Fish mitochondrial genome sequencing: expanding genetic resources to support species detection and biodiversity monitoring using environmental DNA

Abstract

Conservation of aquatic resources is hampered by our limited knowledge of biological diversity and its distribution. Due to challenges with detection of rare or difficult to sample species, and the expansion of genetic technologies, fisheries professionals are supplementing traditional biodiversity field studies with emerging environmental DNA (eDNA) techniques. eDNA is generally evaluated with qPCR (primer- and probe-mediated single species detection), single-gene metabarcoding (PCR primer-mediated diversity analysis) or the emerging technique of multi-gene metagenomics (PCR independent diversity analysis). In each case, techniques are dependent on sequence databases for primer design and/or taxonomic assignment of recovered sequence data. Current reference databases contain limited mitochondrial genome information and are reliant on specific gene fragments, such as COI, which are not always suited for qPCR and metabarcoding marker design needs. To facilitate primer design and enhance taxonomic resolution for eDNA approaches, we describe a suite of order and/or family-specific long-range PCR primers sufficient for sequencing complete mitochondrial genomes. While the intent was to obtain mitochondrial genome data for freshwater fish, primers were designed on sequence alignments from all available species (including marine), and should be broadly applicable within their respective taxonomic group. We have sequenced 205 complete mitochondrial genomes representing 65 species/subspecies from 9 fish families, including novel genomes from 28 species not represented in GenBank at the time of submission. Continued expansion of species representation in mitochondrial genome databases will help move biodiversity assessment from single-gene metabarcoding approaches to multi-gene metagenomics and provide a valuable resource for eDNA applications, molecular ecology and phylogenetics.