Submitted to: Cereal Rusts and Mildews Conference European and Mediterranean Proceedings
Publication Type: Abstract Only
Publication Acceptance Date: 6/1/2004
Publication Date: 7/1/2004
Citation: Chen, X., Ling, P. 2004. Towards cloning wheat genes for resistance to stripe rust and functional genomics of puccinia striiformis f. sp. tritici. 11th International Cereal Rusts and Mildews Conference European and Mediterranean Proceedings. Page A2.10.
Technical Abstract: Wheat stripe rust, caused by Puccinia striiformis f. sp. tritici, is one of the most important diseases of wheat in the United States. Use of genetic resistance is the most preferred approach to control the disease. In order to understand the molecular basis of the host-pathogen interactions, we are conducting research towards cloning and characterizing genes for resistance to strip rust and understanding functional genomics of the pathogen. The dominant gene Yr5 confers resistance to all races of P. striiformis f. sp. tritici identified so far in the United States. Resistance gene analog polymorphism (RGAP) markers that are co-segregating with the Yr5 locus and have high homology with plant resistance genes have been previously identified (Yan et al., 2003). Based on a pair of the co-dominant and co-segregating RGAP markers, sequence tagged site (STS) and cleaved amplified polymorphic sequence (CAPS) markers also were developed, which could enable the incorporation of Yr5 into a wide range of wheat varieties (Chen et al., 2003). To clone Yr5, a genomic Hind III BAC library of hexaploid wheat (2n = 6x) was constructed using the wheat Yr5 near-isogenic line (NIL) (AVS/6*Yr5) developed in the Plant Breeding Institute, the University of Sydney, Australia. The BAC library consists of 410,000 clones with an average insert size of 130 kb, and covers approximately 3.3x wheat genome equivalents. Colony pools and high-density filters of the BAC library were made for identifying resistance clones. The STS markers were used to screen the multi-dimensional BAC clone pools. Twelve positive clones were identified using the Yr5 STS markers. Sub-clone libraries for each of the 12 positive clones were constructed. The average insert-size of sub-clones ranges from 4 kb to 12 kb. Each of the sub-clone libraries covers at least 10X of the original BAC insert. To isolate the expressed sequences from the Yr5 locus, full-length cDNA libraries were constructed from both the pathogen-challenged and non-challenged Yr5 NIL. Each of the libraries consists of about 35,000 clones. The Yr5 STS markers are used to screen the sub-clone and cDNA libraries. To understand the host-pathogen interactions, genomic and functional genomic studies of the wheat stripe rust pathogen were initiated. For physical mapping and gene isolation, a BAC library of P. striiformis f. sp. tritici was constructed with Hind III digested genomic DNA fragments cloned into the pIndigo BAC-5 cloning vector (Epicentre, USA). The BAC library that consists of 43,000 clones with an average insert size of 50 kb covers about 20x of the P. striiformis genome equivalent. To study functional genomics, especially genes involved in pathogenicity and genes of biologically importance, a full-length cDNA library of P. striiformis f. sp. tritici was constructed. This library consists of 17,280 clones representing general mRNA transcripts. An initial characterization and gene analysis were conducted by sequencing about 200 randomly selected clones. The results of the complete cDNA sequencing show approximately 98% of the cDNA clones in this library are of full length. These sequences are used in the BLAST search to characterize the genes.