Polymorphism for immune functions can explain significant variation in health and reproductive success within species. Drastic loss in genetic diversity at such loci constitutes an extinction risk and should be monitored in species of conservation concern. However, effective implementations of genome-wide immune polymorphism sets into high-throughput genotyping assays are scarce. Here, we report the design and validation of a microfluidics-based amplicon sequencing assay to comprehensively capture genetic variation in Alpine ibex (Capra ibex). This species represents one of the most successful large mammal restorations recovering from a severely depressed census size and a massive loss in diversity at the major histocompatibility complex (MHC). We analysed 65 whole-genome sequencing sets of the Alpine ibex and related species to select the most representative markers and to prevent primer binding failures. In total, we designed ~1,000 amplicons densely covering the MHC, further immunity-related genes as well as randomly selected genome-wide markers for the assessment of neutral population structure. Our analysis of 158 individuals shows that the genome-wide markers perform equally well at resolving population structure as RAD-sequencing or low-coverage genome sequencing data sets. Immunity-related loci show unexpectedly high degrees of genetic differentiation within the species. Such information can now be used to define highly targeted individual translocations. Our design strategy can be realistically implemented into genetic surveys of a large range of species. In conclusion, leveraging whole-genome sequencing data sets to design targeted amplicon assays allows the simultaneous monitoring of multiple genetic risk factors and can be translated into species conservation recommendations.

中文翻译：

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用于高通量群体监测高山山羊免疫遗传学的一千个扩增子的稳健测序分析

免疫功能的多态性可以解释物种内健康和繁殖成功的显着差异。此类位点遗传多样性的急剧丧失构成灭绝风险，应在受保护的物种中进行监测。然而，将全基因组免疫多态性设置为高通量基因分型分析的有效实施是稀缺的。在这里，我们报告了基于微流体的扩增子测序分析的设计和验证，以全面捕获高山山羊 ( Capra ibex ) 的遗传变异。）。该物种代表了从人口普查规模严重下降和主要组织相容性复合体 (MHC) 多样性大量丧失中恢复过来的最成功的大型哺乳动物修复之一。我们分析了 65 个高山野山羊和相关物种的全基因组测序集，以选择最具代表性的标记并防止引物结合失败。总的来说，我们设计了约 1,000 个扩增子，密集覆盖 MHC、其他免疫相关基因以及随机选择的全基因组标记，用于评估中性种群结构。我们对 158 个人的分析表明，全基因组标记在解析群体结构方面的表现与 RAD 测序或低覆盖基因组测序数据集相同。免疫相关基因座在物种内表现出出乎意料的高度遗传分化。这些信息现在可用于定义高度靶向的个体易位。我们的设计策略可以实际实施到大范围物种的基因调查中。总之，利用全基因组测序数据集设计靶向扩增子分析可以同时监测多个遗传风险因素，并可以转化为物种保护建议。