多基因DNA条形码鉴定6个鳗鱼物种

Identification of Six Eel Species Using Polygenic DNA Barcoding

建立6种鳗鱼的物种多基因DNA条形码精准鉴定方法。以鳗鱼DNA为模板,采用3对通用引物对6种鳗鱼的3个基因(16S rRNA、Cyt b、COⅠ)部分DNA片段进行聚合酶链式反应扩增、测序,结果6种鳗鱼各获得3条16S r DNA(638~643 bp)、Cyt b(464~466 bp)、COⅠ(705~707 bp)基因部分DNA序列,从中选取各物种序列同源的片段设计3对新引物对6种鳗鱼的16S rRNA、Cyt b、COⅠ基因部分DNA片段进行PCR扩增,其产物大小分别为504~507、400、609 bp,再从各片段中筛选出具有6种鳗鱼物种特异性强的、碱基数分别为262、280、300 bp的3条DNA片段序列,作为6种鳗鱼物种的3个基因的标准DNA条形码,应用DNAMAN V6软件进行同源性分析,并通过Gen Bank数据库的比对验证,制定了供检测实践用的同源率判别指标,建立鳗鱼物种的多基因条形码检测方法。应用该方法对30个待检鳗鱼样品进行检测,结果表明,各样品基于3个基因DNA条形码的比对,符合同源率指标,物种判别结果互相吻合,从而精准判别样品的所属物种。该方法稳定、精准、易于操作,可应用于6种鳗鱼的物种鉴定,值得推广。

This paper develops an accurate method to identify six eel species including Anguilla rostrata,A.anguilla,A.japonica,A.mossambica,A.marmorata,and A.bicolor pacifica using polygenic DNA barcoding.Three pairs of universal primers were used to amplify partial fragments of the 16 S rRNA,Cyt b and COⅠgenes of the eel species with the total genomic DNA from eel as a template and the amplified products were sequenced.As a result,16 S rDNA（638–643 bp）,Cyt b（464–466 bp） and COⅠ（705–707 bp） gene fragments were obtained for each species.Homologous sequences of these genes were found.Three new primer pairs were designed to amplify 638–643,400 and 609 bp fragments of the 16 S rRNA,Cyt b and COⅠ genes by PCR,respectively.The specific 16 S rRNA,Cyt b and COⅠ fragments of 262,280 and 300 bp were selected as standard DNA barcodes for the identification of eel species.Homology analysis was performed using DNAMAN software（version 6） and the homology for species discrimination was developed by aligning the DNA barcodes with the Gen Bank database.Analysis of 30 samples from six eel species by the developed method showed that the species were consistently identified using the DNA barcodes.In conclusion,the method is stable,precise and easy to operate and can be applied to the identification of six species of eels.