Map-based cloning reveals the complex organization of the BnRf locus and leads to the identification of BnRf(b), a male sterility gene, in Brassica napus.

KEY MESSAGE: Sequencing of BAC clones reveals the complex organization of the BnRf locus and allowed us to clone BnRf (b) , which encodes a nucleus-localized chimeric protein BnaA7.mtHSP70-1-like. The male sterility in an extensively used genic male sterility (GMS) line (9012A) in Brassica napus was regarded to be conferred by BnMs3/Bnms3 and the multiallelic BnRf locus including three alleles. We previously mapped BnRf to a 13.8 kb DNA fragment on the B. napus chromosome A7. In the present study, we isolated bacterial artificial chromosome clones individually covering the restorer allele BnRf (a) and the male-sterile allele BnRf (b) , and revealed that the candidate regions of BnRf (a) and BnRf (b) show complex structural variations relative to the maintainer allele BnRf (c). By analyzing the recombination events and the newly developed markers, we delimited BnRf (a) to a 35.9 kb DNA fragment that contained seven predicted open-reading frames (ORFs). However, genetic transformation of the ORF G14 from both the male-sterile and restorer lines into wild-type Arabidopsis plants led to a stable male-sterile phenotype matching a 9012A-derived GMS line (RG206A); moreover, the male sterility caused by G14 could be fully recovered by the restorer gene BnMs3. These facts indicate that BnRf (b) corresponds to G14 while BnRf (a) likely associates with another flanking ORF. G14 encodes a nucleus-localized chimeric protein designated as BnaA7.mtHSP70-1-like. Ectopic expression of G14 in Arabidopsis negatively regulates some vital genes responsible for tapetum degeneration, and delayed programmed cell death of tapetum and led to the developmental arrest of tetrads. Our work not only presents new insights on the hereditary model of sterility control but also lays a solid foundation for dissecting the molecular basis underlying male sterility and restoration in 9012A.