Genetic Analysis And Fine Mapping Of A Lesion Mimic Mutant Spl34 In Rice (Oryza Sativa L.)

Disease lesion mimic mutants spontaneously generate necrotic lesions resembling to which formed in the hypersensitive response under the condition of no adversity stress or pathogen attack.Lesion forming is usually accompanied with programmed cell death,accumulation of reactive oxygen species,up-regulated expression of defense related genes and increased synthesis of signal molecules such as Salicylic acid,jasmonic acid and ethylene.Among the reported lesion mimic mutants,many of them with higher resistance to pathogenic bacteria,therefore the lesion mimic mutants are excellent materials to study programmed cell death and the mechanism of disease resistance in plants.In rice,lesion mimic mutants can provide abundant germplasm resources for human to breed resistant variety,since many of them demonstrated an enhanced resistance to rice blast and bacterial blight.In this study,a lesion mimic mutant spl34,exhibiting stable inheritance was screened from the progeny of indica restorer line Jinhui 10 with seeds treated by ethyl methane sulfonate（EMS）.In order to understand the phenot ypic characteristics of mutants,a series of researches such as identification of morphological phenotype,investigation of agronomic traits,determination of photosynthetic pigment contents,and analysis of histochemical and so on,were conducted systematically.F luorescence microscope was used to observe the fluorescence of chloroplasts.The lesion mimic mutant spl34 was crossed with Xinong 1A with normal phenotype,F1 and F2 were used for genetic analysis and gene mapping,and the main results are as follows.1.Morphological identificationBrown lesions were first observed on the leaf sheath of lower leaves of spl34 at the late tillering stage.Subsequently,brown lesions spread along midrib and then distributed on the entire leaf.At maturity stage,brown lesions spread to the whole leaves and leaves sheath of the plants.The shading assay showed that the formation of lesions on the mutant were induced by light.2.Agronomic traits investigationAt maturity stage,the agronomic traits of wild type and spl34 were investigated.The results showed that,compared with the wild type,both the plant height and the ear length of the spl34 were shorter,and reached a significant level respectively.Grain number per panicle,1000-grain weight and seed setting rate of spl34 were decreased at an extremely remarkable difference.3.Photosynthetic pigment content determinationThe photosynthetic pigments content determination of wild type and spl34 were conducted at heading stage.the results showed that,compared wit h wild type,the photosynthetic pigments content of flag leaves in spl34 were slightly increased but the interactions were not significant.But the photosynthetic pigments content of the second leaves and third leaves in spl34 were lower than that of the wild type at a remarkable or extremely remarkable difference.4.Chloroplasts observation under fluorescence microscopyThe leaf of wild type and spl34 were used to observation under fluorescence microscopy at heading stage and could find that,the red fluorescence of chloroplasts of mutant spl34 was weaker than that of wild type.In addition,the site had formed lesions also with weaker red fluorescence compared with the site had not appeared lesions in spl34.5.Histochemical analysisHistochemical analysis indicated that,there were programmed cell death and hydrogen peroxide accumulated occurred in the sites of lesions produced.6.Physiological and biochemical indexes determinationDetermination of the Physiological and biochemica l indexes of wild type and spl34 demonstrated that,compared with wild type,the activities of antioxidant enzymes such as CAT,POD and SOD were reduced in spl34,whereas the contents of reactive oxygen species（ROS）,such as H₂O₂ and O2-in spl34 were higher.However there was no significant difference in the content of ·OH between spl34 and the wild type.7.Rice blast resistance spectrum determination and resistance identificationRice blast resistance spectrum determination results showed that t he resistance frequency to ZA physiological group of spl34 was higher than that of the wild type,while the frequency of disease resistance to ZB physiological group was lower than that of the wild type.In additional,no difference was observed in the frequency of disease resistance to ZC、ZD、ZE and ZG physiological group between spl34 and the wild type.Results of resistance identification indicated that the disease index of seedling blast and leaf blast and the incidence rate of panicle neck blast of spl34 were slightly higher than that of the wild type,and the difference was not significant.8.Genetic analysis and gene mappingGenetic analysis suggested that the phenotype of spl34 was controlled by a single nuclear recessive gene and was mapped on chromosome 4 between In De l markers LR49 and LR52 with an interval of 200 kb.Sequencing analysis of the candidate genes in the target region revealed that a single base replacement（G3449T）at the 3449 position of the DNA sequence of candidate gene: LOC_Os04g56480,leading to a substitution from tryptophane to cysteine.The target gene spl34 was allelic to HM47.9.Expression of target genes and defense related genesThe q RT-PCR results suggest that,compared with the wild type,the expression level of spl34 gene in lesion mimic mutant was down-regulated.The relative expression levels of pathogenesis-related genes PR1 a,PR1b and NPR1 in the spl34 was significantly higher than that in the wild type,but the relative expression levels of other pathogenesis related genes and the b last resistance genes between spl34 and the wild type were roughly the same.