Environmental DNA (eDNA) analysis enables estimation of species composition of aquatic macro-organisms. Few previous studies have attempted to retrieve entire mt-DNA sequences from eDNA, instead focusing on short sequences of mitochondrial DNA (mt-DNA), which have provided relatively limited sequence resolution for species identification or haplotype detection. A stable protocol to obtain whole mt-DNA sequences would improve resolution and expand applications of eDNA analysis to the evaluation of intraspecific genetic diversity and biogeographical study.
Therefore, we tried to amplify the whole mt-DNA using step-down PCR and previously-reported universal fish primers. The optimal annealing temperature for PCR was examined using eDNA from a fish tank, and the optimal number of PCR cycles was determined with eDNA from a river and a pond in Kyoto, Japan. Finally, long PCR products of river and pond eDNA samples were subjected to metabarcoding to confirm that PCR products derived from fish eDNA. Additionally, the fish species composition detected in this analysis was compared with that of metabarcoding of the original eDNA samples to evaluate detection sensitivity of the step-down PCR.
Results/Conclusions
Using tank-derived eDNA, step-down PCR with an initial annealing temperature of 74 °C followed by stepping down 2 °C for every 5 cycles until reaching 68 °C for the final step produced an amplicon with the target length (ca. 16 kb) without extra gel bands. River and pond samples were then subjected for step-down PCR with varying PCR cycles in the final step, and 35 cycles was found to best amplify target DNA and minimize extra gel bands. Metabarcoding of the river sample revealed 5 and 21 species from the PCR products and original eDNA, respectively. For the pond sample, 4 and 13 species were detected, respectively. Detected species from step-down PCR products were a subset of those from the original eDNA and were biased toward species which were detected from their original eDNA with large numbers of reads.
Overall, this study revealed that fish eDNA in field samples contains mt-DNA at a sufficient concentration for PCR amplification. Although routine sequencing of long amplicons will require further resolution of many technical challenges, our PCR protocol provides a first step for maximizing information gained from aquatic eDNA.