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.

北美水獭（Lontra canadensis）的多个种群正处于受威胁或恢复进程中，但相关研究难度极大：其种群密度偏低、个体难以通过目视识别，且栖息于会加速生物样本降解的水生环境。单核苷酸多态性（Single Nucleotide Polymorphisms, SNPs）可提升我们对难以开展研究的物种的种群统计与遗传参数的监测能力。本研究采用限制性酶切位点相关DNA（Restriction Site Associated DNA, RAD）测序技术，在美国蒙大拿州的水獭种群中发掘出20772个SNPs，其中包含14512个在其全球分布范围内至少一个其他种群中同样具有多态性的基因座。经严格筛选以最小化ascertainment偏倚、筛选出高质量且高杂合度（观测杂合度$H_o = 0.2–0.50$）的SNPs后，我们开发并验证了52组独立的SNP定量PCR（quantitative PCR, qPCR）基因分型检测体系，其中41组在稀释DNA样本中表现优异。该41个基因座在种群归属检验中展现出极强的分辨能力，仅在地理邻近的种群间出现1例错判，错判率为1.6%。本研究开发的SNPs标记不仅能够高效区分个体并准确判定其来源种群，同时在高浓度与稀释浓度DNA样本的基因型结果间，以及原始RAD测序数据与SNP qPCR检测阵列的基因型结果间均表现出高度一致性。