| Powdery mildew(Blumeria graminis f.sp tritici,Bgt)and stripe rust(Puccinia striiformis Westend f.sp tritici,Pst)are the most important diseases of wheat worldwide occurred in the wheat growing areas of the world and serious threat to wheat production and quality.Chinese landrace harbor diverse and abundant disease resistance genes,providing valuable resource for the resistant improved cultivar.In this study,the powdery mildew resistance gene Pm5e was cloned from cultivar Fuzhuang 30.Combining BSR-Seq and comparative genomics analysis,the genetic linkage maps of the stripe rust resistance genes YrMM58 and YrHY1 were constructed.The main results are as following:1.Disease resistance parent Fuzhuang 30 was used to cross with susceptible wheat cultivar Nongda 015 to generate segregating population for fine mapping the powdery mildew resistance gene Pm5e.Using comparative genomics analyses,4 polymorphic markers,linked to Pm5e were developed.The Pm5e gene was mapped in a 0.08 cM genetic interval flanked by markers WGGC13290 and WGGB201.2.Four positive BAC clones were identified from the Wangshuibai BAC libraries using the Pm5e-tagged markers.A 162 kb genomic sequence was assembled through BAC sequencing and used to develop polymorphic markers to map Pm5e in a 0.04 cM genetic interval between markers WGGB2 and WGGB3.Gene annotation revealed 3 candidate genes,Pm5e-1,Pm5e-2 and Pm5e-3 in a 13.1 kb genomic region containing Pm5e.Sequence comparioson indicated that Pm5e-1 and Pm5e-3 genes have same sequences between Fuzhuang 30 and susceptible cultivar Chinese Spring,but two SNPs in Pm5e-2 between the two cultivars.Haplotype ananlysis revealed a A/G SNP in Pm5e-2 is related to the Pm5e gene resistance from Fuzhuang 30.Therefore Pm5e-2 encodes a NB-ARC-LRR protein was determined as the candidate gene of powdery mildew resistance gene Pm5e.3.The genomic DNA sequences of the three candidate genes of Pm5e were amplified and sequenced from EMS-induced powdery mildew susceptible mutants of Fuzhuang 30.Comparison of the sequences revealed that two nonsense(premature stop codon)mutations,one 5 amino acids deletion,one intron-exon boundary splicing site mutation,and two missense(single point)mutations happened on the Pm5e-2 gene in 6 mutants,respectively,suggesting that Pm5e-2 is most likely powdery mildew resistance gene Pm5e.4.The genomic sequence of Pm5e-2 gene from Fuzhuang 30 was cloned into expression vector pCAMBIA1300-Bar and used for functional complemental characterization of the Pm5e gene by Agrobacterium-mediated transformation of susceptible wheat line Fielder.The homozygous Ti and T2 plants of transgenic lines 16-2 and 27-1 showed resistance to Bgt isolate E20,suggesting Pm5e-2 is powdery mildew resistance gene Pm5e.5.Fuzhuang 30 and Xuezao were artificially inoculated with E20,and tested for the expression level of Pm5e at different time points.We found that the expression of Pm5e is inducible,suggesting Pm5e plays an essential role in the function of powdery mildew resistance.6.Chinese wheat lines Mengmai 58 and Huaiyang 1 showed a high-level of resistance to prevailing PST race CYR34(V26)at adult plant stage.Genetic analysis indicated that the stripe rust resistance in Mengmai 58 and Huaiyang 1 are both controlled by a single dominant gene,provisionally designated YrMM58 and YrHY1,respectively.Combining BSR-Seq and comparative genomics analyses,YrMM58 and YrHY1 were mapped to the distal end of chromosome arm 2AS,with the closest marker WGGB148 being 7.7 cM and 3.8 cM apart genetically. |
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