Genetic Analysis And Gene Mapping Of A Rice Lesion Resembling Disease Mutant A344

Plant lesion resembling disease (LRD) is a kind of phenomenon that plant spontaneously generate necrotic lesions resembling disease symptoms without obvious adversity stress, physical injury or pathogen infection. A large number of studies have shown that Ird mutants are powerful tools to research plant PCD and the mechanisms of disease resistance response. Identification of the lrd mutants and cloning and function analysis of the mutant genes will provide important theoretical evidence to reveal the regulatory pathway of plant PCD and the mechanism of disease resistance response. So far, nearly 200 rice lrd mutants have been reported, most of the lrd mutants’character is controlled by a single recessive nuclear gene, a few of them are controlled by a single dominant nuclear gene or double recessive nuclear genes. According to statistics,76 Ird genes have been positioned on the rice chromosomes, of those 18 genes have been cloned.The experimental material of this study was lrd mutant a344, which was identified from R498 EMS-induced mutant library. The main results were as follows:1. The characteristic of a344:from the early tillering, a344 old leaves by leaf sheath to tip, the tender leaves from leaf tip to leaf base gradually spread reddish-brown spots. As the plant growth and development, the formation of brown spots became violently and those spots covered the whole ground part of the plant at last.2. Agronomic traits investigation and seed-test data analysis found that:compared with wild type (R498), the plant height and panicle length of a344 were decreased significantly, and the number of primary branch and secondary branch was significantly reduced, grain length、grain width、1000-grain weight and seed setting rate of a344 were also significantly reduced.3. The histochemical assay indicated that the lesions on a344 leaves were caused by PCD and hydrogen peroxide accumulated in a344 leaves. The Shading assay showed that the lesions on a344 leaves were induced by natural light.4. Determination of photosynthetic pigment during different period showed that since the beginning of tillering stage the mutant a344 formed lesions, the photosynthetic pigment content of a344 has decreased significantly compared with R498. And, at the full heading stage a344 photosynthetic rate was significantly lower than R498.5. By inoculating rice bacterial blight pathogen PXO99 found that the resistance of a344 was obviously improved compared with wild type R498. By inoculating the rice blast races that non-affinity to R498 during the seedling stage, a344 displayed susceptibility to rice blast race FJ089-1 and D7. The disease resistance mechanisms of a344 needs further research.6. Transmission electron microscope observation found that the shape of chloroplast was irregular in the a344 mesophyll cells, part of the internal structure of the chloroplast is very fuzzy, the number of starch grains was decreased, and there were a lot of osmium granules in the chloroplast. Those results indicated that the mutation of a344 lead to chloroplasts dysplasia.7. Genetic analysis suggested that the phenotype of a344 was controlled by a single recessive nuclear gene. Gene a344 was preliminarily mapped on the long arm of rice chromosome 4, between two SSR markers RM5478 and RM1112, genetic distances were 1.5 cM and 1.3 cM. By using Indel markers, gene a344 was physical delimitated to a 46-kb region, and there were 7 ORFs predicted in this region. So far, there was no LRD gene been cloned in this region, thus we speculated a344 was likely to be a new LRD gene in rice.8. Gene sequence alignment results showed that there was a base substitution (G->T) on the fifth exon of a candidate gene LOC_Os04g56480 in the mapping range, causing a amino acids changed (W->L). Coseparation test results showed that the mutant base was coseparated with the LRD phenotype, therefore, we speculated that the mutation in LOC_Os04g56480 was likely to be the reason lead to the mutant phenotype. The identification of the mutant a344 and fine mapping of the gene will provide powerful evidences to the cloning of the gene, gene function analysis and researching of the LRD formation mechanisms.