A potential association between the BM 1500 microsatellite and fat
deposition in beef cattle
Carolyn J. Fitzsimmons, Sheila M. Schmutz, Reynold D. Bergen, John J. McKinnon
Department of Animal and Poultry Science, University of Saskatchewan, Saskatoon S7N 5B5, Canada
Received: 27 October 1997 / Accepted: 23 January 1998
Abstract. The obese gene was hypothesized as a candidate gene
for fat characteristics in beef cattle. The BM 1500 microsatellite,
near the obese gene, was characterized in 158 purebred beef bulls
for which carcass trait information was available. Four breeds were
included in the analyses—Angus, Charolais, Hereford, and Sim-
mental. Four alleles were found. Lengths were approximately 138,
147, 149, and 140 bp with genotypic frequencies of 0.47, 0.44,
0.09, and 0.003 respectively. The carcass traits %rib fat, %rib lean,
average fat, and grade fat were found to be significantly associated
with the different alleles. The presence of the 138-bp allele in the
genotype of an animal is correlated with higher levels of fat,
whereas the 147-bp allele has the opposite effect. The 149-bp
allele was found in low numbers, and a homozygote was never
identified. Hereford and Angus bulls had the greatest frequencies
of 138-bp alleles (Hereford ס 0.57, Angus ס 0.59), while Cha-
rolais and Simmental had a greater proportion of 147-bp alleles
(Charolais ס 0.54, Simmental ס 0.58). This information may aid
cattle producers in selecting cattle for markets that differ in the
amount of fat required.
Since the cloning of the obese (ob) gene and its receptor, there has
been a great deal of research activity surrounding this gene (Zhang
et al. 1994; Tartaglia et al. 1995). The 16-kDa protein product of
the ob gene, leptin, has been denoted as the missing link between
energy regulation, hunger, and adipose tissue (Halaas et al. 1995;
Pelleymounter et al. 1995). Therefore, the obese gene could be a
potential candidate gene controlling some proportion of fat depo-
sition in cattle.
The obese gene has been assigned to Chromosome (Chr) 6 in
mice (Friedman and Leibel 1990) and 7q31.3 in humans (Isse et al.
1995). Recently the ob gene has been mapped to Chr 4 in cattle
(Stone et al. 1996b) and Chr 18 in swine (Neuenschwander et al.
1996; Sasaki et al. 1996).
Stone and associates (1996a) located two polymorphic micro-
satellites within a lambda phage genomic clone containing exons 2
and 3 of the bovine ob gene. BM 1500 and BM 1501 are located
approximately 3.6 and 2.5 kb 3Ј of the stop codon, respectively
(Stone et al. 1996a). We chose to investigate the BM 1500 mic-
rosatellite because it exhibited four alleles and was in close prox-
imity to the obese gene. Therefore, this microsatellite could po-
tentially be linked to polymorphisms within the obese gene that
lead to variation in carcass composition. The intent of this inves-
tigation was to determine whether an association existed between
the alleles of the BM 1500 microsatellite and phenotypic charac-
teristics of beef carcasses.
Materials and methods
The cattle population consisted of 158 yearling bulls representing
four breeds: 61 Angus, 57 Charolais, 18 Simmental, and 22 Hereford.
These animals were part of a previous study designed to predict carcass
traits via real-time ultrasound (Bergen et al. 1996). These bulls were raised
at the Beef Research Unit at the University of Saskatchewan, comparable
to a typical Record Of Performance bull testing program (Bergen et al.
1996). Most of the bulls were purchased from a wide variety of ranches and
were, therefore, considered to be unrelated. A moderate energy ration was
fed, and the bulls were slaughtered within 2 weeks of the 112-day test
(Bergen et al. 1996).
Carcass measurements were collected on the chilled car-
cass through the Agriculture and Agri-Food Canada Blue Tag Program
(Bergen et al. 1996). The carcass data of particular interest to us were
average fat, grade fat, %rib fat, %rib lean, and marbling. Average fat (mm)
is the mean value of fat measurements taken at three points along the 12
rib longissimus dorsi muscle. Grade fat (mm) is the narrowest fat depth
over the 4
quadrant of the longissimus dorsi muscle distal to the spine.
%rib fat and %rib lean content were determined via dissection of a 7-bone
rib (6–12th ribs) collected from the left side of each carcass, with the
remainder comprised of bone. Carcass marbling (intramuscular fat) was
visually assessed with values ranging from 0 (devoid of marbling), 1 (trace
marbling), 2 (slight marbling), to 3 (small marbling; Bergen et al. 1996).
Weight at the start and end of the test period and carcass weights were also
DNA was obtained from whole blood via a phenol/
chloroform extraction performed on an Automated Applied Biosystems
340A Nucleic Acid Extractor.
The primers that were developed for the BM 1500
microsatellite by Stone and colleagues (1996a) were synthesized by and
purchased from UCDNA Services at the University of Calgary, Alberta.
The forward and reverse primers were 5ЈGATGCAGCAGAC-
CAAGTGG3Ј and 5ЈCCCATTGCTAGAACCCAGG3Ј respectively. The
end labeling reaction consisted of 2.0 l of 5× forward reaction buffer, 2.0
P] ATP, 2.0 l of reverse primer (10 pmole/l), 2.5 lofdH
and 1.5 l of kinase. This mixture was then left to incubate in a hot water
bath of 37°C for at least 30 min. The PCR cocktail contained 1 lof1
KCl (wt/gel), 0.2 lof1
Tris (pH 8.3), 0.4 lof10m
dNTPs, 0.8 l
of 50 m
, 10 pmoles of forward and reverse primers, 0.12 lofTaq
DNA polymerase, and 14.4 lofdH
O. This mixture was added to 1 lof
DNA per sample to arrive at a total of 20 l. A further 19 l of mineral oil
was added to each sample before the PCR reaction was run. The PCR
thermocycler used was a Thermolyne Temp ⅐ Tronic®. The program con-
sisted of 3 min at 94°C, followed by a cycle that ran 25 times: 94°C for 45
s, 57°C for 30 s, and ending with 72°C for 45 s. The program concluded
with 4 min at 72°C to help complete unfinished reactions.
After the PCR was completed, 19 l of formamide was added to the
samples, which were denatured at 75°C for 2 min and then put on ice.
Samples were loaded, along with an M13 ladder, in a 6% polyacrylamide
gel and run at 40 Watts for approximately 1 h and 45 min. Then the gel was
Correspondence to: C.J. Fitzsimmons
Mammalian Genome 9, 432–434 (1998).
© Springer-Verlag New York Inc. 1998