基于eDNA技术的舟山近海中国团扇鳐定性与定量分析

Qualitative and quantitative analysis of Platyrhina sinensis based on eDNA methods in Zhoushan offshore

为探讨环境DNA (environmental DNA, eDNA)技术用于中国团扇鳐监测方面的可行性,同时开展基于eDNA技术的舟山近海中国团扇鳐定性与定量分析。本实验将中国团扇鳐与其同属汤氏团扇鳐COI基因片段序列进行比较分析,使用Primer Express 3.0软件设计中国团扇鳐特异性引物与Taq Man探针。在舟山朱家尖近海设计了A、B、C共3个定置网调查站位,定期收集中国团扇鳐样品。于2017年12月19日、2018年4月13日和2018年7月14日分别采集水样(站位A、B1、B2、C1、C2、D),开展eDNA微滴式数字PCR(droplet digital PCR, ddPCR)检测,并将检测结果与水温和采样点进行差异显著性分析。结果显示,不同站位、不同时间的中国团扇鳐eDNA浓度不同,水样采集点对中国团扇鳐eDNA浓度的影响极显著,不同采样点eDNA在不同季节存在极显著差异。研究表明,eDNA检测技术灵敏度高,水温、底质和水深对中国团扇鳐的分布皆有影响。研究结果为其他海域的中国团扇鳐e DNA追踪监测奠定了基础。

Environmental DNA(eDNA)is the general term of DNA released by organisms in ice,soil,air,water,sediment and other environments,which has been widely used in field investigation of aquatic biological resources in recent years.Platyrhina sinensis is mainly distributed in the western area of the North Pacific Ocean,and the coastal area of Zhoushan may be the northern limit of its distribution.In order to explore the feasibility of eDNA technology in the detection of P.sinensis,the qualitative and quantitative detection of P.sinensis in Zhoushan off-shore was carried out based on eDNA technology at the same time.In this study,the COI gene sequences of P.sin-ensis and Platyrhina tangi were compared and analyzed.Primer Express 3.0 software was used to design specific primers and TaqMan probe of P.sinensis.Three fixed net survey stations A,B and C were designed in Zhujiajian offshore,Zhoushan,and samples of P.sinensis were collected regularly.Water samples(stations A,B1,B2,C1,C2 and D)were collected on December 19,2017,April 14,2018 and July 14,2018,respectively,and eDNA droplet digital PCR detection was carried out.Significant difference analysis was conducted between eDNA detec-tion results and water temperature and sampling sites.The results showed that:the concentrations of eDNA of P.sinensis were different at different stations and at different times.The effects of water sampling sites on eDNA concentration of P.sinensis were significant,and there were significant differences in eDNA at different sampling sites in different seasons.This study showed that eDNA detection was sensitive.Water temperature,water depth and substrate quality all affected the distribution of P.sinensis.The results of this study lay a foundation for eDNA tracking and monitoring of P.sinensis in other sea areas.