OdoCocktail-Japan: Primer sets to enrich environmental DNA of Japanese Odonata species

Satoshi Yamamoto , Naoki Katayama, Junsuke Yamasako, Kenji Ito, Yuki G. Baba

Monitoring is essential to conserve and recover biodiversity. Environmental DNA (eDNA)-based metabarcoding analysis has been recognized as a cost-effective and efficient method to monitor species, but it still has limitations, such as insufficient reference databases and lack of primer sets suitable to detect target species. As biological indicators of freshwater habitats, Odonata species have been a monitoring target. As of yet, however, no metabarcoding primer sets can efficiently detect them. In the present study, we developed three primer sets (named OdoCocktail-Japan) to amplify a partial 16S rRNA sequence of Japanese Odonata species and assessed the performance of the primer sets using eDNA samples collected in the field. Each primer set (versions 1, 2, and 3) was designed to cover 203 of the 204 Japanese Odonata species, whose 16S rRNA sequences are available in a public database. The forward and reverse primers of versions 1 and 2 were designed to match four and five regions, respectively. On the other hand, those of version 3 were designed to match a single pair of regions, and the 3′-end base of forward primers was used to identify Odonata species. In silico PCR suggested that OdoCocktail-Japan primer sets cover 60–75% (including 203 Japanese Odonata) of all Odonata sequences available in the public database. The assessment using field samples showed that OdoCocktail-Japan improved the efficiency of Odonata detection in eDNA metabarcoding: the proportion of Odonata reads in high-throughput sequencing reached 32.7–85.6%, with OdoCocktail version 1 enriching Odonata sequences most efficiently among all versions. In addition, detected species were consistent with those encountered during water sampling or historical records for each sampling site. However, when Odonata eDNA was relatively rare or absent in samples, all versions amplified Ephemeroptera and Plecoptera as alternative templates in PCR. Thus, OdoCocktail-Japan primer sets offer high performance with samples from lentic environments, although further improvements are needed for sampling lotic environments, where Ephemeroptera and Plecoptera are usually abundant. We discuss how OdoCocktail-Japan can be modified to detect Odonata species from countries and areas outside Japan.