A novel metabarcoding primer pair for environmental DNA analysis of Cephalopoda (Mollusca) targeting the nuclear 18S rRNA region

Cephalopods are pivotal components of marine food webs, but biodiversity studies are hampered by challenges to sample these agile marine molluscs. Metabarcoding of environmental DNA (eDNA) is a potentially powerful technique to study oceanic cephalopod biodiversity and distribution but has not been applied thus far. We present a novel universal primer pair for metabarcoding cephalopods from eDNA, Ceph18S (Forward: 5 ' -CGC GGC GCT ACA TAT TAG AC-3 ', Reverse: 5 ' -GCA CTT AAC CGA CCG TCG AC-3 '). The primer pair targets the hypervariable region V2 of the nuclear 18S rRNA gene and amplifies a relatively short target sequence of approximately 200 bp in order to allow the amplification of degraded DNA. In silico tests on a reference database and empirical tests on DNA extracts from cephalopod tissue estimate that 44-66% of cephalopod species, corresponding to about 310-460 species, can be amplified and identified with this primer pair. A multi-marker approach with the novel Ceph18S and two previously published cephalopod mitochondrial 16S rRNA primer sets targeting the same region (Jarman et al. 2006 Mol. Ecol. Notes.6, 268-271; Peters et al. 2015 Mar. Ecol.36, 1428-1439) is estimated to amplify and identify 89% of all cephalopod species, of which an estimated 19% can only be identified by Ceph18S. All sequences obtained with Ceph18S were submitted to GenBank, resulting in new 18S rRNA sequences for 13 cephalopod taxa.

Automated summary

A novel universal primer pair, Ceph18S, was developed for metabarcoding cephalopods from environmental DNA, allowing for accurate amplification and identification of a large percentage of cephalopod species. The study estimates that a multi-marker approach using Ceph18S in conjunction with two previously published mitochondrial 16S rRNA primer sets can amplify and identify 89% of all cephalopod species, with 19% uniquely identifiable by Ceph18S. The new primer pair provides a promising method for studying cephalopod biodiversity.