涉案偶蹄目动物DNA条形码构建

【目的】利用线粒体DNA条形码标记鉴定涉案偶蹄目动物,为解决偶蹄目动物残体、制品的鉴定难题提供技术参考。【方法】对16个偶蹄目动物物种共67份样本,采用试剂盒提取DNA,扩增、测定物种的线粒体DNA的COI基因、Cyt b基因片段;在GenBank上Blast搜索进行同源性分析;利用MEGA 7.0软件分别比较这两个基因片段种内和种间遗传距离,构建系统发育树。【结果】在同源性分析中,西伯利亚狍(Capreolus pygargus)的Cyt b基因片段与GenBank中西伯利亚狍、欧洲狍(Capreolus capreolus)基因序列的同源性均为99.64%~99.82%,该片段不能区分西伯利亚狍与欧洲狍这两个近缘种。其余测序结果与数据库中参考序列一致且相似度较高。对于COI片段,种内遗传距离为0~1.4%,种间遗传距离为1.8%~18.8%。对于Cyt b基因片段种内遗传距离为0~0.5%,种间遗传距离为4.0%~15.8%。两个片段的种内遗传差异均小于种间遗传差异,存在较为明显的条形码间隔。COI和Cyt b基因片段的系统发育树中,同物种的不同个体均能形成具有较高支持度的单分支;不同物种之间界限清晰,每个物种的所有个体均能够形成具有较高支持度的单分支。【结论】基于COI和Cyt b基因片段构建的DNA条形码均可用于对涉案偶蹄目动物的物种鉴定。在物种鉴定中,可采用两个片段联合使用的方式,尤其是对于近缘种的鉴定,以降低错误风险。

DNA barcoding of Artiodactyla in cases involved in wildlife for species identification

【Objective】In this study, we aimed to identify Artiodactyla species in cases of wildlife destruction, using mitochondrial DNA barcoding. 【Method】COI and Cyt b sequences in 67 samples from 16 species were amplified. Animal samples were identified using Blast search on the GenBank sequences. Intra-and inter-specific genetic distances were compared, and phylogenetic trees were constructed using MEGA 7.0. 【Result】In a homology analysis, the Cyt b sequences of Capreolus pygargus and Capreolus capreouls were 99.64%-99.82% homologous; this sequence could not distinguish two closely related species, whereas other COI and Cyt b sequences could. The intra-specific genetic distance was 0-1.4% for COI and 0-0.5% for Cyt b, whereas the inter-specific genetic distance was 1.8%-18.8% for COI and 4.0%-15.8% for Cyt b. An obvious barcoding gap was detected in both markers for all species. The phylogenetic tree showed that samples from the same species formed monophyletic groups with high support values. The gaps between different species were clear, and all samples from each species formed a single branch with a high support value.【Conclusion】COI and Cyt b may serve as molecular markers for Artiodactyla identification in cases involving wildlife resources. To minimize the risk of false identification, the two DNA barcodes could be used simultaneously, especially for the closely related species.