| Bacterial blight is one of the most serious rice diseases, causing great damage to rice production. Novel bacterial blight resistance cultivars play an important role in bacterial blight resistance breeding in rice.In this study, a large-scale screening was performed on a rice mutant population of 50,000 T-DNA insertion lines in the field. Plants at five-leaf stage were inoculated with Xanthomonas oryzae pv.oryzae (Xoo) P10 by cutting the leaf top with scissors and sprayed for at least four times. Some candidate bacterial blight resistance mutants were preliminarily screened from the population. Among them, two mutant lines were selected for further researches. At the same time, some other phenotype mutants were observed in the library. To make preparations for mapping the resistance genes in these mutants, high-resolution melting (HRM) was used to genotype the F2 population in rice. Major research results were summarized as follows:1. A total of 125 bacterial blight resistance mutants' M0 lines were screened from the T-DNA mutant library. Simultaneously, some other phenotype mutants were observed, such as leaf colour mutants, lesion mimic mutant, degenerated spikelet, changes of ear stem length, beared and beardless rice, rice glume mutant, dwarf and tall-culm mutant, leaf shape mutant.2. Resistance of 17 mutants' M1 progenies from the 125 lines was identified by the P10 strain. It shows that the M1 progenies of two mutants BBM14 and BBM66 were resistant to P10 strain stably and hereditably. However, no resistance separation was observed among the M1 progenies and transgene PCR detection shows that T-DNA insertion was homozygous, so further researches are needed to determine whether the resistance of BBM14 and BBM66 to the P10 strain was caused by T-DNA insertion was uncertain. The growth curves of the P10 strain in BBM14 and BBM66 leaves were analyzed, it shows that bacterial growth in the two mutants was suppressed compared to the parent zhonghuall, this confirmed the resistance of BBM14 and BBM66 to P10 strain further. Resistant spectrum showed that BBM14 was resistant to the strains P2 (PXO86), P3 (PXO79), P7 (PXO145), P8 (PXO280), and P10 (PXO124), and susceptible to the strains P4 (PXO71), P6 (PXO99). While BBM66 was resistant to the strains P2, P7, P8, P10 and susceptible to the strains P3, P4, P6. Comparing the resistance spectrum of BBM14 and BBM66 to the known bacterial blight resistance genes in this research, it was suggested that there were new bacterial blight resistance genes in the two mutants. The expression of some defense related genes were analyzed in the mutants after inoculated by P10, and it shows that OsPR1b was upregulated after P10 strain inoculation.3. HRM technique was employed for F2 population genotyping in rice and the HRM reaction conditions were optimizated to make preparations for mapping the resistance genes in BBM14 and BBM66 For optimizing the reaction conditions, Twenty-two F2 lines were selected from the cross between the highly resistant cultivar Y73 and the susceptible cultivar IR24. With the 5μl reaction system and normal PCR procedure, a total of 353 STS and SSR markers were used to screen the markers showing polymorphic HRM curves between Y73, IR24 and F1. A total of 103 STS and SSR markers showed polymorphic melting curves among the three parents. These 103 markers were further used to genotype the F2 population. It showed that 12 STS and SSR markers genotyped the mapping population successfully. And the HRM genotyping results were consistent with traditional electrophoresis results completely. To raise the efficiency of STS and SSR markers for HRM genotyping, another two HRM reaction conditions were applied for screening the 103 polymorphic markers. In a 5μ1 reaction system and touchdown-PCR procedure, no screening efficiency improvement was observed. However, in a 10μl reaction system and touchdown-PCR procedure,9 new markers genotyped the mapping population successfully except for the 12 markers, HRM marker screening efficiency increased greatly. Therefore,it is practical for applying HRM in F2 genotyping in rice. And among the three HRM reaction conditions, the 10μl reaction system and touchdown-PCR procedure was more stable and suitable for genotyping in rice.From the above results, it shows that BBM14 and BBM66 were resistant to part of the bacterial blight strains stably. Meanwhile, HRM was useful for F2 population genotyping in rice. So applying HRM for mapping the resistance gene in BBM14 and BBM66 can accelerate not only the gene identification but also the application of HRM genotyping in rice. |
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