Identification Of RGA Markers Linked To The Resistance Gene Yr15 Against Wheat Stripe Rust

Wheat, one of the most important food crops in the world, is always threatened by a lot of diseases. Stripe rust (yellow rust), caused by Puccinia striiformis west. f. sp. tritici, is more universal and more serious. It is hard to control the wheat stripe rust effiectively own to its high viability. However, it is an effective way to breed resistant cultivars conferring major resistance gene by marker-assisted selection, which would accelerate breeding process. In 1910, Yr15 gene founded in Triticum dicoccoide s is one kind of wild wheat in the north of Canada. Many experiments have demonstrated that the resistance gene Yr15 is well against to popular races. As a result, the resistance gene Yr15 of the wheat served as a model is great value to molecular markers-assisted selection of the resistance gene, map clone and cloning resistance genes based on homological sequences. RGA labeling is a technique which can detect the DNA mutation from the genome scale by utilizing the character of the resistance gene conserved region and amplify the genome DNA specifically through man-made synthesing primers. In this experiments, a near-isogenic line (NIL) Yr15/6×Avocet S carrying the resistance gene Yr15 against wheat stripe rust and its susceptible parent Avocet S was served as material to perform RGA analysis. After amplified DNA fragments were separated with 4% denaturing PAGE (polyacrylamide gel electrophoresis) and displayed by silver staining, thirty to seventy bands were detected, which is 5 folds more than that revealed on agarose gels. Of the 442 primer pairs from 49 individual RGA primers that were screened, 2 reproducible polymorphic DNA fragments were found。The results suggested that using denaturing PAGE-silver staining could not only remarkably increase the level of DNA polymorphism detected in wheat, but also improve the repeatability of RGA analysis because of detection of low copy DNA fragments effectively. Second, Of 2 polymorphic DNA fragments, Yr15-R1 fragments derived from primers XaILR-F and PtoFen-S, and Yr15-R2 fragments derived from primers XaILR-R 和Pto-kin2IN, were confirmed to be linked to Yr15 gene by preliminary genetic linkage analysis. Genetic linkage between the PCR products and the target gene was tested on 214 segregating F2 plants derived from a cross between Avocet S and Yr15/6×Avocet S. It was shown that the polymorphic DNA fragment Yr15-R1 was linked to Yr15 gene completely. It was also found that Yr15-R2 was linked to Yr15 gene closely using Mapmaker 3.0 software, and the genetic distance is 2.0cM . Finally, The fragments Yr15-R1 and Yr15-R2, closely linked to the target gene, were recovered from polyacrylamide gel and cloned in pGEM-T easy vector, then sequenced from two ends, and the measured sequence was further compared.