STARCH BRANCHING ENZYME

Abstract

a new starch branching enzyme, and to the gene encoding the enzyme. In particular, the invention provides a new starch branching enzyme type II from wheat, the nucleic acid encoding the enzyme, and constructs comprising the nucleic acid. The invention also relates to a novel method for identification of branching enzyme type II proteins, which is useful for screening wheat germplasm for null or altered alleles of wheat branching enzyme IIb. The novel gene, protein and methods of the invention are useful in production of plants which produce grain with novel properties for food and industrial applications, for example wheat grain containing high amylose or low amylopectin starch.

Description

[0001]This invention relates to a new starch branching enzyme, and to the gene encoding the enzyme. In particular, the invention relates to a new starch branching enzyme type II from wheat. The invention also relates to a novel method for identification of such branching enzyme type II proteins, which is useful for screening wheat germplasm for null or altered alleles of wheat branching enzyme IIb. The novel gene, protein and methods of the invention are useful in production of wheat plants which produce grain with novel properties for food and industrial applications, for, example wheat grain containing high amylose or low amylopectin starch.

BACKGROUND OF THE INVENTION

[0002]In plants, two classes of genes encode starch branching enzymes, known respectively as BEI, and BEII. In the monocotyledonous cereals, there is strong evidence demonstrating that the BEII class contains two independent types of genes, known in maize as BEIIa and BEIIb (Gao et al., 1996; Fisher et al., 1996). In barley, two types of genes have been reported, and shown to be differentially expressed (Sun et al., 1998). An additional class of branching enzyme (50/51 kD) from barley has also been described (Sun et al., 1996).

[0003]In dicotyledonous plants, loss of BEII activity through either mutation (Bhattacharyya et al., 1990) or gene suppression technologies gives rise to starches containing high amylose levels (Safford, 1998, Jobling 1999).

[0004]In monocotyledonous plants, mutations giving rise to high amylose contents are known in maize, rice and barley. In neither rice (Mizuno et al., 1993) nor barley (Schondelmaier et al., 1992) have the known high amylose phenotypes been associated with the BEIIa or BEIIb mutations respectively. However, in maize it is firmly established that the high amylose phenotype is associated with down regulation of the BEIIb gene (Boyer et al., 1980; Boyer and Preiss, 1981, Fisher et al, 1996).

[0005]The impact of down-regulation of BEI has been investigated through antisense inhibition in potato tuber; the down-regulation has been found to alter the properties of the starch, but not its gross structural features, such as the amylose content (Filpse et al., 1996). In wheat, antisense down-regulation of BEI activity has small but significant effects on starch structure (Baga et al, 1999). The branching enzyme I gene from maize has been cloned (Kim et al., 1998), but mutants affecting branching enzyme I activity in maize are not known.

[0006]No mutations specifically reducing BEIIa activity have been reported, and no gene suppression experiments in plants have succeeded in reducing BEIIa activity, although the du1 mutation in maize is known to reduce the expression of both BEIIa and starch synthase III. However, the du1 mutation is now known to be due to mutation of the structural gene for starch synthase III (Gao 1998, Cao 1999).

[0007]In our previous patent application No. PCT/AU98/00743 (WO99/14314), we have described the structure of a BEII gene from wheat, which we have subsequently designated the BEIIa gene.

[0008]In the present application we describe the isolation of a second BEII gene from wheat, which we have designated the BEIIb gene, and discuss the uses to which this gene sequence can be applied. We have surprisingly found that in wheat the expression level of the various branching enzymes is very different to that in maize and barley. In this specification we show that BEIIb in wheat is expressed at low levels in the soluble fraction of the wheat endosperm, and is predominantly found within the starch granule. This indicates that there are important differences in the regulation of gene expression in wheat compared to other cereals, suggesting that the manipulation of the amylose to amylopectin ratio in wheat will involve the manipulation of more than just the BEIIb gene.

[0009]We have also surprisingly found that the BEIIa and BEIIb gene structures are highly conserved with respect to exon size and position, allowing us to prepare DNA-based diagnostics which they can distinguish not only the BEIIa and BEIIb classes of genes, but also the forms of these genes encoded on the A, B and D genomes of wheat, and to identify the BEIIb proteins expressed by the wheat A, B and D genomes, providing an essential tool for the screening of wheat germplasm for null or altered alleles of wheat branching enzyme IIa.

SUMMARY OF THE INVENTION

[0010]In a first aspect, the invention provides an isolated nucleic acid molecule encoding wheat starch branching enzyme IIb (BEIIb).

[0011]Preferably the nucleic acid sequence is a DNA sequence, and may be genomic DNA or cDNA.

[0012]Preferably the nucleic acid molecule has the sequence depicted in FIG. 8 (SEQ ID NO:5), FIG. 9 (SEQ ID NO:6), or SEQ ID NO:10. It will be clearly understood that the invention also encompasses nucleic acid molecules capable of hybridising to these sequences under at least low stringency hybridization conditions, or a nucleic acid molecule with at least 70% sequence identity to at least one of these sequences. Methods for assessing ability to hybridize and % sequence identity are well known in the art. Even more preferably the nucleic acid molecule is capable of hybridizing thereto under high stringency conditions, or has at least 80%, most preferably at least 90% sequence identity. A nucleic acid molecule having at least 70%, preferably at least 90%, more preferably at least 95% sequence identity to one or more of these sequences is also within the scope of the invention.

[0013]Biologically-active untranslated control sequences of genomic DNA are also within the scope of the invention. Thus the invention also provides the promoter of BEIIb.

[0014]In a second aspect of the invention, there is provided a genetic construct comprising a nucleic acid sequence of the invention, a biologically-active fragment thereof, or a fragment thereof encoding a biologically-active fragment of BEIIb operably linked to one or more nucleic acid sequences which are capable of facilitating expression of BEIIb in a plant, preferably a cereal plant. The construct may be a plasmid or a vector, preferably one suitable for use in transformation of a plant. Such a suitable vector is a bacterium of the genus Agrobacterium, preferably Agrobacterium tumefaciens. Methods of transforming cereal plants using Agrobacterium tumefaciens are known; see for example Australian Patent No. 667939 by Japan Tobacco Inc.; Australian Patent No. 687863 by Japan Tobacco Inc.; International Patent Application No. PCT/US97/10621 by Monsanto Company; and Tingay et al (1997).

[0015]In a third aspect, the invention provides a genetic construct for targeting of a desired gene to endosperm of a cereal plant, and/or for modulating the time of expression of a desired gene in endosperm of a cereal plant, comprising a BEIIb promoter, operatively linked to a nucleic acid sequence encoding a desired protein, and optionally also operatively linked to one or more additional targeting sequences and/or one or more 3' untranslated sequences.

[0016]The nucleic acid encoding the desired protein may be in either the sense orientation or in the anti-sense orientation. Alternatively it may be a duplex construct, comprising a portion of the nucleic acid sequence encoding the desired protein in both the sense and anti-sense orientations, operably linked by a spacer sequence. It is contemplated that any desired protein which is encoded by a gene which is capable of being expressed in the endosperm of a cereal plant is suitable for use in the invention. Preferably the desired protein is an enzyme of the starch biosynthetic pathway. For example, the antisense sequences of GBSS, starch debranching enzyme, SBE II, low molecular weight glutenin, or grain softness protein I, may be used. Preferred sequences for use in sense orientation include those of bacterial isoamylase, bacterial glycogen synthase, or wheat high molecular weight glutenin Bx17.

[0017]In a fourth aspect, the invention provides a wheat BEIIb polypeptide, comprising an amino acid sequence encoded by a nucleic acid molecule according to the invention, or a polypeptide having at least 70%, more preferably 80%, even more preferably 90% amino acid sequence identity thereto, and having the biological activity of BEIIb.

[0018]The polypeptide may be designed on the basis of amino acid sequences deduced from the nucleic acid sequences of the invention, or may be generated by expression of the wheat BEIIb nucleic acid molecule in a heterologous system. Suitable heterologous systems are very well known in the art, and the skilled person will readily be able to select a system suitable for the particular purpose desired.

[0019]In a fifth aspect, the invention provides an antibody directed against BEII polypeptide. The antibody may be polyclonal or monoclonal. It will be clearly understood that the invention also encompasses biologically-active antibody fragments, such as Fab, (Fab)₂, and ScFv. Methods for production of antibodies and fragments thereof are very well known in the art.

[0020]The antibodies of the invention may be used for identification and separation of BEIIb proteins, for example by affinity electrophoresis. This greatly facilitates the identification and combination of altered forms of BEIIb via analysis of germplasm, and greatly assists plant breeding. The antibodies of the invention are suitable for use in any affinity-based separation system, preferably using methods which overcome interference by amylases. Suitable methods are known in the art.

[0021]In a sixth aspect, the invention provides a plant cell transformed by a genetic construct according to the invention, or a plant derived from such a cell. Additionally, a transformed plant cell may also comprise one or more null alleles for a gene selected from the group consisting of GBSS, BEIIa, and SSII. Preferably the plant is a cereal plant, more preferably wheat or barley.

[0022]In a seventh aspect, the invention provides a method of modifying the characteristics of starch produced by a plant, comprising the steps of:

[0023]a) increasing the level of expression of BEIIb in the plant, for example by introducing a nucleic acid molecule encoding BEIIb into a host plant, or

[0024]b) decreasing the level of expression of BEIIb in the plant, for example by introducing an anti-sense nucleic acid sequence directed to a nucleic acid molecule encoding BEIIb into a host plant.

[0025]As is well known in the art, over-expression of a gene can be achieved by introduction of additional copies of the gene, and anti-sense sequences can be used to suppress expression of the protein to which the anti-sense sequence is complementary. Other methods of suppressing expression of genes are known in the art, for example co-suppression, RNA duplex formation, or homologous recombination. It would be evident to the person skilled in the art that sense and anti-sense sequences may be chosen depending on the host plant, so as to effect a variety of different modifications of the characteristics of the starch produced by the plant.

[0026]Preferably the plant is a cereal plant, more preferably wheat or barley.

[0027]Preferably the branching of the amylopectin component of starch is modified by either of these procedures. More preferably a plant with high amylose content is produced.

[0028]In an eighth aspect, the invention provides a method of targeting expression of a desired gene to the endosperm of a cereal plant, comprising the step of transforming the plant with a construct according to the invention.

[0029]In a ninth aspect, the invention provides a method of identifying a null or altered allele encoding an enzyme of the starch biosynthetic pathway, comprising the step of subjecting DNA from a plant suspected to possess such an allele to a DNA fingerprinting or amplification assay, which utilizes at least one DNA probe comprising one or more of the nucleic acid molecules of the invention. The nucleic acid molecule may be a genomic DNA or a cDNA, and may comprise the full-length coding sequence or a fragment thereof. Any suitable method for identification of BEIIb sequences may be used, including but not limited to PCR, rolling circle amplification, RFLP, and AFLP. Such methods are well known in the art, and any suitable technique may be used.

[0030]In a tenth aspect, the invention provides a plant comprising one or more BEIIb null alleles, in combination with one or more other null alleles selected from the group consisting of BEIIa, GBSS, SSII and BEI. Optionally the plant may also comprise a BEIIa or BEIIb gene expressed in either the sense or the anti-sense orientation. The null alleles for BEIIa, GBSS SSII and BEI may be identified using methods described in PCT/AU97/00743.

[0031]It will clearly understood that the invention also encompasses products produced from plants according to the invention, including but not limited to whole grain, part grain, flour or starch.

[0032]Because of the very close relationship between Aegilops tauschii, formerly known as Triticum tauschii, and wheat, as discussed in PCT/AU97/00743, results obtained with A. tauschii can be directly applied to wheat with little if any modification. Such modification as may be required represents routine trial and error experimentation. Sequences from these genes can be used as probes to identify null or altered alleles in wheat, which can then be used in plant breeding programes to provide modifications of starch characteristics. The novel sequences of the invention can be used in genetic engineering strategies or to introduce a desired gene into a host plant, or to provide anti-sense sequences for suppression of expression of the BEIIb gene in a host plant, in order to modify the characteristics of starch produced by the plant.

[0033]While the invention is described in detail in relation to wheat, it will be clearly understood that it is also applicable to other cereal plants of the family Gramineae, such as maize, barley and rice.

[0034]Methods for transformation of monocotyledonous plants such as wheat, maize, barley and rice and for regeneration of plants from protoplasts or immature plant embryos are well known in the art. See for example Lazzeri et al, 1991; Jahne et al, 1991 and Wan and Lemaux, 1994 for barley; Wirtzens et al, 1997; Tingay et al, 1997; Canadian Patent Application No. 2092588 by Nehra; Australian Patent Application No. 61781/94 by National Research Council of Canada, and Australian Patents No. 667939 and No. 687863 by Japan Tobacco Co.

[0035]The sequences of ADP glucose pyrophosphorylase from barley (Australian Patent Application No. 65392/94), starch debranching enzyme and its promoter from rice (Japanese Patent Publication No. Kokai 6261787 and Japanese Patent Publication No. Kokai 5317057), and starch debranching enzyme from spinach and potato (Australian Patent Application No. 44333/96) are all known.

BRIEF DESCRIPTION OF THE FIGURES

[0036]FIG. 1 shows the sequence of the SBE9 branching enzyme cDNA encodes SBE IIa, cloned from a wheat cv Rosella cDNA library (SEQ ID NO:1).

[0037]FIG. 2 shows the sequence of the branching enzyme BEIIa gene (SEQ ID NO:2) contained within the F2 lambda clone isolated from an Aegilops tauschii genomic DNA library.

[0038]FIG. 3 shows the results of hybridisation of Aegilops tauschii DNA with probes derived from wSBE II-DA1 type sequences. A. Hybridisation with a probe from SBE9 consisting of exons 5-9. B. Hybridisation with fragment F2.2 (consisting of exons 4-9 and introns 4-8 and part of introns 3 and 9). Enzymes used for the digest were: 1. Bam HI, 2. Dra I, 3. EcoR I, 4. EcoR V. Molecular size markers are indicated.

[0039]FIG. 4 shows the alignment of sequences of Intron 5 fragments from the A, B and D genomes of wheat

[0040]FIG. 5 shows the PCR analysis of A. tauschii genomic clones using Intron V sequences.

[0041]FIG. 6 shows the alignment of a 262 bp PCR fragment amplified from hexaploid wheat using the primers sr913F and WBE2E6R, and a region from the wheat branching enzyme IIb gene wSBE II-DB1.

[0042]FIG. 7 shows the alignment of barley branching enzyme IIb cDNA, wheat branching enzyme IIb cDNA, and SBE9 sequences with the sequence of the wheat (A. tauschii) branching enzyme IIb gene.

[0043]FIG. 8 shows the partial genomic sequence of a branching enzyme IIb gene from A. tauschii (SEQ ID NO:5).

[0044]FIG. 9 shows the sequence of a cDNA for branching enzyme IIb gene from hexaploid wheat (SEQ ID NO:6).

[0045]FIG. 10 shows the sequence alignment of branching enzyme genes. The cDNA sequences used for this analysis were SBE9 (SEQ ID NO:1; FIG. 1), wheat BEIIb cDNA (SEQ ID NO:6; FIG. 9), Y11282, a wheat branching enzyme sequence (Nair et al. 1997), barley BEIIa (Sun et al. 1998), barley BEIIb (Sun et al. 1998), rice BEIII (Mizuno et al. 1993), rice BEIV (Genbank Accession No. E14723) maize BEIIa (Gao et al. 1997) and maize BEIIb (Gao et al. 1997). The observed N-terminal of wheat (Morell et al., 1997; Y11282) is shown in bold. FIG. 11 shows the dendrogram of BE sequences. The sequences analysed were for wheat Y11282 (Nair et al., 1997), SBE 9 (SEQ ID NO:1; (FIG. 1), wheat BEIIb (SEQ ID NO:9; FIG. 9), barley IIa and IIb (Sun et al. 1998), maize IIa (Gao et al. 1997), maize IIb (Fisher et al. 1993), rice III (Mizuno et al. 1993), rice IV (Genbank accession E14723), potato BEI (Khoshnoodi et al. 1997), potato BE II (Cangiano et al 1993), pea BEI and BEII (Burton et al. 1995), E. coli BE (Baecker et al. 1986) and bacillus (Kiel et al 1992). Note that pea BE I and pea BE II sequences correspond to maize BE II and BE I respectively because of differences in nomenclature conventions.

[0046]FIG. 12 shows the comparison of exon/intron structure for the BEIIa and BEIIb genes. (1) wheat branching enzyme IIa gene, wSBE II DA1 (2) maize amylose extender BEIIb gene (3) partial wheat branching enzyme IIb gene, wSBE II DB1 (4) partial barley branching enzyme IIb gene.

[0047]FIG. 13 shows the results of analysis of the expression of mRNA for the BEIIa and BEIIb genes in wheat. Panel (A): Hybridisation of SBE9 probe to lanes 1 to 3 and hybridisation of wheat BEIIb cDNA probe to lanes 4 to 6. Panel (B): mRNA loading for each lane.

[0048]Lanes 1 and 4 contain leaf mRNA; lanes 2 and 5 contain pre-anthesis floret mRNA; lanes 3 and 6 contain mRNA from wheat endosperm collected 15 days after anthesis.

[0049]FIG. 14 shows the results of analysis of wheat endosperm branching enzyme IIa by affinity electrophoresis.

[0050]Samples: Lanes 1,4 and 7 contained 20 μg endosperm soluble protein, lanes 2, 5 and 8 contained 30 μg endosperm soluble protein and lanes 3 and 6 contained 10 μg endosperm soluble protein.

[0051]FIG. 15 shows the results of non-denaturing gel electrophoresis analysis of branching enzymes in the soluble fraction of wheat endosperm.

[0052]Samples were: Lane 1, R6 pre-immune, 1:100; Lane 2, R6 pre-immune, 1:3000; Lane 3, R6, 1:100; Lane 4, R6, 1:1000; Lane 5, R6, 1:3000; Lane 6, 3KLH, 1:2000; Lane 7, 3KLH, 1:5000; Lane 8, R7 pre-immune, 1:1000; Lane 9, R7 pre-immune 1:5000; Lane 10, R7, 1:1000; Lane 11, R7, 1:3000; Lane 12, R7, 1:5000

[0053]FIG. 16 shows the results of affinity electrophoresis separation of branching enzyme IIa forms from diverse wheat germplasm using the gel conditions described in FIG. 11 (Panel C). Panel A. Lane 1, Durati, T. durum; Lane 2 A. tauschii, Accession No. 24242; Lane 3, A. tauschii, Accession No. 24095; Lane 4, A. tauschii, Accession No. 24092; Lane 5, Hartog, Triticum. aestivum; Lane 6, Rosella, T. aestivum; Lane 7, Corrigin, T. aestivum; Lane 8, Bodallin, T. aestivum; Lane 9, Beulah, T. aestivum; Lane 10 Bindawarra, T. aestivum; Lane 11, Barley (Hordeum vulgare). Panel B. Lane 1: Afghanistan 006, Triticum durum; Lane 2, Persia 20, T. aestivum; Lane 3, Afghanistan 8, T. aestivum; Lane 4, Kashmir 4, T. aestivum; Lane 5, Gandum Sockhak, T. aestivum; Lane 6, Warbler, T. aestivum; Lane 7, Bayles, T. aestivum; Lane 8, Kometa; Lane 9, Kashmir 14, T. aestivum; Lane 10, Rosella, T. aestivum; Lane 11, Kashmir 8, T. aestivum; Lane 12, Beijing 10, T. aestivum; Lane 13, Savannah, T. aestivum; Lane 14, Afghanistan 55-623, T. aestivum; Lane 15, Karizik, T. aestivum; Lane 16, Indore E98, T. durum; Lane 17, Iraq 17, T. durum; Lane 18, Seri 82, T. aestivum; Lane 19, Indore 19, T. aestivum.

[0054]FIG. 17 shows the results of two-dimensional separation of the components of the wheat starch granule 88 kD band. The wheat starch granule 88 kDa band was electrophoresed in the first dimension through an SDS-PAGE gel. Lanes were excised, renatured, and placed on top of a non-denaturing PAGE gel and electrophoresed in a second dimension. Two lanes were placed on top of each non-denaturing PAGE gel. (A) protein staining with Coomassie blue reagent (B) Immunoblotting of gels with either 3KLH or R6 antibodies, as indicated on the figure.

[0055]FIG. 18 is a diagrammatic representation of the BEII genes from various species, showing the exon/intron structure. The dark rectangles represent exons.

[0056]FIG. 19 shows the results of PCR amplification of SBE IIb gene from CS nullisomic lines, using the primers ARA 12F and ARA 10R.

[0057]FIG. 20 shows the results of PCR amplification of SBE IIb gene, using the primers ARA 6F and ARA 8R from Triticum spp. Lanes: 1) T. monococcum, 2) T. durum, 3) T. urartu, 4) T. tauschii, 5) CSDT2DS, 6) CSDT2BL-9, 7) CSDT2AS and 8) CS.

[0058]FIG. 21 shows the alignment of the exon 1--intron 1--exon 2 region of the SBE IIb gene from the A, B and D genomes. * indicates that the sequence could not be specifically assigned to the A or B genome.

[0059]FIG. 22 shows the alignment of the BEIIb sequences from each genome.

[0060]FIG. 23 shows the results of PCR amplification of the SBE IIb gene was carried out using the primers ARA 19F and ARA 15R, followed by restriction digestion using Rsa1. Lanes 1) CS, 2) T. monococcum, 3) T. tauschii, 4) CSDT2BL-9, which is missing part of the long arm of chromosome 2B, and 6) CSDT2AS, which is missing the long of chromosome 2A.

[0061]FIG. 24 shows the results of PCR amplification of intron 3 region of SBE IIb from wheat lines, using the primers ARA 19F and ARA 23R followed by Rsa 1 digestion. Lane 12 is the null mutant for the D genome

[0062]FIG. 25 is a schematic representation showing the development of the SBE IIa construct. A) Biogemma vector, pDV03000; B) pBluescript carrying the full length cDNA of SBE IIa; C) SBE IIa construct in pDV03000; D) Sense IIa construct and E) Antisense IIa construct.

[0063]FIG. 26 is a schematic representation of the development of the SBE IIb construct. A) Biogemma vector, pDV03000; B) pGEM-T carrying a 1046 bp fragment of SBE IIb; C) SBE IIb construct in pDV03000; D) Sense IIb construct and E) Antisense IIb construct.

[0064]FIG. 27 is a schematic representation of a SBE II duplex construct. A) SBE sequence inserted in between the promoter and the terminator in its linear form; B) Duplex formation of mRNA within the transgenic plant.

EXAMPLE 1

Isolation of BEII Genes from an A. tauschii Genomic Library and Their Characterisation by PCR

Plant Material

[0065]Aegilops tauschii, CPI 110799, was used for the construction of the genomic library. Previously this accession has been shown to be most like the ancestral D genome donor of wheat, on the basis of the conservation of order of genetic markers (Lagudah et al. 1991). The Triticum aestivum cultivars Rosella, Wyuna and Chinese Spring were used for the construction of different cDNA libraries.

cDNA and Genomic Libraries

[0066]The construction of the cDNA and genomic libraries used in this example was as described in Rahman et al., (1997,1999) and in Li et al. (1999). Conditions for library screening were hybridisation at 25% formamide, 5×SSC, 0.1% SDS, 10× Denhardts, 100 μg/ml salmon sperm DNA at 42° C. for 16 h, followed by washing at 2×SSC, 0.1% SDS at 65° C. for 3×1 h.

Screening of a Wheat cDNA Library

[0067]Screening of a wheat cv Rosella cDNA library prepared from endosperm (mid-stage of development) with the maize SBE I clone (Baba et al., 1991) at low hybridisation stringency led to the isolation of two classes of positive plaques. One class hybridised strongly to the probe, and encoded wheat SBE I (Rahman et al., 1997,1999). The second class was weakly hybridising. The clone with the longest insert from this second class was called SBE 9, and its sequence showed greater identity to SBE II than to SBE I type sequences. This was designated SBE IIa. The sequence of SBE 9 (SEQ ID NO:1) is set out in FIG. 1.

Screening of A. tauschii Genomic Library

[0068]A genomic library constructed from A. tauschii was screened by DNA hybridisation with SBE9, and four positive clones were purified. These were designated F1 to F4. The sequence from positions 537 to 890 of SBE9 was amplified by PCR, and used to screen the A. tauschii library again. Clones isolated from this screening are referred to as G1 and G2 and H1 to H8

(1) Number of BEII Type Genes in Wheat

[0069]The sequence of a branching enzyme gene, designated F2, from Aegilops tauschii was described in WO99/14314, and is given in FIG. 2 (SEQ ID NO:2). A probe generated from F2, designated F2.2, contained sequences from 2704 to 4456 bp of SEQ ID NO:2, and contained exons 4-9, introns 4-8, and parts of intron 3 and 9. Hybridisation of A. tauschii DNA (cut with four different restriction enzymes) with F2.2 revealed only one strongly hybridising band and several very faint bands (FIG. 3, panel B), consistent with the presence of a single BEII type gene in the A. tauschii genome. The cDNA clone, SBE9 (SEQ ID NO:1) has >95% identity to the exon regions of the F2 branching enzyme gene. A region of SBE9 from nucleotides 537 to 890, including exons 5 to 9, was used as a hybridisation probe, and gave a much more complex pattern (FIG. 3, panel A), strongly indicating that there is more than one BEII gene type in the A. tauschii genome.

EXAMPLE 2

PCR Analysis of BEIIa--Intron 5

[0070]PCR primers, sr913F (5' ATC ACT TAC CGA GAA TGG G 3', SEQ ID NO:3) and WBE2E6R (5' CTG CAT TTG GAT TTC AAT TG 3', SEQ ID NO:4) were designed to anneal to Exon 5 and Exon 6 respectively of the wheat F2 gene in order to amplify the intron region (Intron 5) between these exons. Analysis of the products of PCR reactions using these primers shows that the primers amplify fragments of 228 bp from the A-genome of wheat, 226 bp from the D genome and 217 bp from the B genome. These fragments were shown to be amplified from chromosome 2A, 2D and 2B of wheat respectively by analysis of nullisomic/tetrasomic chromosome-engineered lines of wheat. In addition to these fragments, a 262 bp fragment was amplified, and this fragment (designated the 262 bp Universal fragment) was not polymorphic among the chromosome engineered lines tested. The 262 bp Universal fragment and the A, B and D regions from the F2 gene were cloned and sequenced, and the sequence comparison is shown in FIG. 4.

EXAMPLE 3

Classification of the G1-G2 and H1-H10 Genes

[0071]PCR analysis using PCR primers sr913F (5' ATC ACT TAC CGA GAA TGG G 3') and WBE2E6R (5' CTG CAT TTG GAT TTC AAT TG 3') showed that the H1 to H10 lambda clones yielded an approximately 200 bp fragment, and the G1 and G2 clones yielded an approximately 260 bp fragment (FIG. 5). Partial sequencing of G1 and G2 showed that the parts of the sequence analysed had 80% identity with the exons 4 and 5 of wSBE II-DA1, but the intervening intron contained a sequence that showed no homology to any sequence contained within F2.

[0072]However, the G1 and G2 clones from A. tauschii showed 92.7% identity to the sequence of the 262 bp universal fragment amplified and cloned from hexaploid wheat, and an alignment of these sequences is shown in FIG. 6. FIG. 7 shows an alignment of a region corresponding to the 537 to 890 bp region of the SBE9 clone from the cDNAs for barley BEIIb (Sun et al., 1995, Sun et al., 1998), SBE9, wheat BEIIb cDNA with the sequence from clone G1. Further sequencing of G1 led to the isolation of a sequence, shown in FIG. 8 (SEQ ID NO:5), which showed high identity with the sequence reported by Sun et al. (1998) for the 5' end of barley IIb cDNA and the partial sequence for the cognate gene. G1 and G2 therefore contain a gene which is distinct from F2, and which has high homology to barley BEIIb. We have designated this gene wSBE II-DB1.

EXAMPLE 4

Isolation of a Wheat BEIIb cDNA and an Additional Genomic Fragment

[0073]A barley cDNA library was constructed using 5 μg of polyA.sup.+ mRNA (1.67 μg of polyA.sup.+ mRNA from 10, 12 and 15 DPA endosperm tissues were pooled). cDNA was synthesised using the cDNA synthesis system marketed by Life Technology, except that the NotI-(dT)₁₈ primer (Pharmacia Biotech) was used to synthesise the first strand of cDNA. Pfu polymerase was added to the reaction after second strand synthesis to flush the ends of cDNAs. SalI-XhoI adapter (Stratagene) was then added to the cDNAs. cDNAs were ligated to SalI-NotI arms of λZipLox (Life Technology) after digestion of cDNAs with NotI followed by size fractionation (SizeSep 400 spun Column of Pharmacia Biotech). The entire ligation reaction (5 μl) was packaged using Gigapack III Gold packaging extract (Stratagene). The titre of the library was tested by transfecting either the Y1090(ZL) or the LE392 strain of E. coli.

[0074]Primers 1 and 2 (Sun et al. 1998)), were used for PCR amplification of a fragment from a barley cDNA library (Ali et al., 2000) using conditions described in Sun et al. (1998). The identity of this fragment was confirmed by sequence analysis, and the fragment was used as a probe to isolate a cDNA by hybridisation, cDNA from a cDNA library constructed from Chinese Spring (Li et al. 1999).

[0075]This cDNA was designed wBEIIb, and its sequence is shown in FIG. 9 (SEQ ID NO:6). This probe was also used to reprobe the genomic library from A. tauschii referred to above, and a clone, designated G5, was recovered from this screen. Analysis showed that the wBEIIb cDNA sequence showed 98.5% identity and the G5 sequence showed 100% identity to sequences already recovered from G1 and G2. G5 therefore represented the same wSBE II-DB1 gene, and the wBEIIb cDNA is a product of the orthologous gene in hexaploid wheat.

EXAMPLE 5

Relationships Between BEII Sequences

[0076]Deduced amino acid sequences for branching enzymes from various cereals were analysed using the PILEUP program from the GCG suite of programs (Devereux 1984), and an alignment of these sequences is shown in FIG. 10. The PILEUP analysis used a penalty of 12 for insertion of a gap and 0.1 for extending the gap per residue. The cDNA sequences used for this analysis were SBE9 (SEQ ID NO:1; FIG. 1), wheat BEIIb cDNA (SEQ ID NO:6; FIG. 9), Y11282, a wheat branching enzyme sequence (Nair et al.1997), barley BEIIa (Sun et al. 1998), barley BEIIb (Sun et al. 1998); rice BEIII (Mizuno et al. 1993), rice BEIV (Genbank Accession No. E14723) maize BEIIa (Gao et al. 1997) and maize BEIIb (Fisher et al., 1993). The observed N-terminal of wheat (Morell et al., 1997; Y11282) is shown in bold.

[0077]The relationships between branching enzyme sequences are illustrated in FIG. 11, using a dendrogram generated by the PILEUP program. The sequences analysed were for wheat Y11282 (Nair et al., 1997), SBE 9 (FIG. 1), wheat BEIIb (FIG. 9), barley IIa and IIb (Sun et al. 1998), maize BEI (Kim et al, 1998), maize IIa (Gao et al. 1997), maize IIb (Fisher et al. 1993), Arabidopsis BEII (U22428, Fisher et al., 1996), Arabidopsis BEII (U18817, Fisher et al., 1996), rice I (Kawasaki et al., 1993), rice III (Mizuno et al. 1993), rice IV (Genbank accession E14723), potato BEI (Khoshnoodi et al. 1997), potato BE II (Cangiano et al 1993), pea BEI and BEII (Burton et al. 1995), E. coli BE (Baecker et al. 1986) and bacillus (Kiel et al 1992). Note that pea BE I and pea BE II sequences correspond to maize BE II and BE I respectively because of differences in nomenclature conventions.

[0078]On the basis of this comparison, the branching enzyme gene contained on clone F2 was classified as a BEIIa type gene and designated wSBE II-DA1.

EXAMPLE 6

Structure of the wSBE II-DA1 and wSBE II-DB1 Genes

[0079]FIG. 12 shows a comparison of the exon/intron structures of the wheat wSBE II-DA1 and wSBE II-DB1 genes. The structure of the wSBE II-DB1 gene is shown from the beginning of the wheat BEIIb cDNA through to exon 5. Hybridisation results suggest that regions at the 3' end of the wheat BEIIb cDNA are not contained within any of the clones G1,G2 and G5. This is not surprising, as the maize SBE II b gene extends over 16.5 kb and required the isolation of two genomic clones (Kim et al 1998). The positions of the intron/exon boundaries for the first five introns of the wheat BEIIa and BEIIb genes are conserved, as shown in Table 1. The size of the first five introns in wSBE II-DB1 vary considerably in size from the first five introns in wSBE II-DA1.

TABLE-US-00001 TABLE 1 Exon/Intron Structures of Cereal branching Enzyme Genes Exons Introns Wheat Wheat Wheat Wheat wSBE Maize WSBE Barley wSBE Maize WSBE Barley II-DA1 BEIIb II-DB1 BEIIb II-DA1 BEIIb II-DB1 BEIIb 1 123^a 112^a 148^a 121^a 1 327 106 148 105 2 98 146 146 152 2 276 244 663 2064 3 242 155 230 230 3 401 1086 465 388 4 99 99 99 99 4 169 76 74 74 5 43 43 43 43^b 5 152 196 181 6 60 60 60 6 335 499 442 7 81 81 81 7 83 81 79 8 117 117 117 8 288 567 178 9 81 84 84 9 629 775 10 122 122 10 175 751 11 120 120 11 974 4020 12 130 130 12 88 86 13 111 111 13 201 148 14 129 129 14 579 3051 15 104 104 15 841 872 16 145 145 16 1019 457 17 148 148 17 135 144 18 105 101 18 176 226 19 74 78 19 201 266 20 156 156 20 377 448 21 75 75 21 89 96 22 384 84 ^aExon 1 numbering begins from ATG of translation start codon ^bPartial sequence for exon or intron

EXAMPLE 7

Expression Analysis at the mRNA Level

[0080]RNA from endosperm at different developmental stages was obtained from wheat grown in the glasshouse as described in Li et al. (1999). RNA was extracted by the method of Higgins et al. (1976), separated on denaturing formamide gels and blotted onto Hybond N+ paper, essentially as described in Maniatis et al. (1992). Probes were prepared from the extreme 3' ends of SBE9 (bases 2450 to 2640 of SEQ ID NO:1) and wBEIIb cDNA (bases 2700 to 2890 of SEQ ID NO:6) by PCR using the following scheme: 94° C., 2 min, 1 cycle, 94° C., 30 s, 55° C., 30 s, 72° C., 30 s, 36 cycles, 72° C. 5 min, 1 cycle, 25° C., 1 min, 1 cycle. The probes were from the 3' untranslated region, and were specific for either wSBE II-DA1 or wSBE II-DB1 type sequences. An RNA species of about 2.9 kb hybridised to each probe (FIG. 13 Panel B). However, the intensity of hybridisation determined by densitometry, and normalised for differences in RNA loading), indicated that RNA hybridising to the wSBE II-DB1 gene was present at 2.5 to 3 fold lower concentration than RNA hybridising to the wSBE II-DA1 gene.

EXAMPLE 8

Analysis of Branching Enzymes by Affinity Electrophoresis Demonstrates that only BEIIa is Predominant in the Soluble Fraction

[0081]In Morell et al., (1997), we reported that only a single form of branching enzyme II could be identified in the wheat developing endosperm soluble fraction. However, this was on the basis of anion-exchange chromatography, and it remained possible that there were multiple forms, even though they could not be separated by this technique. Matsumoto has developed an affinity electrophoresis method for measuring the interaction of branching enzymes with polysaccharide substrates (Matsumoto et al., 1990), and we have further developed this technique specifically to allow the separation of the branching enzyme IIa forms encoded by each of the three wheat genomes. FIG. 14 shows an immunoblot of a non-denaturing polyacrylamide gel electrophoresis experiment in which the gel matrix contained the β-limit dextrin of maize amylopectin alone (FIG. 14, lanes 1 and 2), showing separation of three forms of branching enzyme IIa. Resolution is slightly enhanced by the addition of the α-amylase inhibitor acarbose (FIG. 14, lanes 3,4 and 5), and substantially enhanced by the addition of α-cyclodextrin (FIG. 14 lanes 6, 7 and 8).

[0082]A non-denaturing gel was prepared, containing a stacking gel composed of 0.125 M Tris-HCl buffer (pH 6.8), 6% acrylamide, 0.06% ammonium persulphate and 0.1% TEMED. The separating gel was composed of three panels. The basic non-denaturing gel mix contained 0.34 M Tris-HCl buffer (pH 8.8), CHAPS (0.05%), glycerol (10.3%), acrylamide (6.2%), 0.06% ammonium persulphate, 0.1% TEMED and the β-limit dextrin of maize amylopectin (0.155%). Panel A (lanes 1 and 2) contained only the basic non-denaturing gel reagents. Panel B (Lanes 3, 4 and 5) contained the basic non-denaturing gel reagents and 0.066 mM acarbose. Panel C (lanes 6, 7 and 8) contained the basic non-denaturing gel reagents and 0.067 mM α-cyclodextrin.

[0083]Following electrophoresis at 100 V for 16 hours at 4° C., the proteins in the separating gel were transferred to nitrocellulose membrane according to Morell et al (1997) and immunoreacted with 1:5000 dilution of 3KLH antibodies (raised against the synthetic peptide AASPGKVLVPDESDDLGC (SEQ ID NO:7) coupled to the keyhole limpet hemocyanin via the heterobifunctional reagent m-Maleimidobenzoyl-N-hydroxysuccinimide ester).

[0084]The use of a β-limit dextrin provides a superior separation because it prevents interference with the separation by endogenous β-amylases in the wheat endosperm tissue, and the use of α-cyclodextrin in the assay further enhances the separation. Without wishing to limit the invention by any proposed mechanism, we believe that this enhancement may result from the inhibition of endogenous wheat endosperm α-amylases.

[0085]The analysis shows that three branching enzyme II proteins are present, and that each of these proteins cross-reacts with antibodies to a synthetic oligopeptide designed from the N-terminal region of the BEIIa protein in a region that shares no homology with the wheat BEIIb protein.

[0086]The soluble fraction of the wheat endosperm was reacted with various antibodies raised against peptides designed on the basis of the sequences of the wheat BEIIa (see FIG. 12) or the wheat BEIIb cDNA. FIG. 15 shows that only 3KLH, raised against the N-terminus of BEIIa, cross-reacted with proteins (marked by arrows) in the soluble fraction which show a specific shift in mobility in the presence of the β-limit dextrin of amylopectin and α-cyclodextrin. Gels were prepared as described in FIG. 14, except that the gel used in Panel A contained the non-denaturing gel mix without the β-limit dextrin of maize amylopectin. Panel B contained the non-denaturing gel mix plus α-cyclodextrin. An extract of developing wheat endosperm was prepared using 3 volumes of extraction buffer per g of tissue, and 140 μl of sample applied per gel. Following electrophoresis at 100 V for 16 hours at 4° C., the proteins in the separating gel were transferred to nitrocellulose membrane according to Morell et al (1997) which was cut into 1 cm strips. The antibodies prepared were 3KLH (see FIG. 11), R6 (raised in rabbit against the synthetic peptide AGGPSGEVMIGC (SEQ ID NO:8) coupled to the keyhole limpet hemocyanin via the heterobifunctional reagent m-Maleimidobenzoyl-N-hydroxysuccinimide ester); pre-immune serum from the R6 rabbit; R7 (raised in rabbit against the synthetic peptide GGTPPSIDGPVQDSDGC (SEQ ID NO:9) coupled to the keyhole limpet hemocyanin via the heterobifunctional reagent m-maleimidobenzoyl-N-hydroxysuccinimide ester) and pre-immune serum from the R7 rabbit.

[0087]As in FIG. 14, the BEIIa protein is separated into three forms (indicated by arrows in FIG. 15, Panel B), by affinity electrophoresis in the presence of β-limit dextrin. In barley (Sun et al., 1997) and maize (Bayer and Preiss 1981) both branching enzymes IIa and IIb are present in the soluble fraction. In some subsequent experiments we have detected low levels of BE IIb in the soluble fraction.

[0088]The separation of the forms of BEIIa encoded by each wheat genome is demonstrated in FIG. 16. In Panel (A) the diploid A. tauschii (lanes 2,3 and 4) and barley line (lane 11) yields a single band. However, the tetraploid T. durum lines (Panel A lane 1, Panel B, lanes 1, 16, and 17) and hexaploid T. aestivum lines (Panel A lanes 5-10, Panel B lanes 2-15, 18-19) give at least 2 bands. Some hexaploid lines (panel A, lane 7 and 9, Panel B lanes 2-6, lanes 8-9, lane 13) yield 2 bands, indicating either that they are null for one genome or that the products of two genomes migrate with identical mobility in the gel system.

[0089]The use of the separation system as a means of screening for wheat genomes with altered or null alleles of branching enzyme IIa is demonstrated by FIG. 14 (Panel B), where different lines are shown to have different numbers and mobilities of branching enzyme IIa proteins.

EXAMPLE 9

Presence of Two Classes of Proteins in the Starch Granule at 88 kDa and their Differential Antibody Binding

[0090]The wheat starch granule contains a number of proteins that have been analysed by SDS-PAGE (Rahman et al., 1995, Denyer et al., 1995, Takaoka et al, Li et al., 1999a, Li et al, 1999b) and two-dimensional gel electrophoresis (Yamamori and Endo, 1996). The following bands have been identified: 60 kDa, GBSS; 75 kDa, SSI; 100 kDa, 108 kDa and 115 kDa, SSII). An 88 kDa band is also observed, and has been shown to be associated with branching enzyme activity (Denyer et al., 1995) and to react to antibodies to maize BEII (Rahman et al., 1995). This protein band was shown to contain at least two protein components.

[0091]This analysis has been extended by purification and analysis of the individual granule proteins. The granule proteins were isolated from 4.7 g of wheat starch granules by boiling in 24 ml of SDS buffer (50 mM Tris-HCl buffer pH 6.8, 10% SDS and 6.25% 2-mercaptothanol) as described by Rahman et al., (1995). Residual granular starch was removed by centrifugation, and granule proteins were separated by applying the supernatant to a 9% SDS-PAGE gel prepared in a Biorad Model 491 Prep Cell apparatus. The SDS gel contained a stacking gel composed of 0.125 M Tris-HCl buffer (pH 6.8), 0.25% SDS, 6% acrylamide, 0.06% ammonium persulphate and 0.1% TEMED and a separating gel containing 0.34 M Tris-HCl buffer (pH 8.8), 0.25% SDS, acrylamide (9%), 0.06% ammonium persulphate, and 0.1% TEMED. The samples were electrophoresised at 60 mAmp constant current for 16 hours, and fractions of ractions (5 ml) collected by a pump operating at 0.5 ml/min. Fractions were analysed by SDS-PAGE, and fractions containing an 88 kDA band precipitated by the addition of 3 volumes of acetone. The precipitate from each 5 ml fraction was collected by centrifugation, the sample dissolved in SDS buffer, and electrophoresed through a standard 8% SDS-PAGE gel. The lane was excised from the gel and renatured in 0.04 M Tris for 2 hours. To generate a two-dimensional separation, the gel was then laid across the top of a second non-denaturing PAGE gel and electrophoresed. Proteins were identified by staining with Coomassie blue (a 50:50 mixture of 2.5% Coomassie Blue R-250 and Coomassie Blue G250 solutions).

[0092]FIG. 17, Panel (A) shows that two proteins were visible after staining, and these were designated 88 kD (U) and 88 kD (L), as indicated by the arrows. Immunoblotting of the two-dimensional gel with peptide antibodies to the N-terminal of BEIIa (3KLH) and to the N-terminus of the wheat BEIIb cDNA sequence (R6; see FIGS. 12 and 13 for details of the antibodies are set out in Example 8) indicated preferential binding of the R6 antibody to 88 kD (U) and preferential binding of 3KLH to 88 kD (L) (FIG. 17, Panel B), providing a provisional assignment of these proteins as BEIIb and BEIIa respectively.

[0093]The proteins were further analysed by digestion with trypsin, and the peptides released were identified by MALDI-TOF analysis at the Australian Proteome Analysis Facility, Macquarie University, Sydney. The results of this analysis, shown in Table 2, demonstrated that 88 kD (U) was the product of the wheat BEIIb gene, and that while the assignment of 88 kD (L) was inconclusive, the results were consistent with the protein being a branching enzyme encoded by either SBE9 or the wheat BEIIb cDNA.

TABLE-US-00002 TABLE 2 (a) Comparison of 88 kD (U) and the predicted protein encoded by the wheat BEIIb cDNA. Matches: 6 MOWSE Score: 4.97e+001 Coverage: 8.85% Matching Peptides: MW Delta Start End Sequence 755.4766 -0.13 320 325 (K) RPKSLR (I) 1337.7092 0.01 453 463 (R) VFNYGNKEVIR (F) 1337.6728 -0.03 703 713 (R) RFDLGDAEFLR (Y) 1508.7623 -0.12 785 799 (K) VVLDSDAGLFGGFGR (I) 1589.6933 -0.08 731 743 (K) YGFMTSDHQYVSR (K) 1692.7049 -0.17 184 198 (R) SDIDEHEGGMDVFSR (G) 1706.8740 -0.04 340 353 (K) INTYANFRDEVLPR (I) (b) Comparison of 88 kD (L) and the predicted proteins encoded by the wheat BEIIb cDNA and SBE9 cDNA. Matches to wheat BEIIb cDNA Matches: 8 MOWSE Score: 1.32e+003 Likelihood: 2.053+003 Coverage: 11.72% Matching Peptides: MW Delta Start End Sequence 819.4603 11.23 464 470 (R)FLLSNAR (W) 1210.5090 -105.27 444 452 (R) GHHWMWDSR (V) 1337.7092 10.53 453 463 (R) VFNYGNKEVIR (F) 1337.6728 -16.68 703 713 (R) RFDLGDAEFLR (Y) 1508.7623 -44.33 785 799 (K) VVLDSDAGLFGGFGR (I) 1573.7446 -16.81 326 339 (R) IYETHVGMSSPEPK (I) 1589.6933 -23.46 731 743 (K) YGFMTSDHQYVSR (K) 1692.7049 -95.07 184 198 (R) SDIDEHEGGMDVFSR (G) 1706.8740 -15.57 340 353 (K) INTYANFRDEVLPR (I) Matches to wheat SBE9 Matches: 6 MOWSE Score: 1.04e+001 Coverage: 8.63% Matching Peptides: MW Delta Start End Sequence 819.4603 11.23 451 457 (R)FLLSNAR (W) 1210.5090 -105.27 431 439 (R) GHHWMWDSR (V) 1508.7875 -27.64 145 156 (K) IYEIDPTLKDFR (S) 1573.7446 -16.81 313 326 (R) IYESHIGMSSPEPK (I) 1599.7641 -9.93 171 185 (R) AAIDQHEGGLEAFSR (G) 1692.8583 -4.45 327 340 (K) INSYANFRDEVLPR (I)

EXAMPLE 10

Sequencing of the SBE IIb Gene

[0094]A partial genomic sequence of the SBEIIb gene was obtained, using clone G5 described in Example 4. So far approximately 8.4 kb of sequence has been obtained. This includes approximately 500 bp upstream of the start codon, presumably comprising the promoter region, and exons 1 to 14 in full. This partial sequence is set out in SEQ ID NO:10. From the sequences of the corresponding maize and Arabidopsis BEII genes, we would expect the gene to contain 22 exons. A comparison between the exon/intron structures of various BEII genes and the wheat BEIIb gene is shown in FIG. 18, and the sizes of the exons in various SBEII genes are compared in Table 3. In this table "Arab" represents Arabidopsis.

TABLE-US-00003 TABLE 3 Sizes of exons in various SBE IIb genes Wheat Maize Barley Wheat Exon no Arab21 Arab22 BEIIa BEIIb BEIIb BEIIb 1 42 124 279 212 121 148 2 253 120 98 146 152 146 3 236 182 243 155 230 230 4 99 99 99 99 99 99 5 43 43 43 43 43 43 6 60 60 60 60 60 7 81 81 81 81 81 8 117 117 117 117 117 9 84 84 84 84 84 10 122 122 122 122 122 11 120 120 120 120 120 12 130 130 130 130 130 13 111 111 111 111 111 14 129 129 129 129 129 15 104 104 104 104 16 145 145 145 145 17 148 148 148 18 101 101 101 19 78 78 78 20 156 156 156 21 75 75 75 22 90 384 304 17 558 18 164

[0095]Using a probe specific for the 3' end of SBE IIb, three clones designated G7, G8 and G9 respectively, have now been isolated from the T. tauschii genomic library, and are being subjected to sequence analysis to provide the 3' region of the gene.

EXAMPLE 11

Development of PCR Primer Sets for the Discrimination of the BEIIb Genes from Each Genome

[0096]A number of primer sets, designed on the basis of comparisons between SBE IIa and SBE IIb genes, were tested on wheat genomic DNA. The sequences of these primers were as follows:

TABLE-US-00004 SEQ ID NO: 11 ARA 12F: 5' CCG TCC TAC ATG ACA CCT GGC CG 3' SEQ ID NO: 12 ARA 10R: 5' CCG CCG GAT CGA GGA GCC GAC GG 3' SEQ ID NO: 13 ARA 6F: 5' GGC GGC GGC GAC GGG ATG GCT GC 3' SEQ ID NO: 14 ARA 8R: 5' CGC CGT CAG GGA TCA TCA CCT CC 3' SEQ ID NO: 15 ARA 19F: 5' CAC CCA TTG TAA TTG GGT ACA CTG 3' SEQ ID NO: 16 ARA 15R 5' TCC ATG CCT CCT TCG TGT TCA TCA 3' SEQ ID NO: 17 ARA 23R 5' CTG CGC ATA AAT CCA AAC TTC TCG 3'

[0097]Targeting the promoter region of SBE IIb using the primers ARA 12F and ARA 13R resulted in the specific amplification of only the D genome gene. Aneuploid analysis using this pair of primers showed that the SBE IIb was located on the long arm of chromosome 2 in wheat, as illustrated in FIG. 19.

[0098]The primers ARA6F and ARA8R, which amplify the exon 1-intron 1-exon 2 region of SBE IIb, could distinguish the D genome from the A and B genomes, as shown in FIG. 20. Sequence analysis of this region indicated that the genes from the A and B genomes completely lack intron 1. This is illustrated in FIG. 21.

EXAMPLE 12

Identification of SBE IIb in Genomes A, B and D

[0099]Sequence analysis of the intron 3 region of SBE IIb, amplified by PCR using the primers ARA 19F and ARA 15R, followed by digestion using the restriction enzyme Rsa1, revealed significant polymorphism amongst the three genomes. This polymorphism, illustrated in the sequence alignment set out in FIG. 22, was utilised to develop genome specific markers which can distinguish between the A, B and D genomes.

[0100]PCR amplification of the SBE IIb gene was carried out using the primers ARA 19F and ARA 15R, followed by restriction digestion using Rsa1. The results of the PCR analysis, shown in FIG. 23, indicate that these primers can distinguish between the three genomes.

[0101]Screening of approximately 600 wheat lines using the genome specific primer pair, ARA 19F and ARA 23R, which amplifies the same region as ARA 19F and ARA 15R, identified one null mutant of the wheat genome. The amplification was performed as described for FIG. 23, and the results are shown in FIG. 24.

EXAMPLE 13

Constructs for Expression of BEII Genes

[0102]Recombinant DNA technology may be used to inhibit or abolish expression of either or both of BE IIa and BE IIb. Three general approaches are used, using transformation of the target plant cells with one of the following types of construct:

[0103]a) `Antisense` constructs of SBE IIa and SBE IIb, in which the desired nucleic acid sequence is inserted into the construct in the opposite direction to the functional gene.

[0104]b) `Sense` constructs of SBE IIa and SBE IIb, in which the desired nucleic acid is inserted in the same direction as the functional gene; this utilises co-suppression events to inhibit the expression of the target gene;

[0105]c) Duplex constructs of SBE IIa and SBE IIb, in which the desired nucleic acid in both the sense and antisense orientations is co-located in the construct on either side of a "spacer" loop formed by an intron sequence.

[0106]In all three cases, the desired nucleic acid is operably linked to a promoter sequence in the construct.

[0107]Sense and antisense constructs have been widely used to modulate gene expression in plants. More recently, it has been shown that the delivery of RNAs with potential to form duplexes is a particularly efficient strategy for generating post-transcriptional gene silencing in transgenic plants (Waterhouse et al., 1998; Smith et al., 2000).

[0108]Transformation of the target wheat cells, or cells of other plants, using these constructs is effected using methods known in the art, such as transformation with Agrobacterium tumefaciens. Once transgenic plants are obtained, they are assessed for the effects of the transgenes on BE IIa and BE IIb expression. For example, in both maize and potato it has been shown that crossing BE II mutations or BE II transgenes into BE I-deficient backgrounds greatly increases amylose content. Wheat BE I null lines, identified using the methods described in WO99/14314, provide a ready source of BE I-deficient genetic material into which BE IIa and BE IIb transgenics can be crossed, in order to extend further the range of starches which can be produced.

[0109]Sense, antisense and duplex constructs of SBE IIa and SBE IIb were generated in the vector pDV03000 (Biogemma Ltd, UK) which carries the high molecular weight gluten promoter (pHMWG) and the Nopaline synthase (Nos) terminator. These constructs are schematically represented in FIGS. 25, 26 and 27. The Biogemma vectors are based on the well-known plasmid pBR322, and comprise a number of restriction sites, as illustrated in FIGS. 25 and 26, for incorporation of desired DNA sequences. The entire desired DNA, plus the promoter and terminator sequences referred to above, can then be excised as a Xho fragment and cloned into a suitable vector, such as Agrobacterium tumefaciens. Those skilled in the art will be aware of other suitable vectors which could be used.

SHE IIa Constructs

[0110]A sense construct of SB IIa was prepared by inserting a 2143 bp fragment of SBE IIa coding sequence in the sense orientation at the EcoR1/Sma1 site of pDV03000. An SBE IIa antisense construct was prepared by inserting 1913 bp of SBE IIa coding sequence in the antisense orientation at the EcoR1/BamH1 site of pDV03000. This is also illustrated in FIG. 25.

SBE IIb Constructs

[0111]A sense construct of SBE IIb was generated by inserting a 1008 bp fragment of the SBE IIb coding sequence in the sense orientation at the EcoR1/Sma1 site of pDV03000. An antisense SBE IIb construct was prepared by inserting a 955 bp sequence of the coding region for SBE IIb at the BamH1/Pst1 site of pDV03000 in the antisense orientation. This is illustrated in FIG. 26.

Duplex Constructs

[0112]A schematic model of a duplex construct is set out in FIG. 27. The duplex construct was prepared using the following protocol, in which all the amplification steps were performed using PCR under conventional conditions.

SBE IIa Duplex

[0113]1) a 468 bp sequence of SBE IIa, which includes the whole of exons 1 and 2 and part of exon 3, with EcoR1 and Kpn1 restriction sites on either side, was amplified to obtain a first fragment (fragment 1);

[0114]2) a second fragment, 512 bp in length, consisting of part of exons 3 and 4, and the whole of intron 3 of SBE IIa, with Kpn1 and Sac1 sites on either side, was amplified to provide fragment 2;

[0115]3) a 528 bp fragment consisting of the complete exons 1, 2 and 3 of SBE IIa, with BamH1 and Sac1 sites on either side, was amplified to provide fragment 3;

[0116]4) fragments 1, 2 and 3 were ligated so that the sequence of fragment 3 was ligated to fragment 2 in the antisense orientation to fragment 1.

SBE IIb Duplex

[0117]1) a 471 bp sequence consisting of the whole of exons 1 and 2 and part of exon 3 of SBE IIb was amplified with EcoR1 and Kpn1 restriction sites on either side to generate fragment 1;

[0118]2) a 589 bp fragment consisting of part of exons 3 and 4 and the whole of intron 3 of SBE IIb, with Kpn1 and Sac1 sites on either side, was amplified to provide fragment 2;

[0119]3) a 528 bp fragment consisting of the complete exons 1, 2 and 3, with BamH1 and Sac1 sites on either side was amplified to provide fragment 3;

[0120]4) fragments 1, 2 and 3 were ligated so that fragment 3 was in the antisense orientation to fragment 1 when ligated to fragment 2.

[0121]The start and end points of the sequences used for making the constructs were as follows:

a) SBE IIa Sense Construct

[0122]Start: 461 bp of Sbe 9 (SBE IIa) cDNA [0123]End: 2603 bp of Sbe 9 (SBE IIa) cDNA

b) SBE IIa Anti-Sense Construct

[0123] [0124]Start: 691 bp of Sbe 9 (SBE IIa) cDNA [0125]End: 2603 bp of Sbe 9 (SBE IIa) cDNA

[0126]This fragment was ligated in the anti-sense orientation.

c) SBE IIb Sense Construct

[0127]Start: 85 bp of SBE IIb cDNA [0128]End: 1085 bp of SBE IIb cDNA

d) SBE IIb Anti-Sense Construct

[0128] [0129]Start: 153 bp of SBE IIb cDNA [0130]End: 1085 bp of SBe IIb cDNA

[0131]This fragment was ligated in the anti-sense orientation.

e) SBE IIa Duplex Construct

i) Fragment 1

[0132]Full exon 1: 1151 bp-1336 bp of SBE IIa genomic sequence

[0133]Full exon 2: 1664 bp-1761 bp of SBE IIa genomic sequence

[0134]Partial exon 3: 2038 bp-2219 bp of SBE IIa genomic sequence

[0135]This fragment had an EcoR1 site (GAATTC) introduced at the start of the exon 1 sequence and a Kpn1 site (GGTACC) introduced at the end of the partial exon 3 sequence.

ii) Fragment 2

[0136]Partial exon 3: 2220 bp-2279 bp of SBE IIa genomic sequence

[0137]Full intron 3: 2280 bp-2680 bp of SBE IIa genomic sequence

[0138]Partial exon 4: 2681 bp-2731 bp of SBE IIa genomic sequence

[0139]This fragment had a Kpn1 site (GGTACC) introduced at the start of the partial exon 3 and a Sac1 site (GAGCTC) introduced at the end of the partial exon 4 sequence.

iii) Fragment 3

[0140]Full exon 1: 1151 bp-1336 bp of SBE IIa genomic sequence

[0141]Full exon 2: 1664 bp-1761 bp of SBE IIa genomic sequence

[0142]Full exon 3: 2038 bp-2279 bp of SBE IIa genomic sequence

[0143]This fragment had a BamH1 site (GGATCC) introduced at the start of the complete exon 1 sequence and a Sac1 site (GAGCTC) introduced at the end of the complete exon 3 sequence.

f) SBE IIb Duplex Construct

i) Fragment 1

[0144]Full exon 1: 489 bp-640 bp of SBE IIb genomic sequence

[0145]Full exon 2: 789 bp-934 bp of SBE IIb genomic sequence

[0146]Partial exon 3: 1598 bp-1770 bp of SBE IIb genomic sequence

[0147]This fragment had an EcoR1 site (GAATTC) introduced at the start of exon 1 and a Kpn1 site (GGTACC) introduced at the end of the partial exon 3 sequence.

ii) Fragment 2

[0148]Partial exon 3: 1771 bp-1827 bp of SBE IIb genomic sequence

[0149]Full intron 3: 1828 bp-2292 bp of SBE IIb genomic sequence

[0150]Partial exon 4: 2293 bp-2359 bp of SBE IIb genomic sequence

[0151]This fragment had a Kpn1 site (GGTACC) introduced at the start of the partial exon 3 sequence and a Sac1 site (GAGCTC) introduced at the end of the partial exon 4 sequence.

iii) Fragment 3

[0152]Full exon 1: 489 bp-640 bp of SBE IIb genomic sequence

[0153]Full exon 2: 789 bp-934 bp of SBE IIb genomic sequence

[0154]Full exon 3: 1598 bp-1827 bp of SBE IIb genomic sequence

[0155]This fragment had a BamH1 site (GGATCC) introduced at the start of exon 1 and a Sac1 site (GAGCTC) introduced at the end of exon 3.

[0156]The SBE IIa and SBE IIb duplexes thus formed were respectively inserted at the EcoR1/BamH1 site of pDV03000.

[0157]Samples of λ phage clones G5 and G9 have been deposited in the Australian Government Analytical Laboratories, acting as an International Depository Authority under the Budapest Treaty on 20 Feb. 2001, under accession numbers NM01/19255 and NM01/19256 respectively.

[0158]It will be apparent to the person skilled in the art that while the invention has been described in some detail for the purposes of clarity and understanding, various modifications and alterations to the embodiments and methods described herein may be made without departing from the scope of the inventive concept disclosed in this specification.

[0159]References cited herein are listed on the following pages, and are incorporated herein by this reference.

REFERENCES

[0160]Ali, S., Holloway B., and Taylor W. C., W. C. 2000 Plant Molecular Biology Report 18 123-132. [0161]Baba, T., K. Kimura, K. Mizuno, H. Etoh, Y. Ishida, O. Shida, and Y. Arai. 1991. Sequence conservation of the catalytic regions of amylolytic enzymes in maize branching enzyme-I. Bioc Biop R 181: 87-94. [0162]Baecker P A, Greenberg E, Preiss J (1986). Biosynthesis of the bacterial glucan: primary structure of Escherichia coli 1,4 alpha D glucan 6-alpha-D-(1,4 alpha-D-glucano)-transferase as deduced from the nucleotide sequence of the glgB gene. J. Biol. Chem. 261, 8738-8743. [0163]Baga, M., A. Repellin, T. Demeke, K. Caswell, N. Leung, E. S. M. Abdel-aal, P. Hucl, and R. N. Chibbar. 1999. Wheat starch modification through biotechnology. STARCH-STARKE 51: 111-116. [0164]Bhattacharyya, M., A. M. Smith, T. H. N. Ellis, C. Hedley, and C. Martin. 1990. The wrinkled-seed character of pea described by Mendel is caused by a transposon-like insertion in a gene encoding starch-branching enzyme. Cell 60: 115-122. [0165]Boyer, C. D., P. A. Damewood, and E. K. G. Simpson. 1980. Effect of gene dosage at high amylose loci on the properties of the amylopectin fractions of the starches. Starch 32: 217-222. [0166]Boyer, C. D. and J. Preiss. 1981. Evidence for independent genetic control of the multiple forms of maize endosperm branching enzymes and starch synthases. Plant Physiology 67: 1141-1145. [0167]Burton, R. A., J. D. Bewley, A. M. Smith, M. K. Bhattacharyya, H. Tatge, S. Ring, V. Bull, W. D. O. Hamilton, and C. Martin. 1995. Starch branching enzymes belonging to distinct enzyme families are differentially expressed during pea embryo development. Plant J. 7: 3-15. [0168]Cangiano G, La Volpe A, Poulsen P, Kreiberg J D (1993) Starch branching enzyme cDNA from Solanum tuberosum. Plant Physiol 102: 1053-1054. [0169]Cao, H. P., J. Imparl-Radosevich, H. P. Guan, P. L. Keeling, M. G. James, and A. M. Myers. 1999. Identification of the soluble starch synthase activities of maize endosperm. Plant Physiology 120: 205-215. [0170]Denyer, K., C. M. Hylton, C. F. Jenner, and A. M. Smith. 1995. Identification of multiple isoforms of soluble and granule-bound starch synthase in developing wheat endosperm. Planta 196: 256-265. [0171]Devereaux, J, Haeberli P, Smithies O (1984) A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res 12: 387-395 [0172]Filpse, E., L. Suurs, and R. G. F. Visser. 1996. Introduction of sense and antisense cDNA for branching enzyme in the amylose-free potato mutant leads to physico-chemical changes in the starch. Planta 198: 340. [0173]Fisher, D. K., M. Gao, K. N. Kim, C. D. Boyer, and M. J. Guiltinan. 1996. Two closely related cDNAs encoding starch branching enzyme from Arabidopsis thaliana. Plant Mol Biol 30: 97-108. [0174]Fisher, D. K., M. Gao, K.-N. Kim, C. D. Boyer, and M. J. Guiltinan. 1996. Allelic analysis of the maize amylose-extender locus suggests that independent genes encode starch-branching enzymes IIa and IIb. Plant Physiology 110: 611-619. [0175]Fisher, D. K., C. D. Boyer, and L. C. Hannah. 1993. Starch branching enzyme II from maize endosperm. Plant Physiology 102: 1045-1046. [0176]Gao, M., D. K. Fisher, K. N. Kim, J. C. Shannon, and M. J. Guiltinan. 1996. Evolutionary conservation and expression patterns of maize starch branching enzyme I and IIb genes suggests isoform specialization. Plant Mol Biol 30: 1223-1232. [0177]Gao, M., J. Wanat, P. S. Stinard, M. G. James, and A. M. Myers. 1998. Characterization of dull1, a maize gene coding for a novel starch synthase. Plant Cell 10: 399-412. [0178]Gao, M., D. K. Fisher, K. N. Kim, J. C. Shannon, and M. J. Guiltinan. 1997. Independent genetic control of maize starch-branching enzymes IIa and IIb--Isolation and characterization of a Sbe2a cDNA. Plant Physiology 114: 69-78. [0179]Higgins T J V, Zwar J A, Jacobsen J V (1976) Gibberellic acid enhances the level of translatable mRNA for a amylase in barley aleurone layers. Nature 260: 166-168. [0180]Jahne A., Lazzeri P. A. and Lorz H. (1991) Regeneration of fertile plants from protoplasts derived from embryonic cell suspensions of barley (Hordeum vulgare L.). Plant Cell Reports 10, 1-6. [0181]Jobling, S. A., G. P. Schwan, R. J. Westcott, C. M. Sidebottom, M. Debet, M. J. Gidley, R. Jeffcoat, and R. Safford. 1999. A minor form of starch branching enzyme in potato (Solanum tuberosum L.) tubers has a major effect on starch structure: cloning and characterisation of multiple forms of SBE A. Plant J 18: 163-171. [0182]Kawasaki, T., K. Mizuno, T. Baba, and H. Shimada. 1993. Molecular analysis of the gene encoding a rice starch branching enzyme. Molecular and General Genetics 237: 10-16. [0183]Khoshnoodi, J., A. Blennow, B. Ek, L. Rask, and H. Larsson. 1996. The multiple forms of starch-branching enzyme I in Solanum tuberosum. Eur J Biochem 242: 148-155. [0184]Kiel J A K W, Boels J M, Beldman G, Venema G. (1992). The glgB gene from the thermophile Bacillus caldolyticus encodes a thermolabile branching enzyme. DNA Seq 3, 221-232. [0185]Kim, K. N., D. K. Fisher, M. Gao, and M. J. Guiltinan. 1998. Genomic organization and promoter activity of the maize starch branching enzyme I gene. Gene 216: 233-243. [0186]Kim, K. N., D. K. Fisher, M. Gao, and M. J. Guiltinan. 1998. Molecular cloning and characterization of the amylose-extender gene encoding starch branching enzyme IIB in maize. Plant Mol Biol 38: 945-956. [0187]Lazzeri, P. A., Brettschneider, R., Luhsrs, R., and Lorz. H. Theor. Appl. Genet. 1991, 81, 437-444. [0188]Lagudah E S, Appels R, McNeil D (1991) The Nor-D3 locus of Triticum tauschii: natural variation and genetic linkage to markers in chromosome 5. Genome 34: 387-395. [0189]Li, Z., S. Rahman, B. Kosar-Hashemi, G. Mouille, R. Appels, and M. K. Morell. 1999. Cloning and characterization of a gene encoding wheat starch synthase I. Theor Appl Genet 98: 1208-1216. [0190]Li, Z. Y., X. S. Chu, G. Mouille, L. L. Yan, B. Kosar-Hashemi, S. Hey, J. Napier, P. Shewry, B. Clarke, R. Appels, M. K. Morell, and S. Rahman. 1999. The localization and expression of the class II starch synthases of wheat. Plant physiology. 120: 1147-1155. [0191]Maniatis T, Fritsch E F, Maniatis J (1982) Molecular cloning. A Laboratory Manual. New York. Cold Spring Harbor Laboratory. [0192]Matsumoto, A., T. Nakajima, and K. Matsuda. 1990. A kinetic study of the interaction between glycogen and Neurospora crassa branching enzyme. J Biochem 107: 123-126. [0193]Mizuno, K., T. Kawasaki, H. Shimada, H. Satoh, E. Kobayashi, S. Okumura, Y. Arai, and T. Baba. 1993. Alteration of the structural properties of starch components by the lack of an isoform of starch branching enzyme in rice seeds. J Biol Chem 268: 19084-19091. [0194]Morell, M. K., A. Blennow, B. Kosar-Hashemi, and M. S. Samuel. 1997. Differential expression and properties of starch branching enzyme isoforms in developing wheat endosperm. Plant Physiology 113: 201-208. [0195]Nair, R. B., M. Baga, G. J. Scoles, K. K. Kartha, and R. N. Chibbar. 1997. Isolation, characterization and expression analysis of a starch branching enzyme II cDNA from wheat. Plant Science 122: 153-163. [0196]Rahman, S., S. Abrahams, D. Abbott, Y. Mukai, M. Samuel, M. Morell, and R. Appels. 1997. A complex arrangement of genes at a starch branching enzyme I locus in the D-genome donor of wheat. Genome 40: 465-474. [0197]Rahman, S., Z. Li, S. Abrahams, D. Abbott, R. Appels, and M. K. Morell. 1999. Characterisation of a gene encoding wheat endosperm starch branching enzyme-I. Theor Appl Genet 98: 156-163. [0198]Rahman, S., B. Kosar-Hashemi, M. S. Samuel, A. Hill, D. C. Abbott, J. H. Skerritt, J. Preiss, R. Appels, and M. K. Morell. 1995. The major proteins of wheat endosperm starch granules. Aust J Plant Phys 22: 793-803. [0199]Safford, R., S. A. Jobling, C. M. Sidebottom, R. J. Westcott, D. Cooke, K. J. Tober, B. H. Strongitharm, A. L. Russell, and M. J. Gidley. 1998. Consequences of antisense RNA inhibition of starch branching enzyme activity on properties of potato starch. Carbohydrate polymers 35: 155-168. [0200]Sathish, P., C. X. Sun, A. Deiber, C. Jansson, and C. X. Sun. Cloning and anti-sense RNA constructs of a starch branching enzyme gene from barley endosperm. Mathis, P. 313-316. 1995. Photosynthesis: from light to biosphere. Volume V. Proceedings of the Xth International Photosynthesis Congress, Montpellier, France, 20-25 Aug. 1995. [0201]Schondelmaier, J., A. Jacobi, G. Fischbeck, and A. Jahoor. 1992. Genetical Studies on the mode of inheritance and localization of the amo1 (High Amylose) gene in barley. Plant Breeding 109: 274-280. [0202]Smith, N. A., Singh S. P., Wang M. B., Stoutjesdijk P. A., Green A. G., Waterhouse P. M. (2000) Nature 407, 319-420. [0203]Sun, C. X., P. Sathish, B. Ek, A. Deiber, C. Jansson, and C. X. Sun. 1996. Demonstration of in vitro starch branching enzyme activity for a 51/50-kDa polypeptide isolated from developing barley (Hordeum vulgare) caryopses. Physiologia. Plantarum. 96: 474-483. [0204]Sun, C. X., P. Sathish, S. Ahlandsberg, A. Dieber, C. Jansson, and C. X. Sun. 1997. Identification of four starch-branching enzymes in barley endosperm: partial purification of forms I, IIa and IIb. New Phytologist. 137: 215-222. [0205]Sun, C. X., P. Sathish, S. Ahlandsberg, C. Jansson, and C. X. Sun. 1998. The two genes encoding starch-branching enzymes IIa and IIb are differentially expressed in barley. Plant Physiology. 118: 37-49. [0206]Takaoka, M., S. Watanabe, H. Sassa, M. Yamamori, T. Nakamura, T. Sasakuma, and H. Hirano. 1997. Structural characterization of high molecular weight starch granule-bound proteins in wheat (Triticum aestivum L). J. Agric. Food Chem. 45: 2929-2934. [0207]Tingay, S., McElroy, D., Kalla, R., Fieg, S., Wang. M, Thornton, S. and Brettel, R. 1997, The Plant Journal, 11, 1369-1376. [0208]Wan Y., and Lemaux, P. G. 1994, Plant Physiology. 104, 37-48.

[0209]Wirtzens, B, Brettel R I S, Murray F., McElroy D, Li Z, Dennis E S. (1998) Comparison of three selectable marker genes for transformation of wheat by microprojectile bombardment. Aust. J. Plant. Physiol. 25, 39-44. [0210]Waterhouse P. M., Graham M. W., Wang M. B. (1998). Virus resistance and gene silencing can be induced by simultaneous expression of sense and antisense RNA. Proc. Natl. Acad. Sci. USA. 95, 13959-13964. [0211]Yamamori, M. and T. R. Endo. 1996. Variation of starch granule proteins and chromosome mapping of their coding genes in common wheat. Theor Appl Genet 93: 275-281.

Sequence CWU 1

5112726DNATriticum sp. 1acgttgctcc cccttctcat cgcttctcaa ttaatatctc catcactcgg ttccgcgctg 60catttcggcc ggcgggttga gtgagatctg ggccactgac cgactcactc gctcgctgcg 120gggatggcga cgttcgcggt gtccggcgcg accctcggtg tggcgcggcc gccggcggcg 180gcgcaacctg aagaattaca gatacctgaa gacatcgagg agcaaacggc tgaagtaaac 240atgacagggg ggactgcaga aaaacttgaa tcttcagaac cgactcaagg cattgtggaa 300acaatcactg atggtgtaac caaaggagtt aaggaactag tcgtggggga gaaaccgcga 360gttgtcccaa aaccaggaga tgggcagaaa atatacgaga ttgacccaac gctgaaagat 420tttcggagcc atcttgacta ccgatacagc gaatacagga gaattcgtgc tgctattgac 480caacatgaag gtggattgga agcattttct cgtggttatg aaaagcttgg atttacccgc 540agtgctgaag gtatcactta ccgagaatgg gctcctggag cgcattctgc agcattagta 600ggtgacttca acaattggaa tccgaatgca gatactatga ccagagatga ttatggtgtt 660tgggagattt tcctccctaa caatgctgat ggatccccag ctattcctca tggctcacgt 720gtaaagatac ggatggatac tccatctggt gtgaaggatt caatttctgc ttggatcaag 780ttctctgtgc aggctccagg tgaaatacca ttcaatggca tatattatga tccacctgaa 840gaggagaagt atgtcttcca acatcctcaa cctaaacgac cagagtcact gaggatttat 900gaatcacaca ttggaatgag cagcccagaa ccgaagataa attcatatgc taattttagg 960gatgaggtgc tgccaagaat taaaaggctt ggatacaatg cagtgcagat aatggcaatc 1020caggagcatt catactatgc gagctttggg taccatgtta ctaatttttt tgcaccaagt 1080agccgttttg gaactccaga ggacttaaaa tccctgatcg atagagcaca tgagcttggt 1140ttgcttgttc ttatggatat tgttcatagt cattcatcaa ataataccct tgacggcttg 1200aatggtttcg atggcactga tacacattac ttccacggtg gtccacgtgg ccatcattgg 1260atgtgggatt ctcgtctatt caactatggg agttgggaag tattgagatt cttactgtca 1320aacgcgagat ggtggcttga agaatataag tttgatggat ttcgatttga tggggtgacc 1380tccatgatgt atactcacca tggattacaa atgacattta ctgggaacta tggcgagtat 1440tttggatttg ctactgatgt tgatgcggta gtttacttga tgctggtcaa cgatctaatt 1500catggacttc atcctgatgc tgtatccatt ggtgaagatg tcagtggaat gcccacattt 1560tgcatccctg ttccagatgg tggtgttggt tttgactatc gcttgcatat ggctgtagca 1620gataaatgga ttgaactcct caagcaaagt gacgaatctt ggaaaatggg tgatattgtg 1680cacaccctaa caaatagaag gtggcttgag aagtgtgtaa cttatgcaga aagtcatgat 1740caagcactag ttggtgacaa gactattgca ttctggttga tggataagga tatgtatgat 1800ttcatggctc tggataggcc ttcaactcct cgcattgatc gtggcatagc attacataaa 1860atgatcaggc ttgtcaccat gggtttaggt ggtgaaggct atcttaactt catgggaaat 1920gagtttgggc atcctgaatg gatagatttt ccaagaggtc cgcaaactct tccaaccggc 1980aaagttctcc ctggaaataa caatagttat gataaatgcc gccgtagatt tgatcttgga 2040gatgcagatt ttcttagata tcatggtatg caagagttcg atcaggcaat gcagcatctt 2100gaggaaaaat atgggtttat gacatctgag caccagtatg tttcacggaa acatgaggaa 2160gataaggtga tcatcttcga aagaggagat ttggtatttg ttttcaactt ccactggagc 2220aatagctttt ttgactaccg tgttgggtgt tccaggcctg ggaagtacaa ggtggcctta 2280gactccgacg atgcactctt tggtggattc agcaggcttg atcatgatgt cgactacttc 2340acaaccgaac atccgcatga caacaggccg cgctctttct cggtgtacac tccgagcaga 2400actgcggtcg tgtatgccct tacagagtaa gaaccagcag ctgcttgtta caaggcaaag 2460agagaactcc agagagctcg tggatcgtga gcgaagcgac gggcaacggc gcgaggctgc 2520tctaagcgcc atgactggga ggggatcgtg cctcttcccc agatgccagg aggagcagat 2580ggataggtag cttgttggtg agcgctcgaa agaaaatgga cgggcctggg tgtttgtcgt 2640gctgcactac cctcctccta tcttgcacat tcccggttgt ttttgtacat ataactaata 2700attgcccgtg cgctcaacgt gaacaa 2726211476DNAAegilops tauschiimodified_base(1722)..(1722)a, c, g, or t 2agaaacacct ccattttaga tttttttttt gttcttttcg gacggtgggt cgtggagaga 60ttagcgtcta gttttcttaa aagaacaggc catttaggcc ctgctttaca aaaggctcaa 120ccagtccaaa acgtctgcta ggatcaccag ctgcaaagtt aagcgcgaga ccaccaaaac 180aggcgcattc gaactggaca gacgctcacg caggagccca gcaccacagg cttgagcctg 240acagcggacg tgagtgcgtg acacatgggg tcatctatgg gcgtcggagc aaggaagaga 300gacgcacatg aacaccatga tgatgctatc aggcctgatg gagggagcaa ccatgcacct 360tttcccctct ggaaattcat agctcacact tttttttaat ggaagcaaga gttggcaaac 420acatgcattt tcaaacaagg aaaattaatt ctcaaaccac catgacatgc aattctcaaa 480ccatgcaccg acgagtccat gcgaggtgga aacgaagaac tgaaaatcaa catcccagtt 540gtcgagtcga gaagaggatg acactgaaag tatgcgtatt acgatttcat ttacatacat 600gtacaaatac ataatgtacc ctacaatttg ttttttggag cagagtggtg tggtcttttt 660tttttacacg aaaatgccat agctggcccg catgcgtgca gatcggatga tcggtcggag 720acgacggaca atcagacact caccaactgc ttttgtctgg gacacaataa atgtttttgt 780aaacaaaata aatacttata aacgagggta ctagaggccg ctaacggcat ggccaggtaa 840acgcgctccc agccgttggt ttgcgatctc gtcctcccgc acgcagcgtc gcctccaccg 900tccgtccgtc gctgccacct ctgctgtgcg cgcgcacgaa gggaggaaga acgaacgccg 960cacacacact cacacacggc acactccccg tgggtcccct ttccggcttg gcgtctatct 1020cctctccccc gcccatcccc atgcactgca ccgtacccgc cagcttccac ccccgccgca 1080cacgttgctc ccccttctca tcgcttctca attaatatct ccatcactcg ggttccgcgc 1140tgcatttcgg ccggcgggtt gagtgagatc tgggcgactg gctgactcaa tcactacgcg 1200gggatggcga cgttcgcggt gtccggcgcg actctcggtg tggcgcgggc cggcgtcgga 1260gtggcgcggg ccggctcgga gcggaggggc ggggcggact tgccgtcgct gctcctcagg 1320aagaaggact cctctcgtac gcctcgctct ctcgaatctc ccccgtctgg ctttggctcc 1380ccttctctct cctctgcgcg cgcatggcct gttcgatgct gttccccaat tgatctccat 1440gagtgagaga gatagctgga ttaggcgatc gcgcttcctg aacctgtatt ttttcccccg 1500cggggaaatg cgttagtgtc acccaggccc tggtgttacc acggctttga tcattcctcg 1560tttcattctg atatatattt tctcattctt tttcttcctg ttcttgctgt aactgcaagt 1620tgtggcgttt tttcactatt gtagtcatcc ttgcattttg caggcgccgt cctgagccgc 1680gcggcctctc cagggaaggt cctggtgcct gacggcgaga gngacgactt ggcaagtccg 1740gcgcaacctg aagaattaca ggtacacaca ctcgtgccgg taaatcttca tacaatcgtt 1800attcacttac caaatgccgg atgaaaccaa ccacggatgc gtcaggtttc gagcttcttc 1860tatcagcatt gtgcagtact gcactgcctt gttcattttg ttagccttgg ccccgtgctg 1920gctcttgggc cactgaaaaa atcagatgga tgtgcattct agcaagaact tcacaacata 1980atgcaccgtt tggggtttcg tcagtctgct ctacaattgc tatttttcgt gctgtagata 2040cctgaagata tcgaggagca aacggcggaa gtgaacatga caggggggac tgcagagaaa 2100cttcaatctt cagaaccgac tcagggcatt gtggaaacaa tcactgatgg tgtaaccaaa 2160ggagttaagg aactagtcgt gggggagaaa ccgcgagttg tcccaaaacc aggagatggg 2220cagaaaatat acgagattga cccaacactg aaagattttc ggagccatct tgactaccgg 2280taatgcctac ccgctgcttt cgctcatttt gaattaaggt cctttcatca tgcaaatttg 2340gggaacatca aagagacaaa gactagggac caccatttca tacagatccc ttcgtggtct 2400gagaatatgc tgggaagtaa atgtataatt gatggctaca atttgctcaa aattgcaata 2460cgaataactg tctccgatca ttacaattaa agagtggcaa actgatgaaa atgtggtgga 2520tgggttatag attttacttt gctaattcct ctaccaaatt cctagggggg aaatctacca 2580gttgggaaac ttagtttctt atctttgtgg cctttttgtt ttggggaaaa cacattgcta 2640aattcgaatg attttgggta tacctcggtg gattcaacag atacagcgaa tacaagagaa 2700ttcgtgctgc tattgaccaa catgaaggtg gattggaagc attttctcgt ggttatgaaa 2760agcttggatt tacccgcagg taaatttaaa gctttattat tatgaaacgc ctccactagt 2820ctaattgcat atcttataag aaaatttata attcctgttt tcccctctct tttttccagt 2880gctgaaggta tcgtctaatt gcatatctta taagaaaatt tatattcctg ttttccccta 2940ttttccagtg ctgaaggtat cacttaccga gaatgggctc cctggagcgc atgttatgtt 3000cttttaagtt ccttaacgag acaccttcca atttattgtt aatggtcact attcaccaac 3060tagcttactg gacttacaaa ttagcttact gaatactgac cagttactat aaatttatga 3120tctggctttt gcaccctgtt acagtctgca gcattagtag gtgacttcaa caattggaat 3180ccaaatgcag atactatgac cagagtatgt ctacagcttg gcaattttcc acctttgctt 3240cataactact gatacatcta tttgtattta tttagctgtt tgcacattcc ttaaagttga 3300gcctcaacta catcatatca aaatggtata atttgtcagt gtcttaagct tcagcccaaa 3360gattctactg aatttagtcc atctttttga gattgaaaat gagtatatta aggatgaatg 3420aatacgtgca acactcccat ctgcattatg tgtgcttttc catctacaat gagcatattt 3480ccatgctatc agtgaaggtt tgctcctatt gatgcagata tttgatatgg tcttttcagg 3540atgattatgg tgtttgggag attttcctcc ctaacaacgc tgatggatcc tcagctattc 3600ctcatggctc acgtgtaaag gtaagctggc caattattta gtcgaggatg tagcattttc 3660gaactctgcc tactaagggt cccttttcct ctctgttttt tagatacgga tggatactcc 3720atccggtgtg aaggattcaa tttctgcttg gatcaagttc tctgtgcagg ctccaggtga 3780aatacctttc aatggcatat attatgatcc acctgaagag gtaagtatcg atctacatta 3840cattattaaa tgaaatttcc agtgttacag ttttttaata cccacttctt actgacatgt 3900gagtcaagac aatacttttg aatttggaag tgacatatgc attaattcac cttctaaggg 3960ctaaggggca accaaccttg gtgatgtgtg tatgcttgtg tgtgacataa gatcttatag 4020ctcttttatg tgttctctgt tggttaggat attccatttt ggccttttgt gaccatttac 4080taaggatatt tacatgcaaa tgcaggagaa gtatgtcttc caacatctca actaaacgac 4140cagagtcact aaggatttat gaatcacaca ttggaatgag cagcccggta tgtcaataag 4200ttatttcacc tgtttctggt ctgatggttt attctatgga ttttctagtt ctgttatgta 4260ctgttaacat attacatggt gcattcactt gacaacctcg attttatttt ctaatgtctt 4320catattggca agtgcaaaac tttgcttcct ctttgtctgc ttgttctttt gtcttctgta 4380agatttccat tgcatttgga ggcagtgggc atgtgaaagt catatctatt ttttttttgt 4440cagagcatag ttatatgaat tccattgttg ttgcaatagc tcggtataat gtaaccatgt 4500tactagctta agatttccca cttaggatgt aagaaatatt gcattggagc gtctccagca 4560agccatttcc taccttatta atgagagaga gacaaggggg gggggggggg gggggttccc 4620ttcattattc tgcgagcgat tcaaaaactt ccattgttct gaggtgtacg tactgcaggg 4680atctcccatt atgaagagga tatagttaat tctttgtaac ctacttggaa acttgagtct 4740tgaggcatcg ctaatatata ctatcatcac aatacttaga ggatgcatct gaanatttta 4800gtgtgatctt gcacaggaac cgaagataaa ttcatatgct aattttaggg atgaggtgtt 4860gccaagaatt aaaaggcttg gatacaatgc agtgcagata atggcaatcc aggagcattc 4920atactatgca agctttgggt attcacacaa tccatttttt tctgtataca cntcttcacc 4980catttggagc tattacatcc taatgcttca tgcacataaa atatttggat ataatccttt 5040attagatata tagtacaact acacttagta ttctgannaa naagatcatt ttattgttgt 5100tggcttgttc caggtaccat gttactaatt tttttgcacc aagtagccgt tttggaactc 5160cagaggactt aaaatccttg atcgatagag cacatgagct tggtttgctt gttcttatgg 5220atattgttca taggtaatta gtccaattta attttagctg ttttactgtt tatctggtat 5280tctaaaggga aattcaggca attatgatac attgtcaaaa gctaagagtg gcgaaagtga 5340aatgtcaaaa tctagagtgg cataaggaaa attggcaaaa actagagtgg caaaaataaa 5400attttcccat cctaaatggc agggccctat cgccgaatat ttttccattc tatataattg 5460tgctacgtga cttctttttt ctcagatgta ttaaaccagt tggacatgaa atgtatttgg 5520tacatgtagt aaactgacag ttccatagaa tatcgttttg taatggcaac acaatttgat 5580gccatagatg tggattgaga agttcagatg ctatcaatag aattaatcaa ctggccatgt 5640actcgtggca ctacatatag tttgcaagtt ggaaaactga cagcaatacc tcactgataa 5700gtggccaggc cccacttgcc agcttcatac tagatgttac ttccctgttg aattcatttg 5760aacatattac ttaaagttct tcatttgtcc taagtcaaac ttctttaagt ttgaccaagt 5820ctattggaaa atatatcaac atctacaaca ccaaattact ttgatcagat taacaatttt 5880tattttatta tattagcaca tctttgatgt tgtagatatc agcacatttt tctatagact 5940tggtcaaata tagagaagtt tgacttagga caaatctaga acttcaatca atttggatca 6000gagggaacat caaataatat agatagatgt caacacttca acaaaaaaat cagaccttgt 6060caccatatat gcatcagacc atctgtttgc tttagccact tgctttcata tttatgtgtt 6120tgtacctaat ctacttttcc ttctacttgg tttggttgat tctatttcag ttgcattgct 6180tcatcaatga ttttgtgtac cctgcagtca ttcgtcaaat aatacccttg acggtttgaa 6240tggtttcgat ggcactgata cacattactt ccacggtggt ccacgcggcc atcattggat 6300gtgggattct cgtctattca actatgggag ttgggaagta tgtagctctg acttctgtca 6360ccatatttgg ctaactgttc ctgttaatct gttcttacac atgttgatat tctattctta 6420tgcaggtatt gagattctta ctgtcaaacg cgagatggtg gcttgaagaa tataagtttg 6480atggatttcg atttgatggg gtgacctcca tgatgtatac tcaccatgga ttacaagtaa 6540gtcatcaagt ggtttcagta acttttttag ggcactgaaa caattgctat gcatcataac 6600atgtatcatg atcaggactt gtgctacgga gtcttagata gttccctagt atgcttgtac 6660aattttacct gatgagatca tggaagattg gaagtgatta ttatttattt tctttctaag 6720tttgtttctt gttctagatg acatttactg ggaactatgg cgaatatttt ggatttgcta 6780ctgatgttga tgcggtagtt tacttgatgc tggtcaacga tctaattcat ggactttatc 6840ctgatgctgt atccattggt gaagatgtaa gtgcttacag tatttatgat ttttaactag 6900ttaagtagtt ttattttggg gatcagtctg ttacactttt tgttaggggt aaaatctctc 6960ttttcataac aatgctaatt tataccttgt atgataatgc atcacttang taatttgaaa 7020agtgcaaggg cattcaagct tacgagcata ttttttgatg gctgtaattt atttgatagt 7080atgcttgttt gggtttttca ataagtggga gtgtgtgact aatgttgtat tatttattta 7140attgcggaag aaatgggcaa ccttgtcaat tgcttcagaa ggctaacttt gattccataa 7200acgctttgga aatgagaggc tattcccaag gacatgaatt atacttcagt gtgttctgta 7260catgtatttg taatagtggt ttaacttaaa ttcctgcact gctatggaat ctcactgtat 7320gttgtnagtg tacacatcca caaacaagta atcctgagct ttcaactcat gagaaaatan 7380gangtccgct tctgccagca ttaactgttc acagttctaa tttgtgtaac tgtgaaattg 7440ttcaggtcag tggaatgcct acattttgca tccctgttcc agatggtggt gttggttttg 7500actaccgcct gcatatggct gtagcagata aatggattga actcctcaag taagtgcagg 7560aatattggtg attacatgcg cacaatgatc tagattacat tttctaaatg gtaaaaagga 7620aaatatgtat gtgaatatct agacatttgc ctgttatcag cttgaatacg agaagtcaaa 7680tacatgattt aaatagcaaa tctcggaaat gtaatggcta gtgtctttat gctgggcagt 7740gtacattgcg ctgtagcagg ccagtcaaca cagttagcaa tattttcaga aacaatatta 7800tttatatccg tatatganga aagttagtat ataaactgtg gtcattaatt gtgttcacct 7860tttgtcctgt ttaaggatgg gcagtaggta ataaatttag ccagataaaa taaatcgtta 7920ttaggtttac aaaaggaata tacagggtca tgtagcatat ctagttgtaa ttaatgaaaa 7980ggctgacaaa aggctcggta aaaaaaactt tatgatgatc cagatagata tgcaggaacg 8040cgactaaagc tcaaatactt attgctacta cacagctgcc aatctgtcat gatctgtgtt 8100ctgctttgtg ctatttagat ttaaatacta actcgataca ttggcaataa taaacttaac 8160tattcaacca atttggtgga taccaganat ttctgccctc ttgttagtaa tgatgtgctc 8220cctgctgctg ttctctgccg ttacaaaagc tgttttcagt tttttgcatc attatttttg 8280tgtgtgagta gtttaagcat gttttttgaa gctgtgagct gttggtactt aatacattct 8340tggaagtgtc caaatatgct gcagtgtaat ttagcatttc tttaacacag gcaaagtgac 8400gaatcttgga aaatgggcga tattgtgcac accctaacaa atagaaggtg gcttgagaag 8460tgtgtaactt atgcagaaag tcatgatcaa gcactagttg gtgacaagac tattgcattc 8520tggttgatgg ataaggtact agctgttact tttggacaaa agaattactc cctcccgttc 8580ctaaatataa gtctttgtag agattccact atggaccaca tagtatatag atgcatttta 8640gagtgtagat tcactcattt tgcttcgtat gtagtccata gtgaaatctc tacagagact 8700tatatttagg aacggaggga gtacataatt gatttgtctc atcagattgc tagtgttttc 8760ttgtgataaa gattggctgc ctcacccatc accagctatt tcccaactgt tacttgagca 8820gaatttgctg aaaacgtacc atgtggtact gtggcggctt gtgaactttg acagttatgt 8880tgcaattttc tgttcttatt tatttgattg cttatgttac cgttcatttg ctcattcctt 8940tccgagacca gccaaagtca cgtgttagct gtgtgatctg ttatctgaat cttgagcaaa 9000ttttattaat aggctaaaat ccaacgaatt atttgcttga atttaaatat acagacgtat 9060agtcacctgg ctctttctta gatgattacc atagtgcctg aaggctgaaa tagttttggt 9120gtttcttgga tgccgcctaa aggagtgatt tttattggat agattcctgg ccgagtcttc 9180gttacaacat aacattttgg agatatgctt agtaacagct ctgggaagtt tggtcacaag 9240tctgcatcta cacgctcctt gaggttttat tatggcgcca tctttgtaac tagtggcacc 9300tgtaaggaaa cacattcaaa aggaaacggt cacatcattc taatcaggac caccatacta 9360agagcaagat tctgttccaa ttttatgagt ttttgggact ccaaagggaa caaaagtgtc 9420tcatattgtg cttataacta cagttgtttt tataccagtg tagttttatt ccaggacagt 9480tgatacttgg tactgtgctg taaattattt atccgacata gaacagcatg aacatatcaa 9540gctctctttg tgcaggatat gtatgatttc atggctctgg ataggcttca actcttcgca 9600ttgatcgtgg catagcatta cataaaatga tcaggcttgt caccatgggt ttaggtggtg 9660aaggctatct taacttcatg ggaaatgagt ttgggcatcc tggtcagtct ttacaacatt 9720attgcattct gcatgattgt gatttactgt aatttgaacc atgcttttct ttcacattgt 9780atgtattatg taatctgttg cttccaagga ggaagttaac ttctatttac ttggcagaat 9840ggatagattt tccaagaggc ccacaaactc ttccaaccgg caaagttctc ccctggaaat 9900aacaatagtt atgataaatg ccgccgtaga tttgatcttg taagttttag ctgtgctatt 9960acattccctc actagatctt tattggccat ttatttcttg atgaaatcat aatgtttgtt 10020aggaaagatc aacattgctt ttgtagtttt gtagacgtta acataagtat gtgttgagag 10080ttgttgatca ttaaaaatat catgattttt tgcagggaga tgcagatttt cttagatatc 10140gtggtatgca agagttcgat caggcaatgc agcatcttga ggaaaaatat ggggtatgtc 10200actggtttgt ctttgttgca taacaagtca cagtttaacg tcagtctctt caagtggtaa 10260aaaaagtgta gaattaattc ctgtaatgag atgaaaactg tgcaaaggcg gagctggaat 10320tgcttttcac caaaactatt ttcttaagtg cttgtgtatt gatacatata ccagcactga 10380caatgtaact gcagtttatg acatctgagc accagtatgt ttcacggaaa catgaggaag 10440ataaggtgat catcctcnaa aagaggagat ttggtatttg ttttcaactt ccactggagc 10500aatagctttt ttgactaccg tgttgggtgt tccaagcctg ggaagtacaa ggtatgcttg 10560ccttttcatt gtccaccctt caccagtagg gttagtgggg gcttctacaa cttttaattc 10620cacatggata gagtttgttg gtcgtgcagc tatcaatata aagaataggg taatttgtaa 10680agaaaagaat ttgctcgagc tgttgtagcc ataggaaggt tgttcttaac agccccgaag 10740cacataccat tcattcatat tatctactta agtgtttgtt tcaatcttta tgctcagttg 10800gactcggtct aatactagaa ctattttccg aatctaccct aaccatccta gcagttttag 10860agcagcccca tttggacaat tggctgggtt tttgttagtt gtgacagttt ctgctatttc 10920ttaatcaggt ggccttggac tctgacgatg cactctttgg tggattcagc aggcttgatc 10980atgatgtcga ctacttcaca accgtaagtc tgggctcaag cgtcacttga ctcgtcttga 11040ctcaactgct tacaaatctg aatcaacttc ccaattgctg atgcccttgc aggaacatcc 11100gcatgacaac aggccgcgct ctttctcggt gtacactccg agcagaactg cggtcgtgta 11160tgcccttaca gagtaagaac cagcagcggc ttgttacaag gcaaagagag aactccagag 11220agctcgtgga tcgtgagcga agcgacgggc aacggcgcga ggctgctcca agcgccatga 11280ctgggagggg atcgtgcctc ttccccagat gccaggagga gcagatggat aggtagcttg 11340ttggtgagcg ctcgaaagaa aatggacggg cctgggtgtt tgttgtgctg cactgaaccc 11400tcctcctatc ttgcacattc ccggttgttt ttgtacatat aactaataat tgcccgtgcg 11460ctcaacgtga aaatcc 11476319DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 3atcacttacc gagaatggg 19420DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 4ctgcatttgg atttcaattg 2053962DNAAegilops tauschii 5ggatccgatc cggctgcggc ggcggcgacg ggatggctgc gccggcattc gcagtttccg 60cggcggggct ggcccggccg tcggctcctc gatccggcgg ggcagagcgg agggggcgcg 120gggtggagct gcagtcgcca tcgctgctct tcggccgcaa caagggcacc cgttcacccc 180gtaattattt gcgccacctt tctcactcac attctctcgt gtattctgtc gtgctcgccc 240ttcgccgacg acgcgtgccg attccgtatc gggctgcggt gttcagcgat cttacgtcgg 300ttccctcctg gtgtggtgat gtctgtaggt gccgtcggcg tcggaggttc tggatggcgc 360gtggtcatgc gcgcgggggg gccgtccggg

gaggtgatga tccctgacgg cggtagtggc 420ggaacaccgc cttccatcga cggtcccgtt cagttcgatt ctgatgatct gaaggtagtt 480ttttttttgc atcgatctga aggtacttga catatactac tgtattaccc tgagtaaata 540ctgccaccat atttttatgg ttcgcttgaa atacctgttt acttgctacg gttttcactt 600tcattgagac gtcggacgaa attcactgaa ttcctataat ttggtagaca ccgaaatata 660tactactcct tccgtcccat aatataagag cgtttttggc accttatatt atagggcgga 720gggagtacct tttaggtcaa aatattgtgg tagtttcaat tgtatacaag aattcaaata 780ttttttttaa aaaaaaatca actaattggt tgagtttcaa gtgaagcgtt ttggtccttt 840ggctgagatg taaaccgaaa tcactgaaat tcatagtagc cgaaacttta atagaactga 900aactcaaaat ctgctatccg gcgaaattct aaagatttgc ttatttcaca cgtaggttgc 960agtacaccct ctttctaatt tattggggaa ggggtattat tatcttgtta gtacctgcct 1020gcatgacaat tgaaatctaa gacaaaacac catatgcgag gcctacacac ggtaggttgg 1080tttacaacta tgtgtgccac agttcgtctg aactttttgt ccttcacatc gtgttaggtt 1140ccattcattg atgatgaaac aagcctacag gatggaggtg aagatagtat ttggtcttca 1200gagacaaatc aggttagtga agaaattgat gctgaagaca cgagcagaat ggacaaagaa 1260tcatctacga gggagaaatt acgcattctg ccaccaccgg gaaatggaca gcaaatatac 1320gagattgacc caacgctccg agactttaag taccatcttg agtatcggta tgcttcgctt 1380ctattgtgtg cactttaaaa acaatttaca gtctttgata agatgtgaat ggctgcttgc 1440tgtgacacga aactcttgaa gttcgtagtc actcttgtgt gttcatggtt ctgaggtaac 1500atggtaaccg aacaaaaata ggaaagtggc aagcactgca atgtgagcta ctgataacca 1560cccattgtaa ttgggtacac tgattaatat atatgtcttc atgggctcta ttttttttca 1620atatctatgc caattgaaca acaatgcttt gtggacgggt gttcttttac cctcttcttc 1680tatcaataga tgatatgcat actcatgcgt atcctacaaa aaattgaaca acaatgccac 1740tttcccccgt gttgcttttg taaggatgaa acacatatgt ccagatcaaa ctatactagc 1800agtctaactg tgccttaatg gatcaaaaac agatatagcc tatacaggag aatacgttca 1860gacattgatg aacacgaagg aggcatggat gtattttccc gcggttacga gaagtttgga 1920tttatgcgca ggtgaaattt cttgactaaa taactatgta tctacctttt ctttgtactc 1980tatcaacatt cctcttccca tgcagcgctg aaggtatcac ttaccgagaa tgggctcctg 2040gagcagatgt acgttcttct aaccatctga tcgtttacct gactatacta attctatctt 2100tcaactaatt gtgaataatt actgctcatc agctatccta aggttgggga ttttgcacct 2160cccagatgaa cagcatatta agtcgcacaa ctagcattat taagaactaa ctcctgcttc 2220caattgcagt ctgcagcatt agttggcgac ttcaacaatt gggatccaaa tgcagaccat 2280atgagcaaag tatgcatgta gtttcacaaa tatatcatat tttctttgta gatttttttt 2340tttagatcgg cttatctatt taaatgtggt tgaatataca ccttatatgt acgttgagct 2400gtaaatatag ttggaagtgt ttaggagtat taaattcact ggactctatt ctttcacttg 2460cctgttgcac gagcccatta ctagatatca atgttgatga tgcttttgtt gtatgaggtc 2520gaagtgaaac atgcatgtta cccttttata taagtaaggt tgcacatgta ttttttatga 2580tctaaacatt atttactgat tttgttcttg caagacacta agcagtttta cataataatg 2640gcgttggagc aggccgactg cacatctgaa ctgtagctcc atgtggttga tatagattac 2700aaatgctcat attcaatgta actgttttca gaatgacctt ggtgtttggg agatttttct 2760gccaaacaat gcagatggtt cgccaccaat tcctcacggc tcacgggtga aggttgtttt 2820cttctccttg ccaacggtgt taggctcagg aacatgtcct gtattactca gaagctcttt 2880tgaacatcta ggtgagaatg gatactccat ctgggataaa ggattcaatt cctgcttgga 2940tcaagtactc cgtgcagact ccaggagata taccatacaa tggaatatat tatgatcctc 3000ccgaagaggt attttacttc atcttctgtg cttttagatt tcagatattt ttattagaag 3060aaaattatga ttttttccct cacgaacctt cccaattgct atttcaagct gtcctactta 3120tttgctgctg gcatcttatt tttctattct ctaaccagtt atgaaattcc ttacatgcat 3180atgcaggaga agtatgtatt caagcatcct caacctaaac gaccaaaatc attgcggata 3240tatgaaacac atgttggcat gagtagcccg gtatttcatc tttaccatgt attccataaa 3300tgaagttagc tatatgcagt tcaaatttat ttacaggttg ttacaatggt atttttgtgt 3360tggtgccctt ctttcgtttt ataagtaaaa aacttatcat aaatttattt gttatgccgc 3420ttggttaata caatctgaaa aatgtaactg tggacaatct agaactagat aatacaaatc 3480tgaaaaaaca tgctggaata gtgtcatttc agtcaactag gatgttttga atgctcaaga 3540gaagtactag tgtgtagcat caaaagctgg tgtccatttg ttcaaatgtt taattaacac 3600tatagtgaaa acaagtaatt gcacaaagaa acaagtaatt gcccaagttc atatgttttt 3660tcactatatt acatgtttca tcaacaattt aattaacctc attccttaca aacatttgta 3720tttacatttg ttcctacata tatagttatt ttatatatca actttataaa tcatgactgt 3780tataattaaa accgatggta tatcaacgat tgagataatt tggcatatgt ggatgaattt 3840tgtggcttgt tatgctcttg ttttaataac ataataaata gattatgctt gttggtagcc 3900tttttacatt aacacatggg caattacttg tttctttgtg caaccaggaa ccaaagatcg 3960ag 396262968DNATriticum sp.modified_base(35)..(35)a, c, g or t 6atggtcgacc tgcaggcggc cgcgaatgca ctagngattt tgacaccaga ccaactggta 60atggtagcga ccggcgctca gctggaattc gcggccgcgt cgaccgtggg tttaagcagg 120agacgaggcg gggtcagttg ggcagttagg ttggatccga tccggctgcg gcggcggcga 180cgggatggct gcgccggcat tcgcagtttc cgcggcgggg ctggcccggc cgtcggctcc 240tcgatccggc ggggcagagc ggagggggcg cggggtggag ctgcagtcgc catcgctgct 300cttcggccgc aacaagggca cccgttcacc ccgtgccgtc ggcgtcggag gttctggatg 360gcgcgtggtc atgcgcgcgg gggggccgtc cggggaggtg atgatccctg acggcggtag 420tggcggaaca ccgccttcca tcgacggtcc cgttcagttc gattctgatg atctgaaggt 480tccattcatt gatgatgaaa caagcctaca ggatggaggt gaagatagta tttggtcttc 540agagacaaat caggttagtg aagaaattga tgctgaagac acgagcagaa tggacaaaga 600atcatctacg agggagaaat tacgcattct gccaccaccg ggaaatggac agcaaatata 660cgagattgac ccaacgctcc gagactttaa gtaccatctt gagtatcgat atagcctata 720caggagaata cgttcagaca ttgatgaaca cgaaggaggc atggatgtat tttcccgcgg 780ttacgagaag tttggattta tgcgcagcgc tgaaggtatc acttaccgag aatgggctcc 840tggagcagat tctgcagcat tagttggcga cttcaacaat tgggatccaa atgcagacca 900tatgagcaaa aatgaccttg gtgtttggga gatttttctg ccaaacaatg cagatggttc 960gccaccaatt cctcacggct cacgggtgaa ggtgcgaatg ggtactccat ctgggacaaa 1020ggattcaatt cctgcttgga tcaagtactc cgtgcagact ccaggagata taccatacaa 1080tggaatatat tatgatcctc ccgaagagga gaagtatgta ttcaagcatc ctcaacctaa 1140acgaccaaaa tcattgcgga tatatgaaac acatgttggc atgagtagcc cggaaccaaa 1200gatcaacaca tatgcaaact tcagggatga ggtgcttcca agaattaaaa gacttggata 1260caatgcagtg caaataatgg caatccaaga gcactcatac tatggaagct ttgggtacca 1320tgttaccaat ttctttgcac caagtagccg ttttgggtcc ccagaagatt taaaatcttt 1380gattgataga gctcacgagc ttggcttggt tgtcctcatg gatgttgttc acagtcacgc 1440gtcaaataat accttggacg ggttgaatgg ttttgatggc acggatacac attacttcca 1500tggcggttca cggggccatc actggatgtg ggattcccgt gtgtttaact atgggaataa 1560ggaagttata aggtttctac tttccaatgc aagatggtgg ctagaggagt ataagtttga 1620tggtttccga ttcgatggcg cgacctccat gatgtatacc catcatggat tacaagtaac 1680ctttacagga agctaccatg aatattttgg ctttgccact gatgtagatg cggtcgttta 1740cttgatgctg atgaatgatc taattcatgg gttttatcct gaagccgtaa ctatcggtga 1800agatgttagt ggaatgccta catttgccct tcctgttcaa gttggtgggg ttggttttga 1860ctatcgctta catatggctg ttgcccgcaa atggattgaa cttctcaaag gaaacgatga 1920agcttgggag atgggtaata ttgtgcacac actaacaaac agaaggtggc tggaaaagtg 1980tgttacttat gctgaaagtc acgatcaagc acttgttgga gacaagacta ttgcattctg 2040gttgatggac aaggatatgt atgatttcat ggcgctgaac ggaccttcga cgcctaatat 2100tgatcgtgga atagcactgc ataaaatgat tagacttatc acaatgggtc taggaggaga 2160gggttatctt aactttatgg gaaatgagtt cgggcatcct gaatggatag actttccaag 2220aggcccacaa gtacttccaa gtggtaagtt catcccagga aacaacaaca gttacgacaa 2280atgccgtcga agatttgacc tgggtgatgc agaatttctt aggtatcatg gtatgcagca 2340gtttgatcag gcaatgcagc atcttgagga aaaatatggt tttatgacat cagaccacca 2400gtacgtatct cggaaacatg aggaagataa ggtgatcgtg tttgaaaaag gggacttggt 2460atttgtgttc aacttccact ggagtagtag ctatttcgac taccgggtcg gctgtttaaa 2520gcctgggaag tacaaggtgg tcttagactc ggacgctgga ctctttggtg gatttggtag 2580gatccatcac actgcagagc acttcacttc tgactgccaa catgacaaca ggccccattc 2640attctcagtg tacactccta gcagaacctg tgttgtctat gctccaatga actaacagca 2700aagtgcagca tacgcgtgcg cgctgttgtt gctagtagca agaaaaatcg tatggtcaat 2760acaaccaggt gcaaggttta ataaggattt ttgcttcaac gagtcctgga tagacaagac 2820aacatgatgt tgtgctgtgt gctcccaatc cccagggcgt tgtgaagaaa acatgctcat 2880ctgtgttatt ttatggatca gcgacgaaac ctcccccaaa tacccctttt ttttttnaaa 2940ggaggatagg cccccggnct ttgcntnn 2968718PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 7Ala Ala Ser Pro Gly Lys Val Leu Val Pro Asp Glu Ser Asp Asp Leu1 5 10 15Gly Cys812PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 8Ala Gly Gly Pro Ser Gly Glu Val Met Ile Gly Cys1 5 10917PRTArtificial SequenceDescription of Artificial Sequence Synthetic peptide 9Gly Gly Thr Pro Pro Ser Ile Asp Gly Pro Val Gln Asp Ser Asp Gly1 5 10 15Cys108381DNATriticum sp.modified_base(4636)..(4636)a, c, g or t 10aagctttgta gccttgcacg ggctccccaa caaactgcct cactcgattg tcaaaaaagt 60aaaaatgatt gtagaaaaaa aaactgactc actcgtcact accctaccgt cctacatgac 120acctggccgc aagacgacgc cgtcctcctg ccgcgcgcgt ccgcgatcac accaccgcaa 180aaaccaaaac ctcttcgccg gtgcgtccca cgctaccatc catgcagccg tccgcccgcg 240cgcgcgttgc ccgcaccacc cgctggcggc caccacgccg ccactctcgc gtgaaggctc 300cgtccgcttc ctcctagttc cactctctct ccgtgctagc agtatatagc atccgccctc 360cgccccctcc caatcttaga acacccctcc ctttgcctcc tcatttcgct cgcgtgggtt 420taagcaggag acgaggcggg gtcagttggg cagttaggtt ggatccgatc cggctgcggc 480ggcggcgacg ggatggctgc gccggcattc gcagtttccg cggcggggct ggcccggccg 540tcggctcctc gatccggcgg ggcagagcgg agggggcgcg gggtggagct gcagtcgcca 600tcgctgctct tcggccgcaa caagggcacc cgttcacccc gtaattattt gcgccacctt 660tctcactcac attctctcgt gtattctgtc gtgctcgccc ttcgccgacg acgcgtgccg 720attccgtatc gggctgcggt gttcagcgat cttacgtcgg ttccctcctg gtgtggtgat 780gtctgtaggt gccgtcggcg tcggaggttc tggatggcgc gtggtcatgc gcgcgggggg 840gccgtccggg gaggtgatga tccctgacgg cggtagtggc ggaacaccgc cttccatcga 900cggtcccgtt cagttcgatt ctgatgatct gaaggtagtt ttttttttgc atcgatctga 960aggtacttga catatactac tgtattaccc tgagtaaata ctgccaccat atttttatgg 1020ttcgcttgaa atacctgttt acttgctacg gttttcactt tcattgagac gtcggacgaa 1080attcactgaa ttcctataat ttggtagaca ccgaaatata tactactcct tccgtcccat 1140aatataagag cgtttttggc accttatatt atagggcgga gggagtacct tttaggtcaa 1200aatattgtgg tagtttcaat tgtatacaag aattcaaata ttttttttaa aaaaaaatca 1260actaattggt tgagtttcaa gtgaagcgtt ttggtccttt ggctgagatg taaaccgaaa 1320tcactgaaat tcatagtagc cgaaacttta atagaactga aactcaaaat ctgctatccg 1380gcgaaattct aaagatttgc ttatttcaca cgtaggttgc agtacaccct ctttctaatt 1440tattggggaa ggggtattat tatcttgtta gtacctgcct gcatgacaat tgaaatctaa 1500gacaaaacac catatgcgag gcctacacac ggtaggttgg tttacaacta tgtgtgccac 1560agttcgtctg aactttttgt ccttcacatc gtgttaggtt ccattcattg atgatgaaac 1620aagcctacag gatggaggtg aagatagtat ttggtcttca gagacaaatc aggttagtga 1680agaaattgat gctgaagaca cgagcagaat ggacaaagaa tcatctacga gggagaaatt 1740acgcattctg ccaccaccgg gaaatggaca gcaaatatac gagattgacc caacgctccg 1800agactttaag taccatcttg agtatcggta tgcttcgctt ctattgtgtg cactttaaaa 1860acaatttaca gtctttgata agatgtgaat ggctgcttgc tgtgacacga aactcttgaa 1920gttcgtagtc actcttgtgt gttcatggtt ctgaggtaac atggtaaccg aacaaaaata 1980ggaaagtggc aagcactgca atgtgagcta ctgataacca cccattgtaa ttgggtacac 2040tgattaatat atatgtcttc atgggctcta ttttttttca atatctatgc caattgaaca 2100acaatgcttt gtggacgggt gttcttttac cctcttcttc tatcaataga tgatatgcat 2160actcatgcgt atcctacaaa aaattgaaca acaatgccac tttcccccgt gttgcttttg 2220taaggatgaa acacatatgt ccagatcaaa ctatactagc agtctaactg tgccttaatg 2280gatcaaaaac agatatagcc tatacaggag aatacgttca gacattgatg aacacgaagg 2340aggcatggat gtattttccc gcggttacga gaagtttgga tttatgcgca ggtgaaattt 2400cttgactaaa taactatgta tctacctttt ctttgtactc tatcaacatt cctcttccca 2460tgcagcgctg aaggtatcac ttaccgagaa tgggctcctg gagcagatgt acgttcttct 2520aaccatctga tcgtttacct gactatacta attctatctt tcaactaatt gtgaataatt 2580actgctcatc agctatccta aggttgggga ttttgcacct cccagatgaa cagcatatta 2640agtcgcacaa ctagcattat taagaactaa ctcctgcttc caattgcagt ctgcagcatt 2700agttggcgac ttcaacaatt gggatccaaa tgcagaccat atgagcaaag tatgcatgta 2760gtttcacaaa tatatcatat tttctttgta gatttttttt tttagatcgg cttatctatt 2820taaatgtggt tgaatataca ccttatatgt acgttgagct gtaaatatag ttggaagtgt 2880ttaggagtat taaattcact ggactctatt ctttcacttg cctgttgcac gagcccatta 2940ctagatatca atgttgatga tgcttttgtt gtatgaggtc gaagtgaaac atgcatgtta 3000cccttttata taagtaaggt tgcacatgta ttttttatga tctaaacatt atttactgat 3060tttgttcttg caagacacta agcagtttta cataataatg gcgttggagc aggccgactg 3120cacatctgaa ctgtagctcc atgtggttga tatagattac aaatgctcat attcaatgta 3180actgttttca gaatgacctt ggtgtttggg agatttttct gccaaacaat gcagatggtt 3240cgccaccaat tcctcacggc tcacgggtga aggttgtttt cttctccttg ccaacggtgt 3300taggctcagg aacatgtcct gtattactca gaagctcttt tgaacatcta ggtgagaatg 3360gatactccat ctgggataaa ggattcaatt cctgcttgga tcaagtactc cgtgcagact 3420ccaggagata taccatacaa tggaatatat tatgatcctc ccgaagaggt attttacttc 3480atcttctgtg cttttagatt tcagatattt ttattagaag aaaattatga ttttttccct 3540cacgaacctt cccaattgct atttcaagct gtcctactta tttgctgctg gcatcttatt 3600tttctattct ctaaccagtt atgaaattcc ttacatgcat atgcaggaga agtatgtatt 3660caagcatcct caacctaaac gaccaaaatc attgcggata tatgaaacac atgttggcat 3720gagtagcccg gtatttcatc tttaccatgt attccataaa tgaagttagc tatatgcagt 3780tcaaatttat ttacaggttg ttacaatggt atttttgtgt tggtgccctt ctttcgtttt 3840ataagtaaaa aacttatcat aaatttattt gttatgccgc ttggttaata caatctgaaa 3900aatgtaactg tggacaatct agaactagat aatacaaatc tgaaaaaaca tgctggaata 3960gtgtcatttc agtcaactag gatgttttga atgctcaaga gaagtactag tgtgtagcat 4020caaaagctgg tgtccatttg ttcaaatgtt taattaacac tatagtgaaa acaagtaatt 4080gcacaaagaa acaagtaatt gcccaagttc atatgttttt tcactatatt acatgtttca 4140tcaacaattt aattaacctc attccttaca aacatttgta tttacatttg ttcctacata 4200tatagttatt ttatatatca actttataaa tcatgactgt tataattaaa accgatggta 4260tatcaacgat tgagataatt tggcatatgt ggatgaattt tgtggcttgt tatgctcttg 4320ttttaataac ataataaata gattatgctt gttggtagcc tttttacatt aacacatggg 4380caattacttg tttctttgtg caaccaggaa ccaaagatcg acacatatgc aaacttcagg 4440gatgaggtgc ttccaagaat taaaagactt ggatacaatg cagtgcaaat aatggcaatc 4500caagagcact catactatgg aagctttggg tagttctctg ggtcgatttc tggttctttt 4560agttatcttt tgtccataga acatatttca actttagcaa ctatactatt atattaactt 4620ttcagctatt gtcttncttt ttcttatgtg agagactgct gcntcttgct acttcctgtg 4680ttctcattca gagtanacat cttatganta gacaactcta tgtngacatt ccggaagtat 4740ncactggctg attcggtcta aaataacata ctgctcagat agccacataa cagtacgatt 4800acacacataa tgaccatgtt tgcatagagt ggcggtagta tgttcctcac catactagca 4860taatgacttg ttatataaga gtatatcata ttaacttctt ttccaatgac atggaagctg 4920taacaacttt caaatcattt ttgtctttta agtgctgctt ttttcctgtt tgacaattaa 4980tacaatacca cttttatgtg tttttacttc tattgcaggt accatgttac caatttcttt 5040gcaccaagta gccgttttgg gtccccagaa gatttaaaat ctttgattga tagagctcac 5100gagcttggct tggttgtcct catggatgtt gttcacaggt acttaatgta atttgaggtt 5160ggcgtgttaa gttcacatta atcttaattc tttatttcaa ttcctatggc ctctctccta 5220gattggaaca gtaaaagcat catccagttt gtataaattg ctaaaagaac attttacatg 5280ttaagtattt tcaattacta tgaaacatat aaatttacat acttattgat tttacgacag 5340aagtaccgat ctcacaagat gaacaattgg ttgatcacat atcatttcat actacaatac 5400aagaaaatga atagagaacg agttaatatt agccttggta aaatcagcaa cttgtttgga 5460aataaagtat agtgatgcca gtgcaaanaa caaggcatca agttggtttc agctcccacg 5520gtcggtgcta gctgtcaagg gtaatttgca cgtagtcgca catagatttg tgtgggagtg 5580gaaagtaacc acagattgtc cgaggaacac gggacacacg tcttagccac aggtttgggc 5640tccccttgat gcgggtagta gctttactcc ttatatgaaa ttatctcaag atagatttca 5700atttggggtt acacttanga actcancaag ttaaggatca actcnctgag ttctatacga 5760ctgatctttg accgagatat cttgatcagg ctaagtanca aaatccaggc cttgagatgt 5820tgaacatgtc cttcattttg ggctgggtgc ccttgggcat aaggtgtngt ccttccttca 5880tgtgcttctt gcagcgtatg acataaacnt cctctgagtt ggtanatgca cggttccctt 5940tgaggaaatc aggggtagtc gcatctnggg aaagttggtc acccangcat ggatcctcng 6000cgcacaccgg gcaaacacgg tgaaaccact tctcctcgac actagctaac ttgacattca 6060agcaaactaa gaatataact ttatntctaa atgaaccgga caccctcctt gtgcctgcac 6120ctacagagta caatgccagt tttggactga actcttgtgt tcatgtatgt gctaatnaca 6180taggttctaa ccatgattct aaatagcgcg ttataactcc actatagtaa tgctatagcg 6240tttanaagat cccgcactaa gggaccttag tccaaataca tgatcaaaca ttttacatag 6300cgcgctatag ctatttaaaa ctatggtcac ccgctaagag gcataactcg ctatttaaaa 6360ctatggttct aacttttaat ctattttatg tcttggtcca aagccccttt ttgttctata 6420gctttacctt tgggttgaga tcacccttaa cccattggta atcctggttg atttactcca 6480tcctttcttg cgtagcttta cttttggttt tttgtttctc acagtcacgc gtcaaataat 6540accttggacg ggttgaatgg ttttgatggc acggatacac attacttcca tggcggttca 6600cggggccatc actggatgtg ggattcccgt gtgtttaact atgggaataa ggaagtatgg 6660gactatagaa tttctattgc catttgttat gtatttatcc attaattaat cctccaaccg 6720atattccaac attgttatct ttatacaggt tataaggttt ctactttcca atgcaagatg 6780gtggctagag gagtataagt ttgatggttt ccgattcgat ggcgcgacct ccatgatgta 6840tacccatcat ggattacaag taattcattg cttgattgtc tttgttctat cttgactacc 6900tgtgcaactt taataagatt acgcctagct aatattttct tttatgttat agtatcaatt 6960tttatttgag cttgaaacct aaattacttt ttttttgaat tgctgcgctc tattttaggt 7020aacctttaca ggaagctacc atgaatattt tggctttgcc actgatgtag atgcggtcgt 7080ttacttgatg ctgatgaatg atctaattca tgggttttat cctgaagccg taactatcgg 7140tgaagatgta agtgtttcta tagtcatctt tcaatatgaa tttgttagaa ctattggtac 7200ttatcttttt tgtagtttag gctattctgt tcattcttac aggaggtgca tacagaagtt 7260gctttagatt ttgaaacgca gtgcacattg tgccattact ttgtagctat atcgagttga 7320gacttgagag ccatggtaat caagttcctg acgtggcatt gcattagata gttgcatgtc 7380taagttcctg acgtggagat agaagaaaga acgcaccccc cgcgtcgctc ctctcagggc 7440gacacgggcg gagccctcac ccccgccgcc acagggagca tccacccttc tcctctcccc 7500tcgccgccgc cggagggcaa agaccgcgcg gcgtcgcggc ggtgggtgcg gcctgggctg 7560gcatctggca gcggcgattt ggcctcccct gcccagaact gtgctgccgc ggtttgtggc 7620agcttgggca tcggcagtgg cccgagtctg cggtggcggc gtgtctggcg tccggaggtg 7680cagcgattgt gcggttgtgt ggctcaggct cggagggcgt gcgggctgcc aggtccggcc 7740agatctggcc tcgagtggct tcgtacgggg cggtggctgt tgcgggtccg tgggccgagg 7800ttcgggtgtg gctgctgctt

gcccggaccg gtggtgcgta acgatgccgg agcagcgtcc 7860tcgggtcgtt gaagtgggcg ctcctccggc agcttcaggt ggtgattcgt cgcagcgggt 7920ggtgcactgg gggtctcggc tgattgtggt gccatggtgg tggtggttgt tggcggtagc 7980aaagtgcctg gtgcacacgg ctanggtttt gcggatggac agacttgatg caatgcctta 8040gggcatagtg aatttcagct aagtacctag caccgacctt ggtcaatgcc accgccgctg 8100gtgtcttagg acgttgttgc ccttgttgga ggcgtgttgt ggagcccctt cacctccatg 8160ggcatttaga tctcgagctc tntgggtgaa aacgccggct ttggctttgg ccggagtggg 8220cggtggcggc gtaaccgtcg ctccccccat gggggtgtag tcttggaggt ctagactcga 8280ggaattcggt accccgggtt cgaaatcgat aagcttggat ccgaagagct cccaacgcgt 8340tggatgcata gcttgagtat tctatagtgt cacctaaata g 83811123DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 11ccgtcctaca tgacacctgg ccg 231223DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 12ccgccggatc gaggagccga cgg 231323DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 13ggcggcggcg acgggatggc tgc 231423DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 14cgccgtcagg gatcatcacc tcc 231524DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 15cacccattgt aattgggtac actg 241624DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 16tccatgcctc cttcgtgttc atca 241724DNAArtificial SequenceDescription of Artificial Sequence Synthetic primer 17ctgcgcataa atccaaactt ctcg 2418226DNATriticum sp. 18atcacttacc gagaatgggc tcctggagcg catgtatgtc ttttaagtct taacagacac 60cttccaattt attgttaatg gtcactattc accaactagc ttactggact tacaaattag 120cttactgaat actgaccagt tactataaat ttatgatctg gcttttgcac cctgttacag 180tctgcagcat tagtaggtga cttcaacaat tgggatccaa atgcag 22619228DNATriticum sp. 19atcacttacc gagaatgggc tcctggagcg catgtacgtc ttttaagtct taacagacac 60cttccaattc attgttaatg gtcacactat tcaccaacta gcttactgga cttacaactt 120agcttactga atactgacca gttgctctaa atttatgatc tggcttttgc atcctgttac 180agtctgcagc attagtaggt gacttcaaca attggaatcc aaatgcag 22820217DNATriticum sp. 20atcacttacc gagaatgggc tcctggagcg catgtacgtc ttaacagaca ccttctaatt 60tattgttaat ggtcactatt caccaactag cttactggac ttacaaaata gcttactgaa 120tactgaccag ttactctaaa tttatgatct ggcttttgga tcctgttaca gtctgcagca 180ttagtaggtg acttcaacaa ttggaatcca aatgcag 21721262DNATriticum sp.modified_base(26)..(26)a, c, g or t 21atcacttacc gagaatgggc tcctgngagc anatgtatgt tcttctgact gtctgatcgt 60ttacctgact atactaattc tatctttcaa ctgcttgtga ataattagtg ctcatctgct 120atcctaaggt tggggatttt gcacttccca gatgaacagc atattaagtt gcacaactan 180ctttatttaa gaactaactc ttgcttccaa ttgcagtctg caacattagt tggcgacttc 240aacaattgga atccaaatgc ag 26222348DNAHordeum vulgare 22tcgcagcgct gaaggtatca cttaccgaga atgggctcct ggagcagatt ctgcagcatt 60agttggcgac ttcaacaatt gggatccaac tgcagaccat atgagcaaaa atgacttggg 120tatttgggag atttttctgc caaacaatgc agatggttcg ccgccaattc ctcatggctc 180acgggtgaag gtgcggatgg atactccatc tgggacaaag gattcaattc ctgcttggat 240caagtactcc gtgcagactc caggagatat accatacaat ggaatatatt atgaccctcc 300tgaagaggag aagtatgtat tcaagcatcc tcaacctaaa cgaccaaa 34823823PRTTriticum sp. 23Met Ala Thr Phe Ala Val Ser Gly Ala Thr Leu Gly Val Ala Arg Pro1 5 10 15Ala Gly Ala Gly Gly Gly Leu Leu Pro Arg Ser Gly Ser Glu Arg Arg 20 25 30Gly Gly Val Asp Leu Pro Ser Leu Leu Leu Arg Lys Lys Asp Ser Ser 35 40 45Arg Ala Val Leu Ser Arg Ala Ala Ser Pro Gly Lys Val Leu Val Pro 50 55 60Asp Gly Glu Ser Asp Asp Leu Ala Ser Pro Ala Gln Pro Glu Glu Leu65 70 75 80Gln Ile Pro Glu Asp Ile Glu Glu Gln Thr Ala Glu Val Asn Met Thr 85 90 95Gly Gly Thr Ala Glu Lys Leu Glu Ser Ser Glu Pro Thr Gln Gly Ile 100 105 110Val Glu Thr Ile Thr Asp Gly Val Thr Lys Gly Val Lys Glu Leu Val 115 120 125Val Gly Glu Lys Pro Arg Val Val Pro Lys Pro Gly Asp Gly Gln Lys 130 135 140Ile Tyr Glu Ile Asp Pro Thr Leu Lys Asp Phe Arg Ser His Leu Asp145 150 155 160Tyr Arg Tyr Ser Glu Tyr Arg Arg Ile Arg Ala Ala Ile Asp Gln His 165 170 175Glu Gly Gly Leu Glu Ala Phe Ser Arg Gly Tyr Glu Lys Leu Gly Phe 180 185 190Thr Arg Ser Ala Glu Gly Ile Thr Tyr Arg Glu Trp Ala Pro Gly Ala 195 200 205His Ser Ala Ala Leu Val Gly Asp Phe Asn Asn Trp Asn Pro Asn Ala 210 215 220Asp Thr Met Thr Arg Asp Asp Tyr Gly Val Trp Glu Ile Phe Leu Pro225 230 235 240Asn Asn Ala Asp Gly Ser Pro Ala Ile Pro His Gly Ser Arg Val Lys 245 250 255Ile Arg Met Asp Thr Pro Ser Gly Val Lys Asp Ser Ile Ser Ala Trp 260 265 270Ile Lys Phe Ser Val Gln Ala Pro Gly Glu Ile Pro Phe Asn Gly Ile 275 280 285Tyr Tyr Asp Pro Pro Glu Glu Glu Lys Tyr Val Phe Gln His Pro Gln 290 295 300Pro Lys Arg Pro Glu Ser Leu Arg Ile Tyr Glu Ser His Ile Gly Met305 310 315 320Ser Ser Pro Glu Pro Lys Ile Asn Ser Tyr Ala Asn Phe Arg Asp Glu 325 330 335Val Leu Pro Arg Ile Lys Arg Leu Gly Tyr Asn Ala Val Gln Ile Met 340 345 350Ala Ile Gln Glu His Ser Tyr Tyr Ala Ser Phe Gly Tyr His Val Thr 355 360 365Asn Phe Phe Ala Pro Ser Ser Arg Phe Gly Thr Pro Glu Asp Leu Lys 370 375 380Ser Leu Ile Asp Arg Ala His Glu Leu Gly Leu Leu Val Leu Met Asp385 390 395 400Ile Val His Ser His Ser Ser Asn Asn Thr Leu Asp Gly Leu Asn Gly 405 410 415Phe Asp Gly Thr Asp Thr His Tyr Phe His Gly Gly Pro Arg Gly His 420 425 430His Trp Met Trp Asp Ser Arg Leu Phe Asn Tyr Gly Ser Trp Glu Val 435 440 445Leu Arg Phe Leu Leu Ser Asn Ala Arg Trp Trp Leu Glu Glu Tyr Lys 450 455 460Phe Asp Gly Phe Arg Phe Asp Gly Val Thr Ser Met Met Tyr Thr His465 470 475 480His Gly Leu Gln Met Thr Phe Thr Gly Asn Tyr Gly Glu Tyr Phe Gly 485 490 495Phe Ala Thr Asp Val Asp Ala Val Val Tyr Leu Met Leu Val Asn Asp 500 505 510Leu Ile His Gly Leu His Pro Asp Ala Val Ser Ile Gly Glu Asp Val 515 520 525Ser Gly Met Pro Thr Phe Cys Ile Pro Val Pro Asp Gly Gly Val Gly 530 535 540Phe Asp Tyr Arg Leu His Met Ala Val Ala Asp Lys Trp Ile Glu Leu545 550 555 560Leu Lys Gln Ser Asp Glu Ser Trp Lys Met Gly Asp Ile Val His Thr 565 570 575Leu Thr Asn Arg Arg Trp Leu Glu Lys Cys Val Thr Tyr Ala Glu Ser 580 585 590His Asp Gln Ala Leu Val Gly Asp Lys Thr Ile Ala Phe Trp Leu Met 595 600 605Asp Lys Asp Met Tyr Asp Phe Met Ala Leu Asp Arg Pro Ser Thr Pro 610 615 620Arg Ile Asp Arg Gly Ile Ala Leu His Lys Met Ile Arg Leu Val Thr625 630 635 640Met Gly Leu Gly Gly Glu Gly Tyr Leu Asn Phe Met Gly Asn Glu Phe 645 650 655Gly His Pro Glu Trp Ile Asp Phe Pro Arg Gly Pro Gln Thr Leu Pro 660 665 670Thr Gly Lys Val Leu Pro Gly Asn Asn Asn Ser Tyr Asp Lys Cys Arg 675 680 685Arg Arg Phe Asp Leu Gly Asp Ala Asp Phe Leu Arg Tyr His Gly Met 690 695 700Gln Glu Phe Asp Gln Ala Met Gln His Leu Glu Glu Lys Tyr Gly Phe705 710 715 720Met Thr Ser Glu His Gln Tyr Val Ser Arg Lys His Glu Glu Asp Lys 725 730 735Val Ile Ile Phe Glu Arg Gly Asp Leu Val Phe Val Phe Asn Phe His 740 745 750Trp Ser Asn Ser Phe Phe Asp Tyr Arg Val Gly Cys Ser Arg Pro Gly 755 760 765Lys Tyr Lys Val Ala Leu Asp Ser Asp Asp Ala Leu Phe Gly Gly Phe 770 775 780Ser Arg Leu Asp His Asp Val Asp Tyr Phe Thr Thr Glu His Pro His785 790 795 800Asp Asn Arg Pro Arg Ser Phe Ser Val Tyr Thr Pro Ser Arg Thr Ala 805 810 815Val Val Tyr Ala Leu Thr Glu 82024768PRTTriticum sp. 24Met Ala Thr Phe Ala Val Ser Gly Ala Thr Leu Gly Val Ala Arg Pro1 5 10 15Pro Ala Ala Ala Gln Pro Glu Glu Leu Gln Ile Pro Glu Asp Ile Glu 20 25 30Glu Gln Thr Ala Glu Val Asn Met Thr Gly Gly Thr Ala Glu Lys Leu 35 40 45Glu Ser Ser Glu Pro Thr Gln Gly Ile Val Glu Thr Ile Thr Asp Gly 50 55 60Val Thr Lys Gly Val Lys Glu Leu Val Val Gly Glu Lys Pro Arg Val65 70 75 80Val Pro Lys Pro Gly Asp Gly Gln Lys Ile Tyr Glu Ile Asp Pro Thr 85 90 95Leu Lys Asp Phe Arg Ser His Leu Asp Tyr Arg Tyr Ser Glu Tyr Arg 100 105 110Arg Ile Arg Ala Ala Ile Asp Gln His Glu Gly Gly Leu Glu Ala Phe 115 120 125Ser Arg Gly Tyr Glu Lys Leu Gly Phe Thr Arg Ser Ala Glu Gly Ile 130 135 140Thr Tyr Arg Glu Trp Ala Pro Gly Ala His Ser Ala Ala Leu Val Gly145 150 155 160Asp Phe Asn Asn Trp Asn Pro Asn Ala Asp Thr Met Thr Arg Asp Asp 165 170 175Tyr Gly Val Trp Glu Ile Phe Leu Pro Asn Asn Ala Asp Gly Ser Pro 180 185 190Ala Ile Pro His Gly Ser Arg Val Lys Ile Arg Met Asp Thr Pro Ser 195 200 205Gly Val Lys Asp Ser Ile Ser Ala Trp Ile Lys Phe Ser Val Gln Ala 210 215 220Pro Gly Glu Ile Pro Phe Asn Gly Ile Tyr Tyr Asp Pro Pro Glu Glu225 230 235 240Glu Lys Tyr Val Phe Gln His Pro Gln Pro Lys Arg Pro Glu Ser Leu 245 250 255Arg Ile Tyr Glu Ser His Ile Gly Met Ser Ser Pro Glu Pro Lys Ile 260 265 270Asn Ser Tyr Ala Asn Phe Arg Asp Glu Val Leu Pro Arg Ile Lys Arg 275 280 285Leu Gly Tyr Asn Ala Val Gln Ile Met Ala Ile Gln Glu His Ser Tyr 290 295 300Tyr Ala Ser Phe Gly Tyr His Val Thr Asn Phe Phe Ala Pro Ser Ser305 310 315 320Arg Phe Gly Thr Pro Glu Asp Leu Lys Ser Leu Ile Asp Arg Ala His 325 330 335Glu Leu Gly Leu Leu Val Leu Met Asp Ile Val His Ser His Ser Ser 340 345 350Asn Asn Thr Leu Asp Gly Leu Asn Gly Phe Asp Gly Thr Asp Thr His 355 360 365Tyr Phe His Gly Gly Pro Arg Gly His His Trp Met Trp Asp Ser Arg 370 375 380Leu Phe Asn Tyr Gly Ser Trp Glu Val Leu Arg Phe Leu Leu Ser Asn385 390 395 400Ala Arg Trp Trp Leu Glu Glu Tyr Lys Phe Asp Gly Phe Arg Phe Asp 405 410 415Gly Val Thr Ser Met Met Tyr Thr His His Gly Leu Gln Met Thr Phe 420 425 430Thr Gly Asn Tyr Gly Glu Tyr Phe Gly Phe Ala Thr Asp Val Asp Ala 435 440 445Val Val Tyr Leu Met Leu Val Asn Asp Leu Ile His Gly Leu His Pro 450 455 460Asp Ala Val Ser Ile Gly Glu Asp Val Ser Gly Met Pro Thr Phe Cys465 470 475 480Ile Pro Val Pro Asp Gly Gly Val Gly Phe Asp Tyr Arg Leu His Met 485 490 495Ala Val Ala Asp Lys Trp Ile Glu Leu Leu Lys Gln Ser Asp Glu Ser 500 505 510Trp Lys Met Gly Asp Ile Val His Thr Leu Thr Asn Arg Arg Trp Leu 515 520 525Glu Lys Cys Val Thr Tyr Ala Glu Ser His Asp Gln Ala Leu Val Gly 530 535 540Asp Lys Thr Ile Ala Phe Trp Leu Met Asp Lys Asp Met Tyr Asp Phe545 550 555 560Met Ala Leu Asp Arg Pro Ser Thr Pro Arg Ile Asp Arg Gly Ile Ala 565 570 575Leu His Lys Met Ile Arg Leu Val Thr Met Gly Leu Gly Gly Glu Gly 580 585 590Tyr Leu Asn Phe Met Gly Asn Glu Phe Gly His Pro Glu Trp Ile Asp 595 600 605Phe Pro Arg Gly Pro Gln Thr Leu Pro Thr Gly Lys Val Leu Pro Gly 610 615 620Asn Asn Asn Ser Tyr Asp Lys Cys Arg Arg Arg Phe Asp Leu Gly Asp625 630 635 640Ala Asp Phe Leu Arg Tyr His Gly Met Gln Glu Phe Asp Gln Ala Met 645 650 655Gln His Leu Glu Glu Lys Tyr Gly Phe Met Thr Ser Glu His Gln Tyr 660 665 670Val Ser Arg Lys His Glu Glu Asp Lys Val Ile Ile Phe Glu Arg Gly 675 680 685Asp Leu Val Phe Val Phe Asn Phe His Trp Ser Asn Ser Phe Phe Asp 690 695 700Tyr Arg Val Gly Cys Ser Arg Pro Gly Lys Tyr Lys Val Ala Leu Asp705 710 715 720Ser Asp Asp Ala Leu Phe Gly Gly Phe Ser Arg Leu Asp His Asp Val 725 730 735Asp Tyr Phe Thr Thr Glu His Pro His Asp Asn Arg Pro Arg Ser Phe 740 745 750Ser Val Tyr Thr Pro Ser Arg Thr Ala Val Val Tyr Ala Leu Thr Glu 755 760 76525736PRTHordeum vulgare 25Gly Glu Met Ala Glu Val Asn Met Thr Gly Gly Ala Ala Glu Lys Leu1 5 10 15Glu Ser Ser Glu Pro Thr Gln Gly Ile Ala Glu Thr Ile Thr Asp Gly 20 25 30Val Thr Lys Gly Val Lys Glu Leu Val Val Gly Glu Lys Pro Gln Val 35 40 45Val Pro Lys Pro Gly Asp Gly Gln Lys Ile Tyr Glu Ile Asp Pro Thr 50 55 60Leu Lys Asp Phe Arg Ser His Leu Asp Tyr Arg Tyr Ser Glu Tyr Lys65 70 75 80Arg Ile Arg Ala Ala Ile Asp Gln His Glu Gly Gly Leu Glu Val Phe 85 90 95Ser Arg Gly Tyr Glu Lys Leu Gly Phe Thr Arg Ser Ala Lys Gly Ile 100 105 110Thr Tyr Arg Glu Trp Ala Pro Gly Ala His Ser Ala Ala Leu Val Gly 115 120 125Asp Phe Asn Asn Trp Asn Pro Asn Ala Asp Thr Met Thr Arg Asp Asp 130 135 140Tyr Gly Val Trp Glu Ile Phe Leu Pro Asn Asn Ala Asp Gly Ser Pro145 150 155 160Ala Ile Pro His Gly Ser Arg Val Lys Ile Arg Met Asp Thr Pro Ser 165 170 175Gly Val Lys Asp Ser Ile Ser Ala Trp Ile Lys Phe Ser Val Gln Ala 180 185 190Pro Gly Glu Ile Pro Phe Asn Gly Ile Tyr Tyr Asp Pro Pro Glu Glu 195 200 205Glu Lys Tyr Val Phe Gln His Pro Gln Pro Lys Arg Pro Glu Ser Leu 210 215 220Arg Ile Tyr Glu Ser His Ile Gly Met Ser Ser Pro Glu Pro Lys Ile225 230 235 240Asn Ser Tyr Ala Asn Phe Arg Asp Glu Val Leu Pro Arg Ile Lys Arg 245 250 255Leu Gly Tyr Asn Ala Val Gln Ile Met Ala Ile Gln Glu His Ser Tyr 260 265 270Tyr Ala Ser Phe Gly Tyr His Val Thr Asn Phe Phe Ala Pro Ser Ser 275 280 285Arg Phe Gly Thr Pro Glu Asp Leu Lys Ser Leu Ile Asp Arg Ala His 290 295 300Glu Leu Gly Leu Leu Val Leu Met Asp Ile Val His Ser His Ser Ser305 310 315 320Asn Asn Thr Leu Asp Gly Leu Asn Gly Phe Asp Gly Thr Asp Thr His 325 330 335Tyr Phe His Gly Gly Pro Arg Gly His His Trp Met Trp Asp Ser Arg 340 345 350Leu Phe Asn Tyr Gly Ser Trp Glu Val Leu Arg Phe Leu Leu Ser Asn 355 360 365Ala Arg Trp Trp Leu Glu Glu Tyr Lys Phe Asp Gly Phe Arg Phe Asp 370

375 380Gly Val Thr Ser Met Met Tyr Thr His His Gly Leu Gln Met Thr Phe385 390 395 400Thr Gly Asn Tyr Gly Glu Tyr Phe Gly Phe Ala Thr Asp Val Asp Ala 405 410 415Val Val Tyr Leu Met Leu Val Asn Asp Leu Ile His Gly Leu Tyr Pro 420 425 430Asp Ala Val Ser Ile Gly Glu Asp Val Ser Gly Met Pro Thr Phe Cys 435 440 445Ile Pro Val Pro Asp Gly Gly Val Gly Phe Asp Tyr Arg Leu His Met 450 455 460Ala Val Ala Asp Lys Trp Ile Glu Leu Leu Lys Gln Ser Asp Glu Ser465 470 475 480Trp Lys Met Gly Asp Ile Val His Thr Leu Thr Asn Arg Arg Trp Leu 485 490 495Glu Lys Cys Val Thr Tyr Ala Glu Ser His Asp Gln Ala Leu Val Gly 500 505 510Asp Lys Thr Ile Ala Phe Trp Leu Met Asp Lys Asp Met Tyr Asp Phe 515 520 525Met Ala Leu Asp Arg Pro Ser Thr Pro Arg Ile Asp Arg Gly Ile Ala 530 535 540Leu His Lys Met Ile Arg Leu Val Thr Met Gly Leu Gly Gly Glu Gly545 550 555 560Tyr Leu Asn Phe Met Gly Asn Glu Phe Gly His Pro Glu Trp Ile Asp 565 570 575Phe Pro Arg Gly Pro Gln Thr Leu Pro Thr Gly Lys Val Leu Pro Gly 580 585 590Asn Asn Asn Ser Tyr Asp Lys Cys Arg Arg Arg Phe Asp Leu Gly Asp 595 600 605Ala Asp Phe Leu Arg Tyr Arg Gly Met Gln Glu Phe Asp Gln Ala Met 610 615 620Gln His Leu Glu Glu Lys Tyr Gly Phe Met Thr Ser Glu His Gln Tyr625 630 635 640Val Ser Arg Lys His Glu Glu Asp Lys Val Ile Ile Phe Glu Arg Gly 645 650 655Asp Leu Val Phe Val Phe Asn Phe His Trp Ser Asn Ser Lys Lys Asp 660 665 670Tyr Arg Val Gly Cys Ser Lys Pro Gly Lys Tyr Lys Val Ala Leu Asp 675 680 685Ser Asp Asp Ala Leu Phe Gly Gly Phe Ser Arg Leu Asp His Asp Val 690 695 700Asp Tyr Phe Thr Thr Glu His Pro His Asp Asn Arg Pro Arg Ser Phe705 710 715 720Ser Val Tyr Thr Pro Ser Arg Thr Ala Val Val Tyr Ala Leu Thr Glu 725 730 73526814PRTZea mays 26Asp Leu Pro Ser Val Leu Phe Arg Arg Lys Asp Ala Phe Ser Arg Thr1 5 10 15Val Leu Ser Cys Ala Gly Ala Pro Gly Lys Val Leu Val Pro Gly Gly 20 25 30Gly Ser Asp Asp Leu Leu Ser Ser Ala Glu Pro Val Val Asp Thr Gln 35 40 45Pro Glu Glu Leu Gln Ile Pro Glu Ala Glu Leu Thr Val Glu Lys Thr 50 55 60Ser Ser Ser Pro Thr Gln Thr Thr Ser Ala Val Ala Glu Ala Ser Ser65 70 75 80Gly Val Glu Ala Glu Glu Arg Pro Glu Leu Ser Glu Val Ile Gly Val 85 90 95Gly Gly Thr Gly Gly Thr Lys Ile Asp Gly Ala Gly Ile Lys Ala Lys 100 105 110Ala Pro Leu Val Glu Glu Lys Pro Arg Val Ile Pro Pro Pro Gly Asp 115 120 125Gly Gln Arg Ile Tyr Glu Ile Asp Pro Met Leu Glu Gly Phe Arg Gly 130 135 140His Leu Asp Tyr Arg Tyr Ser Glu Tyr Lys Arg Leu Arg Ala Ala Ile145 150 155 160Asp Gln His Glu Gly Gly Leu Asp Ala Phe Ser Arg Gly Tyr Glu Lys 165 170 175Leu Gly Phe Thr Arg Ser Ala Glu Gly Ile Thr Tyr Arg Glu Trp Ala 180 185 190Pro Gly Ala Tyr Ser Ala Ala Leu Val Gly Asp Phe Asn Asn Trp Asn 195 200 205Pro Asn Ala Asp Ala Met Ala Arg Asn Glu Tyr Gly Val Trp Glu Ile 210 215 220Phe Leu Pro Asn Asn Ala Asp Gly Ser Pro Ala Ile Pro His Gly Ser225 230 235 240Arg Val Lys Ile Arg Met Asp Thr Pro Ser Gly Val Lys Asp Ser Ile 245 250 255Pro Ala Trp Ile Lys Phe Ser Val Gln Ala Pro Gly Glu Ile Pro Tyr 260 265 270Asn Gly Ile Tyr Tyr Asp Pro Pro Glu Glu Glu Lys Tyr Val Phe Lys 275 280 285His Pro Gln Pro Lys Arg Pro Lys Ser Leu Arg Ile Tyr Glu Ser His 290 295 300Val Gly Met Ser Ser Pro Glu Pro Lys Ile Asn Thr Tyr Ala Asn Phe305 310 315 320Arg Asp Glu Val Leu Pro Arg Ile Lys Lys Leu Gly Tyr Asn Ala Val 325 330 335Gln Ile Met Ala Ile Gln Glu His Ser Tyr Tyr Ala Ser Phe Gly Tyr 340 345 350His Val Thr Asn Phe Phe Ala Pro Ser Ser Arg Phe Gly Thr Pro Glu 355 360 365Asp Leu Lys Ser Leu Ile Asp Lys Ala His Glu Leu Gly Leu Leu Val 370 375 380Leu Met Asp Ile Val His Ser His Ser Ser Asn Asn Thr Leu Asp Gly385 390 395 400Leu Asn Gly Phe Asp Gly Thr Asp Thr His Tyr Phe His Gly Gly Pro 405 410 415Arg Gly His His Trp Met Trp Asp Ser Arg Leu Phe Asn Tyr Gly Ser 420 425 430Trp Glu Val Leu Arg Phe Leu Leu Ser Asn Ala Arg Trp Trp Leu Glu 435 440 445Glu Tyr Lys Phe Asp Gly Phe Arg Phe Asp Gly Val Thr Ser Met Met 450 455 460Tyr Thr His His Gly Leu Gln Val Thr Phe Thr Gly Asn Tyr Gly Glu465 470 475 480Tyr Phe Gly Phe Ala Thr Asp Val Asp Ala Val Val Tyr Leu Met Leu 485 490 495Val Asn Asp Leu Ile Arg Gly Leu Tyr Pro Glu Ala Val Ser Ile Gly 500 505 510Glu Asp Val Ser Gly Met Pro Thr Phe Cys Ile Pro Val Gln Asp Gly 515 520 525Gly Val Gly Phe Asp Tyr Arg Leu His Met Ala Val Pro Asp Lys Trp 530 535 540Ile Glu Leu Leu Lys Gln Ser Asp Glu Tyr Trp Glu Met Gly Asp Ile545 550 555 560Val His Thr Leu Thr Asn Arg Arg Trp Leu Glu Lys Cys Val Thr Tyr 565 570 575Cys Glu Ser His Asp Gln Ala Leu Val Gly Asp Lys Thr Ile Ala Phe 580 585 590Trp Leu Met Asp Lys Asp Met Tyr Asp Phe Met Ala Leu Asp Arg Pro 595 600 605Ser Thr Pro Arg Ile Asp Arg Gly Ile Ala Leu His Lys Met Ile Arg 610 615 620Leu Val Thr Met Gly Leu Gly Gly Glu Gly Tyr Leu Asn Phe Met Gly625 630 635 640Asn Glu Phe Gly His Pro Glu Trp Ile Asp Phe Pro Arg Gly Pro Gln 645 650 655Ser Leu Pro Asn Gly Ser Val Ile Pro Gly Asn Asn Asn Ser Phe Asp 660 665 670Lys Cys Arg Arg Arg Phe Asp Leu Gly Asp Ala Asp Tyr Leu Arg Tyr 675 680 685Arg Gly Met Gln Glu Phe Asp Gln Ala Met Gln His Leu Glu Gly Lys 690 695 700Tyr Glu Phe Met Thr Ser Asp His Ser Tyr Val Ser Arg Lys His Glu705 710 715 720Glu Asp Lys Val Ile Ile Phe Glu Arg Gly Asp Leu Val Phe Val Phe 725 730 735Asn Phe His Trp Ser Asn Ser Tyr Phe Asp Tyr Arg Val Gly Cys Phe 740 745 750Lys Pro Gly Lys Tyr Lys Ile Val Leu Asp Ser Asp Asp Gly Leu Phe 755 760 765Gly Gly Phe Ser Arg Leu Asp His Asp Ala Glu Tyr Phe Thr Ala Asp 770 775 780Trp Pro His Asp Asn Arg Pro Cys Ser Phe Ser Val Tyr Ala Pro Ser785 790 795 800Arg Thr Ala Val Val Tyr Ala Pro Ala Gly Ala Glu Asp Glu 805 81027841PRTOryza sativa 27Met Ala Ser Phe Ala Val Ser Gly Ala Arg Leu Gly Val Val Arg Ala1 5 10 15Gly Gly Gly Gly Gly Gly Gly Gly Gly Pro Ala Ala Arg Ser Gly Gly 20 25 30Val Asp Leu Pro Ser Val Leu Phe Arg Arg Lys Asp Ser Phe Ser Arg 35 40 45Gly Val Val Ser Cys Ala Gly Ala Pro Gly Lys Val Leu Val Pro Gly 50 55 60Gly Gly Ser Asp Asp Leu Leu Ser Ser Ala Glu Pro Asp Val Glu Thr65 70 75 80Gln Glu Gln Pro Glu Glu Ser Gln Ile Pro Asp Asp Asn Lys Val Lys 85 90 95Pro Phe Glu Glu Glu Glu Glu Ile Pro Ala Val Ala Glu Ala Ser Ile 100 105 110Lys Val Val Ala Glu Asp Lys Leu Glu Ser Ser Glu Val Ile Gln Asp 115 120 125Ile Glu Glu Asn Val Thr Glu Gly Val Ile Lys Asp Ala Asp Glu Pro 130 135 140Thr Val Glu Asp Lys Pro Arg Val Ile Pro Pro Pro Gly Asp Gly Gln145 150 155 160Lys Ile Tyr Gln Ile Asp Pro Met Leu Glu Gly Phe Arg Asn His Leu 165 170 175Asp Tyr Arg Tyr Ser Glu Tyr Lys Arg Met Arg Ala Ala Ile Asp Gln 180 185 190His Glu Gly Gly Leu Asp Ala Phe Ser Arg Gly Tyr Glu Lys Leu Gly 195 200 205Phe Thr Arg Ser Ala Glu Gly Ile Thr Tyr Arg Glu Trp Ala Pro Gly 210 215 220Ala Gln Ser Ala Ala Leu Val Gly Asp Phe Asn Asn Trp Asn Pro Asn225 230 235 240Ala Asp Thr Met Thr Arg Asn Glu Tyr Gly Val Trp Glu Ile Ser Leu 245 250 255Pro Asn Asn Ala Asp Gly Ser Pro Ala Ile Pro His Gly Ser Arg Val 260 265 270Lys Ile Arg Met Asp Thr Pro Ser Gly Val Lys Asp Ser Ile Pro Ala 275 280 285Trp Ile Lys Phe Ala Val Gln Ala Pro Gly Glu Ile Pro Tyr Asn Gly 290 295 300Ile Tyr Tyr Asp Pro Pro Glu Glu Glu Lys Tyr Val Phe Gln His Pro305 310 315 320Gln Pro Lys Arg Pro Asn Ser Leu Arg Ile Tyr Glu Ser His Ile Gly 325 330 335Met Ser Ser Pro Glu Pro Lys Ile Asn Thr Tyr Ala Asn Phe Arg Asp 340 345 350Glu Val Leu Pro Arg Ile Lys Lys Leu Gly Tyr Asn Ala Val Gln Ile 355 360 365Met Ala Ile Gln Glu His Ser Tyr Tyr Ala Ser Phe Gly Tyr His Val 370 375 380Thr Asn Phe Phe Ala Pro Ser Ser Arg Phe Gly Thr Pro Glu Asp Leu385 390 395 400Lys Ser Leu Ile Asp Lys Ala His Glu Leu Gly Leu Leu Val Leu Met 405 410 415Asp Ile Val His Ser His Ala Ser Asn Asn Thr Leu Asp Gly Leu Asn 420 425 430Gly Phe Asp Gly Thr Asp Thr His Tyr Phe His Gly Gly Pro Arg Gly 435 440 445His His Trp Met Trp Asp Ser Arg Leu Phe Asn Tyr Gly Ser Trp Glu 450 455 460Val Leu Arg Tyr Leu Leu Ser Asn Ala Arg Trp Trp Leu Glu Glu Tyr465 470 475 480Lys Phe Asp Gly Phe Arg Phe Asp Gly Val Thr Ser Met Met Tyr Thr 485 490 495His His Gly Leu Gln Val Ala Phe Thr Gly Asn Tyr Gly Glu Tyr Phe 500 505 510Gly Phe Ala Thr Asp Val Asp Ala Val Val Tyr Leu Met Leu Val Asn 515 520 525Asp Leu Ile His Gly Leu Tyr Pro Glu Ala Val Ala Ile Gly Glu Asp 530 535 540Val Ser Gly Met Pro Thr Phe Cys Ile Pro Val Gln Asp Gly Gly Val545 550 555 560Gly Phe Asp Tyr Arg Leu His Met Ala Val Pro Asp Lys Trp Ile Glu 565 570 575Leu Leu Lys Gln Ser Asp Glu Tyr Trp Lys Met Gly Asp Ile Val His 580 585 590Thr Leu Thr Asn Arg Arg Trp Ser Glu Lys Cys Val Thr Tyr Ala Glu 595 600 605Ser His Asp Gln Ala Leu Val Gly Asp Lys Thr Ile Ala Phe Trp Leu 610 615 620Met Asp Lys Asp Met Tyr Asp Phe Met Ala Leu Asp Arg Pro Ser Thr625 630 635 640Pro Arg Ile Asp Arg Gly Ile Ala Leu His Lys Met Ile Arg Leu Val 645 650 655Thr Met Gly Leu Gly Gly Glu Gly Tyr Leu Asn Phe Met Gly Asn Glu 660 665 670Phe Gly His Pro Glu Trp Ile Asp Phe Pro Arg Gly Pro Gln Ser Leu 675 680 685Pro Asn Gly Ser Val Leu Pro Gly Asn Asn Tyr Ser Phe Asp Lys Cys 690 695 700Arg Arg Arg Phe Asp Leu Gly Asp Ala Asp Tyr Leu Arg Tyr His Gly705 710 715 720Met Gln Glu Phe Asp Gln Ala Met Gln His Leu Glu Glu Lys Tyr Gly 725 730 735Phe Met Thr Ser Glu His Gln Tyr Ile Ser Arg Lys His Glu Glu Asp 740 745 750Lys Val Ile Ile Phe Glu Arg Gly Asp Leu Val Phe Val Phe Asn Phe 755 760 765His Trp Ser Asn Ser Tyr Phe Asp Tyr Arg Val Gly Cys Leu Lys Pro 770 775 780Gly Lys Tyr Lys Ile Val Leu Asp Ser Asp Asp Gly Leu Phe Gly Gly785 790 795 800Phe Ser Arg Leu Asp His Asp Ala Glu Tyr Phe Thr Ala Asp Trp Pro 805 810 815His Asp Asn Arg Pro Cys Ser Phe Ser Val Tyr Thr Pro Ser Arg Thr 820 825 830Ala Val Val Tyr Ala Leu Thr Glu Asp 835 84028829PRTHordeum vulgare 28Met Ala Ala Pro Ala Phe Ala Val Ser Ala Ala Gly Ile Ala Arg Pro1 5 10 15Ser Ala Arg Arg Ser Ser Gly Ala Glu Pro Arg Ser Leu Leu Phe Gly 20 25 30Arg Asn Lys Gly Thr Arg Phe Pro Arg Ala Val Gly Val Gly Gly Ser 35 40 45Gly Trp Arg Val Val Met Arg Ala Gly Gly Pro Ser Gly Glu Val Met 50 55 60Ile Pro Asp Gly Gly Ser Gly Gly Ser Gly Thr Pro Pro Ser Ile Glu65 70 75 80Gly Ser Val Gln Phe Glu Ser Asp Asp Leu Glu Val Pro Phe Ile Asp 85 90 95Asp Glu Pro Ser Leu His Asp Gly Gly Glu Asp Thr Ile Arg Ser Ser 100 105 110Glu Thr Tyr Gln Val Thr Glu Glu Ile Asp Ala Glu Gly Val Ser Arg 115 120 125Met Asp Lys Glu Ser Ser Thr Val Lys Lys Ile Arg Ile Val Pro Gln 130 135 140Pro Gly Asn Gly Gln Gln Ile Tyr Asp Ile Asp Pro Met Leu Arg Asp145 150 155 160Phe Lys Tyr His Leu Glu Tyr Arg Tyr Ser Leu Tyr Arg Arg Ile Arg 165 170 175Ser Asp Ile Asp Glu Tyr Asp Gly Gly Met Asp Val Phe Ser Arg Gly 180 185 190Tyr Glu Lys Phe Gly Phe Val Arg Ser Ala Glu Gly Ile Thr Tyr Arg 195 200 205Glu Trp Ala Pro Gly Ala Asp Ser Ala Ala Leu Val Gly Asp Phe Asn 210 215 220Asn Trp Asp Pro Thr Ala Asp His Met Ser Lys Asn Asp Leu Gly Ile225 230 235 240Trp Glu Ile Phe Leu Pro Asn Asn Ala Asp Gly Ser Pro Pro Ile Pro 245 250 255His Gly Ser Arg Val Lys Val Arg Met Asp Thr Pro Ser Gly Thr Lys 260 265 270Asp Ser Ile Pro Ala Trp Ile Lys Tyr Ser Val Gln Thr Pro Gly Asp 275 280 285Ile Pro Tyr Asn Gly Ile Tyr Tyr Asp Pro Pro Glu Glu Glu Lys Tyr 290 295 300Val Phe Lys His Pro Gln Pro Lys Arg Pro Lys Ser Leu Arg Ile Tyr305 310 315 320Glu Thr His Val Gly Met Ser Ser Pro Glu Pro Lys Ile Asn Thr Tyr 325 330 335Ala Asn Phe Arg Asp Glu Val Leu Pro Arg Ile Lys Arg Leu Gly Tyr 340 345 350Asn Ala Val Gln Ile Met Ala Ile Gln Glu His Ser Tyr Tyr Gly Ser 355 360 365Phe Gly Tyr His Val Thr Asn Phe Phe Ala Pro Ser Ser Arg Phe Gly 370 375 380Ser Pro Glu Asp Leu Lys Ser Leu Ile Asp Arg Ala His Glu Leu Gly385 390 395 400Leu Leu Val Leu Met Asp Val Val His Ser His Ala Ser Ser Asn Thr 405 410 415Leu Asp Gly Leu Asn Gly Phe Asp Gly Thr Asp Thr His Tyr Phe His 420 425 430Gly Gly Ser Arg Gly His His Trp Met Trp Asp Ser Arg Val Phe Asn 435 440 445Tyr Gly Asn Lys

Glu Val Ile Arg Phe Leu Leu Ser Asn Ala Arg Trp 450 455 460Trp Leu Glu Glu Tyr Lys Phe Asp Gly Phe Arg Phe Asp Gly Ala Thr465 470 475 480Ser Met Met Tyr Thr His His Gly Leu Gln Val Thr Phe Thr Gly Ser 485 490 495Tyr His Glu Tyr Phe Gly Phe Ala Thr Asp Val Asp Ala Val Val Tyr 500 505 510Leu Met Leu Val Asn Asp Leu Ile His Ala Leu Tyr Pro Glu Ala Val 515 520 525Thr Ile Gly Glu Asp Val Ser Gly Met Pro Thr Phe Ala Leu Pro Val 530 535 540Gln Val Gly Gly Val Gly Phe Asp Tyr Arg Leu His Met Ala Val Ala545 550 555 560Asp Lys Trp Ile Glu Leu Leu Lys Gly Ser Asp Glu Gly Trp Glu Met 565 570 575Gly Asn Ile Val His Thr Leu Thr Asn Arg Arg Trp Leu Glu Lys Cys 580 585 590Val Thr Tyr Ala Glu Ser His Asp Gln Ala Leu Val Gly Asp Lys Thr 595 600 605Ile Ala Phe Trp Leu Met Asp Lys Asp Met Tyr Asp Phe Met Ala Leu 610 615 620Asn Gly Pro Ser Thr Pro Asn Ile Asp Arg Gly Ile Ala Leu His Lys625 630 635 640Met Ile Arg Leu Ile Thr Met Ala Leu Gly Gly Glu Gly Tyr Leu Asn 645 650 655Phe Met Gly Asn Glu Phe Gly His Pro Glu Trp Ile Asp Phe Pro Arg 660 665 670Gly Pro Gln Val Leu Pro Thr Gly Lys Phe Ile Pro Gly Asn Asn Asn 675 680 685Ser Tyr Asp Lys Cys Arg Arg Arg Phe Asp Leu Gly Asp Ala Glu Phe 690 695 700Leu Arg Tyr His Gly Met Gln Gln Phe Asp Gln Ala Met Gln His Leu705 710 715 720Glu Glu Lys Tyr Gly Phe Met Thr Ser Asp His Gln Tyr Val Ser Arg 725 730 735Lys His Glu Glu Asp Lys Val Ile Val Phe Glu Lys Gly Asp Leu Val 740 745 750Phe Val Phe Asn Phe His Trp Ser Asn Ser Tyr Phe Asp Tyr Arg Val 755 760 765Gly Cys Leu Lys Pro Gly Lys Tyr Lys Val Val Leu Asp Ser Asp Ala 770 775 780Gly Leu Phe Gly Gly Phe Gly Arg Ile His His Thr Gly Glu His Phe785 790 795 800Thr Asn Gly Cys Gln His Asp Asn Arg Pro His Ser Phe Ser Val Tyr 805 810 815Thr Pro Ser Arg Thr Cys Val Val Tyr Ala Pro Met Asn 820 82529836PRTTriticum sp. 29Met Ala Ala Pro Ala Phe Ala Val Ser Ala Ala Gly Leu Ala Arg Pro1 5 10 15Ser Ala Pro Arg Ser Gly Gly Ala Glu Arg Arg Gly Arg Gly Val Glu 20 25 30Leu Gln Ser Pro Ser Leu Leu Phe Gly Arg Asn Lys Gly Thr Arg Ser 35 40 45Pro Arg Ala Val Gly Val Gly Gly Ser Gly Trp Arg Val Val Met Arg 50 55 60Ala Gly Gly Pro Ser Gly Glu Val Met Ile Pro Asp Gly Gly Ser Gly65 70 75 80Gly Thr Pro Pro Ser Ile Asp Gly Pro Val Gln Phe Asp Ser Asp Asp 85 90 95Leu Lys Val Pro Phe Ile Asp Asp Glu Pro Ser Leu His Asp Gly Gly 100 105 110Glu Asp Thr Ile Trp Ser Ser Glu Thr Asn Gln Val Ser Glu Glu Ile 115 120 125Asp Ala Glu Asp Thr Ser Arg Met Asp Lys Glu Ser Ser Thr Arg Glu 130 135 140Lys Leu Arg Ile Leu Pro Pro Pro Gly Asn Gly Gln Gln Ile Tyr Glu145 150 155 160Ile Asp Pro Thr Leu Arg Asp Phe Lys Tyr His Leu Glu Tyr Arg Tyr 165 170 175Ser Leu Tyr Arg Arg Ile Arg Ser Asp Ile Asp Glu His Glu Gly Gly 180 185 190Met Asp Val Phe Ser Arg Gly Tyr Glu Lys Phe Gly Phe Met Arg Ser 195 200 205Ala Glu Gly Ile Thr Tyr Arg Glu Trp Ala Pro Gly Ala Asp Ser Ala 210 215 220Ala Leu Val Gly Asp Phe Asn Asn Trp Asp Pro Asn Ala Asp His Met225 230 235 240Ser Lys Asn Asp Leu Gly Val Trp Glu Ile Phe Leu Pro Asn Asn Ala 245 250 255Asp Gly Ser Pro Pro Ile Pro His Gly Ser Arg Val Lys Val Arg Met 260 265 270Gly Thr Pro Ser Gly Thr Lys Asp Ser Ile Pro Ala Trp Ile Lys Tyr 275 280 285Ser Val Gln Thr Pro Gly Asp Ile Pro Tyr Asn Gly Ile Tyr Tyr Asp 290 295 300Pro Pro Glu Glu Glu Lys Tyr Val Phe Lys His Pro Gln Pro Lys Arg305 310 315 320Pro Lys Ser Leu Arg Ile Tyr Glu Thr His Val Gly Met Ser Ser Pro 325 330 335Glu Pro Lys Ile Asn Thr Tyr Ala Asn Phe Arg Asp Glu Val Leu Pro 340 345 350Arg Ile Lys Arg Leu Gly Tyr Asn Ala Val Gln Ile Met Ala Ile Gln 355 360 365Glu His Ser Tyr Tyr Gly Ser Phe Gly Tyr His Val Thr Asn Phe Phe 370 375 380Ala Pro Ser Ser Arg Phe Gly Ser Pro Glu Asp Leu Lys Ser Leu Ile385 390 395 400Asp Arg Ala His Glu Leu Gly Leu Val Val Leu Met Asp Val Val His 405 410 415Ser His Ala Ser Asn Asn Thr Leu Asp Gly Leu Asn Gly Phe Asp Gly 420 425 430Thr Asp Thr His Tyr Phe His Gly Gly Ser Arg Gly His His Trp Met 435 440 445Trp Asp Ser Arg Val Phe Asn Tyr Gly Asn Lys Glu Val Ile Arg Phe 450 455 460Leu Leu Ser Asn Ala Arg Trp Trp Leu Glu Glu Tyr Lys Phe Asp Gly465 470 475 480Phe Arg Phe Asp Gly Ala Thr Ser Met Met Tyr Thr His His Gly Leu 485 490 495Gln Val Thr Phe Thr Gly Ser Tyr His Glu Tyr Phe Gly Phe Ala Thr 500 505 510Asp Val Asp Ala Val Val Tyr Leu Met Leu Met Asn Asp Leu Ile His 515 520 525Gly Phe Tyr Pro Glu Ala Val Thr Ile Gly Glu Asp Val Ser Gly Met 530 535 540Pro Thr Phe Ala Leu Pro Val Gln Val Gly Gly Val Gly Phe Asp Tyr545 550 555 560Arg Leu His Met Ala Val Ala Arg Lys Trp Ile Glu Leu Leu Lys Gly 565 570 575Asn Asp Glu Ala Trp Glu Met Gly Asn Ile Val His Thr Leu Thr Asn 580 585 590Arg Arg Trp Leu Glu Lys Cys Val Thr Tyr Ala Glu Ser His Asp Gln 595 600 605Ala Leu Val Gly Asp Lys Thr Ile Ala Phe Trp Leu Met Asp Lys Asp 610 615 620Met Tyr Asp Phe Met Ala Leu Asn Gly Pro Ser Thr Pro Asn Ile Asp625 630 635 640Arg Gly Ile Ala Leu His Lys Met Ile Arg Leu Ile Thr Met Gly Leu 645 650 655Gly Gly Glu Gly Tyr Leu Asn Phe Met Gly Asn Glu Phe Gly His Pro 660 665 670Glu Trp Ile Asp Phe Pro Arg Gly Pro Gln Val Leu Pro Ser Gly Lys 675 680 685Phe Ile Pro Gly Asn Asn Asn Ser Tyr Asp Lys Cys Arg Arg Arg Phe 690 695 700Asp Leu Gly Asp Ala Glu Phe Leu Arg Tyr His Gly Met Gln Gln Phe705 710 715 720Asp Gln Ala Met Gln His Leu Glu Glu Lys Tyr Gly Phe Met Thr Ser 725 730 735Asp His Gln Tyr Val Ser Arg Lys His Glu Glu Asp Lys Val Ile Val 740 745 750Phe Glu Lys Gly Asp Leu Val Phe Val Phe Asn Phe His Trp Ser Ser 755 760 765Ser Tyr Phe Asp Tyr Arg Val Gly Cys Leu Lys Pro Gly Lys Tyr Lys 770 775 780Val Val Leu Asp Ser Asp Ala Gly Leu Phe Gly Gly Phe Gly Arg Ile785 790 795 800His His Thr Ala Glu His Phe Thr Ser Asp Cys Gln His Asp Asn Arg 805 810 815Pro His Ser Phe Ser Val Tyr Thr Pro Ser Arg Thr Cys Val Val Tyr 820 825 830Ala Pro Met Asn 83530741PRTZea mays 30Lys Ala Val Met Val Pro Glu Gly Glu Asn Asp Gly Leu Ala Ser Arg1 5 10 15Ala Asp Ser Ala Gln Phe Gln Ser Asp Glu Leu Glu Val Pro Asp Ile 20 25 30Ser Glu Glu Thr Thr Cys Gly Ala Gly Val Ala Asp Ala Gln Ala Leu 35 40 45Asn Arg Val Arg Val Val Pro Pro Pro Ser Asp Gly Gln Lys Ile Phe 50 55 60Gln Ile Asp Pro Met Leu Gln Gly Tyr Lys Tyr His Leu Glu Tyr Arg65 70 75 80Tyr Ser Leu Tyr Arg Arg Ile Arg Ser Asp Ile Asp Glu His Glu Gly 85 90 95Gly Leu Glu Ala Phe Ser Arg Ser Tyr Glu Lys Phe Gly Phe Asn Ala 100 105 110Ser Ala Glu Gly Ile Thr Tyr Arg Glu Trp Ala Pro Gly Ala Phe Ser 115 120 125Ala Ala Leu Val Gly Asp Val Asn Asn Trp Asp Pro Asn Ala Asp Arg 130 135 140Met Ser Lys Asn Glu Phe Gly Val Trp Glu Ile Phe Leu Pro Asn Asn145 150 155 160Ala Asp Gly Thr Ser Pro Ile Pro His Gly Ser Arg Val Lys Val Arg 165 170 175Met Asp Thr Pro Ser Gly Ile Lys Asp Ser Ile Pro Ala Trp Ile Lys 180 185 190Tyr Ser Val Gln Ala Pro Gly Glu Ile Pro Tyr Asp Gly Ile Tyr Tyr 195 200 205Asp Pro Pro Glu Glu Val Lys Tyr Val Phe Arg His Ala Gln Pro Lys 210 215 220Arg Pro Lys Ser Leu Arg Ile Tyr Glu Thr His Val Gly Met Ser Ser225 230 235 240Pro Glu Pro Lys Ile Asn Thr Tyr Val Asn Phe Arg Asp Glu Val Leu 245 250 255Pro Arg Ile Lys Lys Leu Gly Tyr Asn Ala Val Gln Ile Met Ala Ile 260 265 270Gln Glu His Ser Tyr Tyr Gly Ser Phe Gly Tyr His Val Thr Asn Phe 275 280 285Phe Ala Pro Ser Ser Arg Phe Gly Thr Pro Glu Asp Leu Lys Ser Leu 290 295 300Ile Asp Arg Ala His Glu Leu Gly Leu Leu Val Leu Met Asp Val Val305 310 315 320His Ser His Ala Ser Ser Asn Thr Leu Asp Gly Leu Asn Gly Phe Asp 325 330 335Gly Thr Asp Thr His Tyr Phe His Ser Gly Pro Arg Gly His His Trp 340 345 350Met Trp Asp Ser Arg Leu Phe Asn Tyr Gly Asn Trp Glu Val Leu Arg 355 360 365Phe Leu Leu Ser Asn Ala Arg Trp Trp Leu Glu Glu Tyr Lys Phe Asp 370 375 380Gly Phe Arg Phe Asp Gly Val Thr Ser Met Met Tyr Thr His His Gly385 390 395 400Leu Gln Val Thr Phe Thr Gly Asn Phe Asn Glu Tyr Phe Gly Phe Ala 405 410 415Thr Asp Val Asp Ala Val Val Tyr Leu Met Leu Val Asn Asp Leu Ile 420 425 430His Gly Leu Tyr Pro Glu Ala Val Thr Ile Gly Glu Asp Val Ser Gly 435 440 445Met Pro Thr Phe Ala Leu Pro Val His Asp Gly Gly Val Gly Phe Asp 450 455 460Tyr Arg Met His Met Ala Val Ala Asp Lys Trp Ile Asp Leu Leu Lys465 470 475 480Gln Ser Asp Glu Thr Trp Lys Met Gly Asp Ile Val His Thr Leu Thr 485 490 495Asn Arg Arg Trp Leu Glu Lys Cys Val Thr Tyr Ala Glu Ser His Asp 500 505 510Gln Ala Leu Val Gly Asp Lys Thr Ile Ala Phe Trp Leu Met Asp Lys 515 520 525Asp Met Tyr Asp Phe Met Ala Leu Asp Arg Pro Ser Thr Pro Thr Ile 530 535 540Asp Arg Gly Ile Ala Leu His Lys Met Ile Arg Leu Ile Thr Met Gly545 550 555 560Leu Gly Gly Glu Gly Tyr Leu Asn Phe Met Gly Asn Glu Phe Gly His 565 570 575Pro Glu Trp Ile Asp Phe Pro Arg Gly Pro Gln Arg Leu Pro Ser Gly 580 585 590Lys Phe Ile Pro Gly Asn Asn Asn Ser Tyr Asp Lys Cys Arg Arg Arg 595 600 605Phe Asp Leu Gly Asp Ala Asp Tyr Leu Arg Tyr His Gly Met Gln Glu 610 615 620Phe Asp Gln Ala Met Gln His Leu Glu Gln Lys Tyr Glu Phe Met Thr625 630 635 640Ser Asp His Gln Tyr Ile Ser Arg Lys His Glu Glu Asp Lys Val Ile 645 650 655Val Phe Glu Lys Gly Asp Leu Val Phe Val Phe Asn Phe His Cys Asn 660 665 670Asn Ser Tyr Phe Asp Tyr Arg Ile Gly Cys Arg Lys Pro Gly Val Tyr 675 680 685Lys Val Val Leu Asp Ser Asp Ala Gly Leu Phe Gly Gly Phe Ser Arg 690 695 700Ile His His Ala Ala Glu His Phe Thr Ala Asp Cys Ser His Asp Asn705 710 715 720Arg Pro Tyr Ser Phe Ser Val Tyr Thr Pro Ser Arg Thr Cys Val Val 725 730 735Tyr Ala Pro Val Glu 74031825PRTOryza sativa 31Met Ala Ala Pro Ala Ser Ala Val Pro Gly Ser Ala Ala Gly Leu Arg1 5 10 15Ala Gly Ala Val Arg Phe Pro Val Pro Ala Gly Ala Arg Ser Trp Arg 20 25 30Ala Ala Ala Glu Leu Pro Thr Ser Arg Ser Leu Leu Ser Gly Arg Arg 35 40 45Phe Pro Gly Ala Val Arg Val Gly Gly Ser Gly Gly Arg Val Ala Val 50 55 60Arg Ala Ala Gly Ala Ser Gly Glu Val Met Ile Pro Glu Gly Glu Ser65 70 75 80Asp Gly Met Pro Val Ser Ala Gly Ser Asp Asp Leu Gln Leu Pro Ala 85 90 95Leu Asp Asp Glu Leu Ser Thr Glu Val Gly Ala Glu Val Glu Ile Glu 100 105 110Ser Ser Gly Ala Ser Asp Val Glu Gly Val Lys Arg Val Val Glu Glu 115 120 125Leu Ala Ala Glu Gln Lys Pro Arg Val Val Pro Pro Thr Gly Asp Gly 130 135 140Gln Lys Ile Phe Gln Met Asp Ser Met Leu Asn Gly Tyr Lys Tyr His145 150 155 160Leu Glu Tyr Arg Tyr Ser Leu Tyr Arg Arg Leu Arg Ser Asp Ile Asp 165 170 175Gln Tyr Glu Gly Gly Leu Glu Thr Phe Ser Arg Gly Tyr Glu Lys Phe 180 185 190Gly Phe Asn His Ser Ala Glu Gly Val Thr Tyr Arg Glu Trp Ala Pro 195 200 205Gly Ala His Ser Ala Ala Leu Val Gly Asp Phe Asn Asn Trp Asn Pro 210 215 220Asn Ala Asp Arg Met Ser Lys Asn Glu Phe Gly Val Trp Glu Ile Phe225 230 235 240Leu Pro Asn Asn Ala Asp Gly Ser Ser Pro Ile Pro His Gly Ser Arg 245 250 255Val Lys Val Arg Met Glu Thr Pro Ser Gly Ile Lys Asp Ser Ile Pro 260 265 270Ala Trp Ile Lys Tyr Ser Val Gln Ala Ala Gly Glu Ile Pro Tyr Asn 275 280 285Gly Ile Tyr Tyr Asp Pro Pro Glu Glu Glu Lys Tyr Ile Phe Lys His 290 295 300Pro Gln Pro Lys Arg Pro Lys Ser Leu Arg Ile Tyr Glu Thr His Val305 310 315 320Gly Met Ser Ser Thr Glu Pro Lys Ile Asn Thr Tyr Ala Asn Phe Arg 325 330 335Asp Glu Val Leu Pro Arg Ile Lys Lys Leu Gly Tyr Asn Ala Val Gln 340 345 350Ile Met Ala Ile Gln Glu His Ala Tyr Tyr Gly Ser Phe Gly Tyr His 355 360 365Val Thr Asn Phe Phe Ala Pro Ser Ser Arg Phe Gly Thr Pro Glu Asp 370 375 380Leu Lys Ser Leu Ile Asp Lys Ala His Glu Leu Gly Leu Val Val Leu385 390 395 400Met Asp Val Val His Ser His Ala Ser Asn Asn Thr Leu Asp Gly Leu 405 410 415Asn Gly Phe Asp Gly Thr Asp Thr His Tyr Phe His Ser Gly Ser Arg 420 425 430Gly His His Trp Met Trp Asp Ser Arg Leu Phe Asn Tyr Gly Asn Trp 435 440 445Glu Val Leu Arg Phe Leu Leu Ser Asn Ala Arg Trp Trp Leu Glu Glu 450 455 460Tyr Lys Phe Asp Gly Phe Arg Phe Asp Gly Val Thr Ser Met Met Tyr465 470 475 480Thr His His Gly Leu Gln Val Ala Phe Thr Gly Asn Tyr Ser Glu Tyr 485 490 495Phe Gly Phe Ala Thr Asp Ala Asp Ala Val Val Tyr Leu Met Leu Val 500

505 510Asn Asp Leu Ile His Gly Leu Tyr Pro Glu Ala Ile Thr Ile Gly Glu 515 520 525Asp Val Ser Gly Met Pro Thr Phe Ala Leu Pro Val Gln Asp Gly Gly 530 535 540Val Gly Phe Asp Tyr Arg Leu His Met Ala Val Pro Asp Lys Trp Ile545 550 555 560Glu Leu Leu Lys Gln Ser Asp Glu Ser Trp Lys Met Gly Asp Ile Val 565 570 575His Thr Leu Thr Asn Arg Arg Trp Ser Glu Lys Cys Val Thr Tyr Ala 580 585 590Glu Ser His Asp Gln Ala Leu Val Gly Asp Lys Thr Ile Ala Phe Trp 595 600 605Leu Met Asp Lys Asp Met Tyr Asp Phe Met Ala Leu Asp Arg Pro Ala 610 615 620Thr Pro Ser Ile Asp Arg Gly Ile Ala Leu His Lys Met Ile Arg Leu625 630 635 640Ile Thr Met Gly Leu Gly Gly Glu Gly Tyr Leu Asn Phe Met Gly Asn 645 650 655Glu Phe Gly His Pro Glu Trp Ile Asp Phe Pro Arg Ala Pro Gln Val 660 665 670Leu Pro Asn Gly Lys Phe Ile Pro Gly Asn Asn Asn Ser Tyr Asp Lys 675 680 685Cys Arg Arg Arg Phe Asp Leu Gly Asp Ala Asp Tyr Leu Arg Tyr Arg 690 695 700Gly Met Leu Glu Phe Asp Arg Ala Met Gln Ser Leu Glu Glu Lys Tyr705 710 715 720Gly Phe Met Thr Ser Asp His Gln Tyr Ile Ser Arg Lys His Glu Glu 725 730 735Asp Lys Met Ile Ile Phe Glu Lys Gly Asp Leu Val Phe Val Phe Asn 740 745 750Phe His Trp Ser Asn Ser Tyr Phe Asp Tyr Arg Val Gly Cys Leu Lys 755 760 765Pro Gly Lys Tyr Lys Val Val Leu Asp Ser Asp Ala Gly Leu Phe Gly 770 775 780Gly Phe Gly Arg Ile His His Thr Ala Glu His Phe Thr Ala Asp Cys785 790 795 800Ser His Asp Asn Arg Pro Tyr Ser Phe Ser Val Tyr Ser Pro Ser Arg 805 810 815Thr Cys Val Val Tyr Ala Pro Ala Glu 820 82532248DNATriticum sp. 32tggcggcggc gacgggatgg ctgcgccggc attcgcagtt tccgcggcgg ggctggcccg 60gccgtcggct cctcgatccg gcggggcaga gcggaggggg cgcggggtgg agctgcagtc 120gccatcgctg ctcttcggcc gcaacaaggg cacccgttca ccccgtgccg tcggcgtcgg 180aggttctgga tggcgcgtgg tcatgcgcgc gggggggccg tccggggagg tgatgatccc 240tgacggcg 24833244DNATriticum sp. 33tgcggcgacg ggatggctgc gccggcattc gcagtttccg cggcggggct ggcccggccg 60tcggctcctc gatccggcgg ggcagagcgg agggggcgcg gggtggagct gcagtcgcca 120tcgctgctct tcggccgcaa caagggcacc cgttcacccc gtgccgtcgg cgtcggaggt 180tctggatggc gcgtggtcat gcgcgcgggg gggccgtccg gggaggtgat gatccctgac 240ggcg 24434396DNATriticum sp. 34cggcggcggc gacgggatgg ctgcgccggc attcgcagtt tccgcggcgg ggctggcccg 60gccgtcggct cctcgatccg gcggggcaga gcggaggggg cgcggggtgg agctgcagtc 120gccatcgctg ctcttcggcc gcaacaaggg cacccgttca ccccgtaatt atttgcgcca 180cctttctcac tcacattctc tcgtgtattc tgtcgtgctc gcccttcgcc gacgacgcgt 240gccgattccg tatcgggctg cggtgttcag cgatcttacg tcggttccct cctggtgtgg 300tgatgtctgt aggtgccgtc ggcgtcggag gttctggatg gcgcgtggtc atgcgcgcgg 360gggggccgtc cggggaggtg atgatccctg acggcg 39635510DNAHordeum vulgare 35ttctgccacc accgggaaat ggacagcaaa tatacgagat tgacccaacg ctccgagact 60ttaagtacca tcttgagtat cggtatgctt cgcttctatt gtgtgcactt taaactttaa 120atacaattta cagtctttga taagatgtga atggctgctt gctgtgacac aaaactcttg 180aagttcgtag tcactcttgt gtgttcatgg ctctgaggtg acatggtaac cgaacaaaaa 240taggaaagtg gcaagaactg caatgtgagc taccgataag cacccattgt aattgggtac 300actgattaat atatgtcttg atgggttcta tgttttttca gtatctatgc caattgaaca 360acaatgccac ttcatttccc ctgtgttgct tttgtaagga tgaaacccat atgtccagat 420caaactgtac tagcagtctc actgtgcctt aatggatcaa aaacagatac agcctatata 480ggagaatacg ttcagacatt gatgaacacg 51036345DNAHordeum vulgare 36gcttcgcttc tattgtgtgc actttaaaaa caatttacag tctttgataa gatgtgaatg 60gctgcttgct gtgacacaaa actcttgaag ttcgtagtca ctcttgtgtg ttcatggctc 120tgaggtgaca tggtaaccga acaaaaatag gaaagtggca agaactgcaa tgtgagctac 180cgataagcac ccattgtaat tgggtacact gattaatata tgtcttgatg ggttctatgt 240tttttcagta tctatgccaa ttgaacaaca atgccacttc atttcccctg tgttgctttt 300gtaaggatga aacacatatg tccagatcaa actatactag cagtc 34537591DNAHordeum vulgare 37ttctgccacc accgggaaat ggacagcaaa tatacgagat tgacccaacg ctccgagact 60ttaagtacca tcttgagtat cggtatgctt cgcttctatt gtgtgcactt taaaaacaat 120ttacagtctt tgataagatg tgaatggctg cttgctgtga cacgaaactc ttgaagttcg 180tagtcactct tgtgtgttca tggttctgag gtaacatggt aaccgaacaa aaataggaaa 240gtggcaagca ctgcaatgtg agctactgat aaccacccat tgtaattggg tacactgatt 300aatatatatg tcttcatggg ctctattttt tttcaatatc tatgccaatt gaacaacaat 360gctttgtgga cgggtgttct tttaccctct tcttctatca atagatgata tgcatactca 420tgcgtatcct acaaaaaatt gaacaacaat gccactttcc cccgtgttgc ttttgtaagg 480atgaaacaca tatgtccaga tcaaactata ctagcagtct aactgtgcct taatggatca 540aaaacagata tagcctatac aggagaatac gttcagacat tgatgaacac g 591388PRTTriticum sp. 38Lys Arg Pro Lys Ser Leu Arg Ile1 53913PRTTriticum sp. 39Arg Val Phe Asn Tyr Gly Asn Lys Glu Val Ile Arg Phe1 5 104013PRTTriticum sp. 40Arg Arg Phe Asp Leu Gly Asp Ala Glu Phe Leu Arg Tyr1 5 104117PRTTriticum sp. 41Lys Val Val Leu Asp Ser Asp Ala Gly Leu Phe Gly Gly Phe Gly Arg1 5 10 15Ile4215PRTTriticum sp. 42Lys Tyr Gly Phe Met Thr Ser Asp His Gln Tyr Val Ser Arg Lys1 5 10 154317PRTTriticum sp. 43Arg Ser Asp Ile Asp Glu His Glu Gly Gly Met Asp Val Phe Ser Arg1 5 10 15Gly4416PRTTriticum sp. 44Lys Ile Asn Thr Tyr Ala Asn Phe Arg Asp Glu Val Leu Pro Arg Ile1 5 10 15459PRTTriticum sp. 45Arg Phe Leu Leu Ser Asn Ala Arg Trp1 54611PRTTriticum sp. 46Arg Gly His His Trp Met Trp Asp Ser Arg Val1 5 104716PRTTriticum sp. 47Arg Ile Tyr Glu Thr His Val Gly Met Ser Ser Pro Glu Pro Lys Ile1 5 10 154814PRTTriticum sp. 48Lys Ile Tyr Glu Ile Asp Pro Thr Leu Lys Asp Phe Arg Ser1 5 104916PRTTriticum sp. 49Arg Ile Tyr Glu Ser His Ile Gly Met Ser Ser Pro Glu Pro Lys Ile1 5 10 155017PRTTriticum sp. 50Arg Ala Ala Ile Asp Gln His Glu Gly Gly Leu Glu Ala Phe Ser Arg1 5 10 15Gly5116PRTTriticum sp. 51Lys Ile Asn Ser Tyr Ala Asn Phe Arg Asp Glu Val Leu Pro Arg Ile1 5 10 15

Claims

1-52. (canceled)

53. Wheat grain comprising a null allele of a gene on a long arm of chromosome 2 encoding wheat starch branching enzyme IIb (BEIIb), in combination with one or more null alleles of genes which encode starch branching enzyme IIa (BEIIa), granule bound starch synthase (GBSS), starch synthase II (SSII), or starch branching enzyme I (BEI).

54. The wheat grain of claim 53, wherein the gene encoding BEIIb is a wSBEII gene on a long arm of chromosome 2D.

55. The wheat grain of claim 53, wherein the gene encoding BEIIb corresponds to the partial BEIIb gene present on λ phage clone G5, wherein a sample of G5 has been deposited with the Australian Government Analytical Laboratories under Accession No. NM01/19255.

56. The wheat grain of claim 54, wherein the wSBEII gene comprises introns of the following sizes: intron 1, 148 base pairs; intron 2, 663 base pairs; intron 3, 465 base pairs; intron 4, 74 base pairs; intron 5, 181 base pairs; intron 6, 442 base pairs; intron 7, 79 base pairs; and intron 8, 178 base pairs.

57. The wheat grain of claim 53, wherein the gene encoding BEIIb encodes an RNA corresponding to a cDNA having a nucleotide sequence as set forth in SEQ ID NO: 6.

58. The wheat grain of claim 53, further comprising a null allele of a second gene encoding BEIIb.

59. The wheat grain of claim 53, wherein the grain of the plant comprises an altered amylose-to-amylopectin ratio.

60. The wheat grain of claim 54, further comprising a null allele of a second gene encoding BEIIb.

61. The wheat grain of claim 53, comprising more than one null alleles of genes which encodes BEIIa.

62. The wheat grain of claim 53, which is whole grain.

63. Wheat flour comprising a null allele of a gene on a long arm of chromosome 2 encoding wheat starch branching enzyme IIb (BEIIb), in combination with one or more null alleles of genes which encode starch branching enzyme IIa (BEIIa), granule bound starch synthase (GBSS), starch synthase II (SSII), or starch branching enzyme I (BEI).

64. A food product comprising wheat grain or wheat flour, wherein the wheat grain or wheat flour comprises a null allele of a gene on a long arm of chromosome 2 encoding wheat starch branching enzyme IIb (BEIIb), in combination with one or more null alleles of genes which encode starch branching enzyme IIa (BEIIa), granule bound starch synthase (GBSS), starch synthase II (SSII), or starch branching enzyme I (BEI).

65. The food product of claim 64, which is selected from the group consisting of breads, pasta, noodles, breakfast cereals, snack foods, cakes, pastries, foods containing flour and starch-based sauces.

66. A process of preparing a food product, comprising(i) obtaining wheat grain or wheat flour, wherein the wheat grain or wheat flour comprises a null allele of a gene on a long arm of chromosome 2 encoding wheat starch branching enzyme IIb (BEIIb), in combination with one or more null alleles of genes which encode starch branching enzyme IIa (BEIIa), granule bound starch synthase (GBSS), starch synthase II (SSII), or starch branching enzyme I (BEI), and(ii) processing the wheat grain or wheat flour to prepare the food product.

67. The process of claim 66, wherein the food product is selected from the group consisting of breads, pasta, noodles, breakfast cereals, snack foods, cakes, pastries, foods containing flour and starch-based sauces.

68. A process of preparing a product, comprising (i) obtaining wheat grain or wheat flour, wherein the wheat grain or wheat flour comprises a null allele of a gene on a long arm of chromosome 2 encoding wheat starch branching enzyme IIb (BEIIb), in combination with one or more null alleles of genes which encode starch branching enzyme IIa (BEIIa), granule bound starch synthase (GBSS), starch synthase II (SSII), or starch branching enzyme I (BEI),(ii) isolating starch from the wheat grain or wheat flour, and(iii) preparing the product from the starch.

69. The process of claim 68, wherein the product is a food product.

70. The process of claim 69, wherein the food product is selected from the group consisting of breads, pasta, noodles, breakfast cereals, snack foods, cakes, pastries, foods containing flour and starch-based sauces.

71. The process of claim 68, wherein the product is a non-food product.

72. The process of claim 71, wherein the non-food product is selected from the group consisting of films, coatings, adhesives, building materials, disposable goods and packaging materials.

Images