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可能。然而，GWA研究仅依赖统计关联分析，因此若要对基因功能作出严谨确凿的论断，必须开展功能验证实验。我们结合使用基因敲除平台（涵盖Tnt1逆转座子、发夹RNA干扰载体与CRISPR/Cas9核酸酶），并配合随机化、高重复性的实验设计，对蒺藜苜蓿（Medicago truncatula）既往一项GWA研究中鉴定出的候选基因进行功能评估——这些候选基因被认为与豆科植物与根瘤菌的共生性状变异相关。我们基于统计关联强度、与注释基因模型的邻近性以及根或根瘤的表达特征，筛选出6个强关联单核苷酸多态性（single nucleotide polymorphisms）簇中的10个候选基因并开展评估。我们发现其中3个候选基因对结瘤产量具有统计学显著性影响，且每个基因均通过2个独立突变体得到验证。这3个基因的注释功能显示，它们通过此前未被报道与结瘤过程相关的途径参与了结瘤产量的数量变异调控，包括磷供应与水杨酸相关的防御反应。本研究结果证实，将GWA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。研究结果凸显了GWA作为正向遗传学补充手段，在解析具有生态与经济重要性性状的遗传基础方面的应用潜力。