Fine mapping and combined omics analysis of CMS restorer gene Rfa in pepper (Capsicum annuum L.)

• Male sterile in the CMS line (1310A) is governed by a single recessive gene, with sterility occurring at the tetrad stage. • Using third-genome sequencing (CA59) + BSA-seq, a novel restorer locus ( Rfa ) was fine-mapped to a 311 kb region on chromosome 6, containing three clustered PPR genes as key candidates. • Transcriptome & metabolome integration showed that Rfa mutation disrupts sporopollenin and pollen exine biosynthesis, impairing pathways for phenylpropanoids, flavonoids, cutin, waxes, and saccharides, leading to defective pollen wall development and male sterility. Cytoplasmic male sterility (CMS) is a principal method for inducing heterosis in pepper ( Capsicum annuum L.) and effectively addresses the difficulty of manual emasculation, simplifies seed production, and improves seed purity . Most research on CMS restorer genes in pepper is based on second-generation genomes; therefore, further research is required to elucidate the molecular mechanisms. In this study, the CMS line 1310A and restorer line CA1403 were subjected to genetic analyses of fertility. The results revealed that sterility was controlled by a single recessive gene and anther abortion occurred in the tetrad stage. Using the third-generation genome CA59, combined with BSA-seq and map-based cloning, we identified and finely mapped a new restorer locus, Rfa , to a 311 kb physical segment on chromosome 6. Moreover, three PPR-coding genes in the form of a gene cluster were identified as candidate genes. Combined transcriptome and metabolome analyses showed that the Rfa mutation downregulated key genes for sporopollenin and pollen exine biosynthesis and then interfered with the synthesis and metabolism pathways of phenylpropanoids, cutin, waxes, flavonoids, and saccharides. This resulted in a lack of essential precursors and energy required for pollen exine and tapetum development, eventually leading to male sterility.