High genetic diversity is a key for animal populations to adapt flexibly to environmental changes. Genetic diversity of a population is prone to be deteriorated by habitat fragmentation, infectious diseases and any other factors. Monitoring the genetic diversity of a target population is essential to make an effective conservation plan; however, it takes time and cost because large number of specimens is required. When the target species has extremely low population density, collection of sufficient number of specimens may become impossible; moreover, the research activity itself might be a threat for the population. Here, we propose an environmental DNA (eDNA) based non-invasive method to analyze mitochondrial haplotypes of Ayu (Plecoglossus altivelis altivelis), an Osmeriformes species. First, we developed a primer set which specifically amplifies a part of the control region of mitochondrial DNA of Ayu. Second, we collected eDNA from rearing water of Ayu juvenile from 20 small aquariums each of which contained one individual. Extracted DNA were amplified by PCR and the amplicons were subjected to Sanger sequencing. Finally, eDNA samples, which were extracted from 1) mixed water of 20 aquariums used above and 2) field water from fishing ground of ayu, were subjected to next generation sequencing (NGS).
Results/Conclusions
The haplotypes determined by Sangar sequencing from 20 eDNA samples derived from the rearing water showed 100% matches with the sequence of the Ayu individual in the corresponding aquarium. The haplotypes were classified into seven types and we could also detect all the haplotypes by NGS analysis when the mixture of the 20 rearing water was used as an eDNA source. Moreover, we succeed in the detection of diverse haplotypes from field water samples which were the mixture of DNA from countless individuals in the sampling site. In addition, the result suggested that there might be a correlation between the abundances of Ayu assigned to each haplotype and the amounts of sequence reads of each haplotype determined by NGS. These results ensure that eDNA analysis can also be applied to haplotype detection, i.e. detection of intraspecific genetic variation, not only to species detection from water samples. This non-invasive method provides a practical way of genetic diversity evaluation for rare species to which conventional direct catchment could not be applied.