Characterization, Genetic Analysis And Gene Mapping Of A Light Brown Spotted Leaf Mutant In Rice

Rice spotted-leaf mutants are important and ideal resources in the elucidation of mechanismsunderlying disease resistance and might be useful for broadening the levels of disease resistance in ricebreeding programs. In previous studies, we have isolated a rice spotted-leaf mutant from anEMS-induced IR64mutant bank. After more than nine generations of selfing, the spotted-leaf trait isable to be inherited stably under different environmental conditions. In present study, we carried outinvestigations on the performance of agronomic traits, measurement of photosynthetic pigment content,histo-chemical analysis, disease evaluation of bacterial blight resistance and blast resistance, expressionanalysis of defense-related genes, genetic analysis and gene mapping. The results are as follows:1. The lesions of the mutant appeared on the leaf tips approximately10-14days after sowing andgradually spread downwards to cover the whole leaves. The symptom lasted to the whole lifegrowth duration and belonged to propagation type of mutant. The mutant also showed significantlyshorter plant height, shorter panicle length, lesser number of filled/panicle, lower rate of theseed-setting, smaller of the thousand grain weight than those of the wild type IR64. The headingdate of the mutant was about5days later than that of IR64. The reason for the dwarfism of themutant was due to the shortened fourth internode and fifth internode.2. In summer field conditions, no lesions were observed on the portion of leaves covered with a pieceof aluminum foil with1-2cm length. Furthermore, the lesions appeared once the aluminum foilswere removed. The results indicated that the initiation of the lesions was induced by naturalsunlight.3. The mutant leaf with lesions was stained by the trypan blue incontrast to the wild type IR64.Similarly, the mutant leaf with lesions was stained by diamio benzidine (DAB) while the wild typewas not stained. The results showed that cell death or membrane damage was detected withhydrogen peroxide (H2O2) accumulation in the mutant, indicating the cell death was caused byreactive oxygen species.4. The contents of photosynthetic pigments including chlorophyll a, chlorophyll b and carotenoidswere all apparently reduced compared with the wild type. Cell death detected in the mutant mightbe responsible for the decrease of the contents of photosynthetic pigments.5. Evaluation of bacterial blight resistance indicated that the mutant lbsl1was highly resistant(P=0.01) to race C6while the wild type IR64was susceptible. Resistance of the mutant to racePXO339was enhanced significantly but still in the range of susceptible. The resistance of themutant to race PXO112, PXO341and OS225were enhanced (P=0.05) at the level of moderateresistant as the wild type IR64which was moderate susceptible. And the resistance of the mutant torace C3was enhanced (P=0.05) at the level of highly resistant as the wild type IR64which showedmoderate resistant. As the resistance of the mutant to race ZH173was enhanced (P=0.05) at thelevel of moderate susceptible as the wild type IR64which was highly susceptible. No significancedifference was observed between the mutant and IR64when inoculated with races PXO61, PXO71, PXO79, PXO86, PXO99, PXO145, PXO280and JS97-2. Interestingly, the resistance of the mutantto race C5was significantly weakened at the level of moderate resistant in contrast to the wild typeIR64which was highly resistant. For blast resistance,8Chinese blast isolates CH131, CH15,CH72, CH102, CH188, CH199, js001-20-1-1and TH16were used for inoculation. The resultsshowed that blast resistance of the mutant to all the isolates except CH131was enhanced.6. 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 PR1, PBZ1, PAL, PO-C1andPOX22.3in the leaves with and without lesions. Our results indicated that the expression level ofPBZ1, PAL and PR1was apparently enhanced in the mutant than in the wild type. And to PAL, theexpression level was enhanced apparently in the leaves with lesions. But the expression levels ofPO-C1and POX22.3was lower than those of the wild type. The results indicated that differentdefense pathways might be involved in the reaction of the mutant to blast and bacterial blightpathogens.7. With the observation of F1, genetic analysis of F2and testimony of F3, we can conclude thespotted-leaf phenotype was controlled by a single recessive gene.780F2individuals with lesionsfrom the cross lbsl1/Morobereken was used for gene mapping. The spotted-leaf gene, tentativelynamed lbsl1(t)，was located in the interval of41kb between markers FBH7and FBH21on theshort arm of chromosome6. It is likely a new gene since no other spotted-leaf genes have beenidentified in this region.