A super PPR cluster for restoring fertility revealed by genetic mapping, homocap-seq and de novo assembly in cotton

Key message Rf candidate genes were related to the super D05_ PPR -cluster and verified to be individually nonfunctional. Abstract Restorer of fertility ( Rf ) genes of cytoplasmic male sterility (CMS) is commonly found to be PPR ( pentatricopeptide repeat ) genes, which are mostly located in a cluster of PPR genes with high similarity. Here, Homocap-seq was applied to analyze PPR clusters in ‘three lines,’ and we found broad variations within the D05_ PPR -cluster in a restorer line and deduced that the D05_ PPR -cluster was associated with fertility restoration. Genetic mapping of Rf and Homocap-seq analysis of three genotypes in the F 2 population validated that the D05_ PPR -cluster was the origin of Rf . Three Rf candidates were cloned that were the most actively expressed genes in the D05_ PPR -cluster in the restorer line as revealed by their high-depth amplicons. However, further transgenic experiments showed that none of the candidates could restore fertility of the CMS line independently. Then, the members of the brand-new super D05_ PPR -cluster in the restorer line, containing 14 full-length PPR s and at least 13 PPR homologous sequences, were identified by long-read resequencing, which validated the effectiveness of variation and expression prediction of Homocap-seq. Additionally, we found that several PPR duplications, including 2 of the 3 Rf candidates, had undergone site-specific selection as potentially important anther development-associated genes. Finally, we proposed that multiple PPR s were coordinately responsible for the fertility restoration of the CMS line.