Linkage mapping of fifty-eight new rat microsatellite markers
Vladimir E. Sverdlov,
* Oksana I. Dukhanina,
* Barbara Hoebee,
John P. Rapp
Department of Physiology and Molecular Medicine, Medical College of Ohio, 3035 Arlington Avenue, Toledo, Ohio 43614-5804, USA
Laboratory of Health Effect Research, National Institute of Public Health and the Environment, Bilthoven 3720 BA, The Netherlands
Received: 24 April 1998 / Accepted: 23 June 1998
Abstract. Fifty-eight new anonymous simple sequence repeats
(SSR) were generated and mapped to various rat chromosomes.
Among them two genes (rat homologs for human cadherin-14 and
mouse fibroblast growth factor-related protein) were mapped on
Chromosomes (Chrs) 2 and 11 respectively. The majority of mark-
ers were generated from a small insert genomic library specific to
Chr 11, 13, 14, and 15. Twenty new markers were mapped to Chr
13, which is known to contain a blood pressure quantitative trait
locus (QTL). Several approaches to obtain microsatellite markers
are described. The protocols and newly generated markers should
be useful for ongoing rat genome project.
The rat, Rattus norvegicus, is an important experimental animal
model for many genetic diseases, especially for multifactorial
traits. Although significant progress has been made in developing
the rat linkage map (Jacob et al. 1995; Bihoreau et al. 1997; Andoh
et al. 1998; RatMap World Wide Web at http://rat.gen.gu.se), there
is a need for dense genetic maps of particular chromosomes and
specific chromosomal regions. High-resolution genetic maps are
needed for successful production of congenic strains and for fine
genetic mapping of specific genes. Markers generated by different
groups are valuable because they supplement each other.
The purpose of this work was to develop new rat microsatellite
markers and to test different approaches for marker production in
ongoing efforts for fine mapping of blood pressure quantitative
trait loci (QTL). Among the chromosomes of particular interest
was the rat Chr 13, which was shown to contain a blood pressure
QTL (Rapp et al. 1989; Zhang et al. 1997).
Materials and methods
Preparation and screening of CA-enriched library.
Rat Chr 13 can-
not be isolated by itself by two-dimensional chromosome sorting, but it is
obtained as a fraction containing Chr 11, 13, 14, and 15 (Hoebee et al.
1994). DNA of this fraction was amplified with a degenerate oligonucleo-
tide primer 6MW, CCGACTCGAGNNNNNNATGTGG (Telenius et al.
1992). The DNA was denatured at 95°C for 3 min in the presence of 10
pmoles of biotinylated oligonucleotide (CA)10 followed by incubation for
5 min at room temperature. Streptavidin paramagnetic particles (SA-PMP;
Promega, Madison, Wis.) were washed three times with 75 m
resuspended in 200 l of that solution. The annealed DNA was added to
SA-PMP, the binding was performed for 10 min at room temperature,
followed by washing four times with 75 m
NaCl. The bound DNA was
eluted two times in 25 l of water (total 50 l), and 1 l was used for
amplification with 6MW oligonucleotide in 50 l. The product of ampli-
fication was separated through 1% agarose gel, and the area of the gel
containing DNA ranging from 300 to 1300 bp was excised. The DNA was
extracted from the gel slice with GeneClean kit (Bio-101, Vista, Calif.) and
eluted in 10 l of water. Eluted DNA was ligated into PCR 2.1 vector (TA
Cloning Kit, Invitrogen, San Diego, Calif.) and used for transformation of
INV␣FЈ One Shot competent cells, provided with the kit. Bacteria were
spread onto ampicillin containing LB-plates with a density of 150–200
colonies per 90-mm plate. Screening was performed by colony hybridiza-
P-labeled (CA)15 oligonucleotide as was described previously
(Dukhanina et al. 1997).
Isolation and analysis of microsatellite-containing plasmids.
cause of the low density of colonies per plate, the colonies showing a
positive hybridization signal were picked and grown in LB media for
plasmid isolation with Qiagen plasmid miniprep kit (Qiagen Inc., Chats-
worth, Calif.) without additional screening. Isolated plasmid DNA was
subjected to restriction analysis to determine the sizes of the inserts. DNA
sequencing was done either manually with Thermo sequenase radiolabeled
terminator cycle sequencing kit (Amersham Life Science, Cleveland, Ohio)
or using a kit from Epicentre Technologies (Madison, Wis) and an auto-
mated DNA sequencer Li-COR model 4000L (Lincoln, Neb.). The se-
quence from each clone was compared with the GenBank database to
eliminate those containing repetitive elements (LINE, B1, B2, ID). The
remaining sequences were used for primer design with the program Primer-
Select from the DNASTAR (Madison, Wis.).
Generation of markers by primer walking.
Often we encounter the
situation in which a genetic marker is in a useful map location, but it is not
polymorphic between the rat strains of interest. In order to find informative
markers in such strategic positions, the Genome Walker Kit for the rat
(Clontech Laboratories, Palo Alto, Calif.) was used to find polymorphic
markers adjacent to known markers. This kit consists of the ‘‘libraries’’ of
DNA fragments prepared by digestion with different restrictases and li-
gated with a special adaptor (primer-adaptor). The libraries are used as
templates for PCR with the primer specific for the adaptor (AP1) and a
sequence-specific primer. The first PCR is followed by a second PCR with
nested primers [specific to the adaptor (AP2) and sequence-specific prim-
ers]. Sequence information around microsatellites of known map position
was used to design the two required sequence-specific primers (25–33
nucleotides in length) for walking upstream or downstream from the mi-
crosatellites. The presence of a microsatellite in the newly amplified DNA
was determined by Southern blot-hybridization with a mixture of
labeled (CA)15 and (CT)15 oligonucleotides. The bands showing a hy-
bridization signal were excised from the agarose gel, and the DNA was
purified and cloned into pCR 2.1 vector as described above. The plasmids
obtained were sequenced, and primers around new microsatellites were
designed for the analysis of polymorphisms.
Generation of markers by amplification with microsatellite-
In attempts to find markers in the regions of blood
pressure QTL, we used the modification of the method of primer walking
described by Broude et al. (1997) and Sverdlov et al. (1998). Briefly,
‘‘libraries’’ of uncloned restriction fragments ligated with primer-adaptor
were amplified in separate reactions with the AP1 primer and a second
BV; where B ס A, T, or G; and V ס A, G, or C. We choose
* Authors contributed equally to this work.
Correspondence to: O.I. Dukhanina
The GenBank accession numbers for the nucleotide sequences reported in
this paper are: AF005929, AF045890, AF052311, AF052335–AF052366,
AF052368–AF052378, AF052500, AF052501, AF052731, AF054152–
AF054158, AF054161, AF056086.
Mammalian Genome 9, 816–821 (1998).
© Springer-Verlag New York Inc. 1998