Use of isogenic lines and simultaneous probing to identify DNA markers tightly linked to the tm-2a gene in tomato

Abstract

The Tm-2a gene of tomato confers resistance to the viral pathogen, tobacco mosaic virus. Like many economically important plant genes, Tm-2a has been characterized phenotypically and by classical linkage analysis, yet nothing is known about its gene product. We report here the isolation of two DNA clones which are very tightly linked to the Tm-2a gene. These clones were identified by testing 122 genomic clones as hybridization probes against Southern blots consisting of DNA from pairs of nearly isogenic lines with or without the Tm-2a gene. Screening such a large number of clones in a short period of time was facilitated by co-labeling and simultaneous probing of sets of up to 10 random genomic clones. Tightly linked clones were distinguished by the fact that they exhibited one or more restriction fragment length polymorphisms between the nearly isogenic lines. Tight linkage of the clones with Tm-2a was verified in a segregating F(2) population. Both mapped to the same locus 0.4 +/- 0.4 centimorgans away from Tm-2a and may provide starting points for a genomic ;;walk'' to this gene. Due to the availability of isogenic lines in many plant species, the strategy outlined in this paper should be widely applicable for selecting DNA clones tightly linked to genes of interest.