Isolation And Identification Of The Genomic DNA Fragments Related To Lr35 Resistance To Wheat Leaf Rust

A study based on the homology-based candidate gene method for cloning resistance gene was conducted by scanning genomic DNA in wheat near-isogenic lines (ThLr35) with degenerated oligonucleotide primers designed according to different conserved domains in cloned resistance genes. The main results are as follows:Four oligonucleotide primers were designed based on the conserved NBS-LRR domain of R genes. No specific amplified products ranging from approximately 200-2000bp were obtained from different primer combinations such as RAF1:R2, RAF8.-R2, RAF8.-R5 and RAF1 :R5. In the meanwhile, a fragment of 633bp which was common in four combinations was cloned.lt showed 79% homology to a part of 211kb fragment (AF326781) in GeneBank which is in turn a part of 450kb fragment containing Lr10.Another primer combination RAF3:R4 was designed corresponding to conserved sequence DVKPEN and GTPWYIAPE of protein kinase. Four main fragments were amplified, the optimized reaction system was established. But no product was obtained using primers designed according to the conserved LRR domains of R genes. The result indicates that conserved motif NBS-LRR and STK might exist in wheat genome, LRR might not exist. In other words, wheat resistance genes to leaf rust might not belong to the type of LRR resistance gene, but NBS-LRR and STK.A 597bp PCR product appeared in ThLr35, but not in the susceptible background parent Thatcher using Lr35 ISSR marker as a template. It has a high homology (90%) with a wheat BAC clone (AF459639) and a 211Kb fragment (AF326781), which contains Lr10, implying this gene may be involved in wheat leaf rust resistance gene Lr35.With primers designed according to the sequence of the DNA fragment, twofragments about 1.0kb and 2.0kb were amplified from the genomic DNA of ThLr35. Whether these fragments are related to Lr35 or not should be further testified.