Detection of QTL for milk production on Chromosomes 1 and 6 of
* Yves Plante,
* John P. Gibson
Centre for Genetic Improvement of Livestock, Department of Animal and Poultry Science, University of Guelph, Guelph, Ontario, Canada N1G 2W1
Bova-Can Laboratories, Saskatchewan Research Council, 15 Innovation Blvd., Saskatoon, Saskatchewan, Canada S7N 2X8
Received: 19 November 1999 / Accepted: 31 August 2000
Abstract. Seventy to 75 sons of each of six Holstein sires were
assayed for genotypes at a number of microsatellite loci spanning
Chromosomes (Chrs) 1 and 6. The number of informative loci
varied from three to eight on each chromosome in different sire
families. Linkage order and map distance for microsatellite loci
were estimated using CRI-MAP. Estimates of QTL effect and
location were made by using a least squares interval mapping
approach based on daughter yield deviations of sons for 305-d
milk, fat, protein yield, and fat and protein percentage. Thresholds
for statistical significance of QTL effects were determined from
interval mapping of 10,000 random permutations of the data across
the bull sire families and within each sire family separately.
Across-sire analyses indicated a significant QTL for fat and pro-
tein yield, and fat percentage on Chr 1, and QTL effects on milk
yield and protein percentage that might represent one or two QTL
on Chr 6. Analyses within each sire family indicated significant
QTL effects in five sire families, with one sire possibly being
heterozygous for two QTLs. Statistically significant estimates of
QTL effects on breeding value ranged from 340 to 640 kg of milk,
from 15.6 to 28.4 kg of fat, and 14.4 to 17.6 kg of protein.
It has long been recognized that genetic markers could be used to
detect and track the inheritance of polymorphisms contributing to
genetic variation, known as quantitative trait loci (QTLs). The
discovery of highly polymorphic microsatellite markers (Litt and
Luty 1989) and the subsequent development of reasonably dense
microsatellite linkage maps for the bovine genome (Bishop et al.
1994; Barendse et al. 1994, 1997) have made marker mapping of
QTLs a practical reality. Several studies recently have reported the
presence of significant QTLs affecting milk production traits on
several different chromosomes (e.g., Georges et al. 1995; Spelman
et al. 1996; Lipkin et al. 1998; Zhang et al. 1998; Velmala et al.
1999). The sizes of QTL effects being discovered are more than
sufficient to warrant their use in selection programs, particularly
for the pre-selection of young bulls entering progeny testing (Ka-
shi et al. 1990; Gomez-Raya and Gibson 1993).
We report here on the use of microsatellite markers to map
QTLs on Chr 1 and 6 that contribute to variation in milk produc-
tion traits in six Holstein sire families with a total of 434 sons
progeny tested in Canada. The results confirm the existence of
several QTLs detected in previous studies as well as detecting
QTLs previously unreported.
Materials and methods
In total, 434 sons of six prominent grandsires, with 71–75 sons
per grandsire, were genotyped at eight and seven microsatellite loci on Chr
1 and 6. Least squares interval mapping was performed based on daughter
yield deviations of the sons for 305-day milk, fat, and protein yield, and fat
%, and protein %.
Semen straws (250–500 l) were thawed at room
temperature and emptied into 1.5-ml Eppendorf tubes. The semen was
washed three times in1×SSC,2m
EDTA to remove the cryoprotectant.
Cells were resuspended in 1.0 ml TE buffer (pH 8.0) containing 100 g
Proteinase K and incubated at 65°C for1htolyse potentially contami-
nating epithelial cells. After centrifugation (12,000 rpm for 2 min), the
sperm cells were resuspended and lysed into 500 l of 100 m
(pH 8.0), 10 m
EDTA, 500 m
NaCl, 1% SDS, 2% ␤-mercaptoethanol,
400 g proteinase K, and incubated for 16–24 h at 65°C. The lysates were
extracted three times with phenol–chloroform–iso-amyl alcohol (25:24:1)
and then three times with chloroform–iso-amyl alcohol (24:1). DNA was
recovered by precipitation in the presence of 0.3
sodium acetate and two
volumes of 95% ethanol. DNA was spooled onto a small glass rod, washed
in 70% ethanol, air dried, and resuspended in an appropriate volume of TE
buffer (pH 8.0). DNA concentrations were estimated by spectrophotometry
(O.D. 260 nm). DNA samples were stored at 4°C until needed.
Polymorphic bovine microsatellites were selected based on
their map positions on Chrs 1 and 6. Each marker was assayed individually
for annealing temperature and Mg
concentration. Template DNA (50 ng)
was initially denatured at 94°C for 5 min, followed by the addition of 20
pmol of each primer, 0.5 pmol kinase end-labeled (␥-
each of dNTP, 10 m
Tris-HCl pH 9.0, 1.5–3.0 m
(depending on the primer pair), 50 m
KCl, 0.01% vol/wt gelatin, 0.1%
Triton X-100, and 0.3 unit Taq DNA polymerase (Sigma) in a total volume
of 12.5 l. Samples were subjected to 30 cycles of amplification, each
cycle consisting of 30 s of denaturation at 94°C, 30 s annealing at 55°C–
68°C (depending on the primer pair), and 30 s extension at 72°C. A final
10-min extension step at 72°C was added at the end of the 30 cycles of
amplification to insure complete extension of the PCR products.
Amplification reactions were stopped by the addition of 12.5 lof
sequencing stop buffer and denatured at 94°C for 5 min. Aliquots of 2.0 l
were loaded into 6% denaturing (7
urea) polyacrylamide gels alongside
an M13 sequencing ladder. Sequencing gels were electrophoresed at 90 W
for 1.5–3.0 h depending on the expected size of the amplified microsatellite
alleles. Gels were fixed (15% methanol, 10% acetic acid), air dried, and
exposed to X-ray films overnight.
Three people independently scored allele sizes against an M13 se-
quencing ladder. Data were corrected based on disagreements among the
three scorers, and samples having ambiguous genotypes were either re-
amplified or the genotypes set as unknown.
All QTL mapping analyses were based on daughter
yield deviations (DYD) of sons based on progeny tests that generally
involved from 50 to 100 daughters. DYD were supplied by G. Jansen and
J. Jamrosik of the Canadian Dairy Network based on Canadian calculations
* All authors contributed equally to this paper.
Correspondence to: J.P. Gibson; E-mail: email@example.com
Mammalian Genome 12, 27–31 (2001).
© Springer-Verlag New York Inc. 2001