Improved linkage map and thirty new microsatellite markers for rat
Oksana I. Dukhanina,*
Vladimir E. Sverdlov,*
John P. Rapp
Department of Physiology and Molecular Medicine, Medical College of Ohio, 3035 Arlington Avenue, Toledo, Ohio 43614-5804, USA
National Institute of Public Health and the Environment, Laboratory of Health Effects Research, Bilthoven 3720 BA, The Netherlands
Received: 13 July 1998 / Accepted: 9 September 1998
Abstract. An improved linkage map for rat Chromosome (Chr)
10 with two F
populations was constructed. Thirty new micro-
satellite markers were generated from a Chr 10-specific, small-
insert genomic library and mapped to rat Chr 10. Among them
were the rat homologs for the mouse gene for light and heavy
chains of myeloperoxidase and human neurofibromatosis 1. Eight
newly generated markers (D10Mco62, D10Mco63, D10Mco64,
D10Mco65, D10Mco67, D10Mco68, D10Mco70, and D10Mco74)
were mapped to the region of the rat Chr 10 blood pressure QTL.
The availability of such markers may be instrumental in the search
for genes responsible for the hypertension.
The rat Chr 10 is of special interest because it contains one of the
major blood pressure quantitative trait loci (QTL) identified in
crosses involving stroke-prone, spontaneously hypertensive rats
(SHRSP) (Jacob et al. 1991; Hilbert et al. 1991) and Dahl salt-
sensitive rats (Deng and Rapp 1992, 1995; Dukhanina et al. 1997a;
Garrett et al. 1998). Although the rat linkage map is well devel-
oped (for example, see RatMap World Wide Web database at
http://rat.gen.gu.se), the need for a dense genetic map still persists.
The purposes of this article are to find new markers and further
improve the genetic linkage maps for rat Chr 10 for the F
MNS) and F
(S × LEW) populations in which the existence of
blood pressure QTL was confirmed by congenic strains
(Dukhanina et al. 1997a; Garrett et al. 1998).
Materials and methods
Preparation and screening of CA-enriched library.
DNA of rat Chr
10 was isolated by two-dimensional chromosome sorting and amplified
with a degenerate oligonucleotide primer 6MW, CCGACTC-
GAGNNNNNNATGTGG (Telenius et al. 1992; Hoebee et al. 1994). The
preparation of the microsatellite-enriched library was done essentially as
described previously (Sverdlov et al. 1998b; Dukhanina and Sverdlov
1998). Briefly, the Chr 10-amplified DNA was denaturated at 95°C for 3
min in the presence of 10 pmoles of biotinylated oligonucleotide (CA)
followed by incubation for 5 min at room temperature. Streptavidin para-
magnetic particles (SA-PMP) (Promega, Madison, Wis.) were prepared,
and binding and elution of annealed DNA was performed according to the
manufacturer’s recommendation except that the manufacturer’s binding
and washing buffer was changed from 0.5 × SSC and 0.1 × SSC to a 300-
NaCl solution. The microsatellite-enriched DNA was reamplified and
size-fractionated by agarose electrophoresis. The fraction 300–1300 bp was
extracted from the gel with GeneClean kit (Bio-101, Vista, Calif.) and used
for the preparation of the library. Screening of the library obtained was
performed by colony hybridization with
tide as was described previously (Dukhanina et al. 1997a).
Isolation and analysis of microsatellite-containing plasmids.
hybridization, plasmid DNA was isolated from the colonies showing a
positive hybridization signal with Qiagen plasmid miniprep kit (Qiagen
Inc., Chatsworth, Calif.). DNA sequencing was done by standard ap-
proaches with either the Thermo sequenase radiolabeled terminator cycle
sequencing kit (Amersham Life Science, Cleveland, Ohio) or the sequenc-
ing kit from Epicentre Technologies (Madison, Wis.) and an automated
DNA sequencer Li-COR model 4000L (Lincoln, Neb.). The sequence from
each clone was compared with the GenBank database to eliminate known
sequences and to identify those containing LINE, B1, B2, ID elements. The
remaining sequences were used for primer design with the program Prim-
erSelect from the DNASTAR (Madison, Wis.).
To obtain sequence beyond a microsatellite that was
close to the end of its clone, we used the GenomeWalker Kit for the rat
(Clontech Laboratories, Palo Alto, Calif.). This kit allows amplification of
the specific DNA fragments beyond the known sequence (‘‘primer walk-
ing’’). The use of this kit for obtaining new microsatellites and sequence
beyond microsatellites was described by us earlier (Dukhanina et al. 1997a;
Sverdlov et al. 1998a). As a result of primer walking, several new DNA
fragments adjacent to the microsatellites were obtained and sequenced. A
primer from the region beyond the microsatellite was designed from the
new sequence data, and the polymorphic status of the microsatellite was
Analysis of polymorphism and linkage of new markers.
were designed to give a specific product of amplification under standard
PCR conditions (initial denature at 95°C for 1.5 min followed by 30 cycles
of 95°C for 40 s, 55°C for 40 s, 72°C for 2 min in the PCR buffer
containing 1.5 m
). The ratio of unlabeled/labeled primer was 10:1,
which resulted in a short time of autoradiographic exposure (1–2h).
Rat strains and populations used for mapping.
The markers obtained
were tested for polymorphism among the following inbred rat strains: Dahl
salt-resistant rats (SR/Jr) and Dahl salt-sensitive rats (SS/Jr) were from our
colony at the Medical College of Ohio and will be referred to hereafter as
R and S rats, respectively; Milan normotensive strain (MNS) was obtained
from the Genetic Resource Section of the National Institutes of Health
(Bethesda, Md.); Lewis rats (LEW) were obtained from Charles River
Laboratories; Albino Surgery (AS) was obtained from C. Heatherington,
National Institute for Medical Research, The Rigeway, Mill Hill, UK;
Wistar-Kyoto (WKY), Brown Norway (BN), and Spontaneously Hyper-
tensive rats (SHR) were obtained from Harlan Sprague-Dawley, Indianapo-
The majority of markers were genotyped in F
(S × LEW) and
(S × MNS) populations. Markers D10Mco46 and D10Mco69 were geno-
typed in an F
(S × WKY) population, and markers D10Mco47 and
D10Mco53 were genotyped in an F
(S × BN) population. All these popu-
Correspondence to: J.P. Rapp
The GenBank accession numbers for the nucleotide sequences reported
in this paper are: AF052306-AF052310, AF052312-AF052334, AF054159,
*Authors contributed equally to this work.
Mammalian Genome 10, 26–29 (1999).
© Springer-Verlag New York Inc. 1999