Linkage mapping of quantitative
trait loci (QTLs) 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. 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 (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) rats 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. In addition, two series of AP-RDA with primer combination yielded seven
additional independent markers. All 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 the 47 independent markers were mapped to unique chromosomal positions by
linkage analysis, even though some arbitrary primers had very similar sequences.
AP-RDA was performed using the AP-amplicon from BUF as the tester and that from
ACI as the driver to isolate markers that can be used for genotyping in ACI
x (ACIxBUF)F1 backcross rats. 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 QTLs. (Y. Yoshida et al.Proc. Natl. Acad. Sci. USA, 96, 610-615 (1999).)
