Identification And Gene Fine Mapping Of A Spotted Leaf Mutant W1764 In Rice

Spotted-leaf mutant, also called lesion mimic mutant, is a kind of mutants that can spontaneously form necrotic spots on leaves without pathogen invasion, abiotic or biotic stress. The spots are similar to lesions which caused by hypersensitive response (HR) of infection of pathogen. The studies of spotted-leaf mutant help to reveal to mechanisms of the plant resistance to pathogens and deeply understand the mechanisms of programmed cell death. In this study, the spotted-leaf mutant W1764 was obtained from a transgenic offspring of the indica cultivar "Minghui 86"(Oryza sativa.). We carried out the investigation on phenotypic traits, histochemical analysis, expression analysis of PR and genetic analysis. The main results are as follows:1. Period and characteristics of performance of mutant spots:The brown spots of mutant first appeared on the apex of basal leaf at later tillering stage, and gradually spread to the whole leaf during the growth period of plants. Compared with wide-type (WT), the plant height and seed-setting of mutant showed no obvious difference, but 1000-grain weight of mutant decreased significantly.2. The conditions of performance of mutant spots:In the field, we covered the different parts of new leaves and old leaves with and without spots of mutant using the aluminum foil. The result showed that no spots were observed on the shaded parts of leaves while unshaded parts were covered by brown spots. When aluminum foil was removed, new spots formed in the shaded area of leaves after 4 days. The results demonstrated that the formation of spots was induced by natural sunlight.3. Histochemical analysis:Leaves from W1764 with spots were stained by typan blue in contrast to WT. Leaves from W1764 with spots were stained by diamio benzidine (DAB) but not in WT. The results indicated that the cell death is association with the accumulation of H2O2 at and around spots in mutant, which may induce HR and the formation of necrosis spots.4. Expression analysis of pathogenesis related genes:To detect the expression levels of PR genes in WT and mutant, semi-quantitative PCR(RT-PCR) was carried out for the expression analysis of GF14e, PO-C1, PBZ1,OSNAC4, Cht3, Chtl, PAL and PB1b. Compared with the wild type, the expression levels of PO-C1, PBZ1, OSNAC4, Cht3 and Chtl were significantly increased in the mutant, no obvious differential expression levels of GF14e and PAL were observed, and PB1b was not expressed between mutant and WT. The results revealed that the expression level of some PR genes, such as PO-C1, PBZ1, OSNAC4, Cht3 and Chtl, was activated and increased in the mutant during the formation of necrosis spots.5. Genetic analysis and mapping:According to genetic analysis results, the mutant phenotype was controlled by a single recessive nuclear gene. The F2 population derived from the cross between W1764 and 9311 were used for genetic mapping. Finally,we located spotted-leaf gene on the long arm of chromosome 12 within 16 kb region through map-based cloning approach.