152022-08-30發(fā)布本文件按照GB/T1.1—2020《標(biāo)準(zhǔn)化工作導(dǎo)則第1部分：標(biāo)準(zhǔn)化文件的結(jié)構(gòu)和起草規(guī)則》的規(guī)定本文件主要起草人：JIUHUANFENG(馮I食用向日葵品種鑒定SNP標(biāo)記法GB/T3543.2農(nóng)作物種子檢驗(yàn)規(guī)程扦樣GB/T3543.5農(nóng)作物種子檢驗(yàn)規(guī)程真實(shí)性和品種GB4407.2經(jīng)濟(jì)作物種子第2部分GB/T6682分析實(shí)驗(yàn)室用水規(guī)格和3.13.2單核苷酸多態(tài)性singlenucleotidepoly基因組中基于單個(gè)核苷酸變異引起的DNA3.3競(jìng)爭(zhēng)性等位基因特異性聚合酶鏈?zhǔn)椒磻?yīng)kompetitiveallelespecific3.4能夠最大程度地將供檢品種與對(duì)照品種區(qū)分開來13.5——實(shí)時(shí)熒光定量PCR儀、全自動(dòng)樣品快速研磨儀、高速離心機(jī)、核酸濃度測(cè)定機(jī)、微量移液器、恒溫水浴鍋、高壓滅菌鍋、酸度計(jì)、磁力攪拌器、電子天平、冰箱等；6.1.1所用試劑至少為分析純或分子生物學(xué)純。試劑配制用水應(yīng)符合GB/T6682規(guī)定的一級(jí)水要求。2EDTA?2H26.2溶液配制6.2.10.5mol/LEDTA溶液（pH8.0）：稱取186EDTA溶液（pH8.0）、100mL1.0mol/LTris-HCl(2——供檢樣品與對(duì)照樣品為種子時(shí)，樣品的取樣、分樣應(yīng)符合GB/T3543.2的規(guī)定；——供檢樣品允許采用混合樣檢測(cè)或單株檢測(cè)?；旌蠘又?.2DNA提取a)采集供檢及對(duì)照樣品的種子或幼嫩組織200mg～300mg，置于2.0mL離心管，放入4mm鋼7.3PCR檢測(cè)配制反應(yīng)體系時(shí)，應(yīng)同時(shí)設(shè)置1～2個(gè)無DNA模板的陰性對(duì)照（NTC）。根據(jù)NTC信號(hào)強(qiáng)弱，判斷最適擴(kuò)增循環(huán)數(shù)。若NTC信號(hào)過高，與其它基因型摻雜，則應(yīng)降低擴(kuò)本文件采用的SNP推薦標(biāo)記及KASP引物推薦用量(μL）2×1×Primer_AlleleXPrimer_AlleleY----3注：對(duì)于有異議的樣品，可按GB/T354利用個(gè)SNP引物，采用平臺(tái)進(jìn)行檢測(cè)，與對(duì)照樣品相比，供檢樣品中共檢測(cè)出差異位點(diǎn)數(shù)個(gè)，差異位點(diǎn)的SNP標(biāo)記編號(hào)為，鑒定意496個(gè)推薦標(biāo)記的KASP引物序列及基因組位置信息見表Primer_AlleleX/Ya（5'Primer__AlleleC（5,-3,）1TCTTTACCTGGTCCCACTG1TCATTATCATCTTCTCCGGCATTATCATCTTCTCCGGT1TGACATTTGTTGAATCAAGTGGACATTTGTTGAATCAAGTGCCAGAGCAAAACGGGTTATTTTT1CTGCGAGCTCCTGCATGGT11GTGTAAGATGTGGTAGACG2ATGATTATTTAAAGAAGAAGAACCGATTTAAAGAAGAAGAACCGCATTTCGGTTTTCTTTAAAACTGT2GGAACTTTTCTACGTAAAATTAACCGAAGAACTTTTCTACGTAAAATTAACCGGCATTCGGTATAACTCGTG2GCATATTAACAGGCAAGAACAGCATATTAACAGGCAAGAACAGTCTGTAATGATCTTGCAGC22GGTATAACAACTCATAAGACTTGGTATAACAACTCATAAGACTTGCCGGATTTTGGTTACTTGCACA2GTTCAACAGACGAGTACATCATCATC3CCCAGTTCCAATCAAACGCTAGTACGGGACGATGCTGT3GAAATTCCGGTCCCGCATTGGACTTTGGTTTTGAGGATC5Primer_AlleleC3GGTAGATTAAAGAAACAGGGGTAGATTAAAGAAACAGGCGACAATTTGAGGCTTTTG3TACACTATGATTTCTAATATTTAACAAACTACACTATGATTTCTAATATTTAACAAACTGGGAGGTTTTGAATGTTTGGTGTTGGT3GTTCTTCATGTATTTAACGCCTTTCTTCATGTATTTAACGCCTACGTACGTTACAAGTTAAGAAACTGTAA3TTAGACCTGTATATACTAATCGCATGAGACCTGTATATACTAATCGCATGTACCCCATCTTCTAGAGCTAGC4GGCTTACCATCTTGACATTATGCTTACCATCTTGACATTATGGTATGGCATCAACGAGGTATCCAGA4TTCTCATATCCGCCAACACTCTCATATCCGCCAACACAA4TGCTGAAGACATTCCTAAAATG4GTTCCATTGTGATCTATGTCACCATTGTGATCTATGTCATGCTTCCGGGCTTTCGATAT4AATTAAACTCCCAAATCGGGGTCCAAAACTCCCAAATCGGGGTCCGGCCTCCTTTGCCTTCTTAC4GTTGGCCATTCCATTCATTTTGTTGGCCATTCCATTCATTTTCTGGATAAACTTACAACGATACAA5GGTACTGTAGTTTATTAACACTAACGGTACTGTAGTTTATTAACACTAATCCGTTTATAGCGAACATGCCT5TTCTCATGGGATTCCGCAATATATTAACAGTAGGAACAACACAC5AATCAAGAGGGAAACAAGTGTCTTATCAAGAGGGAAACAAGTGTCTTCGCGAATTACGCGCATTTCTTAT5CAAGGGTTGTATACAACACAAAAGGGTTGTATACAACACAAAGGCGCAAGCTGCAAGTTTCTTTAGCA5TCCGGGTTCAGCATCTTGAAATCCGGGTTCAGCATCTTGAA5GTTGGTGTTCTTTCAGCCTGTTTCAAAGTAACTTAAACCCTCA6Primer_AlleleC6GCATCTTTACCGACACTAAGCCATCTTTACCGACACTAAGCTCGTACCAATTTCGCAAAATCGA6AGAAGCGTCCCAAACGCACGCTCCCCGTTACAAGATTC6GAAGTTGGATGAGGAGGTGGTT6ATGTTGTGTTATGCAAGTGAGAGAGGTTGTGTTATGCAAGTGAGAGACACCTTAACGATATTAACCCGA6TATGCTTAAACATTATGGCTAATGCTTAAACATTATGGCTACAGCCAATAATTACAATTGGTGATCAA6ATCTAACGCTATGGAAGCCTCTGCTAACGCTATGGAAGCCTCTCGCGTAGCCCTTTAAGTG7CAAACCGACTGTTTCCCGGAAACCGACTGTTTCCCGGACTAAGAACTTCAATCCGGT7TACGATGACGTTGTGGAACGGCCTGATTTTAGATGGATCGTC7TGCAACCTTTCCGGACCCAGCAACCTTTCCGGACCCATT7TGCGTAAATGAGACCGATAATCGCGTAAATGAGACCGATAACAGGGGTGAAGCGAAATTCCA7GAACCGGGTAACAAAGACAAGAAACCGGGTAACAAAGACAAGAGTCTTCATTATTTGGGATGATT78CGGACCATTTTGACCCATAATCGGACCATTTTGACCCATAATGGAAACGGGTCAAACAAGATTT8GCCTTCACCTATACAATAGAATTTATGCCTTCACCTATACAATAGAATTTATCATGCTTTTATCTGTTGGGGTA8CCAATATGAATTCCATCCATTCACACAATATGAATTCCATCCATTCACATGGGACTAATTTTGTTGTGGATGGG8GAGAGCGAGTTCACCAACATAAAGAGCGAGTTCACCAACATAA7Primer_AlleleC8GAGTTAAACCTAATTGCAAGCGAGTTAAACCTAATTGCAAGCAGCGAGACCGAGAAAAGAGCC8CGCATTAACTGCTTAGGATCCGCATTAACTGCTTAGGATCCACAACAAGAACACAAGATGTTGT9CGTTCCTTTACTCATCCTAAAGTTCCTTTACTCATCCTAAGTGGAAGTATCTCGATAGC9CATTTGAAAATGGGATGGTGCGATGCATTTAGAGTGCGAGCAT9GCATCATCAAAGAATCGAGCTACATCATCAAAGAATCGAGCAATTAAATTTCATTATGCTAGCTG9TTTGACTGGCCTTACCCTTCGGCACTGAGAGTTAAAACAA9GCTGGTCTAACATCATCCTC9CATCTTGAAGTGGCCCAAAATATCTTGAAGTGGCCCAAAATCTACCTTGTCTTTATAGTTTTAGTTGCAGTCAACCGAAAAAGGACTCGGTCAACCGAAAAAGGACTCGAGCTTACCACCTTGGCATCGCAGCGGAGACCGACACTTTAGCGGAGACCGACACTTTGCAAGTTCATAACAGAGAAGATTTTCAGAAGTTCATAACAGAGAAGATTTTCAGAACCGAAACCTTGTGAAACTTGTTGGACCCATGTTTCGTTTTCTTCCACCCATGTTTCGTTTTCTTCCGCCGGAGAGGATAATACTTACCGCTGACGAAACTCCGTGTAATGTTACCGGAGAGGCCATTGCAAATCCTATGAAACATCGTCCAAATCCTATGAAACATCGTCAGCGCTTGGAGACGCATAACATGAAGTTTGACCAAAAATACTAGAATTCAAAGTTTGACCAAAAATACTAGAATTCAACGACGATTCTTTACCTTCGGACACTATCGCTACACAAGATTACAACGTGCTACACAAGATTACAACG8Primer_AlleleCATCTTTGACACCACCAAACTCTAGCTTTGACACCACCAAACTCTAGTACATCCAAGGTTATGAAGACCAATATTCGGATAACACCCTTCAAATATTCGGATAACACCCTTCAGATCGGATAAAGTCGAAATGGCTTTTTCAGGCTGAGTTGGAAGATCCAGGCTGAGTTGGAAGATCGACGTACAGCAAGTGTCACTCAATCGAAAGAGGCGCAACACAATCGAAAGAGGCGCAACACTTGCCGTTGTATGTGAATTCTTCATCAACTGTTACATAGCCGGCACAATCTTGCAAAATGGCTGATAATGGTTTCCCGGTCGAAGATAATGGTTTCCCGGTCGAAACCGAACCGGCTCCTCCGTAATATGTACTTTAGATACATTTTATTGTAACATATGTACTTTAGATACATTTTATTGTAACGTTCCCCAAGAAAAGTACCATAGTATCAAAGCAGAATGGCGATAACTATCTTTACAGCTTTACAAACATTCAACTATCTTTACAGCTTTACAAACATTCGAAGATGCTGGTGTGATGCACCTGTTCGAACATCGGTAACTGTTCGAACATCGGTAACTGGCAGTTTTGACCGATCGCAGAAACCATAAACTCTATATACATTTCTAGAAACCATAAACTCTATATACATTTCTTAGGTTCATGAGCATGTGTAATTTGACAAGATAACGAAAGATCACACCATAACGAAAGATCACACCGGATGAGAGTTAAGTAGCCAAGATGAGAGTTAAGTAGCCAAGACATCTTGCGTGAAGCAAACATATACAAACTATGAGCATATGCATATCTCAAACTATGAGCATATGCATATCGGATATGATCTGGGATGCTATTTGTACAAAACCTGACATTAAACAGGTACAAAACCTGACATTAAACAGCCTTATACTATCAGAAGTTGAAGCT9Primer_AlleleCGGCCGTGAAGTTGAAGGATAACATCCAAAAGCCAGTGCAAGGCATAATCGAGAGTAAAAAGTTGTAGTTCATAATCGAGAGTAAAAAGTTGTAGTTAGAATAAGAGATTCAGTACTCCACTGGGAAGTCAAATCTCTAGTCACTGAAGTCAAATCTCTAGTCAGTTAGACGAAGACGATAAGGTTAAGACGAAGACGATAAGGTTATGTTCGAAACTGGCAGCTTCATGAGCAACAACAATGGCACAATCTCAACAACAATGGCACAATCTCGTGTGAAGTCTGGTGAATGAGGTAAGGTGTTGAATATTAATGTGTAAGGTGTTGA