A method for assisted screening of wheat with different thousand-grain weight based on the A6886G SNP site in the TaHDA9-2A gene

Abstract

The present invention discloses a method for assisting screening of different thousand-grain weight wheats based on the A6886G SNP site in the TaHDA9-2A gene, comprising the following steps: detecting whether the genotype of the wheat to be tested is genotype I or genotype II based on the TaHDA9-2A gene, the thousand-grain weight of genotype I wheat> the thousand-grain weight of genotype II wheat; the wheat of genotype I is wheat whose genotype is AA homozygous based on the A6886G SNP site; the wheat of genotype II is wheat whose genotype is GG homozygous based on the A6886G SNP site; the A6886G SNP site is the 6886th nucleotide from the 5' end of SEQ ID NO:1 in the wheat genome. Experiments have shown that the wheat thousand-grain weight trait can be screened by detecting the genotype of the wheat to be tested based on the TaHDA9-2A gene. The present invention has important application value in wheat breeding.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a method for assisting in screening wheat with different thousand-grain weights based on the A6886G SNP site in the TaHDA9-2A gene. The A6886G SNP site is the 6886th nucleotide from the 5' end of SEQ ID NO: 1 in the wheat genome. Background Art

[0002] Wheat (Triticum aestivum L.) is one of my country's most important grain crops. Increasing wheat yield per unit area is a key measure to meet the ever-increasing demand for food and a strategic goal for ensuring food security. Number of spikes, number of kernels per spike, and 1000-kernel weight are the three key components of wheat yield. Harmonizing and improving these three factors is crucial for high-yield wheat breeding. Compared with number of spikes and number of kernels per spike, 1000-kernel weight has the greatest impact on wheat yield and is relatively less affected by environmental factors. Therefore, improving 1000-kernel weight is an important approach to increasing wheat yield.

[0003] At present, researchers have located a large number of QTLs regulating wheat thousand-grain weight. For example, Su et al. detected a total of 21 thousand-grain weight QTLs on chromosomes 1A, 1B, 2A, 2B, 2D, 3A, 3B, 4B, 5A, 5B, 6A, 6D and 7A using the "Shixin828xKenong2007" RIL population; MeCartney et al. detected QTL loci located on 1A, 2A, 2B, 3B, 6D and 7A using the "Opata85x W7984" RIL population; Su et al. detected a major thousand-grain weight QTL on chromosome 7A and named it TaTKW-7AL, which explained 19.7% of the phenotypic variation in thousand-grain weight; Huang et al. used the "AC Karma x 87E03" DH population detected QTL loci for thousand-grain weight on chromosomes 2B, 2D, 3B, 4B, 4D, and 6A, with one locus contributing as much as 26.3%. Using a recombinant inbred line population, Li et al. detected QTL loci controlling thousand-grain weight on chromosomes 1D, 3B, 5D, 6A, and 7D. However, because the phenotypic contribution of most QTLs is small and their reproducibility is poor across years and environments, QTLs are difficult to apply to the genetic improvement of wheat thousand-grain weight.

[0004] CAPS markers, also known as PCR-RFLP (polymerase chain reaction for restriction fragment length polymorphism), are a type of co-dominant molecular marker based on PCR that reveals information on restriction length variation of specific PCR fragments. The basic principle is to amplify the target DNA using PCR, and then digest the amplified product with specific endonucleases to cut it into fragments of different sizes, which are then directly resolved on gel electrophoresis. The restriction enzyme sites of different alleles are distributed differently, resulting in DNA fragment bands of different lengths. The advantage is that it avoids the tedious transfer and hybridization steps in RFLP analysis while maintaining the accuracy of RFLP analysis. However, it is relatively rare for SNPs to be located at restriction enzyme sites. Therefore, the dCAPS marker was proposed. That is, based on the CAPS marker, mismatched bases are introduced into the amplification primers, and new restriction enzyme action sites are introduced in combination with the SNP site to produce a polymorphism similar to the CAPS marker. Summary of the Invention

[0005] The purpose of the present invention is to screen or assist in screening wheat with different thousand-grain weights.

[0006] The present invention firstly protects a method for screening or auxiliary screening of wheat with different thousand-grain weights.

[0007] The method for screening or assisting in screening wheat with different thousand-grain weights protected by the present invention may specifically be method 1, which may include the following steps: detecting whether the genotype of the wheat to be tested is genotype I or genotype II based on the TaHDA9-2A gene, and the thousand-grain weight of genotype I wheat is greater than the thousand-grain weight of genotype II wheat;

[0008] The wheat of genotype I is a wheat having a genotype of AA homozygous based on the A6886G SNP site;

[0009] The wheat of genotype II is a wheat having a GG homozygous genotype based on the A6886G SNP site;

[0010] The A6886G SNP site is the 6886th nucleotide from the 5' end of SEQ ID NO: 1 in the wheat genome.

[0011] The method for screening or assisting in screening wheat of different thousand-grain weights protected by the present invention may specifically be method 2, which may include the following steps in sequence:

[0012] (A1) using the genomic DNA of the wheat to be tested as a template, PCR amplification is performed using primer pair A consisting of primer F1 and primer R1 to obtain PCR amplification product P1;

[0013] (A2) using the PCR amplification product P1 as a template, performing PCR amplification with primer pair B consisting of primer F2 and primer R2 to obtain a PCR amplification product P2;

[0014] (A3) digesting the PCR amplification product P2 with the restriction endonuclease EcoRV to obtain a digestion product; and then performing the following evaluation: if the digestion product is a single DNA fragment, the genotype of the wheat to be tested based on the TaHDA9-2A gene is genotype I; if the digestion product is two DNA fragments, the genotype of the wheat to be tested based on the TaHDA9-2A gene is genotype II;

[0015] The thousand-grain weight of wheat of genotype I is greater than that of wheat of genotype II;

[0016] The primer F1 is a single-stranded DNA molecule shown in SEQ ID NO: 4;

[0017] The primer R1 is a single-stranded DNA molecule shown in SEQ ID NO: 5;

[0018] The primer F2 is a single-stranded DNA molecule shown in SEQ ID NO: 6;

[0019] The primer R2 is a single-stranded DNA molecule shown in SEQ ID NO:7.

[0020] The method for screening or assisting in screening wheat of different thousand-grain weights protected by the present invention may specifically be method three, which may include the following steps in sequence:

[0021] (B1) using the genomic DNA of the wheat to be tested as a template, PCR amplification is performed using primer pair A consisting of primer F1 and primer R1 to obtain PCR amplification product P1;

[0022] (B2) using the PCR amplification product P1 as a template, performing PCR amplification on primer pair B consisting of primer F2 and primer R2 to obtain a PCR amplification product P2;

[0023] (B3) digesting the PCR amplification product P2 with the restriction endonuclease EcoRV to obtain a digestion product; and then performing the following evaluation: if the digestion product contains only a 100 bp DNA fragment, the genotype of the wheat to be tested based on the TaHDA9-2A gene is genotype I; if the digestion product contains only a 75 bp DNA fragment and a 25 bp DNA fragment, the genotype of the wheat to be tested based on the TaHDA9-2A gene is genotype II;

[0024] The thousand-grain weight of wheat of genotype I is greater than that of wheat of genotype II;

[0025] The primer F1 is a single-stranded DNA molecule shown in SEQ ID NO: 4;

[0026] The primer R1 is a single-stranded DNA molecule shown in SEQ ID NO: 5;

[0027] The primer F2 is a single-stranded DNA molecule shown in SEQ ID NO: 6;

[0028] The primer R2 is a single-stranded DNA molecule shown in SEQ ID NO:7.

[0029] The method for screening or assisting in screening wheat of different thousand-grain weights protected by the present invention may specifically be method 4, which may include the following steps in sequence:

[0030] (B1) using the genomic DNA of the wheat to be tested as a template, PCR amplification is performed using primer pair A consisting of primer F1 and primer R1 to obtain PCR amplification product P1;

[0031] The primer F1 is a single-stranded DNA molecule shown in SEQ ID NO: 4;

[0032] The primer R1 is a single-stranded DNA molecule shown in SEQ ID NO: 5;

[0033] (2) Sequence the PCR amplification product P1 and then perform the following evaluation:

[0034] If the nucleotide sequence of the PCR amplification product P1 is as shown in SEQ ID NO: 2, positions 6562 to 7444 from the 5' end, the genotype of the wheat to be tested based on the TaHDA9-2A gene is genotype I;

[0035] If the nucleotide sequence of the PCR amplification product P1 is as shown in SEQ ID NO: 3, positions 6562 to 7444 from the 5' end, the genotype of the wheat to be tested based on the TaHDA9-2A gene is genotype II;

[0036] The thousand-grain weight of wheat of genotype I is greater than that of wheat of genotype II.

[0037] The present invention also provides a kit for identifying or assisting in identifying the thousand-grain weight of wheat. The kit may include a substance for detecting whether the genotype of the wheat to be tested is genotype I or genotype II based on the TaHDA9-2A gene;

[0038] The genotype I is the TaHDA9-2A gene with the genotype of AA homozygous based on the A6886G SNP site;

[0039] The genotype II is the TaHDA9-2A gene with a homozygous genotype of GG based on the A6886G SNP site;

[0040] The A6886G SNP site is the 6886th nucleotide from the 5' end of SEQ ID NO: 1 in the wheat genome.

[0041] The kit may specifically be composed of substances for detecting whether the genotype of the wheat to be tested based on the TaHDA9-2A gene is genotype I or genotype II.

[0042] Any of the above-mentioned substances for detecting whether the genotype of the wheat to be tested is genotype I or genotype II based on the TaHDA9-2A gene may include a primer pair A consisting of primer F1 and primer R1 and / or a primer pair B consisting of primer F2 and primer R2;

[0043] The primer F1 is a single-stranded DNA molecule shown in SEQ ID NO: 4;

[0044] The primer R1 is a single-stranded DNA molecule shown in SEQ ID NO: 5;

[0045] The primer F2 is a single-stranded DNA molecule shown in SEQ ID NO: 6;

[0046] The primer R2 is a single-stranded DNA molecule shown in SEQ ID NO:7.

[0047] Any of the above-mentioned materials for detecting whether the genotype of the wheat to be tested is genotype I or genotype II based on the TaHDA9-2A gene can specifically be composed of the primer pair A and the primer pair B.

[0048] Any of the above-mentioned materials for detecting whether the genotype of the wheat to be tested is genotype I or genotype II based on the TaHDA9-2A gene can specifically be composed of the primer pair A.

[0049] Any of the above-mentioned substances for detecting whether the genotype of the wheat to be tested is genotype I or genotype II based on the TaHDA9-2A gene may further include a restriction endonuclease EcoRV.

[0050] Any of the above-mentioned materials for detecting whether the genotype of the wheat to be tested is genotype I or genotype II based on the TaHDA9-2A gene can specifically be composed of the primer pair A, the primer pair B and the restriction endonuclease EcoRV.

[0051] The present invention also protects the use of any of the above-mentioned kits, which may be at least one of (z1) to (z4):

[0052] (z1) Screening or assisting in screening wheat of different thousand-kernel weights;

[0053] (z2) Identify or assist in identifying the thousand-grain weight of wheat;

[0054] (z3) Identifying or assisting in identifying the genotype of the wheat TaHDA9-2A gene;

[0055] (z4) Wheat breeding.

[0056] The present invention also protects the molecular marker shown in SEQ ID NO: 1.

[0057] The present invention also protects the use of the molecular marker represented by SEQ ID NO: 1, which can be at least one of (z1) to (z4):

[0058] (z1) Screening or assisting in screening wheat of different thousand-kernel weights;

[0059] (z2) Identify or assist in identifying the thousand-grain weight of wheat;

[0060] (z3) identifying or assisting in identifying the genotype of the wheat TaHDA9-2A gene;

[0061] (z4) Wheat breeding.

[0062] In the above, when the genotype of the A6886G SNP site in the molecular marker is AA homozygous, the wheat is determined to be genotype I; when the genotype of the A6886G SNP site in the molecular marker is GG homozygous, the wheat is determined to be genotype II; the A6886G SNP site is the 6886th nucleotide from the 5' end of SEQ ID NO:1 in the wheat genome; the thousand-grain weight of the wheat of genotype I is greater than the thousand-grain weight of the wheat of genotype II.

[0063] In the above, the > may specifically be a statistical >.

[0064] Experiments have shown that the method provided by the present invention can be used to detect the genotype of the wheat to be tested based on the TaHDA9-2A gene, and can screen or assist in screening the wheat thousand-grain weight trait. The present invention has important application value in the process of wheat molecular marker-assisted breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] Figure 1 These are the genotype detection results of the TaHDA9-2A gene in some wheat varieties in natural populations. DETAILED DESCRIPTION

[0066] The present invention will be further described in detail below in conjunction with specific embodiments. The examples provided are only for illustrating the present invention and are not intended to limit the scope of the present invention. The examples provided below can serve as a guide for further improvements by those skilled in the art and are not intended to limit the present invention in any way.

[0067] Unless otherwise specified, the experimental methods in the following examples are conventional methods and were performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials and reagents used in the following examples, unless otherwise specified, were all commercially available.

[0068] The wheat materials used in the following examples were all from the China National Crop Germplasm Bank (website: http: / / icscaas.com.cn / jiguoku / zhongzhiku.htm), and material information can be found on the China Crop Germplasm Information Network (website: http: / / icgr.caas.net.cn). Since the wheat materials are all cultivated varieties, they are usually assumed to be highly homozygous plant materials with homozygous genotypes.

[0069] Example 1. Discovery of the A6886G SNP in the wheat TaHDA9-2A gene and establishment of a wheat genotyping method based on the TaHDA9-2A gene

[0070] 1. Discovery of the A6886G SNP in the TaHDA9-2A gene in wheat

[0071] After extensive experiments, the inventors of the present invention discovered a single-nucleotide polymorphism (SNP) site in the wheat TaHDA9-2A gene (nucleotide sequence shown in SEQ ID NO: 1), designated A6886G SNP. The A6886G SNP is located at position 6886 from the 5' end of SEQ ID NO: 1, and the genotypes are AA homozygous and GG homozygous. Because genomic DNA is a double-stranded DNA molecule composed of two reverse-complementary single-stranded DNA molecules, the protein-encoding DNA molecule is generally designated as a sense DNA molecule, and the reverse-complementary DNA molecule to the sense DNA molecule is designated as an antisense DNA molecule. The genotypes of the A6886G SNP are all antisense DNA genotypes.

[0072] Wheat is divided into two genotypes based on the differences in the TaHDA9-2A gene: TaHDA9-2A-A (hereinafter referred to as genotype I) and TaHDA9-2A-G (hereinafter referred to as genotype II). The nucleotide sequence of the TaHDA9-2A gene in genotype I wheat is shown in SEQ ID NO:2. The nucleotide sequence of the TaHDA9-2A gene in genotype II wheat is shown in SEQ ID NO:3.

[0073] 2. Synthesis of Primer Pair A and Primer Pair B for Amplification of Target Sequences Including the A6886G SNP

[0074] Primer pair A and primer pair B were designed and synthesized for amplifying a target sequence including the A6886G SNP. Primer pair A consisted of primer F1 and primer R1. Primer pair B consisted of primer F2 and primer R2.

[0075] The nucleotide sequences of each primer are as follows:

[0076] Primer F1: 5′-GATCGAACGGTCAAAGTTATTGCGCGGG-3′ (SEQ ID NO: 4)

[0077] Primer R1: 5′-GCGTGCCTTGGTTATCTCTTACCCGAGCTC-3′ (SEQ ID NO: 5)

[0078] Primer F2: 5′-CTAGTGAAATGTACTCAGGGGTCTA-3′ (SEQ ID NO: 6)

[0079] Primer R2: 5′-GCCGCAAGTAATAGAACCCTGATAT-3′ (SEQ ID NO: 7)

[0080] The target sequence amplified by primer pair A is shown in SEQ ID NO: 1 at positions 6562 to 7444 from the 5' end.

[0081] 3. Establishment of a wheat genotyping method based on the TaHDA9-2A gene

[0082] 1. Extract the genomic DNA of the wheat to be tested.

[0083] 2. Using the genomic DNA of the wheat to be tested in step 1 as a template, PCR amplification is performed using primer pair A consisting of primer F1 and primer R1 to obtain PCR amplification product P1.

[0084] The reaction system was 10 μL, consisting of 3.6 μL ddH2O, 5 μL 2×Taq enzyme Mix, 0.2 μL primer F1 aqueous solution (concentration of 10 μmol / L), 0.2 μL primer R1 aqueous solution (concentration of 10 μmol / L) and 1 μL genomic DNA of the wheat to be tested (concentration of 20 ng / μL).

[0085] 2×Taq enzyme Mix was a product of Nanjing Novozymes Co., Ltd., with the product catalog number being P131.

[0086] The reaction conditions were as follows: 95°C for 3 min; 95°C for 15 s, 60°C for 15 s, 72°C for 20 s, 32 cycles; 72°C for 10 min; and storage at 16°C.

[0087] 3. After completing step 2, using the diluted solution of PCR amplification product P1 (a mixture of 1 volume part PCR amplification product P1 and 9 volume parts water) as a template, PCR amplification is performed using primer pair B consisting of primer F2 and primer R2 to obtain PCR amplification product P2.

[0088] The reaction system was 10 μL, consisting of 3.6 μL ddH2O, 5 μL 2×Taq enzyme Mix, 0.2 μL primer F2 aqueous solution (concentration of 10 μmol / L), 0.2 μL primer R2 aqueous solution (concentration of 10 μmol / L) and 1 μL dilution of PCR amplification product P1.

[0089] The reaction conditions were as follows: 95°C for 3 min; 95°C for 15 s, 60°C for 15 s, 72°C for 10 s, 32 cycles; 72°C for 10 min; and storage at 16°C.

[0090] 4. The PCR amplification product P2 obtained in step 3 was digested with the restriction endonuclease EcoRV to obtain a digestion product; the digestion product was subjected to 4% agarose gel electrophoresis and the following judgment was made: if the digestion product was band type A (displaying one band of 100 bp), the wheat A6886G SNP to be tested was AA homozygous, that is, the genotype of the wheat to be tested based on the TaHDA9-2A gene was genotype I; if the digestion product was band type B (displaying two bands of 75 bp and 25 bp, respectively), the wheat A6886G SNP to be tested was GG homozygous, that is, the genotype of the wheat to be tested based on the TaHDA9-2A gene was genotype II.

[0091] Example 2: Association Analysis and Verification between Wheat TaHDA9-2A Gene Genotype and Wheat Thousand-Grain Weight

[0092] 1. Genotyping of the TaHDA9-2A gene in wheat in natural populations

[0093] Genotyping of each wheat variety in a natural population was performed using the method described in step 3 of Example 1. The natural population consisted of 298 wheat varieties (all hexaploid). The wheat variety names are detailed in Table 1.

[0094] Some test results can be found in Figure 1 (M is a DNA marker, and the other lanes represent different wheat varieties; G is a wheat variety of genotype II, and A is a wheat variety of genotype I).

[0095] The genotypes of 298 wheat varieties based on the TaHDA9-2A gene are shown in Table 1: the genotypes of 128 wheat varieties based on the TaHDA9-2A gene are genotype II, and the genotypes of 170 wheat varieties based on the TaHDA9-2A gene are genotype I.

[0096] Table 1

[0097]

[0098]

[0099]

[0100]

[0101]

[0102] Note: I is genotype I, II is genotype II.

[0103] 2. Thousand-grain weight trait detection

[0104] 298 wheat varieties were planted in four different environments, and the average thousand-grain weight of the two genotypes of wheat was calculated after harvest.

[0105] The statistical results are shown in Table 2.

[0106] Table 2

[0107]

[0108] Note: P value is the significance level of the association analysis. * " indicates P < 0.05," ** ” indicates P < 0.01.

[0109] 3. Correlation Analysis

[0110] Tassel2.1 software was used to select the single linear model + population structure (GLM + Q) method to conduct an association analysis between the genotype of the wheat TaHDA9-2A gene and 1000-grain weight in the natural population. The results are shown in Table 2.

[0111] The results showed that, in a natural population of 298 wheat varieties, the thousand-grain weight of genotype I wheat was greater than that of genotype II wheat; the ">" represents a statistically significant difference. Studies of natural populations have shown that genotype I is an excellent genotype for increasing thousand-grain weight in wheat.

[0112] The above results indicate that the genotype of the wheat to be tested based on the TaHDA9-2A gene can be used to screen or assist in screening the thousand-grain weight trait of wheat, which has important application value in the process of wheat molecular marker-assisted breeding.

[0113] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be practiced over a wide range of parameters, concentrations, and conditions without departing from the spirit and scope of the present invention and without unnecessary experimentation. Although specific embodiments have been given herein, it should be understood that further modifications may be made to the present invention. In summary, this application is intended to encompass any variations, uses, or improvements to the present invention, including those made by conventional techniques known in the art that depart from the scope of the present invention. Applications of the essential features may be made within the scope of the following claims. <110> Hebei Normal University <120> A method for assisted screening of wheat with different thousand-grain weight based on the A6886G SNP site in the TaHDA9-2A gene <160> 7 <170> PatentIn version 3.5 <210> 1 <211> 7523 <212> DNA <213> Triticum aestivum L. <220> <221> <222> (6886)…(6886) <223> n is a or g <400> 1 agggcgagac cacctcctcc cccgcatctt gcttggattt tgttctagaa ttcctcgtct 60 cttgcttgct gttatggtca gacttctggc ccctggagga attttcgcta cctgctggat 120 acctgttctc atcaagatcc gaatgaccca gccgcccaca agagaaacag aagtatggga 180 tttgttcata ttgtatgtca taagaatcca ccttctttct cctcgccgaa tcaataagca 240 cccatctcct cattggttta ttgacctcca ccgatactct atctcgaagg tagcctccgt 300 tgtgatcaaa cttagcaatc tgaacctctt tatctatcat cctggctatt gggaggctcc 360 atgcatcatc tctcaaatta tagggtaaat taagaaccct agcccaaacc tgcaggcgat 420 cgaacttgac ctcatctggg cgcatgcact cttcaaactc ggccaagatg accgcattct 480 tgttgatgtg ccaaggcgat cctgcccaaa 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cgaagtattc aatgtattca ttgtcaggga tctctgcaaa gaaaatgcca tgtaaaactc 2280 cggacaatgg ataccacta aggagaatt gcttatggag agtagttcta tagggaag 2340 gtattgggac atattggat ttttcacctt acagatcaa gaaagaga 2400 aaagaatac aaaaaatg ccgctatatt gaggattat actagctact gtcactcaa 2460 ccaacaaggt taagggagc agaacaaaca aatgagatac catttgggag ttctgtgtct 2520 agaagaaccc cagtttcaac agcccaacag cgtgctacat tctctttggt gtacccacca 2580 cctcctgtca cctgcatgtt tgaaaatata tgaatatcct agaaagagca aaataatca 2640 tacaatgata gttttctccc ttaccagcag cgggatttta aatttcttaa caaactttac 2700 acattcagca tggcctgttt atggaaacag atcaagttag tagtataat aatagattat 2760 ccgagtgagt tagctcaatg aaactaaagt ccaaaaggacc caaataccat caacgcaaga 2820 aaattctcag aatacacaaa ttcaacagtg gggacaaggt aatcacggtt agtttataca 2880 tctgatgttt tgccaaatct atagtagcac gtaatatcaa gttttagaga gtaccttcta 2940 ttgaaagatt gaaacaccct aggcggtctc gcgccaatga atcagcccca cattgaagaa 3000 caatagcacc tggcagatat gtctcaacaa ctttggcaat aatctacatg ataagcaaca 3060 ttacgtacct caataagagt agacaag aacaagaata acataaagt aatgcacatc 3120 gacttttacc gttttaaaaa ggcgagtaaa gctgctgtca tctatgccat ctttaagtgg 3180 gatgttgatt gcataatatc ttccttccct gtctcctata tccttcaaaa tgacagccag 3240 gtaaattgat atgaagagca catccttaca aaatgaagct aaatttttta aagctctcat 3300 catcattact taacagcacg tgatattgaa ctatttaaaa acaaagcaag atgcaggctc 3360 tgtaagtgca ttttttcag catgttaata aatagaaaag cgcacatttc tgctcatatc 3420 gaaccttgcc attcctctat gtgagttaca aactctagtt gatgagataa ttttgattgg 3480 agaactcgca actcaatcaa agacaattgc gtaaagaaac ccttccactc atacagatgc 3540 acgcataaa tatgtgcaaa atagcatta tggcaaaagt aaagacccaa tacaaaaacg 3600 atgaagtgaa tcttcaaatg atggttcaag catgaccgca agggttgtgt tggtcagttg 3660 acagcttcta ttcttctaag ctcagatgat aatacatgca tcagaatgaa gaacatcctt 3720 tttggcacta ccgcttatca aattagatc ataaacctcc accattccca aatatgatgc 3780 tggctatttg gatagaatac cgcacgaaga ttgtccagta atagagacaa gagttcaatg 3840 ataaaagtca catacttaca tgttctactt ctaggtggac aggagagagt gaagaaaaga 3900 agaaactgaa gtatgtcacg atggttccta aaattattga ttacctccag taccatcagc 3960 tttgagtaat attatgctaa aacccattgt aactcagttt gtaaatgtct cttctatttc 4020 attcaattga gagttcatat tccaacttta atcgacataa gtaaataggg gacaacaaac 4080 accataaaac tcatgcccaa tattgagcac aaactaagat acaaactgaa aataatagcg 4140 atgaactgct gagaaattaa gtaaaagcaa ctacataaaa tgtatattca taaagcttgt 4200 cagtggaaca ttaccttaat atcacctgtg ccaggaaaga acatgtcacc atacttgtgg 4260 aaacttacag tcattaccct gcaaatatgt atagacaaaa tcagattgga aatctaaagg 4320 ctctcaaaga gacagcggca ttactataca gcaacctcac actagacaga gccagagatt 4380 agcaactaaa atgaatatat ggtgataaca aaagagtagg catctatgga ggctctgaag 4440 atcagtgaga cacatcctat caacaattac agtcatgtat tttactatcc ataaaagcat 4500 gaacctgtca gtgaaataga aggcttcttc aactccatct ccatgatgga catcaatgtc 4560 aattagaga accctagcat gatacttgag aagctccaga attcccaaaa ccaggtcatt 4620 aatgtagcag aagcctgacg cctcacactt ctttgcatga tgcagcccac cagcccaatt 4680 atagcaata tcacatgttt tatgatttag tcttcgagcc gcatctgaaa aataaagaaa 4740 tgcttagaga gatcacaaaa cagaaagaag cattggcaat tttcctatta cctagagttc 4800 ctccagcata gatttggcag aactcaaaca aatcatcaaa gaccgggcag tcctctccaa 4860 ggttgtctgg gacaatgatt acaatgacga agtcagataa gtgaagtaag aataccccaa 4920 atacatggag atgtgattt tagcaattcc attagtaca acaggcatat tgtggaaatt 4980 atagcagaca gttcttcatt taagtgcaaa cagctaagct tccatttgga tacaacaatt 5040 tgttgagaga tcagaccatc ttactgtgtt ccgcttctat tttaagcata catccaaaga 5100 cctaagctaa tatagcacta ccttagattc aggtaaatca tttgggagt tttgggttag 5160 gtgtcagaag ttacgcaagt ttgaccaaat gctgctggcg ttggttcat ataaacaact 5220 gtggcgagcc acaaattctt aggcacatgg cacatggtcc actcgtcata cacatgttct 5280 aaccaagttt tgacatgatt ttttacagc taaatataag caagcaatat atgcggcgtg 5340 tattttattg tgaaatagc aaagcctggg agagtagtct gtaaagaga aaatatcat 5400 cattcataca tctagttaat tcacttgcat acagtgctg ggtatcagga gttattcggt 5460 gcaagaatttc cacataatca gcagaatgga actgcgcaag ctctgttgga tatgctttgt 5520 ggggtctctg caaatccaa tgggagcaa aaggtaaa cgacacattt aaacaattca 5580 taattacaa gaaaggggat gaacaataat tcaataccga catatatctc cattttctg 5640 tgagatcat atgatacac gagatggtgt gtcatgcaaa ggcgatgcgg ttcatcggg 5700 tgatttggcc caagtacac attgccaca tcccctgaga tgttcggtca cagaaaagta 5760 accaagtaag caatcataa aggttgaaca gcaaaaaac ttatatagtg ttgaaggtgt 5820 gtggattttc tacaacaac acagttaagg aacttagtcat gagaaccaa tcaatact 5880 agcgcagaaa ttttaccat ttgactcct tatcgaccaa gatgaagca tacactaatg 5940 caattttac atgcaccta cggctagggt tgaacaacc aaggaatag taggccaag 6000 aacagggaca cgccaatttct agttgaacag atcacaatgg cacgtagcat caagtaattt 6060 tccttgtcgc ataaccatag cacaggattc gactgcaatg acccatgctg acgtcttgcc 6120 actacaaatc ggcgaaacaa gcacaacata ccactatgcc cacacagtat ttccccctac 6180 tttcgaaatt gccagatgag acacctagga ggagtaagag taacgaattg cgactagggg 6240 agaactaggt atgaacggtg gttggttggg gggtggggtt gcagaagagg gggcgtaggg 6300 aacataccat cgtagaaata ggatatcctg tctttctcca acatgaccgc gctctcgagc 6360 ggccgcctgc ctctacaggc ggaggcggcg ccaaatgggg gcagccgggg aagccgacag 6420 cgagttcgcg acggcggagg cggaggcggc ggcggaagtt ggggcggtcg tgcgtgagga 6480 ggaaggggaa aaccgagcga ggcgatggct ctggggaacg ttctggaccg gcatggacag 6540 tcaggatcgt gttggtacca tgatcgaacg gtcaaagtta ttgcgcgggt gaattatttc 6600 gcctcgattg gtactcaaaa gtactgggtt ttttttttag ttggaagtac tgctatttct 6660 gattctgtac aaaatactgt attactcccc attagttagt cttcgtcggt ctaaaaacaa 6720 ctgctactca aacgaatgta tgtaacgaaa tacgtctaga tatcttcgct tgagcactag 6780 ttatatttag atatagcgag tattttctga actagtgaaa tgtactcagg ggtctaactt 6840 agctgaacca ttctttggt caacatttt agccgagcct gtaggnacat caggttcta 6900 ttacttgcgg ctggattcaa tttattt aaacatctag gtggctaaat caacatattt 6960 aatttagtat gtatagta gttaataaat gatttatta attttggtcc aatctaaca 7020 caagcaact tgagaccata ccatgttatc tcagcttatg cttctttact cagggaatt 7080 aaattaata ttgtagggg aattagtatt attcatgtgt agccggcatc agaacattgt 7140 tcatgactat atggataagt atgcccacac aaaacaactg aagatcatg gatatcgcct 7200 agagggggtg cataggcgct ttagataat tacggttttag ccttgaacaa atgtgaaata 7260 aaactagttt taatttgtca agcacaaac ctaaattgaa taggctcacc tatgtgcacc 7320 aaaacttat actaagtagg aacaact aagtgatagc aagatatata tcatgtcata 7380 ttatggctat tgcaagtaa agtgcatat taaagagctc gggtaagaga taaccaaggc 7440 acggcggagac gacgttgtat ccccaagttc acacccttgc agatactaat cttcgttttgg 7500 agcggcgtgg aggcacaatg ctt 7523 <210> 2 <211> 7523 <212> DNA <213> Triticum aestivum L. <400> 2 agggcgagac cacctcctcc cccgcatctt gcttggattt tgttctagaa ttcctcgtct 60 cttgcttgct gttatggtca gacttctggc cctggagga atttcgcta cctgctggat 120 acctgtctc atcaagatcc gaatgaccca gccgcccaca agagaaacag aagtatggga 180 tttgttcata ttgtatgtca taagaatcca ccttctttct cctcgccgaa tcaataagca 240 cccatctcct cattggttta ttgacctcca ccgatactct atctcgaagg tagcctccgt 300 tgtgatcaaa cttagcaatc tgaacctctt tatctatcat cctggctatt gggaggctcc 360 atgcatcatc tctcaaatta tagggtaaat taagaaccct agcccaaacc tgcaggcgat 420 cgaacttgac ctcatctggg cgcatgcact cttcaaactc ggccaagatg accgcattct 480 tgttgatgtg ccaaggcgat cctgcccaaa ctctgtcacg gtctcgctcg gactcaaatt 540 cagccacaaa catattcgct ccagtcgagc agaactgcag tcctctaggg tttccccaag 600 ccggtcgcaa agcattgcca atggtgtgta tatgatactg attgcggtac agaaccttac 660 cagccagcaa ccacttcacc ggagcaccat ccagacggtc atctatgacg agaggagtcg 720 cctcctcctc ggatatgtcc agctttccca gcgcttcctc aagtttctc ctcgccgatc 780 tggtattatt cgatcccgag ttcgcctgca tcgtctccgc cgcgggtggt gtagccatca 840 ccccgccga gtccttgggg cggcccgccg atgagatccg cggccgccga tcggtttcca 900 cgtcgaaacc ctagcgccac cagggagccc tagtttcgg ggaaaaagta gtcctctatg 960 aatgttatca attagttgct tttccaaatt caaaaaggcc atacatacaa atacaagaat 1020 aatccaaagg ctaaataaaa aattacgtac gtactgaaat gatggtattt taagtaagta 1080 caaatgtgag atcgtacaat gctgtacaag tcagcaagtg ttacgttctc ccacaatgct 1140 ctccagccta gcgtcagatc ctaaaatgtc aatcagccaa ctattcccca tcccacgatg 1200 ttataaactg aacagcagct gctgcgggaa agactttcag cttccaccta catacatcgt 1260 caagatgccg atctcaatgt ccgccgtcat cgtgatcatt gtcgttgtcg ccttcatagt 1320 actcgtcgtc acgatgaacc tgcttgtctt gggtatgctc tgcatgacat aatacacact 1380 tctgtaagaa aatagaaata tgatggcacg gttcagaagg actggagaca ataccaggca 1440 aggagctggc tgaatgaaga agaatacaac caatttaaga ataaatagat aacaaaatgg 1500 accgcattta cttcatttca ggggaaattt ctcattatgt ggcgcgcaca tgcttcgaga 1560 ggacaatggc gagttagcac ggagaaatca tcaagttttt acttgtccat atgagcactg 1620 caagacatgc cagaggtatt tcacttacga tcaacacgtt catcaggatc cagctcgtct 1680 tcatcaaaat ccgggacata gaaatcgggt ggaacctgca aaataaactc attatgtatc 1740 cggtctctgc tcagaaacaa caaactacac tatcctggtg gaagcaagaa agttaaagac 1800 aacaaaagat gcaaaggcat attatagtct tcacatggta tccatccccg gtttcaattg 1860 ggatgtttca acaatcaaca tgcggacgag aatacaaact aacctcttgc atctgaacgc 1920 caggtgcatg ttgtatggcc cgcaaactct ccattacttg cactttgatt gaactgagat 1980 acgtcttact attcaagttg tcctggtcca ttcatcaata ggaagacaaa tatcatttgc 2040 acatctacac acctctaaag aaagaaattg atattctagt taagcgtacc siacatataa 2100 aaacatgacg caccaacaa cagttaaatc cacaacca ggcaggcata gagttagaaa 2160 gatcagtcag aaggttcat accatgttca gatttggtac ttcaatgta taatctggac 2220 cgaagtattc aatgtattca ttgtcaggga tctctgcaaa gaaaatgcca tgtaaaactc 2280 cggacaatgg ataccacta aggagaatt gcttatggag agtagttcta tagggaag 2340 gtattgggac atattggat ttttcacctt acagatcaa gaaagaga 2400 aaagaatac aaaaaatg ccgctatatt gaggattat actagctact gtcactcaa 2460 ccaacaaggt taagggagc agaaaaaaaatgagatac catttgggg ttctgtgtct 2520 agaagaaccc cagttcac agcccacag cgtgctacat tctctttggt gtaccacca 2580 cctcctgtca cctgcatgtt tgaaaata tgaatatcct agaagagca aaaataatca 2640 tacaatgata gttttctccc taccagcag cgggatttta aatttcttta caactttac 2700 acattcagca tggcctgttt atggaacag atcagttag tagtataat atagattat 2760 ccgagtgagt tagctcaatg aaactaaagt ccaaaaggacc caaataccat caacgcaaga 2820 aaattctcag aatacacaaa ttcaacagtg gggacaaggt aatcacggtt agtttataca 2880 tctgatgttt tgccaaatct atagtagcac gtaatatcaa gttttagaga gtaccttcta 2940 ttgaaagatt gaaacaccct aggcggtctc gcgccaatga atcagcccca cattgaagaa 3000 caatagcacc tggcagatat gtctcaacaa ctttggcaat aatctacatg ataagcaaca 3060 ttacgtacct caataagagt agacaag aacaagaata acataaagt aatgcacatc 3120 gacttttacc gttttaaaaa ggcgagtaaa gctgctgtca tctatgccat ctttaagtgg 3180 gatgttgatt gcatatatc ttccttccct gtctcctata tccttcaaaa tgacagccag 3240 gtaaattgat atgaagagca catccttaca aaatgaagct aaatttttta aagctctcat 3300 catcattact taacagcacg tgatattgaa ctatttaaaa aaaagcaag atgcaggctc 3360 tgtaagtgca tttttttcag catgttaata aatagaaaag cgcacatttc tgctcatatc 3420 gaaccttgcc attcccttat gtgagttaca aactctagtt gatgagataa ttttgattgg 3480 agaactcgca actcaatca agacaattgc gtaagaaac ccttccactc atacagatgc 3540 acgcataaaa tatgtgcaaa atagcatta tggcaaagt aaagacccca tacaaaacg 3600 atgaagtgaa tctcaatg atggttcaag catgaccgca agggttgtgt tggtcagttg 3660 acagcttcta ttctctaag ctcagatgat atacatgca tcagaatgaa gaacatcctt 3720 tttggcacta ccgcttatca atatagatc aaacctcc accattcccca atatgatgc 3780 tggctatttg gatagaatac cgcacgaaga tgtccagta atagacaca gagttcaatg 3840 aaaagtca catacttaca tgttctactt ctaggtggac aggagagt gagaaaga 3900 agaaactgaa gtatgtcacg atggttccta aaattattga taccactccag taccatcagc 3960 tttgagtaat attatgctaa aacccattgt aactcagttt gtaaatgtct cttctatttc 4020 attcaattga gagttcatat tccaacttta atcgacata gtaatagggg gacaac 4080 accataaac tcatgcccaa tattgagcac aaaactgaa aataatagcg 4140 atgaactgct gagaaattta gtaaaagcaa ctacataaaa tgtatattca taagcttgt 4200 cagtggaaca ttaccttaat atcacctgtg ccaggaaaga acatgtcacc atacttgtgg 4260 aaacttacag tcattaccct gcaaatatgt atagacaaaa tcagattgga aatctaaagg 4320 ctctcaaaga gacagcggca ttactataca gcaacctcac actagacaga gccagagatt 4380 agcaactaaa atgaatatat ggtgataaca aaagagtagg catctatgga ggctctgaag 4440 atcagtgaga cacatcctat caacaattac agtcatgtat tttactatcc ataaaagcat 4500 gaacctgtca gtgaaataga aggcttcttc aactccatct ccatgatgga catcaatgtc 4560 aatatagaga accctagcat gatacttgag aagctccaga attcccaaaa ccaggtcatt 4620 aatgtagcag aagcctgacg cctcacactt ctttgcatga tgcagcccac cagcccaatt 4680 aatagcaata tcacatgttt tatgatttag tcttcgagcc gcatctgaaa aataaagaaa 4740 tgcttagaga gatcacaaaa cagaaagaag cattggcaat tttcctatta cctagagttc 4800 ctccagcata gatttggcag aactcaaaca aatcatcaaa gaccgggcag tcctctccaa 4860 ggttgtctgg gacaatgatt acaatgacga agtcagataa gtgaagtaag aataccccaa 4920 atacatggag atgtgattt tagcaattcc attagtaca acaggcatat tgtggaaatt 4980 atagcagaca gttcttcatt taagtgcaaa cagctaagct tccatttgga tacaacaatt 5040 tgttgagaga tcagaccatc ttactgtgtt ccgcttctat tttaagcata catccaaaga 5100 cctaagctaa tatagcacta ccttagattc aggtaaatca tttgggagt tttgggttag 5160 gtgtcagaag ttacgcaagt ttgaccaaat gctgctggcg ttggttcat ataaacaact 5220 gtggcgagcc acaaattctt aggcacatgg cacatggtcc actcgtcata cacatgttct 5280 aaccaagttt tgacatgatt ttttacagc taaatataag caagcaatat atgcggcgtg 5340 tattttattg tgaaatagc aaagcctggg agagtagtct gtaaaagaga aaaatatcat 5400 cattcataca tctagttaat tcacttgcat acaagtgctg ggtatcagga gttattcggt 5460 gcaagaattc cacataatca gcagaatgga actgcgcaag ctctgttgga tatgctttgt 5520 ggggtctctg caaaatccaa tgggagcaaa ataaggtaaa cgacacattt aaacaattca 5580 taattacaaa gaaaggggat gaacaataat tcaataccga catatatctc cattttcttg 5640 tgaagatcat atgataacac gagatggtgt gtcatgcaaa ggcgatgcgg tttcatcggg 5700 5760 accaagtag aatcataaa aggttgaaca gcaaaaaaaac ttatatagtg ttgaaggtgt 5820 gtggatttc tacaaacaac acagttaagg aactagtcat gagaaccaaa tcaaataact 5880 5940 caaattttac aatgcaccta cggctagggt tgaacaactc aaggaaatag taggccaaag 6000 aacagggaca cgccaattct agttgaacag atcacaatgg cacgtagcat caagtaattt 6060 tccttgtcgc ataaccatag cacaggattc gactgcaatg acccatgctg acgtcttgcc 6120 actacaaatc ggcgaaaaa gcaacaata ccactatgcc cacacagtat ttccccctac 6180 6240 agaactaggt atgaacggtg gttggttggg gggtggggtt gcaagaagg gggcgtaggg 6300 aacataccat cgtagaaata ggatatcctg tctttctcca acatgaccgc gctctcgagc 6360 ggccgcctgc ctctacaggc ggaggcggcg ccaaatgggg gcagccgggg aagccgacag 6420 cgagttcgcg acggcggagg cggaggcggc ggcggaagtt ggggcggtcg tgcgtgagga 6480 ggaaggggaa aaccgagcga ggcgatggct ctggggaacg ttctggaccg gcatggacag 6540 tcaggatcgt gttggtacca tgatcgaacg gtcaaagtta ttgcgcgggt gaattatttc 6600 gcctcgattg gtactcaaaa gtactgggtt ttttttttag ttggaagtac tgctatttct 6660 gattctgtac aaaatactgt attactcccc attagttagt cttcgtcggt ctaaaaacaa 6720 ctgctactca aacgaatgta tgtaacgaaa tacgtctaga tatcttcgct tgagcactag 6780 ttatatttag atatagcgag tattttctga actagtgaaa tgtactcagg ggtctaactt 6840 agctgaacca ttcttttggt caaacatttt agccgagcct gtaggaacat cagggttcta 6900 ttacttgcgg ctggattcaa tttattttat aaacatctag gtggctaaat caacatattt 6960 aatttagtat gtatatagta gttaataaat gatatttata attttggtcc aatctaaaca 7020 caagcaaact tgagaccata ccatgttatc tcagcttatg cttctttact cagggaaatt 7080 aaattaata ttgtagggg aattagtatt attcatgtgt agccggcatc agaacattgt 7140 tcatgactat atggataagt atgcccacac aaaacaactg aagatcatg gatatcgcct 7200 agagggggtg cataggcgct ttagataat tacggttttag ccttgaacaa atgtgaaata 7260 aaactagttt taatttgtca agcacaaac ctaaattgaa taggctcacc tatgtgcacc 7320 aaaacttat actaagtagg aacaact aagtgatagc aagatatata tcatgtcata 7380 ttatggctat tgcaagtaa agtgcatat taaagagctc gggtaagaga taaccaaggc 7440 acggcggagac gacgttgtat ccccaagttc acacccttgc agatactaat cttcgttttgg 7500 agccggcgtgg agccacaatg ctt 7523 <210> 3 <211> 7523 <212> DNA <213> Triticum aestivum L. <400> 3 agggcgagac cacctcctcc cccgcatctt gcttggatt tgtttagaa ttcctcgtct 60 cttgcttgct gttatgtca gactctggc cctggagga atttcgcta cctgctggat 120 acctgttctc atcagatcc gatgacccca gccgcccaca agagaacag aagtatggga 180 tttgttcata ttgtatgtca taagaatcca ccttctttct cctcgccgaa tcaataagca 240 cccatctcct cattggttta ttgacctcca ccgatactct atctcgaagg tagcctccgt 300 tgtgatcaaa cttagcaatc tgaacctctt tatctatcat cctggctatt gggaggctcc 360 atgcatcatc tctcaaatta tagggtaaat taagaaccct agcccaaacc tgcaggcgat 420 cgaacttgac ctcatctggg cgcatgcact cttcaaactc ggccaagatg accgcattct 480 tgttgatgtg ccaaggcgat cctgcccaaa ctctgtcacg gtctcgctcg gactcaaatt 540 cagccaaaa catattcgct ccagtcgagc agaactgcag tcctctaggg tttccccaaag 600 ccggtcgcaa agcattgcca atggtgtgta tatgatactg attgcggtac agaaccttac 660 cagccagcaa ccacttcacc ggagcaccat ccagacggtc atctatgacg agaggatcg 720 cctctcctc ggatatgtcc agctttccca gcgcttctc aagtttctcc ctcgccgatc 780 tggtattatt cgatcccgag ttcgcctgca tcgtctccgc cgcgggtggt gtagccatca 840 ccctcgccga gtccttgggg cggcccgccg atgagatccg cggccgccga tcggtttcca 900 cgtcgaaacc ctagcgccac cagggagccc tagtttcgg ggaaaaagta gtcctctatg 960 aatgttatca attagttgct tttccaaatt caaaaaggcc atacatacaa atacaagaat 1020 aatccaaagg ctaaataaaa aattacgtac gtactgaaat gatggtattt taagtaagta 1080 caaatgtgag atcgtacaat gctgtacaag tcagcaagtg ttacgttctc ccacaatgct 1140 ctccagccta gcgtcagatc ctaaaatgtc aatcagccaa ctattcccca tcccacgatg 1200 ttataaactg aacagcagct gctgcgggaa agactttcag cttccaccta catacatcgt 1260 caagatgccg atctcaatgt ccgccgtcat cgtgatcatt gtcgttgtcg ccttcatagt 1320 actcgtcgtc acgatgaacc tgcttgctt gggtatgctc tgcatgacat aatacacact 1380 tctgtaagaa aatagaaata tgatggcacg gttcagaagg actggagaca ataccaggca 1440 aggagctggc tgaatgaaga agaatacaac caatttaaga ataaatagat aacaaaatgg 1500 accgcattta cttcatttca ggggaaattt ccattatgt ggcgcgcaca tgcttcgaga 1560 ggacaatggc gagttagcac ggagaaatca tcaagttttt acttgtccat atgagcactg 1620 cagacatgc cagaggtatt tcacttacga tcacacgtt catcaggatc cagctcgtct 1680 tcatcaaat ccgggacata gaatcggggt ggaacctgca aaataactc attatgtatc 1740 cggtctctgc tcagaaacaaactacac tatcctggtg gagcagaa agttaagac 1800 aacaaaagat gcaaggcat attatagtct tcacatgta tccatccccg gttcaatg 1860 ggatgtttca acaatcaca tgcggacgag atacaact aacctcttgc atctgaacgc 1920 caggtgcatg ttgtatggcc cgcaactct ccattacttg cacttgatt gaactgagat 1980 acgtcttact attcaagttg tcctggtcca ttcatcaata ggaagacaaa tatcatttgc 2040 acatctacac acctctaaag aaagaaattg atattctagt taagcgtacc siacatataa 2100 aaacatgacg caccaacaa cagttaaatc cacaacca ggcaggcata gagttagaaa 2160 gatcagtcag aaggttcat accatgttca gatttggtac ttcaatgta taatctggac 2220 cgaagtattc aatgtattca ttgtcaggga tctctgcaaa gaaaatgcca tgtaaaactc 2280 cggacaatgg ataccacta aggagaatt gcttatggag agtagttcta tagggaag 2340 gtattgggac atattggat ttttcacctt acagatcaa gaaagaga 2400 aaagaatac aaaaaatg ccgctatatt gaggattat actagctact gtcactcaa 2460 ccaacaaggt taagggagc agaaaaaaaatgagatac catttgggg ttctgtgtct 2520 agaagaaccc cagttcac agcccacag cgtgctacat tctctttggt gtaccacca 2580 cctcctgtca cctgcatgtt tgaaaata tgaatatcct agaagagca aaaataatca 2640 tacaatgata gttttctccc taccagcag cgggatttta aatttcttta caactttac 2700 acattcagca tggcctgttt atggaacag atcagttag tagtataat atagattat 2760 ccgagtgagt tagctcaatg aaactaagt ccaaggacc ccgaaccat caaccaat 2820 aaatttctcag atacacaaa ttcacagtg gggacaaggt atcacggtt agtttataca 2880 tctgatgttt tgccaaatct atagtagcac gtaatatcaa gttttagaga gtaccttcta 2940 ttgaaagatt gaacaccct aggcggtctc gcgccaatga atcagcccca cattgaagaa 3000 caatagcacc tggcagatat gtctcaacaa ctttggcaat atctacatg atagcaaca 3060 ttacgtacct caataagagt agacaag aacaagaata acataaagt aatgcacatc 3120 gacttttacc gttttaaaaa ggcgagtaaa gctgctgtca tctatgccat ctttaagtgg 3180 gatgttgatt gcatatatc ttccttccct gtctcctata tccttcaaaa tgacagccag 3240 gtaaattgat atgaagagca catccttaca aaatgaagct aaatttttta aagctctcat 3300 catcattact taacagcacg tgatattgaa ctatttaaaa aaaagcaag atgcaggctc 3360 tgtaagtgca tttttttcag catgttaata aatagaaaag cgcacatttc tgctcatatc 3420 gaaccttgcc attcccttat gtgagttaca aactctagtt gatgagataa ttttgattgg 3480 agaactcgca actcaatcaa agaacaattgc gtaaagaaac ccttccactc atacagatgc 3540 acgcataaaa tatgtgcaaa aatagcatta tggcaaaagt aaagacccaa tacaaaaacg 3600 atgaagtgaa tcttcaaatg atggttcaag catgaccgca agggttgtgt tggtcagttg 3660 3720 tttggcacta ccgcttatca aatatagatc ataaacctcc accattccca aatatgatgc 3780 tggctatttg gatagaatac cgcacgaaga tgtccagta atagacaca gagttcaatg 3840 aaaagtca catacttaca tgttctactt ctaggtggac aggagagt gagaaaga 3900 agaaactgaa gtatgtcacg atggttccta aaattattga taccactccag taccatcagc 3960 tttgagtaat attatgctaa aacccattgt aactcagttt gtaaatgtct cttctatttc 4020 attcaattga gagttcatat tccaacttta atcgacata gtaatagggg gacaac 4080 accataaac tcatgcccaa tattgagcac aaaactgaa aataatagcg 4140 atgaactgct gagaaattta gtaaaagcaa ctacataaaa tgtatattca taagcttgt 4200 cagtggaaca ttaccttaat atcacctgtg ccaggaaaga acatgtcacc atacttgtgg 4260 aaacttacag tcattaccct gcaatatgt atagacaaa tcagattgga atctaaagg 4320 ctctcaaaga ghagcggca ttactataca gcaacctcac actagacaga gccagagatt 4380 agcaactaa atgaatatat gggtgataaca aaagagtagg catctatgga ggctctgaag 4440 atcagtgaga cacatcctat cacaat ac atcatgtat tttactcc ataaaagcat 4500 gaacctgtca gtgaaataga aggcttcttc aactccatct ccatgatgga catcaatgtc 4560 aattagaga accctagcat gatacttgag aagctccaga attcccaaaa ccaggtcatt 4620 aatgtagcag aagcctgacg cctcacactt ctttgcatga tgcagcccac cagcccaatt 4680 atagcaata tcacatgttt tatgatttag tcttcgagcc gcatctgaaa aataaagaaa 4740 tgcttagaga gatcacaaaa cagaaagaag cattggcaat tttcctatta cctagagttc 4800 ctccagcata gatttggcag aactcaaaca aatcatcaaa gaccgggcag tcctctccaa 4860 ggttgtctgg gacaatgatt acaatgacga agtcagataa gtgaagtaag aataccccaa 4920 atacatggag atgtgattt tagcaattcc attagtaca acaggcatat tgtggaaatt 4980 atagcagaca gttcttcatt taagtgcaaa cagctaagct tccatttgga tacaacaatt 5040 tgttgagaga tcagaccatc ttactgtgtt ccgcttctat tttaagcata catccaaaga 5100 cctaagctaa tatagcacta ccttagattc aggtaaatca tttgggagt tttgggttag 5160 gtgtcagaag ttacgcaagt ttgaccaaat gctgctggcg ttggttcat ataaacaact 5220 gtggcgagcc acaaatttctt aggcacatgg cacatgtcc actcgtcata cacatgttct 5280 aaccaagttt tgacatgatt ttttacagc taaataag caagcaatat atgcggcgtg 5340 tattttattg tgaaatagc aaagcctggg agagtagtct gtaaagaga aaatatcat 5400 cattcataca tctagttaat tcacttgcat acagtgctg ggtatcagga gttattcggt 5460 gcaagaatttc cacataatca gcagaatgga actgcgcaag ctctgttgga tatgctttgt 5520 ggggtctctg caaatccaa tgggagcaa aaggtaaa cgacacattt aaacaattca 5580 taattacaa gaaaggggat gaacaataat tcaataccga catatatctc cattttctg 5640 tgagatcat atgatacac gagatggtgt gtcatgcaaa ggcgatgcgg ttcatcggg 5700 tgatttggcc caagtacac attgccaca tcccctgaga tgttcggtca cagaaaagta 5760 accaagtaag caatcataa aggttgaaca gcaaaaaac ttatatagtg ttgaaggtgt 5820 gtggattttc tacaacaac acagttaagg aacttagtcat gagaaccaa tcaatact 5880 agcgcagaaa ttttaccat ttgactcct tatcgaccaa gatgaagca tacactaatg 5940 caaattttac aatgcaccta cggctagggt tgaacaactc aaggaaatag taggccaaag 6000 aacagggaca cgccaattct agttgaacag atcacaatgg cacgtagcat caagtaattt 6060 tccttgtcgc ataaccatag cacaggattc gactgcaatg acccatgctg acgtcttgcc 6120 actacaaatc ggcgaaaaa gcaacaata ccactatgcc cacacagtat ttccccctac 6180 6240 agaactaggt atgaacggtg gttggttggg gggtggggtt gcaagaagg gggcgtaggg 6300 aacataccat cgtagaaata ggatatcctg tctttctcca acatgaccgc gctctcgagc 6360 ggccgcctgc ctctacaggc ggaggcggcg ccaaatgggg gcagccgggg aagccgacag 6420 cgagttcgcg acggcggagg cggaggcggc ggcggaagtt ggggcggtcg tgcgtgagga 6480 ggaaggggaa aaccgagcga ggcgatggct ctggggaacg ttctggaccg gcatggacag 6540 tcaggatcgt gttggtacca tgatcgaacg gtcaaagtta ttgcgcgggt gaattatttc 6600 gcctcgattg gtactcaaaa gtactgggtt ttttttttag ttggaagtac tgctatttct 6660 gattctgtac aaatactgt attactcccc attagttagt cttcgtcggt ctaaaaacaa 6720 ctgctactca aacgaatgta tgtaacgaaa tacgtctaga tatctcgct tgagcactag 6780 ttatatttag atatagcgag tattttctga actagtgaaa tgtactcagg ggtctaactt 6840 agctgaacca ttctttggt caacatttt agccgagcct gtaggacat caggttcta 6900 ttacttgcgg ctggattcaa tttattt aaacatctag gtggctaaat caacatattt 6960 aatttagtat gtatagta gttaataaat gatttatta attttggtcc aatctaaca 7020 caagcaact tgagaccata ccatgttatc tcagcttatg cttctttact cagggaatt 7080 aaattaata ttgtagggg aattagtatt attcatgtgt agccggcatc agaacattgt 7140 tcatgactat atggataagt atgcccacac aaaacaactg aagatcatg gatatcgcct 7200 agagggggtg cataggcgct ttagataat tacggttttag ccttgaacaa atgtgaaata 7260 aaactagttt taatttgtca agcacaaac ctaaattgaa taggctcacc tatgtgcacc 7320 aaaacttat actaagtagg aacaact aagtgatagc aagatatata tcatgtcata 7380 ttatggctat tgcaaagtaa agtgcataat taaagagctc gggtaagaga taaccaaggc 7440 acgcggagac gacgttgtat ccccaagttc acacccttgc agatactaat cttcgtttgg 7500 agcggcgtgg aggcacaatg ctt 7523 <210> 4 <211> 28 <212> DNA <213> Artificial Sequence <400> 4 gatcgaacgg tcaaagttat tgcgcggg 28 <210> 5 <211> 30 <212> DNA <213> Artificial Sequence <400> 5 gcgtgccttg gttatctctt acccgagctc 30 <210> 6 <211> 25 <212> DNA <213> Artificial Sequence <400> 6 ctagtgaaat gtactcaggg gtcta 25 <210> 7 <211> 25 <212> DNA <213> Artificial Sequence <400> 7 gccgcaagta atagaaccct gatat 25

Claims

1. A method for screening or assisting in screening wheat of different thousand-grain weights, comprising the following steps: detecting the wheat to be tested based on TaHDA9-2A Whether the genotype of the gene is genotype I or genotype II, the thousand-grain weight of genotype I wheat is greater than the thousand-grain weight of genotype II wheat; The wheat of genotype I is a wheat having a genotype of AA homozygous based on the A6886G SNP site; The wheat of genotype II is a wheat having a GG homozygous genotype based on the A6886G SNP site; The A6886G SNP site is the 6886th nucleotide from the 5' end of SEQ ID NO: 1 in the wheat genome.

2. A method for screening or assisting in screening wheat of different thousand-grain weights, comprising the following steps in sequence: (A1) Using the genomic DNA of the wheat to be tested as a template, PCR amplification is performed using primer pair A consisting of primer F1 and primer R1 to obtain PCR amplification product P1; (A2) using the PCR amplification product P1 as a template, performing PCR amplification on primer pair B consisting of primer F2 and primer R2 to obtain a PCR amplification product P2; (A3) The PCR amplification product P2 is digested with restriction endonuclease EcoRV to obtain a digestion product; and then the following evaluation is performed: if the digestion product is a DNA fragment, the wheat to be tested is based on TaHDA9-2A The genotype of the gene is genotype I; if the enzyme cleavage product is two DNA fragments, the wheat to be tested is based on TaHDA9-2A The genotype of the gene is genotype II; The thousand-grain weight of wheat of genotype I is greater than that of wheat of genotype II; The primer F1 is a single-stranded DNA molecule shown in SEQ ID NO: 4; The primer R1 is a single-stranded DNA molecule shown in SEQ ID NO: 5; The primer F2 is a single-stranded DNA molecule shown in SEQ ID NO: 6; The primer R2 is a single-stranded DNA molecule shown in SEQ ID NO:

7.

3. A method for screening or assisting in screening wheat of different thousand-grain weights, comprising the following steps in sequence: (B1) Using the genomic DNA of the wheat to be tested as a template, PCR amplification is performed using primer pair A consisting of primer F1 and primer R1 to obtain PCR amplification product P1; (B2) using the PCR amplification product P1 as a template, performing PCR amplification on primer pair B consisting of primer F2 and primer R2 to obtain a PCR amplification product P2; (B3) The PCR amplification product P2 is digested with restriction endonuclease EcoRV to obtain digestion products; and then the following evaluation is performed: if the digestion product contains only a 100 bp DNA fragment, the wheat to be tested is TaHDA9-2A The genotype of the gene is genotype I; if the enzyme digestion product contains only a 75 bp DNA fragment and a 25 bp DNA fragment, the wheat to be tested is based on TaHDA9-2A The genotype of the gene is genotype II; The thousand-grain weight of wheat of genotype I is greater than that of wheat of genotype II; The primer F1 is a single-stranded DNA molecule shown in SEQ ID NO: 4; The primer R1 is a single-stranded DNA molecule shown in SEQ ID NO: 5; The primer F2 is a single-stranded DNA molecule shown in SEQ ID NO: 6; The primer R2 is a single-stranded DNA molecule shown in SEQ ID NO:

7.

4. A method for screening or assisting in screening wheat of different thousand-grain weights, comprising the following steps in sequence: (1) Using the genomic DNA of the wheat to be tested as a template, PCR amplification was performed using primer pair A consisting of primer F1 and primer R1 to obtain the PCR amplification product P1; The primer F1 is a single-stranded DNA molecule shown in SEQ ID NO: 4; The primer R1 is a single-stranded DNA molecule shown in SEQ ID NO: 5; (2) Sequence the PCR amplification product P1 and then make the following evaluation: If the nucleotide sequence of the PCR amplification product P1 is as shown in SEQ ID NO: 2 from the 6562nd to the 7444th position from the 5' end, the wheat to be tested is based on TaHDA9-2A The genotype of the gene is genotype I; If the nucleotide sequence of the PCR amplification product P1 is as shown in SEQ ID NO:3 from the 6562nd to the 7444th position from the 5' end, the wheat to be tested is based on TaHDA9-2A The genotype of the gene is genotype II; The thousand-grain weight of wheat of genotype I is greater than that of wheat of genotype II.

5. Detection of wheat based on TaHDA9-2A The application of the substance for genes of genotype I or genotype II is at least one of (z1) to (z3): (z1) Screening or assisting in the screening of wheat of different thousand-kernel weights; (z2) Identify or assist in identifying wheat thousand-grain weight; (z3) Identification or auxiliary identification of wheat TaHDA9-2A the genotype of a gene; The genotype I is based on the genotype of the A6886G SNP site, which is AA homozygous. TaHDA9-2A Gene; The genotype II is based on the genotype of the A6886G SNP site, which is the GG homozygous type. TaHDA9-2A Gene; The A6886G SNP site is the 6886th nucleotide from the 5' end of SEQ ID NO: 1 in the wheat genome.

6. The use according to claim 5, characterized in that: The detection of wheat to be tested is based on TaHDA9-2A The material for determining whether the genotype of the gene is genotype I or genotype II includes a primer pair A consisting of primer F1 and primer R1 and / or a primer pair B consisting of primer F2 and primer R2; The primer F1 is a single-stranded DNA molecule shown in SEQ ID NO: 4; The primer R1 is a single-stranded DNA molecule shown in SEQ ID NO: 5; The primer F2 is a single-stranded DNA molecule shown in SEQ ID NO: 6; The primer R2 is a single-stranded DNA molecule shown in SEQ ID NO:

7.

7. The use according to claim 6, characterized in that: The detection of wheat to be tested is based on TaHDA9-2A The substance for determining whether the genotype of the gene is genotype I or genotype II also includes the restriction endonuclease EcoRV.

8. The use of the molecular marker represented by SEQ ID NO: 1, which is at least one of (z1)-(z3): (z1) Screening or assisting in the screening of wheat of different thousand-kernel weights; (z2) Identify or assist in identifying wheat thousand-grain weight; (z3) Identification or auxiliary identification of wheat TaHDA9-2A the genotype of a gene; The application is achieved through the A6886G SNP site; the A6886G SNP site is the 6886th nucleotide from the 5' end of SEQ ID NO: 1 in the wheat genome; If the genotype based on the A6886G SNP site is AA homozygous, then TaHDA9-2A The genotype of the gene is genotype I; If the genotype based on the A6886G SNP site is GG homozygous, then TaHDA9-2A The genotype of the gene is genotype II; The thousand-grain weight of genotype I wheat was greater than that of genotype II wheat.

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