Mapping of 55 new rat microsatellite markers from
Laurie A. Shepel,* Hong Lan,* Gerlyn M. Brasic, Megan E. Gheen, Lih-Ching Hsu,** Jill D. Haag,
Michael N. Gould
Department of Human Oncology, University of Wisconsin-Madison, K4/334 CSC, 600 Highland Avenue, Madison, Wisconsin, USA
Received: 11 February 1998 / Accepted: 7 April 1998
Abstract. Fifty-five novel rat microsatellite markers were isolated
from libraries specific for rat chromosomes (Chrs) 1, 2, and 7. The
markers were mapped in three backcross rat populations. Thirty of
these markers mapped to Chrs 1, 2, or 7, while the other 25
mapped to other chromosomes. New markers for two genes, liver-
specific transporter gene (Livtr) and insulin-responsive glucose
transporter (Glut4), were also mapped to rat Chrs 9 and 10, re-
spectively. Three provisionally assigned markers from previous
studies were also confirmed. Detailed methodologies for the gen-
eration and enrichment of clones containing repeat sequences and
for the isolation of chromosome-specific markers are presented,
since they represent unique combinations and modifications of
previous protocols. Such methods and the newly presented mark-
ers should be useful for both specific and general mapping studies
in the rat.
The rat is widely used for biomedical research and as a model for
many human diseases. Since the development and publication of
several rat genetic linkage maps (Bihoreau et al. 1997; Jacob et al.
1995; Serikawa et al. 1992), the rat is now being used for many
genetic studies, particularly for analysis of quantitative trait loci
(QTL) and complex traits. Our laboratory has been using the rat as
a model for the inheritance of factors controlling breast cancer
(Gould 1995; Hsu et al. 1994; Shepel et al. 1998). Prior to the start
of the Rat Genome project (Whitehead Institute/MIT Center for
Genome Research; http://www.genome.wi.mit.edu/rat/public),
which is now expanding the rat genetic map with anonymous
microsatellite or simple sequence repeat (SSR) markers, we also
generated SSR markers in our laboratory to assist in fine-mapping
breast cancer susceptibility genes on Chrs 1, 2, and 7. Such mark-
ers were isolated from chromosome-specific libraries generated
from flow-sorted rat chromosomes, and some of these markers
have been reported previously (Shepel et al. 1997, 1998). The
purpose of the present work is to report the generation and map-
ping of 55 additional, novel, anonymous SSR markers in the rat
genome. The methods used to isolate these markers entail new
combinations and improvements upon previously published pro-
cedures. Additionally, we report the mapping of two genes with
novel SSR markers and confirmation of three provisionally as-
Materials and methods
Animals and genetic crosses.
Copenhagen (COP), Wistar Furth (WF),
Wistar Kyoto (WKY), and Fisher 344 (F344) inbred rats were purchased
from Harlan Sprague-Dawley, Inc. (Indianapolis, Ind.). Three different
genetic backcrosses from other studies (Hsu et al. 1994; Shepel et al. 1998;
Benton, Haag, and Gould, unpublished; Haag et al. 1992; Shepel et al.
1997) were used here for mapping: (i) (WF × COP)F
× WF, n ס 90
(although some genomic regions were mapped in 183 rats); (ii) (WKY ×
× WF, n ס 94; and (iii) (WKY × F344)F
× F344, n ס 79. DNA
samples from individual female backcross rats (as well as parental control
rats) were isolated from tail clips taken at weaning and used for genotype
Source of markers and genetic linkage analysis.
(SSR primer sets) were obtained from Research Genetics (Huntsville,
Ala.), published data, or by collaboration (see Acknowledgments). The
RatMap World Wide Web database (http://rat.gen.gu.se) and the Rat Ge-
nome Project database (see above) contain all previously mapped markers
and original references. Additionally, we generated new SSR markers from
chromosome-specific, small-insert libraries created in our laboratory (see
below). A search of the GenBank database also revealed novel repeat
sequences that were also used to generate new markers. Markers that
yielded differently sized polymerase chain reaction (PCR) products be-
tween different rat strain DNA samples (that is, polymorphic markers)
were then used for linkage analysis.
The genotype of each rat at each marker was determined by PCR as
previously described (Shepel et al. 1998). Two-point linkage analysis was
performed with the MAPMAKER/EXP computer program (Lander et al.
1987; Lincoln and Lander, 1993). Genetic linkage maps were then gener-
ated by multipoint linkage analysis and maximum likelihood methods with
the same program. The Kosambi mapping function (Kosambi 1944) was
used to estimate centimorgan (cM) distances. This function attempts to
model some level of recombinational interference and has been widely
used for genetic mapping in the rat.
Generation of microsatellite markers from chromosome-specific
libraries. Chromosome-specific libraries were generated by using
(i) degenerate oligonucleotide-primed PCR, and (ii) an alternative
method with restriction endonuclease digestion of the chromo-
somes, linker/adapter ligation followed by PCR. The DOP-PCR
method requires only a small quantity of sorted chromosomal ma-
terial with fewer steps and less manipulation than the alternative
method; however, DOP-PCR is prone to background amplification
of any contaminating DNA present in the reaction. With both
methods, enrichment for repeat-containing sequences was accom-
plished with hybridization selection and affinity capture.
1) DOP-PCR for library generation.
Whole chromosomal DNA
template was obtained by flow cytometric sorting of chromosomes from
COP rat embryo cell preparations (Shepel et al. 1994). In that paper, Chr
7 had been identified in peak ‘‘IJ’’ in Fig. 2; however, we subsequently
determined that Chr 7 is actually contained in peak ‘‘H’’ (identified in the
The nucleotide sequence data reported in this paper have been submitted to
GenBank and have been assigned the accession numbers AF045287–
Correspondence to: L.A. Shepel
*Authors contributed equally to this work.
**Present address: University of Utah, Salt Lake City, UT 84112, USA.
Mammalian Genome 9, 622–628 (1998).
© Springer-Verlag New York Inc. 1998