基于叶绿体rpoC1序列在山药分子鉴定中的应用

Application of chloroplast rpoC1 sequence in molecular identification of Chinese yam

目的研究基于rpoC1序列分析,建立山药物种鉴定的新方法。方法利用DNA条形码技术对收集到的7个山药样本提取基因组DNA,以rpoC1基因引物进行PCR扩增、测序,将所得序列在美国国家生物信息中心(National Center for Biotechnology Information,NCBI)数据库中进行BLAST比对,从GenBank获取薯蓣属、木薯属和番薯属rpoC1序列,应用MEGA 7.0软件计算种内和种间的(K2P)遗传距离,并构建邻接(neighbor joining,NJ)系统聚类树。结果7个山药样本rpoC1基因获得成功扩增和测序。山药样本最大种内K2P遗传距离为0.009,远远小于山药与木薯、番薯的种间K2P遗传距离0.104~0.118,也小于山药与盾叶薯蓣和穿龙薯蓣的种间K2P遗传距离0.026~0.035,构建的系统发育树显示山药与盾叶薯蓣、穿龙薯蓣、木薯和番薯单独聚为一类。结论rpoC1序列作为DNA条形码候选序列,能够实现对山药和混伪品的鉴定,同时也适用于食用山药和药用山药物种的区分,rpoC1序列可为山药物种鉴定提供新的分子鉴定方法。

Objective To establish a new molecular method to authenticate Chinese yam by using rpoC1 sequences.Methods Genomic DNA was extracted from 7 Chinese yam samples and was used as templates for PCR amplification with the primers of rpoC1 gene.The sequences were aligned in the National Center for Biotechnology Information(NCBI)database using BLAST.Based on GenBank Nucleotide Database,rpoC1 sequences of Dioscorea,Manihot and Ipomoea were downloaded.K2P distances were calculated using MEGA 7.0 software and neighbor-ioning(NJ)phylogenetic tree was established.Results rpoC1 gene was successfully amplified and sequenced in 7 Chinese yam samples.K2P genetic distance of intraspecific genetic distance of Chinese yam was 0.009,which was far less than the interspecific K2P genetic distance between Chinese yam and Manihot and Ipomoea(0.104-0.118),and also less than the interspecific K2P genetic distance between Chinese yam and Dioscorea zingiberensis and Dioscorea nipponica(0.026-0.035).The results of NJ phylogenetic tree showed that Chinese yam could be separated from Dioscorea zingiberensis or Manihot,and classified into a separate one.Conclusion As a candidate sequence of DNA barcoding,rpoC1 sequence can be used to identify yam and adulterants,and it can also be used to distinguish edible and medicinal yam species.rpoC1 sequence can provide a new molecular method for yam species identification.