Identification Of Makers Liked To The Resistance Gene Yr5 Against Wheat Stripe Rust

Stripe rust (yellow rust), caused by Puccinia striiformis West. f. sp. tritici, is one of the most important diseases on wheat throughout the world. It is an effective way to breed resistant cultivars conferring major resistance gene by maker-assisted selection, which would accelerate breeding program.RAPD analysis was performed between a near-isogenic line (NIL,) 7r5/6+Avocet S carrying the resistance gene Yr5 against wheat stripe rust and its susceptible parent Avocet S, using the YrS gene donor parent Triticum spelta album as control. Amplified DNA fragments were separated with 4% denaturing PAGE (polyacrylamide gel electrophoresis) and displayed by silver staining. Fifty to 100 bands were detected, 5 folds more than that revealed on agarose gels. A total of 240 random primers were screened, and 23 reproducible polymorphic DNA fragments were found. The results suggested that using denaturing PAGE-silver staining could remarkably increase the level of DNA polymorphism detected in wheat, and could improve the repeatability of RAPD analysis because of effective detection of low copy DNA fragments.Of 23 polymorphic DNA fragments, 6 fragments (S1320207, S1348363, S1476395, S1476401, S1397830 and S1380359) respectively derived from 5 primers, were confirmed to be linked to Yr5 gene by preliminary genetic linkage analysis. The fragments S1320207, S1348363 and S1476401, closely Gnked to the target gene, were recovered from polyacrylamide gel and cloned in pGEM-T easy vector, then sequenced from two ends. According to the sequences, two pairs of specific primers were designed. Genetic linkage between the PCR products and the target gene was tested on 121 segregating F2 plants derived from a cross between Avocet S and YrJ/6+Avocet S. It was shown that the polymorphic DNA fragment SC-S1320156 was completely linked to Yr5 gene, and SC-S1348357 was closely linked to Yr5 gene.The total RNA was extracted from wheat leaves respectively collected 12h, 24h and 48h after inoculation and cDNA were prepared. Five degenerated primers were designed according to the P-loop, Kinase-2a, Kinase-r3a, EGF domain of NBS which universally exist in cloned R genes. PCR amplifications were performed with the designed RGA primers and cDNA as templates. Four polymorphic DNA fragments were obtained when anneal temperature was low, only one polymorphic DNA fragment D-4 was found with the same templates and primers when annealtemperaturewas high.PCR products from primer pair N1-E1 were excised from PAGE and used as template. Reamplification was performed with primer pair K2a-E1 and Touch Down PCR was employed. Aspecific DNA band of 550bp was detected, named as RD-1. PCR products from primer pair K2a-E1 were excised from PAGE and used as template. Reamplification was performed with primer pair K3a-E1 and Touch Down PCR was employed. A specific DNA band of 470bp was detected, named as RD-2. Both RD-1 and RD-2 had expected molecular weight. Employing semi-nest primer to amplify target fragment twice, it could improve specificity and be in favor of detecting target fragment.Postgraduate: Chen Xiaohong Mjaor: Plant Molecular BiologySupervisor: Prof. Hu Baozhong Prof. Niu Yongchun...