Quantitative evaluation of intraspecific genetic diversity in a natural fish population using environmental DNA analysis

In this study, to quantitatively evaluate the genetic diversity using HTS-based eDNA analysis, we applied quantitative eDNA metabarcoding method to eDNA samples taken from three rivers in Japan. We targeted a fish species, Ayu (Plecoglossus altivelis altivelis), an abundant and important fishery target in Japanese inland waters. During the library preparation process, a mixture of known copy numbers of internal standard DNAs was added to each eDNA library to quantify the Ayu eDNA copy numbers. The sequence reads of each Ayu haplotype were then converted to the DNA copy numbers based on the relationship between the copy numbers and sequence reads of the internal standard DNAs. In addition, multiple Ayu specimens were collected from each sampling site and determined haplotype by Sanger sequencing.