Subgenome chromosome walking in wheat: A 450-kb physical contig in Triticum monococcum L. spans the Lr10 resistance locus in hexaploid wheat (Triticum aestivum L.)

For many agronomically important plant genes, only their position on a gene tic map is known. In the absence of an efficient transposon tagging system, such genes have to be isolated by map-based cloning. In bread wheat Tritic um aestivum, the genome is hexaploid, has a size of 1.6 x 10(10) bp, and co ntains more than 80% of repetitive sequences. So far, this genome complexit y has not allowed chromosome walking and positional cloning. Here, we demon strate that chromosome walking using bacterial artificial chromosome (BAC) clones is possible in the diploid wheat Triticum monococcum (Am genome). BA C end sequences were mostly repetitive and could not be used for the first walking step. New probes corresponding to rare low-copy sequences were effi ciently identified by low-pass DNA sequencing of the BACs, Two walking step s resulted in a physical contig of 450 kb on chromosome 1A(m)S. Genetic map ping of the probes derived from the BAC contig demonstrated perfect colinea rity between the physical map of T. monococcum and the genetic map of bread wheat on chromosome 1AS. The contig genetically spans the Lr10 leaf rust d isease resistance locus in bread wheat, with 0.13 centimorgans correspondin g to 300 kb between the closest flanking markers. Comparison of the genetic to physical distances has shown large variations within 350 kb of the cont ig. The physical contig can now be used for the isolation of the orthologou s regions in bread wheat. Thus, subgenome chromosome walking in wheat can p roduce large physical contigs and saturate genomic regions to support posit ional cloning.