Development And Application Of The Codominant Markers For The Avirulence Gene AVR-Pik In Rice Blast Vathogenmagnaporthe Oryzae

The rice blast is one of the three major diseases of rice in China,ithas brought severe damage in the output of rice cultivationin our country.Breeding and cultivation of improved resistant varieties is the most economical and effectiveapproach for controlling rice blast.At present,with the help ofmolecular marker technology,we have genetically analyzed rice blast at the level of DNA deeply,the analysis provided important theoretical basis for breeding enduringnew rice varieties with wide resistance.With the development of DNA molecular markers,MAS is widely applied to the research of avirulence genes,and it has become a fast way to identify avirulence genes correctly.In recent years,the excavation and identification of the SNP of avirulence gene,have laid the foundation of DNA molecular markers which based on the SNP of AVR genes.We have found five alleles of avirulence geneAVR-Pik for now(AVR-Pik-A?B?C?D and E)(Yoshida et al.,2009;Kanzaki et al.,2012).This research designed three codominant markersCM-136?CM-200 and CM-234,using the SNP differences(SNP136?SNP139?SNP143?SNP200andSNP234)between the five alleles of AVR-Pik.Five alleles of avirulence geneAVR-Pikcan be divided into three classes by CM-136,they are AVR-Pik-A/B,AVR-Pik-D,AVR-Pik-C/E.AVR-Pik-Cand AVR-Pik-E can be distinguished by CM-200.In the last step,we use CM-234 to distinguish A VR-Pik-Aand A VR-Pik-B.We screened and identified a total of 348M.oryzaeisolate in a particular group using codominant markers,the results revealed that 269 isolatesmay contain A VR-Pik genes,and sixteen of the 348M.oryzaeisolates may contain no one.Then we select five isolates that habor known alleles and put the DNA into PCR reaction,the AGE reveals that alleles in five isolates are consistent with the sequencing detection.Next,we randomly select twenty-four isolates that may habor avirulence geneAVR-Pik,further sequencing detection reveals that the sequences of avirulence geneAVR-Pik in twenty-four avirulence geneAVR-Pik are consistent with the CDS of five alleles of AVR-Pik.Thus we verify the effectiveness of codominant markers.There are sixteen isolates that are not detected avirulence geneAVR-Pik.Three pairs of primers(D1,D2 and D3)that designed based on the sequence of avirulence geneAVR-Pik-D,are used to identify sixteen isolates and one control group isolate called HLJ9-1,the result indicate that these sixteen isolates may not habor avirulence geneAVR-Pik.The identification result of using codominat markes to detect 348 M.oryzaeisolateis important to understand the composition,distribution and occurance frequency of avirulence genes of the fungal flora.The detection results of 269 isolates from seven provinces and identification of strains reveal that the ancestral allele AVR-Pik-Dis the most widely distributed AVR-Pikalleles,it is found in all provinces and IS,the OF of AVR-Pik-D is 50.93%;AVR-Pik-E,developed from AVR-Pik-D,is the second most widely distributed allele,after AVR-Pik-D,it occurs in all sampling provinces except Heilongjiang,Anhhui and Fujian,the OF of AVR-Pik-E is 19.33%.A VR-Pik subsequently evolve A VR-Pik-A and-C,theOF of these two alleles are significantly less thanAVR-Pik-DandAVR-Pik-E;the OF respectively are 7.81%and 1.86%.AVR-Pik-B,whose OF is 20.07%,is the last allele of AVR-Pik,andAVR-Pik-Boccurs only in Hubei,Heilongjiang and IS.AVR-Pik-D can be recognized byPik,Pikp,Pikm,Piks and Pikh;AVR-Pik-E can be recognized by recognized byPik,Pikp and Piks;AVR-Pik-A can be recognized by recognized byPikm;AVR-Pik-C can not be recognized by any alleles of Pit;as for AVR-Pik-B,whether it has any function of avirulence genes remains undiscovered.Using ten resistant materials and 138 isolates that have been identified to do biopsy,AVR-Pik-A,-B,-C,-Dand-Ecan be recognized by different Pik alleles(Pik,Pikp,Pikm,Piks and Pikh),the results indicate that those isolates that habor different AVR-Pikalleles have diffenent pathogenic frequency onto rice materials who have different Pik alleles.The fewer resistant genes are identified,the higher the pathogenic frequency is.For example,the number of Pik alleles that can recognize AVR-Pik-Ais less than AVR-Pik-E,the pathogenic frequency of AVR-Pik-Ais 57.30%,as for AVR-Pik-E,thepathogenic frequency is 40.28%.This research provides basis for the layout of resistant varieties and the selection of resistance genes.