Fine Mapping Of The Powdery Mildew Resistance Gene Pm61 In Wheat Landrace Xuxusanyuehuang

Wheat powdery mildew,caused by Blumeria graminis f.sp.tritici,is a fungal disease that impacts severly on production of wheat?Triticum aestivum?.Identification of wheat powdery mildew resistance gene is required for sustainable improvement in disease resistance.Wheat Xuxusanyuehuang is a wheat landrace from Fengdu,Sichuan Province,showing resistance to powdery mildew.In this study,Xuxusanyuehuang was crossed to the powdery mildew susceptible cultivars Mingxian 169 and Zhongzuo 9504 to develop the genetic mapping populations.To fine map the powdery mildew resistance gene in Xuxusanyuehuang,the comparive genomics analysis and BSR-Seq?bulked segregant analysis and RNA-Seq?techniques were used to develop polymorphic molecular markers for constructing high-resolution genetic linkage map.The following results were obtained.1.Among the 46 Bgt isolates that were collected form different wheat producing regions,Xuxusanyuehuang was resistant to 31 isolates.Fifteen Bgt isolates were used to detect and compare the resistance spectrum of Pm61 and other Pm genes,with the smallest difference from Pm1c.Only 4 Bgt isolates were virulent on Pm61.Thirty-one Bgt isolates were used to test response of Xuxusanyuehuang to powdery mildew.Twenty isolates were avirulent on Xuxusanyuehuang.The resistance to Bgt isolates collected form Beijing,Shandong and Jiangsu is good,and this indicates that it is useful in the resistance to powdery mildew.2.A new powdery mildew resistance gene Pm61 was discovered and mapped by using comparative genomics method.Genetic analysis was carried out by inoculating the F₁,F₂,and F_2:3 populations with Bgt isolates E09 and Bgt1.Resistance to powdery mildew in Xuxusanyuehuang was controlled by a single recessive gene.Analysis with SSR markers identified a gene on chromosome 4AL that was responsible for the disease resistance.More molecular markers were developed using the comparative genomic analysis with the genome sequences of sorghum,rice,Ae.tauschii and Brachypodium distachyon.This gene was located in a genetic region between SSR markers Xicsx65 and Xicsx79.Because it is a new locus that confers resistance to powdery mildew,the powdery mildew resistance gene in Xuxusanyuehuang was officially designated Pm61.3.Development Markers by BSR-Seq Technology for saturation of the Pm61 genetic linkage map.Using the bulked RNA samples from the homozygous resistant and susceptible F_2:3 families,BSR-Seq analysis enriched 80 SNP that were associated with the disease resistance in a 31 Mb genomic interval of the distal end of chromosome arm 4AL.Two SNP markers,Xicsn2 and Xicsn4,were linked to Pm61with genetic distances of 4.26 cM and 0.24 cM from the target gene,corresponding to a 5.34 Mb genomic interval in the Chinese Spring reference genome.4.A genetic linkage map to fine mapping of Pm61 was constructed by using large populations.An large population consisting of 5500 F_2:3 families derived from Xuxusanyuehuang×Zhongzuo 9504 was used to construct the high-density genetic linkage map.Pm61 was finely mapped in a 1.0 cM genetic interval,corresponding to a 693 Kb physical interval in the Chinese Spring reference genome.A total of47 putative coding genes,including 9 genes encoding for disease resistance associated proteins,were annotated.5.Several molecular assisted breeding markers were developed.Two polymorphic KASP markers,Xicsk8 and Xicsk13,were screened from 13 pairs of KASP markers which were transformed by the polymorphic SNP markers with the BSR-Seq method.Six polymorphic SSR markers were screened from 964 pairs of SSR primers designed with the Chinese Spring Reference Genome.The KASP marker Xicsk8 and SSR marker owm104 can be used for molecular marker-assisted selection of the Pm61 gene.