Mitochondrial Genomic Resources for Key North American Species to Support Targeted Environmental DNA Assay Development

Complete mitochondrial genomes (mitogenomes) provide the best opportunity for designing robust species-targeted environmental DNA (eDNA) assays. However, the lack of available full mitogenome sequences for many North American species constrains assay development. While specific mitochondrial markers such as mt-co1 and mt-cytb are commonly used, they often lack the resolution needed to distinguish between closely related or recently diverged species. This study addresses these gaps by generating 324 standardized mitogenomes from 119 ecologically, culturally, or economically significant species across North America. Using the mtGrasp pipeline optimized for short-read sequences, we assembled high-quality mitogenomes with consistent orientation and start sites to support rapid comparative analyses. These genomic resources enabled the development and validation of 125 species-targeted quantitative PCR (qPCR)-based eDNA assays, guided by alignments with sympatric taxa and refined using the unikseq pipeline to identify unique and/or conserved regions across 18 mitochondrial genes. Most of these assays demonstrated high sensitivity and met the Canadian national standards. The availability of complete mitogenome resources is especially relevant for challenging taxa such as rockfish (Genus Sebastes), where high interspecific similarity and intraspecific variability render standard metabarcoding markers insufficient. All data have been publicly deposited in GenBank (BioProject PRJNA1139757) to support broader adoption. By improving taxonomic resolution and reducing cross-amplification risks, these mitogenome resources advance the development of robust eDNA tools for biodiversity assessment, species-at-risk monitoring, and biosecurity applications, thus directly contributing to national and global conservation objectives, including the Kunming-Montreal Global Biodiversity Framework.