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.)
N. Stein et al., 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.), P NAS US, 97(24), 2000, pp. 13436-13441
Citations number
30
Categorie Soggetti
Multidisciplinary
Journal title
PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA
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.