Loss of genetic variation negatively impacts breeding efforts and food security. Genebanks house over 7 million accessions representing vast allelic diversity that is a resource for sustainable breeding. Discovery of DNA variations is an important step in the efficient use of these resources. While technologies have improved and costs dropped, it remains impractical to consider resequencing millions of accessions. Candidate genes are known for most agronomic traits, providing a list of high priority targets. Heterogeneity in seed stocks means that multiple samples from an accession need to be evaluated to recover available alleles. To address this we developed a pooled amplicon sequencing approach and applied it to the out-crossing cereal rye (Secale cereale L.). Using the amplicon sequencing approach 95 rye accessions of different improvement status and worldwide origin, each represented by a pooled sample comprising DNA of 96 individual plants, were evaluated for sequence variation in six candidate genes with significant functions on biotic and abiotic stress resistance, and seed quality. Seventy-four predicted deleterious variants were identified using multiple algorithms. Rare variants were recovered including those found only in a low percentage of seed. We conclude that this approach provides a rapid and flexible method for evaluating stock heterogeneity, probing allele diversity, and recovering previously hidden variation. A large extent of within-population heterogeneity revealed in the study provides an important point for consideration during rye germplasm conservation and utilization efforts.

中文翻译：

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深度采样和混合扩增子测序揭示了异质黑麦品种中隐藏的基因变异

遗传变异的丧失会对育种工作和粮食安全产生负面影响。基因库拥有超过 700 万个种质，代表着巨大的等位基因多样性，是可持续育种的资源。DNA变异的发现是有效利用这些资源的重要一步。虽然技术已经改进并且成本下降，但考虑对数百万种材料进行重新测序仍然不切实际。候选基因以大多数农艺性状而闻名，提供了一系列高优先级目标。种子库存的异质性意味着需要评估来自种质的多个样品以恢复可用的等位基因。为了解决这个问题，我们开发了一种混合扩增子测序方法，并将其应用于异型杂交谷物黑麦（Secale Greeke L.）。使用扩增子测序方法，评估了对生物和非生物胁迫抗性和种子具有重要功能的 6 个候选基因的序列变异，对 95 个具有不同改良状态和全球来源的黑麦品种进行了评估，每个品种均由包含 96 个个体植物 DNA 的混合样本代表。质量。使用多种算法识别出 74 个预测的有害变异。发现了罕见的变异，包括仅在一小部分种子中发现的变异。我们的结论是，这种方法提供了一种快速灵活的方法来评估种群异质性、探测等位基因多样性和恢复先前隐藏的变异。研究中揭示的群体内很大程度的异质性为黑麦种质保护和利用工作提供了重要的考虑点。