基于高通量测序技术的鱼类环境DNA研究中通用引物的筛选验证

Universal primer screening and verification for fish environmental DNA research based on high-throughout sequencing technology

为了筛选出一个或多个适宜研究环境DNA(environmental DNA,eDNA)的鱼类通用引物,本实验选取了5对引物,分别对鱼类线粒体细胞色素b(Cytochromeb,Cytb)基因、16SrDNA和细胞色素c氧化酶Ⅰ(COⅠ)基因部分片段进行扩增。在对两个水样eDNA进行扩增后发现,引物CY45M和1634M均扩增出目的条带,且扩增产物质量适用于后续的高通量测序,而引物CYFM、16ACM和COⅠM均没有扩增出目的条带。继续对另外7个水样eDNA扩增后发现,引物CY45M和1634M在所有水样eDNA中均取得良好的扩增效果。对目的产物进行IlluminaMiseq测序后发现,引物CY45M和1634M扩增产物注释上的鱼类物种均为11种,二者获得的鱼类种类既存在差异也存在重复。综上所述,引物CY45M和1634M都可作为鱼类群落结构eDNA研究的通用引物。

In order to screen one or more applicably universal primers for environmental DNA(eDNA) research based on high- throughout sequencing technology,five pairs of primers of fish mitochondrial genome fragment were chosen from literatures, which could amplify the partial sequences of Cyt b gene,16S rDNA and COⅠ gene.After initial amplification on two water eDNA samples,it was found that CY45M and1634M had better amplification effects and their products could be applied to the subsequent high-throughout sequencing.Meanwhile,no purpose band could be amplified using the other three primers, including CYFM,16ACM and COⅠM.After the next amplification on 7 water eDNA samples using CY45M and1634M,it was found that all the eDNA were amplified by these two pairs of primers, and the PCR products presented clear bright bands by gel electrophoresis.According to the high-throughout sequencing and BLAST results,the sequences gained by both of CY45M and1634M could be annotated on11 fish species.Nevertheless,there were dissimilarity and repeatability upon these fish species.In summary,the CY45M and1634M were both suitable universal primers for study on eDNA of fish community structure in Qiantang River.