A wolf SNP marker panel optimized for non-invasive samples based on the Fludigm SNP genotyping IFC

Across large parts of Europe wolves became extinct due to systematic persecution. Conservation efforts over the last decades, however, enabled the comeback of this elusive carnivore. Since the late 1990s, microsatellites have been used to genotype non-invasive samples (mainly scat), because data for management and conservation, such as population size and dynamics, is difficult to obtain otherwise. This marker system has a number of drawbacks, most notably a lack of straight-forward data exchangeability among laboratories. Recently, single nucleotide polymorphisms (SNPs) have gained attention as molecular markers because they have several advantages compared to microsatellites, e.g., transferability between working groups. In this study, we used a representative set of 79 wolf samples from 12 European countries genotyped on the Affymetrix dog SNP chip (tens of thousands of SNPs). From these we filtered for non-genic SNPs in Hardy-Weinberg and linkage equilibrium with minor allele frequency of at least 0.1. A set of 192 SNPs were then genotyped in 288 reference samples, mainly non-invasive samples, such as scat, hair, and urine. Further, we analysed DNA dilution series isolated from tissue to investigate assay sensitivity, and genotyped pedigreed families to be able to trace potential genotyping errors. Nanofluidic technology from Fluidigm (SNPtype assays used with the 96.96 Genotyping IFC) enabled us to technically multiplex 96 SNPs and genotype 96 samples at a time. Adjustments of the genotyping protocols to facilitate successful application to non-invasive sample material led to the selection 96 SNPs that are fit for the monitoring challenges of the future, i.e., quick and easy genotyping while preserving exchangeability of data between laboratories.