Prediction And Clonging Of Candinate Genes Of Qym.nau-5a.1 Confering Resistance To Wheat Yellow Mosaic Virus In ’Xifeng’ Wheat

Wheat yellow mosaic(WYM)is caused by wheat yellow mosaic virus(WYMV)),which is transmitted by Polymyxa graminis in the soil.WYMV may result in up to 50%yield loss,and is hard to control by chemical application.The application of wheat varieties with WYMV resistance is the most effective and economic approach for disease control.In recent years,the development and utilization of wheat varieties with good WYMV resistance,such as Yangfu wheat varieties and Ningmai 9,has been effective to control the disease in the wheat grown area of the Yangtze River region.However,the identification and utilization of more resistance resources will be helpful to enrich the genetic diversity.In previous research,we identified Xifeng wheat is highly resistant to WYMV,and the major resistance QTL QYm.nau-5A.1 was mapped to the long arm of 5A.Further fine mapping mapped QYm.nau-5A.1 to a region flanked by two markers,Xwmc415.1 and 5EST-440.Within this region,two EST-STS markers,5EST-44 and 5EST-90,co-segregated with QYm.nau-5A.1.The purpose of this study is to narrow down the genetic region for further cloning of the QYm.nau-5A.1.A genome-wide association study(GWAS)by 55K SNP array analysis using a natural wheat population consisted by 417 wheat varieties.Referring to the reference genome sequences,the numbers and gene functions of coding genes within the QYm.nau-5A.1 regions of different species or genomes were compared.Candidate genes for QYm.nau-5A.1 were predicted,and one of the candidates TaCP was cloned and functional analyzed.The main results obtained were as follows.1.GWAS of WYMV resistance using a natural populationA panel consisting of 417 materials,including 385 wheat germplasm and 42 cultivars with known WYMV response,was evaluated for WYMV resistance and genotyped by the 55K SNP array.GWAS identified that 393 SNPs were associated with WYMV resistance.These SNPs were distributed on chromosomes 2A,2B,2D,5A,5D and 7D,respectively.We further found that 13 SNPs on 5A were significantly associated with WYMV resistance.Among the m,6(AX-109342568,AX-109934392,AX-110990493,AX-111115088,AX-110588803 and AX-110941592)were anchored to the 535Mb-537Mb genome region(covering a physical distance of 2.0MB),which were within the previously mapped QYm.nau-5A.1 region(535-539Mb).GWAS indicated the QYm.nau-SA.1 was present in 51 of the 406 varieties.2.Prediction,cloning and functional analysis of candidate genes for QYm.nau-5A.1Referring to the reference genome sequences of hexaploidy wheat(Chinese spring and AiKang 58,both are hexaploidy,genome AABBDD),tetraploid wheat(T.doccocoides,genome AABB)and diploid wheat progenitor(T.uratu,genome AA),48 functional genes were annotated within the 2.0Mb genome region of QYm.nau-5A.1.By Gene Ontology(GO),10 disease resistance related genes were selected as candidates of QYm.nau-5A.1.One of them coding a cysteine protease was cloned from wheat and designated as TaCP.Electric expression analysis showed that TaCP was significantly up-regulated in response to the infection of Blumeria graminis,Puccinia strilformis and Fusarium graminearum.qRT-PCR showed that in response to WYMV infection,the TaCP from Xifeng wheat was significantly up-regulated,while no significant change in susceptible varieties Zhen 9523 and Yangmai 158.These indicated TaCP may play role in WYMV resistance.The sequences of TaCP genes cloned from Xifeng wheat and Zhen9523 were compared.It was found that showed that TaCP had ORF of 1133bp,including 4 exons and 3 introns.It codes a protein of 376 aa.The TaCP from the two varieties had 3 SNPs difference.The P1 protein from the WYMV genome was used as bait to screen the Y2H library of Haynaldia villosa.Both partial and full length TaCP interact interacted with P1.To elucidate the role of TaCP in WYMV resistance,we obtained transgenic plant by over-expressing TaCP in Yangmai 158.PCR identified 9 T0 plants were positive.Their derived T1 lines and T2 lines were evaluated for WYMV resistance.However,all the transgenic plants showed no obvious improved resistance.3.Development of TaCP-based SNP marker and its potential use in marker assisted selection(MAS)The TaCP gene alleles from XiFeng wheat and Zhen9523 has a SNP variation at the 355bp,with a T in XiFeng wheat and an A in Zhen9523.This SNP was also present in sub-genomes B and D of Chinese spring,Ae.tauschii,T.urartu,AiKang58 and Hordeum vulgare.Based on this SNP,a pair of primer pairs was designed and a co-dominant SNP marker 5SNP-R was developed.Amplification of 5SNP-R in a mapping population for WYMV resistance and the natural population showed that it was co-segregated with QTL QYm.nau-5A.1.This indicated that 5SNP-R can be used as a diagnostic marker for marker-assisted selection of QTL QYm.nau-5A.1 in wheat breeding.