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)作物種子檢驗規(guī)程扦樣GB/T3543.5農(nóng)作物種子檢驗規(guī)程真實性和品種GB4407.2經(jīng)濟(jì)作物種子第2部分GB/T6682分析實驗室用水規(guī)格和單核苷酸多態(tài)性singlenucleotidepoly基因組中基于單個核苷酸變異引起的DNA競爭性等位基因特異性聚合酶鏈?zhǔn)椒磻?yīng)kompetitiveallelespecific能夠最大程度地將供檢品種與對照品種區(qū)分開來124.2利用一套多態(tài)性高、穩(wěn)定性好、品種區(qū)別力強(qiáng)、具良好基因型分型質(zhì)量的推薦標(biāo)記，檢測供檢樣品與對照樣品之間的SNP差異位點數(shù)目，從而判——實時熒光定量PCR儀、全自動樣品快速研磨儀、高速離心機(jī)、核酸濃度測定儀、微孔板離心——微量離心管、普通槍頭、384孔PCR板(白色)、封板膜、鋼珠等。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)定的一級水要求。6.2.10.5mol/LEDTA溶液NaOH(約20g)調(diào)pH至8.0,定容至1000mL,高壓滅菌。6.2.21.0mol/LTris-HCl(pH8.0):稱取60.55gTris堿，溶于400mL水中，用HCl調(diào)pH至8.0,定容至500mL,高壓滅菌。6.2.32×CTAB提取液：分別稱取20gCTAB和81.7gNaCl,加水溶解，分別加入40mL0.5mol/L時，按2%(M/V)加入聚乙烯吡咯烷酮K30(PVP-K30)。——供檢樣品與對照樣品為種子時，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或單株檢測?；旌蠘又芶)采集供檢及對照樣品的種子或幼嫩組織200mg～300mg，置于2.0mL離心管，放入4mm鋼配制反應(yīng)體系時，應(yīng)同時設(shè)置1～2個無DNA模板的陰性對照（NTC）。根據(jù)NTC信號強(qiáng)弱，判斷最適擴(kuò)增循環(huán)數(shù)。若NTC信號過高，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：對于有異議的樣品，可按GB/T354利用個SNP引物，采用平臺進(jìn)行檢測，與對照樣品相比，供檢樣品中共檢測出差異位點數(shù)個，差異位點的SNP標(biāo)記編號為，鑒定意496個推薦標(biāo)記的KASP引物序列及基因組位置信息見表Primer_AlleleX/Ya（5'1TCTTTACCTGGTCCCACTG1TCATTATCATCTTCTCCGGCATTATCATCTTCTCCGGT1TGACATTTGTTGAATCAAGTGGACATTTGTTGAATCAAGTGCCAGAGCAAAACGGGTTATTTTT1CTGCGAGCTCCTGCATGGT11GTGTAAGATGTGGTAGACG2ATGATTATTTAAAGAAGAAGAACCGATTTAAAGAAGAAGAACCGCATTTCGGTTTTCTTTAAAACTGT2GGAACTTTTCTACGTAAAATTAACCGAAGAACTTTTCTACGTAAAATTAACCGGCATTCGGTATAACTCGTG2GCATATTAACAGGCAAGAACAGCATATTAACAGGCAAGAACAGTCTGTAATGATCTTGCAGC22GGTATAACAACTCATAAGACTTGGTATAACAACTCATAAGACTTGCCGGATTTTGGTTACTTGCACA2GTTCAACAGACGAGTACATCATCATC3CCCAGTTCCAATCAAACGCTAGTACGGGACGATGCTGT3GAAATTCCGGTCCCGCATTGGACTTTGGTTTTGAGGATC5Primer_AlleleC3GGTAGATTAAAGAAACAGGGGTAGATTAAAGAAACAGGCGACAATTTGAGGCTTTTG3TACACTATGATTTCTAATATTTAACAAACTACACTATGATTTCTAATATTTAACAAACTGGGAGGTTTTGAATGTTTGGTGTTGGT3GTTCTTCATGTATTTAACGCCTTTCTTCATGTATTTAACGCCTACGTACGTTACAAGTTAAGAAACTGTAA3TTAGACCTGTATATACTAATCGCATGAGACCTGTATATACTAATCGCATGTACCCCATCTTCTAGAGCTAGC4GGCTTACCATCTTGACATTATGCTTACCATCTTGACATTATGGTATGGCATCAACGAGGTATCCAGA4TTCTCATATCCGCCAACACTCTCATATCCGCCAACACAA4TGCTGAAGACATTCCTAAAATG4GTTCCATTGTGATCTATGTCACCATTGTGATCTATGTCATGCTTCCGGGCTTTCGATAT4AATTAAACTCCCAAATCGGGGTCCAAAACTCCCAAATCGGGGTCCGGCCTCCTTTGCCTTCTTAC4GTTGGCCATTCCATTCATTTTGTTGGCCATTCCATTCATTTTCTGGATAAACTTACAACGATACAA5GGTACTGTAGTTTATTAACACTAACGGTACTGTAGTTTATTAACACTAATCCGTTTATAGCGAACATGCCT5TTCTCATGGGATTCCGCAATATATTAACAGTAGGAACAACACAC5AATCAAGAGGGAAACAAGTGTCTTATCAAGAGGGAAACAAGTGTCTTCGCGAATTACGCGCATTTCTTAT5CAAGGGTTGTATACAACACAAAAGGGTTGTATACAACACAAAGGCGCAAGCTGCAAGTTTCTTTAGCA5TCCGGGTTCAGCATCTTGAAATCCGGGTTCAGCATCTTGAA5GTTGGTGTTCTTTCAGCCTGTTTCAAAGTAACTTAAACCCTCA6Primer_AlleleC6GCATCTTTACCGACACTAAGCCATCTTTACCGACACTAAGCTCGTACCAATTTCGCAAAATCGA6AGAAGCGTCCCAAACGCACGCTCCCCGTTACAAGATTC6GAAGTTGGATGAGGAGGTGGTT6ATGTTGTGTTATGCAAGTGAGAGAGGTTGTGTTATGCAAGTGAGAGACACCTTAACGATATTAACCCGA6TATGCTTAAACATTATGGCTAATGCTTAAACATTATGGCTACAGCCAATAATTACAATTGGTGATCAA6ATCTAACGCTATGGAAGCCTCTGCTAACGCTATGGAAGCCTCTCGCGTAGCCCTTTAAGTG7CAAACCGACTGTTTCCCGGAAACCGACTGTTTCCCGGACTAAGAACTTCAATCCGGT7TACGATGACGTTGTGGAACGGCCTGATTTTAGATGGATCGTC7TGCAACCTTTCCGGACCCAGCAACCTTTCCGGACCCATT7TGCGTAAATGAGACCGATAATCGCGTAAATGAGACCGATAACAGGGGTGAAGCGAAATTCCA7GAACCGGGTAACAAAGACAAGAAACCGGGTAACAAAGACAAGAGTCTTCATTATTTGGGATGATT78CGGACCATTTTGACCCATAATCGGACCATTTTGACCCATAATGGAAACGGGTCAAACAAGATTT8GCCTTCACCTATACAATAGAATTTATGCCTTCACCTATACAATAGAATTTATCATGCTTTTATCTGTTGGGGTA8CCAATATGAATTCCATCCATTCACACAATATGAATTCCATCCATTCACATGGGACTAATTTTGTTGTGGATGGG8GAGAGCGAGTTCACCAACATAAAGAGCGAGTTCACCAACATAA7Primer_AlleleC8GAGTTAAACCTAATTGCAAGCGAGTTAAACCTAATTGCAAGCAGCGAGACCGAGAAAAGAGCC8CGCATTAACTGCTTAGGATCCGCATTAACTGCTTAGGATCCACAACAAGAACACAAGATGTTGT9CGTTCCTTTACTCATCCTAAAGTTCCTTTACTCATCCTAAGTGGAAGTATCTCGATAGC9CATTTGAAAATGGGATGGTGCGATGCATTTAGAGTGCGAGCAT9GCATCATCAAAGAATCGAGCTACATCATCAAAGAATCGAGCAATTAAATTTCATTATGCTAGCTG9TTTGACTGGCCTTACCCTTCGGCACTGAGAGTTAAAACAA9GCTGGTCTAACATCATCCTC9CATCTTGAAGTGGCCCAAAATATCTTGAAGTGGCCCAAAATCTACCTTGTCTTTATAGTTTTAGTTGCAGTCAACCGAAAAAGGACTCGGTCAACCGAAAAAGGACTCGAGCTTACCACCTTGGCATCGCAGCGGAGACCGACACTTTAGCGGAGACCGACACTTTGCAAGTTCATAACAGAGAAGATTTTCAGAAGTTCATAACAGAGAAGATTTTCAGAACCGAAACCTTGTGAAACTTGTTGGACCCATGTTTCGTTTTCTTCCACCCATGTTTCGTTTTCTTCCGCCGGAGAGGATAATACTTACCGCTGACGAAACTCCGTGTAATGTTACCGGAGAGGCCATTGCAAATCCTATGAAACATCGTCCAAATCCTATGAAACATCGTCAGCGCTTGGAGACGCATAACATGAAGTTTGACCAAAAATACTAGAATTCAAAGTTTGACCAAAAATACTAGAATTCAACGACGATTCTTTACCTTCGGACACTATCGCTACACAAGATTACAACGTGCTACACAAGATTACAACG8Primer_AlleleCATCTTTGACACCACCAAACTCTAGCTTTGACACCACCAAACTCTAGTACATCCAAGGTTATGAAGACCAATATTCGGATAACACCCTTCAAATATTCGGATAACACCCTTCAGATCGGATAAAGTCGAAATGGCTTTTTCAGGCTGAGTTGGAAGATCCAGGCTGAGTTGGAAGATCGACGTACAGCAAGTGTCACTCAATCGAAAGAGGCGCAACACAATCGAAAGAGGCGCAACACTTGCCGTTGTATGTGAATTCTTCATCAACTGTTACATAGCCGGCACAATCTTGCAAAATGGCTGATAATGGTTTCCCGGTCGAAGATAATGGTTTCCCGGTCGAAACCGAACCGGCTCCTCCGTAATATGTACTTTAGATACATTTTATTGTAACATATGTACTTTAGATACATTTTATTGTAACGTTCCCCAAGAAAAGTACCATAGTATCAAAGCAGAATGGCGATAACTATCTTTACAGCTTTACAAACATTCAACTATCTTTACAGCTTTACAAACATTCGAAGATGCTGGTGTGATGCACCTGTTCGAACATCGGTAACTGTTCGAACATCGGTAACTGGCAGTTTTGACCGATCGCAGAAACCATAAACTCTATATACATTTCTAGAAACCATAAACTCTATATACATTTCTTAGGTTCATGAGCATGTGTAATTTGACAAGATAACGAAAGATCACACCATAACGAAAGATCACACCGGATGAGAGTTAAGTAGCCAAGATGAGAGTTAAGTAGCCAAGACATCTTGCGTGAAGCAAACATATACAAACTATGAGCATATGCATATCTCAAACTATGAGCATATGCATATCGGATATGATCTGGGATGCTATTTGTACAAAACCTGACATTAAACAGGTACAAAACCTGACATTAAACAGCCTTATACTATCAGAAGTTGAAGCT9Primer_AlleleCGGCCGTGAAGTTGAAGGATAACATCCAAAAGCCAGTGCAAGGCATAATCGAGAGTAAAAAGTTGTAGTTCATAATCGAGAGTAAAAAGTTGTAGTTAGAATAAGAGATTCAGTACTCCACTGGGAAGTCAAATCTCTAGTCACTGAAGTCAAATCTCTAGTCAGTTAGACGAAGACGATAAGGTTAAGACGAAGACGATAAGGTTATGTTCGAAACTGGCAGCTTCATGAGCAACAACAATGGCACAATCTCAACAACAATGGCACAATCTCGTGTGAAGTCTGGTGAATGAGGTAAGGTGTTGAA