DB11T 507-2007玉米品種純度及真實(shí)性SSR分子檢測(cè)方法

2007北京DB市地方標(biāo)準(zhǔn)DB11/T507—2007SSR-basedMethodtoAssayPurityandGenuinenessinZeamaysL.2發(fā)布1實(shí)施北京市質(zhì)量技術(shù)監(jiān)督局發(fā)布IDBT007前言1DBT007SSR分子標(biāo)記原理，規(guī)定了進(jìn)行玉米單交種和親本自交系的品種純度及真實(shí)性檢測(cè)方義inenessSSRsimplesequencerepeatmarker理設(shè)備及試劑儀器設(shè)備PCR擴(kuò)增儀；DNA序列分析電泳槽；高壓電泳儀(3000V,400mA,400W)；電子天平(感量0.01g,0.001g)；冰箱；微量加樣器(0.5μL~10μL,20μL~200μL,100μL~1000μL)；磁力攪拌器；膠片觀.2試劑四乙酸二鈉(EDTA-Na2)；三羥甲基氨基甲烷(Tris)；鹽酸(HCl，36%)；氫氧化鈉(NaOH)；SR2DBT007Silane)；剝離硅烷(RepelSilane)；無水乙醇；四甲基乙二胺；過硫酸銨；冰醋酸；硝酸銀；甲醛引物篩選DNA擴(kuò)增 作將玻璃板洗凈，用無水乙醇擦洗兩遍，干燥。在長(zhǎng)玻璃板上涂0.5%親和硅烷(BindingSilane)0.5mL，帶凹槽的玻璃板上涂2.0%剝離硅烷(RepelSilane)0.5mL。操作過程中防止兩塊玻璃板互相預(yù)電泳在正極槽(下槽)和負(fù)極槽(上槽)中分別加入1倍TBE緩沖液600ml，拔出梳子，90W恒功率預(yù)電泳15min~20min。點(diǎn)樣6.3.4電泳染色3DBT007SSR析示定取樣SSR分析果判定送檢樣品與標(biāo)準(zhǔn)樣品有一對(duì)以上(包括一對(duì))引物檢測(cè)出差異，判定送檢樣品與標(biāo)準(zhǔn)樣品不屬于典型帶型。4DBT007(規(guī)范性附錄)ADNA提取試劑配制A.1.20.5mol/LEDTA(pH8.0)：取EDTA-Na2186.1g，加入100mL蒸餾水，再加入適量10mol/LNaOH溶液，加熱至完全溶解后，冷卻至室溫，用10mol/L氫氧化鈉溶液調(diào)pH值至8.0，加水定容至1000A.1.4TE緩沖液(pH2.0)：取1mol/LTris-HCl(pH8.0)5mL和0.5mol/LEDTA(pH8.0)1mL，用APCR擴(kuò)增試劑配制TPCTPTTPmmolLLA.2.2SSR引物：用超純水分別配制左引物(L)和右引物(R)終濃度均為40μmol/L的儲(chǔ)存液，等體A凝膠與電泳試劑配制AmLmolLEDTApH)1mL，溴酚蘭0.125g，A.3.210倍TBE緩沖液：稱取三羥甲基氨基甲烷(Tris)108g，硼酸55g，加適量去離子水溶解后，DTApHmLmLLLA.3.5親和硅烷(BindingSilane)緩沖液：取無水乙醇49.75mL和冰醋酸250μL，混合。A.3.60.5%親和硅烷(BindingSilane)：在9.95mL親和硅烷緩沖液加入50μL親和硅烷原液，混勻。AgmL溶解，現(xiàn)用現(xiàn)配。LA銀染試劑配制AmL，加去離子水定容至1000mL。Ag，加去離子水定容至1000mL。A顯影液：取氫氧化鈉30g和甲醛5mL，加去離子水定容至1000mL。5DBT007(資料性附錄)1bnlg439左引物(L):5＇-TTGACATCGCCATCTTGGTGACCA-3＇右引物(R):5＇-TCTTAATGCGATCGTACGAAGTTGTGGAA-3＇2bnlg161左引物(L):GCTTTCGTCATACACACACATTCA-3＇TATTT3bnlg125左引物(L):GGGACAAAAGAAGAAGCAGAG-3＇右引物(R):GAAATGGGACAGAGACAGACAAT-3＇4bnlg198左引物(L):GTTTGGTCTTGCTGAAAAATAAAA-3＇CCTACATTATTATCTC5bnlg238CAT右引物(R):GAGCATGAGCTTGCATATTTCTTGTGG-3＇6bnlg240LAAGAACAGAAGGCATTGATACATAA-3＇右引物(R):TGCAGGTGTATGGGCAGCTA-3＇7bnlg107AGATGGCTTGTTATGG右引物(R):CAACAACAAGTGGCTGGCTAGGGTGAA-3＇8phi034左引物(L):TAGCGACAGGATGGCCTCTTCT-3＇右引物(R):GGGGAGCACGCCTTCGTTCT-3＇9phi053左引物(L):CTGCCTCTCAGATTCAGAGATTGAC-3＇右引物(R):AACCCAACGTACTCCGGCAG-3＇phi056左引物(L):ACTTGCTTGCCTGCCGTTAC-3＇右引物(R):CGCACACCACTTCCCAGAA-3＇phi065左引物(L):AGGGACAAATACGTGGAGACACAG-3＇右引物(R):CGATCTGCACAAAGTGGAGTAGTC-3＇phi072LACCGTGCATGATTAATTTCTCCAGCCTT-3＇右引物(R):ACAGCGCGCAAATGGATTGAACT-3＇phi080左引物(L):CACCCGATGCAACTTGCGTAGA-3＇右引物(R):TCGTCACGTTCCACGACATCAC-3＇phi085左引物(L):AGCAGAACGGCAAGGGCTACT-3＇RTTTGGCACACCACGACGAphi96100左引物(L):AGGAGGACCCCAACTCCTG-3＇右引物(R):TTGCACGAGCCATCGTAT-3＇umc1061左引物(L):AGCAGGAGTACCCATGAAAGTCC-3＇RTATCACAGCACGAAGCGATAGATG-3＇bnlg233LTCTGATATCATAAAGGAGGACCG-3＇右引物(R):GGAGCTTGCGCTTTTTAACA-3＇umc2084左引物(L):ATCGCGACGAGTTAATTCAAACA-3＇T右引物(R):TACGATGTCTTCAGTGTGACACCA-3＇bnlg1450左引物(L):ACAGCTCTTCTTGGCATCGT-3＇右引物(R):GACTTTGCTGGTCAGCTGGT-3＇bnlg1940左引物(L):5CCTTTTGTTTCAGGCCGTTA-3＇右引物(R):CAGCAGCCTGATGATGAACA-3＇6DBT0071bnlg125左引物(L):5＇-GGGACAAAAGAAGAAGCAGAG-3＇右引物(R):5＇-R:GAAATGGGACAGAGACAGACAAT-3＇2bnlg439左引物(L):5＇-TTGACATCGCCATCTTGGTGACCA-3＇右引物(R):5＇-TCTTAATGCGATCGTACGAAGTTGTGGAA-3＇3phi072T右引物(R):5＇-ACAGCGCGCAAATGGATTGAACT-3＇4bnlg162AAAACCTAATAAAGGGA右引物(R):5＇-CAAGTAGCTAGCAGTCATTTGCAGTGT-3＇5bnlg161左引物(L):5＇-GCTTTCGTCATACACACACATTCA-3＇ATATTT6bnlg198左引物(L):5＇-GTTTGGTCTTGCTGAAAAATAAAA-3＇GGCCTACATTATTATCTC7phi116左引物(L):5＇-GCATACGGCCATGGATGGGA-3＇右引物(R):5＇-TCCCTGCCGGGACTCCTG-3＇8phi123左引物(L):5＇-GGAGACGAGGTGCTACTTCTTCAA-3＇右引物(R):5＇-TGTGGCTGAGGCTAGGAATCTC-3＇9phi065左引物(L):5＇-AGGGACAAATACGTGGAGACACAG-3＇右引物(R):5＇-CGATCTGCACAAAGTGGAGTAGTC-3＇umc1040左引物(L):5＇-CATTCACTCTCTTGCCAACTTGA-3＇R5＇-AGTAAGAGTGGGATATTCTGGGAGTT-3＇umc1944左引物(L):5＇-GAAGAAGGATCGCACACATGG-3＇右引物(R):5＇-AGACTGTCGCGCTGTACTATACCC-3＇phi077左引物(L):5＇-GAGAAGAGGATCAGGTTCGTTCCA-3＇右引物(R):5＇-CGCGTTGTACATCTTGCCTGCTT-3＇bnlg240右引物(R):5＇-TGCAGGTGTATGGGCAGCTA-3＇bnlg238右引物(R):5＇-GAGCATGAGCTTGCATATTTCTTGTGG-3＇phi085左引物(L):5＇-AGCAGAACGGCAAGGGCTACT-3＇右引物(R):5＇-TTTGGCACACCACGACGA-3＇左引物(L):5＇-CGTGCTACTACTGCTACAAAGCGA-3＇右引物(R):5＇-AGTCGTTCGTGTCTTCCGAAACT-3＇bnlg1006左引物(L):5＇-GACCAGCGTGTTGATCCC-3＇右引物(R):5＇-GAGACCCCGACTCTCTCTC-3＇bnlg1621左引物(L):5＇-CTCTTCGATCTTTAAGAGAGAGAGAG-3＇右引物(R):5＇-ACACGAGGCACTGGTACTAACG-3＇phi295450左引物(L):5＇-CCTTTTCATGTTGCTTTCCC-3＇右引物(R):5＇-GCCCAATCCTTCCTTCCT-3＇phi053左引物(L):5＇-CTGCCTCTCAGATTCAGAGATTGAC-3＇右引物(R):5＇-AACCCAACGTACTCCGGCAG-3＇7DBT007umc1904左引物(L):5＇-CAGCCACTCGTTTATGGAGGTTTA-3＇右引物(R):5＇-TGTTACTAGTCGATCTGATGCCCA-3＇phi048L:5＇-GCAAACCTTGCATGAACCCGATTGT-3＇右引物(R):5＇-CAAGCGTCCAGCTCGATGATTTC-3＇phi080左引物(L):5＇-CACCCGATGCAACTTGCGTAGA-3＇右引物(R):5＇-TCGTCACGTTCCACGACATCAC-3＇phi099左引物(L):5＇-TACAAAAATCAGGACTGCGAAAAACCCAA-3＇右引物(R):5＇-GTCGGTGTGTGATCCTTCCAC-3＇phi015左引物(L):5＇-GCAACGTACCGTACCTTTCCGA-3＇RACGCTGCATTCAATTACCGGGAAG＇phi037左引物(L):5＇-CCCAGCTCCTGTTGTCGGCTCAGAC-3＇右引物(R):5＇-TCCAGATCCGCCGCACCTCACGTCA-3＇bnlg1306左引物(L):5＇-CACCTTGAAAGCATCCTCGT-3＇右引物(R):5＇-CAAAAACAAATGGCAGCTGA-3＇bnlg1505左引物(L):5＇-GAAAGACAAGGCGAAGTTGG-3＇右引物(R):5＇-GCTTCTGAACTGGATCGGAG-3＇bnlg1884左引物(L):5＇-TTCGGATGCATGTGTAACGT-3＇右引物(R):5＇-CGGAAGTCCCATCTGTTTGT-3＇umc2105左引物(L):5＇-ACATACATAGGCTCCCTTTTTCCG-3＇右引物(R):5＇-TCCCGTGACACTCTCTTTCTCTCT-3＇phi047左引物(L):5＇-GGAGATGCTCGCACTGTTCTC-3＇右引物(R):5＇-CTCCACCCTCTTTGACATGGTATG-3＇bnlg2291左引物(L):5＇-CCTCTCGATGTTCTGAAGCC-3＇右引物(R):5＇-GTCATAACCTTGCCTCCCAA-3＇umc1822TATAATTTTGCAAGCAGAAAGGG右引物(R):