Data from: Validating genome-wide association candidates controlling quantitative variation in nodulation

Genome-wide association (GWA) studies offer the opportunity to identify genes that contribute to naturally occurring variation in quantitative traits. However, GWA relies exclusively on statistical association, so functional validation is necessary to make strong claims about gene function. We used a combination of gene-disruption platforms (Tnt1 retrotransposons, hairpin RNA-interference constructs, and CRISPR/Cas9 nucleases) together with randomized, well-replicated experiments to evaluate the function of genes that an earlier GWA study in Medicago truncatula had identified as candidates contributing to variation in the symbiosis between legumes and rhizobia. We evaluated ten candidate genes found in six clusters of strongly associated single nucleotide polymorphisms, selected on the basis of their strength of statistical association, proximity to annotated gene models, and root or nodule expression. We found statistically significant effects on nodule production for three candidate genes, each validated in two independent mutants. Annotated functions of these three genes suggest their contributions to quantitative variation in nodule production occur through processes not previously connected to nodulation, including phosphorous supply and salicylic acid-related defense response. These results demonstrate the utility of GWA combined with reverse mutagenesis technologies to discover and validate genes contributing to naturally occurring variation in quantitative traits. The results highlight the potential for GWA to complement forward genetics in identifying the genetic basis of ecologically and economically important traits.

全基因组关联（Genome-wide association, GWA）研究为鉴定参与数量性状自然变异的基因提供了重要契机。然而，全基因组关联研究仅依赖统计关联分析，因此若要对基因功能作出严谨论断，功能验证环节必不可少。本研究结合基因敲除平台（Tnt1反转录转座子、发夹RNA干扰载体与CRISPR/Cas9核酸酶）与随机化、高重复实验方案，对蒺藜苜蓿（Medicago truncatula）早前一项全基因组关联研究中鉴定出的候选基因功能进行评估——这些候选基因被认为参与豆科植物与根瘤菌共生过程的变异调控。本研究对6个强关联单核苷酸多态性（single nucleotide polymorphisms, SNP）簇中发现的10个候选基因开展评估，筛选依据包括其统计关联强度、与注释基因模型的间距以及在根系或根瘤中的表达情况。研究发现，3个候选基因对根瘤生成具有统计学意义上的显著影响，且每个基因均通过2个独立突变体完成了验证。上述3个基因的注释功能表明，它们通过此前未与结瘤过程相关联的通路调控根瘤生成的数量变异，相关通路包括磷素供应与水杨酸介导的防御反应。本研究结果证实，将全基因组关联研究与反向诱变技术相结合，可有效发现并验证参与数量性状自然变异的基因。研究结果同时凸显了全基因组关联研究作为正向遗传学补充手段的潜力，可用于解析具有生态与经济重要性的性状的遗传基础。