Fine Mapping Of Broad-spectrum Powdery Mildew Resistance Gene Pm5V And Analysis Of Its Candidate Genes

Powdery mildew,caused by the biotrophic fungus Blumeria graminis f.sp.tritici（Bgt）,is a global disease that poses a serious threat to wheat production.Powdery mildew interferes with photosynthesis,thereby reducing plant growth,heading,grain filling and end-use quality.Resistant cultivars are considered to be a cost-effective and environmentally friendly way of controlling this disease.Although more than 70 genes/alleles（Pm genes）conferring powdery mildew resistance have been identified in wheat and its relatives,most of them are ineffective due to the presence of virulence in the pathogen.Hence,identification of new resistance sources in adapted germplasm is an important and long-term objective in achieving durable and broad-spectrum resistance.Dasypyrum villosum（2n=14,VV）is recognized as an excellent gene pool of new resistance genes because it is a near-nonhost to many wheat pathogens and widespread immunity to multiple diseases,such as strip rust,leaf rust and powdery mildew.Four formally designated powdery mildew resistance genes,Pm21,Pm55,Pm62 and Pm67,have been transferred from D.villosum to wheat by means of compensating Robertsonian translocations（Rob Ts）,confirming that D.villosum is indeed an important source of powdery mildew resistance genes for wheat.In our previous study,a novel broad-spectrum powdery mildew resistance gene Pm5V was genetically mapped on the chromosome arm 5VS of D.villosum by using a F₂segregating population generated between resistant parent TF5V-11（5VS#5）and susseptable parent TF5V-2（5VS#4）at the seedling stage.Pm5V was located between the markers CINAU5VS-10 and CINAU5VS-18.To fine mapping Pm5V gene on 5VS,2167 F₂individuals were used to select the recombinations occured near this resistance gene region.More polymorphism molecular markers around the Pm5V region were developed based on the 5VS genome sequences and BSA-Seq sequences.The NLR genes were selected as the candidated Pm5V gene for further founction analysis.The genetic effect analysis of the near-isogenic lines carrying the Pm5V gene is carried out to clarify the breeding value of Pm5V at the same time.The main contants as following:1.Fine mapping of the broad-spectrum powdery mildew resistance gene Pm5VIn our previous study,a novel broad-spectrum powdery mildew resistance gene Pm5V was genetically mapped on the chromosome arm 5VS of D.villosum by using a F₂segregating population generated between resistant parent TF5V-11（5VS#5）and susseptable parent TF5V-2（5VS#4）at the seedling stage.Pm5V was located between the markers CINAU5VS-10 and CINAU5VS-18,which corresponds to the 43 Mb-55.8 Mb interval on CS 5DS.To fine mapping Pm5V gene on 5VS,2167 F₂ individuals were used to select the recombinations occured near this resistance gene region.Eighteen polymorphism molecular markers around the Pm5V region were developed based on the 5VS genome sequences and BSA-seq sequences.Finally the Pm5V gene was located in the 2.1 Mb interval between the markers CINAU5VS-28 and CINAU5VS-42.The phenotypes of 2（F2-30-20 and F2-30-126）of the 38 recombination plants were inconsistent with the genotype of the Pm2 allele marker on 5VS.At the same time,the Pm2 gene was verified by VIGS experiment.The results indicated that Pm5V gene was not the Pm2 allele on D.villosum.Therefore,the Pm5V gene could be a new powdery mildew resistance gene that has not been cloned in wheat and its relatives.2.Analysis of candidate genes for the broad-spectrum powdery mildew resistance gene Pm5VTo explore the genome sequence of the 5VS chromosome arm of D.villosum,chromosome sorting and next-generation sequencing technologies were used to sorte T5DL.5VS translocated chromosome in CS beckground.A total of about 2.5 million T5DL.5VS#4 chromosomes were sorted using flow cytometer,and the sorting purity was89%.A total of 96.3 Gb effective sequences of the T5DL.5VS chromosome were obtained using the Hiseq2500 sequencing platform,and the sequencing depth was 50×.By assembling and removing the 5DL sequence,a 162.958 Mb 5VS sequences were finally achieved and assembled into 18544 scaffolds with an N50 of 18.3 Kb.Using NLR-Annotator software to analyze the 5VS scaffolds sequence,a total of 31 NLR genes were tapped,of which 17 have alleles on the short arm of the fifth homologous chromosome of the Triticeae family.The remaining 14 have not been aligned to the alleles on wheat chromosomes.They need to be further verified whether they are the unique NLR gene on the 5VS of D.villosum.Based on the genetic mapping interval,the polymorphic molecular markers were developed of NLR genes near the mapping locus.The markers of the two NLR genes（NLR-V1 and NLR-V2）were co-segregated with Pm5V by the analysis of recombinations individuals.Thus,the two NLR genes were regard as the candidated Pm5V genes for further founction analysis.3.Genetic effection of the broad-spectrum powdery mildew resistance gene Pm5VIn order to clarify the breeding value of Pm5V,the genetic effect of the translocated chromosome T5DL.5VS#5 carrying the Pm5V gene was analyzed by using near-isogenic lines.The results showed that the kernel hardness index of the near-isogenic lines carrying the Pm5V gene was significantly lower than that of the background parent NAU0686.All the T5DL.5VS#5 lines were soft grain texture.In addition,there were no significant differences in effective ear per plant,the spike length of the main stem and the thousand-grain weight between the T5DL.5VS#5 translocated lines and the background parent NAU0686.However,the obvious differences on the plant height,spikelet number and number of grains per spike were present between them.The plant height of the T5DL.5VS#5 translocated lines were 5.1cm lower than that of NAU0686,and the spikelet number and the number of grains per spike of the main stem were increased 1.5 and 5.7,respectively.The genetic effection analysis showed that the T5DL.5VS#5 translocation material carrying the Pm5V gene has a significant positive effect in yield-related traits and has good breeding value for wheat improvement.