Data from: Recurrent gene deletions and the evolution of adaptive cyanogenesis polymorphisms in white clover (Trifolium repens L.)

Understanding the molecular evolution of genes that underlie intraspecific polymorphisms can provide insights into the process of adaptive evolution. For adaptive polymorphisms characterized by gene presence/absence (P/A) variation, underlying loci commonly show signatures of long-term balancing selection, with gene-presence and gene-absence alleles maintained as two divergent lineages. We examined the molecular evolution of two unlinked P/A polymorphisms that underlie a well-documented adaptive polymorphism for cyanogenesis (hydrogen cyanide release with tissue damage) in white clover. Both cyanogenic and acyanogenic plants occur in this species, and the ecological forces that maintain this chemical defense polymorphism have been studied for several decades. Using a sample of 65 plants, we investigated the molecular evolution of sequences flanking the two underlying cyanogenesis genes: Ac/ac (controlling the presence/absence of cyanogenic glucosides), and Li/li (controlling the presence/absence of their hydrolyzing enzyme, linamarase). A combination of genome-walking, PCR assays, DNA sequence analysis, and Southern blotting was used to test whether these adaptive P/A polymorphisms show evidence of long-term balancing selection, or whether gene-absence alleles have evolved repeatedly through independent deletion events. For both loci, we detect no signatures of balancing selection in closest flanking genomic sequences. Instead, we find evidence for variation in the size of the deletions characterizing gene-absence alleles. These observations strongly suggest that both of these polymorphisms have been evolving through recurrent gene deletions over time. We discuss the genetic mechanisms that could account for this surprising pattern and the implications of these findings for mechanisms of rapid adaptive evolution in white clover.

解析调控种内多态性的基因的分子演化机制，可为适应性演化过程提供关键洞见。对于以基因存在/缺失（presence/absence, P/A）变异为特征的适应性多态性而言，其所在基因座通常会呈现长期平衡选择的信号，且存在型与缺失型等位基因会被维持为两个分化的演化支系。本研究聚焦白三叶中一类已有充分研究的产氰作用（组织损伤时释放氰化氢）适应性多态性，解析其背后两个不连锁的P/A多态性的分子演化机制。该物种中同时存在产氰与非产氰植株，维持这一化学防御多态性的生态驱动力已被研究数十年。我们以65株植株为样本，对两个产氰相关基因侧翼序列的分子演化展开研究：两个基因座分别为调控氰苷存在/缺失的Ac/ac，以及调控其水解酶——亚麻苦苷酶——存在/缺失的Li/li。我们结合基因组步移、聚合酶链式反应（Polymerase Chain Reaction, PCR）检测、DNA序列分析与Southern印迹杂交技术，检验这两类适应性P/A多态性是否存在长期平衡选择的信号，抑或缺失型等位基因是否通过独立缺失事件反复演化而来。针对两个基因座，我们在其紧邻侧翼的基因组序列中未检测到平衡选择的信号；相反，我们发现表征缺失型等位基因的缺失片段大小存在变异。上述结果强烈表明，这两类多态性均是通过长期反复的基因缺失事件实现演化的。我们将讨论可解释这一意外结果的遗传机制，以及本研究发现对白三叶快速适应性演化机制的启示。