Physical gene mapping of the pig genome

A novel technique termed Direct In situ Single Copy PCR (DISC-PCR) was developed and refined. DISC-PCR involves direct PCR amplification of short unique sequences (100-400 bp) on metaphase chromosome spreads, with non-isotopic detection methods to determine the site of amplification.; DISC-PCR was used to assign and orient six microsatellites (MS) within a linkage group to chromosome 1. There was an excellent concordance between the physical and linkage assignments. Linear order of MS was identical and relative distances were similar; the mapping coverage of chromosome 1 was estimated at 37%. Also, three previously linked microsatellites were assigned to porcine chromosome 13 using DISC-PCR. The chromosome localizations of the MS were identical to that established by linkage data. The coverage of chromosome 13 was estimated at 75%. Thus, DISC-PCR could rapidly assign and order MS markers for those probes isolated from large genomic clones do not exist.; Two MS identified as Sw920 and Sw19 were in a large, previously unassigned linkage group. DISC-PCR was used to localize the two MS to porcine chromosome 10. Another small, unassigned linkage group was assigned to the distal end of chromosome 6 by physical anchoring two MS in this linkage group using fluorescence in situ hybridization (FISH) procedure. The utility of DISC-PCR and FISH indicates effective methods for physical anchoring of previously marker-deficient regions and extends the coverage of linkage groups in the pig genome.; Six microsatellite-containing cosmid probes were used to anchor the MS to their chromosomal positions. FISH is shown here an accurate, rapid means of transferring MS loci from the swine linkage map to the physical map when large genomic clones containing MS are available.; Mapping of type I loci (coding sequences) provides a skeleton of landmark loci in the gene map. It is valuable for comparative mapping and positional cloning. The porcine Oct-2 gene was assigned to chromosome 6 using the DISC-PCR procedure. The Fas gene was mapped to chromosome 14 by FISH.; Therefore, this study illustrates the development of a new technique, DISC-PCR, and the application of DISC-PCR and FISH for physical gene mapping of the pig genome. The power of direct mapping extremely short PCR-based sequences, expressed sequence tagged sites (ESTs) and sequence tagged sites (STSs), by DISC-PCR could expedite the construction of gene maps of mammalian species. Our results contributes to the partial merging of physical and linkage maps into a comprehensive map, thus opens prospects for the future exploration of mapping economically, agriculturally quantitative trait loci (QTLs). This in turn will facilitate the development of more efficient breeding strategies through marker assisted selection.