Data from: Development of a genotype-by-sequencing immunogenetic assay as exemplified by screening for variation in red fox with and without endemic rabies exposure

Pathogens are recognized as major drivers of local adaptation in wildlife systems. By determining which gene variants are favored in local interactions among populations with and without disease, spatially explicit adaptive responses to pathogens can be elucidated. Much of our current understanding of host responses to disease comes from a small number of genes associated with an immune response. High-throughput sequencing (HTS) technologies, such as genotype-by-sequencing (GBS), facilitate expanded explorations of genomic variation among populations. Hybridization-based GBS techniques can be leveraged in systems not well characterized for specific variants associated with disease outcome to “capture” specific genes and regulatory regions known to influence expression and disease outcome. We developed a multiplexed, sequence capture assay for red foxes to simultaneously assess ~300-kbp of genomic sequence from 116 adaptive, intrinsic, and innate immunity genes of predicted adaptive significance and their putative upstream regulatory regions along with 23 neutral microsatellite regions to control for demographic effects. The assay was applied to 45 fox DNA samples from Alaska, where three arctic rabies strains are geographically restricted and endemic to coastal tundra regions, yet absent from the boreal interior. The assay provided 61.5% on-target enrichment with relatively even sequence coverage across all targeted loci and samples (mean = 50×), which allowed us to elucidate genetic variation across introns, exons, and potential regulatory regions (4,819 SNPs). Challenges remained in accurately describing microsatellite variation using this technique; however, longer-read HTS technologies should overcome these issues. We used these data to conduct preliminary analyses and detected genetic structure in a subset of red fox immune-related genes between regions with and without endemic arctic rabies. This assay provides a template to assess immunogenetic variation in wildlife disease systems.

病原体被认为是野生生物系统中局部适应的主要驱动因素。通过明确存在与不存在病害的种群间局部互作中受青睐的基因变异类型，可阐明宿主对病原体的空间特异性适应性响应。目前学界对宿主病害响应的认知，大多源自与免疫反应相关的少数基因。高通量测序（High-throughput sequencing, HTS）技术，例如基因型测序（genotype-by-sequencing, GBS），可推动对种群间基因组变异的更广泛探索。基于杂交的GBS技术可应用于尚未明确与病害结局相关特定变异的研究系统，以“捕获”已知影响基因表达与病害结局的特定基因及调控区域。我们为赤狐开发了一套多重序列捕获检测体系，可同时对116个具备预测适应性意义的适应性、内源性及先天性免疫基因、其推定上游调控区域，以及23个用于控制群体遗传效应的中性微卫星区域的约300千碱基对基因组序列进行评估。该体系被应用于取自阿拉斯加的45份赤狐DNA样本：阿拉斯加境内存在3种地理局限的北极狂犬病毒株，这些毒株仅在沿海苔原区域呈地方性流行，而北方针叶林内陆区域无该病毒分布。该检测体系实现了61.5%的靶向富集率，且所有靶向位点与样本间的测序覆盖度相对均匀（平均覆盖度为50×），由此得以阐明内含子、外显子及潜在调控区域内的遗传变异（共4819个单核苷酸多态性（Single Nucleotide Polymorphism, SNP）位点）。不过该技术在精准描述微卫星变异方面仍存在局限，但长读长HTS技术有望解决上述问题。我们利用这些数据开展了初步分析，在存在与不存在地方性流行北极狂犬病的区域间，于赤狐免疫相关基因的子集内检测到了遗传结构分化。该检测体系可为野生生物病害系统中的免疫遗传变异评估提供参考范式。