Genetic Analysis And Mapping Of Rice(Oryza Sativa L.) Mutant

A male sterility mutant was selected from a M2 population through mutagenizing rice cultivar 9522 with ⁶⁰Co y-ray, which was controlled by a single recessive nuclear locus. Observation of semi-thin section and SEM（scanning electron microscope） showed that the anther development of mutant was normal in the early stage, but abnormal at the end of meiosis with the shrinked microspore, broken germ pore and defective outer wall of anther. Consistently, chemical composition assay showed that the level of total cutin was dramatically reduced in this mutant. As compared with the wild type, almost all of the cutin monomers were significantly decreased in the mutant, especially the long chain fatty acids. Previous study has localized the mutant locus between the two molecular markers, OS409 and XY409-3 on chromosome 4 using map-based cloning, which contains 15 BAC clones. Whole genome resequencing was used to identify the mutant gene and we noted that there was a region which may be deleted in the mutant after alignment sequencing result with the wide type. Finally, we validated the large region deletion in the mutant by PCR and sequence analysis. To further investigate the reason responsible for the mutant phenotype, we identified three candidate genes, CYP87, HOTHEAD and CYP704 after screening the genes in the deletion region by gene expression pattern analysis, microarray analysis, gene co-expression analysis and sequence homology analysis. Further, we conducted complementation test for the candidate genes, and we have found that the CYP87 and HOTHEAD complementation constructs can not rescue the mutant phenotype. Meanwhile, we used the CRISPR-Cas system to knock out the three candidate genes respectively. So far, we have gained the cyp87 homozygous mutants. Interesting, the primary observation showed that the setting rate of cyp87 is decreasing compared with the wild type, which indicates that the CYP87 play a role in the rice anther development process.