Map-based Cloning Of Two Male Sterility Genes In Rice

In flowering plants, male reproductive development is a complex biological process, while the mechanism underlying plant male reproductive development remains less understood. Here we identified a no pollen male sterile mutant, gamyb5, from the mutant library of 60Co-γ-treated indica cultivar Zhonghui8015(Zh8015). There was no significant difference in agronomic traits such as plant type, height and tiller number between gamyb5 mutant and wild-type. But gamyb5 showed slender and white anthers without mature pollen grains. Cross-sections of anther showed the microspore mother cells failed to form functional tetrads and microspores in gamyb5 mutant. Moreover, tapetal cells of gamyb5 became abnormally enlarged and the programmed cell death of tapetum was delayed. gamyb5 as the pollen acceptor was crossed with wild type Zh8015 and a japonica cultivar 02428 respectively. Genetic analysis of both hybridization populations indicated that gamyb5 was controlled by a single recessive nuclear gene which was mapped on the long arm of chromosome1. With developed SSR, Indel markers and F2 mapping population of gamyb5/02428, the gene was fine mapped to a region of 16-kb on the long arm of chromosome 1 between markers ZF-29 and ZF-31. Sequencing analysis of the two ORFs in this region revealed that the Os01g0812000, which encodes a gibberellin-induced MYB transcription factor, had an 8 nucleotide bases deletion in the second extron probably responsible for the male sterility phenotype. Additionally, real-time fluorescent quantitative PCR analysis showed that the expression level of the important regulators UDT1, TDR, CYP703A3 and CYP704B2 in anther decreased significantly in gamyb5. Together, these results suggested GAMYB played key roles in anther meiosis and tapetum programmed cell death.The cyp703a3-3 mutant also from the mutant library of 60Co-γ-treated indica cultivar Zhonghui8015(Zh8015) showed thinness and white anthers without mature pollen grains, behaving male sterility. Scanning electron microscopy(SEM) displayed that the cyp703a3-3 mutant anther epidermis shriveled compared with the wild type. Anther epicuticular ridges appeared as an abnormal three-dimensional knitting pattern and rarely detectable Ubish bodies, leaving only some unknown fragments on the inner surface of the anther locule. Cross-sections of anther showed the microspores failed to become round and vacuole in cyp703a3-3 mutant at stages10. While tapetal cells of cyp703a3-3 became abnormally enlarged and the tapetum did not undergo fitly PCD. Then microspore gradually degraded, resulting in empty anthers cavity without mature pollen grains. Genetic analysis and fine mapping demonstrated the cyp703a3-3 male sterile trait was controlled by a single recessive nuclear gene. The gene was finally mapped to a region of 47.78-kb on the chromosome 8 between markers S15-29 and S15-30. Sequencing analysis manifested 3 nucleotide bases deletion in the first extron of CYP703A3, leading to frame shift, probably caused the male sterility phenotype. Additionally, genetic complementation validated cyp703a3-3 mutant phenotype was restored by wild-type CYP703A3. In conclusion, the CYP703A3 played important role in controlling rice anthers and pollen wall development.