Development and evaluation of PCR primers for environmental DNA (eDNA) metabarcoding of Amphibia

By

Sakata, Masayuki K.
Kawata, Mone U.
Kurabayashi, Atsushi
Kurita, Takaki
Nakamura, Masatoshi
Shirako, Tomoyasu
Kakehashi, Ryosuke
Nishikawa, Kanto
Hossman, Mohamad Yazid
Nishijima, Takashi
Kabamoto, Junichi
Miya, Masaki
Minamoto, Toshifumi

Type

Article

Date of Publication

2022-2-21

Original Publication

Metabarcoding and metagenomics

Volume

6

Pages

1--12

Subjects

Amphibia , biodiversity monitoring , environmental DNA , metabarcoding , universal primer , Аgricultural ecosystem

Contributed by

Pensoft Publishers

Language

English

Published by

Bulgaria Pensoft Publishers 2017-

Identifiers

DOI: https://doi.org/10.3897/mbmg.6.76534

 

Abstract

Biodiversity monitoring is important for the conservation of natural ecosystems in general, but particularly for amphibians, whose populations are pronouncedly declining. However, amphibians’ ecological traits (e.g. nocturnal or aquatic) often prevent their precise monitoring. Environmental DNA (eDNA) metabarcoding – analysis of extra-organismal DNA released into the environment – allows the easy and effective monitoring of the biodiversity of aquatic organisms. Here, we developed and tested the utility of original PCR primer sets. First, we conducted in vitro PCR amplification tests with universal primer candidates using total DNA extracted from amphibian tissues. Five primer sets successfully amplified the target DNA fragments (partial 16S rRNA gene fragments of 160–311 bp) from all 16 taxa tested (from the three living amphibian orders Anura, Caudata and Gymnophiona). Next, we investigated the taxonomic resolution retrieved using each primer set. The results revealed that the universal primer set “Amph16S” had the highest resolution amongst the tested sets. Finally, we applied Amph16S to the water samples collected in the field and evaluated its detection capability by comparing the species detected using eDNA and physical survey (capture-based sampling and visual survey) in multiple agricultural ecosystems across Japan (160 sites in 10 areas). The eDNA metabarcoding with Amph16S detected twice as many species as the physical surveys (16 vs. 8 species, respectively), indicating the effectiveness of Amph16S in biodiversity monitoring and ecological research for amphibian communities.