Identification On Alleles Of Rice Blast Resistance Genes And Mapping And Cloning Of A New Resistance Gene To Rice Blast

Rice was one of the most important food crops in the world, there were many kinds of diseases in rice, rice blast was caused by Magnaporthe grisea which occurred widely in the world. Rice blast was considered as the main disease, it was able to produce serious yield loss when it appeared. The phenomenon which seeds couldn’t be gathered often occoured in mountainous area with the morning dew or hilly region that seriously infected the rice production and food security.Breeding practice had proved that cultivating varieties with lasting resistance was regarded as a primary measure to preventing and controlling rice blast. Identification on rice blast resistance genes in main rice cultivars would contribute to understand the distribution of resistance genes, which could guide breeding and cultivating of disease resistant rice varieties.1. Identification of alleles for rice blast resistance genes in 24 Rice Cultivars from Liaoning ProvinceIn this study,24 cultivars from Liaoning province were used to study the distribution of resistance genes and the function of their alleles. Primers were designed according to the conservative region sequence of 9 genes to amplify the coding region in 24 cultivars. The amplification results from 24 cultivars were compared, which suggested that the 24 cultivars from Liaoning province did not carry the Pi21、Pi36、Pi37、Pit and their resistant alleles. However, Pid2, Pid3, Pita and Piks/Piks/Pikm/Pikp existed in the 24 varieties with different genotypes and mutation frequencies. Among these resistance genes, the resistance alleles of Pid2 and Pid3 were detected in two cultivars. The alleles of Pita were detected in four cultivars and the new mutations did not change the founction of the Pita. The same sequences of Piks/Piks/Pikm/Pikp were not found in the 24 cultivars. After being inoculated with rice blast fungus carried AVR-Pik, Liaojing454 and Shennong265 were all resistant to the pathogens, which showed that the alleles carried by Liaojing454 and Shennong265 may serve resistance functions.19 Piz-t alleles were detected in 24 varieties, the inoculation results showed that most of the mutations caused the loss of the disease resistance function of the alleles.2. Mapping of new rice blast resistance gene Pi65(t) in variety Gangyu129Durable resistant varieties could be obtained through the aggregation with different types of resistance genes. Using the rice blast from Liaoning province,110 rice varieties materials form Liaoning province were conducted on the resistance spectrum analysis. Screening for variety that showed high resistance(Gangyu 129) and susceptible(Liaoxing 1) for two consecutive years in the field,, high resistance variety(Gangyu 129) did not contain any known resistance genes through genotype identification. Using variety Gangyu 129 and Liaoxing 1 to build DH population, through the SLAF-seq technology to map the candidate gene to 30.42-30.98M. The fine mapping experiment continued by recombinant screening, and the candidate genes were further mapped on positioning to 30.42-30.85M. There were 21 candidate genes in this area, RT-PCR result showed that there existed expression difference between variety Gangyuan8 and Liaoxingl in four of these genes. When variety Gangyu129 was inoculated by fungus, the expression level of these four genes increased. This result showed that these four genes may be related to the resistance. Thses genes were Os11g0691700, Os11g0691800, Os11g0691900, Os11g0692100, only gene Os11g0691700 were types of NBS-LRR. NBS-LRR was proved to be related to resistance, and most resistance genes were this type of genes. Therefore, gene Os11g0691700 was a new resistance gene that was mapped on variety Gangyu 129, named as Pi65(t).3. Cloning and vector constructing of resistance alleles and candidate gene Os11g0691700According to the result of genotype identification, Pi9 alleles carried by varieties Liaonong979 and Pita alleles carried by varieties Gangyuan8 are all resistance alleles. The Pi9 resistance alleles and Pita resistance alleles were cloned in two varieties respectively, and were connected to the expression vector; Using SLAF-seq technology, one candidate gene was found to be related to resistance. Cloning Pi65(t) gene and connecting to the expression vector.