Fine mapping and cloning of an intersubspecific reproductive isolation locus MSHB1 in rice

Reproductive isolation between rice subspecies poses a significant barrier to harnessing heterosis in hybrid breeding. Here, we identified and characterized a novel locus, designated Male Sterility-Related Hybrid Breakdown Locus on Chromosome 1 ( MSHB1 ), through the utilization of chromosome segment substitution lines (CSSLs) derived from crosses between an indica cultivar Huanghuazhan (HHZ) and an aromatic cultivar Basmati Surkh 89 − 15 (BAS). A near-isogenic line, CS004, carrying the BAS-derived MSHB1 BAS allele, exhibits complete male sterility, reduced plant height, fewer panicles, and shorter panicle length. Fine mapping localized MSHB1 to a 28.583-kb interval on the short arm of chromosome 1. Genetic complementation confirmed that the MSHB1 allele from HHZ is sufficient to restore fertility. MSHB1 encodes a homolog of Esa1-associated factor 6 (EAF6), which regulates histone acetylation and transcriptional activity. Comparative genomic analyses revealed that functional divergence in promoter activity between duplicated OsEAF6 paralogs ( MSHB1 and MSHB12 ) drives hybrid breakdown. These findings elucidate a molecular mechanism underlying intersubspecific reproductive isolation and highlight MSHB1 as a strategic target for overcoming hybrid sterility to enhance heterosis in rice breeding.