山溪鲵属的水样环境DNA分析方法的建立

Establishment of analysis method for environment DNA of Batrachuperus from water samples

通过优化建立一套符合山溪鲵属物种环境DNA(environmental DNA,e DNA)分析的操作方法,为后期山溪鲵属资源调查及保护提供理论依据。以太白山溪鲵为研究对象,在室内养殖条件下,进行水样过滤及目标物种线粒体细胞色素b(Cytb)基因的扩增,根据测序序列构建系统发育树,来判定水样DNA中目标物种的归属。结果表明:(1)通过酒精直接浸泡、锡箔纸包裹、唾液收集管收集这3种方法对过滤养殖水后的滤膜进行贮存,发现酒精直接浸泡法可增加后期e DNA的提取量;(2)在进行目的片段的PCR扩增时发现高保真Mix的扩增成功率大于普通Mix;(3)应用属间特异引物进行不同物种扩增后,可通过系统发育分析来确定种间的分类。通过优化首次建立了山溪鲵属物种的水样e DNA分析方法,可为后期生活在相同水质且种间分化较小的物种进行e DNA分析提供参考。

This study established the method of environmental DNA(eDNA)analysis of Batrachuperus through a series of optimization,to provide a theoretical basis for resource investigation and protection of Batrachuperus in future research.This study takes Batrachuperus taibaiensis(B.ta)as the research object to filter water from the aquaculture tank,and genus-specific primer used for the amplification of Cytb gene,afterthat phylogenetic tree was conducted according to sequence analysis to determine the belonging of target species in water.The main results as follows:(1)Three membrane storage methods(like membrane directly socked in alcohol,membrane wrapped in tinfoil and membrane collected with saliva collection tube)have been compared,found that membrane directly socked in alcohol can increase the eDNA yield compared with the other;(2)The PCR amplification efficiency of high-fidelity Mix is higher than ordinary in eDNA analysis;(3)Classification of those species that amplified using genus-specific primer were conducted on a phylogenetic analysis.[Conclusion]This study is the first to establish an optimal method for obtaining eDNA of Batrachuperus from water samples,which provides reference for eDNA extraction of other species who lived in similar waters and have less genetically diverse.