The vector is a plasmid, a cosmid, a phage or an expression vector, a transformation vector, shuttle vector or cloning vector; it may be double-stranded or single-stranded, linear or circular; or it may transform a prokaryotic or eukaryotic organism either via integration into its genome or extrachromosomally. The nucleic acid molecule or DNA molecule according to the invention in an expression vector or expression cassette is, preferably, operatively linked with one or more regulatory sequences which allow the transcription and, optionally, the expression in a prokaryotic or eukaryotic cell; (Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001). These regulatory sequences are preferably promoters or terminators--in particular, a transcription initiation starting point, a ribosome binding location, an RNA-processing signal, a transcription termination location, and/or a polyadenylation signal. For example, the nucleic acid molecule is here under the control of a suitable promoter and/or a terminator. Suitable promotors may be constitutive promotors (example: 35S promoter from the "Cauliflower mosaic virus" (Odell et al., Nature 313 (1985), 810-812); those promoters which are pathogenically inducible are especially suitable (example: PR1 promoter from parsley (Rushton et al., EMBO J. 15 (1996), 5,690-5,700)). Particularly suitable pathogenically-inducible promoters are synthetic or chimeric promoters which do not occur in nature, are composed of multiple elements, and contain a minimal promoter, and have at least one cis-regulatory element upstream of the minimal promoter, which at least one cis-regulatory element serves as a binding location for special transcription factors. Chimeric promoters are designed according to the desired requirements and are induced or repressed via different factors. Examples of such promoters are found in WO 00/29592, WO 2007/147395, and WO 2013/091612. For example, a suitable terminator is the nos-terminator (Depicker et al., J. Mol. Appl. Genet. 1 (1982), 561-573). Suitable promoters and terminators may also be the native promoter and the native terminator, whose DNA sequences are reproduced in SEQ ID Nos. 7 and 8. The vectors or expression cassettes additionally contain for conventional indicator/reporter genes or resistance genes for the detection of the transfer of the desired vector or DNA molecule/nucleic acid molecule, and for selection of the individuals that contain these, since a direct detection via the expression of the gene is for the most part rather difficult. Since the nucleic acid molecule according to the invention here itself encodes for a polypeptide which confers resistance to Cercospora leaf spot disease, it is not essential for the expression in plant cells to provide an additional resistance gene; however, it is recommended, in order to allow a rapid selection.
[0188] Examples of indicator/reporter genes are, for example, the luciferase gene and the gene encoding green fluorescent protein (GFP). These, furthermore, also allow tests for the activity and/or regulation of a promoter of the gene. Examples of resistance genes--especially, for plant transformations--are the neomycin phosphotransferase gene, the hygromycin phosphotransferase gene, or the gene encoding phosphinothricin acetyltransferase. Additional positive selection markers may be enzymes which provide the transformed plant a selection advantage over the non-transformed plant--in particular, a nutrient advantage, e.g., the mannose-6-phosphate isomerase or the xylose isomerase. However, this does not preclude additional indicator/reporter genes or resistance genes known to the person skilled in the art. In a preferred embodiment, the vector is a plant vector. Furthermore, the expression cassette may be present as integrated into a plant genome.
[0189] In a further aspect, the present invention relates to cells that include the vectors, recombinant DNA molecules, and/or nucleic acid molecules according to the invention. A cell in the sense of the invention may be a prokaryotic (for example, bacterial) or eukaryotic cell (for example, a plant cell or a yeast cell). The cell is preferably an agrobacterium such as Agrobacterium tumefaciens or Agrobacterium rhizogenes, an Escherichia coli cell, or a plant cell; the plant cell is particularly preferably a cell of a plant of the genus Beta, the species Beta vulgaris, or the subspecies Beta vulgaris subsp. vulgaris. The cell may also be present as a culture. The invention also consequently covers a cell culture which contains such cells. The cell culture is preferably a pure culture or an isolate that contains no cells of another type.
[0190] Known to the person skilled in the art are both numerous methods, such as conjugation or electroporation, with which he may introduce the nucleic acid molecule according to the invention, the recombinant DNA molecule, and/or the vector or the expression cassette of the present invention into an agrobacterium, and methods such as diverse transformation methods (biolistic transformation, agrobacterium-mediated transformation) with which he may introduce the nucleic acid molecule according to the invention, the DNA molecule, and/or the vector of the present invention into a plant cell (Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001.
[0191] Furthermore, the present invention preferably relates to a Cercospora-resistant plant--preferably, a plant of the species Beta vulgaris subsp. vulgaris or a portion thereof--that contains the nucleic acid molecule according to the invention which confers the Cercospora resistance. The Cercospora-resistant plant may contain the nucleic acid molecule according to the invention as a transgene or as an endogene. Within the scope of the invention, for the first time, plants of the subspecies Beta vulgaris subsp. vulgaris were produced which contain the nucleic acid molecule according to the invention. The invention here also includes plants of the subspecies Beta vulgaris subsp. vulgaris which contain the nucleic acid molecule according to the invention as an endogene.
[0192] A portion may thereby be a cell, a tissue, an organ, or a combination of multiple cells, tissues, or organs. A combination of multiple organs is, for example, a blossom or a seed. A Cercospora-resistant plant of the present invention preferably shows a higher resistance to Cercospora--in particular, Cercospora beticola--than a corresponding plant that does not contain the nucleic acid molecule according to the invention (control plant). The control plant ideally has the identical genotype as the transgenic plant, and has been cultured under identical conditions, but does not contain the resistance-conferring nucleic acid molecule. The level of the resistance, e.g., to Cercospora beticola, may be qualitatively established in plants of the genus Beta by determining rating scores. A higher resistance manifests in an improvement in the resistance by at least one rating score, by at least two rating scores, and, preferably, by at least three or more rating scores.
[0193] A plant cell or plant or portion thereof of the present invention that contains the nucleic acid molecule according to the invention--in particular, a plant of the genus Beta--preferably shows a higher resistance to a pathogen--in particular, to Cercospora beticola--than a corresponding plant cell or plant or portion thereof that does not contain the nucleic acid molecule according to the invention, or contains a sensitive allelic variant of the nucleic acid molecule. The level of the resistance, e.g., to Cercospora beticola, may be qualitatively established in plants of the genus Beta by determining rating scores. A higher resistance manifests in an improvement in the resistance by at least one rating score, by at least two rating scores, and, preferably, by at least three or more rating scores.
[0194] In the case of a transgenic plant cell, or plant or portion thereof, this comprises the nucleic acid molecule or DNA molecule according to the invention as a transgene or the vector or the expression cassette of the present invention. Such a transgenic plant cell or plant or portion thereof is, for example, one that is transformed--preferably, stably--with the nucleic acid molecule, DNA molecule according to the invention, or with the vector or the expression cassette of the present invention. In a preferred embodiment, the nucleic acid molecule is operatively linked with one or more regulatory sequences which allow the transcription and, optionally, the expression in the plant cell. The total structure made up of the nucleic acid molecule according to the invention and the regulatory sequence(s) then represents the transgene. Such regulatory sequences are, for example, a promoter or a terminator. Numerous functional promoters and terminators that are applicable in plants are known to the person skilled in the art.
[0195] The invention also includes a vacuole of the cell according to the invention, and the content substances stored therein.
[0196] Furthermore, the invention also relates to the cell extract from a cell--preferably, from a plant cell, particularly preferably, from a cell of Beta vulgaris, and, especially preferably, from a cell of one of the following crops: sugar beet, chard, or beetroot. No plant can be regenerated from the cell extract.
[0197] Likewise encompassed by the invention is a plant genome containing the nucleic acid according to the invention.
[0198] The sugar concentration from the cell extract may thereby be increased relative to a cell that is not a cell according to the invention, but that belongs to the same species or crop. This applies, in particular, under the conditions when infested by Cercospora.
[0199] Also encompassed by the invention is the use of the cell extract for the production of sugar (saccharose) or for the production of juice--preferably, beetroot juice.
[0200] Likewise encompassed by the invention is the sugar--in particular, saccharose--contained in the cells according to the invention and their vacuoles.
[0201] An additional aspect of the invention is seed stock comprising seeds that contain the nucleic acid according to the invention. The nucleic acid according to the invention may be present transgenically or endogenously. The seed stock and the seeds may be technically treated. The invention thus also comprises technically-treated seed stock and technically-treated seeds. The various embodiments of technically-treated seed stock are explained in detail in the following whereby the term seed stock also includes seeds: Technically-treated seed stock may be present in polished form. The outermost layer of the seed is thereby removed, so that the seed assumes a more rounded form. This is helpful in sowing, where an optimally uniform shape leads to a uniform distribution of the seed stock grains. Technically-treated seed stock furthermore encompasses pelleted seed stock. The seed stock is thereby embedded in a pelleting mass that protects the seed stock contained therein and leads to a larger mass, such that the pelleted seed stock shows a greater resistance capability with regard to wind drift and is thus less susceptible to being blown away by the wind, and, at the same time, a more precise positioning during sowing is enabled. In a preferred embodiment of the invention, all pelleted seed stock grains of a batch or unit designated for sale have essentially the same shape and the same mass. Deviations of 5% in diameter and mass are possible. However, the deviations preferably do not exceed 1%. As one of the main components, the pelleting mass may contain for example a mineral compound such as clay, bentonite, kaolin, humus and/or peat, for example. It is possible to add an adhesive material like polyacylamide. Additional possible components are cited in U.S. Pat. No. 4,067,141. Moreover, the pelleting mass may contain additional chemical agents that positively influence the cultivation in practice. These may here be substances that are counted among fertilizing agents. These include compounds rich of one or more of the following elements: Nitrogen, Phosphorus and Potassium (macronutrients). Therefore, the fertilizing ingredients may contain for example Nitrate nitrogen, Ammonium nitrogen, Magnesium Nitrate, Calcium Ammonium Nitrate, Mono Ammonium Phosphate, Mono Potassium Phosphate and Potassium Nitrate. Furthermore, pelleting mass may contain fungicides, insecticides, and/or antifeedants. The fungicides may be thiram and/or hymexazol and/or other fungicides. The insecticide may be a substance from the neonicotinoid group. The substance from the neonicotinoid group is preferably imidacloprid (ATC Code: QP53AX17) and/or clothianidin (CAS number 210880-92-5). Furthermore, the insecticide may also be cyfluthrin (CAS number 68359-37-5), beta-cyfluthrin or tefluthrin. It is worth mentioned that the compound included in the dressing or pelleting mass are taken up by the plant and show systemic effect thereby providing suitable protection of the whole plant. Plants resulting from pelleted seed including one or more pesticides therefore differ from naturally occurring plants and show better performance under biotic stress conditions. In this context the invention also encompasses a mixture of a pelleting mass and a seed according to the invention. Furthermore, the invention also encompasses a method for producing a pelleted seed according to the invention comprising the following steps:
a) providing a sugar beet plant seed comprising the nucleic acid according to the invention, b) embedding the sugar beet plant seed in a pelleting mass, and c) allowing the pelleting mass to dry or drying the pelleting mass, wherein the seed may be optionally a primed or pregerminated seed or the seed may be allowed to be primed during step b).
[0202] The pelleted seed stock is a specific embodiment of dressed seed stock. In this context technically-treated seed stock encompasses also the dressed seed stock. However, the invention is not limited to pelleted seed stock, but, rather, may be applied with any form of dressed seed stock. The invention thus also relates to dressed seed stock, which includes pelleted seed stock, but is not limited to this. Dry dressing, wet dressing, and suspension dressing are thus also encompassed. The dressing may thereby also contain at least one dye (coloring), such that the dressed seed stock may be quickly differentiated from undressed seed stock, and, furthermore, good visibility in the environment is ensured after sowing. The dressing may also contain those agrochemicals which are described in the context of the pilling mass. The invention includes thus such dressed seed stock whereby the dressing contains at least one anti-feedant such as an insecticide and/or at least one fungicide. Optionally, so called electronic dressing (dressing by application of electric energy) may be applied. Electronic dressing is not a dressing in the strict sense of the word but is very suitable to destroy plant pathogens which adhere to the seed or seed stock before planting the seed or seed stock. It is also beneficial that seeds or seed stock which have only been treated by use of electronic dressing (without using agrochemicals) can be fed to animals in case more seed or seed stock is available than needed to till a field.
[0203] An additional form of technically-treated seed stock is encrusted seed stock. What is known as coating is also spoken of in this context as well as of seed stock treated with a coating. The difference to pelleted seed stock is that the seed grains retain their original shape, wherein this method is especially economical. The method is described in EP 0 334 258 A1, for example. An additional form of technically-treated seed stock is sprouted or primed seed stock. Sprouted seed stock is pretreated via a pre-germination, whereas primed seed stock has been pretreated via a priming ("germination"). Pre-germinated and primed seed stock have the advantage of a shorter emergence time. The point in time of the emergence after sowing is, at the same time, more strongly synchronized. This enables better agrotechnical processing during cultivation and especially during the harvest, and, additionally, increases the yield quantity. In pre-germination, the seed stock is germinated until the radicle exits the seed stock shell, and the process is subsequently stopped. In the priming, the process is stopped before the radicle exits the seed stock shell. Compared to pre-germinated seed stock, seed stock that has been subjected to a priming is insensitive to the stress of a re-drying and, after such a re-drying, has a longer storage life in comparison to pre-germinated seed stock, for which a re-drying is generally not advised. In this context, technically pre-treated seed stock also includes primed and re-dried seed stock. The process of pre-germination is explained in U.S. Pat. No. 4,905,411 A. Various embodiments of priming are explained in EP 0 686 340 A1. In addition to this, it is also possible to simultaneously pill and prime seed stock in one process. This method is described in EP 2 002 702 B1. Primed seed stock which is moreover pelleted, is encompassed by the present invention.
[0204] The technically-treated seed stock may additionally be furnished with one or more of the herbicide resistances explained above. This allows a further-improved agrotechnical cultivation, since the technically-treated seed stock may be deployed on a field that has previously been treated with weed killer, and that therefore is weed-free.
[0205] In addition to this, the invention also encompasses a mixture containing the seed stock according to the invention or the seeds according to the invention, and a dressing mass as defined above. The dressing mass is thereby preferably embodied as a pelleting mass, as defined above. With storage of seed stock according to the invention, storage conditions are preferably to be chosen that do not negatively affect the stability or storage life of the seed stock. Fluctuations in humidity may, especially, have a disadvantageous effect here. Part of the invention is a method for the storage of the seed stock in a bag or container that is via simultaneously water-repellent and breathable. Such a bag or container may be designed as a carton or packing. Such a carton or packing may optionally possess an inner vapor barrier. If the carton or packing is designed as a duplex carton, its stability increases. A container, bag, carton or packing comprising the seed stock according to the invention, or technically-treated seed stock according to the invention, is likewise a part of the invention. It is likewise part of the invention to store seed stock according to the invention or technically-treated seed stock according to the invention in such a bag, container, box, packing or carton.
[0206] The present invention also encompasses varieties comprising the resistance gene according to the invention. Furthermore, plants, seeds and seedstock of such a variety are included. The seeds and seedstock of such a variety may be subject to a technical treating as described herein (e.g. pelleting). Suitable sugar beet varieties for the introduction of the resistance gene are for example BTS 7300 N, BTS 2045, BTS 3750, DAPHNA, KORTESSA KWS or SABATINA KWS. Sugar beet plants of the named varieties are also examples of hybrid sugar beet plants. Suitable red beet varieties for the introduction of the resistance gene are for example Jolie, Scarlett (PV-9503) or Diaz wherein Jolie and Diaz are also examples of hybrid red beet plants. Suitable Swiss Chard varieties for the introduction of the resistance gene are for example Fluence, Ion or Tesla/PV-9022. Suitable varieties of Spinacia oleracea (spinach) for the introduction of the resistance gene are for example PV-9210, PV-1194 or La Paz/PV-1237. Hybrid plants take advantage from the heterosis effect.
[0207] In one embodiment, the plant according to the invention is a hybrid plant or a double haploid plant. Hybrid plants and double haploid plants do not occur in nature and cannot be isolated from nature. In a further embodiment of the plant according to the invention, the nucleic acid molecule according to the invention is present in heterozygous or homozygous form. In the case of a hybrid plant, the nucleic acid molecule may also be present in hemizygous form. The invention also encompasses hybrid seeds and double haploid seeds which contain a nucleic acid according to the invention or a polypeptide according to the invention.
[0208] A further embodiment of the present invention comprises a plant--preferably, of the species Beta vulgaris--that is characterized in that the resistance to Cercospora in this plant is further increased. For example, this may be realized by means of "gene stacking," i.e., the resistance is increased using this dose effect. For this, the plants according to the invention that contain the Cercospora resistance-conferring allele are over-transformed with this resistance allele in order to increase the amount of the transcription of the gene in the plant. An alternative approach includes the gene editing/site-directed mutagenesis or TILLING-mediated modification of the native promoter of the resistance-conferring allele, in order to increase its expression rate, or the modification of the resistance-conferring LRR gene allele itself, in order to increase its activity or stability. Such a method for increasing the activity by means of modification of a resistance gene is described in WO 2006/128444 A2, for example, and may be performed by means of the techniques known to the person skilled in the art. An additional approach may include the fusion of the nucleic acid molecule according to the invention with a heterologous promoter that exhibits a higher activity in comparison to the native promoter--in particular, after Cercospora infection.
[0209] An additional embodiment of the present invention relates to a sugar beet plant or a portion thereof or a pelleted seed of such a plant which is harvestable before bolting because no bolting of the sugar beet plant occurs during the first 10, 11, 12, 13, 14 or 15 months after germination and the development of a beet body is finished during this period. Suitable varieties to create a sugar beet plant according to this paragraph by introduction of the resistance according to the invention are for example DAPHNA, KORTESSA KWS or SABATINA KWS.
[0210] In one embodiment of the present invention the sugar beet plant or a portion thereof or a pelleted seed of such a plant has a genome allowing the development of a beet body having a mass summing up to at least 50%, 60%, 70%, 80% or even 90% of the total mass of the full-grown plant. Suitable varieties to create a sugar beet plant according to this paragraph by introduction of the resistance according to the invention are for example DAPHNA, KORTESSA KWS or SABATINA KWS.
[0211] In another embodiment of the present invention the sugar beet plant or a portion thereof or a pelleted seed of such a plant has a genome allowing the development of a beet body having a minimum mass of 200 g, 250 g, 300 g, 350 g, 400 g, 450 g or 500 g and a maximum mass of 1000 g, 1100 g, 1200 g, 1300 g, 1400 g, 1500 g, 1600 g, 1700 g, 1800 g, 1900 g or even 2000 g via photosynthesis. Suitable varieties to create a sugar beet plant according to this paragraph by introduction of the resistance according to the invention are for example DAPHNA, KORTESSA KWS or SABATINA KWS.
[0212] An additional embodiment of the present invention is directed to a sugar beet plant or a portion thereof or a pelleted seed of such a plant wherein the genome of the sugar beet plant allows development of a beet body having a saccharose concentration of at least 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19% or even 20%. Suitable varieties to create a sugar beet plant according to this paragraph by introduction of the resistance according to the invention are for example DAPHNA, KORTESSA KWS or SABATINA KWS.
[0213] In one embodiment of the present invention the sugar beet plant or a portion thereof or a pelleted seed of such a plant includes at least one, at least two, at least three, at least four, at least five, at least ten, at least twenty or even at least thirty mutation(s) relative to SEQ ID No. 1, 2 or 4.
[0214] The method for the production of an organism which comprises a mutated version of the nucleic acid molecule according to the above given embodiment [1] and/or a mutated version of a promoter comprising a nucleic acid sequence selected from the group consisting of (a) SEQ ID NO: 7, (b) a nucleotide sequence, which hybridizes under stringent conditions with a sequence which is complementary to the sequence according to (a), and (c) a nucleotide sequence which is at least 70% identical to a sequence according to SEQ ID NO: 7, wherein the method includes the following steps:
[0215] (I) Provision of an organism or a cell comprising the nucleic acid molecule and/or the promoter
[0216] (II) Increase of the mutation rate of the organism or the cell or mutagenesis of the organism or the cell
[0217] (III) Phenotypic selection of an organism, which as a result of a mutation exhibits an altered resistance or altered resistance level towards Cercospora beticola or Genotypic selection of an organism or a cell which comprises a mutation in the nucleic acid molecule and/or the promoter wherein the mutation has been created via step (II) and optionally
[0218] (IV) Regeneration of the organism from the cell obtained via step (III).
[0219] The organism can be a plant. Preferably the plant is a Beta vulgaris. However, it is also possible to use unicellular organisms as bacteria. The bacterium can be E. coli. If the organism is a plant then the method can be applied in vivo as well as in vitro. If the organism is a plant and the method is applied in vitro, a cell culture of the plant may be established and the increase of the mutation rate or the mutagenization may occur in the cell culture. The increase of the mutation rate encompasses for example the application of mutagenic agents like for example 5-bromouracil or ethylmethane sulfonate (EMS) or the application of physical mutagens like ionizing radiation or UV light. The mutagenization encompasses also the targeted mutagenesis. The targeted mutagenesis can be achieved by precise methods as gene editing (as explained further below). The regeneration of organism out of cells is explained in various standard references of the cell biology. The regeneration of plants is for example explained in the standard reference "Plant biotechnology: comprehensive biotechnology, second supplement" (Michael W. Fowler, Graham Warren, Murray Moo-Young--Pergamon Press--1992). The regeneration of Beta vulgaris out of the cell culture is described in Lindsey & Gallois "Transformation of sugarbeet (Beta vulgaris) by Agrobacterium tumefaciens." Journal of experimental botany 41.5 (1990): 529-536.
[0220] These references also describe how plant cell cultures are established. As explained further above the mutated version of the nucleic acid molecule respectively the promoter characterizes themselves preferably due to the expression rate of the resistance imparting nucleic acid molecule which is increased by the mutation. Such an effect can also rely on the presence of several mutations. For example, it is possible to introduce two, three, four, five or more mutations in the promoter or the nucleic acid molecule.
[0221] By the introduction of mutations thus more resistance imparting protein can be built in the cell or the protein has a better effect. Thereby, the resistance in comparison to a control plant comprising the unaltered nucleic acid according to the invention can be increased for example by at least 1, 2, 3, 4, 5 or more percent. The increase can be measured as explained further below. Moreover, the resistance due to the mutation or mutations can be increased by at least one rating score. The determination of rating scores is explained elsewhere herein. Furthermore, the resistance protein can impart--as a result of the mutations--an altered effect and in some circumstances can exhibit effect against such pathogens which have adapted themselves to the initial resistance mechanism. In this context the invention encompasses also such mutated variants of the nucleic acids according to the invention and mutated variants of the protein according to the invention. Preferably the invention encompasses such variants which do not occur in nature and cannot be isolated from nature to make sure that the pathogen had no opportunity to adapt itself to such variants. The above described method for the production of an organism which comprises a mutated version of the nucleic acid molecule may furthermore include a further step, in which those organisms or respectively plants are identified, which have a further increased resistance due to the mutation or mutations. If an increase of resistance has taken place may be determined by the herein explained rating scores or the measuring of the resistance level.
[0222] Besides the above described method for the production of organisms which comprise a mutated version of the nucleic acid molecule or of the promoter it is also possible to modify the according nucleic acids chemically in an isolated state to achieve the desired effects (as for example those which are described above). The advantage of this approach is that the compounds can be edited even more precisely. For this purpose, the following method is offered:
[0223] Production of a chemically modified nucleic acid molecule according to the above given embodiment [1] and/or a chemically modified promoter comprising a nucleotide sequence which is chosen from
(a) SEQ ID NO: 7;
[0224] (b) a nucleotide sequence which hybridizes under stringent conditions to a nucleotide sequence according to (a); (c) a nucleotide sequence which is at least 70% identical to a sequence according to SEQ ID NO: 7; wherein the method comprises the following steps: (I) Provision of the nucleic acid molecule as stated above in isolated form (II) chemical modification of the nucleic acid molecule or the promoter by one of the following steps:
(IIa) Mutagenization
[0225] (IIb) Gene editing (IIc) Restriction and ligation respectively insertion or deletion.
[0226] Furthermore, chemical modifications can be generated by such approaches as stated elsewhere herein in context of allelic variants. The gene editing given under step (II) above is equal to the term "Genome-Editing". Optionally the chemically modified nucleic acid molecule or the chemically modified promoter can be subsequently introduced into a cell or can be stably integrated. With the help of such a cell, the chemically modified nucleic acid molecule and the modified promoter can be propagated in context of the cell proliferation. They can be subsequently isolated in vast number and expression analyses may be performed. Expression analyses are especially suitable when the chemical modification concerns the promoter. It is possible to harvest the cells and to isolate the chemically modified resistance protein for chemical analyses. If the cell which comprises the chemically modified nucleic acid molecule or the modified promoter is a plant cell, a complete plant may be regenerated out of this cell. The approaches described within this passage can be performed subsequently to the above given method for the production of a modified form of the nucleic acid molecule and/or a modified promoter and the obtained variants are also a part of this invention. Moreover, a plant comprising the chemically modified nucleic acid molecule or the modified promoter are also part of the invention. Thus, the invention is also related to a plant obtained by this method. Furthermore, the invention relates also to the chemically modified nucleic acid molecules obtained by this method and to the encoded polypeptides. These compounds may be optimized versions of the original (not modified) compounds, wherein the resulting resistance level--as explained further above--may be increased by at least by 1, 2, 3, 4, 5, or more percent or may be increased by at least one rating score. In this regard the method for the production of a chemically modified nucleic acid molecule is also a method for the optimization of the nucleic acid molecule. The method for optimization may furthermore contain an additional step, in which those modified variants of the nucleic acid molecule are identified which lead in comparison to the unamended variants to an increased resistance in a plant. In a further embodiment, the plant of the present invention additionally, transgenically or endogenously, comprises a second nucleic acid molecule at a different position in the genome, which encodes a polypeptide that is able to confer a resistance to Cercospora in the plant in which the polypeptide is expressed. For example, one or more of the resistance genes or resistance loci that are described in the prior art may--insofar as they are not already present in the initial genotype--be introduced into the present plant by means of crossing, transformation, homology-directed repair, or homologous recombination in the plant. Among these are, for example, the rhizomania resistance RZ1 (Lewellen, R. T., I. O. Skoyen, and A. W. Erichsen, "Breeding sugar beet for resistance to rhizomania: Evaluation of host-plant reactions and selection for and inheritance of resistance." 50th Winter Congress of the International Institute for Sugar Beet Research, Brussels (Belgium), Feb. 11-12, 1987. IIRB. Secretariat General, 1987), or the rhizomania resistance RZ3 (WO 2014/202044).
[0227] The present invention additionally relates to a method for increasing the resistance to Cercospora in a plant of the species Beta vulgaris, wherein the increase in the resistance takes place without the resistance-conferring gene according to the invention, in comparison to an isogenic plant. The increase in the resistance may take place via integration of the nucleic acid molecule according to the invention into the genome of at least one cell of a plant of the species Beta vulgaris, as well as possible regeneration of a plant from the plant cell. The integration may take place both by means of sexual crossing, e.g., with one of the aforementioned Beta vulgaris subsp. maritima and subsequent selection, or by means of homology-directed repair or homologous recombination. The two latter methods cited are preferably supported by site-directed nucleases which may be selected from, but are not limited to, the following: CRISPR nuclease, including Cas9, CasX, CasY, or Cpf1 nuclease, TALE nuclease, zinc finger nuclease, meganuclease, Argonaut nuclease, restriction endonuclease, including FokI or a variant thereof, recombinase, or two, site-specific, nicking endonucleases. The introduction of the resistance-conferring gene by means of CRISPR-mediated homologous recombination in Beta vulgaris subsp. vulgaris is shown in Example 1.
[0228] Moreover, the invention encompasses also a method of producing an agronomically sugar beet plant of the genus Beta that displays improved resistance to Cercospora beticola, the method comprising introgressing into said plant a chromosomal interval that confers the improved resistance to Cercospora beticola, wherein the chromosomal interval maps to a position between a sequence represented by a marker selected from the group consisting of
s4p4293s01 and s4p4295s01 and a sequence represented by a marker selected from the group consisting of s4p4301s01 and sxh0678s01, characterized in that the chromosomal interval comprises a nucleotide sequence encoding a polypeptide that is able to confer resistance to Cercospora beticola in a plant in which the polypeptide is expressed wherein the nucleotide sequence is selected from (a) a nucleotide sequence encoding a polypeptide having an amino acid sequence according to SEQ ID No. 3; (b) a nucleotide sequence that comprises the DNA sequence according to SEQ ID No. 2; (c) a nucleotide sequence that comprises a DNA sequence selected from the group consisting of SEQ ID No. 1 or SEQ ID No. 53; (d) a nucleotide sequence that hybridizes to a nucleotide sequence which is complementary to the nucleotide sequence according to (a), (b), or (c), under stringent conditions; (e) a nucleotide sequence encoding a polypeptide which, via substitution, deletion, and/or addition of one or more amino acids of the amino acid sequence, differs from a polypeptide encoded by the nucleotide sequence according to (a), (b), or (c); (f) a nucleotide sequence encoding a polypeptide which has an amino acid sequence that is at least 70% identical to an amino acid sequence according to SEQ ID No. 3; (g) a nucleotide sequence that is at least 70% identical to a DNA sequence according to SEQ ID No. 1 or SEQ ID No. 2;
[0229] An alternative approach includes the increase in the expression of the nucleic acid molecule according to the invention in the plant. This may take place via modification of the native promoter, wherein the modification preferably takes place by means of gene editing or site-directed mutagenesis which is mediated via site-directed nucleases, and, optionally, repair models. Examples of such nucleases have already been cited above. The increase in the expression of the nucleic acid molecule according to the invention may likewise take place via fusion of the nucleic acid molecule with a heterologous promoter, which exhibits a higher activity in comparison to the native promoter--in particular, after Cercospora infection. The fusion may likewise take place via site-directed nuclease and repair models, but also by means of direct insertion after double-strand break.
[0230] As has already been mentioned above, a method for increasing the Cercospora resistance, may also result in the increase in the activity and/or stability of the polypeptide according to the invention, via modification of the nucleotide sequence of the nucleic acid molecule according to the invention. Such a method for increasing the activity by means of modification of a resistance gene is described in WO 2006/128444 A2, for example, and may be performed by means of the techniques known to the person skilled in the art. This approach is explained in detail further below.
[0231] Alternatively, a Cercospora-resistant genotype may be produced from a Cercospora-sensitive genotype by means of random or directed mutagenesis of the nucleic acid sequence of the sensitive gene, and thus the Cercospora resistance may be increased. Examples of polymorphisms which differentiate the sensitive allele from the resistant allele are presented in FIG. 1.
[0232] For example, the sensitive allele may be modified via gene mutation by means of TALE nucleases (TALEN's) or zinc finger nucleases (ZFN's), as well as CRISPR/Cas systems, which--among other things--are described by way of example in WO 2014/144155 A1 (Engineering plant genomes using CRISPR/Cas systems) and in Osakabe & Osakabe, Plant Cell Physiol., 56 (2015), 389-400. This may also be achieved via use of the method designated as TILLING (Targeted Induced Local Lesions in Genomes), wherein it is described, e.g., in the German patent application DE 10 2013 101 617, how point mutations are caused in the sensitive gene, and plants are subsequently selected that exhibit a suitable, i.e., resistance-conferring, mutation, e.g., a barley resistant to yellow mosaic virus; see DE 10 2013 101 617 on pp. 4, 8, and 12, in paragraphs
[0014],
[0026], and
[0038]. The TILLING method is also described in detail in the publication by Henikoff et al. (Henikoff et al., Plant Physiol. 135, 2004, 630-636).
[0233] These methods preferably lead to an improvement in the resistance by at least one rating score--particularly preferably, to an improvement in the resistance by at least two, three, or more rating scores. After mutagenesis of the plant cells and subsequent regeneration of plants from the mutagenized plant cells, or mutagenesis of plants, the plants may then be identified that exhibit one or more mutations, as depicted in FIG. 1, in an endogenous nucleic acid molecule. In this context the already mentioned plant according to the invention may be characterized by that the resistance is increased by at least one rating score, preferably by at least two or more rating scores. Alternatively, the resistance of the plants according to the invention may be increased for example by at least 1, 2, 3, 4, 5 or more percent in comparison to a control plant, which does not comprise the nucleic acid according to the invention. The increase can be measured by inoculation of respectively one healthy leaf with an isolate of the pathogen and the determination of the infested surface after 15 days. A reduce of 5% of the infested surface corresponds to an increase of the resistance of 5%. Further parameters for the conduction of the measuring can be derived from the below given embodiment "resistance rest".
[0234] An additional embodiment of the present invention is a method for producing a Cercospora-resistant plant, which may take place via transformation of a plant cell with the nucleic acid molecule according to the invention, the recombinant DNA molecule, or with the vector or the expression cassette, and regeneration of the transgenic plant from the transformed plant cell (see Example 2), as well as, as described above, by means of random or targeted mutagenesis of the nucleic acid sequence of the sensitive gene to generate a Cercospora-resistant genotype, or via crossing and selection, e.g., with one of the aforementioned Beta vulgaris subsp. maritima. Vectors or expression cassettes, as well as methods for transforming plants, have already been described above.
[0235] The method for production of a Cercospora-resistant plant alternatively includes, as described above, the introduction of a site-directed nuclease and a repair matrix into a cell of a plant of the species Beta vulgaris, wherein the site-directed nuclease is able to generate at least one double-strand break of the DNA in the genome of the cell--preferably, upstream and/or downstream of a target region--and the repair matrix comprises the nucleic acid molecule according to the invention. The method furthermore includes the cultivation of this cell under conditions that allow a homology-directed repair or a homologous recombination, wherein the nucleic acid molecule is incorporated from the repair matrix into the genome of the plant. Furthermore, the regeneration of a plant from the modified plant cell is encompassed (see Example 1).
[0236] In a preferred embodiment, the target region is an allelic variant of the nucleic acid molecule according to the invention, wherein the allelic variant encodes a polypeptide which does not confer resistance to Cercospora. In a further preferred embodiment, this allelic variant comprises a nucleotide sequence that encodes a polypeptide with an amino acid sequence according to SEQ ID No. 6 and/or comprises the encoded DNA sequence according to SEQ ID NO: 5 or the genomic DNA sequence according to SEQ ID No. 4.
[0237] As described in connection with the nucleic acid molecule according to the invention, substitutions, deletions, insertions, additions, and/or any other change may be introduced that, either alone or in combinations, do in fact change the nucleotide sequence, but perform the same function as the initial sequence--here, the nucleotide sequence of the allelic variant of the nucleic acid molecule according to the invention. Therefore, in a further embodiment, the invention includes a nucleotide sequence that encodes a polypeptide which represents a derivative of the polypeptide which is encoded by the allelic variant of the nucleic acid molecule according to the invention, or which comprises the amino acid sequence of the allelic variant of the nucleic acid molecule according to the invention. A derived amino acid sequence which has at least one substitution, deletion, insertion, or addition of one or more amino acids, wherein the functionality of the encoded polypeptide/protein is preserved, represents a derivative of the polypeptide. The nucleotide sequence, using conventional methods that are known in the prior art, e.g., via site-directed mutagenesis, PCR-mediated mutagenesis, transposon mutagenesis, genome editing, etc., substitutions, deletions, insertions, additions, and/or any other change, either solely or in combinations with the gene, may thereby be introduced, which do in fact change the nucleotide sequence, but perform the same function as the initial sequence.
[0238] With regard to the amino acid sequence, after modification via an aforementioned method, this also has a common structural domain and/or possesses common functional activity. Nucleotide sequences or amino acid sequences that at least approximately 80%, at least approximately 85%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, at least approximately 99%, or at least approximately 100% identical to the nucleotide sequence or amino acid sequence of the cited allelic variant of the nucleic acid molecule according to the invention are defined here as being sufficiently similar. Accordingly, the present invention includes a nucleotide sequence that is able to hybridize, under stringent conditions, with a nucleotide sequence that is complementary to a nucleotide sequence of the allelic variant of the nucleic acid molecule according to the invention or to the nucleotide sequence that encodes the corresponding amino acid sequence.
[0239] In a further preferred embodiment, the method according to the invention is characterized in that the double strand break occurs in an allelic variant of the nucleic acid molecule according to embodiment [1] or that the at least one double strand break occurs at a position which is at least 10,000 base pairs upstream or downstream of the allelic variant, wherein the allelic variant codes for a polypeptide which does not impart a resistance towards Cercospora.
[0240] For the person skilled in the art, it is obvious that numerous, different sensitive sequences may occur that derive from the nucleic acid molecule according to the invention, but do not confer resistance to Cercospora, such that the sequences listed above (SEQ ID Nos. 4, 5, and 6) should only be considered as an example of sequences, and the present invention is not limited to the aforementioned allelic variant of the nucleic acid molecule according to the invention. Such an allelic variant can comprise a nucleotide sequence, which is selected from:
[0241] (a) a nucleotide sequence encoding a polypeptide having an amino acid sequence according to SEQ ID No. 6;
[0242] (b) a nucleotide sequence that comprises the DNA sequence according to SEQ ID No. 5;
[0243] (c) a nucleotide sequence that comprises a DNA sequence according to SEQ ID No. 4;
[0244] (d) a nucleotide sequence that hybridizes with the complementary sequence according to (a), (b), or (c), under stringent conditions;
[0245] (e) a nucleotide sequence encoding a polypeptide which, via substitution, deletion, and/or addition of one or more amino acids of the amino acid sequence, differs from a polypeptide encoded by the nucleotide sequence according to (a), (b), or (c);
[0246] (f) a nucleotide sequence encoding a polypeptide which has an amino acid sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to an amino acid sequence according to SEQ ID No. 6;
[0247] (g) a nucleotide sequence that is at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to a DNA sequence according to SEQ ID No. 4 or SEQ ID No. 5;
[0248] As described above, with quantitative heredity of QTL, not only is the desired resistance to often introduced into the plant, but, rather, also often unwanted features such as, for example, reduced yield, due to the inheritance of additional genes that are not linked with the positive feature of the resistance. This increasingly occurs if, as in the case of Cercospora resistance, the resistance is inherited in previously available cultivars via many resistance genes with small effect. Therefore, in a preferred embodiment, the introduction of the nucleic acid molecule according to the invention, which already shows, on its own, a dominant resistance effect, or of the vector or the expression cassette, is not linked with the introduction of unwanted features, wherein the yield is, preferably, not negatively affected. Furthermore, encompassed by the invention is the plant that is obtained via such a method.
[0249] Although the QTL analyses with that have previously been known from the prior art could detect actual QTL's, the underlying genomic regions that had shown a QTL effect also mediated the disadvantages described above, which is why "linkage drag" is also discussed in this context. At the same time, the QTL's and the effects connected therewith were not described uniformly in the respective prior art, and merely mediated a weak effect, such that the utilization of these results in the breeding of Cercospora-resistant plants was possible to only a limited extent, and was largely uncertain. Targeted breeding and controlled integration of the resistance gene into the gene pool of the sugar beet are now enabled by means of the identification of the resistance gene described herein. This ensures the breeding and generation of entirely new Cercospora-resistant cultivars that exhibit a high resistance to the pathogen, without negatively affecting the sugar yield.
[0250] The present invention likewise relates to a method for the identification, and possibly the provision, of a plant of the species Beta vulgaris that is resistant to the pathogen Cercospora, characterized in that the method includes a step of the detection of the presence and/or of the expression of a nucleic acid molecule according to the invention or of the polypeptide according to the invention in the plant or a sample/portion thereof. The presence and/or the expression of a nucleic acid molecule according to the invention, or of the polypeptide according to the invention, may be tested by means of standard methods known to the person skilled in the art, e.g., by means of PCR, RT-PCR, or western blot.
[0251] Furthermore, the identification method according to the invention also includes the detection of the nucleic acid molecule according to the invention by means of detection of at least one polymorphism between resistant and sensitive sequences, i.e., between the sequences of the nucleic acid molecule according to the invention and the sequences of the allelic variant of the nucleic acid molecule according to the invention that is described above, using molecular markers that detect one or more polymorphisms. As has already been described above, it is obvious to the person skilled in the art that numerous sensitive sequences exist, i.e., numerous sequences that encode the allelic variant of the nucleic acid molecule according to the invention. One of these is presented by way of example in the sequence comparison with the nucleotide sequence of the nucleic acid molecule according to the invention in FIG. 1. A preferred embodiment of the method according to the invention consequently includes the detection of at least one polymorphism that is presented in FIG. 1 using molecular markers which detect the polymorphisms--in particular, diagnostic polymorphisms. This detection preferably occurs using at least one molecular marker per polymorphism--in particular, per diagnostic polymorphism. It is known to the person skilled in the art which marker techniques are to be applied to detect a corresponding polymorphism, and how molecular markers for this are constructed (see Advances in Seed Science and Technology Vol. I, Vanangamudi et al., 2008). Furthermore, the present invention encompasses molecular markers which describe or detect a polymorphism according to FIG. 1, such as the use of a molecular marker for detection of a polymorphism according to FIG. 1. It is thereby also possible to use markers that do not differentiate between various polymorphisms, as long as the markers are able to detect such a polymorphism as it occurs in the nucleic acid molecule according to the invention, but is not contained the sensitive allelic variant.
[0252] Alternatively, or additionally, the identification method according to the invention includes a step of detecting at least one marker locus in the nucleotide sequence of the nucleic acid molecule according to the invention or in a cosegregating regions thereof. Preferably the cosegregating region is a genomic region in Beta vulgaris which cosegregates with the Cercospora resistance conferred by the polypeptide according to the present invention, or with the nucleic acid molecule according to the present invention, more preferably the cosegregating region comprises and is flanked by markers sxh0678s01 and s4p0264s01, by markers s4p4301s01 and s4p2271s01, by markers s4p4301s01 and s4p4293s01, or by markers s4p4301s01 and s4p4295s01. The detection may thereby take place via a method step in which at least one marker or at least one primer pair binds at the locus according to SEQ ID No. 74 or 75--preferably, at the locus according to SEQ ID No. 76 or 77--and, optionally as a result of this, a signal is generated, e.g., a fluorescence signal or a sequence amplificate. Thus, alternatively or additionally the cosegregating region may comprise a sequence according to SEQ ID NO 74 and/or 75, or SEQ ID NO: 76 and/or 77. Furthermore, the preceding identification methods also represent methods for selection of a plant which exhibits the resistance to Cercospora according to the invention. The method for selection includes a concluding step of selecting a resistant plant.
[0253] In this context, the present invention also includes the development or production of molecular markers that are suitable for detecting the aforementioned polymorphisms between the nucleic acid molecule according to the invention (resistant allele) and the sensitive allelic variant, wherein the markers are preferably suitable for detecting the polymorphisms presented in FIG. 1 or the construction of hybridization probes that specifically bind to the nucleotide sequence of the nucleic acid molecule according to the invention, or the production of a pair of nucleic acid molecules that is suitable for amplifying, in a PCR, a region that is specific to the nucleic acid molecule according to the invention, and thus for detecting these in a plant or plant cell.
[0254] The invention preferably includes a method for producing oligonucleotides of at least 15, 16, 17, 18, 19, or 20--preferably, at least 21, 22, 23, 24, or 25, particularly preferably, at least 30, 35, 40, 45, or 50, and, especially preferably, at least 100, 200, 300, 500 or 1,000--nucleotides in length that specifically hybridize with a nucleotide sequence of the nucleic acid molecule according to the invention or the nucleic acid molecule that is complementary thereto, or a pair of nucleic acid molecules--preferably, in the form of oligonucleotides--that is suitable for attachment as a forward and reverse primer to a region that is specific to the nucleic acid molecule according to the invention, and for amplifying this in a polymerase chain reaction (PCR), or that is suitable for hybridization as a forward and reverse primer to a region in the Beta vulgaris genome that, in Beta vulgaris, has a cosegregation with the Cercospora resistance conferred by the polypeptide according to the invention or with the nucleic acid molecule according to the invention. An example for suitable primers for the detection of a resistance-mediating nucleotide sequence according to the invention are given by SEQ ID NO 98 and SEQ ID NO 99. These two sequences build a primer pair which can be used in the PCR. The invention also includes a kit comprising oligonucleotides or molecular markers according to the invention.
[0255] The method for the production of oligonucleotides initially includes: the comparison of the nucleotide sequence of the nucleic acid molecule according to the invention with the nucleotide sequence of the corresponding nucleic acid molecule that does not confer resistance or of the sensitive allelic variant, which preferably has a nucleotide sequence according to SEQ ID No. 4 or 5; the identification of the sequence differences between the two nucleotide sequences; and the generation of nucleic acid molecules--here, meaning oligonucleotides--that specifically bind to the nucleic acid molecule according to the invention, but not to the nucleic acid molecule that does not mediate resistance.
[0256] Furthermore, the oligonucleotide according to the invention may be connected to a fluorescent dye in order to generate a fluorescence signal, e.g., under excitation via light of the corresponding wavelength. The fluorescent dye may be fluorochrome. The oligonucleotides according to the invention may be coupled with other compounds that are suitable for generating a signal. Such oligonucleotides do not occur in nature and also cannot be isolated from nature. The following is executed to produce such marked oligonucleotides: DNA may be marked bio-orthogonally. For this, DNA may be marked in vivo or in vitro with nucleoside analogs, which, for example, may subsequently be coupled with a fluorophore per Staudinger reaction. In addition to this, DNA may also be chemically provided with fluorophores. Oligonucleotides may be marked via a phosphoramidite synthesis with fluorophores that, for example, are used in QPCR, DNA sequencing, and in situ hybridization. Furthermore, DNA may be generated enzymatically in the course of a polymerase chain reaction with fluorescent nucleotides, or be marked with a ligase or a terminal deoxynucleotidyl transferase. DNA may also be detected indirectly via a biotinylation and fluorescent avidin. For couplings, fluorescein, fluorescent lanthanides, gold nanoparticles, carbon nanotubes, or quantum dots, among other things, are used as fluorophores. One of the most commonly used fluorescent substances is FAM (carboxyfluorescein). Consequently, oligonucleotides and, in particular, primers that possess a FAM marking are encompassed by the invention. FAM is preferably present as 6-FAM, wherein--depending upon the desired wavelength of the emission and excitation--other FAM variants, e.g., 5-FAM, may, however, also be used. Examples of additional fluorescence markers are AlexaFluor, ATTO, Dabcyl, HEX, Rox, TET, Texas Red, and Yakima Yellow. Depending upon the field of use, the oligonucleotides may be furnished with modifications of the bases or of the sugar phosphate spine. Among these are, among others, amino-dT, azide-dT, 2-aminopurine,5-Br-dC, 2'-deoxyinosine (INO), 3'-deoxy-A, C, G, 5-Met-dC, 5-OH-Met-dCN6-Met-dA, and others.
[0257] Furthermore, the present invention also relates to a marker chip ("DNA chip", "assay" or microarray) which contains at least one oligonucleotide according to the invention that is suitable for detection. The marker chip is suitable for application in one or more detection methods according to the invention.
[0258] The invention likewise includes a method for production of the protein according to the invention. The method includes the provision or cultivation of a cell culture which contains the SEQ ID No. 2, and the subsequent expression of the protein encoded by SEQ ID No. 2.
[0259] Furthermore, the present invention also relates to a Cercospora-resistant plant or a portion thereof which was identified, and, if applicable, selected, via a method as described in the preceding. In particular, the present invention relates to a population of plants comprising plants that are available according to one of the methods according to the invention as described in the preceding, and that preferably are resistant to Cercospora leaf spot disease or Cercospora infestation, and are characterized by the presence of a nucleic acid molecule according to the invention. The population preferably has at least 10--preferably, at least 50, more preferably, at least 100, particularly preferably, at least 500, and, particularly in agricultural farming, preferably at least 1,000--plants. The proportion of plants in the population that do not carry the nucleic acid molecule according to the invention and/or are susceptible to Cercospora leaf spot disease is preferably below 25%--preferably, below 20%, more preferably, below 15%, even more preferably, 10%, and, in particular, preferably below 5%, if present at all.
[0260] With the fine mapping described above, the position of the Cercospora resistance-conferring gene in the genome of Beta vulgaris subsp. maritima could be determined, and the gene itself and the surrounding sequence regions could be identified. This in turn represents the basis for the development of DNA hybridization probes or genetic markers in the target region, with the aid of which the Cercospora resistance-mediating gene could be detected, or could be differentiated from the gene that does not confer resistance.
[0261] DNA hybridization probes may be derived from the sequence of the Cercospora resistance-conferring gene and be used for the screening of genomic and/or cDNA banks of the desired organism. The probes may be used to amplify identified homologous genes via the known process ofpolymerase chain reaction (PCR), and to check whether the Cercospora resistance-conferring gene is present endogenously in an organism, or has been successfully introduced as heterologous genetic element.
[0262] The person skilled in the art may here resort to customary hybridization, cloning, and sequencing methods, which, for example, are listed in Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001. The person skilled in the art may also synthesize and use oligonucleotide primers to amplify sequences of the Cercospora resistance-conferring gene. In order to achieve a specific hybridization, such probes should be specific and have at least a length of 15 nucleotides--preferably, at least 20 nucleotides. A detailed guide to hybridization of nucleic acids may be found in Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology--Hybridization with Nucleic Acid Probes, Part 1, Chapter 2, "Overview of principles of hybridization and the strategy of nucleic acid probe assays." Elsevier, New York (1993); and in Current Protocols in Molecular Biology, Chapter 2, Ausubel et al., eds., Greene Publishing and Wiley lnterscience, New York (1995).
[0263] Therefore, a nucleic acid molecule of at least 15, 16, 17, 18, 19, or 20--preferably, at least 21, 22, 23, 24, or 25, particularly preferably, at least 30, 35, 40, 45, or 50, and, especially preferably, at least 100, 200, 300, 500, or 1,000--nucleotides in length is the subject matter of the present invention, wherein this nucleic acid molecule specifically hybridizes with a previously-described nucleotide sequence according to the invention that comprises the Cercospora resistance-conferring gene. This also explicitly encompasses the range of 15 to 35 nucleotides.
[0264] The present invention thus also relates to markers as oligonucleotides--in particular, primer oligonucleotides. These comprise a nucleic acid molecule of at least 15 nucleotides in length that specifically hybridizes with a nucleotide sequence defined as in the preceding.
[0265] In particular, the present invention encompasses a pair of nucleic acid molecules--preferably, in the form of oligonucleotides or a kit containing this pair of oligonucleotides--that is suitable for hybridization as a forward and reverse primer to a region that is specific to the nucleic acid molecule according to the invention, and for amplifying this in a polymerase chain reaction (PCR), or that is suitable as a forward and reverse primer for hybridization to a region in the Beta vulgaris genome that, in Beta vulgaris, exhibits a cosegregation with the Cercospora resistance conferred by the polypeptide according to the invention, or with the nucleic acid molecule according to the invention.
[0266] The following advantages for the breeding and development of new resistant plant lines of the genus Beta may also be achieved via the present invention. Sequence information, as well as the identified polymorphisms which allow a differentiation between resistant and susceptible alleles of the disclosed gene, i.e., between the alleles that confer a Cercospora resistance and the alleles that are not capable of conferring this resistance, make possible the marker development directly in the gene, as described above, as well as in the regions situated upstream and downstream, which represents an important facilitation for the plant breeder--in particular, with regard to the development of optimized elite lines without "linkage drag." Moreover, knowledge about the sequential structure may be used for the identification of additional resistance genes--in particular, against Cercospora--which are homologous or orthologous, for example.
[0267] Therefore, the present invention also encompasses a method for the identification of additional nucleic acid molecules encoding polypeptides or additional proteins that are able to confer a resistance to Cercospora in a plant in which the polypeptide is expressed. The person skilled in the art may thereby use databases, employing suitable search profiles and computer programs for the screening for homologous sequences or for sequence comparisons. Moreover, by means of conventional molecular biology techniques, the person skilled in the art may himself derive additional DNA sequences encoding Cercospora resistance proteins, and use these within the scope of the present invention. For example, suitable hybridization probes may be derived from the sequence of the nucleic acid molecule according to the invention and be used for the screening of genomic and/or cDNA banks of the desired organism. The person skilled in the art may here resort to customary hybridization, cloning, and sequencing methods, which, for example, are listed in Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001. Using known sequences, the person skilled in the art may also synthesize and use oligonucleotide primers to amplify sequences of Cercospora resistance-conferring nucleic acid molecules.
[0268] In one embodiment, the present invention therefore encompasses a method for the identification of a nucleic acid molecule which encodes a polypeptide that is able to confer a resistance to Cercospora in a plant of the species Beta vulgaris in which the polypeptide is expressed. The method thereby includes the comparison of the amino acid sequence of the polypeptide according to the invention which, in Beta vulgaris subsp. vulgaris, confers a Cercospora resistance with amino acid sequences from a sequence database, or with sequences of allelic variants of the polypeptide according to the invention in genotypes of the species Beta vulgaris. Furthermore, the method according to the invention includes the identification of an amino acid sequence or of an allelic variant that is at least 80% identical to the amino acid sequence of the polypeptide according to the invention, as well as the introduction of a nucleic acid molecule encoding the identified amino acid sequence or allelic variant in a plant of the species Beta vulgaris; expression of the nucleic acid molecule in the plant; and, optionally, subsequent verification of the resistance to Cercospora.
[0269] As described in the preceding, additional Cercospora resistance-conferring proteins or their coding genes, i.e., homologs, analogs, and orthologs, that are at least 70%--preferably, at least 80%, particularly preferably, at least 90%, especially preferably, at least 95%, or even 98%--identical to the amino acid sequence of the polypeptide which is encoded by the nucleic acid molecule according to the invention may be identified via classical bioinformatic approaches (database searches and computer programs for screening for homologous sequences).
[0270] The term, homolog(s), thereby means that the genes concerned (from two different plant species) have essentially the same function and a common ancestor, and therefore typically show a significant identity in their nucleic acid or coded amino acid sequences. However, there are also many genes that are homologous to one another, without protein sequences resulting in a meaningful paired alignment. In contrast to this, the term, analog(s), describes genes or proteins that (likewise) have an identical or similar function, but are not created from the same structure, i.e., have no common ancestor. In this case, often, no significant identity can be established in their nucleic acid or encoded amino acid sequence, or, in the best case, in specific functional domains.
[0271] In the context of genome sequencing, homologs are, for annotation, more finely classified. The terms, orthology and paralogy, have been introduced for this. Orthologs are genes that are connected via a speciation event. Paralogs are genes that trace back to a duplication event.
[0272] A gene is, then, fundamentally a homolog or analog or ortholog in the sense of the present invention if it is able to confer Cercospora resistance in a plant. To check, methods, which have already been described in the preceding, known to the person skilled in the art are used, e.g., the amplification of the identified homolog or analog or ortholog by means of PCR, cloning in expression vectors, introduction into the target plant or plant cell, and checking the resistance.
[0273] As described above, the usage disclosed here of the resistant gene allele in cis- or transgenic approaches opens up the possibility of new resistant species of the genus Beta which, using the dose effect, exhibit a higher resistance, or in which a resistance break may be avoided and the resistance development optimized via the stacking of the disclosed gene with other resistance genes. Modifications of the gene by means of tilling or targeted engineering to optimize the codon selection for an increased expression or for the development of new or modified resistance alleles are also possible. According to a preferred embodiment the codon-optimized sequences or the modified resistance alleles are not occurring in nature but are artificial. An example of a modified genomic sequence is provided by SEQ ID No. 94 in which the codon at position 16--18 is modified but the encoded amino acid sequence is unchanged and corresponds to SEQ ID No. 3. An example of a modified cDNA sequence is provided by SEQ ID No. 95 in which the codon at position 55-57 is modified but the encoded amino acid sequence is unchanged and corresponds to SEQ ID No. 3. SEQ ID No. 94 and SEQ ID No. 95 are also examples for hybridizing sequences. An example of a modified resistance conferring allele is given by the amino acid sequence according to SEQ ID No. 96 in which the amino acid valine has been replaced with the amino acid leucine at position 209. The amino acid sequence according to SEQ ID No. 96 is encoded by the modified cDNA according to SEQ ID No. 97. These sequences do not occur in nature but are artificial. When replacing amino acids in for example the resistance-mediating Sequence according to SEQ ID No. 3 it is recommended to exchange amino acids within the following groups:
a) glycine, alanine, valine, leucine, isoleucine b) serine, cysteine, selenocysteine, threonine, methionine c) phenylalanine, tyrosine, tryptophan d) histidine, lysine, arginine e) aspartate, glutamate, asparagine, glutamine.
[0274] The present invention also relates to the use in a plant of the identified Cercospora resistance-conferring gene allele in a genetic or molecular stack with other genetic elements which may confer agronomically advantageous properties. The economic value of cultivated plants may thereby be markedly increased, in that, for example, the yield performance is increased in comparison to plants that possess the same genetics, but have not been furnished with the nucleic acid according to the invention. Furthermore, new crop areas for a plant may be opened up that were not previously accessible to the cultivation of this plant due to biotic factors such as strong pathogen pressure. In particular, the present invention relates to the use of the identified Cercospora resistance-conferring gene allele in methods for controlling an infestation with the pathogen Cercospora beticola in the agricultural or horticultural cultivation of plants of the genus Beta, e.g., encompassing the identification and selection of plants of the genus Beta with the aid of one of the methods described in the preceding and/or the cultivation of the plants so selected or descendants thereof. The present invention thus includes a method for the cultivation of plants of the species Beta vulgaris, including, in a first step, the provision of Cercospora-resistant plants of the species Beta vulgaris according to the invention, or the production of plants of the species Beta vulgaris with the aid of the production method according to the invention, or the identification and selection of plants of the species Beta vulgaris with the aid of the identification method according to the invention that has been described in the preceding; and including, in a second step, the cultivation of the plants from the first step, or the deployment of seed stock of the plants from the first step, or the raising of plants from the first step. The cultivation method thereby counteracts an infestation of the cultivated plants by Cercospora. The cultivation method may be part of a method for producing sugar. The method for the production of sugar includes the steps of the cultivation method, and additionally, as a penultimate step, the harvesting of the cultivated plants, and, as a last step, the extraction of sugar from the aforesaid plants.
[0275] The cultivation method may also be part of a method for producing seed stock. The method for the production of seed stock includes the steps of the cultivation method, and additionally, as a penultimate step, the vernalization of the cultivated plants, and, as a last step, the extraction of seeds from the aforesaid plants.
[0276] The extracted seeds may optionally be pelleted, in order to obtain pelleted seed stock of the species Beta vulgaris. In this instance, it is a method for the production of pelleted seed stock.
[0277] Moreover, the method for the production of seed stock may be designed as a method for the production of Cercospora-resistant seed stock. The method for the production of Cercospora-resistant seed stock includes the steps of the method described above for the production of seed stock, and additionally, as a last step, the verification of the nucleic acid according to the invention according to a method described herein in at least one of the extracted seeds--preferably, in at least 0.1% or in at least 1% of the extracted seeds. The verification is particularly preferably implemented so that the seed remains germinable. This means that the extraction of the DNA required for verification from the seed does not neutralize the germinability of the seed. In such an instance, the verification of the nucleic acid according to the invention may have taken place in an especially large proportion of all extracted seeds. For example, the verification may take place in at least 2%--preferably, at least 3%, particularly preferably, at least 4%--of all extracted seeds.
[0278] The plants according to the invention, their cells, or seeds or seed stock according to the invention may possess additional, agronomically advantageous properties, or be furnished with such. One example is the tolerance or resistance to an herbicide such as glyphosate, glufosinate, or ALS inhibitors. The tolerance to glyphosate or an ALS-inhibitor herbicide is preferred. A specific embodiment of the glyphosate resistance is disclosed in U.S. Pat. No. 7,335,816 B2. Such a glyphosate resistance is, for example, available from seed stock stored at the NCIMB, Aberdeen (Scotland, UK), under the access number, NCIMB 41158 or NCIMB 41159. Such seeds may be used in order to obtain a glyphosate-tolerant sugar beet plant. The glyphosate resistance may also be introduced into other species of the genus Beta via crossing.
[0279] The invention thus also encompasses plants, their cells, or seeds or seed stock, characterized in that these contain the nucleic acid according to the invention, and furthermore in that
a) a DNA fragment of the genomic DNA of the plant, portions, or seeds thereof may be amplified via polymerase chain reaction with a first primer that has the nucleotide sequence of SEQ ID No. 81, and a second primer that has the nucleotide sequence of SEQ ID No. 82, wherein the DNA fragment is at least 95%--preferably, 100%--identical to the nucleotide sequence of SEQ ID No. 83, and/or b) a DNA fragment of the genomic DNA of the plant, portions, or seeds thereof may be amplified via polymerase chain reaction with a first primer that has the nucleotide sequence of SEQ ID No. 84, and a second primer that has the nucleotide sequence of SEQ ID No. 85, wherein the DNA fragment is at least 95% identical--preferably, 100% identical--to the nucleotide sequence of SEQ ID No. 86, and/or c) a DNA fragment of the genomic DNA of the plant, portions, or seeds thereof may be amplified via polymerase chain reaction with a first primer that has the nucleotide sequence of SEQ ID No. 87, and a second primer that has the nucleotide sequence of SEQ ID No. 88, wherein the DNA fragment is at least 95% identical--preferably, 100% identical--to the nucleotide sequence of SEQ ID No. 89.
[0280] A specific embodiment of the ALS-inhibitor herbicide resistance is disclosed in the document, WO2012/049268 A1. For example, such an ALS-inhibitor herbicide resistance is available from a deposit of NCIMB, Aberdeen, UK, under the number NCIMB 41705. Furthermore, such an ALS-inhibitor resistance may be produced via tilling or site-directed mutagenesis, e.g., via gene editing, such as through the use of CRISPR/Cas, CRISPR/Cpf1, TALENS or zinc finger nucleases. The invention thus also encompasses plants, their cells, or seeds or seed stock, characterized in that these contain the nucleic acid according to the invention, and furthermore in that these exhibit a mutation in an endogenous acetolactate synthase gene, wherein the acetolactate synthase gene encodes an acetolactate synthase protein which, as a result of the mutation at position 569, has a different amino acid than tryptophan. As a result of the mutation, the amino acid at position 569 is preferably alanine, glycine, isoleucine, leucine, methionine, phenylalanine, proline, valine, or arginine. Position 569 is preferably defined via the position 569 of SEQ ID No. 90. Furthermore, the specific sequence of the mutated acetolactate synthase gene SEQ ID No. 91 is preferred. The mutated sequence of the acetolactate synthase gene, or the sequence according to SEQ ID No. 91, does not occur in nature and cannot be isolated from nature. Furthermore, the mutation may be present both heterozygously and homozygously in the plants, their cells or seeds, or the seed stock.
[0281] We recommend the homozygous presence of the mutation, since this promotes a more stable or more intensive phenotypical occurrence of the resistance.
[0282] Numerous additional herbicides and their applicability are known to the person skilled in the art from the prior art. He may resort to the prior art in order to achieve knowledge of which genetic elements are to be used in what manner in order to implement a corresponding tolerance in plants.
[0283] Moreover, an herbicide tolerance has the synergistic effect that the occurrence of weeds is reduced via the use of herbicides. This is advantageous in combating Cercospora, because it is known that the conidia (asexual spores) or the pseudostroma (mycelium) of Cercospora beticola can survive for up to 2 years on plant material.
[0284] A further example of an agronomically advantageous property is an additional pathogen resistance, wherein pathogens may be insects, viruses, nematodes, bacteria, or fungi, for example. For example, a broad pathogen defense for a plant may be achieved via combination of different pathogen resistances/tolerances, since genetic elements may exhibit additive effects among one another. For example, numerous resistance genes for this are known to the person skilled in the art as genetic elements. For example, US20160152999A1 discloses an RZ resistance gene against the disease Rhizomania. This disease is caused by the agent, "Beet Necrotic Yellow Vein Virus." Several disease resistances contained in one plant have synergistic effects upon one another. If a plant is infested for the first time by a pathogen, its immune system is normally weakened, and the epidermis as an outer barrier is often damaged, such that the probability of further infections is increased. An additional example of an agronomically advantageous property is cold tolerance or frost tolerance. Plants which exhibit this property may already be sown earlier in the year, or may remain in the field longer, which may lead to increased yields, for example. Here, the person skilled in the art may also resort to the prior art to find suitable genetic elements. Additional examples of agronomically advantageous properties are water usage efficiency, nitrogen usage efficiency, and yield. Genetic elements which may be used to confer such properties might be found in the prior art.
[0285] Furthermore, numerous modifications for pathogen defense are known to the person skilled in the art. In addition to the families of the R-genes that are often described, the Avr/R approach, the Avr gene complementation (WO 2013/127379), the autoactivation of an R-gene (WO 2006/128444), or the HIGS (host-induced gene silencing) approach (e.g., WO2013/050024) may be advantageously used. In particular, the autoactivation of an R-gene might be important to the present invention. For this, a nucleic acid is to be created that encodes an autoactivated resistance protein for generation of a resistance to pathogens in plants. This nucleic acid then has only a limited portion of an NBS-LRR resistance gene, such as the wb-R-gene, which extends downstream from the 5' end of the coding region of the NBS-LRR resistance gene to the beginning of the coding for the NBS domain of the NBS-LRR resistance gene.
[0286] In this context, a method is also encompassed which contains the step of the removal of that region of the nucleic acid according to the invention which encodes the N-terminal region and which begins with the p-loop in the NBS domain, and extends up to the end of the N-terminal region.
[0287] The resistance proteins that are encoded for by such shortened nucleic acids are generally autoactivated, in that these resistance proteins trigger an immune reaction in the plant, even in the absence of the associated pathogen, and thus increase the base immunity of the plant. Furthermore, such a shortened nucleic acid according to the invention, and the polypeptide that is encoded by this, are encompassed.
[0288] Furthermore, the invention also includes the use of the Cercospora resistance-conferring gene allele, identified with a method described above, for combination with one of the preceding modifications, or with a genetic element described in the preceding which may convey in a plant one or more agronomically advantageous properties.
[0289] In addition to relating to the plant according to the invention, the present invention also relates to seeds or descendants, or to an organ, a plant part, a tissue, or a cell thereof in the production of products that are typically produced from sustainable raw materials, such as foodstuffs and animal feed--preferably, sugar or syrup (molasses), wherein the molasses is also used for industrial applications, e.g., in alcohol production or as a growing medium for the production of biotechnological products, in the production of materials or substances for the chemical industry, e.g., refined chemicals, pharmaceuticals or precursors thereof, diagnostics, cosmetics, bioethanol, or biogas. An example of the use of sugar beet as a biogenic raw material in biogas plants is described in the application DE 10 2012 022 178 A1; see, for example, paragraph 10.
[0290] The following examples explain the invention, but without limiting the subject matter of the invention. Unless indicated otherwise, standard molecular biology methods have been used; see, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001, Fritsch et al., Cold Spring Harbor Laboratory Press: 1989; Mayer et al., Immunochemical Methods in Cell and Molecular Biology, eds., Academic Press, London, 1987, and Weir et al., Handbook of Experimental Immunology, Volumes I-IV, Blackwell, eds., 1986.
[0291] Some of the most important sequences according to the invention are explained in detail in the following:
[0292] SEQ ID No. 1: genomic DNA sequence of the Cercospora resistance-conferring gene from Beta vulgaris subsp. maritima.
[0293] SEQ ID No. 2: cDNA sequence of the Cercospora resistance-conferring gene as it does not occur in nature.
[0294] SEQ ID No. 3: amino acid sequence of the Cercospora resistance-conferring protein as it is encoded by SEQ ID No. 1 or SEQ ID No. 2.
[0295] SEQ ID No. 4: genomic DNA sequence of the sensitive Variant of the Cercospora resistance-conferring gene
[0296] SEQ ID No. 5: cDNA of the sensitive Variant of the Cercospora resistance-conferring gene
[0297] SEQ ID No. 6: Amino acid sequence of the sensitive Variant of the Cercospora resistance-conferring gene
[0298] SEQ ID No. 7: native promoter of the Cercospora resistance-conferring gene from Beta vulgaris subsp. maritima.
[0299] SEQ ID No. 8: native terminator of the Cercospora resistance-conferring gene from Beta vulgaris subsp. maritima.
[0300] SEQ ID No. 53: sequence of the locus from Beta vulgaris subsp. maritima containing the Cercospora resistance-conferring gene according to SEQ ID No. 1.
EXAMPLES
[0301] The present invention is also described and demonstrated by way of the following examples. However, the use of these and other examples anywhere in the specification is illustrative only and in no way limits the scope and meaning of the invention or of any exemplified term. Likewise, the invention is not limited to any particular preferred embodiments described here. Indeed, many modifications and variations of the invention may be apparent to those skilled in the art upon reading this specification, and such variations can be made without departing from the invention in spirit or in scope. The invention is therefore to be limited only by the terms of the appended claims along with the full scope of equivalents to which those claims are entitled.
Example 1: Introduction of the Resistance-Conferring Gene by Means of CRISPR-Mediated Homologous Recombination in Beta vulgaris Subsp. Vulgaris
[0302] Design and Selection of the crRNA:
[0303] Suitable crRNA's for Cpf1-mediated induction of double-strand breaks have been designed with the aid of CRISPR RGEN Tools (Park J., Bae S., and Kim J.-S. Cas-Designer: A web-based tool for choice of CRISPR-Cas9 target sites. Bioinformatics 31, 4014-4016 (2015); Bae S., Park J., and Kim J.-S. Cas-OFFinder: A fast and versatile algorithm that searches for potential off-target sites of Cas9 RNA-guided endonucleases. Bioinformatics 30, 1473-1475 (2014)). For this, suitable protospacers were sought within the genomic DNA sequences having a length of 500-1,300 bp which flank the 5'- and 3'-end of the Cercospora resistance gene from Beta vulgaris subsp. maritima. In order to ensure the functionality of the endonuclease Cpf1 from Lachnospiraceae bacterium ND2006 (Lb), protospacers having a length of 24 nt were selected whose genomic binding sequence at the 5'-end was flanked by an essential protospacer adjacent motif (PAM) having the sequence 5'-TTTV-3' (V=G or C or A). Suitable protospacers were selected according to the predetermined quality criteria of the tool and reconciled as to potential off-targets with a reference genome of B. vulgaris subsp. vulgaris. For the continuing tests, crRNA's, exclusively, were selected that, in addition to the actual target sequence, have at most 15 identical bases with a functional PAM. Since the first 18 nt of the protospacer are essential to the detection and cutting of the target sequence, an unwanted cutting within other genomic sequences could be precluded in this way (Tang, X., L. G. Lowder, T. Zhang, A. A. Malzahn, X. Zheng, D. F. Voytas, Z. Zhong, Y. Chen, Q. Ren, Q. Li, E. R. Kirkland, Y. Zhang, and Y. Qi (2017), "A CRISPR-Cpf1 system for efficient genome editing and transcriptional repression in plants." Nat Plants 3: 17018). In this way, four potential crRNA's at the 5'-flanking region (5'crRNA #1-4) and three crRNA's at the 3'-flanking region (3'crRNA #1-3) of the resistance gene could be identified (see Table A).
TABLE-US-00003 TABLE A Selected target sequences within the 5'- and 3'- flanking DNA sequences of the resistance gene in B. vulgaris. The PAM is underlined. Binding at Name of Genomic target sequence with the +/- the crRNA 5'-flanking PAM (underlined) strand 5'crRNA#1 TTTATTTCGATTTCGATTCTTGGATTAT - (SEQ ID No. 16) 5'crRNA#2 TTTCAACCCAGTATCCTTATCCGTCACT - (SEQ ID No. 17) 5'crRNA#3 TTTATTTAAACATGATACGTATCATATT + (SEQ ID No. 18) 5'crRNA#4 TTTAAACATGATACGTATCATATTGAGT + (SEQ ID No. 19) 3'crRNA#1 TTTGTGGGTGGGTGGTTTTCACGTGTGT - (SEQ ID No. 20) 3'crRNA#2 TTTCCCCTCCCTTTGCCGCTGCGAAGTT - (SEQ ID No. 21) 3'crRNA#3 TTTCTTCTTCTTGCTTCCACCATAACAC - (SEQ ID No. 22)
[0304] Cloning of the genetic elements: For the cloning of the cpf1-expression cassette and the crRNA-expression cassette, first, a detection sequence of the restriction enzyme of BbsI that prevents cloning was removed from the target vector pZFNnptII via introduction of a point mutation (T to G). The mutagenesis was performed with a mutagenesis kit according to the specification of the manufacturer, using two mutagenesis primers (see Table B).
TABLE-US-00004 TABLE B Mutagenesis primer used to introduce a point muta- tion (T to G, underlined) to remove the BbsI detection sequence. Name Sequence 5'.fwdarw.3' Mutagenesis primer 1 TCAGTGCAGCCGTCGTCTGAAAACGACA (SEQ ID No. 23) Mutagenesis primer 2 TGTCGTTTTCAGACGACGGCTGCACTGA (SEQ ID No. 24)
[0305] For the expression of the Lbcpf1 gene in B. vulgaris, a DNA sequence codon-optimized for A. thaliana, with 5'-flanking PcUbi promoter sequence from Petroselinum crispum (SEQ ID No. 79) and a 3'-flanking 3A terminator sequence from Pea sp. as a DNA fragment, was synthetically produced. The restriction interface (HindIII) that is relevant to cloning within the Lbcpf1-coding sequence (CDS) [SEQ ID No. 78] were removed via the introduction of a silent mutation (base exchange, without modifying the amino acid sequence), in order to avoid an unintended cutting within the coding region. The codon optimization was performed with the aid of the GeneArt algorithm from Invitrogene/ThermoScientific. In order to enable the transport of the Cpf1 in the cell nucleus, the coding sequence of the nucleus location signal (NLS) of the SV40 was integrated into the cpf1 CDS at the 5'-end, and the NLS of the nucleoplasmin was integrated at the 3'-end. For the ligation in the binary target vector pZFNnptII (FIG. 2), the expression cassette was flanked by two HindIII restriction interfaces and subsequently ligated to pZFNnptlI_LbCpf1. The successful insertion of the PcUbi::Cpf1::TPea expression cassette was verified by means of sequencing, wherein the binding regions of the primers used for the sequencing were situated both in the flanking vector regions and within the expression cassette (see Table C).
TABLE-US-00005 TABLE C Primer used for the sequencing of the PcUbi::Cpf1::TPea expression cassette integrated into pZFNnptII Name Sequence 5'.fwdarw.3' pSeq_CRBM_F1 SEQ ID No. 25 pSeq_CRBM_R1 SEQ ID No. 26 pSeq_CRBM_F2 SEQ ID No. 27 pSeq_CRBM_R2 SEQ ID No. 28 pSeq_CRBM_F3 SEQ ID No. 29 pSeq_CRBM_R3 SEQ ID No. 30 pSeq_CRBM_F4 SEQ ID No. 31 pSeq_CRBM_R4 (SEQ ID No. 32)
[0306] After transcription into the plant cell, the crRNA's should be cut out via two flanking ribozymes. For this, the precursor crRNA was flanked by the coding sequences of a hammerhead ribozyme and an HDV ribozyme (Tang, X., L. G. Lowder, T. Zhang, A. A. Malzahn, X. Zheng, D. F. Voytas, Z. Zhong, Y. Chen, Q. Ren, Q. Li, E. R. Kirkland, Y. Zhang, and Y. Qi (2017), "A CRISPR-Cpf1 system for efficient genome editing and transcriptional repression in plants." Nat Plants 3: 17018).
[0307] For a perfect ligation of the individual protospacers at the coding sequence of the crRNA repeat, two BbsI detection sequences were integrated between crRNA repeat and HDV ribozyme, wherein the overhangs that were used for the cloning were adapted accordingly. In order to ensure an identical expression strength of the cpf1 and the crRNA's, the crRNA ribozyme cassette was bounded, at the 5'-end, by the PcUbi promoter sequence and, at the 3'-end, by [a/the] 3A terminator sequence. For the later ligation in the target vector pZFNnptII_Cpf1, the crRNA expression cassette was flanked by two PstI interfaces and ordered as a synthetic DNA fragment. The protospacers were synthesized as complementary oligonucleotides and annealed according to a standard protocol. The 24-bp-long DNA fragment that was generated in this way was flanked by the 4-nt overhangs that are relevant to the ligation (see Table D).
TABLE-US-00006 TABLE D Sequence of oligonucleotides that were used for the generation of short 24-bp protospacers. The 4-nt overhangs that are used for the ligation are the respective four first nucleotides of each listed sequence. Name of the crRNA Sequence 5'.fwdarw.3' 5'crRNA#1 SEQ ID No. 33 SEQ ID No. 34 5'crRNA#2 SEQ ID No. 35 SEQ ID No. 36 5'crRNA#3 SEQ ID No. 37 SEQ ID No. 38 5'crRNA#4 SEQ ID No. 39 SEQ ID No. 40 3'crRNA#1 SEQ ID No. 41 SEQ ID No. 42 3'crRNA#2 SEQ ID No. 43 SEQ ID No. 44 3'crRNA#3 SEQ ID No. 45 SEQ ID No. 46
[0308] The efficiency of the four crRNA's was tested by means of agrobacteria-mediated gene transfer in leaves of B. vulgaris. The pZFNtDTnptII plasmid was co-transformed in order to check the transformation efficiency. The transformation of the leaf explant took place via vacuum infiltration according to a standard protocol. The fluorescence of the tDT was checked after six days by means of fluorescence microscopy, and leaf explants with heterogeneous fluorescence were discarded. Ten days after infiltration took place, the leaf explants were quick-frozen in liquid nitrogen, pestled, and the genomic DNA was isolated by means of the CTAB method (Clarke, Joseph D., "Cetyltrimethyl ammonium bromide (CTAB) DNA miniprep for plant DNA isolation." Cold Spring Harbor Protocols 2009.3 (2009): pdb-prot5177). The efficiency of the individual crRNA's was determined by an external service provider, using the frequency of the inserted editions (e.g., insertions, deletions, or base exchange) in comparison to unedited sequences in the genomic DNA, by means of NGS.
[0309] As a synthetic DNA construct, the most efficient crRNA's-5'crRNA #3 and 3'crRNA #1--with the previously described ribozymes, promoter, and terminator sequences, were ordered as reverse-oriented expression cassettes. The entire DNA construct was flanked by two PstI restriction interfaces for cloning in the target vector pZFNnptII_LbCpf1. After insertion of the crRNA's has taken place, the LbCpf1 and crRNA expression cassettes were ligated from the vector pZFNnptII_LbCpf1_crRNA into the pUbitDTnptII vector via HindIII.
[0310] As a repair template which should be integrated into the genome of B. vulgaris via homologous recombination, the resistance gene expression cassette was flanked, at the 5'-end, by the 5'crRNA #3 and, at the 3'-end, by the 3'crRNA #1 binding sequence. This enabled the excision of the resistance gene expression cassette from the plasmid via Cpf1. The entire DNA template was synthesized as an 87,326-bp-long synthetic DNA fragment (SEQ ID No. 80) and used directly in the vector backbone for the transformation. The resistance gene plasmid and the pUbitDTnptII_ILbCpf1_crRNA plasmid were introduced into B. vulgaris callus cultures with the aid of a gene cannon.
[0311] The transformation efficiency was determined using the transient tDT fluorescence, one day after the transformation, by means of fluorescence microscopy. The callus cultures were cultivated on shoot induction medium without selection pressure (without Kanamycin), and the regenerated shoots were subsequently checked for the site-directed integration of the resistance-conferring resistance gene cassette. For this, the genomic DNA was isolated by means of CTAB. The integration of the resistance-conferring gene was amplified by means of PCR using the primers pCRBM_F1 according to SEQ ID No. 47 and pCRBM_R1 according to SEQ ID No. 48 (see Table E), and the PCR products were subsequently sequenced with both primers. Shoots, in which the successful insertion of the expression cassette could be verified in this manner, were identified in the following analyses of the integration site of the resistance gene. In order to verify the insertion within the desired target sequence in the genome, the flanking regions of the resistance gene expression cassette were amplified by means of PCR. The binding of a primer here took place within the resistance gene DNA sequence; the binding of the second primer took place outside of the 5'- or 3'-flanking homologous region of the inserted expression cassette (see Table E). The amplified DNA sequences were sequenced using the same primers, and the integration at the desired location was confirmed in this way. In order to preclude the binding of the primers pCRBM_F1 (SEQ ID No. 47), pCRBM_R1 (SEQ ID No. 48), pCRBM_R2 (SEQ ID No. 50) and pCRBM_F3 (SEQ ID No. 51) in sequence-similar regions of the genome, all primer sequences were compared beforehand with the B. vulgaris genome. For the primer pCRBM_F3 (SEQ ID No. 51), it was not possible to select the nucleotide sequence such that a binding to the wild-type sequence could be precluded. Therefore, the 3'-flanking region was amplified in all shoots that tested positive for the resistance gene, and the site-specific insertion was verified exclusively via the subsequent sequencing. The generated PCR product thereby differs by 18 bp from the wild-type sequence. In order to enable the complete sequencing of the amplified sequences, the PCR products were additionally sequenced via a third primer with a binding location within the amplified sequence (pCRBM_S2, pCRBM_S3; see Table E). In order to preclude the nonspecific binding of the primers pCRBM_F1 (SEQ ID No. 47), pCRBM_R1 (SEQ ID No. 48) and pCRBM_R2 (SEQ ID No. 50) within the wild-type genome, the nucleotide sequences were compared with an internal reference genome of B. vulgaris. The primers were additionally tested by means of PCR for the binding in genomic sequences of B. vulgaris wild-type plants.
[0312] In order to preclude the integration of the resistance gene in other regions of the genome, a targeted amplification of the target location was performed (Targeted Locus Amplification, TLA).
TABLE-US-00007 TABLE E Primer used to verify the insertion of the resistance gene expression cassette at the desired integration site. Size of the Name Sequence 5'.fwdarw.3' PCR product Binding pCRBM_F1 SEQ ID No. 47 450 bp within the resistance gene expression cassette pCRBM_R1 SEQ ID No. 48 within the resistance gene expression cassette pCRBM_F2 SEQ ID No. 49 1,140 bp up-strand of the 5'- flanking homologous region pCRBM_R2 SEQ ID No. 50 within the resistance gene promoter sequence pCRBM_S2 SEQ ID No. 66 pCRBM_F3 SEQ ID No. 51 1,280 within the resistance gene terminator sequence pCRBM_R3 SEQ ID No. 52 down-strand of the 3'- flanking homologous region pCRBM_S3 SEQ ID No. 67
[0313] In addition to the verification and the successful insertion of the resistance gene expression cassette into the genome of B. vulgaris, the unwanted integration of plasmid DNA was also checked. For this, genomic DNA, in which the verification had already yielded a successful insertion of the resistance gene at the desired target site, was checked for the presence of plasmid DNA by means of PCR. Sequence regions within the cpf1, the crRNA ribozyme cassette, and the tDT were thereby amplified using the primers listed in Table F, and subsequently sequenced.
TABLE-US-00008 TABLE F Primers used to verify stably-integrated, plasmid-specific sequences in the genome of the regenerated B. vulgaris shoots Size of the Name Sequence 5'.fwdarw.3' PCR product Binding pSeq_LbCpf1_F4 SEQ ID No. 68 214 Cpf1 pSeq_LbCpf1_R3 SEQ ID No. 69 pSeq_Ribozyme_F SEQ ID No. 70 172 crRNA ribozyme pSeq_Ribozyme_R SEQ ID No. 71 cassette pSeq_tDT_F SEQ ID No. 72 400 tDT pSeq_tDT_R SEQ ID No. 73
Example 2: Introduction of the Resistance-Conferring Gene as a Transgene by Means of Gene Transformation in Beta vulgaris Subsp. Vulgaris
[0314] The transgenic approach to the production of Cercospora-resistant plants served not only for the alternative validation of the LRR gene as the resistance-conferring gene, but also as a means of producing transgenic resistance events that confers a novel Cercospora resistance or improve already existing Cercospora resistances.
[0315] The binary vector pZFN-nptII-LRR was generated by means of the following standard cloning procedures: Within the T-DNA of this vector, the cDNA of the resistance gene according to SEQ ID NO 2 was cloned together with its native promoter sequence. The T-DNA furthermore included the neomycin phosphotransferase II (nptII) gene, which confers resistance to a bandwidth of aminoglycoside antibiotics such as kanamycin or paromomycin. These antibiotic resistances were used for the selection of the transgenic plant cells and tissues. The NOS promoter and the pAG7 terminator flanked the nptII gene. The backbone of the binary vector furthermore contained the colEl and the pVSJ origin for the plasmid replication in Escherichia coli or Agrobacterium tumefaciens. The aadA gene confers streptomycin/spectinomycin resistance for bacteria selection. The pZFN-nptII-LRR plasmid was transformed in agrobacterium strain AGL-1 by means of standard procedure.
[0316] The transformation of the sugar beets took place according to Lindsey & Gallois (1990), "Transformation of sugarbeet (Beta vulgaris) by Agrobacterium tumefaciens." Journal of experimental botany 41.5, 529-536.). For this, "micropropagated shoots" of genotype 04E05B1DH5, which did not carry the resistance gene according to the invention, were used as starting material. Shoots were multiplied in the corresponding medium according to Lindsey & Gallois (1990). In order to induce as many meristems as possible, the "shoots" were transferred into a different medium (see Lindsey & Gallois (1990)) and incubated in darkness for several weeks at approximately 30.degree. C. Agrobacterium strain AGL-1 with vector pZFN-nptII-LRR (FIG. 3) was cultured in an additional medium (see Lindsey & Gallois (1990)), additionally provided with corresponding antibiotics for selection. Sections of meristematic tissue based upon the shoot to be treated were incubated with agrobacterium for several hours in an additional medium (see Lindsey & Gallois (1990)). Plant explants and agrobacteria were co-cultivated in darkness for at least 2 days in medium (see Lindsey & Gallois (1990)), and inoculated explants were subsequently incubated in darkness for approximately 2 weeks in an additional medium (see Lindsey & Gallois (1990)). The explants were thereupon further propagated in an additional medium (see Lindsey & Gallois (1990)) and sub-cultivated, in order to enable the selection of the transgenic tissue. In order to conclude the selection phase and to reduce the extent of chimera formation, green "shoots" were transferred to medium H, and all were propagated for 2 weeks. Leaf material was then extracted from the green, growing "shoots" and examined by means of PCT for the presence of the transgene. Suitable "shoots" were rooted in medium I and subsequently transferred to a greenhouse for production of T1 seed stock. Furthermore, leaf material derived from these "shoots" was used to analyse the expression of the transformed resistance gene.
Analysis of the Expression Level
[0317] RNA was isolated from the leafes of the in vitro "shoots" and used within an qRT-PCR. The qRT-PCR was performed according to Weltmeier et al. 2011 (s. background of invention). Measured values were normalized against the reference gene PLT3_075_F09 (s. Weltmeier et al. 2011). The expression was determined by the use of the following primer sequences:
TABLE-US-00009 Size [No. Size of amplification Sequence nucleotides] T.sub.m [C. .degree.] product [No. nucleotides] SEQ ID No. 92 21 59.8 170 SEQ ID No. 93 21 58.9 170
Resistance Test in Sugar Beet after Inoculation with Cercospora beticola Under Greenhouse Conditions:
[0318] A pure Cercospora beticola culture with a known high virulence was propagated on vegetable juice agar in Petri dishes (9 cm diameter) at 20.degree. C. under near-ultraviolet (NUV) light. After 14 days, the surface of the agar on which the mold was grown was flooded with 10 ml of sterile water per Petri dish, and the conidia and mycelium fragments were carefully scraped off with the aid of a subject carrier. An inoculum density of 20,000 conidia/mycelium fragments per ml, plus 0.1% TWEEN 20, was used to inoculate the plants. At the point in time of the inoculation, the plants had been cultivated for 8 to 9 weeks under greenhouse conditions. The top side and underside of the leaves were treated with the inoculum. The plants were subsequently incubated for 5 to 7 days at 25.degree. C., 18 h/6 h light/dark, and approximately 100% humidity. The first Cercospora symptoms on the sugar beet leaves occurred after 12 to 14 hours. An assessment of the symptoms of the individual plants was performed regularly, with the assistance of the assessment of the rating scores shown in Table 1A. The results are shown below.
TABLE-US-00010 TABLE G Results of transgenic verification of the function of the resistance gene according to the invention in transformed plants; LSD = least significant difference; dpi = days post infection Expression level of the resistance Test Number of Average rating score gene according to group Individuals 8dpi 11dpi 13dpi 15dpi Function the invention 1 57 1.60 3.82 5.24 6.46 negative control 0.0 2 38 1.28 3.07 4.42 6.04 transgenic validation 4.6 3 60 1.26 2.83 4.38 6.20 transgenic validation 12.5 4 45 1.50 3.40 4.62 6.18 no validated 0.0 expression in vitro 5 60 1.44 3.62 5.29 6.38 transgenic validation 2.6 6 57 1.68 3.69 5.30 6.52 transgenic validation 3.3 7 66 1.58 3.84 5.43 6.57 transgenic validation 2.9 8 60 1.31 2.81 4.19 5.48 transgenic validation 11.3 9 72 1.25 3.07 4.61 5.97 transgenic validation 26.8 10 60 1.44 3.26 4.77 6.00 transgenic validation 10.7 11 57 1.60 3.83 5.48 6.64 transgenic validation 4.4 12 72 1.34 2.62 3.75 5.19 resistant source plant not determined mean value 58.66 1.44 3.32 4.8 6.13 overall LSD value -- 0.17 0.47 0.48 0.4
[0319] Results of the Transgenic Validation of the Resistance Gene According to the Invention (s. Table G)
[0320] Test group 1 represents a negative control. The genotype is the same as for test groups 2 to 11 but no transformation has taken place. Therefore, no expression could be detected. Test group 4 has been transformed but no expression could be detected. Test groups 2, 3 and 5 to 11 represent transformants which carry the resistance gene according to the invention only due to the transformation. Test group 12 represents a breeding line comprising the resistance gene according to the invention in a non-transgenic version. The rating scores of all lines has been established after inoculating the plant material with Cercospora beticola as described above. Test group 12 shows the highest resistance which is indicated by a final value of 5,19.
[0321] The transgenic lines showed a rating score according to the following table:
TABLE-US-00011 TABLE H Rating scores of the transgenic lines of table G 8 dpi 11 dpi 13 dpi 15 dpi mean value 1.42 3.33 4.87 6.2 transgenic validation mean value 1.315 2.99 4.48 5.91 transgenic validation for lines having an expression level > 10
[0322] Table H shows that the rating scores for transgenic validation groups only. First the mean value for all transgenic test groups (excluding group 4) are shown. Below the rating scores only for those transgenic lines which showed an expression level of at least 10 (groups 3, 8, 9, 10; s. Table G) are given. Here, the final rating score is 5.91. That is a significantly higher resistance than the negative control of group 1 which has a rating score of only 6.46 (least significant difference=0.4; s. Table G). The best transgenic test group (group 8) shows an even better resistance due to a rating score is 5.48 (s. Table G).
[0323] It is worth mentioning that the expression level of transgenic insertions may be influenced by the integration locus. As the expression level was measured in vitro the actual expression level under infection conditions could be higher--especially under when the resistance gene is under control of a pathogen inducible promoter.
[0324] Statistical Evaluation of the Results of the Transgenic Validation
TABLE-US-00012 TABLE I statistic clustering test cluster cluster cluster cluster group 8 dpi 11 dpi 13 dpi 15 dpi 1 ab a a ab 2 e de bc cd 3 e ef c bcd 4 bc bcd bc bcd 5 cd abc a abc 6 a ab a ab 7 ab a a a 8 de ef c e 9 e de bc d 10 cd cd b d 11 ab a a a 12 de f d e
[0325] Table I shows a statistical evaluation of the rating scores contained in Table G. Each letter symbolizes the allocation to a statistical group. For example, it is evident that after the final evaluation (15 dpi) test group 8 (transgenic verification) is in the same cluster as test group 12 (resistant source) but in a different cluster than test group 1 (negative control). According to this test group 8 is significantly different from test group 1 but not significantly different than test group 12.
[0326] In addition, a box-plot analysis has been performed. The illustration of the box-plots is available from FIG. 4-7.
Example 3: Production of a Resistant Sugar Beet Plant According to the Invention on the Basis of Genetic Material Accessed from Beta vulgaris Subsp. Maritima
[0327] The process described hereafter was based on pooling wild beet material to generate a Cercospora resistance genepool. The Beta vulgaris subsp. maritima accessions used as starting material for the breeding program are listed in the following table.
TABLE-US-00013 TABLE 2 Accessions of Beta vulgaris subsp. maritima and their resistance rating scores towards Cercospora used in a breeding program; the first 4 columns on the right present the accession numbers wherein a certain accession may have different accession numbers depending on the deposition facility (USDA GRIN = US Department of Agriculture Germplasm Resources Information Network; IDBBNR = International Database for Beta; DEU001 = Plant Genetic Resource Collection; IPK = Leibnitz-Institut fur Pflanzengenetik und Kulturpflanzenforschung) Resistance rating score for Cercospora GRIN studies SUGAR- SUGAR- SUGAR- SUGAR- SUGAR- SUGAR- BEET_ BEET_ BEET_ BEET_ BEET_ BEET_ SUGAR- SUGAR- CERCO- CERCO- CERCO- CERCO- CERCO- RICHA- Accession denomination BEET_ BEET_ SPORA_ SPORA_ SPORA_ SPORA_ SPORA_ RDSON_ USDA IPK CAC_ CAC_ 1989_ 1992_ 1994_ 1997_ 1999_ 2010_ GRIN IDBBNR DEU 001 CERCO_ WBITALY RUPPEL RUPPEL RUPPEL RUPPEL PANELLA TEST1910 1 PI 120704 5191 1 9 PI 169020 5265 1 8 PI 169023 5268 1 7 PI 169030 5274 1 8 PI 546536 9703 3 8 PI 546539 9706 3 6 PI 518303 5797 4 2 PI 518303 5797 4 2 PI 546534 9701 3 8 PI 590763 4587 4 PI 590766 4591 4 PI 109038 5160 1 5 7 28894 BETA 1521 2195 32375 BETA 1429 3555 48819 64088 BETA 1987 8535 58260 BETA 1987 3358 36542 BETA 1228 3744 51437 BETA 1083 6071 54762 BETA 1655 2649 54832 BETA 992 7103 57737 BETA 1127 8634 62120 BETA 1057 8635 62121 BETA 1447 8636 62122 BETA 1014 8637 62123 BETA 1432 8638 62124 BETA 1090 8640 62126 BETA 1377 8642 62128 BETA 1558 8643 62130 BETA 1348 8644 62131 BETA 1610 2212 28921 BETA 1666 3546 48810 BETA 1304 PI 504196 3 7 8 PI 546409 3 3401 45516 BETA 2174 PI 504245 5726 8 IDBB studies 202 237 238 244 248 PI 109038 5160 28894 BETA 1521 2195 32375 BETA 1429 3555 48819 64088 BETA 2157 8535 58260 BETA 1987 3358 36542 BETA 1228 3744 51437 BETA 1083 6071 54762 BETA 1655 2649 54832 BETA 992 7103 57737 BETA 3 1127 8634 62120 BETA 2 1057 8635 62121 BETA 1447 8636 62122 BETA 1014 8637 62123 BETA 1432 6 8638 62124 BETA 1090 6 8640 62126 BETA 1377 8642 62128 BETA 1558 8643 62130 BETA 8 1348 8644 62131 BETA 1610 2212 28921 BETA 1666 3546 48810 BETA 1304 PI 504196 PI 546409 3401 45516 BETA 2174 PI 504245 5726 IDBB studies 249 250 252 254 259 270 288 288 PI 109038 5160 8 28894 BETA 1521 2195 32375 BETA 1429 3555 48819 4 64088 BETA 2157 8535 58260 BETA 2 1987 3358 36542 BETA 1228 3744 51437 BETA 1083 6071 54762 BETA 1655 2649 54832 BETA 5 992 7103 57737 BETA 3 1127 8634 62120 BETA 5 1057 8635 62121 BETA 1 1447 8636 62122 BETA 3 1014 8637 62123 BETA 3 1432 8638 62124 BETA 2 1090 8640 62126 BETA 2 1377 8642 62128 BETA 2 1558 8643 62130 BETA 4 1348 8644 62131 BETA 2 1610 2212 28921 BETA 7 1666 3546 48810 BETA 3 3 1304 PI 504196 PI 546409 3401 45516 BETA 3 2174 PI 504245 5726 1
[0328] As it is apparent from Table 2 the accessed genetic material had shown previously a non-uniform resistance level against Cercospora and the degree of resistance varied throughout the different studies. For example, the accession "PI 120704" showed a score of 1 in one study and a score of 9 in another study. As this publicly available data seemed to be unreliable, seed material from the accessions has been planted and the resulting plants were screened phenotypically for Cercospora resistance. About 150 partial resistant plants have been selected. However, as the seen resistance in each plant could have been the result of a plenty of genes all having a small contribution the chances to identify a single gene suitable for establishing a resistance or increasing the resistance level in a measurable manner was limited. It was decided to cross the about 150 resistant plants among each other using an open pollination scenario. This approach also allowed for the generation of recombinations within the genetic material. Crossing and selection have been repeated for several generations to improve the resistance level. The best descendants have been cloned and prepared for a genetic mapping approach. The mapping of the herein described resistance was coupled with intensive phenotyping. With the aid of the setup of a population of over 4,000 dividing descendants and the development of special recombination screens, the target region was reduced, and thus ever further isolated, via analysis of informative recombinants (genotypical and phenotypical) in a series of resistance tests. This genetic mapping, as well as the creation of physical maps accompanied by WHG sequencing ("whole genome sequencing"), comparative BAC (Bac-by-Bac) sequencing, and bioinformatic analyses, led to the identification of three recombinant genotypes that confirmed the resistance gene (1 recombinant in the neighboring gene, on the one hand, and 2 recombinants in the neighboring gene, on the other). In light of particular requirements, the inventors placed the highly repetitive structure in the target region, which, among other things, contains tandem repeats with very high sequence homology, which made the marker development, and thus the identification of informative recombinants, enormously more difficult. The following steps were particularly decisive for the location of the genetic structure of the resistance gene:
[0329] development of the markers s4p0264s01, s4p2271s01, sxh0678s01, s4p4293s01, s4p4295s01, s4p4301s01 (see Table 1B).
[0330] Fine mapping coupled with intensive phenotyping. The phenotypes were verified with 90-180 descendants per plant in a greenhouse test, and with intensive statistical methods (for example, t-test, power analysis, etc.).
[0331] BAC clone identification and sequencing from BAC pools of the resistant genotype.
[0332] Sequence evaluation, as well as sequence and protein comparison between RR (i.e., resistant) and ss (i.e., sensitive) genotypes; an unambiguous assembly of the RR and ss sequence data was thereby not always possible, due to the sequence complexity.
[0333] In the framework of the breeding program, the Beta vulgaris subsp. maritima derived resistance was crossed with an elite sugar beet line. Several back crossings via marker assisted selection allowed to transfer the resistance gene in established sugar beet germplasm. Surprisingly, no undesired effects towards sugar yield etc. could be observed. Subsequently, a proof of concept for the resistance gene within sugar beet has been established via transformation and the generation of sugar beets which were transgenic for the resistance gene (s. above). After this successful proof of concept the generated sugar beet germplasm comprising the resistance gene could be used for the generation of a Cercospora resistant sugar beet variety.
Example 4: Screening the Starting Accessions for the Identified Resistance Gene
[0334] After the resistance gene has been identified the genetic source material (accessions according to Table 2) was screened by the help of markers to identify the accession which carried the resistance gene. The number of the analyzed plants per accession was dependent on the availability of seeds and is given in the table below.
TABLE-US-00014 TABLE 3 Number of plants per accession analyzed for the presence of the identified resistance gene Accession denomination USDA GRIN IDBBNR DEU001 IPK Plants [No.] PI 120704 5191 40 PI 169020 5265 40 PI 169023 5268 18 PI 169030 5274 18 PI 546536 9703 50 PI 546539 9706 34 PI 518303 5797 2 PI 518303 5797 16 PI 546534 9701 40 PI 590763 4587 12 PI 590766 4591 28 PI 109038 5160 23 28894 BETA 1521 40 2195 32375 BETA 1429 40 3555 48819 14 64088 BETA 2157 12 8535 58260 BETA 1987 40 3358 36542 BETA 1228 40 3744 51437 BETA 1083 31 6071 54762 BETA 1655 40 2649 54832 BETA 992 12 7103 57737 BETA 1127 40 8634 62120 BETA 1057 18 8635 62121 BETA 1447 40 8636 62122 BETA 1014 40 8637 62123 BETA 1432 37 8638 62124 BETA 1090 11 8640 62126 BETA 1377 40 8642 62128 BETA 1558 40 8643 62130 BETA 1348 40 8644 62131 BETA 1610 40 2212 28931 BETA 1666 36 3546 48810 BETA 1304 40 PI 504196 37 PI 546409 3401 45516 BETA 2174 40 PI 504245 5726 40
[0335] Each of the given plants of each accession has been screened by the use of 572 SNP markers which were located 5' as well as 3' to the resistance gene. Due to the large amount of markers a haplotype pattern could be derived. However, none of the accessions used as starting material showed the haplotype of the line CRBM which carried the identified resistance gene. Most similarities have been found to the accession 48819 (DEU001 denomination)/3555 (IDBBNR) (s. Tables 2 and 3). The following table shows an extract of the entire marker analysis including positions 5' and 3' of the resistance gene.
TABLE-US-00015 TABLE 4 Comparison of a resistant line according to the invention and 14 plants of accession 48819 via SNP marker analysis (del = deletion, ins = insertion, Pos start = starting position of molecular marker on genetic strand, * = Position of resistance gene according to SEQ ID NO 1) Marker s4e562 s4e5 s4p2 s4p2 s4p4 s4p4 sxh6 sxh3 s4p4 s4p8 8s03 628s 272s 273s 291s 293s 264s 116s 295s 772s 02 01 01 01 01 01 01 01 01 Pos 62.81 62.82 62.83 62.83 62.84 62.84 62.84 62.84 62.85 62.86 start CRBM A A A A A A G C A T * comprising SEQ ID NO: 1 48819_1 A G T C G G G C T A 48819_2 A G T C G G G C T A 48819_3 A G T C G G G C A/T A/T 48819_4 A G T C G G G C A/T A/T 48819_5 A G T C G G G C A/T T 48819_6 A G T A/C G A G C A/T A/T 48819_7 A G T C G G A/G A/C NA A 48819_8 A G T A/C G G G A/C NA A 48819_9 A G T C G G G A/C A A/T 48819_10 A G T C G G G C A T 48819_11 A G T C G G G C A T 48819_12 A G T C G G G C A/T T 48819_13 A G T A/C G A G C A/T A/T 48819_14 A G A/T A/C G G G C T A Marker s4p8 s4p430 sxh067 s4p227 s4p430 s4p430 s4e971 s4e789 s4p430 s4p430 783s 1s01 8s01 6s01 5d01 6s01 4s01 5s01 7s01 9s01 01 Pos 62.94 62.94 62.97 62.97 62.98 62.98 62.98 62.98 62.99 63.01 start CRBM C/T A A G del G G C C T comprising SEQ ID NO: 1 48819_1 C A A G ins G G C C T 48819_2 C A A G ins G G C C T 48819_3 C A/T A/C A/G ins A/G C/G C/G C/T C/T 48819_4 C A/T A/C A/G ins A/G C/G C/G C/T C/T 48819_5 C T A/C A ins A C/G G C/T C 48819_6 C A/T C G ins G G C C C/T 48819_7 C T C G ins G G C C C/T 48819_8 C A/T A/C G ins G G NA C C 48819_9 C T C A/G ins A/G C/G G C/T C 48819_10 C T C A ins A C G T C 48819_11 C T C A ins A C G T C 48819_12 C T A/C A ins A C/G G C/T C 48819_13 C A/T C A/G ins A/G C/G C/G C/T C/T 48819_14 C A/T A/C G ins A/G C/G C/G C/T C/T
[0336] The results of the marker analysis (as exemplified by the data given in Table 4) show that the resistance gene according to the invention could not be traced back to one of the accessions according to Table 2. Even plants of the accession 48819 which shared the strongest marker overlap with the resistant line according to the invention had significant differences. Noticeable, was the detection of a deletion within the resistant line whereas accession 48819 showed a deletion at the same position. This could be an indication that a significant genetic restructuring at this locus took place during the generation of the resistance gene according to the invention. This assumption would also explain why it was not possible to trace back the resistance gene back to the starting material of the breeding program.
Example 5: Creation of Cercospora Resistant Seed Stock
[0337] The generated sugar beet germplasm comprising the resistance gene (outcome of Example 3) could be used for the generation of a Cercospora resistant sugar beet variety. For this purpose the gene was transmitted via crossing into a DH parent line which was crossed with a DH parent line originating from the other hybrid breeding pool. The result was a hybrid variety comprising the resistance towards Cercospora according to the present invention. The seeds of the variety were separated from each other (singularized), cleaned and polished. Afterwards, the seeds have been subjected to priming and pelleting as described in EP2002702A1. The resulting seed stock has been filed in a packaging made of cardboard which comprised an interlayer as vapor barrier. The resulting seed stock was suitable for sowing, growing, harvesting and subsequent industrial sugar production.
[0338] While several possible embodiments are disclosed above, embodiments of the present invention are not so limited. These exemplary embodiments are not intended to be exhaustive or to unnecessarily limit the scope of the invention, but instead were chosen and described in order to explain the principles of the present invention so that others skilled in the art may practice the invention. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims.
Sequence CWU
1
1
9913720DNABeta vulgaris 1atgaacatga aaatcctcct tttgtttgtc ttccttcatc
acctccacta cttcatccat 60ggcagaacac ttacagaacg ccaagcttta ctaagtatca
aatctgccat tacttatgat 120tattataact ctctctcctc atggaaaaac acaacacacc
actgcagttg gccatacatc 180acttgctcct cctcttcttc ttcttcttct gttatttctc
tcaacttcac catgttattt 240ctcgaaggaa ttctctcccc tgatataggc ttcctcacca
acctgcaaaa cctctctatt 300cgatctaacc ttttttctgg cccactcccc cattctctct
ctctcctcac ccaactccgc 360tatctcgacg tttcccaaaa cagtttcaca ggtccaatcc
catcttctct ctctctcctc 420acccaactcc gctatctcca cgtttccggc aacagtttca
caggtccaat cccatctttt 480ctctctctcc tcacccaact ccgctatctc gacgtttccg
acaacagttt cacaggtcca 540atcccatctt ctctctctct cctcacccaa ctccgctatc
tcgacgtttc ctacaacaat 600ctaaatggca ctcttccctt atcggtcgtt gagaagatgt
cggagctcag ctaccttaac 660cttaggtata actctttcta cggtgagatt ccaccggagt
ttgggaaact taagaagctt 720gaaacattga atcttggtaa caacactctt tctgggagtc
ttccatctga gttgggttca 780ttaaagagtt tgaaacatat ggacttttct agtaatatgc
tatttggtga gatcccacaa 840tcttattctc ttcttcgaaa cttaatcgat attgatctta
atagaaacaa gttatatggg 900agtatacctg attatattgg agattttccg gagttggaat
cacttttatt agactcgaat 960aacttcacag ggagtatccc acaaaagtta ggtacaaacg
ggaagttgca atatctagat 1020ataagtaaca acaattttag tggtagtttg ccactaagtc
tttgcaaagg agacaaactc 1080caagatctgg acgcatccta taatttgttg gttgggtcaa
ttcctgagag tttgggaagt 1140tgcaagtcac ttgaaggagt gtacatggga aataatttct
taaacgggtc gattcctaag 1200ggcttgtttg ggagtgatgt ttcacttaat gacaaacttc
ttagtggagg tctcgatgag 1260aaattcggtg attgcgttaa tcttcgggac attgatctct
ctaataataa gctatcaggg 1320aagttacctg cgaccatcgg aaactgtatt catcttcggt
ccttgacgct ttataataac 1380acctgtaccg gacgtatccc tcaagagatt agcaagtgta
agcagctaca gaccctcgat 1440ctcagccaaa atcagttctc tggtgtgata cccaatgata
ttacaggtaa gaaagtatat 1500taaacttgtt acttttgaaa atattcgctc tagtttttgt
ttcagttggt ccattctcac 1560tttgtattat tgaaatatat cccaaaaaag taaatataat
tatataaaag aatcttgcta 1620aaaataatat gaattatttt tgtatgtgca aaataatgta
caaatctaac taatttgttg 1680tggataataa tattaattgt gtgaaatagt aaatgtgtgg
agatatataa ctttatttat 1740catattcact caggttttta ggtatttatt atgagttttg
cattggagat atccaacttg 1800acaatagtat ttttgtaata taccaatata taaagattac
tgtacataac caaaatgtat 1860acttttctta tttttataaa cttatatatt cctcttcttt
gtatttatca caacattttt 1920tatacccttt tgcctcatat taatagcaac acttataatt
tatttattta ctttttattt 1980cttggtctat aacctcatct acccacatat gacacaccct
ataaaggacc cacatgatta 2040accaaaatat acaaatatct tcaatgaaat taactttaac
actaatatga taaaaatcat 2100gtcccgcttt ttatcctcta actaagactc tgcataaagg
tatattgcaa ttaatatgag 2160atggaagagg tataataatt atatgatcaa attcctggat
tgaaaaataa atatgagatt 2220aaaagtggta tgtttttggt taaaagaaac tatccataaa
gtatgttttt ggttaaaaga 2280aactatgcaa cataccaatc aaatgtttat acgcttacaa
tttatgtacc acttttttgt 2340cattgttttt ctattgtttg ccatacgtac gttactaaat
catgttgtct tttcacattt 2400taactaacaa taaattacta ttgatacacc aaaaaaatct
atgagcattg gagtacgttg 2460tttgatagaa gcttcgtgct attatttctt gtcaaagaat
ttcatatctc aatatcttct 2520aatttaacaa tctaacgaaa tttttttgac ccaggaaaca
aatccatttg caatctggaa 2580aagatacaaa cacttaaatt atcaaacaat gctttgactg
gtgaaatccc tcattgtgtt 2640ggaaatatcg agctcatagc attatttctc caatcaaaca
aactgaacgg taccataccc 2700gcaaacttct caaagttatg tgattcattg atatatctag
atcttagtga caatcaactc 2760gaaggagttc tacctaagtc cttgtccaaa tgtcaaagtc
tagaactcct aaatgtcggg 2820aacaataggc taagagataa atttccttca tggttagaca
acctcccacg tctccaagtt 2880ttcagtgtgc gttttaacgc cttctacggt cctataacta
gctcaccaaa agttagtcac 2940ccatttccta tgctacaaat tatcgaccta tctaacaata
agttttgtgg caagttgcca 3000agaagatata tcaaaaactt tgcaaccatg cgcaatatga
atgagtctgg tgttgggaat 3060ccacagtacc tgggggactc atcaatatat agtattacgt
actctatggt attgacattc 3120aatgggttac aacaaaaata tgaaaagctt attgtgacga
tgtcgacctt tgatatatcc 3180agcaacaact ttactggaca gattccatat gttatagggg
gattacgctc acttcgtaac 3240cttaatctct ctcataatgt cttaaccggg aacattcctc
catcaattgc aaaattgtct 3300ttgcttcaag atttggacct ttcatcaaac agacttactg
gtcgtatccc tcaagaatta 3360gttagtttaa catttcttgg gagtttcaat gtttcgaaca
atctattgga ggggtctata 3420cctcatggtt tcaacttcga cacgtacaca gctaattcat
accaggggaa tctcgaatta 3480tgtggaaaac cattacctga gtgtggagaa agaagggcaa
aaggcaccac taataatcaa 3540gatgatccta aaaatgataa tgaacgaatg ttgtcgatgt
ccgaaatcgt agttatgggg 3600tttggcagtg gtgtactagt tgggttggct tggggatact
atatgttttc agtgggaaag 3660cccttttggt ttatcaagat ggctagcaaa atggaatcaa
tattgattgg ttttttctga 372022652DNAArtificial SequencecDNA sequence of
the Cercospora resistance- conferring gene 2atgaacatga aaatcctcct
tttgtttgtc ttccttcatc acctccacta cttcatccat 60ggcagaacac ttacagaacg
ccaagcttta ctaagtatca aatctgccat tacttatgat 120tattataact ctctctcctc
atggaaaaac acaacacacc actgcagttg gccatacatc 180acttgctcct cctcttcttc
ttcttcttct gttatttctc tcaacttcac catgttattt 240ctcgaaggaa ttctctcccc
tgatataggc ttcctcacca acctgcaaaa cctctctatt 300cgatctaacc ttttttctgg
cccactcccc cattctctct ctctcctcac ccaactccgc 360tatctcgacg tttcccaaaa
cagtttcaca ggtccaatcc catcttctct ctctctcctc 420acccaactcc gctatctcca
cgtttccggc aacagtttca caggtccaat cccatctttt 480ctctctctcc tcacccaact
ccgctatctc gacgtttccg acaacagttt cacaggtcca 540atcccatctt ctctctctct
cctcacccaa ctccgctatc tcgacgtttc ctacaacaat 600ctaaatggca ctcttccctt
atcggtcgtt gagaagatgt cggagctcag ctaccttaac 660cttaggtata actctttcta
cggtgagatt ccaccggagt ttgggaaact taagaagctt 720gaaacattga atcttggtaa
caacactctt tctgggagtc ttccatctga gttgggttca 780ttaaagagtt tgaaacatat
ggacttttct agtaatatgc tatttggtga gatcccacaa 840tcttattctc ttcttcgaaa
cttaatcgat attgatctta atagaaacaa gttatatggg 900agtatacctg attatattgg
agattttccg gagttggaat cacttttatt agactcgaat 960aacttcacag ggagtatccc
acaaaagtta ggtacaaacg ggaagttgca atatctagat 1020ataagtaaca acaattttag
tggtagtttg ccactaagtc tttgcaaagg agacaaactc 1080caagatctgg acgcatccta
taatttgttg gttgggtcaa ttcctgagag tttgggaagt 1140tgcaagtcac ttgaaggagt
gtacatggga aataatttct taaacgggtc gattcctaag 1200ggcttgtttg ggagtgatgt
ttcacttaat gacaaacttc ttagtggagg tctcgatgag 1260aaattcggtg attgcgttaa
tcttcgggac attgatctct ctaataataa gctatcaggg 1320aagttacctg cgaccatcgg
aaactgtatt catcttcggt ccttgacgct ttataataac 1380acctgtaccg gacgtatccc
tcaagagatt agcaagtgta agcagctaca gaccctcgat 1440ctcagccaaa atcagttctc
tggtgtgata cccaatgata ttacaggaaa caaatccatt 1500tgcaatctgg aaaagataca
aacacttaaa ttatcaaaca atgctttgac tggtgaaatc 1560cctcattgtg ttggaaatat
cgagctcata gcattatttc tccaatcaaa caaactgaac 1620ggtaccatac ccgcaaactt
ctcaaagtta tgtgattcat tgatatatct agatcttagt 1680gacaatcaac tcgaaggagt
tctacctaag tccttgtcca aatgtcaaag tctagaactc 1740ctaaatgtcg ggaacaatag
gctaagagat aaatttcctt catggttaga caacctccca 1800cgtctccaag ttttcagtgt
gcgttttaac gccttctacg gtcctataac tagctcacca 1860aaagttagtc acccatttcc
tatgctacaa attatcgacc tatctaacaa taagttttgt 1920ggcaagttgc caagaagata
tatcaaaaac tttgcaacca tgcgcaatat gaatgagtct 1980ggtgttggga atccacagta
cctgggggac tcatcaatat atagtattac gtactctatg 2040gtattgacat tcaatgggtt
acaacaaaaa tatgaaaagc ttattgtgac gatgtcgacc 2100tttgatatat ccagcaacaa
ctttactgga cagattccat atgttatagg gggattacgc 2160tcacttcgta accttaatct
ctctcataat gtcttaaccg ggaacattcc tccatcaatt 2220gcaaaattgt ctttgcttca
agatttggac ctttcatcaa acagacttac tggtcgtatc 2280cctcaagaat tagttagttt
aacatttctt gggagtttca atgtttcgaa caatctattg 2340gaggggtcta tacctcatgg
tttcaacttc gacacgtaca cagctaattc ataccagggg 2400aatctcgaat tatgtggaaa
accattacct gagtgtggag aaagaagggc aaaaggcacc 2460actaataatc aagatgatcc
taaaaatgat aatgaacgaa tgttgtcgat gtccgaaatc 2520gtagttatgg ggtttggcag
tggtgtacta gttgggttgg cttggggata ctatatgttt 2580tcagtgggaa agcccttttg
gtttatcaag atggctagca aaatggaatc aatattgatt 2640ggttttttct ga
26523883PRTBeta vulgaris 3Met
Asn Met Lys Ile Leu Leu Leu Phe Val Phe Leu His His Leu His1
5 10 15Tyr Phe Ile His Gly Arg Thr
Leu Thr Glu Arg Gln Ala Leu Leu Ser 20 25
30Ile Lys Ser Ala Ile Thr Tyr Asp Tyr Tyr Asn Ser Leu Ser
Ser Trp 35 40 45Lys Asn Thr Thr
His His Cys Ser Trp Pro Tyr Ile Thr Cys Ser Ser 50 55
60Ser Ser Ser Ser Ser Ser Val Ile Ser Leu Asn Phe Thr
Met Leu Phe65 70 75
80Leu Glu Gly Ile Leu Ser Pro Asp Ile Gly Phe Leu Thr Asn Leu Gln
85 90 95Asn Leu Ser Ile Arg Ser
Asn Leu Phe Ser Gly Pro Leu Pro His Ser 100
105 110Leu Ser Leu Leu Thr Gln Leu Arg Tyr Leu Asp Val
Ser Gln Asn Ser 115 120 125Phe Thr
Gly Pro Ile Pro Ser Ser Leu Ser Leu Leu Thr Gln Leu Arg 130
135 140Tyr Leu His Val Ser Gly Asn Ser Phe Thr Gly
Pro Ile Pro Ser Phe145 150 155
160Leu Ser Leu Leu Thr Gln Leu Arg Tyr Leu Asp Val Ser Asp Asn Ser
165 170 175Phe Thr Gly Pro
Ile Pro Ser Ser Leu Ser Leu Leu Thr Gln Leu Arg 180
185 190Tyr Leu Asp Val Ser Tyr Asn Asn Leu Asn Gly
Thr Leu Pro Leu Ser 195 200 205Val
Val Glu Lys Met Ser Glu Leu Ser Tyr Leu Asn Leu Arg Tyr Asn 210
215 220Ser Phe Tyr Gly Glu Ile Pro Pro Glu Phe
Gly Lys Leu Lys Lys Leu225 230 235
240Glu Thr Leu Asn Leu Gly Asn Asn Thr Leu Ser Gly Ser Leu Pro
Ser 245 250 255Glu Leu Gly
Ser Leu Lys Ser Leu Lys His Met Asp Phe Ser Ser Asn 260
265 270Met Leu Phe Gly Glu Ile Pro Gln Ser Tyr
Ser Leu Leu Arg Asn Leu 275 280
285Ile Asp Ile Asp Leu Asn Arg Asn Lys Leu Tyr Gly Ser Ile Pro Asp 290
295 300Tyr Ile Gly Asp Phe Pro Glu Leu
Glu Ser Leu Leu Leu Asp Ser Asn305 310
315 320Asn Phe Thr Gly Ser Ile Pro Gln Lys Leu Gly Thr
Asn Gly Lys Leu 325 330
335Gln Tyr Leu Asp Ile Ser Asn Asn Asn Phe Ser Gly Ser Leu Pro Leu
340 345 350Ser Leu Cys Lys Gly Asp
Lys Leu Gln Asp Leu Asp Ala Ser Tyr Asn 355 360
365Leu Leu Val Gly Ser Ile Pro Glu Ser Leu Gly Ser Cys Lys
Ser Leu 370 375 380Glu Gly Val Tyr Met
Gly Asn Asn Phe Leu Asn Gly Ser Ile Pro Lys385 390
395 400Gly Leu Phe Gly Ser Asp Val Ser Leu Asn
Asp Lys Leu Leu Ser Gly 405 410
415Gly Leu Asp Glu Lys Phe Gly Asp Cys Val Asn Leu Arg Asp Ile Asp
420 425 430Leu Ser Asn Asn Lys
Leu Ser Gly Lys Leu Pro Ala Thr Ile Gly Asn 435
440 445Cys Ile His Leu Arg Ser Leu Thr Leu Tyr Asn Asn
Thr Cys Thr Gly 450 455 460Arg Ile Pro
Gln Glu Ile Ser Lys Cys Lys Gln Leu Gln Thr Leu Asp465
470 475 480Leu Ser Gln Asn Gln Phe Ser
Gly Val Ile Pro Asn Asp Ile Thr Gly 485
490 495Asn Lys Ser Ile Cys Asn Leu Glu Lys Ile Gln Thr
Leu Lys Leu Ser 500 505 510Asn
Asn Ala Leu Thr Gly Glu Ile Pro His Cys Val Gly Asn Ile Glu 515
520 525Leu Ile Ala Leu Phe Leu Gln Ser Asn
Lys Leu Asn Gly Thr Ile Pro 530 535
540Ala Asn Phe Ser Lys Leu Cys Asp Ser Leu Ile Tyr Leu Asp Leu Ser545
550 555 560Asp Asn Gln Leu
Glu Gly Val Leu Pro Lys Ser Leu Ser Lys Cys Gln 565
570 575Ser Leu Glu Leu Leu Asn Val Gly Asn Asn
Arg Leu Arg Asp Lys Phe 580 585
590Pro Ser Trp Leu Asp Asn Leu Pro Arg Leu Gln Val Phe Ser Val Arg
595 600 605Phe Asn Ala Phe Tyr Gly Pro
Ile Thr Ser Ser Pro Lys Val Ser His 610 615
620Pro Phe Pro Met Leu Gln Ile Ile Asp Leu Ser Asn Asn Lys Phe
Cys625 630 635 640Gly Lys
Leu Pro Arg Arg Tyr Ile Lys Asn Phe Ala Thr Met Arg Asn
645 650 655Met Asn Glu Ser Gly Val Gly
Asn Pro Gln Tyr Leu Gly Asp Ser Ser 660 665
670Ile Tyr Ser Ile Thr Tyr Ser Met Val Leu Thr Phe Asn Gly
Leu Gln 675 680 685Gln Lys Tyr Glu
Lys Leu Ile Val Thr Met Ser Thr Phe Asp Ile Ser 690
695 700Ser Asn Asn Phe Thr Gly Gln Ile Pro Tyr Val Ile
Gly Gly Leu Arg705 710 715
720Ser Leu Arg Asn Leu Asn Leu Ser His Asn Val Leu Thr Gly Asn Ile
725 730 735Pro Pro Ser Ile Ala
Lys Leu Ser Leu Leu Gln Asp Leu Asp Leu Ser 740
745 750Ser Asn Arg Leu Thr Gly Arg Ile Pro Gln Glu Leu
Val Ser Leu Thr 755 760 765Phe Leu
Gly Ser Phe Asn Val Ser Asn Asn Leu Leu Glu Gly Ser Ile 770
775 780Pro His Gly Phe Asn Phe Asp Thr Tyr Thr Ala
Asn Ser Tyr Gln Gly785 790 795
800Asn Leu Glu Leu Cys Gly Lys Pro Leu Pro Glu Cys Gly Glu Arg Arg
805 810 815Ala Lys Gly Thr
Thr Asn Asn Gln Asp Asp Pro Lys Asn Asp Asn Glu 820
825 830Arg Met Leu Ser Met Ser Glu Ile Val Val Met
Gly Phe Gly Ser Gly 835 840 845Val
Leu Val Gly Leu Ala Trp Gly Tyr Tyr Met Phe Ser Val Gly Lys 850
855 860Pro Phe Trp Phe Ile Lys Met Ala Ser Lys
Met Glu Ser Ile Leu Ile865 870 875
880Gly Phe Phe44748DNABeta vulgaris 4ttactatgaa caatacccta
atatcattag gttttcccct tctctctcct aagtgccaaa 60ctgccaaccc cctcccatct
ttatttcaat aagagcacca ttaaattatt gtgtaacaaa 120gaccattatt ttaagatcac
taataaggtt gctctaatta ttcctagaat tctagtgaaa 180aaagaaagat aaaagatgaa
catggggtga tgactgatga ctgagagaca acagacaaca 240cttggttgag ttgatatttg
acgcaaagac ttggcgtgtt ggaaggttca ttacacattt 300tatccaagtc aactttgaag
tcttcttagc tagagactaa tagagtgaac gtgttggaag 360gttcatgttc atgacattat
aaaagtaata atagtgaaat ttcacaaagt atttataaac 420ccaggacaga ctcaagagct
ctacttatta tattagtgaa aaacaaacat acacacgaca 480ataacacaac ataaacaata
atgaacatga aaatcctcct tttgtttgtc ttccttcatc 540acctccacta cttcatcaat
ggcagaacac taacagaaca tcaagcttta ctaagtatca 600aatctgccat tactaatgat
acgaatagct atctctcctt atggaaaaac acaacacacc 660actgcagttg gccatacatc
acttgctcct cctcttcttc ttctgtcatt tctctcgata 720tctcctactt agagctcacc
ggaattctct cccctgatat aggcttcctc accaacctcc 780aaaacctcac tattcaatgg
aacgattttt ctggccccct ccccacttct ctctctctcc 840tcacccaact ccgccatctc
gacgtttcct acaacaattt cacaggtcca atcccatctt 900ctctctctct cctcacccaa
ctccgccatc tcgacgtttc cttcaacagt ttcacaggtc 960caatcccatc ttctctctct
ctcctcaccc aactccgcta tctcgacgtt tcccaaaaca 1020gtttcacagg tccaatccca
tcttctctct ctctcctcac ccaactccgc tatctcgacg 1080tttccgacaa cagtttcaca
ggtccaatcc catcttttct ctctctcctc acccaactcc 1140gctatctaga cgtttcctac
aacaatctaa atggcactct tcccttatcg gtcgttgaga 1200tgtcggaact caggtacctt
aaccttaagt ataactcttt ctacggtgag attccaccgg 1260agtttgggaa acttaagaag
cttcaaacat tggatcttgg taacaactat ctttctgggg 1320gtcttccatt tgagttgggt
tcattaaaga gtttgaaata tattgatctt agtataaaca 1380atttatatgg gagtatacct
gattatattg gagattttcc ggagttggaa tcacttttat 1440tagactcgaa taacttcaca
gggagtatcc cacaaaagtt aggtacaaac gggaagttgc 1500aatatctaga tataagtaac
aacaatttta gtgggagttt gccagcaagt ctttgcaaag 1560gagacaaact ccaacatttg
ggagtatccg ataatttgtt ggttgggcca attcctgaga 1620gtttgggaag ttgcaagtca
cttgaagaag tgaacatggg aaataatttc tttaacgggt 1680cgattcctaa gggcttgttt
ggcctcccaa acattattga tgtttcactc aatgacaatc 1740ttcttagcgg aggtctcgat
gagaaatttg gtgattgtgt taatcttttc aacattgatc 1800tctctaataa taagctatca
gggaagttac ctgcgactat tggaaactgt tctaatcttc 1860agttgttgat gcttaatcag
aataacttca ccggaagtat ccctcaagag attagcaagt 1920gtaagcagct acgggccctc
gatctcagcc aaaatcagtt ctctggtgtg atacccaatg 1980atattacagg taagaaagta
tattaaactt gttacttttg aaaatattcg ctctagtttt 2040ctttcagttg gtccattctc
acttttgcat tattgaaata tatccctaaa aaagtaaatg 2100taattatata aaagaatctt
gctcaaaata atatgaatta tttttgtatg tgcaaaataa 2160tgtacaatct aactaatttg
ttgtgaaaaa taatataatt gtgtgaaata gtaaatgtgt 2220ggagatatat aactttattt
atcatattca ctaagggttt taggtatttt actatgactt 2280ttgcattatg gagatatcca
acttgacaat agtatttttg taatatactt cctccgtttc 2340taaataagtg caacatttac
atagtgttta ctattcacag tttaaacttt aattagcttt 2400ggtgatttac attttaggaa
aaacatagtc atgtgggatc ttattagatt cgtctgaatg 2460tgaatttttt taatatcaac
tttttataat ttttacttat tgacaattga agatattaat 2520ggttaaaata atgcattggc
aaacgtgcaa acaagaaatg ttgcacttat ttagaaacgg 2580aggaagtatc atatatgaag
attattgtac ataacacttt tcttattttt ataaactata 2640tattcttctt ctttgtattt
atcacaacac tttttatatc tttgcctcat attaatggca 2700acacttttaa tttatctatt
tactttttat ttcttggtct atagcccatt tacatactta 2760tgacacacct cataaaggac
ccacacgatt aaccaaaata tacaaatatc ttcaatgaaa 2820ttaacttcaa tactaatatg
ataaaaatca tgccccgctg tttatcctca tcctaagact 2880ctgcataaaa ttattatttc
ttgtccatac ttaatcatgt tgtgttttca cattttaact 2940aataataaat tacaattgat
acaccaaaaa actctatgag cattgggtat gttgtttgat 3000agaagcttca tgctattatt
tcttgtcaaa gaatttcata tctcgatatc ttctatacca 3060tctaacgaac aattattttc
tgcaggaaac aaaaccattt gcaattttga agaaattaaa 3120ttacttgatt tatcaaacaa
tattttgacc ggtgaaatcc ctcgttgtct tggaaatact 3180agtactcaac tcgaaacatt
atttcttcaa tcaaacaaac tgaacggtac catacccgca 3240aacttctcaa agttatgtga
ttcattgatg tatctagatc ttagtgacaa tcaactcgaa 3300ggagttctac ctaagtcatt
gtccaaatgt caaaatttga aactcctaaa tgtcgggaac 3360aacaggctaa gagataaatt
tccctcatgg ctagacaacc tcccacatct ccaagttttc 3420agtgtgcgtt tcaatgcctt
ctacggtcct ataactagct catcaaaggt taatcaccca 3480tttcctatgc tacaaattat
cgacctatct aacaatgagt tttgtggcaa gttgccaaga 3540agatatatca aaaattttgc
aaccatgcgc aatatgaatg agtctggtgt tggggatcca 3600cagtacctgg aggactcata
tagtccgtac tctatggtat tgacattcaa tgggttacaa 3660caaaaatatg aaaagcttat
tgtgacgatg tcgacctttg atatatccaa caacaacttt 3720actggacaga ttccatatgt
tataggggga ttacactcac ttcgtaacct taatctctcg 3780cataatgtct taaccgggaa
cattcctcca tcaattgcaa aattgtcttt gcttcaggat 3840ttggaccttt catcaaacag
acttattggt cgtatccctc aagaattagt tagtttaaca 3900tttcttggga gcttcaatgt
ttcgaacaat ctattggagg ggcctatacc tattggtaac 3960aacttcaata cattctcgaa
taattcatac caggggaatg tcggattgtg tggaaaacca 4020ttacctgagt gtggagaaag
aagggcaaaa agcaccacta ataatcaaga tgttcctaaa 4080aatgataatg aacgaatgtt
gtcgatgtcc gaaatcgtag ttatggggtt tggcagtggt 4140gtactagttg ggttggcttg
gggatactat atgttttcag tgggaaagcc cttttggttt 4200atcaagatgg ctagcaaaat
ggaatcaata ttgattggtt ttttctgacc aacaatttgt 4260tagccgatga agagcatcaa
aaccaaaaaa acaaaaaaat tgagtaatat gcatgagtgt 4320gaccttgttt tccaaagttt
agcattacta ttagtgtctc aattcataat aataaaaaaa 4380ttagcttgtt caagatttgt
attttattca aagatttttt atgtctcttg tgcttctttt 4440atcttatata tattttttgt
atggtttgtt tttgtttaat attagtccct ccgctcaaaa 4500tgatctttca cgcttgagat
tggcattaag gtcaagagat gttgctaagc tttagaataa 4560aaaaattcca aatgcataga
gggaaagaaa gcgagacaaa atgttggaga aggcagagta 4620aatgatgtga tggaggataa
atagtagaag tgtgataccg aaagtttgaa aataataagg 4680aattttattt cttgctggca
cttcgttcta gtacaggttt ttggcccttc aaaatgctta 4740taatgtag
474852460DNAArtificial
SequencecDNA sequence of the sensitive variant of the Cercospora
resistance-mediating gene 5atgaacatga aaatcctcct tttgtttgtc ttccttcatc
acctccacta cttcatcaat 60ggcagaacac taacagaaca tcaagcttta ctaagtatca
aatctgccat tactaatgat 120acgaatagct atctctcctt atggaaaaac acaacacacc
actgcagttg gccatacatc 180acttgctcct cctcttcttc ttctgtcatt tctctcgata
tctcctactt agagctcacc 240ggaattctct cccctgatat aggcttcctc accaacctcc
aaaacctcac tattcaatgg 300aacgattttt ctggccccct ccccacttct ctctctctcc
tcacccaact ccgccatctc 360gacgtttcct acaacaattt cacaggtcca atcccatctt
ctctctctct cctcacccaa 420ctccgccatc tcgacgtttc cttcaacagt ttcacaggtc
caatcccatc ttctctctct 480ctcctcaccc aactccgcta tctcgacgtt tcccaaaaca
gtttcacagg tccaatccca 540tcttctctct ctctcctcac ccaactccgc tatctcgacg
tttccgacaa cagtttcaca 600ggtccaatcc catcttttct ctctctcctc acccaactcc
gctatctaga cgtttcctac 660aacaatctaa atggcactct tcccttatcg gtcgttgaga
tgtcggaact caggtacctt 720aaccttaagt ataactcttt ctacggtgag attccaccgg
agtttgggaa acttaagaag 780cttcaaacat tggatcttgg taacaactat ctttctgggg
gtcttccatt tgagttgggt 840tcattaaaga gtttgaaata tattgatctt agtataaaca
atttatatgg gagtatacct 900gattatattg gagattttcc ggagttggaa tcacttttat
tagactcgaa taacttcaca 960gggagtatcc cacaaaagtt aggtacaaac gggaagttgc
aatatctaga tataagtaac 1020aacaatttta gtgggagttt gccagcaagt ctttgcaaag
gagacaaact ccaacatttg 1080ggagtatccg ataatttgtt ggttgggcca attcctgaga
gtttgggaag ttgcaagtca 1140cttgaagaag tgaacatggg aaataatttc tttaacgggt
cgattcctaa gggcttgttt 1200ggcctcccaa acattattga tgtttcactc aatgacaatc
ttcttagcgg aggtctcgat 1260gagaaatttg gtgattgtgt taatcttttc aacattgatc
tctctaataa taagctatca 1320gggaagttac ctgcgactat tggaaactgt tctaatcttc
agttgttgat gcttaatcag 1380aataacttca ccggaagtat ccctcaagag attagcaagt
gtaagcagct acgggccctc 1440gatctcagcc aaaatcagtt ctctggtgtg atacccaatg
atattacaga tcttagtgac 1500aatcaactcg aaggagttct acctaagtca ttgtccaaat
gtcaaaattt gaaactccta 1560aatgtcggga acaacaggct aagagataaa tttccctcat
ggctagacaa cctcccacat 1620ctccaagttt tcagtgtgcg tttcaatgcc ttctacggtc
ctataactag ctcatcaaag 1680gttaatcacc catttcctat gctacaaatt atcgacctat
ctaacaatga gttttgtggc 1740aagttgccaa gaagatatat caaaaatttt gcaaccatgc
gcaatatgaa tgagtctggt 1800gttggggatc cacagtacct ggaggactca tatagtccgt
actctatggt attgacattc 1860aatgggttac aacaaaaata tgaaaagctt attgtgacga
tgtcgacctt tgatatatcc 1920aacaacaact ttactggaca gattccatat gttatagggg
gattacactc acttcgtaac 1980cttaatctct cgcataatgt cttaaccggg aacattcctc
catcaattgc aaaattgtct 2040ttgcttcagg atttggacct ttcatcaaac agacttattg
gtcgtatccc tcaagaatta 2100gttagtttaa catttcttgg gagcttcaat gtttcgaaca
atctattgga ggggcctata 2160cctattggta acaacttcaa tacattctcg aataattcat
accaggggaa tgtcggattg 2220tgtggaaaac cattacctga gtgtggagaa agaagggcaa
aaagcaccac taataatcaa 2280gatgttccta aaaatgataa tgaacgaatg ttgtcgatgt
ccgaaatcgt agttatgggg 2340tttggcagtg gtgtactagt tgggttggct tggggatact
atatgttttc agtgggaaag 2400cccttttggt ttatcaagat ggctagcaaa atggaatcaa
tattgattgg ttttttctga 24606819PRTBeta vulgaris 6Met Asn Met Lys Ile Leu
Leu Leu Phe Val Phe Leu His His Leu His1 5
10 15Tyr Phe Ile Asn Gly Arg Thr Leu Thr Glu His Gln
Ala Leu Leu Ser 20 25 30Ile
Lys Ser Ala Ile Thr Asn Asp Thr Asn Ser Tyr Leu Ser Leu Trp 35
40 45Lys Asn Thr Thr His His Cys Ser Trp
Pro Tyr Ile Thr Cys Ser Ser 50 55
60Ser Ser Ser Ser Val Ile Ser Leu Asp Ile Ser Tyr Leu Glu Leu Thr65
70 75 80Gly Ile Leu Ser Pro
Asp Ile Gly Phe Leu Thr Asn Leu Gln Asn Leu 85
90 95Thr Ile Gln Trp Asn Asp Phe Ser Gly Pro Leu
Pro Thr Ser Leu Ser 100 105
110Leu Leu Thr Gln Leu Arg His Leu Asp Val Ser Tyr Asn Asn Phe Thr
115 120 125Gly Pro Ile Pro Ser Ser Leu
Ser Leu Leu Thr Gln Leu Arg His Leu 130 135
140Asp Val Ser Phe Asn Ser Phe Thr Gly Pro Ile Pro Ser Ser Leu
Ser145 150 155 160Leu Leu
Thr Gln Leu Arg Tyr Leu Asp Val Ser Gln Asn Ser Phe Thr
165 170 175Gly Pro Ile Pro Ser Ser Leu
Ser Leu Leu Thr Gln Leu Arg Tyr Leu 180 185
190Asp Val Ser Asp Asn Ser Phe Thr Gly Pro Ile Pro Ser Phe
Leu Ser 195 200 205Leu Leu Thr Gln
Leu Arg Tyr Leu Asp Val Ser Tyr Asn Asn Leu Asn 210
215 220Gly Thr Leu Pro Leu Ser Val Val Glu Met Ser Glu
Leu Arg Tyr Leu225 230 235
240Asn Leu Lys Tyr Asn Ser Phe Tyr Gly Glu Ile Pro Pro Glu Phe Gly
245 250 255Lys Leu Lys Lys Leu
Gln Thr Leu Asp Leu Gly Asn Asn Tyr Leu Ser 260
265 270Gly Gly Leu Pro Phe Glu Leu Gly Ser Leu Lys Ser
Leu Lys Tyr Ile 275 280 285Asp Leu
Ser Ile Asn Asn Leu Tyr Gly Ser Ile Pro Asp Tyr Ile Gly 290
295 300Asp Phe Pro Glu Leu Glu Ser Leu Leu Leu Asp
Ser Asn Asn Phe Thr305 310 315
320Gly Ser Ile Pro Gln Lys Leu Gly Thr Asn Gly Lys Leu Gln Tyr Leu
325 330 335Asp Ile Ser Asn
Asn Asn Phe Ser Gly Ser Leu Pro Ala Ser Leu Cys 340
345 350Lys Gly Asp Lys Leu Gln His Leu Gly Val Ser
Asp Asn Leu Leu Val 355 360 365Gly
Pro Ile Pro Glu Ser Leu Gly Ser Cys Lys Ser Leu Glu Glu Val 370
375 380Asn Met Gly Asn Asn Phe Phe Asn Gly Ser
Ile Pro Lys Gly Leu Phe385 390 395
400Gly Leu Pro Asn Ile Ile Asp Val Ser Leu Asn Asp Asn Leu Leu
Ser 405 410 415Gly Gly Leu
Asp Glu Lys Phe Gly Asp Cys Val Asn Leu Phe Asn Ile 420
425 430Asp Leu Ser Asn Asn Lys Leu Ser Gly Lys
Leu Pro Ala Thr Ile Gly 435 440
445Asn Cys Ser Asn Leu Gln Leu Leu Met Leu Asn Gln Asn Asn Phe Thr 450
455 460Gly Ser Ile Pro Gln Glu Ile Ser
Lys Cys Lys Gln Leu Arg Ala Leu465 470
475 480Asp Leu Ser Gln Asn Gln Phe Ser Gly Val Ile Pro
Asn Asp Ile Thr 485 490
495Asp Leu Ser Asp Asn Gln Leu Glu Gly Val Leu Pro Lys Ser Leu Ser
500 505 510Lys Cys Gln Asn Leu Lys
Leu Leu Asn Val Gly Asn Asn Arg Leu Arg 515 520
525Asp Lys Phe Pro Ser Trp Leu Asp Asn Leu Pro His Leu Gln
Val Phe 530 535 540Ser Val Arg Phe Asn
Ala Phe Tyr Gly Pro Ile Thr Ser Ser Ser Lys545 550
555 560Val Asn His Pro Phe Pro Met Leu Gln Ile
Ile Asp Leu Ser Asn Asn 565 570
575Glu Phe Cys Gly Lys Leu Pro Arg Arg Tyr Ile Lys Asn Phe Ala Thr
580 585 590Met Arg Asn Met Asn
Glu Ser Gly Val Gly Asp Pro Gln Tyr Leu Glu 595
600 605Asp Ser Tyr Ser Pro Tyr Ser Met Val Leu Thr Phe
Asn Gly Leu Gln 610 615 620Gln Lys Tyr
Glu Lys Leu Ile Val Thr Met Ser Thr Phe Asp Ile Ser625
630 635 640Asn Asn Asn Phe Thr Gly Gln
Ile Pro Tyr Val Ile Gly Gly Leu His 645
650 655Ser Leu Arg Asn Leu Asn Leu Ser His Asn Val Leu
Thr Gly Asn Ile 660 665 670Pro
Pro Ser Ile Ala Lys Leu Ser Leu Leu Gln Asp Leu Asp Leu Ser 675
680 685Ser Asn Arg Leu Ile Gly Arg Ile Pro
Gln Glu Leu Val Ser Leu Thr 690 695
700Phe Leu Gly Ser Phe Asn Val Ser Asn Asn Leu Leu Glu Gly Pro Ile705
710 715 720Pro Ile Gly Asn
Asn Phe Asn Thr Phe Ser Asn Asn Ser Tyr Gln Gly 725
730 735Asn Val Gly Leu Cys Gly Lys Pro Leu Pro
Glu Cys Gly Glu Arg Arg 740 745
750Ala Lys Ser Thr Thr Asn Asn Gln Asp Val Pro Lys Asn Asp Asn Glu
755 760 765Arg Met Leu Ser Met Ser Glu
Ile Val Val Met Gly Phe Gly Ser Gly 770 775
780Val Leu Val Gly Leu Ala Trp Gly Tyr Tyr Met Phe Ser Val Gly
Lys785 790 795 800Pro Phe
Trp Phe Ile Lys Met Ala Ser Lys Met Glu Ser Ile Leu Ile
805 810 815Gly Phe Phe71998DNABeta
vulgarispromoter(1)..(1998)native promoter of the Cercospora resistance-
conferring gene Beta vulgaris subsp. maritima 7gagcatagtg agtgcaaaag
ccatggaagc tagattaaaa aggccatcat tctaagttag 60acaattggaa acaacatcga
gatacacgta cacataaggg ctgctcttct ctattactcc 120ctctgttcct aatcatttgc
ttttttagcg ggttccaaag gcctatgttt gaccactaat 180atatttaaat taaaactggt
gatatatatt aaaagaaaat tatgatgaat ttaacaaaaa 240ccatatatgt tatgtccttt
tttttcctat attaatgaat ttttacagtc aaagttggtg 300aactttgacc caaaaaaaga
aatggagcaa aaaaaaaaaa aaaaaaaaaa aactagggac 360aatgagtaac atttttatct
atgtcttttt aatatgaata tacgtaacaa attctgcaaa 420aatagagata gcaactaata
acacgcatga aaatgacaag ttatattata cctttttttc 480tcaatatatg aatatacgta
acaaattaac tccagtagtt tttagtaaaa ctattagatt 540attgtgtaac atatactctg
gaaatagtac taagatccat tacaatcttt attgagaaat 600ttcctcatgt accccctgag
gtttggcgta atttccaaat acccctcata tttgaggaat 660ttctcaaata ccctgatgtt
tttgtttaga ctcaaaatac ctttactatg gacagtaccc 720taatgtcatt aagttttccc
cttctctctc cccaattttc tctctcctcc cattccccca 780cccactaccc actgcccact
gccaagtagg ggtgtaagtg gattggactg gattggactt 840tgccaaattc aaatccagtc
caaagttttt tggactcgag aaattgagtc caagtccgat 900ccaaatattt tttgagtcca
gtccaatcta gtccgataat tttttcttga gtccgaatcc 960agtccagtcc agtccgatta
ttatatcttt tttcccgatt taggttcaat gattcacaac 1020attttttgag atgcttgagc
atttgacatc tgattcaatt atcaatatcc acaaataaga 1080ttgaaagctt aaattaaagt
aaaatactat gaataaaaag ttgaattaga tgcttacctt 1140gatctaagtt gagaggaagc
atagagactg agaattaatc tgagggacaa atagagaatg 1200cgagagtcga gacagtgagg
tagaaagaaa atgaagagta agaggaagtg agtattaagg 1260actgaggagt aaagtaagat
agaattagtt ggctactagc ctactaatgc agtattgcta 1320gtataattta cttatttaac
aaatggagct aagtgcaata gtttagcgcc aattgacata 1380tttagagaga gaaggctgaa
aaatccaata tttttaaaat agtatcatta tttttaatat 1440atacattata tataaaaata
tttttggact ggactggaca tattggactc caaagggatg 1500agtccaaatc cagacaaaaa
atatttggac ttgaaaattt aagtccgagt ccagtccgaa 1560aaattttcag tccaatccag
tccgacaaat ttggactgga ctggattgga ctctgaactt 1620ttcgtagtcc gcttacaccc
ctactgccaa gtgccaaact gccaaccccc ttttggttga 1680gttgatattt gacgcaaaga
cttggcgtgt tggaaggttc attacacatt ttatccaagt 1740caactttgaa gtcttcttag
ctagagacta gagtgaacgt gttggaaggt tcattacaca 1800ttttatccaa tcaaactttg
aagtcttctt agctagagac tagagtgaac gtgttggaag 1860gttcatgttc atgacattat
aaaagtaata atagtgaaat ttcacaaagt atttataaac 1920ccaggacaga ctcaagagct
ctacttatta ttagtgaaaa acaaacatac acacgacaat 1980aacacaacat aaacaata
199882000DNABeta
vulgaristerminator(1)..(2000)native terminator of the Cercospora
resistance- conferring gene from Beta vulgaris subsp. maritima
8ccaacaattt gttagccgat gaagagcatc aaaaccaaaa aaaacaaaaa aaattgatta
60atatgcatga gtgtgacctt gttttccaaa gtttagcatt actattagtg tctcaattca
120taataataaa aaaattagct tgttcaagat ttgtattttt attcaaagat tttttttgtc
180tcttgtgctt cttttatctt atatatattt tttgtatggt ttgtttttgt ttaatattag
240tccctccgct caaaatgatc tttcacgctt gagattggca ttaaggtcaa gagatgttgc
300taagctttag aataaaaaaa ttccaaatgc atagagggaa agaaagcgag acaaaatgtt
360ggagaaggca gagtaaatga tgtgatggag gataaatagt agaagtgtga taccgaaagt
420ttgaaaataa taaggaattt tatttcttgc tggcactttg ttctagtaca ggtttttagc
480ccttcaaaat gtttataatg tagagtcaaa attaatatcc ttaactagtt tttaagtccg
540ggttatatcc tagatattaa taatattcat ttattagtaa cattttattt tataaatata
600atactaagca ttatttggtt tgctggttaa gactttagtg tatatctatt tctttttttt
660tttattgtat gcgtgtttac ataaactaaa gactataagg gatagtacca cgtggcgcag
720ttccttgctt aggaacgtct tttaatatat taactagtat ttgggcccgg gcgttgctcc
780gggttggtat tgtgtttccg aacatgatgt gcagtttttc ccattcccac taaaatatat
840aaaggaaaac tcaacattta aaagatacaa atataataat atggacactt aaaacatgat
900taaaagttga ttgagatggt aattgtgtca tgttataata gtaagaggtt gcctaattga
960ggttgaggtg gtggagtagt ggtatcgctt cccatctgtt atccctgagg tataaggatc
1020aaacctcata ggactcattt gagtaatttc ccatatcctc ctctcaaatg agtccttttc
1080atctgacaaa aaaaaagagt ctaattttaa attaaaatta gacgatcttt tataaaatcg
1140gcactttctg cacataggtc acaatttttt tgtttctatc tctctgcttt ctttaatttc
1200acagtctcca actctccatc aacatcttac ttattttaga atagatgatg tatggtagta
1260ttaaatggta aagtactaaa gctcctataa tacacagaag cttacatagt atagattcgt
1320acatgagaca aggttacaat atactttctc cgttcttttt atattacaat aattactatt
1380ttaagtagtt tcacatctat tgtaacaatt ccaattttgt tatagaaagc aactttaata
1440attgacaata ttgcccttac tttatcttat taaaaccatc attaattact cactttctct
1500tataaaattg cttttatttt ctaaggatga tttctctcct attctagtta attaaagagt
1560tacttttgtg ctaaactgct catttattcc aaatccttaa aaattgtgtc caaacgtatt
1620gttgtaatat aaaaagaaca gaggtactat tagtttgaat aaattttgat cagattaggt
1680cacctttagg gggcgtttgg ttaggggtat tctggaaagg gtaagggaat caacttactt
1740aattccctta cttgttgttt gtttgctcaa tttaatgatt ccctttaccc accccttact
1800cccaaagtcc tttactctca ttctccccac cccccaaggt ttcacttacc ctttcttgat
1860tcatcattga ccatatcttt gaccacccaa ctaccaccac cacttgacca cctaatcacc
1920taaccaccta attacccaac cactattacc acccaacccc tccacctgcc caccaatcgg
1980caccataact gcccaaccgt
20009911PRTBeta vulgarisPEPTIDE(1)..(911)Konsensus Sequenz aus Abbildung
1VARIANT(20)..(20)N or HVARIANT(27)..(27)H or RVARIANT(39)..(39)N or
YVARIANT(41)..(41)T or YVARIANT(42)..(42)N or YVARIANT(42)..(42)S or
NVARIANT(43)..(43)Y or SVARIANT(47)..(47)L or SVARIANT(75)..(75)D or
NVARIANT(76)..(76)I or FVARIANT(77)..(77)S or TVARIANT(78)..(78)Y or
MVARIANT(80)..(80)E or FVARIANT(82)..(82)T or EVARIANT(99)..(99)T or
SVARIANT(101)..(101)Q or RVARIANT(102)..(102)W or SVARIANT(104)..(104)D
or LVARIANT(111)..(111)T or HVARIANT(121)..(121)H or
YVARIANT(126)..(126)Y or QVARIANT(171)..(171)D or HVARIANT(174)..(174)Q
or GVARIANT(184)..(184)S or FVARIANT(208)..(208)F or
SVARIANT(246)..(246)K or RVARIANT(265)..(265)Q or EVARIANT(268)..(268)D
or NVARIANT(277)..(277)G or SVARIANT(280)..(280)F or
SVARIANT(290)..(290)Y or HVARIANT(318)..(318)S or NVARIANT(319)..(319)I
or RVARIANT(321)..(321)N or KVARIANT(376)..(376)A or
LVARIANT(386)..(386)H or DVARIANT(388)..(388)G or DVARIANT(389)..(389)V
or AVARIANT(391)..(391)D or YVARIANT(397)..(397)P or
SVARIANT(410)..(410)E or GVARIANT(412)..(412)N or YVARIANT(418)..(418)F
or LVARIANT(429)..(429)L or SVARIANT(440)..(440)N or
KVARIANT(457)..(457)F or RVARIANT(458)..(458)N or DVARIANT(478)..(478)S
or IVARIANT(479)..(479)N or HVARIANT(481)..(481)Q or
RVARIANT(482)..(482)L or SVARIANT(484)..(484)M or TVARIANT(486)..(486)N
or YVARIANT(487)..(487)Q or NVARIANT(489)..(489)N or
TVARIANT(490)..(490)F or CVARIANT(493)..(493)S or RVARIANT(505)..(505)R
or QVARIANT(506)..(506)A or TVARIANT(605)..(605)R or
QVARIANT(607)..(607)K or EVARIANT(629)..(629)H or RVARIANT(648)..(648)S
or PVARIANT(651)..(651)N or SVARIANT(666)..(666)E or
KVARIANT(692)..(692)D or NVARIANT(697)..(697)E or GVARIANT(705)..(705)P
or TVARIANT(733)..(733)H or RVARIANT(733)..(733)N or
SVARIANT(748)..(748)H or RVARIANT(785)..(785)I or TVARIANT(811)..(811)P
or SVARIANT(814)..(814)I or HVARIANT(816)..(816)N or
FVARIANT(819)..(819)N or DVARIANT(821)..(821)F or YVARIANT(822)..(822)S
or TVARIANT(823)..(823)N or AVARIANT(823)..(823)N or
AVARIANT(830)..(830)V or LVARIANT(831)..(831)G or EVARIANT(847)..(847)S
or GVARIANT(854)..(854)V or D 9Met Asn Met Lys Ile Leu Leu Leu Phe Val
Phe Leu His His Leu His1 5 10
15Tyr Phe Ile Xaa Gly Arg Thr Leu Thr Glu Xaa Gln Ala Leu Leu Ser
20 25 30Ile Lys Ser Ala Ile Thr
Xaa Asp Xaa Xaa Xaa Xaa Leu Ser Xaa Trp 35 40
45Lys Asn Thr Thr His His Cys Ser Trp Pro Tyr Ile Thr Cys
Ser Ser 50 55 60Ser Ser Ser Ser Ser
Ser Val Ile Ser Leu Xaa Xaa Xaa Xaa Leu Xaa65 70
75 80Leu Xaa Gly Ile Leu Ser Pro Asp Ile Gly
Phe Leu Thr Asn Leu Gln 85 90
95Asn Leu Xaa Ile Xaa Xaa Asn Xaa Phe Ser Gly Pro Leu Pro Xaa Ser
100 105 110Leu Ser Leu Leu Thr
Gln Leu Arg Xaa Leu Asp Val Ser Xaa Asn Asn 115
120 125Phe Thr Gly Pro Ile Pro Ser Ser Leu Ser Leu Leu
Thr Gln Leu Arg 130 135 140His Leu Asp
Val Ser Phe Asn Ser Phe Thr Gly Pro Ile Pro Ser Ser145
150 155 160Leu Ser Leu Leu Thr Gln Leu
Arg Tyr Leu Xaa Val Ser Xaa Asn Ser 165
170 175Phe Thr Gly Pro Ile Pro Ser Xaa Leu Ser Leu Leu
Thr Gln Leu Arg 180 185 190Tyr
Leu Asp Val Ser Asp Asn Ser Phe Thr Gly Pro Ile Pro Ser Xaa 195
200 205Leu Ser Leu Leu Thr Gln Leu Arg Tyr
Leu Asp Val Ser Tyr Asn Asn 210 215
220Leu Asn Gly Thr Leu Pro Leu Ser Val Val Glu Lys Met Ser Glu Leu225
230 235 240Xaa Tyr Leu Asn
Leu Xaa Tyr Asn Ser Phe Tyr Gly Glu Ile Pro Pro 245
250 255Glu Phe Gly Lys Leu Lys Lys Leu Xaa Thr
Leu Xaa Leu Gly Asn Asn 260 265
270Xaa Leu Ser Gly Xaa Leu Pro Xaa Glu Leu Gly Ser Leu Lys Ser Leu
275 280 285Lys Xaa Met Asp Phe Ser Ser
Asn Met Leu Phe Gly Glu Ile Pro Gln 290 295
300Ser Tyr Ser Leu Leu Arg Asn Leu Ile Asp Ile Asp Leu Xaa Xaa
Asn305 310 315 320Xaa Leu
Tyr Gly Ser Ile Pro Asp Tyr Ile Gly Asp Phe Pro Glu Leu
325 330 335Glu Ser Leu Leu Leu Asp Ser
Asn Asn Phe Thr Gly Ser Ile Pro Gln 340 345
350Lys Leu Gly Thr Asn Gly Lys Leu Gln Tyr Leu Asp Ile Ser
Asn Asn 355 360 365Asn Phe Ser Gly
Ser Leu Pro Xaa Ser Leu Cys Lys Gly Asp Lys Leu 370
375 380Gln Xaa Leu Xaa Xaa Ser Xaa Asn Leu Leu Val Gly
Xaa Ile Pro Glu385 390 395
400Ser Leu Gly Ser Cys Lys Ser Leu Glu Xaa Val Xaa Met Gly Asn Asn
405 410 415Phe Xaa Asn Gly Ser
Ile Pro Lys Gly Leu Phe Gly Xaa Pro Asn Ile 420
425 430Ile Asp Val Ser Leu Asn Asp Xaa Leu Leu Ser Gly
Gly Leu Asp Glu 435 440 445Lys Phe
Gly Asp Cys Val Asn Leu Xaa Xaa Ile Asp Leu Ser Asn Asn 450
455 460Lys Leu Ser Gly Lys Leu Pro Ala Thr Ile Gly
Asn Cys Xaa Xaa Leu465 470 475
480Xaa Xaa Leu Xaa Leu Xaa Xaa Asn Xaa Xaa Thr Gly Xaa Ile Pro Gln
485 490 495Glu Ile Ser Lys
Cys Lys Gln Leu Xaa Xaa Leu Asp Leu Ser Gln Asn 500
505 510Gln Phe Ser Gly Val Ile Pro Asn Asp Ile Thr
Gly Asn Lys Ser Ile 515 520 525Cys
Asn Leu Glu Lys Ile Gln Thr Leu Lys Leu Ser Asn Asn Ala Leu 530
535 540Thr Gly Glu Ile Pro His Cys Val Gly Asn
Ile Glu Leu Ile Ala Leu545 550 555
560Phe Leu Gln Ser Asn Lys Leu Asn Gly Thr Ile Pro Ala Asn Phe
Ser 565 570 575Lys Leu Cys
Asp Ser Leu Ile Tyr Leu Asp Leu Ser Asp Asn Gln Leu 580
585 590Glu Gly Val Leu Pro Lys Ser Leu Ser Lys
Cys Gln Xaa Leu Xaa Leu 595 600
605Leu Asn Val Gly Asn Asn Arg Leu Arg Asp Lys Phe Pro Ser Trp Leu 610
615 620Asp Asn Leu Pro Xaa Leu Gln Val
Phe Ser Val Arg Phe Asn Ala Phe625 630
635 640Tyr Gly Pro Ile Thr Ser Ser Xaa Lys Val Xaa His
Pro Phe Pro Met 645 650
655Leu Gln Ile Ile Asp Leu Ser Asn Asn Xaa Phe Cys Gly Lys Leu Pro
660 665 670Arg Arg Tyr Ile Lys Asn
Phe Ala Thr Met Arg Asn Met Asn Glu Ser 675 680
685Gly Val Gly Xaa Pro Gln Tyr Leu Xaa Asp Ser Ser Ile Tyr
Ser Ile 690 695 700Xaa Tyr Ser Met Val
Leu Thr Phe Asn Gly Leu Gln Gln Lys Tyr Glu705 710
715 720Lys Leu Ile Val Thr Met Ser Thr Phe Asp
Ile Ser Xaa Asn Asn Phe 725 730
735Thr Gly Gln Ile Pro Tyr Val Ile Gly Gly Leu Xaa Ser Leu Arg Asn
740 745 750Leu Asn Leu Ser His
Asn Val Leu Thr Gly Asn Ile Pro Pro Ser Ile 755
760 765Ala Lys Leu Ser Leu Leu Gln Asp Leu Asp Leu Ser
Ser Asn Arg Leu 770 775 780Xaa Gly Arg
Ile Pro Gln Glu Leu Val Ser Leu Thr Phe Leu Gly Ser785
790 795 800Phe Asn Val Ser Asn Asn Leu
Leu Glu Gly Xaa Ile Pro Xaa Gly Xaa 805
810 815Asn Phe Xaa Thr Xaa Xaa Xaa Asn Ser Tyr Gln Gly
Asn Xaa Xaa Leu 820 825 830Cys
Gly Lys Pro Leu Pro Glu Cys Gly Glu Arg Arg Ala Lys Xaa Thr 835
840 845Thr Asn Asn Gln Asp Xaa Pro Lys Asn
Asp Asn Glu Arg Met Leu Ser 850 855
860Met Ser Glu Ile Val Val Met Gly Phe Gly Ser Gly Val Leu Val Gly865
870 875 880Leu Ala Trp Gly
Tyr Tyr Met Phe Ser Val Gly Lys Pro Phe Trp Phe 885
890 895Ile Lys Met Ala Ser Lys Met Glu Ser Ile
Leu Ile Gly Phe Phe 900 905
91010201DNABeta vulgaris 10agagcagatt ggcatacttr tgaatattct cactggctat
taaattctca gaagaaaaat 60caacaccaag attatgacat gcttgtgcaa agacacaccc
rgtcatgaat gcatcatagc 120cagcttcatg cttagcccca gagttccaat ttgaggayct
gcaagaaaac atgggagtaa 180gatggtttca cataaaacat g
20111201DNABeta vulgaris 11gggtttcttc gaagtttgat
tttgttacat ttttcaaaga gaaattagtt gttgatgttg 60aataatgatg ataagtagtt
agggttcgta gtaaggtgga sgaragagaa aatggcgtca 120ctctgayrag cttcttcatt
ttgttcttct tccttagctc tgttttcagt cactgcgcca 180tttttttttt aaaaggaaga t
20112134DNABeta vulgaris
12caagcacaaa atcaaataat gagaatcaca ctatccaaag aaaatttcca tccacattta
60tccaacacar ttatctctct tttacaccca aattatgtca accaaaaaca staaaacaag
120tgagtgcagt agct
13413134DNABeta vulgaris 13taagtaaaaa gtggtaaaag aattaccaaa arcgcacara
ataaattaat tagytggatw 60taactawtta acctattcct tttttctgtc gctataacta
cttttgctta acttattgat 120ggtttgatcg ttga
13414150DNABeta vulgaris 14ttataatgta gagtcaaaat
taatatcctt aactagtttt taagtccggg ttatatccta 60gatattwata atattcattt
attagtaaca ttttatttta taaatataat actaagcatt 120atttggtttg ctggttaaga
ctttagtgta 15015201DNABeta vulgaris
15acatctacac tgggagactg ataaggacgt ttgcagatgt caagtatggg aatcatcatc
60taacatgggt ggagattgtg tacaatgtta tttcattcat mgtggcaata attaccattg
120ttgcgtttac tgtatatgcc aagagagcct tcgaagaact taagagggca gaagctaagg
180aggatcgaga agaagaaacc t
2011628DNAArtificial Sequencegenomic target sequence with 5'-flanking PAM
(4 bp) 5'crRNA # 1 16tttatttcga tttcgattct tggattat
281728DNAArtificial Sequencegenomic target
sequence with 5'-flanking PAM (4 bp) 5'crRNA # 2 17tttcaaccca
gtatccttat ccgtcact
281828DNAArtificial Sequencegenomic target sequence with 5'-flanking PAM
(4 bp) 5'crRNA # 3 18tttatttaaa catgatacgt atcatatt
281928DNAArtificial Sequencegenomic target sequence
with 5'-flanking PAM (4 bp) 5'crRNA # 4 19tttaaacatg atacgtatca
tattgagt 282028DNAArtificial
Sequencegenomic target sequence with 5'-flanking PAM (4 bp) 3'crRNA
# 1 20tttgtgggtg ggtggttttc acgtgtgt
282128DNAArtificial Sequencegenomic target sequence with 5'-flanking
PAM (4 bp) 3'crRNA # 2 21tttcccctcc ctttgccgct gcgaagtt
282228DNAArtificial Sequencegenomic target
sequence with 5'-flanking PAM (4 bp) 3'crRNA # 3 22tttcttcttc
ttgcttccac cataacac
282328DNAArtificial SequenceMutagenesis primer 1 23tcagtgcagc cgtcgtctga
aaacgaca 282428DNAArtificial
SequenceMutagenesis primer 2 24tgtcgttttc agacgacggc tgcactga
282520DNAArtificial SequencepSeq_CRBM4_F1
25agcgcaacgc aattaatgtg
202620DNAArtificial SequencepSeq_CRBM4_R1 26gatgaagctg aggtagtacc
202721DNAArtificial
SequencepSeq_CRBM4_F2 27aggaaggtta gcaagctcga g
212820DNAArtificial SequencepSeq_CRBM4_R2
28tctcgtcgac cttctggatg
202921DNAArtificial SequencepSeq_CRBM4_F3 29atgctgagta cgatgacatc c
213022DNAArtificial
SequencepSeq_CRBM4_R3 30tagacctgct tctcaacctt ca
223120DNAArtificial SequencepSeq_CRBM4_F4
31accactcact cctcgataag
203221DNAArtificial SequencepSeq_CRBM4_R4 32aacgacaatc tgatcgggta c
213328DNAArtificial
SequenceOligonucleotide fw for the generation of short 24-bp
protospacers (5'crRNA # 1) 33agattttcga tttcgattct tggattat
283428DNAArtificial SequenceOligonucleotide rev
for the generation of short 24-bp protospacers (5'crRNA # 1)
34ggccataatc caagaatcga aatcgaaa
283528DNAArtificial SequenceOligonucleotide fw for the generation of
short 24-bp protospacers (5'crRNA # 2) 35agataaccca gtatccttat
ccgtcact 283628DNAArtificial
SequenceOligonucleotide rev for the generation of short 24-bp
protospacers (5'crRNA # 2) 36ggccagtgac ggataaggat actgggtt
283728DNAArtificial SequenceOligonucleotide fw
for the generation of short 24-bp protospacers (5'crRNA # 3)
37agattttaaa catgatacgt atcatatt
283828DNAArtificial SequenceOligonucleotide rev for the generation of
short 24-bp protospacers (5'crRNA # 3) 38ggccaatatg atacgtatca tgtttaaa
283928DNAArtificial
SequenceOligonucleotide fw for the generation of short 24-bp
protospacers (5'crRNA # 4) 39agataacatg atacgtatca tattgagt
284028DNAArtificial SequenceOligonucleotide rev
for the generation of short 24-bp protospacers (5'crRNA # 4)
40ggccactcaa tatgatacgt atcatgtt
284128DNAArtificial SequenceOligonucleotide fw for the generation of
short 24-bp protospacers (3'crRNA # 1) 41agattgggtg ggtggttttc
acgtgtgt 284228DNAArtificial
SequenceOligonucleotide rev for the generation of short 24-bp
protospacers (3'crRNA # 1) 42ggccacacac gtgaaaacca cccaccca
284328DNAArtificial SequenceOligonucleotide fw
for the generation of short 24-bp protospacers (3'crRNA # 2)
43agatccctcc ctttgccgct gcgaagtt
284428DNAArtificial SequenceOligonukleotid rev fuer die Generierung
kurzer 24-bp Protospacer (3'crRNA#2) 44ggccaacttc gcagcggcaa
agggaggg 284528DNAArtificial
SequenceOligonucleotide fw for the generation of short 24-bp
protospacers (3'crRNA # 3) 45agatttcttc ttgcttccac cataacac
284628DNAArtificial SequenceOligonucleotide rev
for the generation of short 24-bp protospacers (3'crRNA # 3)
46ggccgtgtta tggtggaagc aagaagaa
284724DNAArtificial SequencePrimer pCRBM4_F1 47cacattttat ccaatcaaac tttg
244823DNAArtificial
SequencePrimer pCRBM4_R1 48ccttcgagaa ataacatggt gaa
234925DNAArtificial SequencePrimer pCRBM4_F2
49gtacagtgac ggataaggat actgg
255025DNAArtificial SequencePrimer pCRBM4_R2 50ttagtggtca aacataggcc
tttgg 255123DNAArtificial
SequencePrimer pCRBM4_F3 51agtaagaggt tgcctaattg agg
235223DNAArtificial SequencePrimer pCRBM4_R3
52ttgccgctgc gaagttccct ctc
235342480DNABeta vulgaris 53aaatgataca ggggtatatt tgactctatg aatttcagaa
atctaatcaa atttgctaag 60cttccaatga ttctactaag ccctacaaat tacaagaatt
agttactttc atctctctgt 120cggcttcaga accagaagtg tacaatatct tgtcaaacaa
actctgctta gaggagctct 180ttcgatcatc ttttttcgat ttggaagttc ccggtgatag
gattgacatt gctgttttct 240cggtcaattc ttctggatct tggttctgtc catctatctc
tggctccatt aatctggtct 300tccaattaat tccgatagcc tcagcttgct ctgcaaacaa
gacctttgag atcggggagc 360tgcagatatc cttataaact tcataaccag cagcacaggt
tttcccacct tccaacaact 420ttgataaagg atgtaggaga gagatagaat catcactcgt
ttctaaccta tccttcaagg 480caaggaagtt aacagccaag tctgccttac taaactgaac
aaatactgca gtttcatcca 540agttatagat gcaagcaact gagtatatac caaacacttt
acagatgcat tgttttatgt 600cacttgcctt gagttttggg gagaatcccc aaatcaaaac
tatgttagga tgcaaaatgt 660taagaaacct ccttttagct gaactgataa cgggaatttc
attcatatca ccagtgctta 720gattgatcac atctccactg ttccaactaa gatagagcag
attggcatac ttgtgaatat 780tctcactggc tattaaattc tcagaagaaa aatcaacacc
aagattatga catgcttgtg 840caaagacaca cccggtcatg aatgcatcat agccagcttc
atgcttagcc ccagagttcc 900aatttgagga cctgcaagaa aacatgggag taagatggtt
tcacataaaa catgtgtaga 960agtgcagtga acactggcga aaacaatcta attttacgaa
ttcattcact cactcagctt 1020caaattaagt ttccccttta tttagggtgc cccaaaaaga
tacactcttc tgtttacctt 1080ctctctccaa gcgaccaatc ttttctctct tctccaacat
cgttttcttt ttctctctct 1140acccactatc cattttgtcc tcctacattt gataactatt
cttaatctcc aagaaaatcc 1200aatgtgtgaa ataattacgg gacagggagt atacagaagc
agcccccttg ccaatatagt 1260ttacaaatta ccctcagaat taggcttacc tttcccaaag
gagcaataaa ttcaaacaaa 1320tctaaaaggt acaaggcatt aagtgccgaa cctcatgtca
tcaacctgga cctccacctt 1380cacacatgga tgtacaccac cattagagga ttgtccagag
gctatctcag ggcacaacag 1440agaaaatgct gaggccaatg acgtgctggc tttattcaag
aatttttgaa ggctcgtgtc 1500tgcattcaaa agtattttcg tgtcgacaac atgaggaaaa
tacttgtgga tctcgagaac 1560aaactcttca acagttgatg gaagaggacc aaagaattta
tggtaaatat gtgccatatc 1620tgcaaaaaat tataatggat aagatgacaa gaaaagatac
taggaaggcc ttcaagtaca 1680aatattatat catgatgctg gacgaccgat gctcccacaa
ttatgtttgt taccaaatgc 1740ttcgaaggat aattactaaa ttatgtgaat ggtggttacc
aagtgtcccg gaccatgcaa 1800taacttctcc tttcagtgac caacaagaag aagacgtacc
taaaaagcaa ttgtgaccta 1860caattagctt cttttcagca gcgagaaggt caaggacatg
ccggaaacct gcagctgctt 1920ttattttgcg agttgcttgc tggtgagacc catacttcac
ctcctcctac aagaacaaac 1980agaacaatca cacatgcaga aagttcccca cataccaagt
tgctgtctgc taaacactga 2040aactaactta tctctacaaa caatgaagga agttcctcac
cagaaggttg atcttatcat 2100tgtcagattc tacaaaaaca ataagcttct gcaagatggc
actgccgtca tgagcacaca 2160caaaaacaag atccttgaag tgcttccttg taacctgtaa
ttgcagatca ttagtatata 2220ttcaagatgt tataaattta ttgaaaagca gcgtctaaaa
caataaaagt catgcttaag 2280gcatagagcg atagagcata gacacttcag agtttaataa
gagcaaatac tccaggagaa 2340cataaatata tttcatatca caaatcctag taccaactgg
caacggctaa ctgccaattt 2400atgtactgct caaaaaggcc aagcatctaa aagatggctt
aaaagtcgga ttttataaga 2460aagtcgtcac atgattgcta ttacattgac atatcaaagg
tcaaatgctg aaatttggtt 2520cagcttgata tatattaagc atacaaacga tacgttgaca
agaaagccta acaagacatg 2580aagcatcagg cacataacat tcaaaagatt accaattcaa
tcaacttcag ctgatgagaa 2640gtaaatccat tcaatcgaag agcaggacgc atactaaaaa
atatggtttc aaattgttgt 2700ttagattcaa cagcacttgg aagctcggca tttctgttct
gtagcaacat atcatgccaa 2760tcactgagcc gaattttcat ccgttcggag aataaaatat
cagctacatt gcccaaaggt 2820aaatctctaa cttcattaga atatgcccat ttcccgttat
atactgaatt caagcgactc 2880aaagcctcgt cttcctgtcg tctagataaa taagacacgc
ctgagattat acaatgtgta 2940aatttaccac aataatgaca tcatactgac aaaatctcaa
acaaatagtt ctaataaagt 3000catgttatct gaaatttcta tgaataggaa attgaacaaa
accttcatgc ttttttccaa 3060ctaaaattga catcttctac attaccttca tgtatgcatg
cattgaagtc aaactggtat 3120tttgccaaga agtcaatcga agttgtttgg cacaggaatt
catatgatgg gccatcagtg 3180ggaagctctt gacgtggaaa tatataaaaa ttatgcctga
caatgaacca ttgtaaaatt 3240attagatgga gtatctctat ttattgttta cagccaattg
agcttttaac aattactata 3300ggtagtgttt ggaaacttgt atttcatttc aaataatgga
attgaaatct ggaatttaaa 3360gtttgtattt caattcctaa tcactgtttt gtaaaggggg
tttgatagaa gagagagaaa 3420tagaggttta atggaggaga gagaaaaagt gtgggtttac
taaaaaaaag agaaataaat 3480attagaaagt gtgggtttac tcatagagtt gggatatgta
tgaggagaga attttcaaat 3540gccaaggtaa tagcttgaat gacaaattta ataatttcaa
attccatgtc atccaaacaa 3600tagatttcat ccaaatccaa gatttgaaat gaaatcttgc
tatccaaaca tatcataaat 3660taattagtaa tttagacttg ctttctgctg cacttactta
tggaaataat tttacttcag 3720tccttaaata acccgcaatt tacatcaaag gcactaatat
aaacacctag ttacgaaatg 3780gaaatatcag atatacctgt aaaagtaaag aaacaaaaat
acaaccctga gcatgaaggt 3840atccttcaaa agtgcaatat ctgcatactt agaaccggaa
ttagaagtgc gaatgcagac 3900aataaccatc ccaggatcag aaacgtccaa gaaagttgag
attcatcatc cattctcttt 3960agcaaattta tgaactctaa tatataaatc ataccccccc
ccccatccaa aagcaattgt 4020caagctgcct gaacccctca taatttagga tacaacaaag
taatcctaaa agacccttta 4080caatactagt actcgggtat ttccacaatc ttctcatcat
tgaatccaaa gcattgcatt 4140tgaagaaatc aaatcataat ccattactat attagagcaa
aatctatgtc attatagtat 4200tggagagcaa gtatgactat taccccttta cactaggcaa
aacacattgt cacaatgcta 4260acttagtcat taaccaatat caatatggga ctgtggatat
tcataaaatc gaagtttttc 4320gcttgctcat aaactatctt tcattccagc acagtacaag
agagaaaaga cagcattttc 4380atacacttct ttctttagtt caaattcaca cagcagcaaa
aaattcactt cttcatagct 4440ttagctcagc aaacaaagca caaagcatgc aattactctc
acacatagca caccaaaaaa 4500acaaaaacca ctaaaaattc acacaaaaaa aaccaacaaa
aattccatcg caatttcaac 4560aatcaaaaca atcttctaag ttaaaaagag agataaagat
gagaagaaaa actaacggat 4620gagcaacgaa ggaattcttc gaagaatccc atcgaaatgg
acaaacacca aattgaacaa 4680cggcgaattt ctcagcagaa tctttcattt taaggtatcg
aacatcgtgc cgatcaaact 4740cgaacgattc gcgccaaggt gagcttgtaa ttccagtcat
ttcgagatca atggcgacaa 4800aatcggcaga ttttacatgc gtagtgaggt caattagggt
ttcttcgaag tttgattttg 4860ttacattttt caaagagaaa ttagttgttg atgttgaata
atgatgataa gtagttaggg 4920ttcgtagtaa ggtggaggaa agagaaaatg gcgtcactct
gacaagcttc ttcattttgt 4980tcttcttcct tagctctgtt ttcagtcact gcgccatttt
tttaaaaaaa aggaagatga 5040acaaagcaaa tattgaaccc aaattttgta attttggccc
actttatatg tacccctccg 5100tttcaaaata tggagcacgc cgcacacacg acatttaggg
tcgaattttg aacattcttc 5160aagatgatct aatggtataa tctctataat ttatatgtgg
catattataa taagagtttt 5220atgaagtcaa aaagtggatg tcatatattt aatgcatggt
aagtttttcc taaatctgta 5280tactagggta acatacatat gttgacttga agtatatata
attcttgtag tataaatatg 5340gctttggcca taagtagtaa tacacaacaa ctagaaaaat
tgaaatcagt ccactgttat 5400cttgtactct ataattttct gtttcctttt gtttcgcaac
aaagacatat ttgtggtgaa 5460agataatttt cgtaaattga atgacttata ttttgaaata
aagagagtat taggtaaggt 5520tacgtgcttt tcgcttgaat ttgttagacc tcaaatgtat
atgtgattag aacggattgg 5580ctctagtttt tattttatag aagtatatat gcatttttct
tagagcacac tcgaaattac 5640tttcggatag atatattcgg gaaaaaaaga ggttgaaggg
aagttcatca ataattatgg 5700taaaggaaaa aggacatcgt tacaattcta aattctagat
aggatgtgat gataatccaa 5760aagtcatctg aaaaactaaa caagtccaag atgctaatga
ttcgagtaga gattgaatga 5820gtgaccctaa ggattgtcaa ccctcttatt ctaacgtgtg
taaaagaatt gacaactcta 5880agagttactc aaacattttt cgattcgagt ggttaatata
ccaatttgaa actattgaca 5940ggagttattt taatgagtat aatggtcaat ggagcactga
attccatctc acatagtcac 6000atatttcatc tcaagttctg atgatttcaa acattgaaaa
aagatgatac aagcaattaa 6060ttcctaggga aacatattgt ggttttcatg gatacaagag
tgagaataaa tcaaaactta 6120ggctctaaca tttcttttct ctactagtaa ttgctaatta
tatcaattca attgtcagtg 6180taatcagtta atcaccaaat ctcttgtata gtcagtaaac
tatacactgt ttagtcctct 6240ggattttgcc cggtcgaatt atgcagcata accaaacttt
gaagtttagt acttcctttg 6300cacccaagtt agcttcacgg cccctgcctt ctggtggatg
gtcaccctat gctttgagca 6360ttctctgcaa tgcgcacgat attcaatgag aacgtcgcct
tgaaaatcta aattgcaact 6420aaaaattaga ttgaaatgaa acccacaaga gttgtttttc
tgagtagttg gtgtagaatt 6480cacaagtctt gctccattgt ttgaagatat gaagacaata
atgtgctatg taaagtgcag 6540ccgctagcta acagtggaag tggaaacttg atcattttac
actcgcacaa gcgaaagctc 6600ggctgacgtt gcaaactgaa gaaaaacctc tcaaaccaat
tcgacttttg ctcaaagttg 6660caaactaaag aaaaaggctg aatgcaaagc aagttcacca
atgaacaata gatcggtgtt 6720ggcctgaggc cacatcaagt gaagttgcct aattgcggcc
ctctcatctg ttcacaggaa 6780tcattttcca tatagaatca ctccaaaata aaagagcaaa
gctgcaccag atgcagaagc 6840ataactttca agacaactga tgacagataa atagcaaaag
aatgcttaag aaatgatcaa 6900aattgaatgg ctctggaatt acctcatcag ctgattttcc
tttctctcta tctctctatc 6960tctttactcg tctatggagc taccacatca catggcgttt
catatgcttt ctgccgtcga 7020actagacgtg cagcaaaagc tccatccatt gaatgcttca
ctgggcatga gcgataaaac 7080ccatcttcag ttaaaaagtc agatggaaca tatctgctta
cagaatctct ttggaagtcc 7140tatcacccaa caaagaatat attaaaatag agaaggagaa
aagaacgtat ctatctgtca 7200gcatccatat gaggtggaaa ctaggagtac tatataaagc
cagtgcagta gctcctaccg 7260gatgtctaag aaggaaggca gaaaccctat cttcgttttc
ttcaagatca atggagcagg 7320tactgtacac aagcacgcca tctggtttga ccagcctgta
gatgttaaac aatcccacag 7380acaaaaggga ataatatgag tgaaacaagt caacaggggg
aaataaccaa taattctagg 7440actgtcaaac tcaagctctt caaaaacaaa gatagctctt
aatctcactt gcaagcagca 7500tcgaacagct cgtcctgcaa cttctttagc tcttccatat
cctctgactt tctattccaa 7560cgcaaatccg gcctcttcag caaaaatata aagttggaac
aaggctctta gatacaagaa 7620ctgaaaaacc ttcgacatat aatggactct atcaagggca
caatgacaaa ttctaaacat 7680gagcatgtat atcaataaaa tactaagaac cctttcaatg
gtactgctag aaggtttatt 7740gctacacttt ttagtacacc atctataggt tttatagtac
catcaaaatg gttcatggtg 7800ccataagaaa attttatgta tttatggtac tatctaccat
atctaatttt ctctgtaaaa 7860atgtatttgt agatagagac cacgagttcc tcttttagat
actgactttt ttttttctac 7920atgatggcca acagacttct caaacaaaaa gaaaaagaaa
atatttagat aatatgagca 7980acaaaatagc aaccacctac ttttgatagt acacccaggc
ccgaacaagg aacatctaaa 8040agaactttat caaacttcga agtgttgctg tcctgaaaag
aatagaaagt aactgcttca 8100acaaagaaga agaggcagaa agcaaagcta gtacgcattt
tgcaatgact tactgaaaag 8160gagcgaagat cagcatggat gcaagtgatc acattatcaa
cacgctgcag cttggctgtt 8220tcttcaagta tccgtaaccg acctttattt atgtccattg
ctgatatcat acctgaaaag 8280ctacacattt agaatgcaga accagcatca ttggtagtta
agttatcact ataccttggc 8340cattcaagcg agatgccatg aagagtgtct tccctccagg
agcagcacag caatcaatga 8400tgtgatcacc aggctgtgga tccagaacag aaacagctag
acctgcagcg aagtatagat 8460gtaaacttgg gttgggctgt cacatttttt cacatcttat
cttcctttct attctttcaa 8520aactgaggag aaatggttgg gatttctata aacgtgagaa
aaatggcatc agattagatg 8580gttttactgc atgaaaaaaa ttgaatgtgt ttcggcatca
cattactaca aggtcaaaag 8640cactatcttt gaaaatgtag gacataatgg gacagagatg
tgctgacctg cactctcatc 8700ctggactgag cataaacctt cttttagaag tccagtttgt
atcacaatct aggatatgag 8760aaaacaactc aagatgtaat tgctcctaag atatcaatca
tttcataata aacataaaag 8820ttattattac aagacagcac ctgcatccca cttctgatgc
agacaaagtc atccaaatgc 8880aaggaaggct catgcgggac ctgcggacaa agtgttgtga
tgcgcataga tatcgaaaga 8940aggccctgta tagcactaat gagataagat tcagtaacct
tcagcatgtt gagcttcaca 9000acaaggtcat ctcgagttaa tccttttgca atattggccc
tagtaccaag aaaaccatat 9060gtattaacaa gagaaaagtg gcatagggat ctttatgact
taggtaagca gttggcaatt 9120agagagaata aaacccccaa acctcaagct gaaactcgga
acactattgt tccacatcat 9180caatttgata gctccttctt gcccaagata cttggtccac
cgtcttacca tccactgaaa 9240ggaaggtatt aaaagggaga aaagactcgt cagcatagaa
aattgtacat cttaaatttt 9300agaagtatag caccatcttc aggcatcagt caacgtaaat
aaataccaca tctacaaata 9360gaaccatact ttctggacag tcgggatcat gagcacagac
aatactgcat gattattgcc 9420tcgtattctc atgtatacaa gtatatgtaa cattaaatag
cagtatttct tgagaaactc 9480accacgggat gggaataagt tgtagcaagg gcacgtgctt
gtgaacgatc atcaccctcc 9540aatttgggta caggaaggga gtcattatcc tataaagaga
aacagctttt gttttcaacc 9600atatcaagac aaacagttta ttaaactata aacaacaaca
atacacatgc acacacctac 9660tgggaacaag atatatacta ctgataagta ttttctgatt
gaagaaaaaa aatctcattt 9720atttgcaaat atagatttaa tgacaagaaa gctttgaacc
ttaaggaaaa ctagctttcg 9780gaggatccca ttcaccatgt ttcctgcgcc tggtctaaga
gcatacttgg caagattcac 9840attctgcaat ataatccaac agtaagaaca cgacatggat
ttagactcaa gtctctgaac 9900ctatagaaca agtaaaatta gatcttatct catttgacaa
tttaaaatta gatagtgcaa 9960tattctgcag ttataagact tcatgtgtgc atactgcaca
agtcatctta aaggtgttat 10020taaagcttta attgccattt gacatcccct tgctcaactt
tagcatgttt ttaggctaca 10080acaatacgca ctgtctacat ggacatacaa attacaagcg
tatggaaaag caataagcgc 10140aaggaagtct tcagccagaa actctctatg agtccaacaa
tatgcaacta aatatccaag 10200taccgtgaat gagtaagaac taacctcgtc aacaacagca
tatggtggca tttccagttt 10260cacaatctca tagcatccaa tcctgaggat ctaaaattaa
agataaatca atacacaaca 10320tatgatatgg gtcggagcgt atataacaag tatagcaact
acatttgaac agataacagc 10380ctttgagaca ataaggaact ccgacattcc agtatatgcc
agatttcata tctttagctc 10440taaattgcca cgcaaaatgt tattgggcaa tatacctgta
gcaggagagg ttccatgttc 10500ctaaaggagc tttcatcatg gcatgaagaa acaataagat
aatccagata ttttctccaa 10560cgaattgaac caccaacaat gtcagtgacc tacaaagaca
agttgtcaac ttaaaacttt 10620tgaagcgtca tttcacttct gtagaccaat acaaaagcta
ctactgcttt acatcataaa 10680acctttagtc cttaggttca tctgattggc aaaaaaggtc
cagatgcaag aaaagcaagt 10740agctgtaatg ctgtattata tcagcattat tcagaacaga
ataataaata tctacagatt 10800ttgggtggaa gcttgatgat agagtatctc cacaaagaga
actcgcttga gtcccaactc 10860ccaaatctac ttttttggag tcacattatc agtcattttt
tctggactct tataggaata 10920gtgtgctatg taatgattta tggagcaggg gcatttcatg
aatagcttta taagttagta 10980tgggtgtctt ggggaataag ttaaagggtt agttagaggg
aagaagtaca acatatatat 11040agagcttttg taagaagggt ggttatgttg aaaatagatg
agaaattggg tgagctcata 11100gtagttcaat ttggactttg ggagagaatt aagcctcttg
aaagcttgaa tatcatttac 11160atttgttgtt tttactctta ttaatcaacc aaagttcatt
ttcttccttt aatttctcca 11220ttttagcact atgatttgtc caagctaagt gatttcttag
catagtgcac agtgtagtat 11280atcggagaac tcatttgagt cctgaaaggt cccacaagtt
acatttttcc tactactact 11340tgcaccaaaa caataagcat cattaagaca ttgtcactgg
tccttcttag gttcttttgg 11400aggggattcc tcagatgggg gaggcaccca tgaaggaaca
tgttaccaag caatgggaca 11460atgcaaaatg caccaataca gtagcttcac ttcattgatt
gcatctatgt cacggaaaac 11520tgaagaaaga agcaacacct caactttatc caggacagat
atccactaac ctaggatgca 11580agcttgagac tatttagcaa ttgcctctgg gatattaaat
cagattacga ctatatttct 11640acagttattg cttaagaaaa aggtacgatt tgaagcttgg
gaagaaagag aacaagagta 11700aaagaccaat ctgagatctc tttcatccag gtctctggtg
cgaaatccaa gagtcctctc 11760aacatattcc atctcattgt tccctgaacc ctttcctctc
tcatttagaa gatcagcgaa 11820ggcaccacca aactctatcc gcatcaatct cacagcagcc
actggattta cacatgaaag 11880caaaccagga gaaccataaa aatcacaaca aacttcctga
tagcctactc actagcatca 11940accattgtgt tcagcctaaa atgagcggct gttttcaatt
gaacagcaac ttacatggac 12000cactgcataa aagtgatttc ttaatccaga caaacaaaaa
tgtttacttc aaccaactga 12060atttgcatca gctcattagt gatttgacaa gttctaattt
atgtatcaac aaacaagacc 12120atatagctag gaaacaagag gcttaggcta agcttaatgc
gtgaacaatg ttagatttca 12180acctatcagc actgtggata actgcaaact gcgacttaaa
taaggaagat aaaggaactg 12240aatatgcaat ttcaaggtgc tcagcatttg aatcaacagt
tacttcagat aattcagaac 12300ataaaagatt tgaacattct aaggctacct catgattgca
agcaatgtta cctgattcgc 12360taaccctcac aagccacaag ccaaagaagc aatttggtaa
atggttcatg gtacaactgt 12420tcgcttttgg actaatctaa caatactagg tggtaaatta
tgttcccata tctattacca 12480taatgtacag caaattaggc agcactaatt ccaaatgacc
caacaaaaaa agaggaagaa 12540aatccaaaaa ttcaagccaa catatgcact aaaattacaa
gcacaaaatc aaataatgag 12600aatcacacta tccaaagaaa atttccatcc acatttatcc
aacacaatta tctctctttt 12660acacccaaat tatgtcaacc aaaaacacta aaacaagtga
gtgcagtagc ttcacatcaa 12720agaatatcaa tcacaaacac cacataataa aatttcaact
cctgcccaaa caaaaaaaat 12780ataaagaaaa aaaaacagca aaatttcaaa gataaaatag
aaaaaaaaaa atcaaaatac 12840agggggaaaa aaagtaaatt taccagctct atgaggcgaa
acctgcaaat tcagcttctg 12900ggttttctct gaaatatcaa gcacaataac cagcaattaa
aaaaaattat aaataaaatt 12960aaaaagaaaa gattgataat taaaatcaaa agagagcaat
ttaaagcaca atcctttttt 13020taccattttt tctgggagga agagcatcct tcgttttggg
tttagacgaa aaaaatgaga 13080gttgttgtat ttgtgcgcat gagtgatcat tgctggaaat
gaaagtggga aagtggtaaa 13140tgagtgcttt gtgaaattgg gttttgagga aaagtagaaa
gaagaagaag ggtcgatgtc 13200agagaagaga gagagtggat ggaaagtagt gatgattgcc
tccattgttg ccggtgaagt 13260gagctttctg caaatatttc actggactag ttttttttag
cagataacgc taaaacagag 13320aaagatgttc ggttaatttt aatttttgga catttaaatg
actattcaat atgtttcaac 13380cttttttttt taaaacaaag gaacaatact agtattagat
tacgttaatg tttagtacat 13440ccaatactta tgtgtgtttg acctaactta aaatcgtaag
ttgtttaaaa tgtcggtgtc 13500ttgtttttaa gagatatcat acttactatc tttggttttt
actcttccat tgttaacaga 13560aactgtattt atttgggtaa ggggtttgag tgaattcctg
taagtatgag aaagttttga 13620gtgaagcaag agaaagagag aagaaaggaa cttcgagtga
agattgagag aaacaacagt 13680tagtgggaac tgttgttggg aacttgagtt taggagctca
ggttgtaccc cgagagaatt 13740aataggtttg taacagagtc ggtggcctat tatagtggaa
agtttgagtc aaaatccatt 13800gtggccgatg tcgtttcttc ttattgggcc taggaagttt
ttcctcgcta aaatttcctg 13860tgttcccatt gtgtgttcct tagctagctt tcaattccgc
aaaaagttac gtttattctc 13920tcactataat tcacccccct cttatagtgc tcatattata
caacaattga tatcaaagca 13980ggaactctaa aaatacagaa atcatgttga gttcaagatc
ttggaaaata tgaatactac 14040agaaaaactg gaagaaaggt actctactca gagaccaccg
atgttcaatg gcaaattcta 14100cacaaactgg aagaactgaa tgaagatctt catcaaagcc
gacaaatatc aggtttgtag 14160aatcatagag gcaggcgatt ttgaagtcac taccactaat
gacacatatg aggtaattcc 14220taaattcata actcatttcg ataaagtata tttcgaaaag
ttggaaatta acgttcttgc 14280tattaaactg cttcattgtg gtcttagacc tcatgaacac
aatcatgtca tgggatgcaa 14340aatcgcaaaa caaatttggg atcttcttga agtcactcat
gaaggtacgg gtaaagttaa 14400gagatcaaaa atcgatcttt taatgaatca atatgaactt
tttcaaatga aatataagga 14460gtccactcaa gagatgttta cacgctttac taatactatt
aatgagctaa cctctcttgg 14520aaaagaaatt acatatgatg aacaggtaag aaaggtccca
aggatcgttg gatggctaag 14580gttacgcctt acaaaaaact aaggacttta cgaagttcaa
tccggaacaa cttactggct 14640cccttatgac tcacgagcta cacttggaca ctgagaatgg
tgacttgtcc aaacagaagt 14700cgattgcctt gaaagccatt tttgtcatac cgtcaattaa
ttaagtaaaa agtggtaaaa 14760gaattaccaa aaacgcacaa aataaattaa ttagttggat
ataactaatt aacctattcc 14820ttttttctgt cgctataact acttttgctt aacttattga
tggtttgatc gttgaatcca 14880agttttctcc acccacaaag atattataga ctttacttta
aaaggtacga taaataatgt 14940ttaatcaggt atgcatcaac cttgaaatta ttaatttatt
aagatcaaat tatgcatatt 15000tatattaaac gtacaggact tgtgcacaat ccatggatga
tattgtagat tttgttgtaa 15060aggagttagg gacaaatgat gttgaattaa gaatgatgag
gaacaacatt gaggtaccta 15120atggcataca agattatgtg gtaacaaagg tgaagaagtt
ggttgtacca ggcaatacag 15180cagcggcaag ccatatatag gatgagctac cataccctta
tgttgtgaac tattgtcacc 15240accaacaaga cattggtcat tacgacatca ctttagttga
ggaatgataa acctcttttt 15300gctagatatt tgcaaacatc tagcagataa agaggaataa
aacactattt atatttcatg 15360aacactattt gttagttgca tgaacactat ttttagttac
acgaacacta gttttagtag 15420catcatgaac actatttttt agcatcggaa ttttcacgac
tactttttgg tttgactgac 15480actctgcaat tttcgagata actttttggt gatatgggtc
ccatgaaata gaagatttat 15540atttcatgaa cactatttgt tagttgcatg aacaatattt
ttagttacac gaacactagt 15600tttagtagca tgaacactat tttttagcat cggaatcttt
gcgactactt tttggtttga 15660ctgacacttt gcaattttcg agataacttt tttgtttgac
tgacaactat ttcctatata 15720tattgacagt tttacccctg ttagatgttt gcaaacatct
agcaaaaaga ggtttatcat 15780tcctccactt tagttagccc aacctccagt aacgccatcc
agaccactgt cgtttgtcac 15840tacgacactt acgcttggca accctatgtc ctagcccttc
gatacctcga tatccgtccg 15900ggcaatgtcc ccagtttgtc acttctctgc cattaatgac
atattttgga gtatcaaacc 15960caactccaag tatatatcgc aacatggctc agtaaagaga
gtcatataat catgacgtag 16020tttctatatg ccatcctacg tagtatcttg taacatgaat
aacagcctgg tttgcaggtt 16080gatggtacat ggtataaatt ggtattactc cctccggtct
ttattagttt aatcctttct 16140tttgtacaga gttataggag aaataatatt gtgggtcata
gaaggaaaga gaaattatta 16200ttttatgtta aagttgaatg tatgtgtgat gaaaagttag
tagtcccatt tcaaaataga 16260aaaaaaaaag gtaaactaat aagggacatc ccaaaaagga
atacgggtaa actaataaat 16320atccatgcag gttgttggta catggtacat gaagccgtcc
aaaaccttca aaagcagtaa 16380gtcctgctgc tatgccatat tcaaatattc aactccaaaa
aaaaaaaaaa aaaaaatcaa 16440aaatccgctt ttcagcgaaa atataggaaa taatccaaga
atcgaaatcg aaataaagtc 16500atgatgcaag tttggagagc tgaagttaca ctatatcgga
gtacttactc aaatgttgat 16560tagtactccg tgcgtttgaa gtaaagtcac atatggagta
gttccaagct aggttgtaca 16620gtgacggata aggatactgg gttgaaaagg tgaacgtcga
gatttatacg tgtatttatt 16680taaacaggat acgtatcata ttgggttctc atacgcgtac
cagctgtgac ttagaaaaat 16740taaccacgct atataggttc caagccctca tgattacctt
ttcatagtgt aaatttcatg 16800tagttgaatg gtgggaatcc aatcacaaaa acactgcagg
taatggaaat gttccaactt 16860tttccaagca ttttaaaata agacatgtga ttactaatta
gggcgtgttc ggcaacagta 16920attgtggtga tagtttttag ctgtgagagt agttgttagc
tgtgctatta gcttttagtg 16980gttggtgtgt agctgttagc tgttagatgt ccaagtagcg
gtgtaaaata ttgatgttcg 17040gtaaaagaag ctgtcaaagt agctgtctaa gaataactag
ttaaaaattc aaataaaact 17100ttaacatata atttatacac cactaaaagc tacccaaaag
ctacaaattg tagcttttga 17160caaacactac taaaacacta cttgtaccac taaaagctac
ttacaccact atcttgccaa 17220acactcttat tttttctaat tagtgttttg acctagtcaa
gacactaaaa gctacttaaa 17280aagcttgtgc cgaacatgcc aattctgaac caaggaacaa
actataacaa aaaagtgcta 17340tgtgaaactt ttgtaggcaa cagaagtaag gcatttttgg
aatgtactaa caaatccgta 17400ttaagacttg tacatgaaaa ttaccgtggt aacatttacc
cacacttcct cattcacgta 17460ctccgattca ttcttataag ggcataaccg cataaggcac
atcaagatcc atgtatctaa 17520tagtttaatt tgcctctgtg tttctgtatt aacaatgagc
atagtgagtg caaaagccat 17580ggaagctaga ttaaaaaggc catcattcta agttagacaa
ttggaaacaa catcgagata 17640cacgtacaca taagggctgc tcttctctat tactccctct
gttcctaatc atttgctttt 17700ttagcgggtt ccaaaggcct atgtttgacc actaatatat
ttaaattaaa actggtgata 17760tatattaaaa gaaaattatg atgaatttaa caaaaaccat
atatgttatg tccttttttt 17820tcctatatta atgaattttt acagtcaaag ttggtgaact
ttgacccaaa aaaagaaatg 17880gagcaaaaaa aaaaaaaaaa aaaaaaaact agggacaatg
agtaacattt ttatctatgt 17940ctttttaata tgaatatacg taacaaattc tgcaaaaata
gagatagcaa ctaataacac 18000gcatgaaaat gacaagttat attatacctt tttttctcaa
tatatgaata tacgtaacaa 18060attaactcca gtagttttta gtaaaactat tagattattg
tgtaacatat actctggaaa 18120tagtactaag atccattaca atctttattg agaaatttcc
tcatgtaccc cctgaggttt 18180ggcgtaattt ccaaataccc ctcatatttg aggaatttct
caaataccct gatgtttttg 18240tttagactca aaataccttt actatggaca gtaccctaat
gtcattaagt tttccccttc 18300tctctcccca attttctctc tcctcccatt cccccaccca
ctacccactg cccactgcca 18360agtaggggtg taagtggatt ggactggatt ggactttgcc
aaattcaaat ccagtccaaa 18420gttttttgga ctcgagaaat tgagtccaag tccgatccaa
atattttttg agtccagtcc 18480aatctagtcc gataattttt tcttgagtcc gaatccagtc
cagtccagtc cgattattat 18540atcttttttc ccgatttagg ttcaatgatt cacaacattt
tttgagatgc ttgagcattt 18600gacatctgat tcaattatca atatccacaa ataagattga
aagcttaaat taaagtaaaa 18660tactatgaat aaaaagttga attagatgct taccttgatc
taagttgaga ggaagcatag 18720agactgagaa ttaatctgag ggacaaatag agaatgcgag
agtcgagaca gtgaggtaga 18780aagaaaatga agagtaagag gaagtgagta ttaaggactg
aggagtaaag taagatagaa 18840ttagttggct actagcctac taatgcagta ttgctagtat
aatttactta tttaacaaat 18900ggagctaagt gcaatagttt agcgccaatt gacatattta
gagagagaag gctgaaaaat 18960ccaatatttt taaaatagta tcattatttt taatatatac
attatatata aaaatatttt 19020tggactggac tggacatatt ggactccaaa gggatgagtc
caaatccaga caaaaaatat 19080ttggacttga aaatttaagt ccgagtccag tccgaaaaat
tttcagtcca atccagtccg 19140acaaatttgg actggactgg attggactct gaacttttcg
tagtccgctt acacccctac 19200tgccaagtgc caaactgcca accccctttt ggttgagttg
atatttgacg caaagacttg 19260gcgtgttgga aggttcatta cacattttat ccaagtcaac
tttgaagtct tcttagctag 19320agactagagt gaacgtgttg gaaggttcat tacacatttt
atccaatcaa actttgaagt 19380cttcttagct agagactaga gtgaacgtgt tggaaggttc
atgttcatga cattataaaa 19440gtaataatag tgaaatttca caaagtattt ataaacccag
gacagactca agagctctac 19500ttattattag tgaaaaacaa acatacacac gacaataaca
caacataaac aataatgaac 19560atgaaaatcc tccttttgtt tgtcttcctt catcacctcc
actacttcat ccatggcaga 19620acacttacag aacgccaagc tttactaagt atcaaatctg
ccattactta tgattattat 19680aactctctct cctcatggaa aaacacaaca caccactgca
gttggccata catcacttgc 19740tcctcctctt cttcttcttc ttctgttatt tctctcaact
tcaccatgtt atttctcgaa 19800ggaattctct cccctgatat aggcttcctc accaacctgc
aaaacctctc tattcgatct 19860aacctttttt ctggcccact cccccattct ctctctctcc
tcacccaact ccgctatctc 19920gacgtttccc aaaacagttt cacaggtcca atcccatctt
ctctctctct cctcacccaa 19980ctccgctatc tccacgtttc cggcaacagt ttcacaggtc
caatcccatc ttttctctct 20040ctcctcaccc aactccgcta tctcgacgtt tccgacaaca
gtttcacagg tccaatccca 20100tcttctctct ctctcctcac ccaactccgc tatctcgacg
tttcctacaa caatctaaat 20160ggcactcttc ccttatcggt cgttgagaag atgtcggagc
tcagctacct taaccttagg 20220tataactctt tctacggtga gattccaccg gagtttggga
aacttaagaa gcttgaaaca 20280ttgaatcttg gtaacaacac tctttctggg agtcttccat
ctgagttggg ttcattaaag 20340agtttgaaac atatggactt ttctagtaat atgctatttg
gtgagatccc acaatcttat 20400tctcttcttc gaaacttaat cgatattgat cttaatagaa
acaagttata tgggagtata 20460cctgattata ttggagattt tccggagttg gaatcacttt
tattagactc gaataacttc 20520acagggagta tcccacaaaa gttaggtaca aacgggaagt
tgcaatatct agatataagt 20580aacaacaatt ttagtggtag tttgccacta agtctttgca
aaggagacaa actccaagat 20640ctggacgcat cctataattt gttggttggg tcaattcctg
agagtttggg aagttgcaag 20700tcacttgaag gagtgtacat gggaaataat ttcttaaacg
ggtcgattcc taagggcttg 20760tttgggagtg atgtttcact taatgacaaa cttcttagtg
gaggtctcga tgagaaattc 20820ggtgattgcg ttaatcttcg ggacattgat ctctctaata
ataagctatc agggaagtta 20880cctgcgacca tcggaaactg tattcatctt cggtccttga
cgctttataa taacacctgt 20940accggacgta tccctcaaga gattagcaag tgtaagcagc
tacagaccct cgatctcagc 21000caaaatcagt tctctggtgt gatacccaat gatattacag
gtaagaaagt atattaaact 21060tgttactttt gaaaatattc gctctagttt ttgtttcagt
tggtccattc tcactttgta 21120ttattgaaat atatcccaaa aaagtaaata taattatata
aaagaatctt gctaaaaata 21180atatgaatta tttttgtatg tgcaaaataa tgtacaaatc
taactaattt gttgtggata 21240ataatattaa ttgtgtgaaa tagtaaatgt gtggagatat
ataactttat ttatcatatt 21300cactcaggtt tttaggtatt tattatgagt tttgcattgg
agatatccaa cttgacaata 21360gtatttttgt aatataccaa tatataaaga ttactgtaca
taaccaaaat gtatactttt 21420cttattttta taaacttata tattcctctt ctttgtattt
atcacaacat tttttatacc 21480cttttgcctc atattaatag caacacttat aatttattta
tttacttttt atttcttggt 21540ctataacctc atctacccac atatgacaca ccctataaag
gacccacatg attaaccaaa 21600atatacaaat atcttcaatg aaattaactt taacactaat
atgataaaaa tcatgtcccg 21660ctttttatcc tctaactaag actctgcata aaggtatatt
gcaattaata tgagatggaa 21720gaggtataat aattatatga tcaaattcct ggattgaaaa
ataaatatga gattaaaagt 21780ggtatgtttt tggttaaaag aaactatcca taaagtatgt
ttttggttaa aagaaactat 21840gcaacatacc aatcaaatgt ttatacgctt acaatttatg
taccactttt ttgtcattgt 21900ttttctattg tttgccatac gtacgttact aaatcatgtt
gtcttttcac attttaacta 21960acaataaatt actattgata caccaaaaaa atctatgagc
attggagtac gttgtttgat 22020agaagcttcg tgctattatt tcttgtcaaa gaatttcata
tctcaatatc ttctaattta 22080acaatctaac gaaatttttt tgacccagga aacaaatcca
tttgcaatct ggaaaagata 22140caaacactta aattatcaaa caatgctttg actggtgaaa
tccctcattg tgttggaaat 22200atcgagctca tagcattatt tctccaatca aacaaactga
acggtaccat acccgcaaac 22260ttctcaaagt tatgtgattc attgatatat ctagatctta
gtgacaatca actcgaagga 22320gttctaccta agtccttgtc caaatgtcaa agtctagaac
tcctaaatgt cgggaacaat 22380aggctaagag ataaatttcc ttcatggtta gacaacctcc
cacgtctcca agttttcagt 22440gtgcgtttta acgccttcta cggtcctata actagctcac
caaaagttag tcacccattt 22500cctatgctac aaattatcga cctatctaac aataagtttt
gtggcaagtt gccaagaaga 22560tatatcaaaa actttgcaac catgcgcaat atgaatgagt
ctggtgttgg gaatccacag 22620tacctggggg actcatcaat atatagtatt acgtactcta
tggtattgac attcaatggg 22680ttacaacaaa aatatgaaaa gcttattgtg acgatgtcga
cctttgatat atccagcaac 22740aactttactg gacagattcc atatgttata gggggattac
gctcacttcg taaccttaat 22800ctctctcata atgtcttaac cgggaacatt cctccatcaa
ttgcaaaatt gtctttgctt 22860caagatttgg acctttcatc aaacagactt actggtcgta
tccctcaaga attagttagt 22920ttaacatttc ttgggagttt caatgtttcg aacaatctat
tggaggggtc tatacctcat 22980ggtttcaact tcgacacgta cacagctaat tcataccagg
ggaatctcga attatgtgga 23040aaaccattac ctgagtgtgg agaaagaagg gcaaaaggca
ccactaataa tcaagatgat 23100cctaaaaatg ataatgaacg aatgttgtcg atgtccgaaa
tcgtagttat ggggtttggc 23160agtggtgtac tagttgggtt ggcttgggga tactatatgt
tttcagtggg aaagcccttt 23220tggtttatca agatggctag caaaatggaa tcaatattga
ttggtttttt ctgaccaaca 23280atttgttagc cgatgaagag catcaaaacc aaaaaaaaca
aaaaaaattg attaatatgc 23340atgagtgtga ccttgttttc caaagtttag cattactatt
agtgtctcaa ttcataataa 23400taaaaaaatt agcttgttca agatttgtat ttttattcaa
agattttttt tgtctcttgt 23460gcttctttta tcttatatat attttttgta tggtttgttt
ttgtttaata ttagtccctc 23520cgctcaaaat gatctttcac gcttgagatt ggcattaagg
tcaagagatg ttgctaagct 23580ttagaataaa aaaattccaa atgcatagag ggaaagaaag
cgagacaaaa tgttggagaa 23640ggcagagtaa atgatgtgat ggaggataaa tagtagaagt
gtgataccga aagtttgaaa 23700ataataagga attttatttc ttgctggcac tttgttctag
tacaggtttt tagcccttca 23760aaatgtttat aatgtagagt caaaattaat atccttaact
agtttttaag tccgggttat 23820atcctagata ttaataatat tcatttatta gtaacatttt
attttataaa tataatacta 23880agcattattt ggtttgctgg ttaagacttt agtgtatatc
tatttctttt tttttttatt 23940gtatgcgtgt ttacataaac taaagactat aagggatagt
accacgtggc gcagttcctt 24000gcttaggaac gtcttttaat atattaacta gtatttgggc
ccgggcgttg ctccgggttg 24060gtattgtgtt tccgaacatg atgtgcagtt tttcccattc
ccactaaaat atataaagga 24120aaactcaaca tttaaaagat acaaatataa taatatggac
acttaaaaca tgattaaaag 24180ttgattgaga tggtaattgt gtcatgttat aatagtaaga
ggttgcctaa ttgaggttga 24240ggtggtggag tagtggtatc gcttcccatc tgttatccct
gaggtataag gatcaaacct 24300cataggactc atttgagtaa tttcccatat cctcctctca
aatgagtcct tttcatctga 24360caaaaaaaaa gagtctaatt ttaaattaaa attagacgat
cttttataaa atcggcactt 24420tctgcacata ggtcacaatt tttttgtttc tatctctctg
ctttctttaa tttcacagtc 24480tccaactctc catcaacatc ttacttattt tagaatagat
gatgtatggt agtattaaat 24540ggtaaagtac taaagctcct ataatacaca gaagcttaca
tagtatagat tcgtacatga 24600gacaaggtta caatatactt tctccgttct ttttatatta
caataattac tattttaagt 24660agtttcacat ctattgtaac aattccaatt ttgttataga
aagcaacttt aataattgac 24720aatattgccc ttactttatc ttattaaaac catcattaat
tactcacttt ctcttataaa 24780attgctttta ttttctaagg atgatttctc tcctattcta
gttaattaaa gagttacttt 24840tgtgctaaac tgctcattta ttccaaatcc ttaaaaattg
tgtccaaacg tattgttgta 24900atataaaaag aacagaggta ctattagttt gaataaattt
tgatcagatt aggtcacctt 24960tagggggcgt ttggttaggg gtattctgga aagggtaagg
gaatcaactt acttaattcc 25020cttacttgtt gtttgtttgc tcaatttaat gattcccttt
acccacccct tactcccaaa 25080gtcctttact ctcattctcc ccacccccca aggtttcact
taccctttct tgattcatca 25140ttgaccatat ctttgaccac ccaactacca ccaccacttg
accacctaat cacctaacca 25200cctaattacc caaccactat taccacccaa cccctccacc
tgcccaccaa tcggcaccat 25260aactgcccaa ccgtcgccca atcaagccac ccaaccggca
ccataaccgc ccaaccaagc 25320cacccaaccg gcaccagaaa ttgtaccaag ctacccacac
acgtgaaaac cacccaccca 25380caagccctag aaaaaatgga agaatcgaga gaaagggagg
ggagagaaaa gatgcagcga 25440ctagaagggg agggggagga tgtgacggca aggggagagg
gaacttcgca gcggcaaagg 25500gaggggaaac gtcgcgtcgg caaagggcta aggtggaatt
gacggggttg cagcaacaag 25560gggagggcat ggagacgtcg taaccgcaag gggaggggca
gcggcagtgg aactggggtg 25620gagaggggta gtggcggcac tagggtgtgg gagaggtggc
gggggatatc aagagagggg 25680ggatatggtg gtgttatggt ggaagcaaga agaagaaaga
ggaaagacaa tgtactaacc 25740aaacaacaca ttaaatctaa gggttttggt ttcctttccc
catctacccc tttcttgatt 25800ccattccctt taccccttta caaccaaact cccccttagt
ttttactact tataaccttc 25860aattttggct gttttttgtg acatttttta cttctccgag
cctggtcata ttttctcccg 25920aaacatttcg aggaaagtcg aagtgacttg tgaagttgtg
cgggtgcttg gcaccatttg 25980tgttgcctcg aaaagcatct gaatacccca tttattcctt
tctcctgaaa cccaaaatta 26040cctcgcaata aacgaaaaga tatccatata tttgttccaa
gccacatgac tcctttccaa 26100cgacctccca tgtgaccatg tccttagaag gcatcccgtg
gcgttcgaag ctcggacccc 26160cggaaagtcc gaaagtgtgt attataactt tcaattttgg
ctgtttttgg gatatttttt 26220acttcttcgg gccttgtcat attttctctc gaaacattca
taggattgtc aatgtgactt 26280gtaagttgta acgttgcacg ggtgcttggc acaatttgca
ttgcctcgaa aagcctctga 26340acaccccatt tgttcatttc tcgtgaaatc caaaattgcc
tcgaaaaaaa cgtaaaggca 26400tccacatatt cgttccaagc cacataactc atttccaatg
acctcccata gagtccgtag 26460ctcggacccc aggaaagtcc aaaaacgtgt actataacct
tcaattttgg ctgtttttgg 26520gacatgtttg gacttcaccg gcctggtcat attatcttcc
gaagcattcc tacaaaatcc 26580gacgagacta gtaacgttgt tacgcgggtg cttgacacca
tatgtgttgc cttagaaagc 26640ctttaaacac cccatttgtt catttttcgt gaaacccaaa
attgtcccga aatgaacata 26700aatgcatcca tgtattcgtt gcaagccaca tgatttcttt
ccaatgacct cccatatcct 26760taggaggcat gcatcatgtg gcgttcggcg agcgggtctc
gggaaagtcc gaaagcctgt 26820gttataacct tcaattttgg ctatttttgg gacatttttg
gcctttttca agcgtgttca 26880tattttctcc cgaagcattc ctaggttagg cgatgtgact
tgtaaagcgt gggtacttgg 26940caccattttc tttgcctcga aaagtctttg agcaccacat
ttgttcattt ctcgtgaaat 27000tcaaaattgc ctcgaaatga acgtaaagac attcacatat
tcattccaag ccacacatga 27060ctcctttcca atgacctccc aagcccctag gagtcgtccc
gtggcgttcg gatccggagc 27120tcgggccccc gagaatgtcc gaaaccgtgt attatgacct
tcaatttttg ctgtttttgg 27180aacatttttt gacttctctg ggctggtcat attttctccc
gaaacatttg taggactacc 27240gacgtgactt gtaatgttgc gtgggtgctt ggcacaattt
gcattgcctc gaaaaacctt 27300taaacaccgc atttgttcat ttctcgtgac acccaaaact
gcctcgaaat gaacgtaaag 27360gcatccatat attcgtttca tgccacatga ctcctttcca
ctgacctccc atgtccctag 27420aaagcacccc atatccgaaa gcttgtatta taaccttcaa
ttttggctgt ttttgggaca 27480cttggacttt ttcggttcgt tcatattttc tctcgaaatg
ttcctagaaa aggtgacgtg 27540agttgtaacg ttgcgcgggt acatggaacc atttgccttg
cctcgaaaaa cctctgaaca 27600ccgcatttgt tcatttctcg tgaaactcat aattacctca
aaatgaacgt aaatgcatcc 27660atatattttt tccaagccac ttgactctta tccaatgaca
ttctatgtcc ttagaaggca 27720ctgcttgtcg tccataattc gggccaggga aatgtatgaa
agtgtgtatt ataaccttca 27780attttggctg tttttgagac aattttttac ttctccggga
ctggtcatat tttctcccga 27840aaaaatactt cgagtgccga cgtgacttgt aacgtcgcgc
ggatgcttga caccatttgt 27900gttacctcga aaagcctttg aacaccacat ttgttcattt
ctcgtgaaac ccaaaattgc 27960ctcgaaatga acgtaaaggc atccacatat ttgttccaag
ccacatgact catttccaat 28020tctctcccat gtccctagga ggcatcccgt ggcgttcgga
gctcggaccc tgggaaagtc 28080cgaaagcgtg tattataacc ttcaattttg gctgtttttg
ggtcattttt tgacgtctct 28140tggcttggtc atattttgtg ccgaaacatt cccaggattg
ccgacttgac ttgtaacatt 28200gctcgagtgc ttggcacaat ttgcattgcc tcaaaaagac
tctaaacacc ccatttgttc 28260atttctcggg aaacccaaaa ttacctcgaa atgaacgtaa
aggcatccac atattcgttc 28320catgccacat gactcttttc caatgacctc ccatgtccct
aggaggcatc ccatggcatt 28380cggagctcga acactgggaa agtccgaaag cgtgtattgt
aaccttcaat tttggttgtt 28440tgtgggacat ttttgggctt ctccgggcct ggccatattt
tctcccgaaa cgttccttgg 28500aaagccgaag tgagttgtaa cattgcacgg gtgtttggca
ccattagtgt tgcctcgaaa 28560agcctttaac caacccattt gttcatttct cgtgaaacct
aaaactgcct cgaaatgaac 28620gtaaatgcat ccacatattc gttccaagcc acatgactcc
tttccaatga ccttccaggc 28680ccctaggagt catcttgtgg cgtttggagc tcagtccccg
gtaaagtctg aaagcgtgta 28740ttataacctt caattttggt tgtttttaag acattatttg
acttctccgg gactgggcat 28800attatctccc gaaacattac taggagtgcc gacgtgactt
gtaacgccgc gtgggtgctt 28860ggcgcaattg tgttgcctcg aaaagccatt gaacaccccc
atttgttcat ttctcgagaa 28920acccaaaatt gcctcgaaat gaatgtaaag gcatcgacat
attcattcca agccacatgg 28980ctcatttcca atgacctccc atatccctag gtgtacaccc
catttgtctg atgttataat 29040agcaagaggt cacgggttca aatcttgtta caagctaatt
ttacttttgt taattgacat 29100gacttatgta cacattggac aattatagtg gagtaacaaa
ggtgacatgt gacgcgtata 29160cattatcaca cacgtctttt aatatatttg tatagatcta
gatttaagag taattttttt 29220aatgcgcaat acttggccaa tttcttctgt atcaaatcat
aggtctttgg ttggttcata 29280agagtaaaga ccaaaataat aatctgaact gcaaaaattt
tctccaagag ttaaaagttt 29340gtataagtta gattaaaaaa attaatgaca tatgatgtag
ttggacatta aatatgtaag 29400tttagaagta attgtgttaa cataaaaaaa gattcgatta
taacataaaa actaaagaaa 29460cacaaaggcg ccgtacaaca atcaatatta cccaagtccc
ctcattaata ttaagggatg 29520acctagctcg tacatattta attatctttg aaaattcgtt
gttcagactt gctagttgct 29580attctatatt tgtatattca ttaatcaatt tttcaatatg
tgagcattta cattttaaac 29640tagagcaaat attgtctctt ttactatttt gttgttgtca
aattttcaaa aataaattgc 29700tcaaatactt ttcctagtga cataaaaaat agagcaaata
atcaaacagt agcagaccca 29760ggaactttta cataatgtag acggcataat gtgttaattt
ttgcttcttt tttctaatat 29820catccaataa cacaattctg cttctattag tttgtagttt
cagatgatga tacccaaaca 29880ataagaccaa gcaacaaatt gataagattt tgcttctctt
tcttccactt ggtgtaactg 29940taacagcttt gaagtttaac ttcagtaatc agttgcatat
ttggcatatg atcaaaacaa 30000tcaaattatt atgtatggaa aagcaaaaaa cttccaggtt
tccatctgaa caaggaggcc 30060aagagggtgg aagcaagcaa ggatatatga tcataaaatc
ctatgaatat gatgtacaaa 30120ccttttctac tgcaattagg taacctaaat gataccacct
aggaacagca acaacttatt 30180tacagcacta aacctaaatc aggttaaagt taatcagacc
accatgtatc tgggtggtct 30240ctcgagggaa agcgtctcca tctgtatccg ggtaacagag
gtttcttctt ctcgatcctc 30300cttggcttct gccctcttaa gttcttcgaa ggctctcttg
gcatatacag taaacgcaac 30360aatggtaatt attgccacta tgaatgaaat aacattgtac
acaatctcca cccatgttag 30420atgatgattc ccatacttga catctgcgaa cgtccttatc
agtctcccac tgcaaatgaa 30480tgctatcagc gtcaatattc gagataccaa ctcatttaac
tattgaattg ccaaaaacag 30540atatctttga ccatatattt gttactaaaa ataacgattg
ataatgtgaa actatcactg 30600atagatttaa aagaactttt ataaaagtat agtttctcta
atgtataact gcagaaaata 30660gaatggggta gacaaatgaa gtaattgttt tgaagaatgc
aaaaggtcaa ttcagtaata 30720cttttatacg tgattggggg aagcattaaa aatcccttct
aagataaaga tgacctcatt 30780ggcaatggaa tcgacatcca cagacccttg cattagaaca
gagtggaagt ttctgtgaac 30840ttacgtgtag atgtaaagaa aagcttctgg caccatccct
gcaattgatc cccatagata 30900aggccaaaac gtcatacttg tcaccacaac tgcgtagttg
aagatagtat agggaaatgg 30960tgaaacccta aagagtgcca ccacgcggaa ctgatgaaac
cagctacctt cggcagcaag 31020cctaagcata gcagccttat ccggccatct ttgcaaccat
tgctaacaag gtacaaaaac 31080ataaacattg tggacttaat tagacaagaa agttaaatta
aaatcaacat tagataatca 31140ataaatcaaa tgtaagcagg gaacatattt cttacatgga
ttctatcccg gaagagcaat 31200ccaagtaaat agggaagaat cattccaata gtagttccaa
ccatgattat cacaaaacca 31260agaccataac caaagatcat gcctgcaagc cacatggatg
ggccagaagg aatcagaaat 31320acagggaaga ttgctaggga agtaacaagg accacagcaa
gaaccggacg gccaaaggca 31380gtggcttccc attgcatcat tggaacaaga acctgcagag
aaagtaccaa aaactttgag 31440gcaaaaattt cctgcttgta tattgcaaaa agtagtacag
cgaaggcatt ccgtgcagaa 31500tggcttatag attggaaata cggagaacaa tgcaactata
agcacaggcc catctcttga 31560cttttgggac aataacatgg acccccagat tgatttataa
gttctcacac catagctaga 31620ttttgttgga actttcataa atcatagtga cataagtata
gcataatatt catgccttcg 31680acagaagttt tcgcatatgg taaggctact attgaaaaaa
ttcccttgtg tttgaagtac 31740gcataaaaat atctagtggc agtcaaccaa ataaaacatt
ctaggagtcc ctcaaaaaat 31800taaagagtca tcagttcaga agactttaat atcaatactt
tctattatcc gggtttggca 31860tgcagtaaat ttcatgagaa aaggaaaaat cagctatttg
attatataag gaactaattc 31920ggatgtatca ctaagctttc catcgactgg aacatcggga
gctagtctcc aatactcgtc 31980aaggatctaa cataaacatc ttctccgcaa tcaaaaagcc
aaggtcacat acatctaggc 32040ctctgtctca ttctgatggc atggtatgat gcaagttaga
caacactatt atttggcaga 32100tgacacttag gggtctaata tttaagctca ttcaagataa
tcaagtaatc aagttcaatc 32160tcaaggtttc agttgcgcta aaaaatgtaa tacttggctc
attcagaatt agtttgttga 32220agctggttgg tatttgcttc atttgttaat ggaaccaggc
tcataaacaa gctttcatta 32280ggctaaactt atttaacaaa atcaaaagct taatactata
atttttgata ggatttcttt 32340tgggcagtta tacatgagta atgaacaagc tctacacaat
cttttttaat gaacaagctt 32400taatcgagct agggtacgtt ctattcaact tattggacct
gaacttattg gaacttatct 32460gaactgaact tattgaacct gaactgaact tattggaact
tattaaacct gattggacct 32520gattcaactt attggacctg attgaacctg attggaactt
attggacctg attgaacctg 32580attgacctta ttggacctta ttggaactta ttgaccctga
ttgaaactta ttagacctta 32640ttggacctga ttgaaactta ttagacctta ttgaacctga
ttgaaactta tttgacctta 32700ttagacaaaa acattattat tattattgtt attattatta
ttattattat tattattatt 32760attattatta ttattattat tattattatt gttaacctga
ttgataacat ttatatcttt 32820catagttatt agtaacgaaa acatgttatc tctagttatt
caaagacgaa ttgcaaaata 32880ttgtaataat aataataata atatattatt attattatta
ttgttaacct taattatttg 32940accatgatta taatattatt caatagcaat atgaataatc
aaataataga caataataca 33000agtataatac tatacattgt ggtactttaa taaaaaaatt
ctaataataa cataatcagc 33060taatagtaat atgaataata aaataataga cataatacaa
ataaataata aaataataga 33120cataatacag ataaataata aaataattta cactaataca
agtataatac tatataatca 33180ttgtggtact ttaattaaaa ttctaataat aacataatcc
gctaatagtg atatgaaatt 33240atgaataaca aaatagtgga caataataca aatgtttatt
aaacattgac tatttggacc 33300ttattggacc ttattagacc tgattggaac ttattggacc
ttattagacc tgattggaac 33360ttattgcacc tgattggaac ttattacacc tgattggaac
ttattgcacc tgattggaac 33420ttattgcacc tgattggaac ttattgcact tattagacct
tattgcaact tatctgaact 33480tatctgaact tattggacct gaaacttaat tttttaagtt
gaacagaacg cacccctagt 33540atccacgaac atagttagtt gttcatcgac aagggtgtta
attccttgac tataaaaaaa 33600atatctgcta atatgtcctc cataccatgt cttgatctga
ttcccaaaat cacgtgtttt 33660cgtgtctggt gaccacgttg ctagacatgg aagacaggtc
taattgttca gtttcaagtc 33720aggttgatta aacatatgtt agcaatatac aatcattatt
agtcaaacta attcaactcg 33780ggtttggttt gattcaggtt atgtcgagga tcaggtccaa
atcgggttaa tccttccagg 33840tcaaatatat ctaagtctgt tttgccaaag tctacttttt
gtatccgtgt ccatgctaaa 33900tgacaaacaa aaagcagctt ttaccaagct cgaatcagat
ttgttcgctt aaagagtcac 33960ttcgctcatt tacagcaaca attaaaggac aaaacattgt
ccattcaact acttacggat 34020attaacttat tggcaactgc tagcgtaata aggcaatcaa
cagcactcgg cctcaataat 34080gaacctacaa ggagtccaat gaccaataca aattatcact
ggcatcatct agcacgacaa 34140tctcttaact ctaagagtct aagtgccttg acatacaaaa
gtattccttt taaaagtacc 34200cccgtgtgga tattctgcca agcaaatgca atcgatacac
ccaattaggg cttttccatt 34260atgagtcctc agagcctcag attgtaaaac aggtcagtaa
aagaggaaaa tagtatttga 34320ttcttttgct aaacccttgg atataagaat ggtgacttgt
attgtcacgc caagcttctt 34380tcataaaagc tgatcatatt attatatgag agttctgagt
ttcaaggtcc gcattcgatc 34440taactagaca tcacttccaa ttaaagttga gaaacgaaac
taggtgtcct ctttgtttcc 34500caaaggtgaa ctttagatac ttattataag catattttgt
tatgaatcgg gctaaggaga 34560gggctactct tggtattgca taattagtta attacttagt
agtagcttga ggaataagga 34620agcaagtaag ttagaggaaa gagtatgaaa atctgctata
aagtgaggag aggagggata 34680gaaggataat cacaaaatta ttgagttaac tttggtttta
gttgcttagg ttgggagtgt 34740ccagccactc gaatgtcttg ggactgtaaa caccattgtt
catgatctaa ttgcatcaat 34800attacaatta actcatttct cttcttatcc atattcatct
tcttacaatc acaactattt 34860ccagatcatc catccaaatc ttcatccact tgccttagtt
tctactccag atttcagtct 34920attacaaatt gatttctaca atatgtcaat tcatcacaaa
ttatcatgtt ttctgaacaa 34980aagttcactg tttcaggaca aatacagaaa gaactacttt
gatgcttaga acagatatat 35040tgtaaaattg tattcggaat ttgggataca actggagaag
atatgaataa ataggcattc 35100agggagctca gaaaaacaga ccgtgccata tggtgctctg
ctgcataaca ggaaataatg 35160gataaagtat gaataacgtt ataacttctt aaaaacctag
atgacaagta ttttggttgc 35220tttttattat tggtaggcaa ggagaatact caacaacagt
ttagccttaa actgcttctt 35280atttctcctc ttcccctttt tcctgatgat ttggggttgt
cactcagttc ttttacctct 35340catttccagg tactttagag ttatattaca caaaggattg
caagagaaga acaggtcgcc 35400ctggcatgca ctcagaaagt atacgaccct tcacaggaaa
tgtggtgctc caagacttat 35460atctcaggct ctcatgagtc atgtcaagga ccatctttaa
tcatttgtat tctaggtttc 35520tcaggcgatg cggtgtgctg gtgtgtctct ccctcccact
tgagtgtgtg tattgtttgt 35580gcccctaagt ttttatctta acaatcacta ctagtcaatt
agtcattacc aaccctaccc 35640acctctcttg ttactgttgt tcttggagat atttcatata
tgtcagctta gaacttatat 35700tacgtttctt attacatatt ctcttaagct cgcgcacata
ctctgtgatc gaagggatcc 35760atattagtta tcttttagtg gagttgttgt gaaaaaagac
tgcatagaaa aattaagata 35820gctcatagtt gtaaatgtaa ttgaactttt agattgatag
ccttgaggct gcttgcattg 35880aaccaaccaa attcagccag gctagtctat gcctctttgg
tgtcacctgg taggttgaat 35940ttgtgtagct gtagttctac aagagactga tttaaaaatg
ttttcgcact gaaacagctt 36000aaaccacaaa acaggaaagt gcagaacaaa ctccagaaaa
tggtgcagaa cataccttct 36060caaaaaggaa aggaactccc cattttaaca gtacgaggac
aactgctaca gcactaatgg 36120aggagatcaa gattttgatc caccagatga aggattctga
tcttgtttca gcctgagaat 36180gtaaggttga agcttcaggc ctctttgtaa tagcagatgt
caccagacta acaaattcac 36240tgtcgtcttg catagcaggc ccaacatcta tgtcatgctt
agttagctcc attgaatttg 36300gcatctccaa gagatctcaa gagctgccca aaaagacggt
acaatattat gagcatacat 36360gacatgatga caacccataa agaatatcat aacctgtcac
attttttatt caaagttcaa 36420cagccctctt acaacatgat tgagaatgga ggggaagaga
gagagagttg gtctcagaca 36480ttgatcacat aatcatttca attagtttta aaggtgctca
tgaaatagaa ctagtgtctt 36540aagctggaga cttctgtatt tttcatggtt ttagattatc
aatcatattc ttagaatctt 36600tgatctctag aactctttcc tttcctccca atattttttc
cactttgtct tttgttaatt 36660acggcttcgc tgcaggcctg caataaatct tttaaatttt
tacagatact atgtagagtt 36720gtatacataa gctctaatct gaagacgatt ggtttcgatg
ctagttaata caaataaata 36780tattatggat ataatatgca gtaaattggg ccatgggcac
cagggacaac ttagacaagt 36840atagtgcaac taccaggaaa tttaagctgg gtacctctga
ttcatcatgc tggttgataa 36900tattattgct tccacaagtg ttcgctacgg ctcaaccaaa
ctaagtcaca actcacaagc 36960tgcacaaccc aactgacaat tatcgcctat tgtctaagct
atacattaca ttaccccaat 37020gccacaacgt ggctcacgcc taggcatggt aaggaagttc
agatgtacgc agccttaccc 37080ttttaataac aaagaggctg tttccaggtg acccttaaat
cttaattgca aacaccatct 37140gctgcttcac ataaataagc gacttcaaaa ttgtaaatta
aagaatttga atgcaaattg 37200tgtgaaaaac aactccatca agaatccatt aagcacgctt
tactattagt atcaataata 37260ggaaaccctt atatcccttt tgacgaaggc acacatgcaa
cactaatgtg tccttataaa 37320cttcatgaaa gtatatctct acgaaaccct tttagtctta
tgtgattctt taagtgtcca 37380actgatgatt ggttacaagg tatttagccc aaagtagcat
ttcagagaga tggtgtagaa 37440tgagtagctt ataaaccgag gttgaggtgt aatcctaata
aattaggaac taataccaca 37500agagagatgg acatgtagag atacaatata gtacagaata
agattatttg aaatcttttt 37560accagggaaa ctccagaggt gttccataaa acacaatacc
atataactgg gagatcaata 37620ttttagatta aaaaatataa aaatctattt gggttgagta
tatagttggt tagtccaata 37680atatataaat ttataaggtg gaggtcttcg gtatatgaca
ttccaaattt gagtatcaaa 37740tgatatatat ggttttccat acttgaatcc cttttcatgt
actacctctg tttcaaatta 37800atagttacac ttacactttt cacgcatgcc aatgcagaac
tttgaggaca tatatcttta 37860gttttgtatt tgtaaaaatt ataaaaagta catattaata
aaatacatat taatacgaat 37920ctaacaagat cccacatgac tatgatttta ttcacgtata
aatcacaaac gagggtcaaa 37980atgcaattgt gaatagtgta aaatgtcaaa gtgtaactat
taatttgaaa cggaggtagt 38040atgtgtttat gcaacacttt tcctttttcc ctttttgcta
tttagtaatt tatgtaaaat 38100acttccattg acccaaaagt tgggtgatta tagtttacat
ctatcattat tatttatcat 38160tactatagat tattcaccat tgtaatcaac tttataaaag
tatacacagg taactcagga 38220gtcaggggtg ctgggccaaa cacttttata gtttaaggtg
aaaaatctcg agaatcttct 38280cctgccacgc aaaatgagtg ttcttccact ttaaagatgt
tataacactt atcttaacct 38340actattcgta aataacactt atcttaacct actattcgtc
aagacatact tgcttcatct 38400cactaagaac gtcttagttt tcatttgaaa ttcgtaccag
aaagattcac ttcaaatcta 38460tttattttta gataaattgt tattaaaaac gacgaagaaa
cgtcagagga caacaaatcc 38520tctaaactcc aaattataag tgagtccaac tatgttgacg
taaggtaatt agagtatcca 38580taaaagccct ggccgctttg gcccacaaag cagcttagaa
tactacccaa ccccaaatat 38640aatcaatcag gtgaggaagc tcgcaacaga tgcgagagtt
ccactccaat caaaggcacc 38700agaacatagc catcgacatc ttctcttctt tacccccctt
gaaaccaaca gatcttaagg 38760aagtccacta gtgaacaagg acataaccac tactcatgtg
gaatgccaat cagcctctgt 38820caaagggaag tccattagtg aacaaggaca tacccactgc
tcaaggtagt catgtggaaa 38880ttggaatccc aatcagcctt tgtcaaaagg aataagccac
atcgcaatga agaaaaaggt 38940gcaaaccaga tttattgcat ctccaacacg acataaatat
cgagaatgag gcctttactg 39000acaaaggaac tctggatttc caatttccac tgagcattgg
actcagttga gaagtaattg 39060gtcttgctag attctgttta cgcacatact cttaatgata
aataaatgta acaggccaat 39120tggtctggaa aaaaacagtt gataaaaggc tagtttgggc
cttggggata aatataatct 39180ggtatgagtt aataaatttc tgtttaaggt aaagagaatg
tgttatgtgg gataatttaa 39240tcaagaaaat cttagtaaga tggaggtagt ctaacttcca
ttcctcaaaa tgtgtaattc 39300cttataaaat cagtcagcct ctagatacat agttagcaaa
aatggaaggt atagaagtgg 39360gggtgaggga agaggaagga aagagaaccg cgatcaatca
tattgttcgt gctcaagttt 39420gagttgtgcc tatagctagt tagagtttgt ctatttcatt
gtttttggtc agtgttcata 39480ttctgagtgt catcgtgttt gggttctaga atgctccttt
tcctaatgtc gacatttctc 39540cactttactc tagaaaaatg atctcattgt agccattcca
gcttcaattt taatggatac 39600taagatccct ttcaggaaca atgttaaggt agatgttagt
gttttaacag ccatgtggat 39660gttagtgtct agaacgagtg gtcaaaacac tactagcctc
aaaatattgt gatcagtctg 39720aaaactctat gttagatggt tgcttttttt ggtaggttcg
cttgttttgg ggggttagct 39780ttgtttattt tcttcacaat ttgcccttaa acttttcaca
aaatctacaa ttgaagattc 39840ttaaatagat aacagacgtg tcagctactt caacagctaa
ttgtacgaaa aagttcagct 39900accttgaaac caaaccacta acagctagta cagtttgttt
ctactattac atttatctaa 39960tataacagct agtatttagt ccaacgatgt ataatatcaa
tgaaatggaa ctaatctgta 40020aattggacct taggcataag agtcgagttg agcaggtaca
ctccaatcac caagttattt 40080aagcttaaaa tgtctaactt ccaatgctgt ttgacgatac
tcattgccaa gtgtttgtta 40140cagatcaacc aagcaaataa agcaacaagt gaacagctgc
actagtaccc aactgcgaat 40200tttcgtcgat tgccaagtgc atgtctggga cacaatacca
tcatgtccat acccattacc 40260ttgcttagcc agctatcgta atccataaca cataaaaacc
aacaaagtct tgatagtttc 40320acaaatcaaa atgttcactt ttcattccaa ccaaaacaag
caataaatct cttcatccat 40380actcacaaga agaacaatct ctcacactac ccacttgatt
agtaaaaacc ccaatcaaaa 40440acaaaatcca acccacataa acaaatcaaa tttagtaact
acccataaac tcaaaaacct 40500caaatcacaa taccaataaa agagatatac aatcaatcaa
aaaaaataca acaacagcta 40560aacaaataac atcataaact aaagttattc attttatttc
ctaactagag atcaattaag 40620cagcataaaa caacatcact aattcaagtt aataatcatc
aaattctata ctataaaaca 40680tacatacctt accaaaacta cccagctgaa aattagggta
gagctccaga aatcccggcg 40740aaaaatccgg tgagaaattc agctaaattt gaaaacttct
ttaggttaag tagtgtacac 40800gatgaattga agatttttac aagcatatga aaatggtggt
tgaaattgaa atgggggttt 40860ttgaaaattg ttgcgacgcg taaaagtgga aaaaaaaaag
gagagaatca aagaaatgag 40920caagtttttg taggtgggtt tactgttgtt gcttttgttt
gtgcacatta ctgactattc 40980ttaattcttc catgcgtgtg ggggtgaagg aattgttttc
ctaagttgtt tagccacttc 41040atagagtcat tggatttgaa taatctaggg aataatgatc
atgtgtttag tgtatctata 41100aattataatt tatgtatgta tattgtatat gtggtgaggc
atagaggaca aggtctaaga 41160ggaatagagg attgtgaggg agtgtttcat gcttttaaga
atgatgagtc attgagtgta 41220ttaagttata agtagtattt gatcgagtag taaagtttgt
atcacgtaaa tcagagtgat 41280aattaggaat tgggatttgc tcaagtggtg agttttccca
tctttccgag caaggtttct 41340agggttcaat tcctacctca agcatttcct tgggatttaa
ggggacggct cagaggaatt 41400cttcttacca atattttaaa aaaaaaaaaa ttaagagtgg
taatttagtt cagatcctac 41460ctttatccgg ttcgaaacga cttcaagaaa aaaaaatccg
acatcgttta aaatttttta 41520cttccgactc atttaatccg cctccaactt tgaaacaagt
agtcttattt cttttatgtt 41580aagaaaattt gccaaaaaaa ccctttttaa agtccagttt
tgcgaaaaaa aaaaacctta 41640taaagcattc tttgtgaaaa caaaccaaaa agtaaattat
ttttgcaaaa tgaaacctaa 41700tctcattttt cggttttgac catggacttt tcgacattga
ccacttctat ttatcttctt 41760cctccataat cacagcctag ccaccactac caacacctgc
cgctagcccc cacaacctgc 41820acccccacaa cctccatcca ccccctcaag cggcaacccc
ccttattccc atacgcggca 41880accctacacc ttatcctcca cccccctccg cccttacctt
ttctcctctc ccttcttccc 41940tccatcaccc ctccccactc tcttctccct ttgcccccca
tcgttgcacc acccataatc 42000cctctctgta accccctctc ctcgcagctc cccctccctc
ccagccaagg ttgaaaaatt 42060acagaggcag tcgcatatgg ggatggggga ctatcgtcta
aggggtggag agagggtttg 42120ggggctgctg gtgggggtgg ggtaggctga atgtggtggg
ggctgagggt ggggggtgaa 42180ggtggggctg caggtcgggc tggcggtatg gagaaagaag
ggaaatagaa gtggttaaca 42240ccggaaagtc catgatcaac accgaaaaat gaaattaggt
ttcatcttgc aaaaataatt 42300tattactttt tgatttgttt tcgcaaagaa tgctttataa
ggttttttcg cataacattt 42360agacttttat catccctctt agatttgaca catattatac
gaattatact aaaaagactc 42420cttatagtaa ttcgactaat gttttattaa aatgaacctt
tagaataact cgggtaatat 4248054201DNABeta vulgaris 54agagcagatt ggcatacttr
tgaatattct cactggctat taaattctca gaagaaaaat 60caacaccaag attatgacat
gcttgtgcaa agacacaccc agtcatgaat gcatcatagc 120cagcttcatg cttagcccca
gagttccaat ttgaggayct gcaagaaaac atgggagtaa 180gatggtttca cataaaacat g
20155201DNABeta vulgaris
55agagcagatt ggcatacttr tgaatattct cactggctat taaattctca gaagaaaaat
60caacaccaag attatgacat gcttgtgcaa agacacaccc ggtcatgaat gcatcatagc
120cagcttcatg cttagcccca gagttccaat ttgaggayct gcaagaaaac atgggagtaa
180gatggtttca cataaaacat g
20156201DNABeta vulgaris 56gggtttcttc gaagtttgat tttgttacat ttttcaaaga
gaaattagtt gttgatgttg 60aataatgatg ataagtagtt agggttcgta gtaaggtgga
cgaragagaa aatggcgtca 120ctctgayrag cttcttcatt ttgttcttct tccttagctc
tgttttcagt cactgcgcca 180tttttttttt aaaaggaaga t
20157201DNABeta vulgaris 57gggtttcttc gaagtttgat
tttgttacat ttttcaaaga gaaattagtt gttgatgttg 60aataatgatg ataagtagtt
agggttcgta gtaaggtgga ggaragagaa aatggcgtca 120ctctgayrag cttcttcatt
ttgttcttct tccttagctc tgttttcagt cactgcgcca 180tttttttttt aaaaggaaga t
20158134DNABeta vulgaris
58caagcacaaa atcaaataat gagaatcaca ctatccaaag aaaatttcca tccacattta
60tccaacacag ttatctctct tttacaccca aattatgtca accaaaaaca staaaacaag
120tgagtgcagt agct
13459134DNABeta vulgaris 59caagcacaaa atcaaataat gagaatcaca ctatccaaag
aaaatttcca tccacattta 60tccaacacaa ttatctctct tttacaccca aattatgtca
accaaaaaca staaaacaag 120tgagtgcagt agct
13460134DNABeta vulgaris 60taagtaaaaa gtggtaaaag
aattaccaaa arcgcacara ataaattaat tagytggatw 60taactattta acctattcct
tttttctgtc gctataacta cttttgctta acttattgat 120ggtttgatcg ttga
13461134DNABeta vulgaris
61taagtaaaaa gtggtaaaag aattaccaaa arcgcacara ataaattaat tagytggatw
60taactaatta acctattcct tttttctgtc gctataacta cttttgctta acttattgat
120ggtttgatcg ttga
13462150DNABeta vulgaris 62ttataatgta gagtcaaaat taatatcctt aactagtttt
taagtccggg ttatatccta 60gatatttata atattcattt attagtaaca ttttatttta
taaatataat actaagcatt 120atttggtttg ctggttaaga ctttagtgta
15063150DNABeta vulgaris 63ttataatgta gagtcaaaat
taatatcctt aactagtttt taagtccggg ttatatccta 60gatattaata atattcattt
attagtaaca ttttatttta taaatataat actaagcatt 120atttggtttg ctggttaaga
ctttagtgta 15064201DNABeta vulgaris
64acatctacac tgggagactg ataaggacgt ttgcagatgt caagtatggg aatcatcatc
60taacatgggt ggagattgtg tacaatgtta tttcattcat cgtggcaata attaccattg
120ttgcgtttac tgtatatgcc aagagagcct tcgaagaact taagagggca gaagctaagg
180aggatcgaga agaagaaacc t
20165201DNABeta vulgaris 65acatctacac tgggagactg ataaggacgt ttgcagatgt
caagtatggg aatcatcatc 60taacatgggt ggagattgtg tacaatgtta tttcattcat
agtggcaata attaccattg 120ttgcgtttac tgtatatgcc aagagagcct tcgaagaact
taagagggca gaagctaagg 180aggatcgaga agaagaaacc t
2016621DNAArtificial SequencePrimer pCRBM4_S2
66gtagttgaat ggtgggaatc c
216721DNAArtificial SequencePrimer pCRBM4_S3 67caatattgcc cttactttat c
216820DNAArtificial
SequencePrimer pSeq_LbCpf1_F4 68accactcact cctcgataag
206922DNAArtificial SequenceSEQ 70
pSeq_LbCpf1_R3 69tagacctgct tctcaacctt ca
227021DNAArtificial SequencePrimer pSeq_Ribozyme_F
70tgcagcggat ccaaattact g
217118DNAArtificial SequencePrimer pSeq_Ribozyme_R 71cctggtccca ttcgccat
187220DNAArtificial
SequencePrimer pSeq_tDT_F 72ttacaagaag ctgtccttcc
207320DNAArtificial SequencePrimer pSeq_tDT_R
73gtactgttcc acgatggtgt
207419956DNABeta vulgaris 74aaatgataca ggggtatatt tgactctatg aatttcagaa
atctaatcaa atttgctaag 60cttccaatga ttctactaag ccctacaaat tacaagaatt
agttactttc atctctctgt 120cggcttcaga accagaagtg tacaatatct tgtcaaacaa
actctgctta gaggagctct 180ttcgatcatc ttttttcgat ttggaagttc ccggtgatag
gattgacatt gctgttttct 240cggtcaattc ttctggatct tggttctgtc catctatctc
tggctccatt aatctggtct 300tccaattaat tccgatagcc tcagcttgct ctgcaaacaa
gacctttgag atcggggagc 360tgcagatatc cttataaact tcataaccag cagcacaggt
tttcccacct tccaacaact 420ttgataaagg atgtaggaga gagatagaat catcactcgt
ttctaaccta tccttcaagg 480caaggaagtt aacagccaag tctgccttac taaactgaac
aaatactgca gtttcatcca 540agttatagat gcaagcaact gagtatatac caaacacttt
acagatgcat tgttttatgt 600cacttgcctt gagttttggg gagaatcccc aaatcaaaac
tatgttagga tgcaaaatgt 660taagaaacct ccttttagct gaactgataa cgggaatttc
attcatatca ccagtgctta 720gattgatcac atctccactg ttccaactaa gatagagcag
attggcatac ttgtgaatat 780tctcactggc tattaaattc tcagaagaaa aatcaacacc
aagattatga catgcttgtg 840caaagacaca cccggtcatg aatgcatcat agccagcttc
atgcttagcc ccagagttcc 900aatttgagga cctgcaagaa aacatgggag taagatggtt
tcacataaaa catgtgtaga 960agtgcagtga acactggcga aaacaatcta attttacgaa
ttcattcact cactcagctt 1020caaattaagt ttccccttta tttagggtgc cccaaaaaga
tacactcttc tgtttacctt 1080ctctctccaa gcgaccaatc ttttctctct tctccaacat
cgttttcttt ttctctctct 1140acccactatc cattttgtcc tcctacattt gataactatt
cttaatctcc aagaaaatcc 1200aatgtgtgaa ataattacgg gacagggagt atacagaagc
agcccccttg ccaatatagt 1260ttacaaatta ccctcagaat taggcttacc tttcccaaag
gagcaataaa ttcaaacaaa 1320tctaaaaggt acaaggcatt aagtgccgaa cctcatgtca
tcaacctgga cctccacctt 1380cacacatgga tgtacaccac cattagagga ttgtccagag
gctatctcag ggcacaacag 1440agaaaatgct gaggccaatg acgtgctggc tttattcaag
aatttttgaa ggctcgtgtc 1500tgcattcaaa agtattttcg tgtcgacaac atgaggaaaa
tacttgtgga tctcgagaac 1560aaactcttca acagttgatg gaagaggacc aaagaattta
tggtaaatat gtgccatatc 1620tgcaaaaaat tataatggat aagatgacaa gaaaagatac
taggaaggcc ttcaagtaca 1680aatattatat catgatgctg gacgaccgat gctcccacaa
ttatgtttgt taccaaatgc 1740ttcgaaggat aattactaaa ttatgtgaat ggtggttacc
aagtgtcccg gaccatgcaa 1800taacttctcc tttcagtgac caacaagaag aagacgtacc
taaaaagcaa ttgtgaccta 1860caattagctt cttttcagca gcgagaaggt caaggacatg
ccggaaacct gcagctgctt 1920ttattttgcg agttgcttgc tggtgagacc catacttcac
ctcctcctac aagaacaaac 1980agaacaatca cacatgcaga aagttcccca cataccaagt
tgctgtctgc taaacactga 2040aactaactta tctctacaaa caatgaagga agttcctcac
cagaaggttg atcttatcat 2100tgtcagattc tacaaaaaca ataagcttct gcaagatggc
actgccgtca tgagcacaca 2160caaaaacaag atccttgaag tgcttccttg taacctgtaa
ttgcagatca ttagtatata 2220ttcaagatgt tataaattta ttgaaaagca gcgtctaaaa
caataaaagt catgcttaag 2280gcatagagcg atagagcata gacacttcag agtttaataa
gagcaaatac tccaggagaa 2340cataaatata tttcatatca caaatcctag taccaactgg
caacggctaa ctgccaattt 2400atgtactgct caaaaaggcc aagcatctaa aagatggctt
aaaagtcgga ttttataaga 2460aagtcgtcac atgattgcta ttacattgac atatcaaagg
tcaaatgctg aaatttggtt 2520cagcttgata tatattaagc atacaaacga tacgttgaca
agaaagccta acaagacatg 2580aagcatcagg cacataacat tcaaaagatt accaattcaa
tcaacttcag ctgatgagaa 2640gtaaatccat tcaatcgaag agcaggacgc atactaaaaa
atatggtttc aaattgttgt 2700ttagattcaa cagcacttgg aagctcggca tttctgttct
gtagcaacat atcatgccaa 2760tcactgagcc gaattttcat ccgttcggag aataaaatat
cagctacatt gcccaaaggt 2820aaatctctaa cttcattaga atatgcccat ttcccgttat
atactgaatt caagcgactc 2880aaagcctcgt cttcctgtcg tctagataaa taagacacgc
ctgagattat acaatgtgta 2940aatttaccac aataatgaca tcatactgac aaaatctcaa
acaaatagtt ctaataaagt 3000catgttatct gaaatttcta tgaataggaa attgaacaaa
accttcatgc ttttttccaa 3060ctaaaattga catcttctac attaccttca tgtatgcatg
cattgaagtc aaactggtat 3120tttgccaaga agtcaatcga agttgtttgg cacaggaatt
catatgatgg gccatcagtg 3180ggaagctctt gacgtggaaa tatataaaaa ttatgcctga
caatgaacca ttgtaaaatt 3240attagatgga gtatctctat ttattgttta cagccaattg
agcttttaac aattactata 3300ggtagtgttt ggaaacttgt atttcatttc aaataatgga
attgaaatct ggaatttaaa 3360gtttgtattt caattcctaa tcactgtttt gtaaaggggg
tttgatagaa gagagagaaa 3420tagaggttta atggaggaga gagaaaaagt gtgggtttac
taaaaaaaag agaaataaat 3480attagaaagt gtgggtttac tcatagagtt gggatatgta
tgaggagaga attttcaaat 3540gccaaggtaa tagcttgaat gacaaattta ataatttcaa
attccatgtc atccaaacaa 3600tagatttcat ccaaatccaa gatttgaaat gaaatcttgc
tatccaaaca tatcataaat 3660taattagtaa tttagacttg ctttctgctg cacttactta
tggaaataat tttacttcag 3720tccttaaata acccgcaatt tacatcaaag gcactaatat
aaacacctag ttacgaaatg 3780gaaatatcag atatacctgt aaaagtaaag aaacaaaaat
acaaccctga gcatgaaggt 3840atccttcaaa agtgcaatat ctgcatactt agaaccggaa
ttagaagtgc gaatgcagac 3900aataaccatc ccaggatcag aaacgtccaa gaaagttgag
attcatcatc cattctcttt 3960agcaaattta tgaactctaa tatataaatc ataccccccc
ccccatccaa aagcaattgt 4020caagctgcct gaacccctca taatttagga tacaacaaag
taatcctaaa agacccttta 4080caatactagt actcgggtat ttccacaatc ttctcatcat
tgaatccaaa gcattgcatt 4140tgaagaaatc aaatcataat ccattactat attagagcaa
aatctatgtc attatagtat 4200tggagagcaa gtatgactat taccccttta cactaggcaa
aacacattgt cacaatgcta 4260acttagtcat taaccaatat caatatggga ctgtggatat
tcataaaatc gaagtttttc 4320gcttgctcat aaactatctt tcattccagc acagtacaag
agagaaaaga cagcattttc 4380atacacttct ttctttagtt caaattcaca cagcagcaaa
aaattcactt cttcatagct 4440ttagctcagc aaacaaagca caaagcatgc aattactctc
acacatagca caccaaaaaa 4500acaaaaacca ctaaaaattc acacaaaaaa aaccaacaaa
aattccatcg caatttcaac 4560aatcaaaaca atcttctaag ttaaaaagag agataaagat
gagaagaaaa actaacggat 4620gagcaacgaa ggaattcttc gaagaatccc atcgaaatgg
acaaacacca aattgaacaa 4680cggcgaattt ctcagcagaa tctttcattt taaggtatcg
aacatcgtgc cgatcaaact 4740cgaacgattc gcgccaaggt gagcttgtaa ttccagtcat
ttcgagatca atggcgacaa 4800aatcggcaga ttttacatgc gtagtgaggt caattagggt
ttcttcgaag tttgattttg 4860ttacattttt caaagagaaa ttagttgttg atgttgaata
atgatgataa gtagttaggg 4920ttcgtagtaa ggtggaggaa agagaaaatg gcgtcactct
gacaagcttc ttcattttgt 4980tcttcttcct tagctctgtt ttcagtcact gcgccatttt
tttaaaaaaa aggaagatga 5040acaaagcaaa tattgaaccc aaattttgta attttggccc
actttatatg tacccctccg 5100tttcaaaata tggagcacgc cgcacacacg acatttaggg
tcgaattttg aacattcttc 5160aagatgatct aatggtataa tctctataat ttatatgtgg
catattataa taagagtttt 5220atgaagtcaa aaagtggatg tcatatattt aatgcatggt
aagtttttcc taaatctgta 5280tactagggta acatacatat gttgacttga agtatatata
attcttgtag tataaatatg 5340gctttggcca taagtagtaa tacacaacaa ctagaaaaat
tgaaatcagt ccactgttat 5400cttgtactct ataattttct gtttcctttt gtttcgcaac
aaagacatat ttgtggtgaa 5460agataatttt cgtaaattga atgacttata ttttgaaata
aagagagtat taggtaaggt 5520tacgtgcttt tcgcttgaat ttgttagacc tcaaatgtat
atgtgattag aacggattgg 5580ctctagtttt tattttatag aagtatatat gcatttttct
tagagcacac tcgaaattac 5640tttcggatag atatattcgg gaaaaaaaga ggttgaaggg
aagttcatca ataattatgg 5700taaaggaaaa aggacatcgt tacaattcta aattctagat
aggatgtgat gataatccaa 5760aagtcatctg aaaaactaaa caagtccaag atgctaatga
ttcgagtaga gattgaatga 5820gtgaccctaa ggattgtcaa ccctcttatt ctaacgtgtg
taaaagaatt gacaactcta 5880agagttactc aaacattttt cgattcgagt ggttaatata
ccaatttgaa actattgaca 5940ggagttattt taatgagtat aatggtcaat ggagcactga
attccatctc acatagtcac 6000atatttcatc tcaagttctg atgatttcaa acattgaaaa
aagatgatac aagcaattaa 6060ttcctaggga aacatattgt ggttttcatg gatacaagag
tgagaataaa tcaaaactta 6120ggctctaaca tttcttttct ctactagtaa ttgctaatta
tatcaattca attgtcagtg 6180taatcagtta atcaccaaat ctcttgtata gtcagtaaac
tatacactgt ttagtcctct 6240ggattttgcc cggtcgaatt atgcagcata accaaacttt
gaagtttagt acttcctttg 6300cacccaagtt agcttcacgg cccctgcctt ctggtggatg
gtcaccctat gctttgagca 6360ttctctgcaa tgcgcacgat attcaatgag aacgtcgcct
tgaaaatcta aattgcaact 6420aaaaattaga ttgaaatgaa acccacaaga gttgtttttc
tgagtagttg gtgtagaatt 6480cacaagtctt gctccattgt ttgaagatat gaagacaata
atgtgctatg taaagtgcag 6540ccgctagcta acagtggaag tggaaacttg atcattttac
actcgcacaa gcgaaagctc 6600ggctgacgtt gcaaactgaa gaaaaacctc tcaaaccaat
tcgacttttg ctcaaagttg 6660caaactaaag aaaaaggctg aatgcaaagc aagttcacca
atgaacaata gatcggtgtt 6720ggcctgaggc cacatcaagt gaagttgcct aattgcggcc
ctctcatctg ttcacaggaa 6780tcattttcca tatagaatca ctccaaaata aaagagcaaa
gctgcaccag atgcagaagc 6840ataactttca agacaactga tgacagataa atagcaaaag
aatgcttaag aaatgatcaa 6900aattgaatgg ctctggaatt acctcatcag ctgattttcc
tttctctcta tctctctatc 6960tctttactcg tctatggagc taccacatca catggcgttt
catatgcttt ctgccgtcga 7020actagacgtg cagcaaaagc tccatccatt gaatgcttca
ctgggcatga gcgataaaac 7080ccatcttcag ttaaaaagtc agatggaaca tatctgctta
cagaatctct ttggaagtcc 7140tatcacccaa caaagaatat attaaaatag agaaggagaa
aagaacgtat ctatctgtca 7200gcatccatat gaggtggaaa ctaggagtac tatataaagc
cagtgcagta gctcctaccg 7260gatgtctaag aaggaaggca gaaaccctat cttcgttttc
ttcaagatca atggagcagg 7320tactgtacac aagcacgcca tctggtttga ccagcctgta
gatgttaaac aatcccacag 7380acaaaaggga ataatatgag tgaaacaagt caacaggggg
aaataaccaa taattctagg 7440actgtcaaac tcaagctctt caaaaacaaa gatagctctt
aatctcactt gcaagcagca 7500tcgaacagct cgtcctgcaa cttctttagc tcttccatat
cctctgactt tctattccaa 7560cgcaaatccg gcctcttcag caaaaatata aagttggaac
aaggctctta gatacaagaa 7620ctgaaaaacc ttcgacatat aatggactct atcaagggca
caatgacaaa ttctaaacat 7680gagcatgtat atcaataaaa tactaagaac cctttcaatg
gtactgctag aaggtttatt 7740gctacacttt ttagtacacc atctataggt tttatagtac
catcaaaatg gttcatggtg 7800ccataagaaa attttatgta tttatggtac tatctaccat
atctaatttt ctctgtaaaa 7860atgtatttgt agatagagac cacgagttcc tcttttagat
actgactttt ttttttctac 7920atgatggcca acagacttct caaacaaaaa gaaaaagaaa
atatttagat aatatgagca 7980acaaaatagc aaccacctac ttttgatagt acacccaggc
ccgaacaagg aacatctaaa 8040agaactttat caaacttcga agtgttgctg tcctgaaaag
aatagaaagt aactgcttca 8100acaaagaaga agaggcagaa agcaaagcta gtacgcattt
tgcaatgact tactgaaaag 8160gagcgaagat cagcatggat gcaagtgatc acattatcaa
cacgctgcag cttggctgtt 8220tcttcaagta tccgtaaccg acctttattt atgtccattg
ctgatatcat acctgaaaag 8280ctacacattt agaatgcaga accagcatca ttggtagtta
agttatcact ataccttggc 8340cattcaagcg agatgccatg aagagtgtct tccctccagg
agcagcacag caatcaatga 8400tgtgatcacc aggctgtgga tccagaacag aaacagctag
acctgcagcg aagtatagat 8460gtaaacttgg gttgggctgt cacatttttt cacatcttat
cttcctttct attctttcaa 8520aactgaggag aaatggttgg gatttctata aacgtgagaa
aaatggcatc agattagatg 8580gttttactgc atgaaaaaaa ttgaatgtgt ttcggcatca
cattactaca aggtcaaaag 8640cactatcttt gaaaatgtag gacataatgg gacagagatg
tgctgacctg cactctcatc 8700ctggactgag cataaacctt cttttagaag tccagtttgt
atcacaatct aggatatgag 8760aaaacaactc aagatgtaat tgctcctaag atatcaatca
tttcataata aacataaaag 8820ttattattac aagacagcac ctgcatccca cttctgatgc
agacaaagtc atccaaatgc 8880aaggaaggct catgcgggac ctgcggacaa agtgttgtga
tgcgcataga tatcgaaaga 8940aggccctgta tagcactaat gagataagat tcagtaacct
tcagcatgtt gagcttcaca 9000acaaggtcat ctcgagttaa tccttttgca atattggccc
tagtaccaag aaaaccatat 9060gtattaacaa gagaaaagtg gcatagggat ctttatgact
taggtaagca gttggcaatt 9120agagagaata aaacccccaa acctcaagct gaaactcgga
acactattgt tccacatcat 9180caatttgata gctccttctt gcccaagata cttggtccac
cgtcttacca tccactgaaa 9240ggaaggtatt aaaagggaga aaagactcgt cagcatagaa
aattgtacat cttaaatttt 9300agaagtatag caccatcttc aggcatcagt caacgtaaat
aaataccaca tctacaaata 9360gaaccatact ttctggacag tcgggatcat gagcacagac
aatactgcat gattattgcc 9420tcgtattctc atgtatacaa gtatatgtaa cattaaatag
cagtatttct tgagaaactc 9480accacgggat gggaataagt tgtagcaagg gcacgtgctt
gtgaacgatc atcaccctcc 9540aatttgggta caggaaggga gtcattatcc tataaagaga
aacagctttt gttttcaacc 9600atatcaagac aaacagttta ttaaactata aacaacaaca
atacacatgc acacacctac 9660tgggaacaag atatatacta ctgataagta ttttctgatt
gaagaaaaaa aatctcattt 9720atttgcaaat atagatttaa tgacaagaaa gctttgaacc
ttaaggaaaa ctagctttcg 9780gaggatccca ttcaccatgt ttcctgcgcc tggtctaaga
gcatacttgg caagattcac 9840attctgcaat ataatccaac agtaagaaca cgacatggat
ttagactcaa gtctctgaac 9900ctatagaaca agtaaaatta gatcttatct catttgacaa
tttaaaatta gatagtgcaa 9960tattctgcag ttataagact tcatgtgtgc atactgcaca
agtcatctta aaggtgttat 10020taaagcttta attgccattt gacatcccct tgctcaactt
tagcatgttt ttaggctaca 10080acaatacgca ctgtctacat ggacatacaa attacaagcg
tatggaaaag caataagcgc 10140aaggaagtct tcagccagaa actctctatg agtccaacaa
tatgcaacta aatatccaag 10200taccgtgaat gagtaagaac taacctcgtc aacaacagca
tatggtggca tttccagttt 10260cacaatctca tagcatccaa tcctgaggat ctaaaattaa
agataaatca atacacaaca 10320tatgatatgg gtcggagcgt atataacaag tatagcaact
acatttgaac agataacagc 10380ctttgagaca ataaggaact ccgacattcc agtatatgcc
agatttcata tctttagctc 10440taaattgcca cgcaaaatgt tattgggcaa tatacctgta
gcaggagagg ttccatgttc 10500ctaaaggagc tttcatcatg gcatgaagaa acaataagat
aatccagata ttttctccaa 10560cgaattgaac caccaacaat gtcagtgacc tacaaagaca
agttgtcaac ttaaaacttt 10620tgaagcgtca tttcacttct gtagaccaat acaaaagcta
ctactgcttt acatcataaa 10680acctttagtc cttaggttca tctgattggc aaaaaaggtc
cagatgcaag aaaagcaagt 10740agctgtaatg ctgtattata tcagcattat tcagaacaga
ataataaata tctacagatt 10800ttgggtggaa gcttgatgat agagtatctc cacaaagaga
actcgcttga gtcccaactc 10860ccaaatctac ttttttggag tcacattatc agtcattttt
tctggactct tataggaata 10920gtgtgctatg taatgattta tggagcaggg gcatttcatg
aatagcttta taagttagta 10980tgggtgtctt ggggaataag ttaaagggtt agttagaggg
aagaagtaca acatatatat 11040agagcttttg taagaagggt ggttatgttg aaaatagatg
agaaattggg tgagctcata 11100gtagttcaat ttggactttg ggagagaatt aagcctcttg
aaagcttgaa tatcatttac 11160atttgttgtt tttactctta ttaatcaacc aaagttcatt
ttcttccttt aatttctcca 11220ttttagcact atgatttgtc caagctaagt gatttcttag
catagtgcac agtgtagtat 11280atcggagaac tcatttgagt cctgaaaggt cccacaagtt
acatttttcc tactactact 11340tgcaccaaaa caataagcat cattaagaca ttgtcactgg
tccttcttag gttcttttgg 11400aggggattcc tcagatgggg gaggcaccca tgaaggaaca
tgttaccaag caatgggaca 11460atgcaaaatg caccaataca gtagcttcac ttcattgatt
gcatctatgt cacggaaaac 11520tgaagaaaga agcaacacct caactttatc caggacagat
atccactaac ctaggatgca 11580agcttgagac tatttagcaa ttgcctctgg gatattaaat
cagattacga ctatatttct 11640acagttattg cttaagaaaa aggtacgatt tgaagcttgg
gaagaaagag aacaagagta 11700aaagaccaat ctgagatctc tttcatccag gtctctggtg
cgaaatccaa gagtcctctc 11760aacatattcc atctcattgt tccctgaacc ctttcctctc
tcatttagaa gatcagcgaa 11820ggcaccacca aactctatcc gcatcaatct cacagcagcc
actggattta cacatgaaag 11880caaaccagga gaaccataaa aatcacaaca aacttcctga
tagcctactc actagcatca 11940accattgtgt tcagcctaaa atgagcggct gttttcaatt
gaacagcaac ttacatggac 12000cactgcataa aagtgatttc ttaatccaga caaacaaaaa
tgtttacttc aaccaactga 12060atttgcatca gctcattagt gatttgacaa gttctaattt
atgtatcaac aaacaagacc 12120atatagctag gaaacaagag gcttaggcta agcttaatgc
gtgaacaatg ttagatttca 12180acctatcagc actgtggata actgcaaact gcgacttaaa
taaggaagat aaaggaactg 12240aatatgcaat ttcaaggtgc tcagcatttg aatcaacagt
tacttcagat aattcagaac 12300ataaaagatt tgaacattct aaggctacct catgattgca
agcaatgtta cctgattcgc 12360taaccctcac aagccacaag ccaaagaagc aatttggtaa
atggttcatg gtacaactgt 12420tcgcttttgg actaatctaa caatactagg tggtaaatta
tgttcccata tctattacca 12480taatgtacag caaattaggc agcactaatt ccaaatgacc
caacaaaaaa agaggaagaa 12540aatccaaaaa ttcaagccaa catatgcact aaaattacaa
gcacaaaatc aaataatgag 12600aatcacacta tccaaagaaa atttccatcc acatttatcc
aacacaatta tctctctttt 12660acacccaaat tatgtcaacc aaaaacacta aaacaagtga
gtgcagtagc ttcacatcaa 12720agaatatcaa tcacaaacac cacataataa aatttcaact
cctgcccaaa caaaaaaaat 12780ataaagaaaa aaaaacagca aaatttcaaa gataaaatag
aaaaaaaaaa atcaaaatac 12840agggggaaaa aaagtaaatt taccagctct atgaggcgaa
acctgcaaat tcagcttctg 12900ggttttctct gaaatatcaa gcacaataac cagcaattaa
aaaaaattat aaataaaatt 12960aaaaagaaaa gattgataat taaaatcaaa agagagcaat
ttaaagcaca atcctttttt 13020taccattttt tctgggagga agagcatcct tcgttttggg
tttagacgaa aaaaatgaga 13080gttgttgtat ttgtgcgcat gagtgatcat tgctggaaat
gaaagtggga aagtggtaaa 13140tgagtgcttt gtgaaattgg gttttgagga aaagtagaaa
gaagaagaag ggtcgatgtc 13200agagaagaga gagagtggat ggaaagtagt gatgattgcc
tccattgttg ccggtgaagt 13260gagctttctg caaatatttc actggactag ttttttttag
cagataacgc taaaacagag 13320aaagatgttc ggttaatttt aatttttgga catttaaatg
actattcaat atgtttcaac 13380cttttttttt taaaacaaag gaacaatact agtattagat
tacgttaatg tttagtacat 13440ccaatactta tgtgtgtttg acctaactta aaatcgtaag
ttgtttaaaa tgtcggtgtc 13500ttgtttttaa gagatatcat acttactatc tttggttttt
actcttccat tgttaacaga 13560aactgtattt atttgggtaa ggggtttgag tgaattcctg
taagtatgag aaagttttga 13620gtgaagcaag agaaagagag aagaaaggaa cttcgagtga
agattgagag aaacaacagt 13680tagtgggaac tgttgttggg aacttgagtt taggagctca
ggttgtaccc cgagagaatt 13740aataggtttg taacagagtc ggtggcctat tatagtggaa
agtttgagtc aaaatccatt 13800gtggccgatg tcgtttcttc ttattgggcc taggaagttt
ttcctcgcta aaatttcctg 13860tgttcccatt gtgtgttcct tagctagctt tcaattccgc
aaaaagttac gtttattctc 13920tcactataat tcacccccct cttatagtgc tcatattata
caacaattga tatcaaagca 13980ggaactctaa aaatacagaa atcatgttga gttcaagatc
ttggaaaata tgaatactac 14040agaaaaactg gaagaaaggt actctactca gagaccaccg
atgttcaatg gcaaattcta 14100cacaaactgg aagaactgaa tgaagatctt catcaaagcc
gacaaatatc aggtttgtag 14160aatcatagag gcaggcgatt ttgaagtcac taccactaat
gacacatatg aggtaattcc 14220taaattcata actcatttcg ataaagtata tttcgaaaag
ttggaaatta acgttcttgc 14280tattaaactg cttcattgtg gtcttagacc tcatgaacac
aatcatgtca tgggatgcaa 14340aatcgcaaaa caaatttggg atcttcttga agtcactcat
gaaggtacgg gtaaagttaa 14400gagatcaaaa atcgatcttt taatgaatca atatgaactt
tttcaaatga aatataagga 14460gtccactcaa gagatgttta cacgctttac taatactatt
aatgagctaa cctctcttgg 14520aaaagaaatt acatatgatg aacaggtaag aaaggtccca
aggatcgttg gatggctaag 14580gttacgcctt acaaaaaact aaggacttta cgaagttcaa
tccggaacaa cttactggct 14640cccttatgac tcacgagcta cacttggaca ctgagaatgg
tgacttgtcc aaacagaagt 14700cgattgcctt gaaagccatt tttgtcatac cgtcaattaa
ttaagtaaaa agtggtaaaa 14760gaattaccaa aaacgcacaa aataaattaa ttagttggat
ataactaatt aacctattcc 14820ttttttctgt cgctataact acttttgctt aacttattga
tggtttgatc gttgaatcca 14880agttttctcc acccacaaag atattataga ctttacttta
aaaggtacga taaataatgt 14940ttaatcaggt atgcatcaac cttgaaatta ttaatttatt
aagatcaaat tatgcatatt 15000tatattaaac gtacaggact tgtgcacaat ccatggatga
tattgtagat tttgttgtaa 15060aggagttagg gacaaatgat gttgaattaa gaatgatgag
gaacaacatt gaggtaccta 15120atggcataca agattatgtg gtaacaaagg tgaagaagtt
ggttgtacca ggcaatacag 15180cagcggcaag ccatatatag gatgagctac cataccctta
tgttgtgaac tattgtcacc 15240accaacaaga cattggtcat tacgacatca ctttagttga
ggaatgataa acctcttttt 15300gctagatatt tgcaaacatc tagcagataa agaggaataa
aacactattt atatttcatg 15360aacactattt gttagttgca tgaacactat ttttagttac
acgaacacta gttttagtag 15420catcatgaac actatttttt agcatcggaa ttttcacgac
tactttttgg tttgactgac 15480actctgcaat tttcgagata actttttggt gatatgggtc
ccatgaaata gaagatttat 15540atttcatgaa cactatttgt tagttgcatg aacaatattt
ttagttacac gaacactagt 15600tttagtagca tgaacactat tttttagcat cggaatcttt
gcgactactt tttggtttga 15660ctgacacttt gcaattttcg agataacttt tttgtttgac
tgacaactat ttcctatata 15720tattgacagt tttacccctg ttagatgttt gcaaacatct
agcaaaaaga ggtttatcat 15780tcctccactt tagttagccc aacctccagt aacgccatcc
agaccactgt cgtttgtcac 15840tacgacactt acgcttggca accctatgtc ctagcccttc
gatacctcga tatccgtccg 15900ggcaatgtcc ccagtttgtc acttctctgc cattaatgac
atattttgga gtatcaaacc 15960caactccaag tatatatcgc aacatggctc agtaaagaga
gtcatataat catgacgtag 16020tttctatatg ccatcctacg tagtatcttg taacatgaat
aacagcctgg tttgcaggtt 16080gatggtacat ggtataaatt ggtattactc cctccggtct
ttattagttt aatcctttct 16140tttgtacaga gttataggag aaataatatt gtgggtcata
gaaggaaaga gaaattatta 16200ttttatgtta aagttgaatg tatgtgtgat gaaaagttag
tagtcccatt tcaaaataga 16260aaaaaaaaag gtaaactaat aagggacatc ccaaaaagga
atacgggtaa actaataaat 16320atccatgcag gttgttggta catggtacat gaagccgtcc
aaaaccttca aaagcagtaa 16380gtcctgctgc tatgccatat tcaaatattc aactccaaaa
aaaaaaaaaa aaaaaatcaa 16440aaatccgctt ttcagcgaaa atataggaaa taatccaaga
atcgaaatcg aaataaagtc 16500atgatgcaag tttggagagc tgaagttaca ctatatcgga
gtacttactc aaatgttgat 16560tagtactccg tgcgtttgaa gtaaagtcac atatggagta
gttccaagct aggttgtaca 16620gtgacggata aggatactgg gttgaaaagg tgaacgtcga
gatttatacg tgtatttatt 16680taaacaggat acgtatcata ttgggttctc atacgcgtac
cagctgtgac ttagaaaaat 16740taaccacgct atataggttc caagccctca tgattacctt
ttcatagtgt aaatttcatg 16800tagttgaatg gtgggaatcc aatcacaaaa acactgcagg
taatggaaat gttccaactt 16860tttccaagca ttttaaaata agacatgtga ttactaatta
gggcgtgttc ggcaacagta 16920attgtggtga tagtttttag ctgtgagagt agttgttagc
tgtgctatta gcttttagtg 16980gttggtgtgt agctgttagc tgttagatgt ccaagtagcg
gtgtaaaata ttgatgttcg 17040gtaaaagaag ctgtcaaagt agctgtctaa gaataactag
ttaaaaattc aaataaaact 17100ttaacatata atttatacac cactaaaagc tacccaaaag
ctacaaattg tagcttttga 17160caaacactac taaaacacta cttgtaccac taaaagctac
ttacaccact atcttgccaa 17220acactcttat tttttctaat tagtgttttg acctagtcaa
gacactaaaa gctacttaaa 17280aagcttgtgc cgaacatgcc aattctgaac caaggaacaa
actataacaa aaaagtgcta 17340tgtgaaactt ttgtaggcaa cagaagtaag gcatttttgg
aatgtactaa caaatccgta 17400ttaagacttg tacatgaaaa ttaccgtggt aacatttacc
cacacttcct cattcacgta 17460ctccgattca ttcttataag ggcataaccg cataaggcac
atcaagatcc atgtatctaa 17520tagtttaatt tgcctctgtg tttctgtatt aacaatgagc
atagtgagtg caaaagccat 17580ggaagctaga ttaaaaaggc catcattcta agttagacaa
ttggaaacaa catcgagata 17640cacgtacaca taagggctgc tcttctctat tactccctct
gttcctaatc atttgctttt 17700ttagcgggtt ccaaaggcct atgtttgacc actaatatat
ttaaattaaa actggtgata 17760tatattaaaa gaaaattatg atgaatttaa caaaaaccat
atatgttatg tccttttttt 17820tcctatatta atgaattttt acagtcaaag ttggtgaact
ttgacccaaa aaaagaaatg 17880gagcaaaaaa aaaaaaaaaa aaaaaaaact agggacaatg
agtaacattt ttatctatgt 17940ctttttaata tgaatatacg taacaaattc tgcaaaaata
gagatagcaa ctaataacac 18000gcatgaaaat gacaagttat attatacctt tttttctcaa
tatatgaata tacgtaacaa 18060attaactcca gtagttttta gtaaaactat tagattattg
tgtaacatat actctggaaa 18120tagtactaag atccattaca atctttattg agaaatttcc
tcatgtaccc cctgaggttt 18180ggcgtaattt ccaaataccc ctcatatttg aggaatttct
caaataccct gatgtttttg 18240tttagactca aaataccttt actatggaca gtaccctaat
gtcattaagt tttccccttc 18300tctctcccca attttctctc tcctcccatt cccccaccca
ctacccactg cccactgcca 18360agtaggggtg taagtggatt ggactggatt ggactttgcc
aaattcaaat ccagtccaaa 18420gttttttgga ctcgagaaat tgagtccaag tccgatccaa
atattttttg agtccagtcc 18480aatctagtcc gataattttt tcttgagtcc gaatccagtc
cagtccagtc cgattattat 18540atcttttttc ccgatttagg ttcaatgatt cacaacattt
tttgagatgc ttgagcattt 18600gacatctgat tcaattatca atatccacaa ataagattga
aagcttaaat taaagtaaaa 18660tactatgaat aaaaagttga attagatgct taccttgatc
taagttgaga ggaagcatag 18720agactgagaa ttaatctgag ggacaaatag agaatgcgag
agtcgagaca gtgaggtaga 18780aagaaaatga agagtaagag gaagtgagta ttaaggactg
aggagtaaag taagatagaa 18840ttagttggct actagcctac taatgcagta ttgctagtat
aatttactta tttaacaaat 18900ggagctaagt gcaatagttt agcgccaatt gacatattta
gagagagaag gctgaaaaat 18960ccaatatttt taaaatagta tcattatttt taatatatac
attatatata aaaatatttt 19020tggactggac tggacatatt ggactccaaa gggatgagtc
caaatccaga caaaaaatat 19080ttggacttga aaatttaagt ccgagtccag tccgaaaaat
tttcagtcca atccagtccg 19140acaaatttgg actggactgg attggactct gaacttttcg
tagtccgctt acacccctac 19200tgccaagtgc caaactgcca accccctttt ggttgagttg
atatttgacg caaagacttg 19260gcgtgttgga aggttcatta cacattttat ccaagtcaac
tttgaagtct tcttagctag 19320agactagagt gaacgtgttg gaaggttcat tacacatttt
atccaatcaa actttgaagt 19380cttcttagct agagactaga gtgaacgtgt tggaaggttc
atgttcatga cattataaaa 19440gtaataatag tgaaatttca caaagtattt ataaacccag
gacagactca agagctctac 19500ttattattag tgaaaaacaa acatacacac gacaataaca
caacataaac aataatgaac 19560atgaaaatcc tccttttgtt tgtcttcctt catcacctcc
actacttcat ccatggcaga 19620acacttacag aacgccaagc tttactaagt atcaaatctg
ccattactta tgattattat 19680aactctctct cctcatggaa aaacacaaca caccactgca
gttggccata catcacttgc 19740tcctcctctt cttcttcttc ttctgttatt tctctcaact
tcaccatgtt atttctcgaa 19800ggaattctct cccctgatat aggcttcctc accaacctgc
aaaacctctc tattcgatct 19860aacctttttt ctggcccact cccccattct ctctctctcc
tcacccaact ccgctatctc 19920gacgtttccc aaaacagttt cacaggtcca atccca
199567519206DNABeta vulgaris 75ccaacaattt gttagccgat
gaagagcatc aaaaccaaaa aaaacaaaaa aaattgatta 60atatgcatga gtgtgacctt
gttttccaaa gtttagcatt actattagtg tctcaattca 120taataataaa aaaattagct
tgttcaagat ttgtattttt attcaaagat tttttttgtc 180tcttgtgctt cttttatctt
atatatattt tttgtatggt ttgtttttgt ttaatattag 240tccctccgct caaaatgatc
tttcacgctt gagattggca ttaaggtcaa gagatgttgc 300taagctttag aataaaaaaa
ttccaaatgc atagagggaa agaaagcgag acaaaatgtt 360ggagaaggca gagtaaatga
tgtgatggag gataaatagt agaagtgtga taccgaaagt 420ttgaaaataa taaggaattt
tatttcttgc tggcactttg ttctagtaca ggtttttagc 480ccttcaaaat gtttataatg
tagagtcaaa attaatatcc ttaactagtt tttaagtccg 540ggttatatcc tagatattaa
taatattcat ttattagtaa cattttattt tataaatata 600atactaagca ttatttggtt
tgctggttaa gactttagtg tatatctatt tctttttttt 660tttattgtat gcgtgtttac
ataaactaaa gactataagg gatagtacca cgtggcgcag 720ttccttgctt aggaacgtct
tttaatatat taactagtat ttgggcccgg gcgttgctcc 780gggttggtat tgtgtttccg
aacatgatgt gcagtttttc ccattcccac taaaatatat 840aaaggaaaac tcaacattta
aaagatacaa atataataat atggacactt aaaacatgat 900taaaagttga ttgagatggt
aattgtgtca tgttataata gtaagaggtt gcctaattga 960ggttgaggtg gtggagtagt
ggtatcgctt cccatctgtt atccctgagg tataaggatc 1020aaacctcata ggactcattt
gagtaatttc ccatatcctc ctctcaaatg agtccttttc 1080atctgacaaa aaaaaagagt
ctaattttaa attaaaatta gacgatcttt tataaaatcg 1140gcactttctg cacataggtc
acaatttttt tgtttctatc tctctgcttt ctttaatttc 1200acagtctcca actctccatc
aacatcttac ttattttaga atagatgatg tatggtagta 1260ttaaatggta aagtactaaa
gctcctataa tacacagaag cttacatagt atagattcgt 1320acatgagaca aggttacaat
atactttctc cgttcttttt atattacaat aattactatt 1380ttaagtagtt tcacatctat
tgtaacaatt ccaattttgt tatagaaagc aactttaata 1440attgacaata ttgcccttac
tttatcttat taaaaccatc attaattact cactttctct 1500tataaaattg cttttatttt
ctaaggatga tttctctcct attctagtta attaaagagt 1560tacttttgtg ctaaactgct
catttattcc aaatccttaa aaattgtgtc caaacgtatt 1620gttgtaatat aaaaagaaca
gaggtactat tagtttgaat aaattttgat cagattaggt 1680cacctttagg gggcgtttgg
ttaggggtat tctggaaagg gtaagggaat caacttactt 1740aattccctta cttgttgttt
gtttgctcaa tttaatgatt ccctttaccc accccttact 1800cccaaagtcc tttactctca
ttctccccac cccccaaggt ttcacttacc ctttcttgat 1860tcatcattga ccatatcttt
gaccacccaa ctaccaccac cacttgacca cctaatcacc 1920taaccaccta attacccaac
cactattacc acccaacccc tccacctgcc caccaatcgg 1980caccataact gcccaaccgt
cgcccaatca agccacccaa ccggcaccat aaccgcccaa 2040ccaagccacc caaccggcac
cagaaattgt accaagctac ccacacacgt gaaaaccacc 2100cacccacaag ccctagaaaa
aatggaagaa tcgagagaaa gggaggggag agaaaagatg 2160cagcgactag aaggggaggg
ggaggatgtg acggcaaggg gagagggaac ttcgcagcgg 2220caaagggagg ggaaacgtcg
cgtcggcaaa gggctaaggt ggaattgacg gggttgcagc 2280aacaagggga gggcatggag
acgtcgtaac cgcaagggga ggggcagcgg cagtggaact 2340ggggtggaga ggggtagtgg
cggcactagg gtgtgggaga ggtggcgggg gatatcaaga 2400gaggggggat atggtggtgt
tatggtggaa gcaagaagaa gaaagaggaa agacaatgta 2460ctaaccaaac aacacattaa
atctaagggt tttggtttcc tttccccatc tacccctttc 2520ttgattccat tccctttacc
cctttacaac caaactcccc cttagttttt actacttata 2580accttcaatt ttggctgttt
tttgtgacat tttttacttc tccgagcctg gtcatatttt 2640ctcccgaaac atttcgagga
aagtcgaagt gacttgtgaa gttgtgcggg tgcttggcac 2700catttgtgtt gcctcgaaaa
gcatctgaat accccattta ttcctttctc ctgaaaccca 2760aaattacctc gcaataaacg
aaaagatatc catatatttg ttccaagcca catgactcct 2820ttccaacgac ctcccatgtg
accatgtcct tagaaggcat cccgtggcgt tcgaagctcg 2880gacccccgga aagtccgaaa
gtgtgtatta taactttcaa ttttggctgt ttttgggata 2940ttttttactt cttcgggcct
tgtcatattt tctctcgaaa cattcatagg attgtcaatg 3000tgacttgtaa gttgtaacgt
tgcacgggtg cttggcacaa tttgcattgc ctcgaaaagc 3060ctctgaacac cccatttgtt
catttctcgt gaaatccaaa attgcctcga aaaaaacgta 3120aaggcatcca catattcgtt
ccaagccaca taactcattt ccaatgacct cccatagagt 3180ccgtagctcg gaccccagga
aagtccaaaa acgtgtacta taaccttcaa ttttggctgt 3240ttttgggaca tgtttggact
tcaccggcct ggtcatatta tcttccgaag cattcctaca 3300aaatccgacg agactagtaa
cgttgttacg cgggtgcttg acaccatatg tgttgcctta 3360gaaagccttt aaacacccca
tttgttcatt tttcgtgaaa cccaaaattg tcccgaaatg 3420aacataaatg catccatgta
ttcgttgcaa gccacatgat ttctttccaa tgacctccca 3480tatccttagg aggcatgcat
catgtggcgt tcggcgagcg ggtctcggga aagtccgaaa 3540gcctgtgtta taaccttcaa
ttttggctat ttttgggaca tttttggcct ttttcaagcg 3600tgttcatatt ttctcccgaa
gcattcctag gttaggcgat gtgacttgta aagcgtgggt 3660acttggcacc attttctttg
cctcgaaaag tctttgagca ccacatttgt tcatttctcg 3720tgaaattcaa aattgcctcg
aaatgaacgt aaagacattc acatattcat tccaagccac 3780acatgactcc tttccaatga
cctcccaagc ccctaggagt cgtcccgtgg cgttcggatc 3840cggagctcgg gcccccgaga
atgtccgaaa ccgtgtatta tgaccttcaa tttttgctgt 3900ttttggaaca ttttttgact
tctctgggct ggtcatattt tctcccgaaa catttgtagg 3960actaccgacg tgacttgtaa
tgttgcgtgg gtgcttggca caatttgcat tgcctcgaaa 4020aacctttaaa caccgcattt
gttcatttct cgtgacaccc aaaactgcct cgaaatgaac 4080gtaaaggcat ccatatattc
gtttcatgcc acatgactcc tttccactga cctcccatgt 4140ccctagaaag caccccatat
ccgaaagctt gtattataac cttcaatttt ggctgttttt 4200gggacacttg gactttttcg
gttcgttcat attttctctc gaaatgttcc tagaaaaggt 4260gacgtgagtt gtaacgttgc
gcgggtacat ggaaccattt gccttgcctc gaaaaacctc 4320tgaacaccgc atttgttcat
ttctcgtgaa actcataatt acctcaaaat gaacgtaaat 4380gcatccatat attttttcca
agccacttga ctcttatcca atgacattct atgtccttag 4440aaggcactgc ttgtcgtcca
taattcgggc cagggaaatg tatgaaagtg tgtattataa 4500ccttcaattt tggctgtttt
tgagacaatt ttttacttct ccgggactgg tcatattttc 4560tcccgaaaaa atacttcgag
tgccgacgtg acttgtaacg tcgcgcggat gcttgacacc 4620atttgtgtta cctcgaaaag
cctttgaaca ccacatttgt tcatttctcg tgaaacccaa 4680aattgcctcg aaatgaacgt
aaaggcatcc acatatttgt tccaagccac atgactcatt 4740tccaattctc tcccatgtcc
ctaggaggca tcccgtggcg ttcggagctc ggaccctggg 4800aaagtccgaa agcgtgtatt
ataaccttca attttggctg tttttgggtc attttttgac 4860gtctcttggc ttggtcatat
tttgtgccga aacattccca ggattgccga cttgacttgt 4920aacattgctc gagtgcttgg
cacaatttgc attgcctcaa aaagactcta aacaccccat 4980ttgttcattt ctcgggaaac
ccaaaattac ctcgaaatga acgtaaaggc atccacatat 5040tcgttccatg ccacatgact
cttttccaat gacctcccat gtccctagga ggcatcccat 5100ggcattcgga gctcgaacac
tgggaaagtc cgaaagcgtg tattgtaacc ttcaattttg 5160gttgtttgtg ggacattttt
gggcttctcc gggcctggcc atattttctc ccgaaacgtt 5220ccttggaaag ccgaagtgag
ttgtaacatt gcacgggtgt ttggcaccat tagtgttgcc 5280tcgaaaagcc tttaaccaac
ccatttgttc atttctcgtg aaacctaaaa ctgcctcgaa 5340atgaacgtaa atgcatccac
atattcgttc caagccacat gactcctttc caatgacctt 5400ccaggcccct aggagtcatc
ttgtggcgtt tggagctcag tccccggtaa agtctgaaag 5460cgtgtattat aaccttcaat
tttggttgtt tttaagacat tatttgactt ctccgggact 5520gggcatatta tctcccgaaa
cattactagg agtgccgacg tgacttgtaa cgccgcgtgg 5580gtgcttggcg caattgtgtt
gcctcgaaaa gccattgaac acccccattt gttcatttct 5640cgagaaaccc aaaattgcct
cgaaatgaat gtaaaggcat cgacatattc attccaagcc 5700acatggctca tttccaatga
cctcccatat ccctaggtgt acaccccatt tgtctgatgt 5760tataatagca agaggtcacg
ggttcaaatc ttgttacaag ctaattttac ttttgttaat 5820tgacatgact tatgtacaca
ttggacaatt atagtggagt aacaaaggtg acatgtgacg 5880cgtatacatt atcacacacg
tcttttaata tatttgtata gatctagatt taagagtaat 5940ttttttaatg cgcaatactt
ggccaatttc ttctgtatca aatcataggt ctttggttgg 6000ttcataagag taaagaccaa
aataataatc tgaactgcaa aaattttctc caagagttaa 6060aagtttgtat aagttagatt
aaaaaaatta atgacatatg atgtagttgg acattaaata 6120tgtaagttta gaagtaattg
tgttaacata aaaaaagatt cgattataac ataaaaacta 6180aagaaacaca aaggcgccgt
acaacaatca atattaccca agtcccctca ttaatattaa 6240gggatgacct agctcgtaca
tatttaatta tctttgaaaa ttcgttgttc agacttgcta 6300gttgctattc tatatttgta
tattcattaa tcaatttttc aatatgtgag catttacatt 6360ttaaactaga gcaaatattg
tctcttttac tattttgttg ttgtcaaatt ttcaaaaata 6420aattgctcaa atacttttcc
tagtgacata aaaaatagag caaataatca aacagtagca 6480gacccaggaa cttttacata
atgtagacgg cataatgtgt taatttttgc ttcttttttc 6540taatatcatc caataacaca
attctgcttc tattagtttg tagtttcaga tgatgatacc 6600caaacaataa gaccaagcaa
caaattgata agattttgct tctctttctt ccacttggtg 6660taactgtaac agctttgaag
tttaacttca gtaatcagtt gcatatttgg catatgatca 6720aaacaatcaa attattatgt
atggaaaagc aaaaaacttc caggtttcca tctgaacaag 6780gaggccaaga gggtggaagc
aagcaaggat atatgatcat aaaatcctat gaatatgatg 6840tacaaacctt ttctactgca
attaggtaac ctaaatgata ccacctagga acagcaacaa 6900cttatttaca gcactaaacc
taaatcaggt taaagttaat cagaccacca tgtatctggg 6960tggtctctcg agggaaagcg
tctccatctg tatccgggta acagaggttt cttcttctcg 7020atcctccttg gcttctgccc
tcttaagttc ttcgaaggct ctcttggcat atacagtaaa 7080cgcaacaatg gtaattattg
ccactatgaa tgaaataaca ttgtacacaa tctccaccca 7140tgttagatga tgattcccat
acttgacatc tgcgaacgtc cttatcagtc tcccactgca 7200aatgaatgct atcagcgtca
atattcgaga taccaactca tttaactatt gaattgccaa 7260aaacagatat ctttgaccat
atatttgtta ctaaaaataa cgattgataa tgtgaaacta 7320tcactgatag atttaaaaga
acttttataa aagtatagtt tctctaatgt ataactgcag 7380aaaatagaat ggggtagaca
aatgaagtaa ttgttttgaa gaatgcaaaa ggtcaattca 7440gtaatacttt tatacgtgat
tgggggaagc attaaaaatc ccttctaaga taaagatgac 7500ctcattggca atggaatcga
catccacaga cccttgcatt agaacagagt ggaagtttct 7560gtgaacttac gtgtagatgt
aaagaaaagc ttctggcacc atccctgcaa ttgatcccca 7620tagataaggc caaaacgtca
tacttgtcac cacaactgcg tagttgaaga tagtataggg 7680aaatggtgaa accctaaaga
gtgccaccac gcggaactga tgaaaccagc taccttcggc 7740agcaagccta agcatagcag
ccttatccgg ccatctttgc aaccattgct aacaaggtac 7800aaaaacataa acattgtgga
cttaattaga caagaaagtt aaattaaaat caacattaga 7860taatcaataa atcaaatgta
agcagggaac atatttctta catggattct atcccggaag 7920agcaatccaa gtaaataggg
aagaatcatt ccaatagtag ttccaaccat gattatcaca 7980aaaccaagac cataaccaaa
gatcatgcct gcaagccaca tggatgggcc agaaggaatc 8040agaaatacag ggaagattgc
tagggaagta acaaggacca cagcaagaac cggacggcca 8100aaggcagtgg cttcccattg
catcattgga acaagaacct gcagagaaag taccaaaaac 8160tttgaggcaa aaatttcctg
cttgtatatt gcaaaaagta gtacagcgaa ggcattccgt 8220gcagaatggc ttatagattg
gaaatacgga gaacaatgca actataagca caggcccatc 8280tcttgacttt tgggacaata
acatggaccc ccagattgat ttataagttc tcacaccata 8340gctagatttt gttggaactt
tcataaatca tagtgacata agtatagcat aatattcatg 8400ccttcgacag aagttttcgc
atatggtaag gctactattg aaaaaattcc cttgtgtttg 8460aagtacgcat aaaaatatct
agtggcagtc aaccaaataa aacattctag gagtccctca 8520aaaaattaaa gagtcatcag
ttcagaagac tttaatatca atactttcta ttatccgggt 8580ttggcatgca gtaaatttca
tgagaaaagg aaaaatcagc tatttgatta tataaggaac 8640taattcggat gtatcactaa
gctttccatc gactggaaca tcgggagcta gtctccaata 8700ctcgtcaagg atctaacata
aacatcttct ccgcaatcaa aaagccaagg tcacatacat 8760ctaggcctct gtctcattct
gatggcatgg tatgatgcaa gttagacaac actattattt 8820ggcagatgac acttaggggt
ctaatattta agctcattca agataatcaa gtaatcaagt 8880tcaatctcaa ggtttcagtt
gcgctaaaaa atgtaatact tggctcattc agaattagtt 8940tgttgaagct ggttggtatt
tgcttcattt gttaatggaa ccaggctcat aaacaagctt 9000tcattaggct aaacttattt
aacaaaatca aaagcttaat actataattt ttgataggat 9060ttcttttggg cagttataca
tgagtaatga acaagctcta cacaatcttt tttaatgaac 9120aagctttaat cgagctaggg
tacgttctat tcaacttatt ggacctgaac ttattggaac 9180ttatctgaac tgaacttatt
gaacctgaac tgaacttatt ggaacttatt aaacctgatt 9240ggacctgatt caacttattg
gacctgattg aacctgattg gaacttattg gacctgattg 9300aacctgattg accttattgg
accttattgg aacttattga ccctgattga aacttattag 9360accttattgg acctgattga
aacttattag accttattga acctgattga aacttatttg 9420accttattag acaaaaacat
tattattatt attgttatta ttattattat tattattatt 9480attattatta ttattattat
tattattatt attattgtta acctgattga taacatttat 9540atctttcata gttattagta
acgaaaacat gttatctcta gttattcaaa gacgaattgc 9600aaaatattgt aataataata
ataataatat attattatta ttattattgt taaccttaat 9660tatttgacca tgattataat
attattcaat agcaatatga ataatcaaat aatagacaat 9720aatacaagta taatactata
cattgtggta ctttaataaa aaaattctaa taataacata 9780atcagctaat agtaatatga
ataataaaat aatagacata atacaaataa ataataaaat 9840aatagacata atacagataa
ataataaaat aatttacact aatacaagta taatactata 9900taatcattgt ggtactttaa
ttaaaattct aataataaca taatccgcta atagtgatat 9960gaaattatga ataacaaaat
agtggacaat aatacaaatg tttattaaac attgactatt 10020tggaccttat tggaccttat
tagacctgat tggaacttat tggaccttat tagacctgat 10080tggaacttat tgcacctgat
tggaacttat tacacctgat tggaacttat tgcacctgat 10140tggaacttat tgcacctgat
tggaacttat tgcacttatt agaccttatt gcaacttatc 10200tgaacttatc tgaacttatt
ggacctgaaa cttaattttt taagttgaac agaacgcacc 10260cctagtatcc acgaacatag
ttagttgttc atcgacaagg gtgttaattc cttgactata 10320aaaaaaatat ctgctaatat
gtcctccata ccatgtcttg atctgattcc caaaatcacg 10380tgttttcgtg tctggtgacc
acgttgctag acatggaaga caggtctaat tgttcagttt 10440caagtcaggt tgattaaaca
tatgttagca atatacaatc attattagtc aaactaattc 10500aactcgggtt tggtttgatt
caggttatgt cgaggatcag gtccaaatcg ggttaatcct 10560tccaggtcaa atatatctaa
gtctgttttg ccaaagtcta ctttttgtat ccgtgtccat 10620gctaaatgac aaacaaaaag
cagcttttac caagctcgaa tcagatttgt tcgcttaaag 10680agtcacttcg ctcatttaca
gcaacaatta aaggacaaaa cattgtccat tcaactactt 10740acggatatta acttattggc
aactgctagc gtaataaggc aatcaacagc actcggcctc 10800aataatgaac ctacaaggag
tccaatgacc aatacaaatt atcactggca tcatctagca 10860cgacaatctc ttaactctaa
gagtctaagt gccttgacat acaaaagtat tccttttaaa 10920agtacccccg tgtggatatt
ctgccaagca aatgcaatcg atacacccaa ttagggcttt 10980tccattatga gtcctcagag
cctcagattg taaaacaggt cagtaaaaga ggaaaatagt 11040atttgattct tttgctaaac
ccttggatat aagaatggtg acttgtattg tcacgccaag 11100cttctttcat aaaagctgat
catattatta tatgagagtt ctgagtttca aggtccgcat 11160tcgatctaac tagacatcac
ttccaattaa agttgagaaa cgaaactagg tgtcctcttt 11220gtttcccaaa ggtgaacttt
agatacttat tataagcata ttttgttatg aatcgggcta 11280aggagagggc tactcttggt
attgcataat tagttaatta cttagtagta gcttgaggaa 11340taaggaagca agtaagttag
aggaaagagt atgaaaatct gctataaagt gaggagagga 11400gggatagaag gataatcaca
aaattattga gttaactttg gttttagttg cttaggttgg 11460gagtgtccag ccactcgaat
gtcttgggac tgtaaacacc attgttcatg atctaattgc 11520atcaatatta caattaactc
atttctcttc ttatccatat tcatcttctt acaatcacaa 11580ctatttccag atcatccatc
caaatcttca tccacttgcc ttagtttcta ctccagattt 11640cagtctatta caaattgatt
tctacaatat gtcaattcat cacaaattat catgttttct 11700gaacaaaagt tcactgtttc
aggacaaata cagaaagaac tactttgatg cttagaacag 11760atatattgta aaattgtatt
cggaatttgg gatacaactg gagaagatat gaataaatag 11820gcattcaggg agctcagaaa
aacagaccgt gccatatggt gctctgctgc ataacaggaa 11880ataatggata aagtatgaat
aacgttataa cttcttaaaa acctagatga caagtatttt 11940ggttgctttt tattattggt
aggcaaggag aatactcaac aacagtttag ccttaaactg 12000cttcttattt ctcctcttcc
cctttttcct gatgatttgg ggttgtcact cagttctttt 12060acctctcatt tccaggtact
ttagagttat attacacaaa ggattgcaag agaagaacag 12120gtcgccctgg catgcactca
gaaagtatac gacccttcac aggaaatgtg gtgctccaag 12180acttatatct caggctctca
tgagtcatgt caaggaccat ctttaatcat ttgtattcta 12240ggtttctcag gcgatgcggt
gtgctggtgt gtctctccct cccacttgag tgtgtgtatt 12300gtttgtgccc ctaagttttt
atcttaacaa tcactactag tcaattagtc attaccaacc 12360ctacccacct ctcttgttac
tgttgttctt ggagatattt catatatgtc agcttagaac 12420ttatattacg tttcttatta
catattctct taagctcgcg cacatactct gtgatcgaag 12480ggatccatat tagttatctt
ttagtggagt tgttgtgaaa aaagactgca tagaaaaatt 12540aagatagctc atagttgtaa
atgtaattga acttttagat tgatagcctt gaggctgctt 12600gcattgaacc aaccaaattc
agccaggcta gtctatgcct ctttggtgtc acctggtagg 12660ttgaatttgt gtagctgtag
ttctacaaga gactgattta aaaatgtttt cgcactgaaa 12720cagcttaaac cacaaaacag
gaaagtgcag aacaaactcc agaaaatggt gcagaacata 12780ccttctcaaa aaggaaagga
actccccatt ttaacagtac gaggacaact gctacagcac 12840taatggagga gatcaagatt
ttgatccacc agatgaagga ttctgatctt gtttcagcct 12900gagaatgtaa ggttgaagct
tcaggcctct ttgtaatagc agatgtcacc agactaacaa 12960attcactgtc gtcttgcata
gcaggcccaa catctatgtc atgcttagtt agctccattg 13020aatttggcat ctccaagaga
tctcaagagc tgcccaaaaa gacggtacaa tattatgagc 13080atacatgaca tgatgacaac
ccataaagaa tatcataacc tgtcacattt tttattcaaa 13140gttcaacagc cctcttacaa
catgattgag aatggagggg aagagagaga gagttggtct 13200cagacattga tcacataatc
atttcaatta gttttaaagg tgctcatgaa atagaactag 13260tgtcttaagc tggagacttc
tgtatttttc atggttttag attatcaatc atattcttag 13320aatctttgat ctctagaact
ctttcctttc ctcccaatat tttttccact ttgtcttttg 13380ttaattacgg cttcgctgca
ggcctgcaat aaatctttta aatttttaca gatactatgt 13440agagttgtat acataagctc
taatctgaag acgattggtt tcgatgctag ttaatacaaa 13500taaatatatt atggatataa
tatgcagtaa attgggccat gggcaccagg gacaacttag 13560acaagtatag tgcaactacc
aggaaattta agctgggtac ctctgattca tcatgctggt 13620tgataatatt attgcttcca
caagtgttcg ctacggctca accaaactaa gtcacaactc 13680acaagctgca caacccaact
gacaattatc gcctattgtc taagctatac attacattac 13740cccaatgcca caacgtggct
cacgcctagg catggtaagg aagttcagat gtacgcagcc 13800ttaccctttt aataacaaag
aggctgtttc caggtgaccc ttaaatctta attgcaaaca 13860ccatctgctg cttcacataa
ataagcgact tcaaaattgt aaattaaaga atttgaatgc 13920aaattgtgtg aaaaacaact
ccatcaagaa tccattaagc acgctttact attagtatca 13980ataataggaa acccttatat
cccttttgac gaaggcacac atgcaacact aatgtgtcct 14040tataaacttc atgaaagtat
atctctacga aaccctttta gtcttatgtg attctttaag 14100tgtccaactg atgattggtt
acaaggtatt tagcccaaag tagcatttca gagagatggt 14160gtagaatgag tagcttataa
accgaggttg aggtgtaatc ctaataaatt aggaactaat 14220accacaagag agatggacat
gtagagatac aatatagtac agaataagat tatttgaaat 14280ctttttacca gggaaactcc
agaggtgttc cataaaacac aataccatat aactgggaga 14340tcaatatttt agattaaaaa
atataaaaat ctatttgggt tgagtatata gttggttagt 14400ccaataatat ataaatttat
aaggtggagg tcttcggtat atgacattcc aaatttgagt 14460atcaaatgat atatatggtt
ttccatactt gaatcccttt tcatgtacta cctctgtttc 14520aaattaatag ttacacttac
acttttcacg catgccaatg cagaactttg aggacatata 14580tctttagttt tgtatttgta
aaaattataa aaagtacata ttaataaaat acatattaat 14640acgaatctaa caagatccca
catgactatg attttattca cgtataaatc acaaacgagg 14700gtcaaaatgc aattgtgaat
agtgtaaaat gtcaaagtgt aactattaat ttgaaacgga 14760ggtagtatgt gtttatgcaa
cacttttcct ttttcccttt ttgctattta gtaatttatg 14820taaaatactt ccattgaccc
aaaagttggg tgattatagt ttacatctat cattattatt 14880tatcattact atagattatt
caccattgta atcaacttta taaaagtata cacaggtaac 14940tcaggagtca ggggtgctgg
gccaaacact tttatagttt aaggtgaaaa atctcgagaa 15000tcttctcctg ccacgcaaaa
tgagtgttct tccactttaa agatgttata acacttatct 15060taacctacta ttcgtaaata
acacttatct taacctacta ttcgtcaaga catacttgct 15120tcatctcact aagaacgtct
tagttttcat ttgaaattcg taccagaaag attcacttca 15180aatctattta tttttagata
aattgttatt aaaaacgacg aagaaacgtc agaggacaac 15240aaatcctcta aactccaaat
tataagtgag tccaactatg ttgacgtaag gtaattagag 15300tatccataaa agccctggcc
gctttggccc acaaagcagc ttagaatact acccaacccc 15360aaatataatc aatcaggtga
ggaagctcgc aacagatgcg agagttccac tccaatcaaa 15420ggcaccagaa catagccatc
gacatcttct cttctttacc ccccttgaaa ccaacagatc 15480ttaaggaagt ccactagtga
acaaggacat aaccactact catgtggaat gccaatcagc 15540ctctgtcaaa gggaagtcca
ttagtgaaca aggacatacc cactgctcaa ggtagtcatg 15600tggaaattgg aatcccaatc
agcctttgtc aaaaggaata agccacatcg caatgaagaa 15660aaaggtgcaa accagattta
ttgcatctcc aacacgacat aaatatcgag aatgaggcct 15720ttactgacaa aggaactctg
gatttccaat ttccactgag cattggactc agttgagaag 15780taattggtct tgctagattc
tgtttacgca catactctta atgataaata aatgtaacag 15840gccaattggt ctggaaaaaa
acagttgata aaaggctagt ttgggccttg gggataaata 15900taatctggta tgagttaata
aatttctgtt taaggtaaag agaatgtgtt atgtgggata 15960atttaatcaa gaaaatctta
gtaagatgga ggtagtctaa cttccattcc tcaaaatgtg 16020taattcctta taaaatcagt
cagcctctag atacatagtt agcaaaaatg gaaggtatag 16080aagtgggggt gagggaagag
gaaggaaaga gaaccgcgat caatcatatt gttcgtgctc 16140aagtttgagt tgtgcctata
gctagttaga gtttgtctat ttcattgttt ttggtcagtg 16200ttcatattct gagtgtcatc
gtgtttgggt tctagaatgc tccttttcct aatgtcgaca 16260tttctccact ttactctaga
aaaatgatct cattgtagcc attccagctt caattttaat 16320ggatactaag atccctttca
ggaacaatgt taaggtagat gttagtgttt taacagccat 16380gtggatgtta gtgtctagaa
cgagtggtca aaacactact agcctcaaaa tattgtgatc 16440agtctgaaaa ctctatgtta
gatggttgct ttttttggta ggttcgcttg ttttgggggg 16500ttagctttgt ttattttctt
cacaatttgc ccttaaactt ttcacaaaat ctacaattga 16560agattcttaa atagataaca
gacgtgtcag ctacttcaac agctaattgt acgaaaaagt 16620tcagctacct tgaaaccaaa
ccactaacag ctagtacagt ttgtttctac tattacattt 16680atctaatata acagctagta
tttagtccaa cgatgtataa tatcaatgaa atggaactaa 16740tctgtaaatt ggaccttagg
cataagagtc gagttgagca ggtacactcc aatcaccaag 16800ttatttaagc ttaaaatgtc
taacttccaa tgctgtttga cgatactcat tgccaagtgt 16860ttgttacaga tcaaccaagc
aaataaagca acaagtgaac agctgcacta gtacccaact 16920gcgaattttc gtcgattgcc
aagtgcatgt ctgggacaca ataccatcat gtccataccc 16980attaccttgc ttagccagct
atcgtaatcc ataacacata aaaaccaaca aagtcttgat 17040agtttcacaa atcaaaatgt
tcacttttca ttccaaccaa aacaagcaat aaatctcttc 17100atccatactc acaagaagaa
caatctctca cactacccac ttgattagta aaaaccccaa 17160tcaaaaacaa aatccaaccc
acataaacaa atcaaattta gtaactaccc ataaactcaa 17220aaacctcaaa tcacaatacc
aataaaagag atatacaatc aatcaaaaaa aatacaacaa 17280cagctaaaca aataacatca
taaactaaag ttattcattt tatttcctaa ctagagatca 17340attaagcagc ataaaacaac
atcactaatt caagttaata atcatcaaat tctatactat 17400aaaacataca taccttacca
aaactaccca gctgaaaatt agggtagagc tccagaaatc 17460ccggcgaaaa atccggtgag
aaattcagct aaatttgaaa acttctttag gttaagtagt 17520gtacacgatg aattgaagat
ttttacaagc atatgaaaat ggtggttgaa attgaaatgg 17580gggtttttga aaattgttgc
gacgcgtaaa agtggaaaaa aaaaaggaga gaatcaaaga 17640aatgagcaag tttttgtagg
tgggtttact gttgttgctt ttgtttgtgc acattactga 17700ctattcttaa ttcttccatg
cgtgtggggg tgaaggaatt gttttcctaa gttgtttagc 17760cacttcatag agtcattgga
tttgaataat ctagggaata atgatcatgt gtttagtgta 17820tctataaatt ataatttatg
tatgtatatt gtatatgtgg tgaggcatag aggacaaggt 17880ctaagaggaa tagaggattg
tgagggagtg tttcatgctt ttaagaatga tgagtcattg 17940agtgtattaa gttataagta
gtatttgatc gagtagtaaa gtttgtatca cgtaaatcag 18000agtgataatt aggaattggg
atttgctcaa gtggtgagtt ttcccatctt tccgagcaag 18060gtttctaggg ttcaattcct
acctcaagca tttccttggg atttaagggg acggctcaga 18120ggaattcttc ttaccaatat
tttaaaaaaa aaaaaattaa gagtggtaat ttagttcaga 18180tcctaccttt atccggttcg
aaacgacttc aagaaaaaaa aatccgacat cgtttaaaat 18240tttttacttc cgactcattt
aatccgcctc caactttgaa acaagtagtc ttatttcttt 18300tatgttaaga aaatttgcca
aaaaaaccct ttttaaagtc cagttttgcg aaaaaaaaaa 18360accttataaa gcattctttg
tgaaaacaaa ccaaaaagta aattattttt gcaaaatgaa 18420acctaatctc atttttcggt
tttgaccatg gacttttcga cattgaccac ttctatttat 18480cttcttcctc cataatcaca
gcctagccac cactaccaac acctgccgct agcccccaca 18540acctgcaccc ccacaacctc
catccacccc ctcaagcggc aacccccctt attcccatac 18600gcggcaaccc tacaccttat
cctccacccc cctccgccct taccttttct cctctccctt 18660cttccctcca tcacccctcc
ccactctctt ctccctttgc cccccatcgt tgcaccaccc 18720ataatccctc tctgtaaccc
cctctcctcg cagctccccc tccctcccag ccaaggttga 18780aaaattacag aggcagtcgc
atatggggat gggggactat cgtctaaggg gtggagagag 18840ggtttggggg ctgctggtgg
gggtggggta ggctgaatgt ggtgggggct gagggtgggg 18900ggtgaaggtg gggctgcagg
tcgggctggc ggtatggaga aagaagggaa atagaagtgg 18960ttaacaccgg aaagtccatg
atcaacaccg aaaaatgaaa ttaggtttca tcttgcaaaa 19020ataatttatt actttttgat
ttgttttcgc aaagaatgct ttataaggtt ttttcgcata 19080acatttagac ttttatcatc
cctcttagat ttgacacata ttatacgaat tatactaaaa 19140agactcctta tagtaattcg
actaatgttt tattaaaatg aacctttaga ataactcggg 19200taatat
19206762000DNABeta vulgaris
76tacgtaacaa attctgcaaa aatagagata gcaactaata acacgcatga aaatgacaag
60ttatattata cctttttttc tcaatatatg aatatacgta acaaattaac tccagtagtt
120tttagtaaaa ctattagatt attgtgtaac atatactctg gaaatagtac taagatccat
180tacaatcttt attgagaaat ttcctcatgt accccctgag gtttggcgta atttccaaat
240acccctcata tttgaggaat ttctcaaata ccctgatgtt tttgtttaga ctcaaaatac
300ctttactatg gacagtaccc taatgtcatt aagttttccc cttctctctc cccaattttc
360tctctcctcc cattccccca cccactaccc actgcccact gccaagtagg ggtgtaagtg
420gattggactg gattggactt tgccaaattc aaatccagtc caaagttttt tggactcgag
480aaattgagtc caagtccgat ccaaatattt tttgagtcca gtccaatcta gtccgataat
540tttttcttga gtccgaatcc agtccagtcc agtccgatta ttatatcttt tttcccgatt
600taggttcaat gattcacaac attttttgag atgcttgagc atttgacatc tgattcaatt
660atcaatatcc acaaataaga ttgaaagctt aaattaaagt aaaatactat gaataaaaag
720ttgaattaga tgcttacctt gatctaagtt gagaggaagc atagagactg agaattaatc
780tgagggacaa atagagaatg cgagagtcga gacagtgagg tagaaagaaa atgaagagta
840agaggaagtg agtattaagg actgaggagt aaagtaagat agaattagtt ggctactagc
900ctactaatgc agtattgcta gtataattta cttatttaac aaatggagct aagtgcaata
960gtttagcgcc aattgacata tttagagaga gaaggctgaa aaatccaata tttttaaaat
1020agtatcatta tttttaatat atacattata tataaaaata tttttggact ggactggaca
1080tattggactc caaagggatg agtccaaatc cagacaaaaa atatttggac ttgaaaattt
1140aagtccgagt ccagtccgaa aaattttcag tccaatccag tccgacaaat ttggactgga
1200ctggattgga ctctgaactt ttcgtagtcc gcttacaccc ctactgccaa gtgccaaact
1260gccaaccccc ttttggttga gttgatattt gacgcaaaga cttggcgtgt tggaaggttc
1320attacacatt ttatccaagt caactttgaa gtcttcttag ctagagacta gagtgaacgt
1380gttggaaggt tcattacaca ttttatccaa tcaaactttg aagtcttctt agctagagac
1440tagagtgaac gtgttggaag gttcatgttc atgacattat aaaagtaata atagtgaaat
1500ttcacaaagt atttataaac ccaggacaga ctcaagagct ctacttatta ttagtgaaaa
1560acaaacatac acacgacaat aacacaacat aaacaataat gaacatgaaa atcctccttt
1620tgtttgtctt ccttcatcac ctccactact tcatccatgg cagaacactt acagaacgcc
1680aagctttact aagtatcaaa tctgccatta cttatgatta ttataactct ctctcctcat
1740ggaaaaacac aacacaccac tgcagttggc catacatcac ttgctcctcc tcttcttctt
1800cttcttctgt tatttctctc aacttcacca tgttatttct cgaaggaatt ctctcccctg
1860atataggctt cctcaccaac ctgcaaaacc tctctattcg atctaacctt ttttctggcc
1920cactccccca ttctctctct ctcctcaccc aactccgcta tctcgacgtt tcccaaaaca
1980gtttcacagg tccaatccca
2000772000DNABeta vulgaris 77ccaacaattt gttagccgat gaagagcatc aaaaccaaaa
aaaacaaaaa aaattgatta 60atatgcatga gtgtgacctt gttttccaaa gtttagcatt
actattagtg tctcaattca 120taataataaa aaaattagct tgttcaagat ttgtattttt
attcaaagat tttttttgtc 180tcttgtgctt cttttatctt atatatattt tttgtatggt
ttgtttttgt ttaatattag 240tccctccgct caaaatgatc tttcacgctt gagattggca
ttaaggtcaa gagatgttgc 300taagctttag aataaaaaaa ttccaaatgc atagagggaa
agaaagcgag acaaaatgtt 360ggagaaggca gagtaaatga tgtgatggag gataaatagt
agaagtgtga taccgaaagt 420ttgaaaataa taaggaattt tatttcttgc tggcactttg
ttctagtaca ggtttttagc 480ccttcaaaat gtttataatg tagagtcaaa attaatatcc
ttaactagtt tttaagtccg 540ggttatatcc tagatattaa taatattcat ttattagtaa
cattttattt tataaatata 600atactaagca ttatttggtt tgctggttaa gactttagtg
tatatctatt tctttttttt 660tttattgtat gcgtgtttac ataaactaaa gactataagg
gatagtacca cgtggcgcag 720ttccttgctt aggaacgtct tttaatatat taactagtat
ttgggcccgg gcgttgctcc 780gggttggtat tgtgtttccg aacatgatgt gcagtttttc
ccattcccac taaaatatat 840aaaggaaaac tcaacattta aaagatacaa atataataat
atggacactt aaaacatgat 900taaaagttga ttgagatggt aattgtgtca tgttataata
gtaagaggtt gcctaattga 960ggttgaggtg gtggagtagt ggtatcgctt cccatctgtt
atccctgagg tataaggatc 1020aaacctcata ggactcattt gagtaatttc ccatatcctc
ctctcaaatg agtccttttc 1080atctgacaaa aaaaaagagt ctaattttaa attaaaatta
gacgatcttt tataaaatcg 1140gcactttctg cacataggtc acaatttttt tgtttctatc
tctctgcttt ctttaatttc 1200acagtctcca actctccatc aacatcttac ttattttaga
atagatgatg tatggtagta 1260ttaaatggta aagtactaaa gctcctataa tacacagaag
cttacatagt atagattcgt 1320acatgagaca aggttacaat atactttctc cgttcttttt
atattacaat aattactatt 1380ttaagtagtt tcacatctat tgtaacaatt ccaattttgt
tatagaaagc aactttaata 1440attgacaata ttgcccttac tttatcttat taaaaccatc
attaattact cactttctct 1500tataaaattg cttttatttt ctaaggatga tttctctcct
attctagtta attaaagagt 1560tacttttgtg ctaaactgct catttattcc aaatccttaa
aaattgtgtc caaacgtatt 1620gttgtaatat aaaaagaaca gaggtactat tagtttgaat
aaattttgat cagattaggt 1680cacctttagg gggcgtttgg ttaggggtat tctggaaagg
gtaagggaat caacttactt 1740aattccctta cttgttgttt gtttgctcaa tttaatgatt
ccctttaccc accccttact 1800cccaaagtcc tttactctca ttctccccac cccccaaggt
ttcacttacc ctttcttgat 1860tcatcattga ccatatcttt gaccacccaa ctaccaccac
cacttgacca cctaatcacc 1920taaccaccta attacccaac cactattacc acccaacccc
tccacctgcc caccaatcgg 1980caccataact gcccaaccgt
2000785488DNAArtificial SequenceCodon-optimized
LBcpf1 78aagcttatcg atgtcgacag gccttaaggg ccagatcccc cgggctgcag
gaattcgatc 60tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat
taatgtgagt 120tagctcactc attaggcacc ccaggcttta cactttatgc ttccggctcg
tatgttgtgt 180ggaattgtga gcggataaca atttcacaca ggaaacagct atgacatgat
tacgaattca 240aaaattacgg atatgaatat aggcatatcc gtatccgaat tatccgtttg
acagctagca 300acgattgtac aattgcttct ttaaaaaagg aagaaagaaa gaaagaaaag
aatcaacatc 360agcgttaaca aacggccccg ttacggccca aacggtcata tagagtaacg
gcgttaagcg 420ttgaaagact cctatcgaaa tacgtaaccg caaacgtgtc atagtcagat
cccctcttcc 480ttcaccgcct caaacacaaa aataatcttc tacagcctat atatacaacc
cccccttcta 540tctctccttt ctcacaattc atcatctttc tttctctacc cccaatttta
agaaatcctc 600tcttctcctc ttcattttca aggtaaatct ctctctctct ctctctctct
gttattcctt 660gttttaatta ggtatgtatt attgctagtt tgttaatctg cttatcttat
gtatgcctta 720tgtgaatatc tttatcttgt tcatctcatc cgtttagaag ctataaattt
gttgatttga 780ctgtgtatct acacgtggtt atgtttatat ctaatcagat atgaatttct
tcatattgtt 840gcgtttgtgt gtaccaatcc gaaatcgttg atttttttca tttaatcgtg
tagctaattg 900tacgtataca tatggatcta cgtatcaatt gttcatctgt ttgtgtttgt
atgtatacag 960atctgaaaac atcacttctc tcatctgatt gtgttgttac atacatagat
atagatctgt 1020tatatcattt tttttattaa ttgtgtatat atatatgtgc atagatctgg
attacatgat 1080tgtgattatt tacatgattt tgttatttac gtatgtatat atgtagatct
ggactttttg 1140gagttgttga cttgattgta tttgtgtgtg tatatgtgtg ttctgatctt
gatatgttat 1200gtatgtgcag cgaattcggc gcgccatggc tcctaagaag aagaggaagg
ttagcaagct 1260cgagaagttt accaactgct acagcctctc taagaccctc aggttcaagg
ctatccctgt 1320gggaaagacc caagagaata tcgacaacaa gaggctcctc gtcgaggatg
agaagagagc 1380tgaagattac aagggcgtga agaagctcct cgacaggtac tacctcagct
tcatcaacga 1440tgtgctccac agcatcaagc tcaagaacct caacaactac atcagcctct
tccgtaagaa 1500aaccaggacc gagaaagaga acaaagagct tgagaacctc gagatcaacc
tccgtaaaga 1560gatcgccaag gctttcaagg gaaacgaggg atacaagagc ctcttcaaga
aggatattat 1620cgagacaatc ctgcctgagt tcctggacga taaggatgag atcgctctcg
tgaacagctt 1680caacggattc actactgcct tcaccggatt cttcgacaac agggaaaaca
tgttcagcga 1740agaggccaag agcacctcta tcgctttcag atgcatcaac gagaacctca
cgcgttacat 1800cagcaacatg gacatcttcg agaaggtgga cgccatcttc gataagcacg
aggtgcaaga 1860aatcaaagag aagatcctca acagcgacta cgacgtcgag gacttttttg
aaggggagtt 1920cttcaacttc gttctcaccc aagagggcat cgacgtgtac aacgctatta
tcggaggatt 1980cgtgaccgag tctggggaga agattaaggg actcaacgag tacatcaacc
tgtacaacca 2040gaaaacgaag cagaagctcc cgaagttcaa gccgctctac aagcaggttc
tctctgatcg 2100tgagagcctc tcattttacg gtgagggtta cacctctgac gaggaagtgc
ttgaggtttt 2160ccgtaacacc ctcaacaaga acagcgagat cttctcgtcc atcaagaagt
tggagaaact 2220tttcaagaac ttcgacgagt acagcagcgc tgggatcttc gttaagaacg
gacctgctat 2280cagcaccatc agcaaggata ttttcggcga gtggaacgtg atcagggaca
agtggaatgc 2340tgagtacgat gacatccacc tcaagaagaa ggctgtcgtc actgagaagt
acgaggatga 2400caggcgtaag tcgttcaaga agatcggctc tttcagcctc gagcagcttc
aagaatacgc 2460tgatgctgat ctcagcgtgg tcgagaagct caaagagatc atcatccaga
aggtcgacga 2520gatctacaag gtgtacgggt cctctgagaa gttgttcgat gctgatttcg
tcctcgagaa 2580gagtctgaag aagaacgacg ctgtcgtcgc gatcatgaag gatttgctcg
acagcgtgaa 2640gtccttcgag aactatatca aggccttctt cggagagggc aaagagacta
atagggacga 2700gtctttctac ggggatttcg tgctcgctta cgatatcctc ctcaaggtgg
accatatcta 2760cgacgccatc agaaactacg tgacccagaa gccttacagc aaggacaagt
tcaagttgta 2820ctttcagaac ccgcagttca tgggcggatg ggacaaagac aaagagacag
attacagggc 2880caccatcctc aggtacgggt ctaagtacta cctggccatc atggacaaga
aatacgccaa 2940gtgcctccaa aagatcgaca aggatgacgt gaacgggaac tatgagaaga
tcaactacaa 3000gctccttccg ggaccgaaca agatgcttcc taaggtgttc ttcagcaaga
aatggatggc 3060ctactacaac ccgtctgagg acatccagaa aatctacaag aacgggacct
tcaagaaagg 3120cgacatgttc aacctcaacg actgccacaa gctcatcgat ttcttcaagg
acagcatctc 3180gcgttacccg aagtggtcta acgcttacga ctttaacttc agcgagacag
aaaagtacaa 3240ggatatcgcc gggttctacc gtgaggttga ggaacagggt tacaaggtta
gcttcgagag 3300cgcctccaag aaagaggttg acaagttggt cgaagagggc aagctctaca
tgttccagat 3360ctataacaag gacttctccg acaagagcca cggaactcct aacctccata
cgatgtactt 3420caagctgctt ttcgacgaga acaaccacgg gcagatcaga ctttctggtg
gtgctgaact 3480cttcatgcgt agggcctcac tcaagaaaga agagttggtt gttcacccgg
ccaactctcc 3540aatcgctaac aagaatcctg acaacccgaa aaagaccacc acgctgtctt
acgacgtcta 3600caaggacaaa aggttcagcg aggaccagta cgagcttcat atcccgatcg
ctatcaacaa 3660gtgcccgaag aacatcttca agatcaatac cgaggtgagg gtgctgctca
agcacgatga 3720taacccttac gtgatcggaa tcgatcgtgg tgagagaaac ctcctctaca
tcgttgtggt 3780ggacggaaag ggaaacatcg tcgagcagta cagcctgaac gagattatca
acaatttcaa 3840cggcatcagg atcaagaccg actaccactc actcctcgat aagaaagaaa
aagagcgttt 3900cgaggccagg cagaactgga cttctatcga aaacatcaaa gagttgaagg
ccggctacat 3960ctctcaggtg gtgcataaga tctgcgagct ggtggaaaag tacgatgctg
tgatcgctct 4020tgaggacctc aactctgggt tcaagaacag tagagtgaag gttgagaagc
aggtctacca 4080aaagttcgag aagatgctca tcgacaagct caactacatg gtggacaaaa
agagcaaccc 4140ttgcgctacc ggtggtgctc ttaagggata ccagatcacg aacaagttcg
agtccttcaa 4200gagcatgagc acccagaacg gcttcatctt ctatatccct gcttggctca
ccagcaagat 4260cgatccttct actggtttcg tgaacctgct caagaccaag tacacctcga
tcgccgacag 4320caagaagttc atctcgtctt tcgacaggat catgtacgtg ccggaagagg
atcttttcga 4380gttcgctctc gactataaga acttcagcag gaccgacgcc gactacatta
agaagtggaa 4440gctctactcc tacgggaacc gtatcaggat cttccgaaat ccgaagaaaa
acaacgtgtt 4500cgactgggaa gaagtgtgcc tcacctctgc ctacaaagaa ctgttcaaca
agtacggcat 4560caactaccag cagggtgata tcagggctct tttgtgcgag cagagcgaca
aggcattcta 4620cagctcattc atggccctca tgtctctcat gctccagatg aggaactcta
tcaccggaag 4680gaccgatgtg gacttcctta tctctccggt caagaactct gacgggatct
tctacgacag 4740ccgtaactat gaggctcaag agaacgctat cctgccgaag aatgctgatg
caaacggggc 4800ttacaacatt gcgagaaagg ttctctgggc tatcgggcag tttaagaaag
cggaagatga 4860gaagctcgac aaggtgaaga tcgccatctc caacaaagag tggcttgagt
acgctcagac 4920ctccgttaag cacaagaggc ctgctgctac taagaaagct ggccaggcca
aaaagaagaa 4980gtgaggcgcg ccgagctcca ggcctcccag ctttcgtccg tatcatcggt
ttcgacaacg 5040ttcgtcaagt tcaatgcatc agtttcattg cccacacacc agaatcctac
taagtttgag 5100tattatggca ttggaaaagc tgttttcttc tatcatttgt tctgcttgta
atttactgtg 5160ttctttcagt ttttgttttc ggacatcaaa atgcaaatgg atggataaga
gttaataaat 5220gatatggtcc ttttgttcat tctcaaatta ttattatctg ttgtttttac
tttaatgggt 5280tgaatttaag taagaaagga actaacagtg tgatattaag gtgcaatgtt
agacatataa 5340aacagtcttt cacctctctt tggttatgtc ttgaattggt ttgtttcttc
acttatctgt 5400gtaatcaagt ttactatgag tctatgatca agtaattatg caatcaagtt
aagtacagta 5460taggcttgag ctccctagga tcaagctt
548879989DNAPetroselinum crispum 79aattcgaatc caaaaattac
ggatatgaat ataggcatat ccgtatccga attatccgtt 60tgacagctag caacgattgt
acaattgctt ctttaaaaaa ggaagaaaga aagaaagaaa 120agaatcaaca tcagcgttaa
caaacggccc cgttacggcc caaacggtca tatagagtaa 180cggcgttaag cgttgaaaga
ctcctatcga aatacgtaac cgcaaacgtg tcatagtcag 240atcccctctt ccttcaccgc
ctcaaacaca aaaataatct tctacagcct atatatacaa 300cccccccttc tatctctcct
ttctcacaat tcatcatctt tctttctcta cccccaattt 360taagaaatcc tctcttctcc
tcttcatttt caaggtaaat ctctctctct ctctctctct 420ctgttattcc ttgttttaat
taggtatgta ttattgctag tttgttaatc tgcttatctt 480atgtatgcct tatgtgaata
tctttatctt gttcatctca tccgtttaga agctataaat 540ttgttgattt gactgtgtat
ctacacgtgg ttatgtttat atctaatcag atatgaattt 600cttcatattg ttgcgtttgt
gtgtaccaat ccgaaatcgt tgattttttt catttaatcg 660tgtagctaat tgtacgtata
catatggatc tacgtatcaa ttgttcatct gtttgtgttt 720gtatgtatac agatctgaaa
acatcacttc tctcatctga ttgtgttgtt acatacatag 780atatagatct gttatatcat
tttttttatt aattgtgtat atatatatgt gcatagatct 840ggattacatg attgtgatta
tttacatgat tttgttattt acgtatgtat atatgtagat 900ctggactttt tggagttgtt
gacttgattg tatttgtgtg tgtatatgtg tgttctgatc 960ttgatatgtt atgtatgtgc
agctgaacc 989808726DNAArtificial
SequenceResistance gene expression cassette 80tttatttaaa catgatacgt
atcatattga gtactcatac gcgtaccagc tgtgacttag 60aaaaattaac cacgctatat
aggttccaag ccctcatgat taccttttca tagtgtaaat 120ttcatgtagt tgaatggtgg
gaatccaatc acaaaaacac tgcaggtaat ggaaatgttc 180caactttttc caagcatttt
aaaataagac atgtgattac taattagggc gtgttcggca 240acagtaactg tggtgatagt
ttttagctgt gagaatagtt gttagctgtg ctgttagctt 300ttagtggttg gtgtgtaact
gttagctgtt agatgtccaa gtagcggtgt aaaatattga 360tgttcgataa aagaagctgt
caaagtagct gtttaagaat aactagttat aaattcaaat 420aaatctttaa tatataattt
atacaccact aaaagctacc caaaagctac aatctaccca 480aaagctacaa tctacccaaa
agctacaaat tgtagctttt gacaaacact actaaaacac 540tacttgtacc actaaaagct
acttacacca ctatcttgcc aaacgctctt attttttcta 600attagtgttt tgacctaatc
aagacactaa aagctactta aaaagcttgt gccgaacacg 660ccaattctga accaaggaac
aaactataac aaaaaagtgc tatgtggaac ttttgtaggc 720aacagaagta aggcattttt
ggaatgtact aacaaatccg tattaagact tgtacatgaa 780aattaccgtg gtaacatttg
cccacacttc ctcattcacg tactccgatt cattctgata 840aggcacatca agatccatgt
atctaatagt ttaatttgcc tctgtgtttc tgtattaaca 900atgagcatag tgagtgcaaa
agccatggaa gctagattaa aaaggccatc attctaagtt 960agacaattgg aaacaacatc
gagatacacg tacacataag ggctgctctt ctctattact 1020ccctctgttc ctaatcattt
gcttttttag cgggttccaa aggcctatgt ttgaccacta 1080atatatttaa attaaaactg
gtgatatata ttaaaagaaa attatgatga atttaacaaa 1140aaccatatat gttatgtcct
tttttttcct atattaatga atttttacag tcaaagttgg 1200tgaactttga cccaaaaaaa
gaaatggagc aaaaaaaaaa aaaaaaaaaa aaaactaggg 1260acaatgagta acatttttat
ctatgtcttt ttaatatgaa tatacgtaac aaattctgca 1320aaaatagaga tagcaactaa
taacacgcat gaaaatgaca agttatatta tacctttttt 1380tctcaatata tgaatatacg
taacaaatta actccagtag tttttagtaa aactattaga 1440ttattgtgta acatatactc
tggaaatagt actaagatcc attacaatct ttattgagaa 1500atttcctcat gtaccccctg
aggtttggcg taatttccaa atacccctca tatttgagga 1560atttctcaaa taccctgatg
tttttgttta gactcaaaat acctttacta tggacagtac 1620cctaatgtca ttaagttttc
cccttctctc tccccaattt tctctctcct cccattcccc 1680cacccactac ccactgccca
ctgccaagta ggggtgtaag tggattggac tggattggac 1740tttgccaaat tcaaatccag
tccaaagttt tttggactcg agaaattgag tccaagtccg 1800atccaaatat tttttgagtc
cagtccaatc tagtccgata attttttctt gagtccgaat 1860ccagtccagt ccagtccgat
tattatatct tttttcccga tttaggttca atgattcaca 1920acattttttg agatgcttga
gcatttgaca tctgattcaa ttatcaatat ccacaaataa 1980gattgaaagc ttaaattaaa
gtaaaatact atgaataaaa agttgaatta gatgcttacc 2040ttgatctaag ttgagaggaa
gcatagagac tgagaattaa tctgagggac aaatagagaa 2100tgcgagagtc gagacagtga
ggtagaaaga aaatgaagag taagaggaag tgagtattaa 2160ggactgagga gtaaagtaag
atagaattag ttggctacta gcctactaat gcagtattgc 2220tagtataatt tacttattta
acaaatggag ctaagtgcaa tagtttagcg ccaattgaca 2280tatttagaga gagaaggctg
aaaaatccaa tatttttaaa atagtatcat tatttttaat 2340atatacatta tatataaaaa
tatttttgga ctggactgga catattggac tccaaaggga 2400tgagtccaaa tccagacaaa
aaatatttgg acttgaaaat ttaagtccga gtccagtccg 2460aaaaattttc agtccaatcc
agtccgacaa atttggactg gactggattg gactctgaac 2520ttttcgtagt ccgcttacac
ccctactgcc aagtgccaaa ctgccaaccc ccttttggtt 2580gagttgatat ttgacgcaaa
gacttggcgt gttggaaggt tcattacaca ttttatccaa 2640gtcaactttg aagtcttctt
agctagagac tagagtgaac gtgttggaag gttcattaca 2700cattttatcc aatcaaactt
tgaagtcttc ttagctagag actagagtga acgtgttgga 2760aggttcatgt tcatgacatt
ataaaagtaa taatagtgaa atttcacaaa gtatttataa 2820acccaggaca gactcaagag
ctctacttat tattagtgaa aaacaaacat acacacgaca 2880ataacacaac ataaacaata
atgaacatga aaatcctcct tttgtttgtc ttccttcatc 2940acctccacta cttcatccat
ggcagaacac ttacagaacg ccaagcttta ctaagtatca 3000aatctgccat tacttatgat
tattataact ctctctcctc atggaaaaac acaacacacc 3060actgcagttg gccatacatc
acttgctcct cctcttcttc ttcttcttct gttatttctc 3120tcaacttcac catgttattt
ctcgaaggaa ttctctcccc tgatataggc ttcctcacca 3180acctgcaaaa cctctctatt
cgatctaacc ttttttctgg cccactcccc cattctctct 3240ctctcctcac ccaactccgc
tatctcgacg tttcccaaaa cagtttcaca ggtccaatcc 3300catcttctct ctctctcctc
acccaactcc gctatctcca cgtttccggc aacagtttca 3360caggtccaat cccatctttt
ctctctctcc tcacccaact ccgctatctc gacgtttccg 3420acaacagttt cacaggtcca
atcccatctt ctctctctct cctcacccaa ctccgctatc 3480tcgacgtttc ctacaacaat
ctaaatggca ctcttccctt atcggtcgtt gagaagatgt 3540cggagctcag ctaccttaac
cttaggtata actctttcta cggtgagatt ccaccggagt 3600ttgggaaact taagaagctt
gaaacattga atcttggtaa caacactctt tctgggagtc 3660ttccatctga gttgggttca
ttaaagagtt tgaaacatat ggacttttct agtaatatgc 3720tatttggtga gatcccacaa
tcttattctc ttcttcgaaa cttaatcgat attgatctta 3780atagaaacaa gttatatggg
agtatacctg attatattgg agattttccg gagttggaat 3840cacttttatt agactcgaat
aacttcacag ggagtatccc acaaaagtta ggtacaaacg 3900ggaagttgca atatctagat
ataagtaaca acaattttag tggtagtttg ccactaagtc 3960tttgcaaagg agacaaactc
caagatctgg acgcatccta taatttgttg gttgggtcaa 4020ttcctgagag tttgggaagt
tgcaagtcac ttgaaggagt gtacatggga aataatttct 4080taaacgggtc gattcctaag
ggcttgtttg ggagtgatgt ttcacttaat gacaaacttc 4140ttagtggagg tctcgatgag
aaattcggtg attgcgttaa tcttcgggac attgatctct 4200ctaataataa gctatcaggg
aagttacctg cgaccatcgg aaactgtatt catcttcggt 4260ccttgacgct ttataataac
acctgtaccg gacgtatccc tcaagagatt agcaagtgta 4320agcagctaca gaccctcgat
ctcagccaaa atcagttctc tggtgtgata cccaatgata 4380ttacaggtaa gaaagtatat
taaacttgtt acttttgaaa atattcgctc tagtttttgt 4440ttcagttggt ccattctcac
tttgtattat tgaaatatat cccaaaaaag taaatataat 4500tatataaaag aatcttgcta
aaaataatat gaattatttt tgtatgtgca aaataatgta 4560caaatctaac taatttgttg
tggataataa tattaattgt gtgaaatagt aaatgtgtgg 4620agatatataa ctttatttat
catattcact caggttttta ggtatttatt atgagttttg 4680cattggagat atccaacttg
acaatagtat ttttgtaata taccaatata taaagattac 4740tgtacataac caaaatgtat
acttttctta tttttataaa cttatatatt cctcttcttt 4800gtatttatca caacattttt
tatacccttt tgcctcatat taatagcaac acttataatt 4860tatttattta ctttttattt
cttggtctat aacctcatct acccacatat gacacaccct 4920ataaaggacc cacatgatta
accaaaatat acaaatatct tcaatgaaat taactttaac 4980actaatatga taaaaatcat
gtcccgcttt ttatcctcta actaagactc tgcataaagg 5040tatattgcaa ttaatatgag
atggaagagg tataataatt atatgatcaa attcctggat 5100tgaaaaataa atatgagatt
aaaagtggta tgtttttggt taaaagaaac tatccataaa 5160gtatgttttt ggttaaaaga
aactatgcaa cataccaatc aaatgtttat acgcttacaa 5220tttatgtacc acttttttgt
cattgttttt ctattgtttg ccatacgtac gttactaaat 5280catgttgtct tttcacattt
taactaacaa taaattacta ttgatacacc aaaaaaatct 5340atgagcattg gagtacgttg
tttgatagaa gcttcgtgct attatttctt gtcaaagaat 5400ttcatatctc aatatcttct
aatttaacaa tctaacgaaa tttttttgac ccaggaaaca 5460aatccatttg caatctggaa
aagatacaaa cacttaaatt atcaaacaat gctttgactg 5520gtgaaatccc tcattgtgtt
ggaaatatcg agctcatagc attatttctc caatcaaaca 5580aactgaacgg taccataccc
gcaaacttct caaagttatg tgattcattg atatatctag 5640atcttagtga caatcaactc
gaaggagttc tacctaagtc cttgtccaaa tgtcaaagtc 5700tagaactcct aaatgtcggg
aacaataggc taagagataa atttccttca tggttagaca 5760acctcccacg tctccaagtt
ttcagtgtgc gttttaacgc cttctacggt cctataacta 5820gctcaccaaa agttagtcac
ccatttccta tgctacaaat tatcgaccta tctaacaata 5880agttttgtgg caagttgcca
agaagatata tcaaaaactt tgcaaccatg cgcaatatga 5940atgagtctgg tgttgggaat
ccacagtacc tgggggactc atcaatatat agtattacgt 6000actctatggt attgacattc
aatgggttac aacaaaaata tgaaaagctt attgtgacga 6060tgtcgacctt tgatatatcc
agcaacaact ttactggaca gattccatat gttatagggg 6120gattacgctc acttcgtaac
cttaatctct ctcataatgt cttaaccggg aacattcctc 6180catcaattgc aaaattgtct
ttgcttcaag atttggacct ttcatcaaac agacttactg 6240gtcgtatccc tcaagaatta
gttagtttaa catttcttgg gagtttcaat gtttcgaaca 6300atctattgga ggggtctata
cctcatggtt tcaacttcga cacgtacaca gctaattcat 6360accaggggaa tctcgaatta
tgtggaaaac cattacctga gtgtggagaa agaagggcaa 6420aaggcaccac taataatcaa
gatgatccta aaaatgataa tgaacgaatg ttgtcgatgt 6480ccgaaatcgt agttatgggg
tttggcagtg gtgtactagt tgggttggct tggggatact 6540atatgttttc agtgggaaag
cccttttggt ttatcaagat ggctagcaaa atggaatcaa 6600tattgattgg ttttttctga
ccaacaattt gttagccgat gaagagcatc aaaaccaaaa 6660aaaacaaaaa aaattgatta
atatgcatga gtgtgacctt gttttccaaa gtttagcatt 6720actattagtg tctcaattca
taataataaa aaaattagct tgttcaagat ttgtattttt 6780attcaaagat tttttttgtc
tcttgtgctt cttttatctt atatatattt tttgtatggt 6840ttgtttttgt ttaatattag
tccctccgct caaaatgatc tttcacgctt gagattggca 6900ttaaggtcaa gagatgttgc
taagctttag aataaaaaaa ttccaaatgc atagagggaa 6960agaaagcgag acaaaatgtt
ggagaaggca gagtaaatga tgtgatggag gataaatagt 7020agaagtgtga taccgaaagt
ttgaaaataa taaggaattt tatttcttgc tggcactttg 7080ttctagtaca ggtttttagc
ccttcaaaat gtttataatg tagagtcaaa attaatatcc 7140ttaactagtt tttaagtccg
ggttatatcc tagatattaa taatattcat ttattagtaa 7200cattttattt tataaatata
atactaagca ttatttggtt tgctggttaa gactttagtg 7260tatatctatt tctttttttt
tttattgtat gcgtgtttac ataaactaaa gactataagg 7320gatagtacca cgtggcgcag
ttccttgctt aggaacgtct tttaatatat taactagtat 7380ttgggcccgg gcgttgctcc
gggttggtat tgtgtttccg aacatgatgt gcagtttttc 7440ccattcccac taaaatatat
aaaggaaaac tcaacattta aaagatacaa atataataat 7500atggacactt aaaacatgat
taaaagttga ttgagatggt aattgtgtca tgttataata 7560gtaagaggtt gcctaattga
ggttgaggtg gtggagtagt ggtatcgctt cccatctgtt 7620atccctgaga tataaggatc
aaacctcata ggactcattt gagtaatttc ccatatcctc 7680ctctcaaatg agtccttttc
atctgacaaa aaaaaatgtc taattttaaa ttaaaattag 7740acgatctttt ataaaatcgg
cactttctgc acataggtca caattttttt gtttctatct 7800ctctgctttc tttaattcta
cagtctccaa ctctccatca acatcttact tattttagaa 7860tagatgatgt atggtagtat
taaatggtaa agtactaaag ctcctataat acacagaagc 7920ttacatagta tagattcgta
catgagacaa ggttacaata tactttctcc gttcttttta 7980tattacaata attactattt
taagtagttt cacatctatt gtaacaattc caattttgtt 8040atagaaagca actttaataa
ttgacaatat tgcccttact ttatcttatt aaaaccatca 8100ttaattactc actttctctt
ataaaattgc ttttattttc taaggataat ttctctccta 8160ttctagttaa ttaaagagtt
acttttgtgc taaactgctc atttgttcca aatccttaaa 8220aattgtgtcc aaacgcattg
ttgtaatata aaaagaacag aggtactatt agtttgaata 8280aattttgatc ggattaggtc
acctttaggg ggcgtttggt taggggtatt ctggaaacgg 8340taagggaatc aacttactta
attcccttac ttgttgtttg tttgctcaat ttaatgattc 8400cctttaccca ccccttactc
ccaaagtcct ttactctcat tccccccacc ccccaaggtt 8460tcacttaccc tttcttgatt
catcattgac catatctttg accacccaac taccaccacc 8520acttgaccac ctaatcacct
aaccacctaa cccaaccact attaccaccc aacccctcca 8580cctgcccacc aatcggcacc
agaactgccc aaccgtcgcc caatcaagcc acccaaccgg 8640caccataacc gcccaaccaa
gccacccaac cggcaccaga aattgtacca agctacccac 8700acacgtgaaa accacccacc
cacaaa 87268120DNAArtificial
SequencePrimer 81atgttatctt taccacagtt
208224DNAArtificial SequencePrimer 82gtccctaaat gaaatacgta
aaac 24833706DNAArtificial
SequencePCR Product 83atgttatctt taccacagtt tgttgctctg acacaaccgg
taaatgcatt ggcctttgtt 60tttgatggca tcaactttgg agcatctgat tttgcatatt
cagccttttc catggtaatt 120cttttacaag aattttcatt ctttcttaag tataaacact
tagcttggga caaacttctg 180atcctatttc ttaatttttg caggtgatgg tggctgttat
gagcattttg tgtttgatgt 240ttctttcttc tcattacggt tttattggga tctgggtggc
tctaactatt tacatgagcc 300tccgcgcgtt tgctgaaggc gggaaacgac aatctgatcc
ccatcaagct tgagctcagg 360atttagcagc attccagatt gggttcaatc aacaaggtac
gagccatatc actttattca 420aattggtatc gccaaaacca agaaggaact cccatcctca
aaggtttgta aggaagaatt 480ctcagtccaa agcctcaaca aggtcagggt acagagtctc
caaaccatta gccaaaagct 540acaggagatc aatgaagaat cttcaatcaa agtaaactac
tgttccagca catgcatcat 600ggtcagtaag tttcagaaaa agacatccac cgaagactta
aagttagtgg gcatctttga 660aagtaatctt gtcaacatcg agcagctggc ttgtggggac
cagacaaaaa aggaatggtg 720cagaattgtt aggcgcacct accaaaagca tctttgcctt
tattgcaaag ataaagcaga 780ttcctctagt acaagtgggg aacaaaataa cgtggaaaag
agctgtcctg acagcccact 840cactaatgcg tatgacgaac gcagtgacga ccacaaaaga
attccctcta tataagaagg 900cattcattcc catttgaagg atcatcagat actcaaccaa
tccttctaga agatctaagc 960ttatcgataa gcttgatgta attggaggaa gatcaaaatt
ttcaatcccc attcttcgat 1020tgcttcaatt gaagtttctc cgatggcgca agttagcaga
atctgcaatg gtgtgcagaa 1080cccatctctt atctccaatc tctcgaaatc cagtcaacgc
aaatctccct tatcggtttc 1140tctgaagacg cagcagcatc cacgagctta tccgatttcg
tcgtcgtggg gattgaagaa 1200gagtgggatg acgttaattg gctctgagct tcgtcctctt
aaggtcatgt cttctgtttc 1260cacggcgtgc atgcttcacg gtgcaagcag ccgtccagca
actgctcgta agtcctctgg 1320tctttctgga accgtccgta ttccaggtga caagtctatc
tcccacaggt ccttcatgtt 1380tggaggtctc gctagcggtg aaacccgtat caccggtctt
ttggaaggtg aagatgttat 1440caacactggt aaggctatgc aagctatggg tgccagaatc
cgtaaggaag gtgatacttg 1500gatcattgat ggtgttggta acggtggact ccttgctcct
gaggctcctc tcgatttcgg 1560taacgctgca actggttgcc gtttgactat gggtcttgtt
ggtgtttacg atttcgatag 1620cactttcatt ggtgacgctt ctctcactaa gcgtccaatg
ggtcgtgtgt tgaacccact 1680tcgcgaaatg ggtgtgcagg tgaagtctga agacggtgat
cgtcttccag ttaccttgcg 1740tggaccaaag actccaacgc caatcaccta cagggtacct
atggcttccg ctcaagtgaa 1800gtccgctgtt ctgcttgctg gtctcaacac cccaggtatc
accactgtta tcgagccaat 1860catgactcgt gaccacactg aaaagatgct tcaaggtttt
ggtgctaacc ttaccgttga 1920gactgatgct gacggtgtgc gtaccatccg tcttgaaggt
cgtggtaagc tcaccggtca 1980agtgattgat gttccaggtg atccatcctc tactgctttc
ccattggttg ctgccttgct 2040tgttccaggt tccgacgtca ccatccttaa cgttttgatg
aacccaaccc gtactggtct 2100catcttgact ctgcaggaaa tgggtgccga catcgaagtg
atcaacccac gtcttgctgg 2160tggagaagac gtggctgact tgcgtgttcg ttcttctact
ttgaagggtg ttactgttcc 2220agaagaccgt gctccttcta tgatcgacga gtatccaatt
ctcgctgttg cagctgcatt 2280cgctgaaggt gctaccgtta tgaacggttt ggaagaactc
cgtgttaagg aaagcgaccg 2340tctttctgct gtcgcaaacg gtctcaagct caacggtgtt
gattgcgatg aaggtgagac 2400ttctctcgtc gtgcgtggtc gtcctgacgg taagggtctc
ggtaacgctt ctggagcagc 2460tgtcgctacc cacctcgatc accgtatcgc tatgagcttc
ctcgttatgg gtctcgtttc 2520tgaaaaccct gttactgttg atgatgctac tatgatcgct
actagcttcc cagagttcat 2580ggatttgatg gctggtcttg gagctaagat cgaactctcc
gacactaagg ctgcttgatg 2640agctcaagaa ttcgagctcg gtaccggatc ctctagctag
agctttcgtt cgtatcatcg 2700gtttcgacaa cgttcgtcaa gttcaatgca tcagtttcat
tgcgcacaca ccagaatcct 2760actgagtttg agtattatgg cattgggaaa actgtttttc
ttgtaccatt tgttgtgctt 2820gtaatttact gtgtttttta ttcggttttc gctatcgaac
tgtgaaatgg aaatggatgg 2880agaagagtta atgaatgata tggtcctttt gttcattctc
aaattaatat tatttgtttt 2940ttctcttatt tgttgtgtgt tgaatttgaa attataagag
atatgcaaac attttgtttt 3000gagtaaaaat gtgtcaaatc gtggcctcta atgaccgaag
ttaatatgag gagtaaaaca 3060cttgtagttg taccattatg cttattcact aggcaacaaa
tatattttca gacctagaaa 3120agctgcaaat gttactgaat acaagtatgt cctcttgtgt
tttagacatt tatgaacttt 3180cctttatgta attttccaga atccttgtca gattctaatc
attgctttat aattatagtt 3240atactcatgg atttgtagtt gagtatgaaa atatttttta
atgcatttta tgacttgcca 3300attgattgac aacatgcatc aatcgacctg cagccactcg
aagcggccgc cactcgagtg 3360gtggccgcat cgatcgtgaa gtttctcatc taagccccca
tttggacgtg aatgtagaca 3420cgtcgaaata aagatttccg aattagaata atttgtttat
tgctttcgcc tataaatacg 3480acggatcgta atttgtcgtt ttatcaaaat gtactttcat
tttataataa cgctgcggac 3540atctacattt ttgaattgaa aaaaattggt aattactctt
tctttttctc catattgacc 3600atcatactca ttgctgatcc atgtagattt cccggacatg
aagccattta caattgaata 3660tatcctaagt aaaacctcat aggttttacg tatttcattt
agggac 37068426DNAArtificial SequencePrimer 84cgctgcggac
atctacattt ttgaat
268528DNAArtificial SequencePrimer 85agttaacttt ccacttatcg gggcactg
2886751DNAArtificial SequencePCR Product
86cgctgcggac atctacattt ttgaattgaa aaaaaattgg taattactct ttctttttct
60ccatattgac catcatactc attgctgatc catgtagatt tcccggacat gaagccattt
120acaattgaat atatcctaag taaaacctca taggttttac gtatttcatt tagggactaa
180aatggtttag gataattact ttagctaaca taagataata aataaataaa taaataaaaa
240taaaatggtt gtagataaat aaggaaatca ataatgaata tgagtgtgag tgataggacg
300ggaatgggaa acttttacac tactttaacg ctattgaacg agtatgagta tgttataaac
360gtaaaatgtt ttatgtgtta gacaatggcc tcaagtgaaa gtgaccctat taatggagga
420aatgcaaacc acgagtctga ggtcacgctc gaagaaatga gggcaaggat cgacgcattg
480cgtagcgacc ctgtttttgg agatgccacg ggagatgcta gtgataaccg aatggattta
540atgaggttga tgatgatgga gcttttacaa ggaaatcgac aaaggcctag aactgaacaa
600gaagagtgct caaacatgtt caagaggttt tcggctcata agcccccaac ttatgatgga
660aagccagacc ccactgagtt tgaagaatgg ctcaacggca tggaaaaatt gttcgatgcc
720acccagtgcc ccgataagtg gaaagttaac t
7518730DNAArtificial SequencePrimer 87gctctgacac aaccggtaaa tgcattggcc
308829DNAArtificial SequencePrimer
88gcagattctg ctaacttgcg ccatcggag
29891042DNAArtificial SequencePCR Product 89gctctgacac aaccggtaaa
tgcattggcc tttgtttttg atggcatcaa ctttggagca 60tctgattttg catattcagc
cttttccatg gtaattcttt tacaagaatt ttcattcttt 120cttaagtata aacacttagc
ttgggacaaa cttctgatcc tatttcttaa tttttgcagg 180cgatggtggc tgttatgagc
attttgtgtt tgatgtttct ctcttctcat tacggtttta 240ttgggatctg ggtggctcta
actatttaca tgagcctccg cgcgtttgct gaaggcggga 300aacgacaatc tgatccccat
caagcttgag ctcaggattt agcagcattc cagattgggt 360tcaatcaaca aggtacgagc
catatcactt tattcaaatt ggtatcgcca aaaccaagaa 420ggaactccca tcctcaaagg
tttgtaagga agaattctca gtccaaagcc tcaacaaggt 480cagggtacag agtctccaaa
ccattagcca aaagctacag gagatcaatg aagaatcttc 540aatcaaagta aactactgtt
ccagcacatg catcatggtc agtaagtttc agaaaaagac 600atccaccgaa gacttaaagt
tagtgggcat ctttgaaagt aatcttgtca acatcgagca 660gctggcttgt ggggaccaga
caaaaaagga atggtgcaga attgttaggc gcacctacca 720aaagcatctt tgcctttatt
gcaaagataa agcagattcc tctagtacaa gtggggaaca 780aaataacgtg gaaaagagct
gtcctgacag cccactcact aatgcgtatg acgaacgcag 840tgacgaccac aaaagaattc
cctctatata agaaggcatt cattcccatt tgaaggatca 900tcagatactg aaccaatcct
tctagaagat ctaagcttat cgataagctt gatgtaattg 960gaggaagatc aaaattttca
atccccattc ttcgattgct tcaattgaag tttctccgat 1020ggcgcaagtt agcagaatct
gc 104290665PRTBeta vulgaris
90Met Ala Ala Thr Phe Thr Asn Pro Thr Phe Ser Pro Ser Ser Thr Pro1
5 10 15Leu Thr Lys Thr Leu Lys
Ser Gln Ser Ser Ile Ser Ser Thr Leu Pro 20 25
30Phe Ser Thr Pro Pro Lys Thr Pro Thr Pro Leu Phe His
Arg Pro Leu 35 40 45Gln Ile Ser
Ser Ser Gln Ser His Lys Ser Ser Ala Ile Lys Thr Gln 50
55 60Thr Gln Ala Pro Ser Ser Pro Ala Ile Glu Asp Ser
Ser Phe Val Ser65 70 75
80Arg Phe Gly Pro Asp Glu Pro Arg Lys Gly Ser Asp Val Leu Val Glu
85 90 95Ala Leu Glu Arg Glu Gly
Val Thr Asn Val Phe Ala Tyr Pro Gly Gly 100
105 110Ala Ser Met Glu Ile His Gln Ala Leu Thr Arg Ser
Lys Thr Ile Arg 115 120 125Asn Val
Leu Pro Arg His Glu Gln Gly Gly Val Phe Ala Ala Glu Gly 130
135 140Tyr Ala Arg Ala Thr Gly Lys Val Gly Val Cys
Ile Ala Thr Ser Gly145 150 155
160Pro Gly Ala Thr Asn Leu Val Ser Gly Leu Ala Asp Ala Leu Leu Asp
165 170 175Ser Val Pro Leu
Val Ala Ile Thr Gly Gln Val Pro Arg Arg Met Ile 180
185 190Gly Thr Asp Ala Phe Gln Glu Thr Pro Ile Val
Glu Val Thr Arg Ser 195 200 205Ile
Thr Lys His Asn Tyr Leu Val Leu Asp Val Glu Asp Ile Pro Arg 210
215 220Ile Val Lys Glu Ala Phe Phe Leu Ala Asn
Ser Gly Arg Pro Gly Pro225 230 235
240Val Leu Ile Asp Leu Pro Lys Asp Ile Gln Gln Gln Leu Val Val
Pro 245 250 255Asp Trp Asp
Arg Pro Phe Lys Leu Gly Gly Tyr Met Ser Arg Leu Pro 260
265 270Lys Ser Lys Phe Ser Thr Asn Glu Val Gly
Leu Leu Glu Gln Ile Val 275 280
285Arg Leu Met Ser Glu Ser Lys Lys Pro Val Leu Tyr Val Gly Gly Gly 290
295 300Cys Leu Asn Ser Ser Glu Glu Leu
Arg Arg Phe Val Glu Leu Thr Gly305 310
315 320Ile Pro Val Ala Ser Thr Leu Met Gly Leu Gly Ser
Tyr Pro Cys Asn 325 330
335Asp Glu Leu Ser Leu His Met Leu Gly Met His Gly Thr Val Tyr Ala
340 345 350Asn Tyr Ala Val Asp Lys
Ala Asp Leu Leu Leu Ala Phe Gly Val Arg 355 360
365Phe Asp Asp Arg Val Thr Gly Lys Leu Glu Ala Phe Ala Ser
Arg Ala 370 375 380Lys Ile Val His Ile
Asp Ile Asp Ser Ala Glu Ile Gly Lys Asn Lys385 390
395 400Gln Pro His Val Ser Ile Cys Ala Asp Val
Lys Leu Ala Leu Arg Gly 405 410
415Met Asn Lys Ile Leu Glu Ser Arg Ile Gly Lys Leu Asn Leu Asp Phe
420 425 430Ser Lys Trp Arg Glu
Glu Leu Gly Glu Gln Lys Lys Glu Phe Pro Leu 435
440 445Ser Phe Lys Thr Phe Gly Asp Ala Ile Pro Pro Gln
Tyr Ala Ile Gln 450 455 460Val Leu Asp
Glu Leu Thr Asn Gly Asn Ala Ile Ile Ser Thr Gly Val465
470 475 480Gly Gln His Gln Met Trp Ala
Ala Gln His Tyr Lys Tyr Arg Asn Pro 485
490 495Arg Gln Trp Leu Thr Ser Gly Gly Leu Gly Ala Met
Gly Phe Gly Leu 500 505 510Pro
Ala Ala Ile Gly Ala Ala Val Ala Arg Pro Asp Ala Val Val Val 515
520 525Asp Ile Asp Gly Asp Gly Ser Phe Ile
Met Asn Val Gln Glu Leu Ala 530 535
540Thr Ile Arg Val Glu Asn Leu Pro Val Lys Ile Met Leu Leu Asn Asn545
550 555 560Gln His Leu Gly
Met Val Val Gln Trp Glu Asp Arg Phe Tyr Lys Ala 565
570 575Asn Arg Ala His Thr Tyr Leu Gly Asn Pro
Ser Lys Ser Ala Asp Ile 580 585
590Phe Pro Asp Met Leu Lys Phe Ala Glu Ala Cys Asp Ile Pro Ser Ala
595 600 605Arg Val Ser Asn Val Ala Asp
Leu Arg Ala Ala Ile Gln Thr Met Leu 610 615
620Asp Thr Pro Gly Pro Tyr Leu Leu Asp Val Ile Val Pro His Gln
Glu625 630 635 640His Val
Leu Pro Met Ile Pro Ser Gly Ala Gly Phe Lys Asp Thr Ile
645 650 655Thr Glu Gly Asp Gly Arg Thr
Ser Tyr 660 665911998DNAArtificial
SequenceAcetolactate synthase with mutation 91atggcggcta ccttcacaaa
cccaacattt tccccttcct caactccatt aaccaaaacc 60ctaaaatccc aatcttccat
ctcttcaacc ctcccctttt ccacccctcc caaaacccca 120actccactct ttcaccgtcc
cctccaaatc tcatcctccc aatcccacaa atcatccgcc 180attaaaacac aaactcaagc
accttcttct ccagctattg aagattcatc tttcgtttct 240cgatttggcc ctgatgaacc
cagaaaaggg tccgatgtcc tcgttgaagc tcttgagcgt 300gaaggtgtta ccaatgtgtt
tgcttaccct ggtggtgcat ctatggaaat ccaccaagct 360ctcacacgct ctaaaaccat
ccgcaatgtc ctccctcgcc atgaacaagg cggggttttc 420gccgccgagg gatatgctag
agctactgga aaggttggtg tctgcattgc gacttctggt 480cctggtgcta ccaacctcgt
atcaggtctt gctgacgctc tccttgattc tgtccctctt 540gttgccatca ctggccaagt
tccacgccgt atgattggca ctgatgcttt tcaggagact 600ccaattgttg aggtgacaag
gtctattact aagcataatt atttagtttt ggatgtagag 660gatattccta gaattgttaa
ggaagccttt tttttagcta attctggtag gcctggacct 720gttttgattg atcttcctaa
agatattcag cagcaattgg ttgttcctga ttgggatagg 780ccttttaagt tgggtgggta
tatgtctagg ctgccaaagt ccaagttttc gacgaatgag 840gttggacttc ttgagcagat
tgtgaggttg atgagtgagt cgaagaagcc tgtcttgtat 900gtgggaggtg ggtgtttgaa
ttctagtgag gagttgagga gatttgttga gttgacaggg 960attccggtgg ctagtacttt
gatggggttg gggtcttacc cttgtaatga tgaactgtct 1020cttcatatgt tggggatgca
cgggactgtt tatgccaatt atgcggtgga taaggcggat 1080ttgttgcttg ctttcggggt
taggtttgat gatcgtgtga ccgggaagct cgaggcgttt 1140gctagccgtg ctaagattgt
gcatattgat attgactctg ctgagattgg gaagaacaag 1200cagccccatg tgtccatttg
tgctgatgtt aaattggcat tgcggggtat gaataagatt 1260ctggagtcta gaatagggaa
gctgaatttg gatttctcca agtggagaga agaattaggt 1320gagcagaaga aggaattccc
actgagtttt aagacatttg gggatgcaat tcctccacaa 1380tatgccattc aggtgcttga
tgagttgacc aatggtaatg ctattataag tactggtgtt 1440gggcagcacc aaatgtgggc
tgcgcagcat tacaagtaca gaaaccctcg ccaatggctg 1500acctctggtg ggttgggggc
tatggggttt gggctaccag ccgccattgg agctgcagtt 1560gctcgaccag atgcagtggt
tgtcgatatt gatggggatg gcagttttat tatgaatgtt 1620caagagttgg ctacaattag
ggtggaaaat ctcccagtta agataatgct gctaaacaat 1680caacatttag gtatggttgt
ccaattggaa gataggttct ataaagctaa ccgggcacat 1740acataccttg gaaacccttc
caaatctgct gatatcttcc ctgatatgct caaattcgct 1800gaggcatgtg atattccttc
tgcccgtgtt agcaacgtgg ctgatttgag ggccgccatt 1860caaacaatgt tggatactcc
agggccgtac ctgctcgatg tgattgtacc gcatcaagag 1920catgtgttgc ctatgattcc
aagtggtgcc ggtttcaagg ataccattac agagggtgat 1980ggaagaacct cttattga
19989221DNABeta vulgaris
92cctgagagtt tgggaagttg c
219323DNABeta vulgaris 93atgtcccgaa gattaacgca atc
23943720DNAArtificial Sequencemodified genomic
sequence of the gene mediating resistance towards cercospora
94atgaacatga aaatcttact tttgtttgtc ttccttcatc acctccacta cttcatccat
60ggcagaacac ttacagaacg ccaagcttta ctaagtatca aatctgccat tacttatgat
120tattataact ctctctcctc atggaaaaac acaacacacc actgcagttg gccatacatc
180acttgctcct cctcttcttc ttcttcttct gttatttctc tcaacttcac catgttattt
240ctcgaaggaa ttctctcccc tgatataggc ttcctcacca acctgcaaaa cctctctatt
300cgatctaacc ttttttctgg cccactcccc cattctctct ctctcctcac ccaactccgc
360tatctcgacg tttcccaaaa cagtttcaca ggtccaatcc catcttctct ctctctcctc
420acccaactcc gctatctcca cgtttccggc aacagtttca caggtccaat cccatctttt
480ctctctctcc tcacccaact ccgctatctc gacgtttccg acaacagttt cacaggtcca
540atcccatctt ctctctctct cctcacccaa ctccgctatc tcgacgtttc ctacaacaat
600ctaaatggca ctcttccctt atcggtcgtt gagaagatgt cggagctcag ctaccttaac
660cttaggtata actctttcta cggtgagatt ccaccggagt ttgggaaact taagaagctt
720gaaacattga atcttggtaa caacactctt tctgggagtc ttccatctga gttgggttca
780ttaaagagtt tgaaacatat ggacttttct agtaatatgc tatttggtga gatcccacaa
840tcttattctc ttcttcgaaa cttaatcgat attgatctta atagaaacaa gttatatggg
900agtatacctg attatattgg agattttccg gagttggaat cacttttatt agactcgaat
960aacttcacag ggagtatccc acaaaagtta ggtacaaacg ggaagttgca atatctagat
1020ataagtaaca acaattttag tggtagtttg ccactaagtc tttgcaaagg agacaaactc
1080caagatctgg acgcatccta taatttgttg gttgggtcaa ttcctgagag tttgggaagt
1140tgcaagtcac ttgaaggagt gtacatggga aataatttct taaacgggtc gattcctaag
1200ggcttgtttg ggagtgatgt ttcacttaat gacaaacttc ttagtggagg tctcgatgag
1260aaattcggtg attgcgttaa tcttcgggac attgatctct ctaataataa gctatcaggg
1320aagttacctg cgaccatcgg aaactgtatt catcttcggt ccttgacgct ttataataac
1380acctgtaccg gacgtatccc tcaagagatt agcaagtgta agcagctaca gaccctcgat
1440ctcagccaaa atcagttctc tggtgtgata cccaatgata ttacaggtaa gaaagtatat
1500taaacttgtt acttttgaaa atattcgctc tagtttttgt ttcagttggt ccattctcac
1560tttgtattat tgaaatatat cccaaaaaag taaatataat tatataaaag aatcttgcta
1620aaaataatat gaattatttt tgtatgtgca aaataatgta caaatctaac taatttgttg
1680tggataataa tattaattgt gtgaaatagt aaatgtgtgg agatatataa ctttatttat
1740catattcact caggttttta ggtatttatt atgagttttg cattggagat atccaacttg
1800acaatagtat ttttgtaata taccaatata taaagattac tgtacataac caaaatgtat
1860acttttctta tttttataaa cttatatatt cctcttcttt gtatttatca caacattttt
1920tatacccttt tgcctcatat taatagcaac acttataatt tatttattta ctttttattt
1980cttggtctat aacctcatct acccacatat gacacaccct ataaaggacc cacatgatta
2040accaaaatat acaaatatct tcaatgaaat taactttaac actaatatga taaaaatcat
2100gtcccgcttt ttatcctcta actaagactc tgcataaagg tatattgcaa ttaatatgag
2160atggaagagg tataataatt atatgatcaa attcctggat tgaaaaataa atatgagatt
2220aaaagtggta tgtttttggt taaaagaaac tatccataaa gtatgttttt ggttaaaaga
2280aactatgcaa cataccaatc aaatgtttat acgcttacaa tttatgtacc acttttttgt
2340cattgttttt ctattgtttg ccatacgtac gttactaaat catgttgtct tttcacattt
2400taactaacaa taaattacta ttgatacacc aaaaaaatct atgagcattg gagtacgttg
2460tttgatagaa gcttcgtgct attatttctt gtcaaagaat ttcatatctc aatatcttct
2520aatttaacaa tctaacgaaa tttttttgac ccaggaaaca aatccatttg caatctggaa
2580aagatacaaa cacttaaatt atcaaacaat gctttgactg gtgaaatccc tcattgtgtt
2640ggaaatatcg agctcatagc attatttctc caatcaaaca aactgaacgg taccataccc
2700gcaaacttct caaagttatg tgattcattg atatatctag atcttagtga caatcaactc
2760gaaggagttc tacctaagtc cttgtccaaa tgtcaaagtc tagaactcct aaatgtcggg
2820aacaataggc taagagataa atttccttca tggttagaca acctcccacg tctccaagtt
2880ttcagtgtgc gttttaacgc cttctacggt cctataacta gctcaccaaa agttagtcac
2940ccatttccta tgctacaaat tatcgaccta tctaacaata agttttgtgg caagttgcca
3000agaagatata tcaaaaactt tgcaaccatg cgcaatatga atgagtctgg tgttgggaat
3060ccacagtacc tgggggactc atcaatatat agtattacgt actctatggt attgacattc
3120aatgggttac aacaaaaata tgaaaagctt attgtgacga tgtcgacctt tgatatatcc
3180agcaacaact ttactggaca gattccatat gttatagggg gattacgctc acttcgtaac
3240cttaatctct ctcataatgt cttaaccggg aacattcctc catcaattgc aaaattgtct
3300ttgcttcaag atttggacct ttcatcaaac agacttactg gtcgtatccc tcaagaatta
3360gttagtttaa catttcttgg gagtttcaat gtttcgaaca atctattgga ggggtctata
3420cctcatggtt tcaacttcga cacgtacaca gctaattcat accaggggaa tctcgaatta
3480tgtggaaaac cattacctga gtgtggagaa agaagggcaa aaggcaccac taataatcaa
3540gatgatccta aaaatgataa tgaacgaatg ttgtcgatgt ccgaaatcgt agttatgggg
3600tttggcagtg gtgtactagt tgggttggct tggggatact atatgttttc agtgggaaag
3660cccttttggt ttatcaagat ggctagcaaa atggaatcaa tattgattgg ttttttctga
3720952652DNAArtificial Sequencemodified cDNA sequence of the gene
mediating resistance towards cercosporavariation(55)..(57)
95atgaacatga aaatcctcct tttgtttgtc ttccttcatc acctccacta cttcatacat
60ggcagaacac ttacagaacg ccaagcttta ctaagtatca aatctgccat tacttatgat
120tattataact ctctctcctc atggaaaaac acaacacacc actgcagttg gccatacatc
180acttgctcct cctcttcttc ttcttcttct gttatttctc tcaacttcac catgttattt
240ctcgaaggaa ttctctcccc tgatataggc ttcctcacca acctgcaaaa cctctctatt
300cgatctaacc ttttttctgg cccactcccc cattctctct ctctcctcac ccaactccgc
360tatctcgacg tttcccaaaa cagtttcaca ggtccaatcc catcttctct ctctctcctc
420acccaactcc gctatctcca cgtttccggc aacagtttca caggtccaat cccatctttt
480ctctctctcc tcacccaact ccgctatctc gacgtttccg acaacagttt cacaggtcca
540atcccatctt ctctctctct cctcacccaa ctccgctatc tcgacgtttc ctacaacaat
600ctaaatggca ctcttccctt atcggtcgtt gagaagatgt cggagctcag ctaccttaac
660cttaggtata actctttcta cggtgagatt ccaccggagt ttgggaaact taagaagctt
720gaaacattga atcttggtaa caacactctt tctgggagtc ttccatctga gttgggttca
780ttaaagagtt tgaaacatat ggacttttct agtaatatgc tatttggtga gatcccacaa
840tcttattctc ttcttcgaaa cttaatcgat attgatctta atagaaacaa gttatatggg
900agtatacctg attatattgg agattttccg gagttggaat cacttttatt agactcgaat
960aacttcacag ggagtatccc acaaaagtta ggtacaaacg ggaagttgca atatctagat
1020ataagtaaca acaattttag tggtagtttg ccactaagtc tttgcaaagg agacaaactc
1080caagatctgg acgcatccta taatttgttg gttgggtcaa ttcctgagag tttgggaagt
1140tgcaagtcac ttgaaggagt gtacatggga aataatttct taaacgggtc gattcctaag
1200ggcttgtttg ggagtgatgt ttcacttaat gacaaacttc ttagtggagg tctcgatgag
1260aaattcggtg attgcgttaa tcttcgggac attgatctct ctaataataa gctatcaggg
1320aagttacctg cgaccatcgg aaactgtatt catcttcggt ccttgacgct ttataataac
1380acctgtaccg gacgtatccc tcaagagatt agcaagtgta agcagctaca gaccctcgat
1440ctcagccaaa atcagttctc tggtgtgata cccaatgata ttacaggaaa caaatccatt
1500tgcaatctgg aaaagataca aacacttaaa ttatcaaaca atgctttgac tggtgaaatc
1560cctcattgtg ttggaaatat cgagctcata gcattatttc tccaatcaaa caaactgaac
1620ggtaccatac ccgcaaactt ctcaaagtta tgtgattcat tgatatatct agatcttagt
1680gacaatcaac tcgaaggagt tctacctaag tccttgtcca aatgtcaaag tctagaactc
1740ctaaatgtcg ggaacaatag gctaagagat aaatttcctt catggttaga caacctccca
1800cgtctccaag ttttcagtgt gcgttttaac gccttctacg gtcctataac tagctcacca
1860aaagttagtc acccatttcc tatgctacaa attatcgacc tatctaacaa taagttttgt
1920ggcaagttgc caagaagata tatcaaaaac tttgcaacca tgcgcaatat gaatgagtct
1980ggtgttggga atccacagta cctgggggac tcatcaatat atagtattac gtactctatg
2040gtattgacat tcaatgggtt acaacaaaaa tatgaaaagc ttattgtgac gatgtcgacc
2100tttgatatat ccagcaacaa ctttactgga cagattccat atgttatagg gggattacgc
2160tcacttcgta accttaatct ctctcataat gtcttaaccg ggaacattcc tccatcaatt
2220gcaaaattgt ctttgcttca agatttggac ctttcatcaa acagacttac tggtcgtatc
2280cctcaagaat tagttagttt aacatttctt gggagtttca atgtttcgaa caatctattg
2340gaggggtcta tacctcatgg tttcaacttc gacacgtaca cagctaattc ataccagggg
2400aatctcgaat tatgtggaaa accattacct gagtgtggag aaagaagggc aaaaggcacc
2460actaataatc aagatgatcc taaaaatgat aatgaacgaa tgttgtcgat gtccgaaatc
2520gtagttatgg ggtttggcag tggtgtacta gttgggttgg cttggggata ctatatgttt
2580tcagtgggaa agcccttttg gtttatcaag atggctagca aaatggaatc aatattgatt
2640ggttttttct ga
265296883PRTArtificial Sequencemodified protein sequence of the gene
mediating resistance towards cercosporaVARIANT(209)..(209) 96Met Asn Met
Lys Ile Leu Leu Leu Phe Val Phe Leu His His Leu His1 5
10 15Tyr Phe Ile His Gly Arg Thr Leu Thr
Glu Arg Gln Ala Leu Leu Ser 20 25
30Ile Lys Ser Ala Ile Thr Tyr Asp Tyr Tyr Asn Ser Leu Ser Ser Trp
35 40 45Lys Asn Thr Thr His His Cys
Ser Trp Pro Tyr Ile Thr Cys Ser Ser 50 55
60Ser Ser Ser Ser Ser Ser Val Ile Ser Leu Asn Phe Thr Met Leu Phe65
70 75 80Leu Glu Gly Ile
Leu Ser Pro Asp Ile Gly Phe Leu Thr Asn Leu Gln 85
90 95Asn Leu Ser Ile Arg Ser Asn Leu Phe Ser
Gly Pro Leu Pro His Ser 100 105
110Leu Ser Leu Leu Thr Gln Leu Arg Tyr Leu Asp Val Ser Gln Asn Ser
115 120 125Phe Thr Gly Pro Ile Pro Ser
Ser Leu Ser Leu Leu Thr Gln Leu Arg 130 135
140Tyr Leu His Val Ser Gly Asn Ser Phe Thr Gly Pro Ile Pro Ser
Phe145 150 155 160Leu Ser
Leu Leu Thr Gln Leu Arg Tyr Leu Asp Val Ser Asp Asn Ser
165 170 175Phe Thr Gly Pro Ile Pro Ser
Ser Leu Ser Leu Leu Thr Gln Leu Arg 180 185
190Tyr Leu Asp Val Ser Tyr Asn Asn Leu Asn Gly Thr Leu Pro
Leu Ser 195 200 205Val Leu Glu Lys
Met Ser Glu Leu Ser Tyr Leu Asn Leu Arg Tyr Asn 210
215 220Ser Phe Tyr Gly Glu Ile Pro Pro Glu Phe Gly Lys
Leu Lys Lys Leu225 230 235
240Glu Thr Leu Asn Leu Gly Asn Asn Thr Leu Ser Gly Ser Leu Pro Ser
245 250 255Glu Leu Gly Ser Leu
Lys Ser Leu Lys His Met Asp Phe Ser Ser Asn 260
265 270Met Leu Phe Gly Glu Ile Pro Gln Ser Tyr Ser Leu
Leu Arg Asn Leu 275 280 285Ile Asp
Ile Asp Leu Asn Arg Asn Lys Leu Tyr Gly Ser Ile Pro Asp 290
295 300Tyr Ile Gly Asp Phe Pro Glu Leu Glu Ser Leu
Leu Leu Asp Ser Asn305 310 315
320Asn Phe Thr Gly Ser Ile Pro Gln Lys Leu Gly Thr Asn Gly Lys Leu
325 330 335Gln Tyr Leu Asp
Ile Ser Asn Asn Asn Phe Ser Gly Ser Leu Pro Leu 340
345 350Ser Leu Cys Lys Gly Asp Lys Leu Gln Asp Leu
Asp Ala Ser Tyr Asn 355 360 365Leu
Leu Val Gly Ser Ile Pro Glu Ser Leu Gly Ser Cys Lys Ser Leu 370
375 380Glu Gly Val Tyr Met Gly Asn Asn Phe Leu
Asn Gly Ser Ile Pro Lys385 390 395
400Gly Leu Phe Gly Ser Asp Val Ser Leu Asn Asp Lys Leu Leu Ser
Gly 405 410 415Gly Leu Asp
Glu Lys Phe Gly Asp Cys Val Asn Leu Arg Asp Ile Asp 420
425 430Leu Ser Asn Asn Lys Leu Ser Gly Lys Leu
Pro Ala Thr Ile Gly Asn 435 440
445Cys Ile His Leu Arg Ser Leu Thr Leu Tyr Asn Asn Thr Cys Thr Gly 450
455 460Arg Ile Pro Gln Glu Ile Ser Lys
Cys Lys Gln Leu Gln Thr Leu Asp465 470
475 480Leu Ser Gln Asn Gln Phe Ser Gly Val Ile Pro Asn
Asp Ile Thr Gly 485 490
495Asn Lys Ser Ile Cys Asn Leu Glu Lys Ile Gln Thr Leu Lys Leu Ser
500 505 510Asn Asn Ala Leu Thr Gly
Glu Ile Pro His Cys Val Gly Asn Ile Glu 515 520
525Leu Ile Ala Leu Phe Leu Gln Ser Asn Lys Leu Asn Gly Thr
Ile Pro 530 535 540Ala Asn Phe Ser Lys
Leu Cys Asp Ser Leu Ile Tyr Leu Asp Leu Ser545 550
555 560Asp Asn Gln Leu Glu Gly Val Leu Pro Lys
Ser Leu Ser Lys Cys Gln 565 570
575Ser Leu Glu Leu Leu Asn Val Gly Asn Asn Arg Leu Arg Asp Lys Phe
580 585 590Pro Ser Trp Leu Asp
Asn Leu Pro Arg Leu Gln Val Phe Ser Val Arg 595
600 605Phe Asn Ala Phe Tyr Gly Pro Ile Thr Ser Ser Pro
Lys Val Ser His 610 615 620Pro Phe Pro
Met Leu Gln Ile Ile Asp Leu Ser Asn Asn Lys Phe Cys625
630 635 640Gly Lys Leu Pro Arg Arg Tyr
Ile Lys Asn Phe Ala Thr Met Arg Asn 645
650 655Met Asn Glu Ser Gly Val Gly Asn Pro Gln Tyr Leu
Gly Asp Ser Ser 660 665 670Ile
Tyr Ser Ile Thr Tyr Ser Met Val Leu Thr Phe Asn Gly Leu Gln 675
680 685Gln Lys Tyr Glu Lys Leu Ile Val Thr
Met Ser Thr Phe Asp Ile Ser 690 695
700Ser Asn Asn Phe Thr Gly Gln Ile Pro Tyr Val Ile Gly Gly Leu Arg705
710 715 720Ser Leu Arg Asn
Leu Asn Leu Ser His Asn Val Leu Thr Gly Asn Ile 725
730 735Pro Pro Ser Ile Ala Lys Leu Ser Leu Leu
Gln Asp Leu Asp Leu Ser 740 745
750Ser Asn Arg Leu Thr Gly Arg Ile Pro Gln Glu Leu Val Ser Leu Thr
755 760 765Phe Leu Gly Ser Phe Asn Val
Ser Asn Asn Leu Leu Glu Gly Ser Ile 770 775
780Pro His Gly Phe Asn Phe Asp Thr Tyr Thr Ala Asn Ser Tyr Gln
Gly785 790 795 800Asn Leu
Glu Leu Cys Gly Lys Pro Leu Pro Glu Cys Gly Glu Arg Arg
805 810 815Ala Lys Gly Thr Thr Asn Asn
Gln Asp Asp Pro Lys Asn Asp Asn Glu 820 825
830Arg Met Leu Ser Met Ser Glu Ile Val Val Met Gly Phe Gly
Ser Gly 835 840 845Val Leu Val Gly
Leu Ala Trp Gly Tyr Tyr Met Phe Ser Val Gly Lys 850
855 860Pro Phe Trp Phe Ile Lys Met Ala Ser Lys Met Glu
Ser Ile Leu Ile865 870 875
880Gly Phe Phe972652DNAArtificial Sequencemodified cDNA of the gene
mediating resistance towards cercosporavariation(625)..(627)
97atgaacatga aaatcctcct tttgtttgtc ttccttcatc acctccacta cttcatccat
60ggcagaacac ttacagaacg ccaagcttta ctaagtatca aatctgccat tacttatgat
120tattataact ctctctcctc atggaaaaac acaacacacc actgcagttg gccatacatc
180acttgctcct cctcttcttc ttcttcttct gttatttctc tcaacttcac catgttattt
240ctcgaaggaa ttctctcccc tgatataggc ttcctcacca acctgcaaaa cctctctatt
300cgatctaacc ttttttctgg cccactcccc cattctctct ctctcctcac ccaactccgc
360tatctcgacg tttcccaaaa cagtttcaca ggtccaatcc catcttctct ctctctcctc
420acccaactcc gctatctcca cgtttccggc aacagtttca caggtccaat cccatctttt
480ctctctctcc tcacccaact ccgctatctc gacgtttccg acaacagttt cacaggtcca
540atcccatctt ctctctctct cctcacccaa ctccgctatc tcgacgtttc ctacaacaat
600ctaaatggca ctcttccctt atcgctcgtt gagaagatgt cggagctcag ctaccttaac
660cttaggtata actctttcta cggtgagatt ccaccggagt ttgggaaact taagaagctt
720gaaacattga atcttggtaa caacactctt tctgggagtc ttccatctga gttgggttca
780ttaaagagtt tgaaacatat ggacttttct agtaatatgc tatttggtga gatcccacaa
840tcttattctc ttcttcgaaa cttaatcgat attgatctta atagaaacaa gttatatggg
900agtatacctg attatattgg agattttccg gagttggaat cacttttatt agactcgaat
960aacttcacag ggagtatccc acaaaagtta ggtacaaacg ggaagttgca atatctagat
1020ataagtaaca acaattttag tggtagtttg ccactaagtc tttgcaaagg agacaaactc
1080caagatctgg acgcatccta taatttgttg gttgggtcaa ttcctgagag tttgggaagt
1140tgcaagtcac ttgaaggagt gtacatggga aataatttct taaacgggtc gattcctaag
1200ggcttgtttg ggagtgatgt ttcacttaat gacaaacttc ttagtggagg tctcgatgag
1260aaattcggtg attgcgttaa tcttcgggac attgatctct ctaataataa gctatcaggg
1320aagttacctg cgaccatcgg aaactgtatt catcttcggt ccttgacgct ttataataac
1380acctgtaccg gacgtatccc tcaagagatt agcaagtgta agcagctaca gaccctcgat
1440ctcagccaaa atcagttctc tggtgtgata cccaatgata ttacaggaaa caaatccatt
1500tgcaatctgg aaaagataca aacacttaaa ttatcaaaca atgctttgac tggtgaaatc
1560cctcattgtg ttggaaatat cgagctcata gcattatttc tccaatcaaa caaactgaac
1620ggtaccatac ccgcaaactt ctcaaagtta tgtgattcat tgatatatct agatcttagt
1680gacaatcaac tcgaaggagt tctacctaag tccttgtcca aatgtcaaag tctagaactc
1740ctaaatgtcg ggaacaatag gctaagagat aaatttcctt catggttaga caacctccca
1800cgtctccaag ttttcagtgt gcgttttaac gccttctacg gtcctataac tagctcacca
1860aaagttagtc acccatttcc tatgctacaa attatcgacc tatctaacaa taagttttgt
1920ggcaagttgc caagaagata tatcaaaaac tttgcaacca tgcgcaatat gaatgagtct
1980ggtgttggga atccacagta cctgggggac tcatcaatat atagtattac gtactctatg
2040gtattgacat tcaatgggtt acaacaaaaa tatgaaaagc ttattgtgac gatgtcgacc
2100tttgatatat ccagcaacaa ctttactgga cagattccat atgttatagg gggattacgc
2160tcacttcgta accttaatct ctctcataat gtcttaaccg ggaacattcc tccatcaatt
2220gcaaaattgt ctttgcttca agatttggac ctttcatcaa acagacttac tggtcgtatc
2280cctcaagaat tagttagttt aacatttctt gggagtttca atgtttcgaa caatctattg
2340gaggggtcta tacctcatgg tttcaacttc gacacgtaca cagctaattc ataccagggg
2400aatctcgaat tatgtggaaa accattacct gagtgtggag aaagaagggc aaaaggcacc
2460actaataatc aagatgatcc taaaaatgat aatgaacgaa tgttgtcgat gtccgaaatc
2520gtagttatgg ggtttggcag tggtgtacta gttgggttgg cttggggata ctatatgttt
2580tcagtgggaa agcccttttg gtttatcaag atggctagca aaatggaatc aatattgatt
2640ggttttttct ga
26529821DNABeta vulgaris 98cgtttccggc aacagtttca c
219921DNABeta vulgaris 99agagagagag aggagtgggt t
21
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