Discovery of 20,000 RAD-SNPs and development of a 52-SNP array for monitoring river otters

Many North American river otter (Lontra canadensis) populations are threatened or recovering but are difficult to study because they occur at low densities, it is difficult to visually identify individuals, and they inhabit aquatic environments that accelerate degradation of biological samples. Single nucleotide polymorphisms (SNPs) can improve our ability to monitor demographic and genetic parameters of difficult to study species. We used restriction site associated DNA (RAD) sequencing to discover 20,772 SNPs present in Montana, USA, river otter populations, including 14,512 loci that were also variable in at least one other population range-wide. After applying careful filtering criteria meant to minimize ascertainment bias and identify high quality, highly heterozygous (H (o) = 0.2-0.50) SNPs, we developed and tested 52 independent SNP qPCR genotyping assays, including 41 that performed well with diluted DNA. The 41 loci provided high power for population assignment tests with only 1 misassignment (1.6 %) between closely neighboring populations. Our SNPs showed high power to differentiate individuals and assign them to population of origin, as well as strong concordance of genotypes from high and diluted concentrations of DNA, and between original RAD and the SNP qPCR array.