Characterization, Genetic Analysis And Gene Mapping Of A Novel Rice Spotted-Leaf Mutant HM143with Broad-spectrum Resistance To Xanthomonas Oryzae Pv. Oryzae

A spotted-leaf mutant of rice HM143was isolated from an EMS-induced IR64mutant bank. Themutant has been selfed for nine generations and the spotted-leaf trait has been stably maintained indifferent environmental conditions. In this study, we present the results of phenotyping, measurement ofphotosynthetic pigment content, histo-chemical analysis, disease evalution of bacterial blight resistanceand blast resistance, expression analysis of defense-related genes, genetic analysis and gene mapping.The results are as follows:1. Irregular brown spots/lesions were observed on the mutant leaves approximately3weeks aftersowing under natural summer field conditions both in Hainan and Hangzhou, China. The lesionsthen spread downwards along the leaf ribs or leaf margins and could reach the leaf sheaths in someplants. The mutant HM143belongs to a propagation type of lesions during the whole growthperiod.1000-grain weight and seed-setting rate were significantly different between the mutant andthe wild type while no obvious differences were found on plant height, panicle length, number ofpanicles, number of filled grain per plant, total number of grain per plant and number of filledgrain per panicle.2. The wild type leaves and the mutant leaves without lesions were clear without dark blue stain aftertrypan-blue staining while the mutant leaves with lesions exhibited dark blue stains in the lesionarea, indicating the presence of cell death or irreversible membrane damage at the lesion sites.HM143leaves with apparent lesions and the wild type IR64were stained with DAB, ahistochemical indicator of the generation and accumulation of reactive oxygen species （ROS） incells. The results shows that no accumulation of H₂O₂in the wild-type but apparent red brownstaining were observed in the sites of necrosis in the mutant. It indicate that the accumulation ofH₂O₂may be responsible for the cell death and produce of the spot.3. No matter in tillering stage or in the heading stage, the contents of photosynthetic pigmentsincluding chlorophyll a, chlorophyll b and carotenoid were no obvious differences between wildtype IR64and mutant HM143without lesions，while significantly decrease in mutant HM143withlesions.4. To know whether the reaction patterns to bacterial blight pathogens are changed in the mutant wecarried out inoculation using15races of Xoo. The results showed that the mutant was resistant toall15races ranging from moderate resistant （MR） to highly resistant （HR） while the wild type wasresistant to5races （4MR,1HR） and susceptible to10races （6HS,4MS）. The control wassusceptible to all15races （5MS,10HS）. Statistical analysis showed that resistance to13out of15Xoo races was significantly enhanced in the mutant in comparison to the wild type. In general, ourresults indicated that the mutant conferred a broad-spectrum resistance to Xoo. To evaluatewhether the lesion mimic was co-segregated with bacterial blight resistance.35F₂individuals withlesions and72F₂individuals with normal green leaves derived from the cross HM143/IR64were inoculated using race HB-17. The results showed that lesion mimic plants were much moreresistant than those of normal leaf plants.35lesion mimic plants showed a lesion length rangingfrom1.5-3.7cm with an average lesion length of2.5±0.8cm, while the72normal leaf plantshad a lesion length ranged from2.7-6.8cm with an average lesion length of4.0±1.2cm whichwas significantly different from that of lesion mimic plants at the level of P=0.01. The resultclearly indicated that the lesion mimic trait was co-segregated with bacterial blight resistance.5. To examine the level of expression of pathogenesis-related genes in the mutant and wild type.Semi-quantitative RT-PCR was carried out for the analysis of POX22.3, PAL, POC1, PBZ1andPR1. The results indicated that the expression level of PAL, POC1and PBZ1was apparentlyenhanced in the mutant than in the wild type, while the expression level of POX22.3and PR1wereno obvious differences between the mutant and the wild type. To PAL and PBZ1, the expressionlevels were enhanced in the mutant with lesions than in the mutant without lesions.6. To determine the genetic control of the spotted-leaf trait, F1plants, F₂individuals and F3lines wereanalyzed. The results showed that the mutation was controlled by a single recessive gene.1023spotted-leaf F₂individuals derived from HM143/R9308were used for mapping. Finally themutation was delimited to a region with a physical distance of52kb on the long arm ofchromosome4, tentatively named spl^HM143. SplHM143is likely a novel rice spotted-leaf gene since noother similar genes have been identified near the chromosomal region.