Genome-Wide Association Analyses Reveal the Genetic Basis of EMS Mutagenesis Efficiency in Rice

Ethyl methanesulfonate (EMS) mutagenesis is widely used to generate genetic diversity in crops, but its efficiency is strongly genotype-dependent, and which underlying mechanisms remain poorly understood. Here, a large-scale phenotypic analysis of 420 diverse accessions of rice (Oryza sativa L.) is performed, revealing extensive variation in EMS tolerance (survival rate) and mutagenesis efficiency (mutation frequency). The Aus subpopulation consistently outperformed others for both traits. A genome-wide association study (GWAS) identified a major locus, SRD7, linked to reduced survival rate under EMS stress. Within this locus, the Rc gene, a key regulator of proanthocyanidins biosynthesis, is identified as the candidate causal factor. Haplotype analysis showed that functional Rc alleles confer high EMS tolerance, a conclusion further validated using transgenic knockout lines. Using near-isogenic lines (NILs), it is confirmed that Rc not only improves seed survival after EMS treatment, but also unexpectedly increases genome-wide mutation frequency. Mechanistic studies demonstrated that Rc enhances the antioxidant capacity of seeds by elevating CAT, SOD, and POD activities, while reducing H₂O₂ accumulation, thereby alleviating EMS-induced oxidative damage. The findings establish Rc as a pleiotropic regulator that enhances EMS mutagenesis efficiency, providing a feasible strategy for accelerating the development of improved rice germplasm.