Rapid communication: polymerase chain reaction-restriction fragment length
polymorphism in the bovine growth hormone gene.
M M Unanian, S K DeNise, H M Zhang and R L Ax
J ANIM SCI 1994, 72:2203.
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Rapid Communication: Polymerase Chain Reaction-Restriction
Fragment Length Polymorphism in the
Bovine Growth Hormone Gene
M. M. Unanianl, S. K. DeNise2, H. M. Zhang, and R. L. Ax
Department of Animal Sciences, University of Arizona, Tucson 85721
Polymorphism. A polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was
detected within the bovine growth hormone gene with
the restriction enzyme HaeIII.
Source and Description. The amplified fragment
was 441 bp in length, containing the fifth exon and 3'
flanking region of the bovine growth hormone gene.
PCR
Primer
Sequences.
Primer
1: 5'ACGCGCTGCTCAAGAAC-3';
Primer
2:
5'GGCTGGAACTAAGAACC-3'.
Methods of Detection. Genomic DNA was extracted
from semen samples and amplified by PCR. The PCR
reaction mixture contained l x Taq polymerase buffer,
1.4 mM magnesium chloride, .4 mM deoxynucleoside
triphosphates, .8 p M primers, .5 unit Perfect Match
Polymerase Enhancer (Stratagene), 3.0 units Taq
polymerase, and 250 ng of bovine genomic DNA in a
final volume of 50 pL. The PCR conditions were 1cycle
of 1.5 rnin at 97"C, 1 rnin at 58"C, and 1min at 72"C,
followed by 34 cycles of 1 min at 94"C, 1 min at 58"C,
and 1 min at 72°C.
Products of PCR were digested with HaeIII and
separated on 4% agarose gels. The digestion mixture
contained 10 pL of PCR product and 4 units of HaeIII
(New England Biolabs, Beverly, MA).
Description of Polymorphism. Three distinct patterns (designated EE, EF, and FF) were observed in
this PCR-RFLP analysis. The E allele product exhibited three fragments of 268 bp, 102 bp, and 71 bp,
as expected based on known sequences (Gordon,
1983). The F allele, in addition to the same
268-bp and 102-bp fragments, had an observable band
of about 50 bp in length instead of the 71-bp band
(Figure 1).
Frequencies. Genotypic frequencies for 184 bulls
from eight breeds were .03 for the FF genotype, .08 for
the EF genotype, and .89 for the EE genotype. The F
allele was identified only in Brahman, Holstein, and
Wagyu crossbreeds.
Chromosomal Location. Growth hormone has been
mapped to bovine chromosome 19 (Womack et al.,
1989).
Literature Cited
Gordon, D. F., D. P. Quick, C. R. Erwin, J. E. Donelson, and R. A.
Maurer. 1983. Nucleotide sequence of bovine growth hormone
chromosomal gene. Mol. Cell. Endocrinol. 33:81.
Wornack, J. E., D. W. Threadgill, Y. D. Moll, L. K. Faber, M. L.
Foreman, A. B. Dietz, T. C. Tobin, L. C. Skow, S. M. Zneimer,
D. S. Gallagher, and D. S. Rogers. 1989. Syntenic mapping of 37
loci in cattle. Cytogenet. Cell Genet. 51:1109.
Key Words: Cattle, Somatotropin, Genetic Markers,
PCR, RFLP
J. Anim. Sci. 1994. 72:2203
441 bp
208 bp
102 bp
U
EE FF EF M
Figure 1. Bovine GH441-HaeIII polymorphism patterns. Genotypes are assigned on the bottom and sizes
of digested fragments are given on the right side. An
undigested sample and a DNA size marker are indicated
by U and M, respectively.
'The authors wish to acknowledge the contribution of K. C.
Maddock for her technical assistance. This work was supported in
part by Sire Power, Inc. Dr. Unanian was supported as a visiting
scientist by EMBRAPA-CPPSE-CNPq Fellowship.
2To whom correspondence should be addressed.
Received April 8, 1994.
Accepted May 23, 1994.
2203
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