淡水鱼类环境DNA宏条形码引物的筛选及其在千岛湖的应用OACSTPCD

In recent years,environmental DNA(eDNA)technology for monitoring fish diversity have been used worldwide due to its high sensitivity,harmlessness to target organisms and low cost.However,the effectiveness of eDNA methods depends on the se-lection of metabarcoding primers.Although some environmental DNA metabarcoding primers for fish are available,few studies have comprehensively evaluated the detection effectiveness of these primers.In this study,29 pairs of fish eDNA metabarcoding primers from COI,Cytb,12S rRNA and 16S rRNA genes(including 2 pairs designed in this study and 27 pairs cited from previous litera-tures)were evaluated.A preliminary analysis was performed in silico PCR,followed by further validation of the 17 primer pairs that worked well by high-throughput sequencing.The results showed that the primers with good results in silico PCR did not neces-sarily perform well in high-throughput sequencing,indicating that the selection of primers cannot rely on silico PCR alone.The me-tabarcoding primers with longer amplification fragment length did not obtain better amplification results.Most of the primers with good performance were in the 200-300 bp fragment length range.In contrast to COI and Cytb,12S rRNA primers and 16S rRNA primers had good amplification effects and were suitable for use as environmental DNA metabarcoding for fish diversity studies.Meanwhile,given the incompleteness of the current fish DNA barcode database and the different characteristics of different prim-ers,amplification using multiple primer pairs would greatly increase the detection probability of species and the credibility of eDNA studies.This study demonstrates the potential of eDNA in assessing biodiversity,which will help future eDNA studies of fish and thus provides a reference for the conservation of fish diversity.