Characterization of 463 Type I markers suitable for dog
Catherine Priat,* Zhihua H. Jiang,*
** Corinna Renier, Catherine Andre´, Francis Galibert
Laboratoire de Biochimie et Biologie Mole´culaire, UPR41CNRS “Recombinaisons Ge´ne´tiques”, Faculte´deMe´decine, 2 avenue du Professeur Le´on
Bernard, 35043 Rennes cedex, France
Received: 5 January 1999 / Accepted: 13 April 1999
Abstract. In total, 463 canine gene markers were identified and
characterized to serve as reagents in canine genome map projects.
These markers are distributed over 221 canine gene markers, 139
TOASTs (Traced Orthologous Sequence Tags), 27 canine
TOASTs, and 76 huESTs (human Expressed Sequence Tags). Out
of 310 canine gene markers, 59%–84% were successfully ampli-
fied on dog DNA, the highest rates of success being observed
when the exon/intron structure is known. Concerning TOASTs and
human ESTs, of the 225 and 300 markers analyzed, 62% and 25%
respectively were able to produce a dog positive amplification. As
part of an ongoing project to map the canine genome using a
dog/hamster radiation hybrid panel, these markers were tested for
their specificity on dog versus hamster DNA. Thus 61%, 21%, and
12% of dog gene markers, TOASTs, and huESTs met the criteria
required for radiation hybrid mapping, respectively. All of these
463 canine gene markers, however, are available and will be of
value to any other mapping strategies.
Of all mammals, Canis familiaris is without any doubt the species
that presents the highest level of polymorphism from both a mor-
phological and behavioral point of view. Numerous controlled
crossbreeds, carried out by breeders over the last few hundred
years, have led to the creation of more than 300 different canine
breeds. These breeding practices, implying a high level of inbreed-
ing, have contributed to the specific fixation within a given breed
of particular alleles that determine the phenotypic characteristics
of that breed (Serpell 1995). This results in a decrease in genetic
variability in each breed and also in the segregation of alleles
conferring susceptibility to recessive genetic diseases (Denis
1997). Many of these are often very close to those encountered in
human medicine, such as Duchenne’s muscular dystrophy, which
is linked to the X Chromosome (Chr) (Sharp et al. 1992), or
haemophilia (Mauser 1996). Genetic analysis of these breeds could
thus prove useful to identify candidate genes to facilite diagnosis
and therapy in human (Ostrander and Giniger 1997; Galibert et al.
Regardless of the animal species under consideration, drawing
up an integrated genomic map with Type I (gene) and Type II
(microsatellite) markers is a necessary step to identify and char-
acterize genes and alleles that determine pathological, morphologi-
cal, and behavioral characteristics. Genomic markers, mostly mi-
crosatellites, have already been characterized (Ostrander et al.
1993, 1995; Holmes et al. 1993, 1995; Francisco et al. 1996) and
mapped with either genetic linkage (Lingaas et al. 1997; Mellersh
et al. 1997; Werner et al. 1997; Neff et al. 1999) or radiation hybrid
strategies (Priat et al. 1998).
In the present study, 463 Type I markers were characterized.
These markers constitute essential tools for mapping the canine
genome with a view to identifying candidate genes and to per-
forming comparative genomic studies. The development of these
markers—canine genes, TOASTs (Traced Orthologous Amplified
Sequence Tags), canine TOASTs and huESTs (human Expressed
Sequence Tags)—was carried out by different strategies. Firstly,
the canine gene sequences present in databases were used to de-
velop more than 200 specific canine gene markers. Unfortunately,
the number of known canine gene sequences is fairly low com-
pared with human and mouse. In the light of this, TOAST markers
were developed by comparing different orthologous genes, one at
least being known at the genomic level, and primers were designed
from the conserved regions. This strategy revealed universal prim-
ers that were subsequently used to generate canine genomic mark-
ers (Jiang et al. 1998), after adjusting the amplification parameters
on dog DNA and sequencing the amplified product. Finally, hu-
man ESTs (huESTs), which have been developed during the map-
ping of the human genome (Gyapay et al. 1996), represent an
important source of orthologous markers. Since most of these have
already been localized within the human, murine, or pig maps,
their mapping in the dog allows comparative genomic studies to be
carried out and, consequently, the identification of conserved syn-
For mapping these markers on a dog/hamster radiation hybrid
panel, their ability to generate a dog-specific PCR product was
tested on a mixture of both canine and hamster DNA. Approxi-
mately half of the markers met this criterion, and 208 of these were
successfully mapped on the dog/hamster RHDF
panel (Priat et
al. 1998; Vignaux et al. 1999 a, b).
It should be noted that all 463 markers examined are suitable
for dog genome mapping, with techniques involving only dog
DNA, such as physical mapping methods.
Materials and methods
Identification and characterization of canine gene markers.
data generated by the PRIMER 0.5 program (Genetic Computer Group,
1994), forward and reverse primers were designed on the base of canine
sequences present in Genbank database. The selection criteria were as
follows: a primer length from 18 to 25 bp, GC content exceeding 50%,
optimal Tm of 60°–65°C, and production of PCR products of 100–300 bp.
Optimal PCR conditions were determined with 50 ng of canine DNA in a
total volume of 10 l containing 15 ng of each primer, 200
KCl, 10 m
Tris-HCl, and 0.5 U Taq Polymerase (Perkin
Elmer). Amplifications were performed in PTC-200 thermocyclers (MJ Re-
search, Cambridge, Mass.) with the following program: denaturation
* Both authors contributed equally to the work.
** Present address: Centre for Genetic Improvement of Livestock, De-
partment of Animal and Poultry Science, University of Guelph, Guelph,
Ontario, N1G 2W1, Canada.
Correspondence to: C. Andre´
Mammalian Genome 10, 803–813 (1999).
© Springer-Verlag New York Inc. 1999