Development of the arbitrarily primed-representational difference analysis method and chromosomal mapping of isolated high throughput rat genetic markers (arbitrarily primed-PCRypolymorphic markersyrat genome)

Linkage mapping of quantitative trait loci requires analysis of a large number of animals. Although genetic markers isolated by representational difference anal- ysis (RDA) and its modifications meet the needs, the number of these markers has been limited. In the present study, we established the arbitrarily primed (AP)-RDA method to isolate virtually an unlimited number of the high throughput genetic markers. A representation of the genome, an AP- amplicon, was prepared by AP-PCR with a single primer or with a combination of primers using genomic DNA of the ACIyN (ACI) or BUFyNac (BUF) rat as a template. By subtracting the AP-amplicon of ACI from that of BUF, a total of 40 polymorphic and independent markers were isolated in seven series of AP-RDA using a single primer. Two series of AP-RDA with primer combination yielded seven additional independent markers. All of the markers gave clear positivey negative signals by hybridization of a filter where AP- amplicons from F2 rats of ACI and BUF were dot-blotted at a high density without any concentration or purification. All of the 47 independent markers were mapped to unique chromo- somal positions by linkage analysis, even though some arbi- trary primers had very similar sequences. The markers were also informative between other strains of rats. Simultaneous hybridization of multiple filters made it possible to genotype a large number of rats simultaneously for multiple genetic loci. The AP-RDA method promises isolation of a large number of high throughput genetic markers in any species and is expected to facilitate linkage mapping of subtle quantitative trait loci.