长江上游鱼类通用引物的筛选与验证

为了筛选出适合使用eDNA技术对长江上游鱼类多样性进行研究的通用引物,本研究选择了6对引物,分别为：Mifish-U、AcMDB07、Teleo、Teleo2、 Fish16S1和FishCB,对长江上游常见的20种鱼类和3种其他水域鱼类肌肉组织提取的DNA扩增后结果显示,6对引物均能扩增出全部23种鱼类,但引物Mifish-U的扩增效果最好。进一步使用引物Mifish-U对长江上游屏山县、涪陵区和巫山县3个采样点的水样eDNA进行高通量测序,结果显示引物Mifish-U能扩增出研究使用的20种长江上游鱼类,其辨别度较高。使用引物Mifish-U对室内养殖3种鱼类的水样eDNA进行高通量测序后定性定量分析,结果显示黄颡鱼和鲤的生物量与序列数相关性显著,鲫的生物量与序列数相关性不显著。综上,引物Mifish-U更适合作为长江上游鱼类多样性研究的通用引物。

Screening and verification of universal primers for fish in the upper reaches of the Yangtze River

In the "ten-year fishing ban" period of the Yangtze River, the traditional survey of the biodiversity of the Yangtze River methods have been restricted.There is an urgent need for a detection method that is low-damaging, more efficient and does not contradict current policies of assessing the fish diversity in the upper Yangtze River. In this study, six pairs of primers, Mifish-U, AcMDB07, Teleo, Teleo2, Fish16S1 and FishCB, were selected to screen out the general primers suitable for studying the fish diversity in the upper Yangtze River using the eDNA technology .The amplification of DNA extracted from muscle tissues of 20 fish species commonly found in the upper Yangtze River and three fish species from other waters showed that all six primer pairs could amplify all 23 fish species, but the best amplification was achieved with primer Mifish-U. Further high-throughput sequencing of eDNA of water sample from three sampling sites in the upper reaches of the Yangtze River, Ping Shan County, Fuling District and Wushan County, using primer Mifish-U, showed that the primer Mifish-U could amplify 20 species of fish from the upper reaches of the Yangtze River with high discrimination. Qualitative and quantitative analysis was performed after high-throughput sequencing of eDNA from water sample of three species of fish cultured indoors using primers Mifish-U. The results showed that the biomass of the yellow jawed fish and the carp correlated significantly with the sequence number. In conclusion, the primer Mifish-U is more suitable as a general primer for fish diversity studies in the upper Yangtze River. This study could provide a reference for the use of eDNA technology to illustrate fish diversity in the upper Yangtze River.