Construction of a high-throughput rat genetic mapping system with
466 arbitrarily primed-representational difference analysis markers
Satoshi Yamashita, Yukinari Yoshida, Ayako Kurahashi, Takashi Sugimura, Toshikazu Ushijima
Carcinogenesis Division, National Cancer Center Research Institute, 1-1 Tsukiji 5-chome, Chuo-ku, Tokyo 104-0045, Japan
Received: 28 April 2000 / Accepted: 14 June 2000
Abstract. Linkage mapping of quantitative trait loci (QTLs) re-
quires genetic markers that can be efficiently genotyped for a large
number of individuals. To isolate genetic markers suitable for this
purpose, we previously established the arbitrarily primed RDA
(AP-RDA) method. Dot-blotting AP-PCR products (AP-
amplicons) onto filters at a high density and hybridization of the
filters with the AP-RDA markers made it possible to genotype a
large number of individuals simultaneously for multiple loci. In
this study, by using 25 primers or primer combinations, we iso-
lated a total of 419 AP-RDA markers by subtracting the AP-
amplicon of BUF rats from that of ACI rats, and vice versa. By
combining 47 previously isolated markers, a rat genetic map was
drawn with 466 AP-RDA markers. Between two given strains of
rats other than ACI and BUF, the average informativeness of the
markers was 38%. As for the intercross of ACI and BUF rats, 12
selected primers served to genotype 259 loci. In addition, the
amounts and quality of genomic DNA to be used for AP-PCR were
examined to guarantee reliable genotyping. Now, initial genome
scanning of the rat for linkage analysis can be performed effi-
ciently using this mapping system with AP-RDA markers.
Linkage mapping of quantitative trait loci (QTLs), which control
quantitative traits such as blood pressure, blood glucose level, and
cancer susceptibility, is a growing and important field. Some ex-
amples include the analysis of 321 mice that revealed QTLs in-
volved in airway hyperresponsiveness (De Sanctis et al. 1995) and
the analysis of 879 mice that revealed QTLs involved in emotion-
ality (Flint et al. 1995). These indicate that a qualitative change of
findings obtained by genetic analysis can be brought about by a
leap in the number of animals that can be analyzed. However, the
microsatellite markers, which are serving as the de facto standard
for polymorphic markers these days (Dietrich et al. 1996; Jacob et
al. 1995; Watanabe et al. 1999), require an individual PCR reaction
and subsequent electrophoresis for each animal for each locus.
Therefore, the labor and cost of genotyping increase linearly with
the increase in the number of animals to be genotyped.
From this point of view, we previously developed the arbi-
trarily primed-representational difference analysis (AP-RDA)
method (Fig. 1) and isolated 47 rat AP-RDA markers (Yoshida et
al. 1999). AP-RDA markers are isolated by subtracting the AP-
PCR product (AP-amplicon) of one strain (driver) from that of
another strain (tester) with a genomic subtraction method, RDA
(Lisitsyn and Wigler 1993). Therefore, the markers are derived
from the sequences present in the tester but not in the driver. These
markers detect the presence and absence of hybridization signals in
the AP-amplicons from the tester and driver strains, respectively,
and the presence or absence of signals in each animal of a back-
cross or intercross population. The first advantage of AP-RDA
markers is that a large number of animals can be genotyped with-
out electrophoresis by hybridizing a small piece of filter on which
their AP-amplicons have been dot-blotted at a high density. The
second advantage is that one AP-amplicon can serve to genotype
multiple loci, which means that the number of PCR reactions can
be greatly reduced by using AP-RDA markers. In our initial study,
one AP-amplicon served to genotype an average of 9.4 loci
(Yoshida et al. 1999). The third advantage is that unlimited num-
bers of AP-RDA markers can be isolated by changing the sequence
of the primers for AP-PCR.
To achieve efficient genotyping of a large number of individu-
als, a complete genetic map consisting of AP-RDA markers is
necessary. In the present study, we newly isolated 419 AP-RDA
markers. By integrating our 47 previous markers, a rat genetic
linkage map was constructed with 466 AP-RDA markers. The
informativeness of the markers in various rat strains and the qual-
ity of DNA for accurate genotyping were also examined. The
genetic mapping system reported here will achieve efficient ge-
nome scanning of the rat.
Materials and methods
Animals and DNA extraction.
ACI/N (ACI), BUF/Nac (BUF), and
Jcl:Wistar (Wistar) strains were purchased from Japan Clea Inc. (Tokyo,
Japan); Donryu (DON) from Charles River, Japan (Yokohama, Japan);
BN/CrlBR (BN) and F344/CrCrlBR (F344) from Charles River Italia
(Como, Italy); LEW/SsNHsd (LEW) from Harlan UK Limited (Oxon,
UK); and Crl:CD(SD) from Charles River (Wilmington, Mass.). DNAs of
LEC and WKAH were provided by M.C. Yoshida (Hokkaido University).
DNA of WTC/Kyo (WTC) was provided by T. Serikawa (Kyoto Univer-
sity). Genomic DNA was extracted from the liver by the phenol and chlo-
roform extraction method, or from the tail by GENEXTRACTOR TA-100
(Takara Shuzo Co., Kyoto, Japan).
Isolation of AP-RDA markers.
AP-RDA was performed as previously
described (Yoshida et al. 1999) with the primers summarized in Table 1.
To prepare AP-amplicons, 400 ng of genomic DNA was amplified in a
400-l reaction mixture by AP-PCR with 3 min initial denaturation at 94°C
and 35 cycles of 1 min denaturation at 94°C, 1 min annealing at 40°C, and
2 min extension at 72°C. Amplicons of both tester and driver were digested
with BamHI endonuclease (New England Biolabs, Beverly, Mass.), and
purified by gel-filtration chromatography (CHROMA SPIN+TE-200;
CLONTECH Laboratories, Palo Alto, Calif.). JBam adaptor (Lisitsyn and
Wigler 1993) was ligated only to the tester amplicon. The tester DNA with
the JBam adaptor was mixed with an excess amount of the driver DNA.
The DNA mixture was denatured and then allowed to reanneal. The rean-
nealed product was subjected to amplification by PCR. The second cycle
of competitive hybridization was performed by switching the JBam adaptor
to the NBam adaptor.
The PCR product after the second cycle of competitive hybridiza-
tion was inserted into the BamHI site of pBluescript II KS (+) phagemid
vector (Stratagene, La Jolla, Calif.). After transformation, inserts
Correspondence to: T. Ushijima; E-mail: firstname.lastname@example.org
Mammalian Genome 11, 982–988 (2000).
© Springer-Verlag New York Inc. 2000