Development of the arbitrarily primed-representational difference analysismethod and chromosomal mapping of isolated high throughput rat genetic markers

Linkage mapping of quantitative trait loci requires analysis of a large num ber of animals. Although genetic markers isolated by representational diffe rence analysis (RDA) and its modifications meet the needs, the number of th ese markers has been limited. In the present study, we established the arbi trarily 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 o f 40 polymorphic and independent markers were isolated in seven series of A P-RDA using a single primer. Two series of AP-RDA with primer combination y ielded seven additional independent markers. All of the markers gave clear positive/ negative signals by hybridization of a filter where AP-amplicons from F-2 rats of ACI and BUF were dot-blotted at a high density without any concentration or purification. All of the 47 independent markers were mapp ed to unique chromosomal positions by linkage analysis, even though some ar bitrary primers had very similar sequences, The markers were also informati ve between other strains of rats. Simultaneous hybridization of multiple fi lters made it possible to genotype a large number of rats simultaneously fo r multiple genetic loci, The AP-RDA method promises isolation of a large nu mber of high throughput genetic markers in any species and is expected to f acilitate linkage mapping of subtle quantitative trait loci.