Fine-Mapping Of Rice Bacterial Blight Resistance Gene X349 And Map-Based Gene Cloning Of Two Mosaic Spot Lesion And Premature Leaf Senescence Mutants In Rice

Rice bacterial blight（BB）,caused by Xanthomonas oryzae pv.oryzae（Xoo）,is a devastating disease affecting the high yield of rice in China.Planting disease resistant varieties is an economic,effective and environment-friendly control method in management of BB.Therefore,it is very important to continuously identify and explore new resources of resistance genes and analyze their resistance mechanisms.In this study,we screened a new BB resistant mutant x349 and two lesion mimic mutants（msl-1 and msl-2）from the susceptible indica rice cultivar JG30 treated with EMS.The main research results on theses mutants are as follows:Firstly,we used 31 Xoo strains from China and abroad to test the BB resistance spectrum of the mutant x349 at maximum tillering stage,and found that x349 showed high resistance to all the tested strains,indicating that the mutant x349 is a broad-spectrum resistant material to bacterial blight.In this study,2290 individuals of F₂population from the cross between JG88（a BB susceptible japonica rice variety）and x349 were used to mapping the BB resistance of x349.by suing Indel molecular markers.The target gene was initially located on the long arm of rice chromosome 2 between molecular markers ID28 and ID37.We further developed new Indel molecular markers and found nine markers located at the end of chromosome 2 and closely linked to x349.According to analyses of the individual plants,markers ID45,ID80,ID84 and ID90 were located on the same side of the target gene,while markers ID48,ID51,ID62 and ID74 were located on the other side of the gene.At the same time,a co-segregated marker ID69 between ID90 and ID74 was identified.Based the position of each marker on chromosome 2,the resistance gene x349 was located between ID90 and ID74,and the physical distance was about 90.6kb.The candidate genes in this region were predicted,and two candidate genes were associated with disease resistance.Both of the lesion mimic mutants（LMMs）,msl-1 and msl-2,showed similar mosaic spot lesions at seedling stage,but they displayed different phenotypes along with development of the plants.At tillering stage,larger orange spots appeared on leaves of msl-2,while only small reddish-brown spots exhibit on leaves of msl-1.At heading stage,the msl-2 plants were completely dead,while the msl-1plants were still alive even if showed apparent premature senility.For both the mutants,the mosaic spot lesion formation was induced by light;DAB and trypan blue staining showed reactive oxygen species were enriched at the lesion sites of the mutants;SOD and CAT activities in the scavenging enzyme system were decreased compared with the wild type.In addition,degraded chloroplasts,decreased photosynthetic pigment content,down-regulated expression of genes associated with chloroplast synthesis/photosynthesis and up regulated expression of genes related to senescence were detected in the mutants,but the abnormality of msl-2 was more serious than that of msl-1 in general.We constructed the F₂mapping population by crossing 02428 with mutants msl-1 and msl-2respectively.The target gene was located between the molecular markers S1 and L4 on chromosome 12,and the physical distance was about 58 kb.Map-based cloning revealed that the lesion mimic and premature senescence traits of both the mutants were controlled by recessive mutated alleles of the SL（Sekiguchi lesion）gene,which encodes the CYP71P1 protein belonging to cytochrome P450monooxygenase family.The difference of mutation sites and mutation types（SNP caused single amino acid change and SNP-caused early termination of translation）led to the different phenotypes in severity between msl-1 and msl-2.Taken together,this work revealed that the CYP71P1 is involved in regulation of both premature senescence and cell death in rice,and its different mutation sites and mutation types could cause different phenotypes in terms of severity.