Wheat Exogenous Resistance To Yellow Dwarf Disease Gene Fragment Cloning And Identification

Primers were designed according to the conserved sequences of cloned resistance genes in plants. Using the method of candidate genes based on homologous sequences, the primers were used to screen the Th. Intermedium, wheat-77z. Intermedium and the susceptible wheat materials. A specifically amplified band, which was co-segregated with BYDV resistance, was obtained. It was about 538bp and converted to a SCAR (sequence characterized amplified region) marker, named as TirgaZ1. The marker was used to screen HW642 genomic TAC library and resulted in four positive clones, which were Tl, T2, T3 and T4.Restriction enzyme digestion analysis showed that the four clones were divided into two kinds, one of which included Tl, T2. T3 and the other included T4. Then the hybridization analysis was performed after the digestion products of Tl and T4 were transferred to a nylon membrane, with the RGA fragment TirgaZl as a probe. Obviously different hybridized bands were shown on the digestion products by different enzymes. And the same digestion products also demonstrated 1-2 identical bands and 1-3 different bands. This indicates that the two clones are perhaps partly overlapped and are on the two sides of a contig. Sequencing analysis showed that Tl contained a conserved domain of resistance genes. Therefore, the two clones might be resistance gene candidates for R gene fragment TirgaZl containing NBS-like sequence.Using the DNA of BYDV-resistant Th. Intermedium, HW642 and susceptible wheat parents as probes, Southern hybridizations were carried out on the digestion products of Tl and T4. Specifically hybridized bands with Th. Intermedium and HW642 but not with wheat parents were shown on the digestion products by Xhol, EcoRV, oRI and Pstl. The results further demonstrate that Tl and T4 are BYDV-resistance gene candidates derived from 7XL of Th. Intermedium.To screen the Bdv₍3-rrelated RGAP markers and RGA fragments, degenerated primers were designed according to the conserved sequence of cloned resistance genes in plants and used in the method of resistance gene analog polymorphism (RGAP). After the first screening with the wheat-Th. intermedium translocation line HW642 containing a BYDV-resistance gene Bdv₂ and the susceptible recurrent wheat parents Zhong8601 and further analysis using different resistance materials with Bdv₂: and susceptible materials without Bdv₂. only 2 out of 187 primer combinations designated as cf9F/PtoKin2IN (Tgp-1) and PtoF/XLRRinv2 (Tgp-2). 350bp and 210bp respectively, could stably amplify two specific band related to resistance gene Bdv₂. This proved the validation of Tgp-1 and Tgp-2 as RGAP markers for Bdv₂. The linkage analysis using the F₂ progeny also shows that the two markers are co-segregated with Bdv₂. The specifically amplified bands were excised from the gels of SDS-PAGE for a second PCR and the products were cloned and sequenced. The sequencing results indicate that Tgp-hso and Tgp-2₂₁₀ are parts of the ORF of certain genes and they are new gene fragments. Based on the two sequences, new primers were designed and Tgp-I₃₅₀ was successfully converted to PCR marker SC-gpl. which was also proved to be co-segregated with Bdv₂ in F₂ progeny analysis.