Development of a SNP Amplicon Panel for Invasive Deer Faecal Pellets and the Effects of Environmental Exposure on...

Non-invasive genetic sampling techniques hold great promise for wildlife monitoring but pose considerable challenges for obtaining genetic data. For faecal samples, mixed DNA from many organisms, low concentration, reaction inhibitors, and DNA degradation over time often hinder success. We aimed to develop a high-throughput, cost-effective faecal genotyping technique for monitoring invasive sambar deer (Cervus unicolor). We developed a multiplexed amplicon panel targeting 371 single nucleotide polymorphism (SNP) markers previously genotyped using restriction-site associated DNA sequencing, and three sex markers. To evaluate panel performance in faecal samples, we undertook a degradation trial assessing the effects of time (0, 2, 4, 7, and 14 days) on genotyping success under three conditions (outdoors on exposed ground, outdoors protected from rain and UV, and indoors). Fresh faecal samples performed equally to tissue, but environmental exposure rapidly degraded DNA. On average, exposed pellets were viable up to 4 days, with ~200 SNPs successfully genotyped, while protected samples were still viable after 14 days. In the field, we recommend sampling when there has been no rain for several days. We demonstrate that our method reliably ascertains individuals, sex, and species, with sufficient concordance among sample types and genotyping methods to support integration of faecal-derived genotypes with existing datasets. Using existing reduced-representation tissue sample data, we further demonstrate that the 200 SNPs reliably genotyped can resolve population structure and first-order kinship, aiding informed management. More broadly, the insights for cost-effective faecal DNA genotyping with optimised sample collection will benefit monitoring of both native wildlife and invasive species.