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

• Published in: Theoretical and applied genetics 2022-02, Vol.135 (2), p.637-652
• Main Authors: Gao, Bin, Ren, Gaofeng, Wen, Tianwang, Li, Haiping, Zhang, Xianlong, Lin, Zhongxu
• Format: Article
• Language: English
• Subjects: Agriculture; Binding proteins; Biochemistry; Biomedical and Life Sciences; Biotechnology; Candidates; Chromosome Mapping; Cytoplasm; Cytoplasmic male sterility; Fertility; Fertility - genetics; Gene mapping; Genetic analysis; Genetic aspects; Genotypes; Gossypium; Life Sciences; Male sterility; Methods; Original Article; Plant Biochemistry; Plant Breeding/Biotechnology; Plant Genetics and Genomics; Plant Infertility - genetics
• ISSN: 0040-5752; 1432-2242
• DOI: 10.1007/s00122-021-03990-0

Key message Rf candidate genes were related to the super D05_ PPR -cluster and verified to be individually nonfunctional. 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.