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1、DNA分子標記1/50隨機擴增RAPD,ISSR,單核苷酸突變檢驗SNP限制性酶切RFLP,PCR-RFLP,TRFP2/50RAPDRAPDrandomamplifiedpolymorphicDNAWilliamsetal.1990原理：隨機擴增引物：任意序列的10個堿基的寡核苷酸片段模板：未知序列的基因組DNA3/504/50RAPD實驗流程DNA提取PCR擴增產(chǎn)物檢測數(shù)據(jù)記錄5/50RAPD很難判斷帶的有無背景影響嚴重Ｑ：帶的亮度差異很大？凝膠分辨率？6/50RAPD7/50RAPD陰性對照出帶陽性對照where8/50RAPD反應(yīng)體系BufferMg2+dNTPDNA聚合酶引
2、物DNAH2OMg2+濃度較高核酸外切酶活性較低單引物，序列短純度，濃度純度9/50RAPD反應(yīng)條件94℃36℃72℃引物退火溫度太低循環(huán)次數(shù)10/50RAPD陰性對照陽性對照可重復(fù)性11/50RAPD數(shù)據(jù)記錄0–1矩陣顯性標記有有有有無有１１１１０１12/50RAPD缺陷可重復(fù)性低顯性標記13/50RAPDRAPDrandomamplifiedpolymorphicDNA10堿基Williamsetal.1990AP-PCRarbitraryprimerPCR20,30堿基Welshetal.1990DAFDNAamplificationfingerprinting5,8個堿基Ca
3、etano-Anollesetal.199114/50隨機擴增RAPD,ISSR,單核苷酸突變檢驗SNP限制性酶切RFLP,PCR-RFLP,TRFP15/50ISSRISSRinter-simplesequencerepeatZietkiewiczetal.1994原理：隨機擴增引物：20bp左右5’-BDB(ACA)5-3’16/5017/50ISSR50mMofKCl,1.5mMofMgCl2,0.25mMofdNTPs,2%formamide,0.2μMofprimer,0.5mMofspermidine,0.8UofTaqDNApolymeraseenzyme(Banglo
4、reGenei,India)and20ngofDNAper25μlreactionInitialdenaturationfor5minat94°C,eachcyclecomprised1-mindenaturationat94°C,45sannealingat49°C,2-minextensionat72°Cwithafinalextensionfor5minat72°Cattheendof45cycles.Mostofthepatternswithextremelygoodpolymorphismandusefulinformationwereoftenaccompaniedwit
5、habackgroundsmear.Toreducethissmear,2%formamidewasusedinthereaction.Allthepatternsgeneratedwererepeatedatleastthreetimesinordertoobtainreproducibledata.Joshietal.2000TheorApplGenet100:131118/50ISSR19/50ISSR可重復(fù)性高于RAPD隨機擴增顯性標記20/50SNPSNPsinglenucleotidepolymorphisms5’–GGATCAGTACTGACTCAG–3’5’–GGAT
6、CAGTAATGACTCAG–3’21/50SNPSNP變異來源轉(zhuǎn)換transition一種嘧啶置換另一種嘧啶C?T一種嘌呤置換另一種嘌呤A?G顛換transversion嘌呤與嘧啶互換，C?A，A?T等缺失/插入22/50SNP的檢測方法23/50SNP檢測－SNaPshot24/50SNP檢測－SNaPshot單引物擴增25/50多重PCR: multiplexprimersextensionarraysSNP檢測－SNaPshot26/50SNP檢測－突變錯配擴增檢驗180bp210bp共顯性標記27/50SNP優(yōu)點特異性的共顯性基因有功能意義缺點引物設(shè)計困難28/50顯性與共
7、顯性23411112101180bp210bpRAPD顯性1231AAABBBSNP共顯性29/50花柱卷曲性的適應(yīng)意義AAControlCutaaAAAAAa共顯性標記的應(yīng)用30/50顯性標記的應(yīng)用種群遺傳多樣性和遺傳結(jié)構(gòu)2345611011121111031000131/50隨機擴增與特異性擴增可重復(fù)性——數(shù)據(jù)的可靠性有條帶無條帶特異性擴增擴增成功擴增不成功非特異性擴增擴增成功？32/50隨機擴增RAPD,ISSR,單核苷酸突變檢驗SNP限制性酶切RF
