Fine mapping and candidate gene analysis of a novel fertility restorer gene for C-type cytoplasmic male sterility in maize (Zea mays L.).

A novel strong fertility restorer gene Rf12 for C-type cytoplasmic male sterility of maize was finely mapped on chromosome 2. Its best candidate gene Zm00001d007531 is predicted to encode a p-type PPR protein. The lack of strong restorer gene of maize CMS-C greatly limits its application in hybrid seed production. Therefore, the cloning of maize CMS-C novel strong restorer genes is necessary. In this study, a strong restorer line ZH91 for maize CMS-C was found, and the novel restorer gene named Rf12 in ZH91 had been mapped in a 146 kb physical interval on maize chromosome 2. Using the third-generation high-throughput sequencing (ONT), the whole genome sequence of ZH91 was got, and with integrating the annotation information of the reference genome B73_RefGen_v4 and B73_RefGen_v5, four candidate genes were predicted in ZH91 within the mapping region. Then using gene cloning, stranded specific RNA sequencing, qRT-PCR analysis and subcellular localization, Zm00001d007531 was identified as the most likely candidate gene of Rf12. Zm00001d007531 encodes a p-type PPR protein with 19 PPR motifs and targets mitochondria and chloroplast. Stranded specific RNA sequencing and qRT-PCR results both show that the expression of Zm00001d007531 between anthers of near-isogenic lines C478 and C478 was significantly difference in pollen mother cell stage. And the result of sequence alignment for Zm00001d007531 gene in 60 materials showed that there are twelve SNPs in CDS region of Zm00001d007531 were tightly linked to the fertility. The finding of a novel strong restorer germplasm resource ZH91 for maize CMS-C can greatly promote the application of maize CMS-C line in maize hybrid seeds production, and the identification of candidate gene Zm00001d007531 can accelerate the backcrossing process of maize CMS-C strong restorer gene Rf12 to some extent.