DIRECT-DETECTION AND ISOLATION OF RESTRICTION LANDMARK GENOMIC SCANNING (RLGS) SPOT DNA MARKERS TIGHTLY LINKED TO A SPECIFIC TRAIT BY USINGTHE RLGS SPOT-BOMBING METHOD

We have developed a technique for isolating DNA markers tightly linked to a target region that is based on RLGS, named RLGS spot-bombing (RL GS-SB). RLGS-SB allows us to scan the genome of higher organisms quick ly and efficiently to identify loci that are linked to either a target region or gene of interest. The method was initially tested by analyz ing a C57BL/6-Gus(S) mouse congenic strain, We identified 33 variant m arkers out of 10,565 total loci in a 4.2-centimorgan (cM) interval sur rounding the Gus locus in 4 days of laboratory work. The validity of R LGS-SB to find DNA markers linked to a target locus was also tested on pooled DNA from segregating backcross progeny bg analyzing the spot i ntensity of already mapped RLGS loci. Finally, we used RLGS-SB to iden tify DNA markers closely linked to the mouse reeler (rl) locus on chro mosome 5 by phenotypic pooling, A total of 31 RLGS loci were identifie d and mapped to the target region after screening 8856 loci. These 31 loci were mapped within 11.7 cM surrounding rl, The average density of RLGS loci located in the rl region was 0.38 cM. Three loci were close ly linked to rl showing a recombination frequency of 0/340, which is < 1 cM from rl. Thus, RLGS-SB provides an efficient and rapid method for the detection and isolation of polymorphic DNA markers linked to a tr ait or gene of interest.