Fine mapping and candidate gene analysis of Rf21(t) for the fertility restoration of Chinsurah Boro II-type cytoplasmic male sterile Oryza sativa (ssp. japonica) lines

Abstract The combination of Chinsurah Boro II (BT)-type cytoplasmic male sterility (CMS) and Rf1, the main fertility restorer gene (Rf) for CMS-BT, has been extensively utilized for the production of three-line commercial japonica hybrid seeds. The identification of new Rf genes holds significance for the breeding BT-type restorer lines, aiming to enhance the heterosis level of BT-type japonica hybrids. In this present study, ‘02428’, a wide-compatibility japonica variety, was observed to partially restore fertility to BT-type CMS lines. Genetic analysis revealed that ‘02428’ carries a dominant Rf gene, Rf21(t), responsible for the fertility restoration of BT-type CMS lines. Leveraging BSA resequencing technology and molecular markers, the Rf21(t) locus was identified, and localized within a candidate interval of 6-12.5Mb on chromosome 2. Using the iso-cytoplasmic restorer populations, Rf21(t) was ultimately mapped to an interval of approximately 77 kb, encompassing 12 predicted genes, including LOC_Os02g17360, encoding a PPR domain-containing protein and LOC_Os02g17380 (Rf2), a cloned Rf for Lead rice-type CMS. Comparative sequence analysis, gene expression profiling, and gene knockout experiments confirmed LOC_Os02g17360 and LOC_Os02g17380 as the most likely candidates of Rf21(t). Furthermore, Rf21(t) showed the dosage effect on the fertility restoration of BT-type CMS lines. This newly identified Rf21(t) represents a valuable genetic resource for the breeding of BT-type japonica restorer lines. Our findings offer practical insights for breeders interested in advancing BT-type japonica hybrid development.