Screening And Cloning Of Stripe Rust Resistance Related Genes To Yr26 In Common Wheat (Triticum Aestivum L.)

Wheat stripe rust,which is caused by Puccinia striiformisf.sp.Tritici(Pst),is one of the most serious diseases adversrly affecting winter cereal production across the world.Urediniospores can spread by wind over thousands of kilometres from the initial infection region in the northerwest of China to the east of China.The wide distribution and frequent occurrence of Pst epidemics in China makes it emergent to find new strategies to preventing stripe rust.New germplasm exploring and varieties development showing high and broad spectrum resistance to stripe rust is the most effective and environment friendly way to prevent and control this disease.Yr26,a stripe rust resistance gene showing high resistance to most of the races of Pst in China,was a gene identified from the T.aestivum-H.villosa 6VS/6AL translocation lines which was created by Cytogenetics Institute,Nanjing agricultural University.Yr26 was initially located on the chromosome 1BL of C-1BL-6-0.32 through the genetic analysis with the low density markers by Wang(2008).Then,a new high density genetic map was constructed involved Yr26,and the gene located by 6 co-segregated markers and 2 flanking marker with genetic distance of 0.08cM and 0.17cM,respectively.Because the Yr26 is located in the pericentromeric region of chromosome,a chromosme region with low recombination frequency,which makes the the map based cloning great challenging.This study was conducted to isolate the resistance related genes and the candidates genes of Yr26 by comprehensive analysis of the differentially expressed genes displayed by genechip and the fine mapping information.1.Exploration of the resistance related genes of Yr26 from the diffrentially expressed genes by virus induced gene silencing system(VIGS)Using genechip hybridization,the gene expression data of eight samples was obtained,inclucing susceptible cv.Yangmai158 and resistant cv.92R137 and cv.R236 before Pst inoculation and after inoculation for 12 and 36 hours.The differentially expressed 17 genes which were specifically upregulated in the stripe rust resistant materials 92R137 and R236 were identified.The 17genes not only showed different expression pattern between the susceptible and resistant materials after Pst inoculation at 12 h and 36 h,but also showed different expression pattern between the resistanct materials before inoculation and after Pst inoculation at 12h and 36h.The putative function analysis showed that Ta.23165.1.S1_at encoded an SNF1 type serine,Ta.16040.1.A1_at encoded a GTP binding protein OBGC1 and TaAffx.114048.1.S1_at encoded a heat shock protein(HP).The BSMV-VIGS was conducted to find out whether these three genes participated in the stripe rust resistance pathway.The results showed that there is no feasibal colonyboth on the control and the gene silenced leaves,however,according to histological observation under the microscope,there were significant differences in the necrosis area aroud the infection sites,numbers of the secondary hyphae branches and length of hypha between the gene silenced leaves and the control.The result suggested that these differentially expressed genes really took part in the resistance regulation in the Yr26 containing materials.2.Screening of candidate gene of Yr26 based on the fine mapping and the gene expression patternTotally 5757 differentially expressed genes were used for Blastn search in the D genome,and the 443 probes had homologous in the 1D chromosome,among which 120 were located in the region covered by two nearest flanking markers CON4 and CON 12 of Yr26.The 120 probes were used again for Blastn search in the IWGSC database,and the result showed that there were 17 genes in 1BL,including the transcription factor and disease resistance protein.3.Cloning and characterization of candidate gene TaRLP2 geneTa.4479.2.S1-a-at was found to be an LRR receptor like gene in the 17 genes mentioned above which was located in the putative Yr26 containing region covered by two flanking markers.Bioinformatics analysis helped us to find the individual copys of this gene in the 1AL,1BL and 1BL chromosome.The conserved primers were designed and used to amplify the whole length of the genes from the resistant materials,(including 92R137,PR and ?-80)and the susceptible materials(Yangmai 158,PS and Zhongyin1286)from the 1AL,1BL and 1BL chromosome respectively,and then the gene from the B genome of resistant and susceptible materials was obtained by using the primers specific to the sequence of the 1BL chromosome.The sequence analysis showed that these sequences were putative LRR recetor genes,so designated as TaRLP2.The multiple sequence alignment showed that the sequence from the resistant materials were identical,and the sequence from the susceptible were identical,however,there were several SNPs,such as C at the 31,255 and 270 position in the susceptible materials were changed to the T in the susceptible materials,the G at the 62 changed to C,the A at the 120 changed to C,and the G at the 279 changed to T.The amino acids anlignment showed that there were two amino acid differences between the susceptible materialsand the resistant materials,for example,at the 16 position the hydrophobicprolinein the susceptible materials changed to hydrophilicglycine in the resistant materials,and at the 31 position the hydrophilicserinein the susceptible materials changed to hydrophobicalaninein the resistant materials.The two difference positions were located in the singal peptide,which perhaps influent the transport of the protein in the cell.In this study,a number of Pst induced genes were screened and several of resistance related genes were isolated and functional analysed using VIGS.A candidate gene of Yr26 gene was also cloned and characterized.The isolated candidate gene and those genes located on theYr26 containing region are valuable for the further reseach to clone the Yr26 gene using the new developed populations.