Linkage mapping of rat Chromosome 5 markers generated from
Hong Lan, Laurie A. Shepel, Jill D. Haag, Michael N. Gould
McArdle Laboratory for Cancer Research, University of Wisconsin Medical School, Madison, Wisconsin, 53706, USA
Received: 17 December 1998 / Accepted: 17 February 1999
Abstract. Seventy-six novel microsatellite markers with various
simple sequence repeat (SSR) motifs are reported in this paper.
They were generated on the basis of non-radioactive library
screening procedures from flow-sorted rat Chromosome (Chr)
5-specific DNA, and were mapped in three rat backcross popula-
tions. Fifty-four of these markers mapped to Chr 5, while the other
22 mapped to other chromosomes of the rat genome. The marker
D3Uwm8 is a new microsatellite marker for the rat syndecan 4
(ryudocan) gene. A genotyping protocol based on agarose gel elec-
trophoresis is also provided in this paper.
Rat Chr 5 is known to carry several interesting quantitative trait
loci (QTL), including the loci for blood pressure (Deng et al. 1994;
Zhang et al. 1997), polycystic kidney disease (Bihoreau et al.
1997a), and ischaemic stroke (Jeffs et al. 1997). It is believed that
identification of genes controlling such traits, by way of positional
cloning, will elucidate the possible molecular mechanisms or path-
ways affected in human diseases.
A dense genetic map is a prerequisite for positional cloning.
Today the majority of rat genetic markers (including Mgh, Mit, and
Rat markers) come from the Rat Genome Project at the Whitehead
Institute for Biomedical Research/MIT Center for Genome Re-
search (http://www.genome.wi.mit.edu/rat/public). Before the Rat
Genome Project started in 1996, however, several research groups
including our laboratory had started to generate SSR markers for
QTL mapping, such as the recently published Wox markers
(Gauguier et al. 1996; Bihoreau et al. 1997b), Arb markers (http://
www.nih.gov/niams/scientific/ratgbase), Mco markers (Gu et al.
1996; Deng et al. 1997a, 1997b), Uwm markers (Shepel et al. 1997,
1998a, 1998b), and some site-specific markers (for example, Ker-
shaw et al. 1995). Here we report 54 new anonymous SSR markers
on rat Chr 5 that were generated from Chr 5-specific libraries.
These markers add substantially to the total number of existing
markers and are potentially useful for fine-mapping and positional
cloning of genes on rat Chr 5.
Materials and methods
Construction and screening of rat Chr 5-specific libraries.
sorted rat Chr 5 DNA (Shepel et al. 1994) was amplified according to a
modified protocol of degenerate oligonucleotide-primed PCR (DOP-PCR)
developed in our laboratory (Primers 1 and 2 and Protocol 2; Shepel et al.
1998b). The PCR products were subjected to either one of the following
two strategies: 1. select for (CA)
repeats by hybridization/affinity capture,
subsequently clone into pAMP10 vector (Life Technologies Inc., Gaithers-
burg, Md.), then screen by Colony PCR, as described before (Shepel et al.
1998b); 2. directly clone into pAMP10 vector and screen for (CA)
other simple sequence repeats by non-radioactive colony hybridization as
detailed below. Positive clones resulting from both strategies were se-
quenced to generate microsatellite markers.
The colony hybridization protocol was based on the DIG Nucleic Acid
Labeling and Detection System of Boehringer-Mannheim Corp. (India-
napolis, Ind.). Briefly, clones were grown at 37°C overnight on 150-mm
LB agar plates containing 100 g/ml ampicillin, and colonies were copied
onto nylon transfer membranes (Boehringer-Mannheim). The membranes
were heated in a microwave oven at high power for 150 s (Jones et al.
1989), followed by treatment with denaturation and neutralization solutions
(Sambrook et al. 1989). DNA was then fixed with the Auto Cross Link
setting of UV Stratalinker 2400 (Stratagene, La Jolla, Calif.).
Oligonucleotides of SSRs, (CA)
, and (ATTT)
were synthesized at the Uni-
versity of Wisconsin Biotechnology Center (Madison, Wis.). The repeat
motifs were chosen based on their abundance in the rat genome (Beckmann
and Weber 1992). One hundred pmol of each oligonucleotide were labeled
with digoxigenin with the DIG Oligonucleotide 3Ј-end Labeling Kit (Boeh-
ringer-Mannheim). Fifty pmol of each of these 16 end-labeled oligonucle-
otides were mixed and used as combined probes for membrane hybridiza-
tion. Hybridizations were performed at 42°C overnight in 10 ml DIG Easy
Hyb (Boehringer-Mannheim), a formamide-free hybridization solution.
After hybridization, the membranes were washed2×5minin2×SSC,
0.1% SDS at room temperature and2×15minin0.1×SSC, 0.1% SDS
at 42°C under constant agitation. Positive clones were visualized with the
DIG Nucleic Acid Detection Kit (Boehringer-Mannheim) according to the
manufacturer’s procedure. A washing temperature of 42°C was used in-
stead of the conventional 68°C in order to retain background signals for all
the colonies, which procedure helps to locate positive clones on the plates.
A further step of size selection was performed for positive clones.
Clones were transferred into 100 l LB liquid culture in 96-well culture
plates (Corning Costar Corp., Cambridge, Mass.). For each clone, 1 lof
overnight culture was used directly as DNA template for colony PCR with
M13 forward and reverse primers to estimate the insert size. Clones with
an insert size smaller than 150 bp were abandoned because there was not
enough flanking sequence to design PCR primers. Clones larger than 1 kb
were also abandoned. In addition, when multiple clones showed identical
insert sizes, only one of them was sequenced in order to eliminate possible
Generating PCR primers.
Plasmid DNA of positive clones was ex-
tracted by the alkaline-lysis method with the QIAGEN plasmid purification
system (Qiagen Inc., Valencia, Calif.). DNA sequences of the clone inserts
were determined with the PRISM Dye-Terminator fluorescent sequencing
system (PE Applied Biosystems, Foster City, CA). DNA sequencing was
performed at the University of Wisconsin Biotechnology Center. The insert
sequence data were analyzed with the Wisconsin Package Version 8.2
[Genetics Computer Group (GCG), Madison, Wis]. In order to eliminate
duplicate clones, a FASTA search was performed for each clone against a
local database containing all the clone sequences from this study. For
unique clones, PCR primers were designed to span the SSRs by use of the
Wisconsin Package software. All primers were synthesized at Research
Genetics (Huntsville, Ala.), from which primers are available to the public.
The nucleotide sequence data reported in this paper have been submitted to
GenBank and have been assigned the accession numbers AF096515-
Mammalian Genome 10, 687–691 (1999).
© Springer-Verlag New York Inc. 1999