Fine Mapping Of A Wheat Stripe Rust Resistance Gene In Chinese Differential Host Jubilejina Ⅱ

Wheat stripe rust, caused by Puccinia striiformis f. sp. tritici (Pst), has been one of the mostdestructive diseases of wheat in China. It could cause great wheat yield losses and decrease wheatproduct quality. The pathogen is an obligate fungus. The effective identification of the virulence gene(s)in races of Pst is achieved with the aid of resistance gene(s) of a set of wheat differentials. Therefore, itwas important to find out the genetic traits and defense characteristics of the resistance genes ofdifferential varieties. Through resistance gene mapping and transcriptome sequencing, results would beused in the characterization of race-specific resistance of differential varieties. The differential varietyJubilejina Ⅱ has special identification ability for the races ofPst found in China. It had importanttheoretical significance and application value to genetically analyze and map the resistance gene(s) anddesignate it in Jubilejina Ⅱ.In this study, we used classic hybridization analysis and constructed F2mapping population byhybridization of Mingxian169as recurrent female parent and N9line of the near-isogenic line (NIL)Mingxian169*6/YrJu4as male parent. SSR and EST-SSR methods were used and primers weredesigned on the base of colinearity analysis between wheat and rice. Transcriptome sequencing analysisof Mingxian169and N9line of Mingxian169*6/YrJu4was conducted with de novo sequencingtechnology. The PCR products with the DNA templates of gene donor Jubilejina Ⅱ, recurrent parentMingxian169and N9line was analyzed with polyacrylamide gel electrophoresis for fine mapping ofthe resistance gene YrJu4. Mingxian169and N9line of Mingxian169*6/YrJu4were inoculated with thePst race CYR17. The total RNA of Mingxian169and N9line of Mingxian169*6/YrJu4after beinginoculated with CYR17for0h,6h,12h,24h and48h was isolated for the transcriptome sequencinganalysis. The results were shown as follows:1.There were31pairs of primers selected could stably amplify special DNA bands betweenMingxian169and Mingxian169*6/YrJu4, such as pTtksuG5, CFD6, CFD168, WMC702, WMS445,CFA2058, GPW2111, GPW2125, PSP3153, GPW7379, BE407000, BE423182, BE444378,BE497393, BE606324, BG606824, R2-10-2, R3-7, R6-1-2, R8-1-2, R11-6-2, R12-3,6a,12b,18a,19b,25a,40a,47a,49b and54b. Seven primers (GPW7379,49b, GPW2125, R2-10-2, BE423182,WMC702,WMS445) were found to be genetically linked with YrJu4after detection with20resistantand20susceptible plants and354F2plants. The genetic distance between the7markers above andYrJu4was17.1cM,14.6cM,14.1cM,13.5cM,4.1cM,11.7cM,15.2cM, respectively. Xbe423182andXwmc170(Wang Dongmei,2013) were the flanking markers of YrJu4in the region of4.6cM. Thelocation of YrJu4on wheat chromosome2A was Xgpw7379—X49b—Xgpw2125—XR2-10-2—YrJu4—Xbe423182—Xwmc702—Xgwm445. YrJu4in Jubilejina Ⅱwas different from other wheat stripe rustresistance genes located on chromosome2A. We suggested that it should be designated as Yr60.A fine mapping population of4253F2plants was constructed. Using classic hybridization analysismethod, Mingxian169, Jubilejina II, N9line of Mingxian169*6/YrJu4and the F2plants wereinoculated with CYR17and phenotype scoring was conducted.The results showed that the resistance of Mingxian169*6/YrJu4to CYR17②was controlled by a pair of recessive genes.2.The transcriptome sequencing results revealed that the isolated mRNA was of high quality and241947unigenes of which N50value was above1021were found totally, and their average length was640nt. The transcriptome results would enrich the wheat transcription data. After being blasted with Nrdatabase, the most annotated unigenes were hitted in the databases of Hordeum vulgare, Brachypodiumdistachyon and Oryza sativa japonica.19404unigenes were annotated and classified into24categoriesin KOG database, and the first three categories were related to signal transduction mechanisms,posttranslational modification and protein turnover, chaperones, and general function prediction only.21496unigenes were annotated in KEGG database. The first three pathways were ribosome (ko03010),plant-pathogen interaction (ko04626) and starch and sucrose metabolism (ko00500). Besides,49019unigenes were annotated in3categories and65subclasses of GO database.The expression pattern differences between Mingxian169and N9line of Mingxian169*6/YrJu4at6h and12h after being inoculated with CYR17were analyzed and46up-regulated unigenes and39down-regulated unigenes were found. We took12h after inoculation as the start point and Mingxian169as control, and found21unigenes continuously up-regulated and31unigenes continuouslydown-regulated from12h to48h post inoculation. These results could provide important information forfurther investigation of the regulatory pathways of resistance gene YrJu4.