Development of a panel of SNP loci in the emblematic southern damselfly (Coenagrion mercuriale) using a hybrid method: pitfalls and recommendations for large-scale SNP genotyping in a non-model endangered species.

Genomic markers are essential tools for studying species of conservation concern, yet non-model species often lack a reference genome. Here we describe a methodology for identifying and genotyping thousands of SNP loci in the southern damselfly (Coenagrion mercuriale), a bioindicator of freshwater stream quality classified as near-threatened, with locally declining populations. We used a hybrid approach combining reduced representation sequencing and target enrichment. First, we identified putative SNP loci using ddRADseq and de novo assembly. Then, single primer enrichment technology targeted 6,000 of these SNPs across 1,920 individuals. Challenges encountered included sequence recapture failure, coverage depth discrepancies, and aberrant FIS values. We provide recommendations to address such issues. After multiple filtering steps, 2,092 SNPs were retained and used to analyse the genetic structure of 131 individuals belonging to 11 populations in France, comparing central and marginal populations. Genetic differentiation was lower among central populations, with no sign of inbreeding. As compared to microsatellite loci, SNPs exhibited greater resolution in detecting fine-scaled genetic structure, identifying putative hybrids in adjacent populations. In this study, we emphasise the difficulties of large-scale SNP genotyping in non-model species via a hybrid method that ultimately did not offer the expected cost and time saving compared to classical ddRAD approaches. However, SNPs showed greater power than previously available markers in identifying conservation units or admixture events, and the panel of reusable probes we describe here offers the potential to improve conservation efforts through future diachronic studies or finer estimations of key parameters like effective population size.