Fine Mapping Of Wheat Powdery Mildew Resistance Gene Pm4 And Molecular Genetic Characterization Of The Powdery Mildew Resistance Gene In Dunhuachunmai

Powdery mildew,caused by Blumeria graminis f.sp.tritici(Bgt),is a devastating wheat foliar disease and causes severe yield losses in wheat production worldwide.Many powdery mildew resistance genes have been identified but due to host-pathogen co-evolution,the race-specific resistance of these genes in wheat cultivars can be quickly overcome by new virulent races of this pathogen.Thus,discovering and utilizing new sources of powdery mildew resistance is a constant task for wheat powdery mildew resistance breeding programs.Genetic and molecular characterization and the subsequent cloning of resistance genes are not only essential for effecctive molecular breeding but also important to understand the mechanism of resistance and evolution of resistance genes.Wheat powdery mildew resistance genes Pm4e and Pm4a were previously mapped to larger genetic intervals of 6.7 cM and 12.7 cM,respectively,at the multi R gene Pm4 locus on chromosome 2AL.In this study,Pm4e and Pm4a were delimited to a 0.19 cM and 0.14 cM genetic interval,respectively,flanked by Xwgrc763 and Xwgrc865,through employment of larger segregating populations,and enrichment of the genetic interval with markers developed based on Chinese Spring(C.S.)survey sequence.In this interval,Pm4e and Pm4a co-segregated with a few markers,some of which were either D29/Pm4a-NIL-dominant or Y 158-dominant,implying great sequence variations in the interval between D29/Pm4a-NIL and Y158.Most of these co-segregation markers couldn't differentiate the Pm4 alleles from each other.Survey of 55 wheat cultivars with four co-dominant markers showed that the Pm4e-cosegrgating loci always coexist.Annotation of the Pm4e interval corresponding C.S.sequence revealed more than a dozen resistance gene analogs clustered in a 2.4 Mb region,although C.S.is susceptible to the Pm4e-avirulent isolate Bgt2.This study has established the foundation for map-based cloning of Pm4e.Moreover,some of the co-dominant markers developed in this study could help in markers-assisted transfer of Pm4e into elite cultivars.Tetraploid wheat Khapli was reported to contain several powdery mildew resistance genes with Pm4a being the first identified and mapped.In order toaccelerate the map-based cloning process of the Pm14 locus,previously we evaluated the response of Khapli mutant libraries,generated either by fast neutron(FN)or ethylmethane sulfonate(EMS)treatment,to powdery mildew and found two FN and three EMS mutants showed susceptibility.In this study,these five mutants along with Pm4a-NILwere subjected to isolate Bgt2(avirulent on Khapli)at seedling stage and mixed isolates of Bgt at adult stage.The cross of Khapli with two FN mutants mft58 and mft59,respectively,produced resistant F1 hybrids and the segregation of their subsequent F2 progenies showed that mutations in a single dominant gene in Khapli might rendered these two mutants susceptible.Moreover,some of the Pm4a co-segregating markers could not amplify their respective bands from one mutant mft58,indicating that a large fragment deletion might happen at the Pm4 locus.The association of the phenotype with the presence or absence of target band in the F2 population further confirmed that the deletion of the fragment,which carried Pm4a,might cause susceptibility of mft58.The mutants generated in this study will be a useful source for identification and functional validation of candidate gene/genes at the Pm4 locus.Dunhuachunmai,an elite Chinese wheat landrace(Triticum aestivum L.),showed resistance to powdery mildew both in field and greenhouse.To reveal the genetic basis of this resistance,a genetic analysis was conducted using an F2 population,derived from the cross of Dunhuachunmai with a susceptible cultivar Yangmai4,after inoculation with avirulent isolates Bgt2.The results indicated that one dominant gene controls the resistance,which was mapped to a region carrying Pm2a on chromosome 5DS.This gene,designated as Pm2x,was delimited to 1.2 cM genetic interval flanked by markers Xcfd81 and Xwgrc1912 on the distal and proximal sides,respectively and co-segregated with markers Xwgrc1880 and Xwgrc1881 designed based on the Chinese Spring survey sequence.Cloning of the Pm2 allele in Dunhuachunmai indicated that it indeed carries Pm2a.The markers derived from Pm2a sequence co-segregated with Pm2x in Dunhuachunmai.The powdery mildew resistance gene in Dunhuachunmai,Pm2x,conferred the same resistance spectrum as that of Pm2a.The markers developed in this study can help in determining the allelic relationships of the genes mapped at the Pm2 locus and transferring the Pm2 to other cultivars or to pyramid it with other genes for more durable resistance.