CN111778353B - SNP molecular marker for identifying common wheat variety and SNP molecular marker detection method - Google Patents
SNP molecular marker for identifying common wheat variety and SNP molecular marker detection method Download PDFInfo
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Abstract
The invention relates to the field of agricultural molecular detection, in particular to an SNP molecular marker combination for wheat variety identification and an SNP molecular marker detection method. The 96 SNP locus combinations detect 192 allelic variation in 1463 examined wheat varieties in China, have higher resolution ratio on the wheat varieties, can stably, repeatedly and specifically amplify 96 SNP primers and primer combinations thereof, and are convenient to popularize and apply.
Description
Technical Field
The invention relates to the field of agricultural molecular detection, in particular to an SNP molecular marker for identifying common wheat varieties and an SNP molecular marker detection method.
Background
Wheat is the widest planting range and the widest consumption range in China, and relates to the largest grain crop in three crops. China is the largest wheat producing country and consuming country in the world, and has a great influence on the international wheat market. In recent years, illegal behaviors such as pushing of varieties before examination, making and selling of fake and inferior seeds, license-plate-covering infringement and the like are prominent, disorder of seed markets is caused, the continuous and stable development of the seed industry health in China is seriously influenced, and the rights and interests of breeders and the benefits of farmers are damaged. The occurrence of the behaviors is also the problem of variety authenticity identification after the root is concluded. Therefore, the establishment of the rapid identification standard of the wheat variety authenticity molecules is a technology which is urgently needed by the country.
The variety authenticity is to compare the inspected sample with the standard sample corresponding to the variety name, and to identify whether the inspected sample is consistent with the standard sample. The variety authenticity identification adopts a morphological method (cell planting identification), a protein electrophoresis method and the like in the past, and because the distinguishing capability is very limited, particularly along with the progress of breeding technology, the difference between varieties is smaller and smaller, and the methods can not meet the requirements of accurate and rapid identification at all. At present, two molecular marker detection technologies of SSR and SNP are mainly promoted internationally for variety differentiation. The recent domestic and foreign practices prove that the SNP molecular marker detection method has the advantages of simplicity, economy, high efficiency, short detection period, sharable data and the like, has the advantages of accurate result, large scale and high automation degree, is an effective means for solving seed quality identification, can provide effective technical support for seed market supervision, and ensures that the seed supervision work is successfully carried out according to law. As the SNP marking technology develops for more than 10 years, the current technology and platform are mature and have the conditions of general implementation. Therefore, the technical specification of the wheat variety authenticity SNP molecular marker detection is formulated, the seed quality of the variety is ensured from the source, the due effect of the variety is exerted, and the method has very important practical significance for ensuring national grain safety and maintaining the healthy, continuous and stable development of the wheat variety industry.
Disclosure of Invention
The invention aims to provide application of 96 wheat SNP molecular markers.
Still another object of the present invention is to provide a method for detecting SNP molecular markers for identifying wheat varieties.
According to a specific embodiment of the invention, the application of the following SNP locus combinations for identifying wheat varieties is provided, wherein the SNP loci are selected from the combination comprising the SNP loci WSNP01-WSNP 96.
According to the specific implementation mode of the invention, whether the sample to be detected is a real approved variety or not is determined by comparing the detection results of the 96 SNP sites of the sample to be detected and the national wheat approval standard sample.
The invention determines 96 SNP locus combinations by screening, the physical positions of the 96 SNP loci are determined based on the whole genome sequence comparison of the Chinese spring of the wheat variety, the version number of the whole genome sequence of the Chinese spring of the wheat variety is IWGSC RefSeqv1.0, and the number of the 96 SNP loci is WSNP01-WSNP 96.
The invention provides 96 SNP locus combinations which can be used for identifying the authenticity of wheat varieties, wheat molecular breeding or assisting the specific identification of the wheat varieties.
Still another object of the present invention is to provide a method for performing authenticity verification of wheat varieties.
According to the specific embodiment of the invention, 96 SNP locus numbers, chromosomes where SNPs are located, specific physical positions (referring to China spring genome sequence, version number is IWGSC RefSeqv1.0), and deoxynucleotide information for authenticating common wheat varieties are as follows:
WSNP01 is located at position 27215962 on chromosome 1A, and its deoxynucleotide is A or G;
WSNP02 is located at position 41817136 on chromosome 1A, and its deoxynucleotide is T or C;
WSNP03 is located at position 495285832 on chromosome 1A, and its deoxynucleotide is T or G;
WSNP04 is located at position 587992062 on chromosome 1B, and its deoxynucleotide is T or C;
WSNP05 is located at position 340381039 on chromosome 1B, and its deoxynucleotide is T or C;
WSNP06 is located at position 20306381 on chromosome 1D, and its deoxynucleotide is C or G;
WSNP07 is located at position 288318891 on chromosome 1D, and its deoxynucleotide is A or G;
WSNP08 is located at position 427323196 on chromosome 1D, and its deoxynucleotide is A or G;
WSNP09 is located at position 43371103 on chromosome 2A, and its deoxynucleotide is T or C;
WSNP10 is located at position 36393269 on chromosome 2A, and its deoxynucleotide is A or G;
WSNP11 is located at position 174150002 on chromosome 2A, and its deoxynucleotide is A or G;
WSNP12 is located at position 41105877 on chromosome 2A, and its deoxynucleotide is T or C;
WSNP13 is located at position 12003721 on chromosome 2B, and its deoxynucleotide is T or C;
WSNP14 is located at position 47427272 on chromosome 2B, and its deoxynucleotide is T or C;
WSNP15 is located at position 108910271 on chromosome 2B, and its deoxynucleotide is T or G;
WSNP16 is located at position 652578839 on chromosome 2B, and its deoxynucleotide is C or G;
WSNP17 is located at position 697446904 on chromosome 2B, and its deoxynucleotide is A or G;
WSNP18 is located at position 773821814 on chromosome 2B, and its deoxynucleotide is A or G;
WSNP19 is located at position 28783896 on 2D chromosome, and its deoxynucleotide is T or C;
WSNP20 is located at position 422881650 on 2D chromosome, and its deoxynucleotide is T or C;
WSNP21 is located at position 431959521 on 2D chromosome, and its deoxynucleotide is A or G;
WSNP22 is located at position 458920669 on 2D chromosome, and its deoxynucleotide is T or C;
WSNP23 is located at position 551877542 on 2D chromosome, and its deoxynucleotide is T or G;
WSNP24 is located at position 615715449 on 2D chromosome, and its deoxynucleotide is A or G;
WSNP25 is located at position 25908324 on chromosome 3A, and its deoxynucleotide is A or G;
WSNP26 is located at position 481678769 on chromosome 3A, and its deoxynucleotide is A or G;
WSNP27 is located at position 735293187 on chromosome 3A, and its deoxynucleotide is T or G;
WSNP28 is located at position 597917383 on chromosome 3A, and its deoxynucleotide is A or G;
WSNP29 is located at position 509365938 on chromosome 3B, and its deoxynucleotide is A or G;
WSNP30 is located at position 627720241 on chromosome 3B, and its deoxynucleotide is A or G;
WSNP31 is located at position 680735249 on chromosome 3B, and its deoxynucleotide is T or C;
WSNP32 is located at position 749862710 on chromosome 3B, and its deoxynucleotide is A or C;
WSNP33 is located at position 819302391 on chromosome 3B, and its deoxynucleotide is A or T;
WSNP34 is located at position 56675266 on the 3D chromosome, and its deoxynucleotide is T or C;
WSNP35 is located at position 537495536 on 3D chromosome, and its deoxynucleotide is T or G;
WSNP36 is located at position 578749369 on 3D chromosome, and its deoxynucleotide is A or G;
the WSNP37 is located at 611664812 th position on a 3D chromosome, and the deoxynucleotide of the WSNP37 is A or G;
WSNP38 is located at position 584425606 on chromosome 4A, and its deoxynucleotide is T or G;
WSNP39 is located at position 116644494 on chromosome 4A, and its deoxynucleotide is A or G;
WSNP40 is located at position 532224647 on chromosome 4A, and its deoxynucleotide is A or G;
WSNP41 is located at position 669197675 on chromosome 4A, and its deoxynucleotide is A or G;
WSNP42 is located at position 36960078 on chromosome 4B, and its deoxynucleotide is T or C;
WSNP43 is located at position 527111678 on chromosome 4B, and its deoxynucleotide is A or G;
WSNP44 is located at position 179840525 on chromosome 4B, and its deoxynucleotide is A or G;
WSNP45 is located at position 636757506 on chromosome 4B, and its deoxynucleotide is C or G;
WSNP46 is located at position 11870112 on the 4D chromosome, and its deoxynucleotide is A or G;
WSNP47 is located at position 364033217 on the 4D chromosome, and its deoxynucleotide is A or C;
WSNP48 is located at position 118605639 on the 4D chromosome, and its deoxynucleotide is T or C;
WSNP49 is located at position 367933139 on the 4D chromosome, and its deoxynucleotide is A or G;
WSNP50 is located at position 448113730 on chromosome 5A, and its deoxynucleotide is A or C;
WSNP51 is located at position 478883696 on chromosome 5A, and its deoxynucleotide is A or G;
WSNP52 is located at position 455550448 on chromosome 5A, and its deoxynucleotide is A or G;
WSNP53 is located at position 500625774 on chromosome 5A, and its deoxynucleotide is T or G;
WSNP54 is located at position 591302258 on chromosome 5A, and its deoxynucleotide is A or G;
WSNP55 is located at position 695003171 on chromosome 5A, and its deoxynucleotide is T or C;
WSNP56 is located at position 6594624 on chromosome 5B, and its deoxynucleotide is T or C;
WSNP57 is located at position 11080396 on chromosome 5B, and its deoxynucleotide is A or G;
WSNP58 is located at position 281675472 on chromosome 5B, and its deoxynucleotide is A or G;
WSNP59 is located at position 485426049 on chromosome 5B, and its deoxynucleotide is T or C;
WSNP60 is located at position 82481988 on the 5D chromosome, and its deoxynucleotide is T or C;
WSNP61 is located at position 302000445 on chromosome 5D, and its deoxynucleotide is A or G;
WSNP62 is located at position 370064912 on chromosome 5D, and its deoxynucleotide is A or G;
the WSNP63 is located at 495427294 th position on a 5D chromosome, and the deoxynucleotide of the WSNP63 is A or G;
WSNP64 is located at position 51949801 on chromosome 6A, and its deoxynucleotide is T or C;
WSNP65 is located at position 617805673 on chromosome 6A, and its deoxynucleotide is A or G;
WSNP66 is located at position 615440555 on chromosome 6A, and its deoxynucleotide is T or C;
WSNP67 is located at position 77112398 on chromosome 6A, and its deoxynucleotide is T or C;
WSNP68 is located at position 445560400 on chromosome 6A, and its deoxynucleotide is A or G;
WSNP69 is located at position 457660545 on chromosome 6A, and its deoxynucleotide is A or C;
WSNP70 is located at position 579446448 on chromosome 6A, and its deoxynucleotide is T or C;
WSNP71 is located at position 22553387 on chromosome 6B, and its deoxynucleotide is A or G;
WSNP72 is located at position 27687594 on chromosome 6B, and its deoxynucleotide is T or C;
WSNP73 is located at position 158129211 on chromosome 6B, and its deoxynucleotide is T or C;
WSNP74 is located at position 227062800 on chromosome 6B, and its deoxynucleotide is A or G;
WSNP75 is located at position 439018758 on chromosome 6B, and its deoxynucleotide is T or C;
WSNP76 is located at position 630873069 on chromosome 6B, and its deoxynucleotide is T or C;
WSNP77 is located at position 659565617 on chromosome 6B, and its deoxynucleotide is A or G;
WSNP78 is located at position 682326705 on chromosome 6B, and its deoxynucleotide is A or G;
WSNP79 is located at position 86651424 on chromosome 6D, and its deoxynucleotide is T or G;
WSNP80 is located at position 133466400 on chromosome 6D, and its deoxynucleotide is T or C;
WSNP81 is located at position 351234720 on chromosome 6D, and its deoxynucleotide is T or C;
the WSNP82 is located at 438410250 th position on a 6D chromosome, and the deoxynucleotide of the WSNP82 is T or C;
WSNP83 is located at position 4471905419 on chromosome 6D, and its deoxynucleotide is A or G;
WSNP84 is located at position 537642742 on chromosome 7A, and its deoxynucleotide is T or C;
WSNP85 is located at position 679816107 on chromosome 7A, and its deoxynucleotide is A or G;
WSNP86 is located at position 707833467 on chromosome 7A, and its deoxynucleotide is A or C;
WSNP87 is located at position 67705483 on chromosome 7B, and its deoxynucleotide is G or C;
WSNP88 is located at position 605748172 on chromosome 7B, and its deoxynucleotide is C or G;
WSNP89 is located at position 678124448 on chromosome 7B, and its deoxynucleotide is A or G;
WSNP90 is located at position 721402941 on chromosome 7B, and its deoxynucleotide is A or C;
WSNP91 is located at position 87479998 on chromosome 7D, and its deoxynucleotide is A or C;
WSNP92 is located at position 349365912 on chromosome 7D, and its deoxynucleotide is T or C;
WSNP93 is located at position 411596748 on chromosome 7D, and its deoxynucleotide is T or C;
WSNP94 is located at position 430012769 on chromosome 7D, and its deoxynucleotide is T or C;
the WSNP95 is located at 452686835 th position on a 7D chromosome, and the deoxynucleotide of the WSNP95 is G or C;
WSNP96 is located at position 508179540 on chromosome 7D and its deoxynucleotide is T or C.
According to the wheat variety authenticity SNP molecular marker detection method, the allele specific primers for detecting the genotypes of the 96 SNP loci comprise the following components:
WSNP01-FAM：5’GGAGAACAAAGCAGAAAAGCTGCAA3’
WSNP01-HEX：5’GAGAACAAAGCAGAAAAGCTGCAG3’
WSNP 01-general: 5 'GTGAACATTCAGGATATTTGTGCTGCATA 3';
WSNP02-FAM：5’CGTAGTCCTCCAGAAAATCG3’
WSNP02-HEX：5’GCTCGTAGTCCTCCAGAAAATCA3’
WSNP 02-general: 5 'GGATCTGGACGGCTTCCCACTT 3'
WSNP03-FAM：5’AGATGTTGAGAACACTTTCGTCCC3’
WSNP03-HEX：5’GAGATGTTGAGAACACTTTCGTCCA3’
WSNP 03-general: 5 'CAGAGAAGAGGGCGTCAGCGTA 3'
WSNP04-FAM：5’GGCAACTCTAACAAACACACTCC3”
WSNP04-HEX：5’AGGGCAACTCTAACAAACACACTCT3’
WSNP 04-general: 5 'GACGAACTCGGCGGGACCATTT 3'
WSNP05-FAM：5’ACCGGCGACTCTCAAGTTGATG3’
WSNP05-HEX：5’AACCGGCGACTCTCAAGTTGATA3’
WSNP 05-general: 5 'CGGGAAACAATGCTCATAGGGACAA 3'
WSNP06-FAM：5’GTAGGATATCGCGGAAGACCAC3’
WSNP06-HEX：5’GTAGGATATCGCGGAAGACCAG3’
WSNP 06-general: 5 'GTGATCTAGCTAGGTTGTCGGCTAA 3'
WSNP07-FAM：5’AAACATGTAAAACTAACTTCACATACCCA3’
WSNP07-HEX：5’ACATGTAAAACTAACTTCACATACCCG3’
WSNP 07-general: 5 'GACCCTTCGAGACCCTACCCAT 3'
WSNP08-FAM：5’AACACATTCAGAGAATATGACTAGGG3’
WSNP08-HEX：5’ATAACACATTCAGAGAATATGACTAGGA3’
WSNP 08-general: 5 'GCCTGAGCGGGCGTTTGGTAA 3'
WSNP09-FAM：5’GCATACATTCCTCCTGTTCGAC3’
WSNP09-HEX：5’GTGCATACATTCCTCCTGTTCGAT3’
WSNP 09-general: 5 'ATTGCCAAGGCGTAGTTTGAGATATTCAT 3'
WSNP10-FAM：5’GTAGAATTAGTCGAGGACCAAGAG3’
WSNP10-HEX：5’GGTAGAATTAGTCGAGGACCAAGAA3’
WSNP 10-general: 5 'AAAACGCGACCGTGATTGGGATCTT 3'
WSNP11-FAM：5’GGACCAGAATCACCAACGAATAAAG3’
WSNP11-HEX：5’GGACCAGAATCACCAACGAATAAAA3’
WSNP 11-general: 5 'TTTAGCTGCAGGGTTTATCACTGATGATT 3'
WSNP12-FAM：5’CCTCCCTCCCGACCTCTG3’
WSNP12-HEX：5’CCCTCCCTCCCGACCTCTA3’
WSNP 12-general: 5 'CTAGTTGGGCCGAGGCAGTTGAA 3'
WSNP13-FAM：5’GTCGAATGGATTTTCCTCGAGCA3’
WSNP13-HEX：5’GTCGAATGGATTTTCCTCGAGCG3’
WSNP 13-general: 5 'TGAGCATCAAAACTCACTCTGTTCAGAA 3'
WSNP14-FAM：5’CCCCTCGGCAACTCCGTGAA3’
WSNP14-HEX：5’CCCTCGGCAACTCCGTGAG3’
WSNP 14-general: 5 'GGTAGTCTATTGAACTCGATCGATATGTA 3'
WSNP15-FAM：5’GATCAATTCTCTTATGCTTGCCAAAGAA3’
WSNP15-HEX：5’ATCAATTCTCTTATGCTTGCCAAAGAC3’
WSNP 15-general: 5 'GAACACTATATGTTCTGGTGGAAGGAAA 3'
WSNP16-FAM：5’ATGATGCATAAAGTTTGATTGCTTCCC3’
WSNP16-HEX：5’ATGATGCATAAAGTTTGATTGCTTCCG3’
WSNP 16-general: 5 'CCATTAAGAAATGAAGTTGGCAATGGGAT 3'
WSNP17-FAM：5’AAGAGGAGAACAGAGAGCGATAGAA3’
WSNP17-HEX：5’GAGGAGAACAGAGAGCGATAGAG3’
WSNP 17-general: 5 'CTCCCAGAGCCAGCCGCCAA 3'
WSNP18-FAM：5’AGTTGAAAACACCGAACCAGCGA3’
WSNP18-HEX：5’GTTGAAAACACCGAACCAGCGG3’
WSNP 18-general: 5 'GGTGAGAGCAAGGATCCCAATCTAT 3'
WSNP19-FAM：5’CATGAGATCCTAAATCAGAGTCGCA3’
WSNP19-HEX：5’ATGAGATCCTAAATCAGAGTCGCG3’
WSNP 19-general: 5 'GGTCAAGTTTTCTGGACCCCATAATTATT 3'
WSNP20-FAM：5’ATTGAGTCGGCACAACATCTCAAG3’
WSNP20-HEX：5’ATATTGAGTCGGCACAACATCTCAAA3’
WSNP 20-general: 5 'TGGTGCTTGCGATGACTTCGTCAAT 3'
WSNP21-FAM：5’CCTGACGCTTCCACGCTGAAC3’
WSNP21-HEX：5’ACCTGACGCTTCCACGCTGAAT3’
WSNP 21-general: 5 'TGGCTCAAGTTGATCGGCCAGAAAT 3'
WSNP22-FAM：5’TTATATGTTTGAAGTAGATGTTGCACG3’
WSNP22-HEX：5’CTTTATATGTTTGAAGTAGATGTTGCACA3’
WSNP 22-general: 5 'ACATCACCTGTAAGAGTTCCTTTGTCAA 3'
WSNP23-FAM：5’CAACGATTCTCTCGCCTTACATAC3’3’
WSNP23-HEX：5’GCAACGATTCTCTCGCCTTACATAA3’
WSNP 23-general: 5 'GGACTGGAATCAATTTTATGTACTCCCTT 3'
WSNP24-FAM：5’AGTACCTTATAGACGACCAGACACT3’
WSNP24-HEX：5’ACCTTATAGACGACCAGACACC3’
WSNP 24-general: 5 'GTTTAGACTGCAATCCTCCATCGGAA 3'
WSNP25-FAM：5’CGATTGAATCTCAAGCGAACAAACG3’
WSNP25-HEX：5’GCGATTGAATCTCAAGCGAACAAACA3’
WSNP 25-general: 5 'TGTAGACATCTAGCAACCCCCTGAA 3'
WSNP26-FAM：5’GGTTCGTGCAGCCCGTTCG3’
WSNP26-HEX：5’AAGGTTCGTGCAGCCCGTTCA3’
WSNP 26-general: 5 'GCGCCATCCTGGACTGCTTCAA 3'
WSNP27-FAM：5’GGGCGTAAAGGTAAGAAAGACTC3’
WSNP27-HEX：5’ACGGGCGTAAAGGTAAGAAAGACTT3’
WSNP 27-general: 5 'TAGGCAAATGCGAGTGTCAATCAATGTTT 3'
WSNP28-FAM：5’AAAGTGGAGCTACAGGAGAAAATGATA3’
WSNP28-HEX：5’AGTGGAGCTACAGGAGAAAATGATC3’
WSNP 28-general: 5 'GTCGGCTAAAAGGTGTTGGCTTTGTA 3'
WSNP29-FAM：5’GTGGTGCAGGACCAAGAGTC3’
WSNP29-HEX：5’CTGTGGTGCAGGACCAAGAGTT3’
WSNP 29-general: 5 'CTCTGACTGCTGCATTGAATTGTCAATAA 3'
WSNP30-FAM：5’GGTAAGCTTTGCCTAGCTTTCCTAT3’
WSNP30-HEX：5’GTAAGCTTTGCCTAGCTTTCCTAC3’
WSNP 30-general: 5 'GTTTGTCCAACTAAGATAGAGTATGGGAA 3'
WSNP31-FAM：5’CATGCCACTGATAATGTGGACGC3’
WSNP31-HEX：5’ACATGCCACTGATAATGTGGACGT3’
WSNP 31-general: 5 'CTACTCATCGGAACCAACTCTACTCAA 3'
WSNP32-FAM：5’GGGGAACTTTCCACCTGATCATG3’
WSNP32-HEX：5’GGGGAACTTTCCACCTGATCATT3’
WSNP 32-general: 5 'GCCTGTTTTCCCGGCACGGAAT 3'
WSNP33-FAM：5’GAATACGGCGCCTTTCGTGCA3’
WSNP33-HEX：5’GAATACGGCGCCTTTCGTGCT3’
WSNP 33-general: 5 'GCTTACACGTCTAGCTATGATGCCAA 3'
WSNP34-FAM：5’ATTGCTCCTGTTCCCAGCAAGG3’
WSNP34-HEX：5’CATTGCTCCTGTTCCCAGCAAGA3’
WSNP 34-general: 5 'AAATTGGTGGAGCTAAGGAACTATGGAAA 3'
WSNP35-FAM：5’GTAAGGGACACACCTCCATCCAT3’
WSNP35-HEX：5’AAGGGACACACCTCCATCCAG3’
WSNP 35-general: 5 'ATAGCTAGATTGTCATCTGGCCAAACAAA 3'
WSNP36-FAM：5’AAGGGCGTTTATTTATGCTTGAAGACA3’
WSNP36-HEX：5’GGGCGTTTATTTATGCTTGAAGACG3’
WSNP 36-general: 5 'TAAACTAGTCGACTTCTTTTCCACGACAA 3'
WSNP37-FAM：5’CGTTTGCGTTCAGGTCGTGC3’
WSNP37-HEX：5’GTCGTTTGCGTTCAGGTCGTGT3’
WSNP 37-general: 5 'GGCAAGTCCTCCGAGGCTCAT 3'
WSNP38-FAM：5’GCATAAGGGTTATCACCAAGTCG3’
WSNP38-HEX：5’CTGCATAAGGGTTATCACCAAGTCA3’
WSNP 38-general: 5 'CAGGTACTTTATGTGAAAGCTCTTGAGAT 3'
WSNP39-FAM：5’GATAACAGCGTTTTCCTGACGAGA3’
WSNP39-HEX：5’ATAACAGCGTTTTCCTGACGAGG3’
WSNP 39-general: 5 'CCATCCCCAACCATAGGACCGAT 3'
WSNP40-FAM：5’AGTCCTTTCTTAAGCACGGCAAC3’
WSNP40-HEX：5’CAGTCCTTTCTTAAGCACGGCAAA3’
WSNP 40-general: 5 'AATTTCATATGCCTGGAGAAGCATCCTT 3'
WSNP41-FAM：5’CTTGGGTGACAAAATGGTTGTACAA3’
WSNP41-HEX：5’CTTGGGTGACAAAATGGTTGTACAG3’
WSNP 41-general: 5 'ATGGTTTGAGTTAACTTGCACAACGGAAT 3'
WSNP42-FAM：5’ATGCTGATAGATCATTTGGCAGTA3’
WSNP42-HEX：5’CTATGCTGATAGATCATTTGGCAGTG3’
WSNP 42-general: 5 'CAATGGCAGTTGCACTATTTGTTGGTATA 3'
WSNP43-FAM：5’CCTTTGACCCGCTGGTGGG3’
WSNP43-HEX：5’GCCTTTGACCCGCTGGTGGA3’
WSNP 43-general: 5 'GCTCGGCCCAGGATGCTACTA 3'
WSNP44-FAM：5’TGATCATGCGCTTTCCCACTGAT3’
WSNP44-HEX：5’GATCATGCGCTTTCCCACTGAC3’
WSNP 44-general: 5 'GCCCTTGCCCCATCGTCTTGAT 3'
WSNP45-FAM：5’AGATTCCTGGAGCTGCGTTTGC3’
WSNP45-HEX：5’AGATTCCTGGAGCTGCGTTTGG3’
WSNP 45-general: 5 'AGTTGAATTTGGCGGACGTGAGGTT 3'
WSNP46-FAM：5’CACAGGGATACCGAAACATGGA3’
WSNP46-HEX：5’CACAGGGATACCGAAACATGGG3’
WSNP 46-general: 5 'TTGTTACCGATTTTGTTCTCTTTTGTCGTT 3'
WSNP47-FAM：5’AGGTTTCCGAATCTAATGATCCG3’
WSNP47-HEX：5’CTAGGTTTCCGAATCTAATGATCCA3’
WSNP 47-general: 5 'GGCAGCATCACACTTCATGCTGATT 3'
WSNP48-FAM：5’ATAGAGCTTTGGCCTTCTACAATTGA3’
WSNP48-HEX：5’AGAGCTTTGGCCTTCTACAATTGC3’
WSNP 48-general: 5 'GGATCTGTTTGGTTTATGCCTGTAATGTA 3'
WSNP49-FAM：5’GTACCTCATTGCGTTTAATGGTAGCA3’
WSNP49-HEX：5’ACCTCATTGCGTTTAATGGTAGCG3’
WSNP 49-general: 5 'GACACGCGTCATCGAGTTTCACAAT 3'
WSNP50-FAM：5’GGAGATCTGCTAGAGACGCCT3’
WSNP50-HEX：5’GGAGATCTGCTAGAGACGCCG3’
WSNP 50-general: 5 'GGATGCCATGCCATGAGTTAGTGTT 3'
WSNP51-FAM：5’CAAGGGTTGAATGACCATGACTAC3’
WSNP51-HEX：5’CAAGGGTTGAATGACCATGACTAT3’
WSNP 51-general: 5 'GAAACGTCTTCGTACATGTGGTCCTA 3'
WSNP52-FAM：5’TATGTTGGCTGAAACCAAGCCTTC3’
WSNP52-HEX：5’TTATGTTGGCTGAAACCAAGCCTTT3’
WSNP 52-general: 5 'CTCACGGCACTTTAAATATTCCATCACTA 3'
WSNP53-FAM：5’AACCCTGTGTAACCTGCTATGACT3’
WSNP53-HEX：5’CCCTGTGTAACCTGCTATGACG3’
WSNP 53-general: 5 'TTTCCCAACACTAAAGCAGAGAGAAGAAA 3'
WSNP54-FAM：5’AAGGAAGCACGAACACCATAAGGAA3’
WSNP54-HEX：5’GGAAGCACGAACACCATAAGGAG3’
WSNP 54-general: 5 'GGCCAAGGATCCAATGGTCGCAT 3'
WSNP55-FAM：5’GTTTTATCACTGACGGTGCCTGC3’
WSNP55-HEX：5’GTTTTATCACTGACGGTGCCTGT3’
WSNP 55-general: 5 'CTTGTCAACCAACACCCAACTTATTACTA 3'
WSNP56-FAM：5’GCGTTCGAATTAAGGAGAGAATCAG3’
WSNP56-HEX：5’GGCGTTCGAATTAAGGAGAGAATCAA3’
WSNP 56-general: 5 'CAGTGTCGTAGGCTGGTCGCTT 3'
WSNP57-FAM：CTTGTGTCGGACAGTCAGGAG3’
WSNP57-HEX：5’ATCTTGTGTCGGACAGTCAGGAA3’
WSNP 57-general: 5 'GTTGATTGTCCCGTCACAGCGTTTA 3'
WSNP58-FAM：5’CAATCATATTGTTTTGGCGAGGAGC3’
WSNP58-HEX：5’AACAATCATATTGTTTTGGCGAGGAGT3’
WSNP 58-general: 5 'CAGAGCTGAACCCCACTGGTGTA 3'
WSNP59-FAM：5’CAAATGATAATGGACTCTGGTATAGTC3’
WSNP59-HEX：5’CAAATGATAATGGACTCTGGTATAGTT3’
WSNP 59-general: 5 'CTGGGACATCGTGGTTTCGTGTAAA 3'
WSNP60-FAM：5’AGGCTGAGCAAGAGTTCTTCTG3’
WSNP60-HEX：5’GTAGGCTGAGCAAGAGTTCTTCTA3’
WSNP 60-general: 5 'TATTCTTCTGCTCGACGTCGCACTT 3'
WSNP61-FAM：5’GCGGAATTCAGTCAGTGTTGCTC3’
WSNP61-HEX：5’GCGGAATTCAGTCAGTGTTGCTT3’
WSNP 61-general: 5 'GTTTAGATCCAAACCAATCCCCGGAA 3'
WSNP62-FAM：5’GGATTTTGAAGGCTCTAGAGATCGT3’
WSNP62-HEX：5’TTGAAGGCTCTAGAGATCGC3’
WSNP 62-general: 5 'GGATCCTCCGGGGCCGGTT 3'
WSNP63-FAM：5’ATGAGAGCATTGGAGAGTTTCCAC3’
WSNP63-HEX：5’TATGAGAGCATTGGAGAGTTTCCAT3’
WSNP 63-general: 5 'CCAGCCCACGCATATCCCCTTT 3'
WSNP64-FAM：5’GATTTCCTTCCTTTTTGTCCCTCAC3’
WSNP64-HEX：5’GATTTCCTTCCTTTTTGTCCCTCAT3’
WSNP 64-general: 5 'GCATATTTCAGGTTTACTTGCCAATCGTA 3'
WSNP65-FAM：5’ATTGTTGGTGGTCTACAGTACCTCA3’
WSNP65-HEX：5’GTTGGTGGTCTACAGTACCTCG3’
WSNP 65-general: 5 'CTGCAAAGGAAATATCTTGTCGAGTGATA 3'
WSNP66-FAM：5’GAACTTTCGTGATCGGTCAATGACT3’
WSNP66-HEX：5’AACTTTCGTGATCGGTCAATGACC3’
WSNP 66-general: 5 'CTAAACCCGTCATAAACACCCGGAT 3'
WSNP67-FAM：5’CCTTCCTCTCCGTGATCGTCAT3’3’
WSNP67-HEX：5’CTTCCTCTCCGTGATCGTCAG3’3’
WSNP 67-general: 5 'GGTTTGCCGGCTAGACCACGTA 3'
WSNP68-FAM：5’ACTTGATTGCTCGCTCCGAGATT3’
WSNP68-HEX：5’CTTGATTGCTCGCTCCGAGATC3’
WSNP 68-general: 5 'CCGAAGCGGATGAAGTGGTAGTTAA 3'
WSNP69-FAM：5’TCACGCGACTCCAGCAACTG3’
WSNP69-HEX：5’CTTCACGCGACTCCAGCAACTA3’
WSNP 69-general: 5 'GGGGCCTACGATTGAGGAAGTAAAA 3'
WSNP70-FAM：5’CGACAGCCGAGGGAACACAC3’
WSNP70-HEX：5’ATCGACAGCCGAGGGAACACAT3’
WSNP 70-general: 5 'GTCAGTCCGTCGTGTCTCTATATCAT 3'
WSNP71-FAM：5’AGAGGACTTGGTAAGATTAAAACTCTC3’
WSNP71-HEX：5’GAGAGGACTTGGTAAGATTAAAACTCTT3’
WSNP 71-general: 5 'GTTGCAAGTAATGCTAGGAGGTGCTT 3'
WSNP72-FAM：5’AAGGGCCTACTGAATACAGCTTCAT3’
WSNP72-HEX：5’GGGCCTACTGAATACAGCTTCAC3’
WSNP 72-general: 5 'CGCTCTTTTGCGCCTTATGTTCCTT 3'
WSNP73-FAM：5’ACGGGCATGAAAAGCTGGTCG3’
WSNP73-HEX：5’CACGGGCATGAAAAGCTGGTCA3’
WSNP 73-general: 5 'GTTGGACTATTCAGATTATTACACAGCCAA 3'
WSNP74-FAM：5’GATGCTCGCTTAGATCCACCAT3’
WSNP74-HEX：5’GATGCTCGCTTAGATCCACCAC3’
WSNP 74-general: 5 'TCCAAACCAAGGATCTAGTATTTCCTCTT 3'
WSNP75-FAM：5’GAACAAATTAGAGATGCTATTAAGTGGG3’
WSNP75-HEX：5’GAACAAATTAGAGATGCTATTAAGTGGA3’
WSNP 75-general: 5 'CCAAGTTATCGTGGCGTACAAGACAT 3'
WSNP76-FAM：5’CATCTCTTCACGGTACATCAACTCAT3’
WSNP76-HEX：5’ATCTCTTCACGGTACATCAACTCAC3’
WSNP 76-general: 5 'GTGGCCATCCGACAGAAAAAGTATTAATT 3'
WSNP77-FAM：5’CAGTCGCCTGATGTCTAGCTA3’
WSNP77-HEX：5’CAGTCGCCTGATGTCTAGCTG3’
WSNP 77-general: 5 'GAGGATGAAAGAGGAATGGCTGTCTT 3'
WSNP78-FAM：5’ACTTATTGACGCCTCCGTTCAAAC3’
WSNP78-HEX：5’CACTTATTGACGCCTCCGTTCAAAT3’
WSNP 78-general: 5 'CCTATCGTTCGCCTTTTTCCACAGTT 3'
WSNP79-FAM：5’GATAGGCTTTGTGAATCGCCCAG3’
WSNP79-HEX：5’GATAGGCTTTGTGAATCGCCCAT3’
WSNP 79-general: 5 'GCGTCCAAAACCGGAGGTCTAAAAA 3'
WSNP80-FAM：5’ATCATCCCTTGGACGTCAAGCG3’
WSNP80-HEX：5’CATCATCCCTTGGACGTCAAGCA3’
WSNP 80-general: 5 'ATGTCTCCTTCACTGGGTGCATCAA 3'
WSNP81-FAM：5’CAGATGTCACCAATACCGAGGG3’
WSNP81-HEX：5’CAGATGTCACCAATACCGAGGA3’
WSNP 81-general: 5 'CTCTATCTGCTGGTTTGAACTATCCTTT 3'
WSNP82-FAM：5’CCATCGCAGCCCAATAGAAAATTGTA3’
WSNP82-HEX：5’CATCGCAGCCCAATAGAAAATTGTG3’
WSNP 82-general: 5 'CGTTACCGTCGGGCCTTCTTCAT 3'
WSNP83-FAM：5’ATGGGCCTGTTGAACTAGCCAC3’
WSNP83-HEX：5’CATGGGCCTGTTGAACTAGCCAT3’
WSNP 83-general: 5 'GGAGTAAATCGCATAGAACCACCATAAAT 3'
WSNP84-FAM：5’GCCGCTTGTGCGCATTTCTTTC3’
WSNP84-HEX：5’GCCGCTTGTGCGCATTTCTTTT3’
WSNP 84-general: 5 'CAAAAAAACCAGGGTCTGCGATCTGTA 3'
WSNP85-FAM：5’GATTTGTGCAGACGGGCTAGCT3’
WSNP85-HEX：5’TGTGCAGACGGGCTAGCC3’
WSNP 85-general: 5 'ACATCCTCCCTGCGATCGCCAA 3'
WSNP86-FAM：5’CGCACGGGACTCTGTCCACA3’
WSNP86-HEX：5’GCACGGGACTCTGTCCACC3’
WSNP 86-general: 5 'GGACTGTCTAGTTGTTGCAATCGCAT 3'
WSNP87-FAM：5’GCTCTGGAAGAAGTCGAC3’
WSNP87-HEX：5’GCTGCTCTGGAAGAAGTCGAG3’
WSNP 87-general: 5 'GTGGAATATGAATTTAACACCTATGGCTAA 3'
WSNP88-FAM：5’TTATGTGATCTCTTTCAGGTTACATAG3’
WSNP88-HEX：5’CTTTATGTGATCTCTTTCAGGTTACATAC3’
WSNP 88-general: 5 'AAATGGTGAAGCAAGTGCAGGCCAT 3'
WSNP89-FAM：5’GATGAACGTGTTTGTGATGTTGATCTA3’
WSNP89-HEX：5’ATGAACGTGTTTGTGATGTTGATCTG3’
WSNP 89-general: 5 'TCATACAAGCAGCGCAGCAACGTAA 3'
WSNP90-FAM：5’GCCAAAAGCCCAACACAGAGGA3’
WSNP90-HEX：5’CCAAAAGCCCAACACAGAGGC3’
WSNP 90-general: 5 'GCTTCAAGCTGTGGATCGCTATCTA 3'
WSNP91-FAM：5’ACCCTAGTGCAGATCAAATCAACCT3’
WSNP91-HEX：5’CCTAGTGCAGATCAAATCAACCG3’
WSNP 91-general: 5 'TACCTGACTGATTTCGCCCCTCATT 3'
WSNP92-FAM：5’TGCCCTTTCAGGTACTACGAC3’
WSNP92-HEX：5’CTTGCCCTTTCAGGTACTACGAT3’
WSNP 92-general: 5 'GTCTTGGCTCGACAGGCGAGTT 3'
WSNP93-FAM：5’CAGGGAAAGATCATTAACGTCAACC3’
WSNP93-HEX：5’GCAGGGAAAGATCATTAACGTCAACT3’
WSNP 93-general: 5 'GTCCCGACTTCGAATTAATGAAGCCAT 3'
WSNP94-FAM：5’CAACCCAAAAGTATTAAAGCCGGC3’
WSNP94-HEX：5’ATCAACCCAAAAGTATTAAAGCCGGT3’
WSNP 94-general: 5 'CAACGAAAACCAGTTTGAGGGGAGAT 3'
WSNP95-FAM：5’AGAACGGAATACCCATGTTTACCC3’
WSNP95-HEX：5’AGAACGGAATACCCATGTTTACCG3’
WSNP 95-general: 5 'CCTGATAAGACATGGAGGTTTTCTCTTAA 3'
WSNP96-FAM：5’AGGGAATTCTTCTACTTTTCTAAACCG3’
WSNP96-HEX：5’TAGGGAATTCTTCTACTTTTCTAAACCA3’
WSNP 96-general: 5 'GTTCAGTGTTGGGTCCGTCTGTAAA 3'.
The invention has the beneficial effects that:
the 96 SNP locus combinations and the corresponding specific KASP primer combinations have the following characteristics in the identification of the bred wheat varieties:
1. 192 allelic variation of 96 SNP markers are detected in 1463 examined wheat varieties in China, the average MAF value of each site is 0.42, the average PIC value of each marker is 0.38, and the SNP markers have higher resolution on the wheat varieties;
2. the distribution frequency of allelic variation of each locus in the bred wheat variety is relatively uniform;
3. the positioning information and the physical position information are provided, and the unit point and the single copy are provided;
4. 96 SNP primers and primer combinations thereof can be stably, repeatedly and specifically amplified, and are convenient to popularize and apply;
5. the 96 SNP loci are finally screened and determined after all the currently known varieties in China are subjected to genotyping;
6. 96 SNP loci have no interlocking relation;
7. 96 SNP loci are basically and uniformly distributed on 21 pairs of chromosomes of wheat;
8. a 96 SNP site compatible chip platform, a Boo microfluidic chip platform, a fixed point sequencing platform and an LGC-SNpline platform based on KASP technology;
9. the cost of 96 SNP loci is low, large-scale detection is easy to realize, authenticity identification and SNP fingerprint construction of 1536 samples can be completed by applying the SNP marker and the primer combination in an indoor laboratory for 1 day, and the cost of manpower, material resources, financial resources, time and land resources is greatly saved.
Drawings
FIG. 1 shows the chromosome distribution of 8061 expansion sites screened according to the embodiment of the present invention;
FIG. 2 is a statistical plot of the 8061 extended sites MAF screened according to an embodiment of the present invention;
FIG. 3 is a statistical chart of PIC values of 8061 extended sites screened according to an embodiment of the present invention;
FIG. 4 shows a chromosome distribution of 420 candidate core loci screened according to an embodiment of the invention;
FIG. 5 is a statistical plot of MAF values for 420 primary core sites screened according to an embodiment of the present invention;
FIG. 6 shows the variety recognition rates for different combinations of loci according to embodiments of the invention.
FIG. 7 is a graph of 75 developed cultivars cluster analysis, wherein (a) cluster analysis was based on SNP markers; (b) clustering analysis based on SSR markers; the names of the varieties corresponding to the figure numbers are shown in Table 1, and the red circles indicate the varieties with closer relativity.
Detailed Description
The wheat variety authenticity SNP molecular marker detection method according to the embodiment of the invention comprises the following steps:
(1) extracting DNA of seeds or other tissues or organs of the inspected sample and the standard sample, wherein the mixed DNA sample at least contains more than 30 different individuals, and the more the selected individuals are, the better the mixed DNA sample represents 100% of genetic variation of the variety;
(2) quality of DNASolving the following steps: detecting the quality of the DNA in the step (1) by using an ultraviolet spectrophotometer, wherein OD is required 260/280 The value is between 1.8 and 2.0, OD 260 / 230 The value is above 2.0, when RNA is not removed, the DNA concentration of the working solution is adjusted to 30 ng/. mu.L, when RNA is removed, the DNA concentration of the working solution is adjusted to 20 ng/. mu.L, and the solution is reserved at 4 ℃;
(3) taking the working solution DNA sample in the step (2) as a template, configuring a reaction system by using the SNP primer, and designing a sample plate: DNA is respectively added into a 96-well plate, each plate is designed with two NTC (sterilized water without template control), two positive internal references (a DH strain homozygous for Chinese spring and genome DNA) and one heterozygous internal reference (DNA samples of the two positive internal references are mixed in equal proportion), a sample to be detected and a corresponding standard sample are respectively placed on the same 384-well plate or 1536-well plate to ensure that genotype data are obtained simultaneously, the DNA samples are distributed into a PCR micropore plate (the 384-well plate or the 1536-well plate), and sample information is edited in Kraken software;
(4) and (4) carrying out PCR amplification on the reaction system prepared in the step (3), wherein the PCR amplification can be carried out on a water bath PCR instrument or a conventional PCR instrument.
(5) Analyzing the genotypes of the sample to be detected and the standard sample, detecting a PCR product by using a fluorescent micropore plate scanner after PCR amplification is finished, and analyzing the genotypes of the sample to be detected and the standard sample by using Klaster caller software;
(6) and (3) analyzing data, and directly recording the genotypes of the to-be-detected sample and the standard sample at each site by using Kraken software. Firstly, observing whether the NTC (non-template contrast) is at the origin, if the NTC drifts out of a limited range, the sample is possibly polluted, the data is not available, then observing whether the internal reference genotype is correct, if the internal reference genotype is incorrect, the data is not available, selecting the data with definite typing, less missing data and compact clustering, and submitting the data;
(7) and (C) counting and recording results, wherein in the step A, two independent parallel tests are adopted for SNP fingerprint construction of the samples, all the samples are subjected to one-time whole-genotyping by using 96 markers, the fingerprint data of the two times are consistent and are taken as the real fingerprints of the samples to be put in storage, and a third test method is adopted when the fingerprint data of the two times are inconsistent. And B, adopting a 96-mark one-time whole genotyping mode and two independent parallel test methods for authenticity detection, taking the genotyping results of the two independent parallel tests as the genotypes of the samples to be tested, recording the genotyping results, counting the number of differential sites of the samples to be tested and the standard samples, and verifying the primer numbers of the differential sites.
Example 1 determination of molecular markers of the invention
1) Screening of wheat variety identification expansion site
190 representative samples were scanned using the SNP chip, and the analysis was performed based on the genotype data of 84661 SNP sites in the 190 samples. And screening out single copy SNP markers, removing duplication, performing Linkage Disequilibrium (LD) analysis, calculating the LD value among the markers, reserving the markers with r2 less than 0.8, and forming a wheat variety identification expansion site by 8061 SNPs in total together with 79 functional markers. The chromosome distribution of 8061 expansion sites is shown in FIG. 1, and the number of SNP markers on the D chromosome set is small, and is 2054 in total. The MAF value distribution of 8061 extended sites is shown in FIG. 2, and the MAF value (minimum allelic variation frequency) of 77% of sites is higher than 0.2 (FIG. 2). The statistical chart of PIC values of 8061 extended sites is shown in FIG. 3, and the average value of PIC values (polymorphic information content) is 0.3, which can better reflect the genetic diversity among varieties.
2) Screening of wheat variety identification primary selection core locus
And (3) screening SNP markers with MAF >0.2 and uniform distribution from the extension loci to serve as initial selection core loci, wherein the total number of the SNP markers is 420, and each chromosome is provided with 20 SNP markers. The chromosome distribution of 420 candidate core loci is shown in FIG. 4. The MAF values for 420 candidate core sites ranged from 0.24-0.5, with 77% of sites having MAF values above 0.4 (FIG. 5). The PIC values varied over a range of 0.3-0.41, with an average value of 0.37 each.
3) SNP locus screening suitable for wheat variety fingerprint construction
Obtaining the flanking sequences of 420 SNP sites, designing and synthesizing KASP primers, selecting 95 representative varieties from 187 samples and a negative control (H) 2 O), 96 samples in total, were genotyped. 282 of 420 sites were successfully converted into KASP primers at 67% conversion efficiency, while some primers had poor specificity, resulting in poor specificityThe amplified product can not carry out accurate genotyping on the variety, so the transformation is difficult. And 1433 standard samples are used for evaluation, and 112 library establishing sites with good repeatability, stability and genotyping quality are determined and are used for constructing a standard sample DNA fingerprint database. The chromosome distribution of 112 database construction sites has the maximum D chromosome group markers, and the total number of the D chromosome group markers is 42; A. and 35 each of the B chromosome sets. The 6 th homologous group marks the maximum, 30 in total; 20 in total from the 5 th homologous group; the number of 1 st homologous group is only 7. Wherein the number of markers on chromosome 6B is at most, 12 in total; the second 10 chromosomes of 5A; there is only one marker on each of chromosomes 1A and 1B.
4) Core locus combined screening suitable for wheat variety authenticity identification
And analyzing the optimal core site combination number based on 1433 wheat standard sample fingerprint data of 112 SNP sites. The results show that the identification efficiency of the combined varieties at different sites is significantly different, and finally 96 core sites are determined to be used as the first-choice marker for variety identity identification (table 1), as shown in fig. 6, the variety identification rate of 96 SNP sites is 99.16%, and the average gene diversity and polymorphism information index are 0.5265 and 0.4355, respectively.
TABLE 1 polymorphic information of SNP sites (markers) for variety authenticity identification
Example 2 SNP marker for wheat variety authenticity identification and SNP fingerprint database construction
The 96 SNP marker combinations in the embodiment 1 are adopted to construct the SNP fingerprints of 1463 examined wheat variety standard samples in China, 1433 non-same-name wheat varieties are remained after the same-name varieties are removed, and clustering analysis is carried out according to 1433 fingerprint data, and the result shows that 99.16% of examined wheat varieties can be distinguished by utilizing 96 SNP markers, so that the wheat variety authenticity identification by utilizing the set of markers and the construction of the standard sample SNP fingerprint database have scientificity, accuracy, feasibility, effectiveness and operability.
Example 3 SNP markers for genetic diversity and specificity identification of wheat varieties
Fingerprints are respectively constructed on 75 representative bred varieties (table 2) selected from ten ecological wheat areas by utilizing SNP (single nucleotide polymorphism) and SSR (simple sequence repeat) technologies, the PowermakerV3.25 software is utilized to analyze the genetic diversity indexes of SNP and SSR sites, the Nei's (1983) genetic distance between the varieties is calculated, UPOVE (upstream clustering analysis) is carried out, and the results show that clustering results are relatively consistent by utilizing different marking technologies (as shown in figure 7), such as the varieties with the numbers of 14 and 70, 48 and 68, 34 and 71, 56 and 51 and 1, 63 and 69 are preferentially clustered together in a clustering graph. The SNP marker technology can be used for wheat genetic diversity analysis and cluster analysis as well as SSR marker technology, and the SNP marker technology and the SSR marker technology have the same effect, so that the SNP marker can assist the screening work of specificity identification of large sample amount at the early stage, the waste of land is reduced, and a large amount of cost such as manpower, material resources and financial resources is saved.
Table 275 parts materials information
Sequence listing
<110> agriculture and forestry academy of sciences of Beijing City
<120> SNP molecular marker for identifying common wheat variety and SNP molecular marker detection method
<160> 30
<170> SIPOSequenceListing 1.0
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ggagaacaaa gcagaaaagc tgcaa 25
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gagaacaaag cagaaaagct gcag 24
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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gtgaacattc aggatatttg tgctgcata 29
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
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cgtagtcctc cagaaaatcg 20
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<213> Artificial Sequence (Artificial Sequence)
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gctcgtagtc ctccagaaaa tca 23
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<213> Artificial Sequence (Artificial Sequence)
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<213> Artificial Sequence (Artificial Sequence)
<400> 7
agatgttgag aacactttcg tccc 24
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<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
gagatgttga gaacactttc gtcca 25
<210> 9
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
cagagaagag ggcgtcagcg ta 22
<210> 10
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
ggcaactcta acaaacacac tcc 23
<210> 11
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 11
agggcaactc taacaaacac actc 24
<210> 12
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 12
gacgaactcg gcgggaccat tt 22
<210> 13
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 13
accggcgact ctcaagttga tg 22
<210> 14
<211> 23
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 14
<210> 15
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 15
cgggaaacaa tgctcatagg gacaa 25
<210> 16
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 16
gtaggatatc gcggaagacc ac 22
<210> 17
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 17
gtaggatatc gcggaagacc ag 22
<210> 18
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 18
gtgatctagc taggttgtcg gctaa 25
<210> 19
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 19
aaacatgtaa aactaacttc acataccca 29
<210> 20
<211> 27
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 20
acatgtaaaa ctaacttcac atacccg 27
<210> 21
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 21
gacccttcga gaccctaccc at 22
<210> 22
<211> 26
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 22
aacacattca gagaatatga ctaggg 26
<210> 23
<211> 28
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 23
ataacacatt cagagaatat gactagga 28
<210> 24
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 24
gcctgagcgg gcgtttggta a 21
<210> 25
<211> 22
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 25
gcatacattc ctcctgttcg ac 22
<210> 26
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 26
gtgcatacat tcctcctgtt cgat 24
<210> 27
<211> 29
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 27
attgccaagg cgtagtttga gatattcat 29
<210> 28
<211> 24
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 28
gtagaattag tcgaggacca agag 24
<210> 29
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 29
ggtagaatta gtcgaggacc aagaa 25
<210> 30
<211> 25
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 30
aaaacgcgac cgtgattggg atctt 25
Claims (1)
1. A method for identifying the authenticity of a common wheat variety, wheat molecular breeding or assisting in the specific identification of a wheat variety, comprising the steps of:
extracting DNA of a sample to be detected and a standard sample;
carrying out PCR amplification on DNA of a detection sample and a standard sample, and determining the genotype of the detection sample at the following 96 SNP sites, wherein the number of the SNP sites, the chromosome where the SNP is located, the specific physical position and the deoxynucleotide information are as follows:
WSNP01 is located at position 27215962 on chromosome 1A, and its deoxynucleotide is A or G;
WSNP02 is located at position 41817136 on chromosome 1A, and its deoxynucleotide is T or C;
WSNP03 is located at position 495285832 on chromosome 1A, and its deoxynucleotide is T or G;
WSNP04 is located at position 587992062 on chromosome 1B, and its deoxynucleotide is T or C;
WSNP05 is located at position 340381039 on chromosome 1B, and its deoxynucleotide is T or C;
WSNP06 is located at position 20306381 on chromosome 1D, and its deoxynucleotide is C or G;
WSNP07 is located at position 288318891 on chromosome 1D, and its deoxynucleotide is A or G;
WSNP08 is located at position 427323196 on chromosome 1D, and its deoxynucleotide is A or G;
WSNP09 is located at position 43371103 on chromosome 2A, and its deoxynucleotide is T or C;
WSNP10 is located at position 36393269 on chromosome 2A, and its deoxynucleotide is A or G;
WSNP11 is located at position 174150002 on chromosome 2A, and its deoxynucleotide is A or G;
WSNP12 is located at position 41105877 on chromosome 2A, and its deoxynucleotide is T or C;
WSNP13 is located at position 12003721 on chromosome 2B, and its deoxynucleotide is T or C;
WSNP14 is located at position 47427272 on chromosome 2B, and its deoxynucleotide is T or C;
WSNP15 is located at position 108910271 on chromosome 2B, and its deoxynucleotide is T or G;
WSNP16 is located at position 652578839 on chromosome 2B, and its deoxynucleotide is C or G;
WSNP17 is located at position 697446904 on chromosome 2B, and its deoxynucleotide is A or G;
WSNP18 is located at position 773821814 on chromosome 2B, and its deoxynucleotide is A or G;
WSNP19 is located at position 28783896 on 2D chromosome, and its deoxynucleotide is T or C;
WSNP20 is located at position 422881650 on 2D chromosome, and its deoxynucleotide is T or C;
the WSNP21 is located at 431959521 th position on a 2D chromosome, and the deoxynucleotide of the WSNP21 is A or G;
WSNP22 is located at position 458920669 on 2D chromosome, and its deoxynucleotide is T or C;
WSNP23 is located at position 551877542 on 2D chromosome, and its deoxynucleotide is T or G;
WSNP24 is located at position 615715449 on 2D chromosome, and its deoxynucleotide is A or G;
WSNP25 is located at position 25908324 on chromosome 3A, and its deoxynucleotide is A or G;
WSNP26 is located at position 481678769 on chromosome 3A, and its deoxynucleotide is A or G;
WSNP27 is located at position 735293187 on chromosome 3A, and its deoxynucleotide is T or G;
WSNP28 is located at position 597917383 on chromosome 3A, and its deoxynucleotide is A or G;
WSNP29 is located at position 509365938 on chromosome 3B, and its deoxynucleotide is A or G;
WSNP30 is located at position 627720241 on chromosome 3B, and its deoxynucleotide is A or G;
WSNP31 is located at position 680735249 on chromosome 3B, and its deoxynucleotide is T or C;
WSNP32 is located at position 749862710 on chromosome 3B, and its deoxynucleotide is A or C;
WSNP33 is located at position 819302391 on chromosome 3B, and its deoxynucleotide is A or T;
WSNP34 is located at position 56675266 on the 3D chromosome, and its deoxynucleotide is T or C;
WSNP35 is located at position 537495536 on 3D chromosome, and its deoxynucleotide is T or G;
WSNP36 is located at position 578749369 on 3D chromosome, and its deoxynucleotide is A or G;
WSNP37 is located at position 611664812 on 3D chromosome, and its deoxynucleotide is A or G;
WSNP38 is located at position 584425606 on chromosome 4A, and its deoxynucleotide is T or G;
WSNP39 is located at position 116644494 on chromosome 4A, and its deoxynucleotide is A or G;
WSNP40 is located at position 532224647 on chromosome 4A, and its deoxynucleotide is A or G;
WSNP41 is located at position 669197675 on chromosome 4A, and its deoxynucleotide is A or G;
WSNP42 is located at position 36960078 on chromosome 4B, and its deoxynucleotide is T or C;
WSNP43 is located at position 527111678 on chromosome 4B, and its deoxynucleotide is A or G;
WSNP44 is located at position 179840525 on chromosome 4B, and its deoxynucleotide is A or G;
WSNP45 is located at position 636757506 on chromosome 4B, and its deoxynucleotide is C or G;
WSNP46 is located at position 11870112 on the 4D chromosome, and its deoxynucleotide is A or G;
the WSNP47 is located at 364033217 th position on a 4D chromosome, and the deoxynucleotide of the WSNP47 is A or C;
WSNP48 is located at position 118605639 on the 4D chromosome, and its deoxynucleotide is T or C;
WSNP49 is located at position 367933139 on the 4D chromosome, and its deoxynucleotide is A or G;
WSNP50 is located at position 448113730 on chromosome 5A, and its deoxynucleotide is A or C;
WSNP51 is located at position 478883696 on chromosome 5A, and its deoxynucleotide is A or G;
WSNP52 is located at position 455550448 on chromosome 5A, and its deoxynucleotide is A or G;
WSNP53 is located at position 500625774 on chromosome 5A, and its deoxynucleotide is T or G;
WSNP54 is located at position 591302258 on chromosome 5A, and its deoxynucleotide is A or G;
WSNP55 is located at position 695003171 on chromosome 5A, and its deoxynucleotide is T or C;
WSNP56 is located at position 6594624 on chromosome 5B, and its deoxynucleotide is T or C;
WSNP57 is located at position 11080396 on chromosome 5B, and its deoxynucleotide is A or G;
WSNP58 is located at position 281675472 on chromosome 5B, and its deoxynucleotide is A or G;
WSNP59 is located at position 485426049 on chromosome 5B, and its deoxynucleotide is T or C;
WSNP60 is located at position 82481988 on the 5D chromosome, and its deoxynucleotide is T or C;
WSNP61 is located at position 302000445 on chromosome 5D, and its deoxynucleotide is A or G;
WSNP62 is located at position 370064912 on chromosome 5D, and its deoxynucleotide is A or G;
the WSNP63 is located at 495427294 th position on a 5D chromosome, and the deoxynucleotide of the WSNP63 is A or G;
WSNP64 is located at position 51949801 on chromosome 6A, and its deoxynucleotide is T or C;
WSNP65 is located at position 617805673 on chromosome 6A, and its deoxynucleotide is A or G;
WSNP66 is located at position 615440555 on chromosome 6A, and its deoxynucleotide is T or C;
WSNP67 is located at position 77112398 on chromosome 6A, and its deoxynucleotide is T or C;
WSNP68 is located at position 445560400 on chromosome 6A, and its deoxynucleotide is A or G;
WSNP69 is located at position 457660545 on chromosome 6A, and its deoxynucleotide is A or C;
WSNP70 is located at position 579446448 on chromosome 6A, and its deoxynucleotide is T or C;
WSNP71 is located at position 22553387 on chromosome 6B, and its deoxynucleotide is A or G;
WSNP72 is located at position 27687594 on chromosome 6B, and its deoxynucleotide is T or C;
WSNP73 is located at position 158129211 on chromosome 6B, and its deoxynucleotide is T or C;
WSNP74 is located at position 227062800 on chromosome 6B, and its deoxynucleotide is A or G;
WSNP75 is located at position 439018758 on chromosome 6B, and its deoxynucleotide is T or C;
WSNP76 is located at position 630873069 on chromosome 6B, and its deoxynucleotide is T or C;
WSNP77 is located at position 659565617 on chromosome 6B, and its deoxynucleotide is A or G;
WSNP78 is located at position 682326705 on chromosome 6B, and its deoxynucleotide is A or G;
WSNP79 is located at position 86651424 on chromosome 6D, and its deoxynucleotide is T or G;
WSNP80 is located at position 133466400 on chromosome 6D, and its deoxynucleotide is T or C;
WSNP81 is located at position 351234720 on chromosome 6D, and its deoxynucleotide is T or C;
WSNP82 is located at position 438410250 on chromosome 6D, and its deoxynucleotide is T or C;
WSNP83 is located at position 4471905419 on chromosome 6D, and its deoxynucleotide is A or G;
WSNP84 is located at position 537642742 on chromosome 7A, and its deoxynucleotide is T or C;
WSNP85 is located at position 679816107 on chromosome 7A, and its deoxynucleotide is A or G;
WSNP86 is located at position 707833467 on chromosome 7A, and its deoxynucleotide is A or C;
WSNP87 is located at position 67705483 on chromosome 7B, and its deoxynucleotide is G or C;
WSNP88 is located at position 605748172 on chromosome 7B, and its deoxynucleotide is C or G;
WSNP89 is located at position 678124448 on chromosome 7B, and its deoxynucleotide is A or G;
WSNP90 is located at position 721402941 on chromosome 7B, and its deoxynucleotide is A or C;
WSNP91 is located at position 87479998 on chromosome 7D, and its deoxynucleotide is A or C;
WSNP92 is located at position 349365912 on chromosome 7D, and its deoxynucleotide is T or C;
WSNP93 is located at position 411596748 on chromosome 7D, and its deoxynucleotide is T or C;
WSNP94 is located at position 430012769 on chromosome 7D, and its deoxynucleotide is T or C;
WSNP95 is located at position 452686835 on chromosome 7D, and its deoxynucleotide is G or C;
WSNP96 is located at position 508179540 on chromosome 7D, and its deoxynucleotide is T or C;
the allele-specific primers for detecting the genotypes of the 96 SNP sites consist of:
WSNP01-FAM：5’GGAGAACAAAGCAGAAAAGCTGCAA3’
WSNP01-HEX：5’GAGAACAAAGCAGAAAAGCTGCAG3’
WSNP 01-general: 5 'GTGAACATTCAGGATATTTGTGCTGCATA 3';
WSNP02-FAM：5’CGTAGTCCTCCAGAAAATCG3’
WSNP02-HEX：5’GCTCGTAGTCCTCCAGAAAATCA3’
WSNP 02-general: 5 'GGATCTGGACGGCTTCCCACTT 3'
WSNP03-FAM：5’AGATGTTGAGAACACTTTCGTCCC3’
WSNP03-HEX：5’GAGATGTTGAGAACACTTTCGTCCA3’
WSNP 03-general: 5 'CAGAGAAGAGGGCGTCAGCGTA 3'
WSNP04-FAM：5’GGCAACTCTAACAAACACACTCC3’3’
WSNP04-HEX：5’AGGGCAACTCTAACAAACACACTCT
WSNP 04-general: 5 'GACGAACTCGGCGGGACCATTT 3'
WSNP05-FAM：5’ACCGGCGACTCTCAAGTTGATG3’
WSNP05-HEX：5’AACCGGCGACTCTCAAGTTGATA3’
WSNP 05-general: CGGGAAACAATGCTCATAGGGACAA 3'
WSNP06-FAM：5’GTAGGATATCGCGGAAGACCAC3’
WSNP06-HEX：5’GTAGGATATCGCGGAAGACCAG3’
WSNP 06-general: 5 'GTGATCTAGCTAGGTTGTCGGCTAA 3'
WSNP07-FAM：5’AAACATGTAAAACTAACTTCACATACCCA3’
WSNP07-HEX：5’ACATGTAAAACTAACTTCACATACCCG3’
WSNP 07-general: 5 'GACCCTTCGAGACCCTACCCAT 3'
WSNP08-FAM：5’AACACATTCAGAGAATATGACTAGGG3’
WSNP08-HEX：5’ATAACACATTCAGAGAATATGACTAGGA3’
WSNP 08-general: 5 'GCCTGAGCGGGCGTTTGGTAA 3'
WSNP09-FAM：5’GCATACATTCCTCCTGTTCGAC3’
WSNP09-HEX：5’GTGCATACATTCCTCCTGTTCGAT3’
WSNP 09-general: 5 'ATTGCCAAGGCGTAGTTTGAGATATTCAT 3'
WSNP10-FAM：5’GTAGAATTAGTCGAGGACCAAGAG3’
WSNP10-HEX：5’GGTAGAATTAGTCGAGGACCAAGAA3’
WSNP 10-general: 5 'AAAACGCGACCGTGATTGGGATCTT 3'
WSNP11-FAM：5’GGACCAGAATCACCAACGAATAAAG3’
WSNP11-HEX：5’GGACCAGAATCACCAACGAATAAAA3’
WSNP 11-general: 5 'TTTAGCTGCAGGGTTTATCACTGATGATT 3'
WSNP12-FAM：5’CCTCCCTCCCGACCTCTG3’
WSNP12-HEX：5’CCCTCCCTCCCGACCTCTA3’
WSNP 12-general: 5 'CTAGTTGGGCCGAGGCAGTTGAA 3'
WSNP13-FAM：5’GTCGAATGGATTTTCCTCGAGCA3’
WSNP13-HEX：5’GTCGAATGGATTTTCCTCGAGCG3’
WSNP 13-general: 5 'TGAGCATCAAAACTCACTCTGTTCAGAA 3'
WSNP14-FAM：5’CCCCTCGGCAACTCCGTGAA3’
WSNP14-HEX：5’CCCTCGGCAACTCCGTGAG3’
WSNP 14-general: 5 'GGTAGTCTATTGAACTCGATCGATATGTA 3'
WSNP15-FAM：5’GATCAATTCTCTTATGCTTGCCAAAGAA3’
WSNP15-HEX：ATCAATTCTCTTATGCTTGCCAAAGAC3’
WSNP 15-general: 5 'GAACACTATATGTTCTGGTGGAAGGAAA 3'
WSNP16-FAM：5’ATGATGCATAAAGTTTGATTGCTTCCC3’
WSNP16-HEX：5’ATGATGCATAAAGTTTGATTGCTTCCG3’
WSNP 16-general: 5 'CCATTAAGAAATGAAGTTGGCAATGGGAT 3'
WSNP17-FAM：5’AAGAGGAGAACAGAGAGCGATAGAA3’
WSNP17-HEX：5’GAGGAGAACAGAGAGCGATAGAG3’
WSNP 17-general: 5 'CTCCCAGAGCCAGCCGCCAA 3'
WSNP18-FAM：5’AGTTGAAAACACCGAACCAGCGA3’
WSNP18-HEX：5’GTTGAAAACACCGAACCAGCGG3’
WSNP 18-general: 5 'GGTGAGAGCAAGGATCCCAATCTAT 3'
WSNP19-FAM：5’CATGAGATCCTAAATCAGAGTCGCA3’
WSNP19-HEX：5’ATGAGATCCTAAATCAGAGTCGCG3’
WSNP 19-general: 5 'GGTCAAGTTTTCTGGACCCCATAATTATT 3'
WSNP20-FAM：5’ATTGAGTCGGCACAACATCTCAAG3’
WSNP20-HEX：ATATTGAGTCGGCACAACATCTCAAA3’
WSNP 20-general: 5 'TGGTGCTTGCGATGACTTCGTCAAT 3'
WSNP21-FAM：5’CCTGACGCTTCCACGCTGAAC3’
WSNP21-HEX：5’ACCTGACGCTTCCACGCTGAAT3’
WSNP 21-general: 5 'TGGCTCAAGTTGATCGGCCAGAAAT 3'
WSNP22-FAM：5’TTATATGTTTGAAGTAGATGTTGCACG3’
WSNP22-HEX：5’CTTTATATGTTTGAAGTAGATGTTGCACA3’
WSNP 22-general: 5 'ACATCACCTGTAAGAGTTCCTTTGTCAA 3'
WSNP23-FAM：5’CAACGATTCTCTCGCCTTACATAC3’3’
WSNP23-HEX：5’GCAACGATTCTCTCGCCTTACATAA3’
WSNP 23-general: 5 'GGACTGGAATCAATTTTATGTACTCCCTT 3'
WSNP24-FAM：5’AGTACCTTATAGACGACCAGACACT3’
WSNP24-HEX：5’ACCTTATAGACGACCAGACACC3’
WSNP 24-general: 5 'GTTTAGACTGCAATCCTCCATCGGAA 3'
WSNP25-FAM：5’CGATTGAATCTCAAGCGAACAAACG3’
WSNP25-HEX：5’GCGATTGAATCTCAAGCGAACAAACA3’
WSNP 25-general: 5 'TGTAGACATCTAGCAACCCCCTGAA 3'
WSNP26-FAM：5’GGTTCGTGCAGCCCGTTCG3’
WSNP26-HEX：5’AAGGTTCGTGCAGCCCGTTCA3’
WSNP 26-general: 5 'GCGCCATCCTGGACTGCTTCAA 3'
WSNP27-FAM：5’GGGCGTAAAGGTAAGAAAGACTC3’
WSNP27-HEX：5’ACGGGCGTAAAGGTAAGAAAGACTT3’
WSNP 27-general: 5 'TAGGCAAATGCGAGTGTCAATCAATGTTT 3'
WSNP28-FAM：5’AAAGTGGAGCTACAGGAGAAAATGATA3’
WSNP28-HEX：5’AGTGGAGCTACAGGAGAAAATGATC3’
WSNP 28-general: 5 'GTCGGCTAAAAGGTGTTGGCTTTGTA 3'
WSNP29-FAM：5’GTGGTGCAGGACCAAGAGTC3’
WSNP29-HEX：5’CTGTGGTGCAGGACCAAGAGTT3’
WSNP 29-general: 5 'CTCTGACTGCTGCATTGAATTGTCAATAA 3'
WSNP30-FAM：5’GGTAAGCTTTGCCTAGCTTTCCTAT3’
WSNP30-HEX：5’GTAAGCTTTGCCTAGCTTTCCTAC3’
WSNP 30-general: 5 'GTTTGTCCAACTAAGATAGAGTATGGGAA 3'
WSNP31-FAM：5’CATGCCACTGATAATGTGGACGC3’
WSNP31-HEX：5’ACATGCCACTGATAATGTGGACGT3’
WSNP 31-general: 5 'CTACTCATCGGAACCAACTCTACTCAA 3'
WSNP32-FAM：5’GGGGAACTTTCCACCTGATCATG3’
WSNP32-HEX：5’GGGGAACTTTCCACCTGATCATT3’
WSNP 32-general: 5 'GCCTGTTTTCCCGGCACGGAAT 3'
WSNP33-FAM：5’GAATACGGCGCCTTTCGTGCA3’
WSNP33-HEX：5’GAATACGGCGCCTTTCGTGCT3’
WSNP 33-general: 5 'GCTTACACGTCTAGCTATGATGCCAA 3'
WSNP34-FAM：5’ATTGCTCCTGTTCCCAGCAAGG3’
WSNP34-HEX：5’CATTGCTCCTGTTCCCAGCAAGA3’
WSNP 34-general: 5 'AAATTGGTGGAGCTAAGGAACTATGGAAA 3'
WSNP35-FAM：5’GTAAGGGACACACCTCCATCCAT3’
WSNP35-HEX：5’AAGGGACACACCTCCATCCAG3’
WSNP 35-general: 5 'ATAGCTAGATTGTCATCTGGCCAAACAAA 3'
WSNP36-FAM：5’AAGGGCGTTTATTTATGCTTGAAGACA3’
WSNP36-HEX：5’GGGCGTTTATTTATGCTTGAAGACG3’
WSNP 36-general: 5 'TAAACTAGTCGACTTCTTTTCCACGACAA 3'
WSNP37-FAM：5’CGTTTGCGTTCAGGTCGTGC3’
WSNP37-HEX：5’GTCGTTTGCGTTCAGGTCGTGT3’
WSNP 37-general: 5 'GGCAAGTCCTCCGAGGCTCAT 3'
WSNP38-FAM：5’GCATAAGGGTTATCACCAAGTCG3’
WSNP38-HEX：5’CTGCATAAGGGTTATCACCAAGTCA3’
WSNP 38-general: 5 'CAGGTACTTTATGTGAAAGCTCTTGAGAT 3'
WSNP39-FAM：5’GATAACAGCGTTTTCCTGACGAGA3’
WSNP39-HEX：5’ATAACAGCGTTTTCCTGACGAGG3’
WSNP 39-general: 5 'CCATCCCCAACCATAGGACCGAT 3'
WSNP40-FAM：5’AGTCCTTTCTTAAGCACGGCAAC3’
WSNP40-HEX：5’CAGTCCTTTCTTAAGCACGGCAAA3’
WSNP 40-general: 5 'AATTTCATATGCCTGGAGAAGCATCCTT 3'
WSNP41-FAM：5’CTTGGGTGACAAAATGGTTGTACAA3’
WSNP41-HEX：5’CTTGGGTGACAAAATGGTTGTACAG3’
WSNP 41-general: 5 'ATGGTTTGAGTTAACTTGCACAACGGAAT 3'
WSNP42-FAM：5’ATGCTGATAGATCATTTGGCAGTA3’
WSNP42-HEX：5’CTATGCTGATAGATCATTTGGCAGTG3’
WSNP 42-general: 5 'CAATGGCAGTTGCACTATTTGTTGGTATA 3'
WSNP43-FAM：5’CCTTTGACCCGCTGGTGGG3’
WSNP43-HEX：5’GCCTTTGACCCGCTGGTGGA3’
WSNP 43-general: 5 'GCTCGGCCCAGGATGCTACTA 3'
WSNP44-FAM：5’TGATCATGCGCTTTCCCACTGAT3’
WSNP44-HEX：5’GATCATGCGCTTTCCCACTGAC3’
WSNP 44-general: 5 'GCCCTTGCCCCATCGTCTTGAT 3'
WSNP45-FAM：5’AGATTCCTGGAGCTGCGTTTGC3’
WSNP45-HEX：5’AGATTCCTGGAGCTGCGTTTGG3’
WSNP 45-general: 5 'AGTTGAATTTGGCGGACGTGAGGTT 3'
WSNP46-FAM：5’CACAGGGATACCGAAACATGGA3’
WSNP46-HEX：5’CACAGGGATACCGAAACATGGG3’
WSNP 46-general: 5 'TTGTTACCGATTTTGTTCTCTTTTGTCGTT 3'
WSNP47-FAM：5’AGGTTTCCGAATCTAATGATCCG3’
WSNP47-HEX：5’CTAGGTTTCCGAATCTAATGATCCA3’
WSNP 47-general: 5 'GGCAGCATCACACTTCATGCTGATT 3'
WSNP48-FAM：5’ATAGAGCTTTGGCCTTCTACAATTGA3’
WSNP48-HEX：5’AGAGCTTTGGCCTTCTACAATTGC3’
WSNP 48-general: 5 'GGATCTGTTTGGTTTATGCCTGTAATGTA 3'
WSNP49-FAM：5’GTACCTCATTGCGTTTAATGGTAGCA3’
WSNP49-HEX：5’ACCTCATTGCGTTTAATGGTAGCG3’
WSNP 49-general: 5 'GACACGCGTCATCGAGTTTCACAAT 3'
WSNP50-FAM：5’GGAGATCTGCTAGAGACGCCT3’
WSNP50-HEX：5’GGAGATCTGCTAGAGACGCCG3’
WSNP 50-general: 5 'GGATGCCATGCCATGAGTTAGTGTT 3'
WSNP51-FAM：5’CAAGGGTTGAATGACCATGACTAC3’
WSNP51-HEX：5’CAAGGGTTGAATGACCATGACTAT3’
WSNP 51-general: 5 'GAAACGTCTTCGTACATGTGGTCCTA 3'
WSNP52-FAM：5’TATGTTGGCTGAAACCAAGCCTTC3’
WSNP52-HEX：5’TTATGTTGGCTGAAACCAAGCCTTT3’
WSNP 52-general: 5 'CTCACGGCACTTTAAATATTCCATCACTA 3'
WSNP53-FAM：5’AACCCTGTGTAACCTGCTATGACT3’
WSNP53-HEX：5’CCCTGTGTAACCTGCTATGACG3’
WSNP 53-general: 5 'TTTCCCAACACTAAAGCAGAGAGAAGAAA 3'
WSNP54-FAM：5’AAGGAAGCACGAACACCATAAGGAA3’
WSNP54-HEX：5’GGAAGCACGAACACCATAAGGAG3’
WSNP 54-general: 5 'GGCCAAGGATCCAATGGTCGCAT 3'
WSNP55-FAM：5’GTTTTATCACTGACGGTGCCTGC3’
WSNP55-HEX：5’GTTTTATCACTGACGGTGCCTGT3’
WSNP 55-general: 5 'CTTGTCAACCAACACCCAACTTATTACTA 3'
WSNP56-FAM：5’GCGTTCGAATTAAGGAGAGAATCAG3’
WSNP56-HEX：5’GGCGTTCGAATTAAGGAGAGAATCAA3’
WSNP 56-general: 5 'CAGTGTCGTAGGCTGGTCGCTT 3'
WSNP57-FAM：CTTGTGTCGGACAGTCAGGAG3’
WSNP57-HEX：5’ATCTTGTGTCGGACAGTCAGGAA3’
WSNP 57-general: 5 'GTTGATTGTCCCGTCACAGCGTTTA 3'
WSNP58-FAM：5’CAATCATATTGTTTTGGCGAGGAGC3’
WSNP58-HEX：5’AACAATCATATTGTTTTGGCGAGGAGT3’
WSNP 58-general: 5 'CAGAGCTGAACCCCACTGGTGTA 3'
WSNP59-FAM：5’CAAATGATAATGGACTCTGGTATAGTC3’
WSNP59-HEX：5’CAAATGATAATGGACTCTGGTATAGTT3’
WSNP 59-general: 5 'CTGGGACATCGTGGTTTCGTGTAAA 3'
WSNP60-FAM：5’AGGCTGAGCAAGAGTTCTTCTG3’
WSNP60-HEX：5’GTAGGCTGAGCAAGAGTTCTTCTA3’
WSNP 60-general: 5 'TATTCTTCTGCTCGACGTCGCACTT 3'
WSNP61-FAM：5’GCGGAATTCAGTCAGTGTTGCTC3’
WSNP61-HEX：5’GCGGAATTCAGTCAGTGTTGCTT3’
WSNP 61-general: 5 'GTTTAGATCCAAACCAATCCCCGGAA 3'
WSNP62-FAM：5’GGATTTTGAAGGCTCTAGAGATCGT3’
WSNP62-HEX：5’TTGAAGGCTCTAGAGATCGC3’
WSNP 62-general: 5 'GGATCCTCCGGGGCCGGTT 3'
WSNP63-FAM：5’ATGAGAGCATTGGAGAGTTTCCAC3’
WSNP63-HEX：5’TATGAGAGCATTGGAGAGTTTCCAT3’
WSNP 63-general: 5 'CCAGCCCACGCATATCCCCTTT 3'
WSNP64-FAM：5’GATTTCCTTCCTTTTTGTCCCTCAC3’
WSNP64-HEX：5’GATTTCCTTCCTTTTTGTCCCTCAT3’
WSNP 64-general: 5 'GCATATTTCAGGTTTACTTGCCAATCGTA 3'
WSNP65-FAM：5’ATTGTTGGTGGTCTACAGTACCTCA3’
WSNP65-HEX：5’GTTGGTGGTCTACAGTACCTCG3’
WSNP 65-general: 5 'CTGCAAAGGAAATATCTTGTCGAGTGATA 3'
WSNP66-FAM：5’GAACTTTCGTGATCGGTCAATGACT3’
WSNP66-HEX：5’AACTTTCGTGATCGGTCAATGACC3’
WSNP 66-general: 5 'CTAAACCCGTCATAAACACCCGGAT 3'
WSNP67-FAM：5’CCTTCCTCTCCGTGATCGTCAT3’3’
WSNP67-HEX：5’CTTCCTCTCCGTGATCGTCAG3’3’
WSNP 67-general: 5 'GGTTTGCCGGCTAGACCACGTA 3'
WSNP68-FAM：5’ACTTGATTGCTCGCTCCGAGATT3’
WSNP68-HEX：5’CTTGATTGCTCGCTCCGAGATC3’
WSNP 68-general: 5 'CCGAAGCGGATGAAGTGGTAGTTAA 3'
WSNP69-FAM：5’TCACGCGACTCCAGCAACTG3’
WSNP69-HEX：5’CTTCACGCGACTCCAGCAACTA3’
WSNP 69-general: 5 'GGGGCCTACGATTGAGGAAGTAAAA 3'
WSNP70-FAM：5’CGACAGCCGAGGGAACACAC3’
WSNP70-HEX：5’ATCGACAGCCGAGGGAACACAT3’
WSNP 70-general: 5 'GTCAGTCCGTCGTGTCTCTATATCAT 3'
WSNP71-FAM：5’AGAGGACTTGGTAAGATTAAAACTCTC3’
WSNP71-HEX：5’GAGAGGACTTGGTAAGATTAAAACTCTT3’
WSNP 71-general: 5 'GTTGCAAGTAATGCTAGGAGGTGCTT 3'
WSNP72-FAM：5’AAGGGCCTACTGAATACAGCTTCAT3’
WSNP72-HEX：5’GGGCCTACTGAATACAGCTTCAC3’
WSNP 72-general: 5 'CGCTCTTTTGCGCCTTATGTTCCTT 3'
WSNP73-FAM：5’ACGGGCATGAAAAGCTGGTCG3’
WSNP73-HEX：5’CACGGGCATGAAAAGCTGGTCA3’
WSNP 73-general: 5 'GTTGGACTATTCAGATTATTACACAGCCAA 3'
WSNP74-FAM：5’GATGCTCGCTTAGATCCACCAT3’
WSNP74-HEX：5’GATGCTCGCTTAGATCCACCAC3’
WSNP 74-general: 5 'TCCAAACCAAGGATCTAGTATTTCCTCTT 3'
WSNP75-FAM：5’GAACAAATTAGAGATGCTATTAAGTGGG3’
WSNP75-HEX：5’GAACAAATTAGAGATGCTATTAAGTGGA3’
WSNP 75-general: 5 'CCAAGTTATCGTGGCGTACAAGACAT 3'
WSNP76-FAM：5’CATCTCTTCACGGTACATCAACTCAT3’
WSNP76-HEX：5’ATCTCTTCACGGTACATCAACTCAC3’
WSNP 76-general: 5 'GTGGCCATCCGACAGAAAAAGTATTAATT 3'
WSNP77-FAM：5’CAGTCGCCTGATGTCTAGCTA3’
WSNP77-HEX：5’CAGTCGCCTGATGTCTAGCTG3’
WSNP 77-general: 5 'GAGGATGAAAGAGGAATGGCTGTCTT 3'
WSNP78-FAM：5’ACTTATTGACGCCTCCGTTCAAAC3’
WSNP78-HEX：5’CACTTATTGACGCCTCCGTTCAAAT3’
WSNP 78-general: 5 'CCTATCGTTCGCCTTTTTCCACAGTT 3'
WSNP79-FAM：5’GATAGGCTTTGTGAATCGCCCAG3’
WSNP79-HEX：5’GATAGGCTTTGTGAATCGCCCAT3’
WSNP 79-general: 5 'GCGTCCAAAACCGGAGGTCTAAAAA 3'
WSNP80-FAM：5’ATCATCCCTTGGACGTCAAGCG3’
WSNP80-HEX：5’CATCATCCCTTGGACGTCAAGCA3’
WSNP 80-general: 5 'ATGTCTCCTTCACTGGGTGCATCAA 3'
WSNP81-FAM：5’CAGATGTCACCAATACCGAGGG3’
WSNP81-HEX：5’CAGATGTCACCAATACCGAGGA3’
WSNP 81-general: 5 'CTCTATCTGCTGGTTTGAACTATCCTTT 3'
WSNP82-FAM：5’CCATCGCAGCCCAATAGAAAATTGTA3’
WSNP82-HEX：5’CATCGCAGCCCAATAGAAAATTGTG3’
WSNP 82-general: 5 'CGTTACCGTCGGGCCTTCTTCAT 3'
WSNP83-FAM：5’ATGGGCCTGTTGAACTAGCCAC3’
WSNP83-HEX：5’CATGGGCCTGTTGAACTAGCCAT3’
WSNP 83-general: 5 'GGAGTAAATCGCATAGAACCACCATAAAT 3'
WSNP84-FAM：5’GCCGCTTGTGCGCATTTCTTTC3’
WSNP84-HEX：5’GCCGCTTGTGCGCATTTCTTTT3’
WSNP 84-general: 5 'CAAAAAAACCAGGGTCTGCGATCTGTA 3'
WSNP85-FAM：5’GATTTGTGCAGACGGGCTAGCT3’
WSNP85-HEX：5’TGTGCAGACGGGCTAGCC3’
WSNP 85-general: 5 'ACATCCTCCCTGCGATCGCCAA 3'
WSNP86-FAM：5’CGCACGGGACTCTGTCCACA3’
WSNP86-HEX：5’GCACGGGACTCTGTCCACC3’
WSNP 86-general: 5 'GGACTGTCTAGTTGTTGCAATCGCAT 3'
WSNP87-FAM：5’GCTCTGGAAGAAGTCGAC3’
WSNP87-HEX：5’GCTGCTCTGGAAGAAGTCGAG3’
WSNP 87-general: 5 'GTGGAATATGAATTTAACACCTATGGCTAA 3'
WSNP88-FAM：5’TTATGTGATCTCTTTCAGGTTACATAG3’
WSNP88-HEX：5’CTTTATGTGATCTCTTTCAGGTTACATAC3’
WSNP 88-general: 5 'AAATGGTGAAGCAAGTGCAGGCCAT 3'
WSNP89-FAM：5’GATGAACGTGTTTGTGATGTTGATCTA3’
WSNP89-HEX：5’ATGAACGTGTTTGTGATGTTGATCTG3’
WSNP 89-general: 5 'TCATACAAGCAGCGCAGCAACGTAA 3'
WSNP90-FAM：5’GCCAAAAGCCCAACACAGAGGA3’
WSNP90-HEX：5’CCAAAAGCCCAACACAGAGGC3’
WSNP 90-general: 5 'GCTTCAAGCTGTGGATCGCTATCTA 3'
WSNP91-FAM：5’ACCCTAGTGCAGATCAAATCAACCT3’
WSNP91-HEX：5’CCTAGTGCAGATCAAATCAACCG3’
WSNP 91-general: 5 'TACCTGACTGATTTCGCCCCTCATT 3'
WSNP92-FAM：5’TGCCCTTTCAGGTACTACGAC3’
WSNP92-HEX：5’CTTGCCCTTTCAGGTACTACGAT3’
WSNP 92-general: 5 'GTCTTGGCTCGACAGGCGAGTT 3'
WSNP93-FAM：5’CAGGGAAAGATCATTAACGTCAACC3’
WSNP93-HEX：5’GCAGGGAAAGATCATTAACGTCAACT3’
WSNP 93-general: 5 'GTCCCGACTTCGAATTAATGAAGCCAT 3'
WSNP94-FAM：5’CAACCCAAAAGTATTAAAGCCGGC3’
WSNP94-HEX：5’ATCAACCCAAAAGTATTAAAGCCGGT3’
WSNP 94-general: 5 'CAACGAAAACCAGTTTGAGGGGAGAT 3'
WSNP95-FAM：5’AGAACGGAATACCCATGTTTACCC3’
WSNP95-HEX：5’AGAACGGAATACCCATGTTTACCG3’
WSNP 95-general: 5 'CCTGATAAGACATGGAGGTTTTCTCTTAA 3'
WSNP96-FAM：5’AGGGAATTCTTCTACTTTTCTAAACCG3’
WSNP96-HEX：5’TAGGGAATTCTTCTACTTTTCTAAACCA3’
WSNP 96-general: 5 'GTTCAGTGTTGGGTCCGTCTGTAAA 3'.
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