Denis Filloux

Positional cloning of the rice male sterility gene ms-IR36, widely used in the inter-crossing phase of recurrent selection schemes

The monogenetic recessive male-sterile gene ms-IR36 is widely used to facilitate the inter-crossing phase of recurrent selection in rice (Oryza sativa), but its segregation within the progeny disturbs other breeding phases. Marker-assisted early identification of msms and Msms seedlings would help overcome this drawback. Using successively bulked segregant analysis and large F2 populations, we mapped the ms-IR36 gene to a 33-kb region on the short arm of chromosome 2 that includes 10 candidate genes. Sequencing of these candidates together with checking rice genome annotations and expression databases allowed the target to be narrowed down to one candidate gene already isolated and characterized as the tapetum degeneration retardation (TDR) gene and reported to be involved in tapetal programmed cell death. Comparison of the sequence of the TDR gene between male-sterile (MS) and male-fertile (MF) IR36 plants detected one non-synonymous nucleotide substitution affecting the active domain of the encoded protein. Perfect co-segregation was observed between polymorphism at this nucleotide (SNP) and the MS/MF phenotype of 946 F2 plants. Spatial modelling of the active domain of the candidate protein reinforced the candidate status of the only SNP identified. Histological characterization of anther development in MS IR36 revealed defects identical to the ones observed in mutants used for the isolation and characterization of the TDR gene: delayed/non-degradation of tapetum tissue and collapse of the haploid microspores. We concluded that ms-IR36 corresponded to the TDR gene with a different mutation from the earlier one described in the same gene. No significant linkage drag was associated with ms-IR36. A SNP-based marker that enables simple early identification of MS plants and MF plants with the Msms genotype was designed.