基于线粒体DNA和核DNA遗传标记的中华鲟种属鉴定

Species Identification of Acipenser sinensis Based on Mitochondrial DNA and Nuclear DNA Genetic Markers

目的 构建一种基于线粒体DNA和核DNA遗传标记的中华鲟及其潜在杂交种的鉴定方法。方法 根据美国国立生物技术信息中心(National Center for Biotechnology Information, NCBI)已公布的中华鲟cytb基因序列设计引物,并选用鲟鱼单拷贝核基因fam43a扩增引物对中华鲟DNA进行扩增与测序,其中对fam43a基因扩增产物进行TA克隆测序。通过BLAST序列相似性检索确定样本来源,并基于邻接法构建鲟形目系统进化树。结果 中华鲟样本测序所得cytb和fam43a基因序列长度分别为728 bp和730 bp,且10个克隆群所测fam43a基因序列完全一致。基本局部比对搜索工具(basic local alignment search tool, BLAST)序列相似分析显示,所测cytb与fam43a基因序列均与GenBank数据库中的中华鲟相似性最高,相似性分别为99.86%和100%。系统进化分析显示,中华鲟样本所测得序列均与GenBank数据库中的中华鲟DNA序列聚类在一起,且与鲟形目其他物种位于不同分支。结论 联合使用线粒体DNA和核DNA遗传标记能够快速准确地鉴定中华鲟及其潜在杂交种,该方法能够满足法医日常检案的鉴定需求,为中华鲟的拯救和保护提供科学技术支持。

Objective To establish a method for the identification of Acipenser sinensis and its potential hybrids based on mitochondrial DNA and nuclear DNA markers.Methods Primers were designed according to the cytb gene sequence of Acipenser sinensis published in NCBI,and the single-copy nuclear gene fam43a amplification primers of sturgeon were used to amplify and sequence the DNA of Acipenser sinensis.The amplified product of fam43a gene was TA cloned and sequenced.The source of the sample was determined by BLAST sequence similarity search,and the phylogenetic tree of Acipenseridae was constructed based on the neighbor-joining method.Results The lengths of cytb and fam43a gene sequences obtained by sequencing the Acipenser sinensis samples were 728 bp and 730 bp,respectively.The fam43a gene sequences of the 10 clonal populations were completely consistent.The analysis of BLAST sequence similarity search showed that the measured cytb and fam43a gene sequences had the highest similarity with the Acipenser sinensis in the GenBank database,which were 99.86%and 100%,respectively.Phylogenetic analysis showed that the sequences of Acipenser sinensis all clustered with the DNA sequences of Acipenser sinensis in the GenBank database,and belonged to different clades with other species of Acipenseridae.Conclusion The combined use of mitochondrial DNA and nuclear DNA markers can quickly and accurately identify Acipenser sinensis and its potential hybrids.This method can meet the needs of forensic identification cases and provide technical support for the rescue and protection of Acipenser sinensis.