Revealing DNA sequence variation within the Lolium perenne genepool is important for genetic analysis and development of breeding applications. We reviewed current literature on plant development to select candidate genes in pathways that control agronomic traits, and identified 503 orthologues in L. perenne. Using targeted resequencing, we constructed a comprehensive catalogue of genomic variation for a L. perenne germplasm collection of 736 genotypes derived from current cultivars, breeding material and wild accessions. To overcome challenges of variant calling in heterogeneous outbreeding species, we used two complementary strategies to explore sequence diversity. First, four variant calling pipelines were integrated with the VariantMetaCaller to reach maximal sensitivity. Additional multiplex amplicon sequencing was used to empirically estimate an appropriate precision threshold. Second, a de novo assembly strategy was used to reconstruct divergent alleles for each gene. The advantage of this approach was illustrated by discovery of 28 novel alleles of LpSDUF247, a polymorphic gene co-segregating with the S-locus of the grass self-incompatibility system. Our approach is applicable to other genetically diverse outbreeding species. The resulting collection of functionally annotated variants can be mined for variants causing phenotypic variation, either through genetic association studies, or by selecting carriers of rare defective alleles for physiological analyses.

中文翻译：

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克服变异调用中的挑战：探索多年生黑麦草（Lolium perenne）植物发育候选基因的序列多样性。

揭示黑麦草基因库内的DNA序列变异对于遗传分析和育种应用的发展很重要。我们回顾了有关植物发育的最新文献，以选择控制农艺性状的途径中的候选基因，并鉴定了百日草中的503个直向同源物。使用针对性的重测序，我们为来自当前品种，育种材料和野生种的736个基因型的紫苏种质建立了完整的基因组变异目录。为了克服异质近交物种中变异调用的挑战，我们使用了两种互补策略来探索序列多样性。首先，四个变体调用管道与VariantMetaCaller集成在一起，以达到最大灵敏度。使用附加的多重扩增子测序来凭经验估计适当的精度阈值。其次，使用从头组装策略为每个基因重建不同的等位基因。通过发现LpSDUF247的28个新的等位基因可以说明这种方法的优势，LpSDUF247是与草自交不亲和系统的S基因座共分离的多态性基因。我们的方法适用于其他遗传多样性近交种。可以通过遗传关联研究或通过选择罕见的缺陷等位基因的携带者进行生理分析来挖掘功能注释的变异体的最终集合，以寻找引起表型变异的变异体。通过发现LpSDUF247的28个新的等位基因可以说明这种方法的优势，LpSDUF247是与草自交不亲和系统的S基因座共分离的多态性基因。我们的方法适用于其他遗传多样性近交种。可以通过遗传关联研究或通过选择罕见的缺陷等位基因的携带者进行生理分析来挖掘功能注释的变异体的最终集合，以寻找引起表型变异的变异体。通过发现LpSDUF247的28个新的等位基因可以说明这种方法的优势，LpSDUF247是与草自交不亲和系统的S基因座共分离的多态性基因。我们的方法适用于其他遗传多样性近交种。可以通过遗传关联研究或通过选择罕见的缺陷等位基因的携带者进行生理分析来挖掘功能注释的变异体的最终集合，以寻找引起表型变异的变异体。