High-generation Rice Breeding Materials Resistance To Rice Blast Gene Composition Expression And Correlation Analysis With Resistance

Rice is the main food crop in most areas of China.Rice production is closely related to the lives of people.However,the growth of rice is often affected by rice blast.Once the rice blast outbreaks,it is difficult to control.In addition,due to the rice blast fungus easily mutation,the selected blast-resistant rice varieties quickly lose their resistance after a few years cultivation.Thus,the blast-resistant variety screening is an urgent task in rice cultivation.At present,most of the research is devoted to the identification of resistance to rice blast through assisted selection of rice blast resistance materials,and the analysis of the presence or absence of resistance genes and the composition of artificially cultivated rice blast resistance varieties with higher resistance and wider resistance spectrum.However,few studies have been conducted on whether or not the genes for resistance to rice blast are expressed at a low level after being infected by the rice blast fungus.In this study,ten cloned rice blast resistance genes that were mainly present in rice varieties in Jiangsu Province were used as experimental materials.The composition and expression level of rice blast resistance genes were detected and identified by PCR technique and real-time fluorescence quantitative PCR technique,respectively.Combined with the results of inoculation and identification of rice blast resistance against rice blast,the composition and expression profiles of rice blast resistance genes in 68 high-generation rice breeding materials provided by Jiangsu Runyang Seed Industry Co.,Ltd were investigated to elucidate the correlation between the resistance and the gene expression level.The main results are as follows:1.Identification of blast resistance genes in high-generation rice materialsUsing PCR amplification technology to detect the profile of 10 rice blast resistance genes in 68 high-generation rice materials,the results showed that 6 rice blast resistance genes were widely present in the high-generation rice materials selected by the company.The proportion of rice blast resistance genes in the tested rice materials was as follows:94.1%contained Pi-i gene;91.2%contained Pik-h gene;and those containing Pia gene,Pib gene,Pita gene,Piz-t gene accounted for 89.7%,72.1%,61.8%,61.8%,respectively,while the proportion that containing Pik-p,Pik-m,Pik,Pil was relatively low,accounting for 38.2%,29.4%,25.0%,and 16.2%,respectively.The number of materials containing 8-10 rice blast resistance genes is relatively small.The results of gene profile identification showed that the majority of 68 high-generation rice materials contained 5 or more rice blast resistance genes,accounting for 83.8%.Among them,5 blast resistance genes,6 blast resistance genes and 7 blast resistance genes accounted for 23.5%,26.5%,23.5%of all test materials,respectively.2.Development of double-gene simultaneous detection technology of rice blast resistance geneIn order to improve the blast resistance genes identification efficiency in rice and to reduce the identification cost,two rice blast resistance genes Pik-h and Pik with large differences in the gene length were selected to construct a detection system that could simultaneously detect two rice blast resistance genes in the same reaction system.The double-gene detection technology of Pik-h and Pik was successfully constructed for the first time in this study3.Resistance identification of high-generation rice material inoculated with blast fungusIn this study,the resistance to leaf blast and to ear neck blast in 68 rice materials was identified by the inoculation method of the rice blast fungus spore solution(leaf blast with wound spray method and ear neck blast with spike neck bag injection method).The identification results of resistance to leaf blast showed that among 68 rice materials,41 showed resistance to leaf blast while 27 were sensitive to leaf blast.Among them,1 showed high resistance,accounting for 1.47%of all tested materials;28 had resistance,accounting for 41.18%;12 had medium resistance,accounting for 17.65%;17 had moderate susceptibility accounting for 25.00%;5 were susceptible,accounting for 7.35%;and 5 were highly susceptible,accounting for 7.35%.The results of identification of resistance to panicle neck blast showed that among 68 tested rice,17 were resistant to panicle blast and 51 were susceptible to panicle blast.Among them,5 showed high resistance,accounting for 7.35%of the total materials tested;5 were resistant,accounting for 7.35%;7 were moderate resistance,accounting for 10.29%;21 had moderate susceptibility,accounting for 30.88%;16 were susceptible,accounting for 23.53%;14 were highly susceptible,accounting for 20.59%.By comparing the resistance to leaf blast and to ear neck blast,the identification results showed that the number of materials resistant to leaf blast in 68 tested rice materials was much higher than the number of materials resistant to ear blast.4.Correlation analysis between rice blast resistance gene composition and resistance in high generation rice materialsIn this study,it was found that,regardless of leaf blast or panicle neck blast,among 68 high-generation rice materials,the proportion of the number of rice blast resistance materials increased linearly with the number of rice blast resistance genes in the corresponding materials.The resistance of rice blast were higher with the higher number of rice blast resistance genes.The identification of 10 blast resistance genes in 68 high-generation rice materials and the correlation analysis of resistance to leaf blast and ear neck blast showed that:The rice blast resistance genes Pita,Pia,Pik-h,Pi-i were positively correlated with leaf blast and ear neck blast resistance(P<0.05).The correlation coefficients of the four genes against leaf blast resistance were0.522,0.364,0.420 and 0.322,respectively.The correlation coefficients of resistance to panicle neck blast were 0.410,0.398,0.449 and 0.428,respectively.The other 6 genes Pib,Pik,Pik-m,Pik-p,Pi1,Piz-t showed weak correlation with the resistance to leaf blast and ear-neck blast.5.Correlation analysis between rice blast resistance gene expression and resistance in high generation rice materialsIn this study,real-time fluorescence quantitative PCR technology was used to detect and compare the differences at the expression levels of the resistance to rice blast genes among the materials of resistance to leaf blast and ear blast.The results showed that the expression levels of Pia,Pib,Pi-i,Pik-h,Piz-t and Pik-p genes in leaf blast resistance and ear neck blast resistant materials were significantly up-regulated after inoculating with Magnaporthe grisea.However,the expression levels of rice blast resistance genes in leaf blast and neck blast materials were either no significant difference or significantly reduced in susceptible materials.This result indicated that the significant up-regulation of rice blast resistance gene expression was positively correlated with the rice resistance to blast disease,that is,rice blast resistance material could actively respond to the infection of rice blast fungus by up-regulating the expression of rice blast resistance gene.Therefore,molecular identification based on the expression level of rice blast resistance genes was an important aspect in evaluating the rice resistance to blast.