Screening And Identification Of The Candidate Gene Of Wheat Powdery Mildew Resistance Gene Pm40

Wheat powdery mildew is caused by obligate parasitic Blumeria graminis f.Sp.Tritici(Bgt).With environmental conditions becoming more suitable for the growth of powdery mildew in wheat in recent years,powdery mildew has become one of the most serious diseases affecting wheat yield and quality.Reasonable use of resistant genes and cultivation of resistant varieties are the most economical,effective,safe and environmental protection measures to control powdery mildew in wheat.The powdery mildew resistance gene Pm40 was derived from the hybrid progeny of common wheat and Thinopyrum intermedium.It was found that several wheat lines carrying Pm40 maintained immune resistance to different physiological species of powdery mildew in different regions and under different environments.Moreover,the wheat line carrying Pm40 has excellent agronomic characters.Therefore,the main objectives of this study is further to identify the resistance of Pm40 to the physiological species of the newly prevalent powdery mildew fungus,to screnn its candidate genes,to develop molecular markers closely linked to the Pm40 gene.It is of great significance to enrich the resistance source of powdery mildew and maintain the stability of wheat yield in China.The important results of this study are as follows:1.The resistance of wheat materials L658,L958 and their parents YU25 and MY11 to the newly popular physiological race E20 and field mixed races of wheat powdery mildew were determined by combining greenhouse and field identification.The results showed that the wheat material L658 and YU25 with Pm40 showed immune resistance in both greenhouse and field,while the wheat material L958 and MY11 without Pm40 showed severe infection.Further cytological observation further showed that the leaf cells of L658 showed significant hypersensitive response at 72 hours post-inoculation with Bgt,while there was no similar response at that time point in L958.2.Based on the published genetic map of the Pm40 gene and the Chinese spring wheat reference genome,the molecular markers flanking the Pm40 gene have been mapping to the reference genome of Chinese spring wheat.It was confirmed that the 37 Mb physical region of the Pm40 gene contained 185 genes.Through further functional annotation of these genes,it was confirmed that the 185 genes included 1 gene encoding CC-NBS-LRR protein and 7 genes encoding receptor protein kinase.3.We analyzed the sequence variation and expression changes of all genes in the region of Pm40 gene at the transcriptional level by the available transcriptome data of both L658 carrying Pm40 and L958 lacking Pm40 after inoculation with Bgt 15.We detected 458 high-quality variants within the 37 Mb physical region of Pm40 gene.Through the annotation of these variants,it was further found that they had a great influence on the protein sequences of 34 genes.These variants and the genes affected by the mutation sites,as well as the differently expressed genes detected in L658 and L958 after inoculation with Bgt were all concentrated in the 8Mb region close to the marker BF291338.We screened out Traes CS7B01G164000 and Traes CS7B01G164600 as possible Pm40 candidate genes in this region.4.We cloned Pm40 candidate gene from wheat line L658 carrying Pm40 and its susceptible sister line L958.Comparison of the cloned gene sequences showed that the sequence of Traes CS7B01G164600 in wheat line L658 and L958 was identical,and the sequence of Traes CS7B01G164000 has two missense mutations in L658 and L958.The physicochemical properties of amino acids corresponding to missense mutations are quite different.Therefore,we speculate that Traes CS7B01G164000 is more likely to be a candidate gene for Pm40.5.The AS-PCR markers were developed according to the sequence differences between the cloned candidate gene Traes CS7B01G164000 in L658 and L958,to identify the mutation site in the higher generation sister line of L658,and the expression changes of the cloned candidate gene after inoculation with Bgt were verified by q RT-RCR.Through co-segregation and expression analysis of candidate genes,it was found that the Traes CS7B01G164000 was cosegregation with powdery mildewy resistance and differently expressed in wheat materials L658 and L958.Finally,the gene Traes CS7B01G164000 was identified as the candidate gene for Pm40.