VertU: universal multilocus primer sets for eDNA metabarcoding of vertebrate diversity, evaluated by both artificial and natural cases

Environmental DNA (eDNA) metabarcoding is a powerful tool for monitoring biodiversity in natural ecosystems. The accuracy and efficacy of eDNA metabarcoding surveys rely heavily on the PCR primers for the amplification of target sequences. Here, we developed three sets of new universal PCR primers targeting the mitochondrial 12S, 16S, and COI genes, named V12S-U, V16S-U, and VCOI-U, respectively. They work for all vertebrate groups including fishes, amphibians, reptiles, birds, and mammals, with amplification length ranged about 200-250 bp, facilitating the use of short-read sequencing platforms such as Illumina HiSeq. The species detection success of the three markers was all over 90%, evaluated both by in-silico PCR and by amplifying mock DNA and zoo eDNA with known species compositions. To evaluate the real performance of the three markers, we applied them to water samples of four different natural environments. We detected a total of 409 vertebrate operation taxonomic units (OTUs) , comprising 218 species, 201 genera, and 96 families distributed in five major vertebrate groups. Our results indicated that utilizing multiple barcode markers can achieve better species detection rates than using only a single marker, which is especially important for monitoring large biological communities such as vertebrates. The new primer sets presented here enable researchers to detect the presence of species of different vertebrate groups in a single eDNA metabarcoding analysis, which simplifies the workflow and reduces the cost. It has the potential to be used as an alternative (or complementary) tool for future eDNA metabarcoding studies targeting vertebrates.