Development of the arbitrarily primed-representational difference analysis method and chromosomal mapping of isolated...

Linkage mapping of quantitative trait loci requires analysis of a large number of animals. Although genetic markers isolated by representational difference analysis (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 ACI/N (ACI) or BUF/Nac (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 positive/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 chromosomal positions by linkage analysis, even though some arbitrary 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.