Cloning Of Fragments Related To Resistance Genes In Wheat By CDNA Capturation And Suppression Subtractive Hybridization

Wheat yellow dwarf(WYD) caused by barley yellow dwarf virus(BYDV) is a severe wheat disease. The distal segment of the long arm of the Thinopyrum intermedium chromosome 7Ai1(7X) carries a BYDV resistance gene Bdv2. This segment was transferred to the distal region of the long arm of wheat chromosome 7D in the YW642 of translocation lines by using the ph1b mutant to induce homoeologous pairing. It is found that the resistance gene Bdv2 from Thinopyrum intermedium is a dominant single gene.Cloning of the resistance gene to BYDV is very important for elucidating resistance mechanism and breeding resistant cultivar. In this research, cDNA capture, suppression subtractive hybridization (SSH) and screening of transformation-competent artificial chromosome genome library of wheat-Thinopyrum intermedium Translocation line YW642 were carried out to clone candidates or fragments relating to the BYDV resistance gene.An oligonucleotide probe that was designed according to the conserved domains of nucleotide binding site(NBS) of some plant resistance genes was used to screen cDNA library of YW642 using Genetrapper?cDNA Posistive Selection System(Invitrogen?, the cDNA clones with the conserved domain of plant resistance genes in the library should be enriched, and the positive clones were identified by colony PCR. 3 positive clones were screened out from 450 clones.The sequences of the 3 clones were queried to GenBank, the clone 4-1 is similar to T.aestivum L. mRNA for ubiquitin, 4-2 similar to Pisum sativum mRNA for Isovaleryl-CoA dehydrogenase gene, and the clone 5-3 similar to T.aestivum ribulose-1,5-bisphosphate carboxylase/oxygenase (RUBISCO) gene.Near isogenic pools was constructed by mixing 10 resistant individual plant of F2 generation of YW642/Zhong8601 as resistant pool and 10 susceptible individual plant as susceptible pool. Suppression subtractive hybridization (SSH) was carried out by using resistant pool as tester and susceptible pool as driver. SSH DNA library was constructed which includes 960 clones, the range of insert size is 100bp to 1200bp and the average size is 350bp. The forward and reverse SSH products were used as probe to screen the library to remove the false positive clones. 8 positive clones were screened out from 192 clones from the library, the positive ratio of the library is about 4.2%. One clone TSH-1 was proved to be special to resistant pool by Southern hybridization. The sequences of the clone was queried to GenBank but no similar sequence was founded, so the TSH-1 is a new DNA fragment.A sequence-characterized amplified region (SCAR) marker SC-gpl co-segregated with the Bdv2 gene was used to screen a TAC library of YW642 by pooled PCR. From 1,440 primary pools, a positive pool 2G4 was detected, a secondary pool 2G4-6 and two third pools 2G4-6-52 and 2G4-6-53 were detected in succession.