Identification And Cloning Of The Candidate Genes Of Wheat Stripe Rust Resistance Gene Yr26

Wheat stripe rust is a serious wheat fungal disease caused by the infection of Puccinia striiformis f.Sp.tritici(Pst).Wheat stripe rust has a wide range of hazards in China and poses a serious threat to wheat production.Therefore,development and cultivating of resistant varieties is the most effective measure for preventing and controlling stripe rust.Yr26,a stripe rust resistance gene,was identified from the T.aestivum-H.villosa 6VS/6AL translocation lines which was created by Cytogenetics Institute in Nanjing University of Agriculture.The gene was once effectively resistant to the dominant race of stripe rust in China and has played a significant role in production,but in recent years it has been found that its resistance is overcome by new races.Ma(2001)found that the Yr26 gene was a dominant monomeric genes and was localized on the chromosome 1B.Yr26 may be derived from one of the initial hybrid parents,conical wheat,y-80,according to pedigree analysis.Yr26 was further located on the deletion line C-1BL-6-0.32 of wheat IB chromosome by Wang(2008).Then,a high density genetic map of Yr26 was constructed by Zhang(2013),and the two closest flanking markers were CON-4 and CON-12.The genetic distances between Yr26 and the two closely linked markers were 0.08 cM and 0.17 cM,respectively.Yr26 was mapped near centromeric region of wheat 1B chromosome,a region with low recombination rate,which brings great difficulties to the map-based cloning.In this study,we try to use Expression profile gene chip,SNP chip and Transcriptome sequencing technology to screen Yr26 candidate gene and disease resistance related gene through comparative genomics analysis with the help of the fine mapping information.1.Screening candidate resistance genes on the chromosomal segments where Yr26 gene is located using SNP chip and comparative genomicsIn this study,21 markers selected by Zhang(2013)were compared with the Aegilops tauschii DD genome database.This group of markers help to identify the collinearity of Yr26 genomic region to the range of 46.534cM-50.758cM in Aegilops tauschii 1D chromosome.The above analysis laid the foundation for the development of marker and the prediction of candidate genes in the Yr26 region.In this study,we used the sequences information of collinearity of Yr26 in Aegilops tauschii 1D chromosome to identify the putative R genes,and two specific genes with R gene domains and 16 SNP markers that have polymorphism between resistant and susceptible parents were screened in the obtained 90KSNP chip data,and another 30 specific genes with R gene domains were screened in the obtained 660KSNP chip data from the same material.The expression of these 30 genes was further analyzed,two genes were screened to up-regulated expression in the resistant material 92R137,and were not expression in the susceptible material Yangmai 158 after inoculated with Pst race CYR32.2.Screening candidate resistance genes on the chromosomal segments where Yr26 gene is located using 92R137 transcriptome data and comparative genomicsIn this study,we use the information of Chinese spring 1A/1B/1D genome database and Yr26 fine mapping information to define the collinearity of Yr26 region on 1A/1B/1D chromosome.According to the genetic map constructed by Zhang(2013),the collinear physical region of Yr26 gene on Chinese spring 1B chromosome was determined to the 127-200Mb region,the collinear physical region on Chinese spring 1A chromosome was determined to the 38-92Mb region and the collinear physical region on Chinese Spring 1D chromosome was determined to the 27-34Mb region.The material 92R137 used in this study carried the stripe rust resistance gene Yr26 and the powdery mildew resistance gene Pm21,which was an excellent germplasm to resistant to stripe rust and powdery mildew.In the three 92R137 transcriptome databases obtained,a total of 882 NBS-LRR genes were obtained.Six candidate genes were screened according to the colinear region of Yr26 on Aegilops tauschii 1D chromosome.Based on the collinearity of Yr26 region on Chinese spring 1B,another 12 candidate genes were screened.These 18 genes can used to develop markers and cloning analysis.3.Functional analysis of wheat stripe rust resistance related gene TaRLP2We used gene chip technology to obtain the gene expression profile that concerning resistance cv.92R137 and cv.R236 and susceptible cv.Yangmail58 before Pst inoculation and after inoculation for 12 and 36 hours.Totally 5757 differentially expressed genes were screened and then were used for Blastn search in the DD genome database by Xing(2015).Ta.4479.2.S1_a_at(TaRLP2)that locating in the region covered by two nearest flanking markers CON-4 and CON-12 of Yr26 and encoding the LRR receptor protein was screened.This gene ’was only significantly up-regulated expression in the resistant material after inoculated with CYR32.And then primers were designed for homologous cloning and sequence analysis.The results showed that there were 6 SNP differences between the sequence of resistant material 92R137 and susceptible material Yangmai 158,and further analysis found that the varieties containg Yr26 showed the same SNP polymorphism as 92R137,while the varieties without Yr26 showed the same SNP polymorphism as Yangmai158.In order to verify the function of the TaRLP2,we used the gene gun transformation technology to overexpress the gene in susceptible material Yangmai 158 and used virus-induced gene silencing technology to silence the gene in resistant materials 92R137.The results showed that TaRLP2 gene was involved in the Yr26 gene-mediated stripe rust resistance.