Blast Resistant And Susceptible Rice Identification Technique System Development,Integration And Application

The rice blast disease is one of the major diseases that limit the high yield of rice.Due to the variability of the rice blast fungus,the disease-resistance of rice is usually lost the disease resistance in a short period,resulting in abandonment in argriculture practice.Therefore,the fast and effective screening and utilization of high blast-resistant varieties has always been hotspot of research worldwide.Among which,molecular identification of rice blast resistant genes is the important aspects of auxiliary selection of blast resistant rice materials.In view of the insufficiency that,to date the molecular identification of rice blast resistant genes mainly uses DNA as a template to qualitatively detect the presence or absence of resistance genes,can't identify the resistance gene expression level in rice during blast infection.In this study,the major resistance genes in rice varieties in Jiangsu Province,which has been cloned,was selected as the target genes.A system including integration of the gene identification related to blast the resistance,detection of blast resistance gene expression and the identification of pathogen inoculation was established.The results are as follows:1.Cultivation of Magnaporthe grisea and comparison of inoculation methodsIn this study,the preserved rice blast fungus was first cultivated to obtain rice blast fungus spore liquid.When the rice was cultured to 3-leavies 1 bud,inoculation was carried out by spraying,wounding,and induction.The results showed that wounding caused greater damage to rice and lead to rice wither and affected the test result.Based on this results,we used spraying to inoculate blast in seedling.2.Development,integration and application of resistance gene identification methodsIn this study,a multiplex PCR system for the simultaneous detection of rice blast resistance genes Pikh and Pil was developed,and a multiplex PCR system for simultaneous detection of rice blast resistance gene Pita and Pib was integrated.Because Pikm is composed of two closely-linked NBS-LRR genes with independent functions,a third method was established to detect Pikm gene including Pikm1-TS and Pikm2-TS.Using these three test systems,the resistance genes Pita,Pib,Pikh,Pil,and Pikm were detected in 114 hybrid rice materials selected and bred by Jiangsu Runyang Seed Co.,Ltd,to determine the distribution of rice blast resistance genes in these rice lines.The study results showed that among 114 lines 81 had rice blast resistance genes Pita,106 lines had Pib,35 lines had Pikh,13 lines had Pikm,and only 3 lines had Pi1,and They accounted for 71%,92.9%,30.7%,11.4%,and 2.6%of the tested materials,respectively.These results indicated that three rice blast resistance genes including Pib,Pita,and Pikh,are widely present in these hybrids rice materials.Why such high frenquency of blast-resistant genes pesents in these materials?That the breeding target of this company focuses on blast resistance rice might be the most reasonable answer.3.Inoculation of Magnaporthe grisea to identify panicle blast resistanceThe field rice was inocated with rice blast fungus and the infecting rate was investigated after 7 days of inocation.Artificial inocation of blast in rice ear was conducted.The identification results of panicle blast inocation showed that:among these 114 materials,2 lines manifested highly resistance,27 lines manifested resistance,32 lines manifested moderate resistance,33 lines showed susceptible,20 lines showed highly susceptible.They accounted for 1.7%,23.7%,28.1%,28.9%,and 17.5%of the total number of identifications,respectively.The correlation analysis between blast-resistant genes and panicle blast resistance showed that Pita and Pikh genes showed significant positive correlation(P<0.05),and the correlation coefficients were 0.297 and 0.239,respectively.Pib,Pil and Pikm genes showed no significant correlation with the resistance of panicle blast.4.Evaluation of rice blast resistance gene expressionIn this study,the semi-quantitative and convenient detection technology for the expression level of rice blast resistance genes was developed to meet with the need of most breeding units which usually have no fluorescent quantitative PCR instruments.Using Ubq5 as an internal reference gene,Pib,Pita,and Pikh were selected as resistance genes for detecting.In the 11 materials sampled,although the Pita was constitutively expressed,its expression was still induced by Magnaporthe grisea in the resistant material.Pikh and Pib genes were inducible expression.Pikh and Pib genes were up-regulated in rice blast resistance materials after inoculation of rice blast fungus,while Pikh and Pib genes were down-regulated in rice blast disease materials.In summary,the expression of the rice blast resistance genes Pib,Pita and Pikh in rice blast resistance materials was induced by the inoculation of rice blast fungus,but this expression induction was not obvious in the susceptible rice materials,even the expression of Pikh and Pib gene were down-regulated.This aspect shows that the rice blast resistance gene expression level can be used to evaluate the blast resistance of rice varieties,and on the other hand,present an rational interpretation why some rice including blast-resistant genes show non-resistance.5.Establishment of prediction method for the infestion by rice blastBased on the achivement of transcriptomics,Barwin gene was selected as a marker for rice blast infection.The semi-quantitative RT-PCR assay method was developed,and the results showed that,in both blast-resistant and blast-susceptible materials,the Barwin genes expression leavel all increased gradually with the prolongation of rice blast fungus infection.Upon these results,it can be speculated that the Barwin gene expression level can be use to predict the infection of rice blast.6.Establishment of marker genes identification method in rice blast susceptible materialsBased on the results of transcriptomic,two candidate marker genes,chitinase 3 and glucan endo-?-1,3-glucosidase,were selected to identify potential susceptible rice material.To elucidate the gene expression profile of these two genes,we designed primers and established a semi-quantitative RT-PCR detection method to detect the expression of these two genes.The test results showed that the expression of these two genes was significantly induced by the rice blast fungus in the susceptible rice material,and no expression or low expression level was found in the disease-resistant material.Same as we expected,these two genes can be act as the marker genes to identify the susceptible material under inocation with rice blast fungus.