基于DNA条形码的蕲蛇、乌梢蛇、金钱白花蛇及混伪品等32种的鉴别

Thirty-two Species of Agkistrodon acutus,Zaocys dhumnades,Bungarus multicinctus,and Adulterants Identified Based on DNA Barcode

目的:利用DNA条形码线粒体细胞色素C氧化酶亚基Ⅰ(COⅠ)基因鉴别蕲蛇、乌梢蛇、金钱白花蛇及其混伪品共32种蛇类。方法:收集蕲蛇、乌梢蛇、金钱白花蛇及其混伪品,对样品DNA进行聚合酶链式反应(PCR)扩增,扩增产物电泳后进行双向测序。应用Codon CodeAligner11.0.1软件对峰图质量进行比对、拼接,利用MEGA11.0软件对各物种碱基进行分析,构建邻接(NJ)系统聚类树并计算遗传距离。结果:NJ系统聚类树显示,蕲蛇、乌梢蛇、金钱白花蛇及其混伪品共32种均独立聚为一支,眼镜蛇科与蝰科聚为一支,并与游蛇科并列聚类。游蛇科黄环林蛇、颈棱蛇与眼镜蛇科聚为一支,游蛇科赤链华游蛇、环纹华游蛇、草腹链蛇与蝰科聚为一支,锦蛇属玉斑锦蛇、黑眉锦蛇、百花锦蛇未聚为一支。结论:利用DNA条形码COⅠ基因能够准确鉴定蕲蛇、乌梢蛇、金钱白花蛇及其混伪品共32种蛇类,赤链华游蛇、环纹华游蛇、草腹链蛇、黄环林蛇、颈棱蛇、玉斑锦蛇、百花锦蛇、黑眉锦蛇、宽吻水蛇、铅色水蛇、中国沼蛇、渔游蛇的种属归类有待进一步研究。

Objective:The cytochrome C oxidase subunitⅠ(COⅠ)gene of DNA barcode was used to identify 32 snake species,such as Agkistrodon acutus(A.acutus),Zaocys dhumnades(Z.dhumnades),Bungarus multicinctus(B.multicinctus),and adulterants.Methods:The samples of A.acutus,Z.dhumnades,B.multicinctus,and adulterants were collected.The DNA of the samples was amplified by polymerase chain reaction(PCR),and the amplified products were electrophoresed and sequenced in two directions.CodonCodeAligner 11.0.1 software was used to compare and concatenate the quality of peak maps.MEGA 11.0 software was used to analyze the bases of each species,construct the clustering tree of the adjacency(NJ)system,and calculate the genetic distance.Results:NJ system clustering tree displays that 32 snake species,such as A.acutus,Z.dhumnades,B.multicinctus,and adulterants are gathered independently.The Elapidae and Viperidae were grouped together and clustered together with the Colubridae.Boiga dendrophila(B.dendrophila)and Pseudagkistrodon rudis(P.rudis)get together with the Elapidae.The Trimerodytes aequifasciatus(T.aequifasciatus),Trimerodytes annularis(T.annularis),Amphiesma stolata(A.stolata),and the Viperidae gather together.Elaphe carinata(E.carinata),Elaphe taeniura(E.carinata),and Elaphe moellendorffi(E.moellendorff)are not clustered together.Conclusion:Thirty-two species of snake,such as A.acutus,Z.dhumnades,B.multicinctus,and adulterants,can be accurately identified using DNA barcode and COⅠgene.The species and genus classification of the T.annularis,T.aequifasciatus,A.stolata,B.dendrophila,P.rudis,E.carinata,E.carinata,E.moellendorffi,E.taeniura,Homalopsis buccata,Hypsiscopus plumbea,Myrrophis chinensis,and Fowlea piscator need to be further studied.