BioSMART ISSN: 1411-321X

Volume 1, Nomor 2 Oktober 1999

Halaman: 8-12

Genetic Variation in Composite and Purebreed Hereford Populations

SUTARNO1, A.J. LYMBERY2,3

1
Jurusan Biologi FMIPA UNS Surakarta
2
Agriculture Western Australia. PO BOX 1231, Bunbury, WA 6231, Australia
3
Vet. Biology, Murdoch University, Murdoch WA 6150 Australia

ABSTRACTS

Crossbreeding systems have been widely applied in commercial animal production, due to their many advantages over purebreeding, especially in
exploiting heterosis. Purebred Hereford and synthetic (composite) breeds were used in this project. The objectives of the project were to investigate
polymorphism in the growth hormone gene and mitochondrial DNA in the composite and purebred Hereford herds from the Wokalup selection
experiment, and compare genetic diversity in the growth hormone gene and mitochondrial DNA in the both herds. Polymorphisms were found both
in growth hormone gene and mitochondrial DNA. Extensive genetic diversity was found in both breeds of cattle in the growth hormone gene and
mitochondrial DNA. As expected, restriction site variation in the two growth hormone regions or in the regions of mitochondrial DNA surveyed
was not independent, confirming restricted recombination between sites. Although there was a trend for greater expected heterozygosity at the
growth hormone locus in the composite than in the Hereford population, this trend was not statistically significant.

Key Words: genetic diversity, composite, hereford

INTRODUCTION Composite breeds are expected to respond more

In commercial animal production, crossbreeding
systems have been widely applied due to their many
advantages over purebreeding, especially in exploiting
heterosis. The theoretical and empirical effects of
crossbreeding are well established, particularly in the
exploitation of non-additive genetic effects to improve
mean levels of performance (Gregory & Cundiff, 1980;
Koch et al., 1985). One drawback of continuous
crossbreeding schemes is the complexity of management
required to crossbreed every generation.
Gregory and Cundiff (1980) suggested that composite
or synthetic breeds of livestock provide an attractive
alternative to continuous crossbreeding systems, since
they are expected to combine ease of management with
the utilization of heterosis and additive genetic
differences between breeds. Many studies in beef cattle
have shown that advanced generations of composite
breeds retain heterosis for many quantitative production
traits at about the expected theoretical level of retained
heterozygosity (Gregory et al., 1991a; 1991b; 1991c;
1991d; 1992). There have been no empirical studies,
however, of single gene heterozygosity in composite
breeds.
Crossbreeding has been applied in many livestock
industries, including beef cattle, dairy cattle, pigs, meat
sheep and wool sheep. In beef cattle, Hammond et al.
(1992) suggested that the best possible formation of
composites may be between Bos taurus x Bos indicus
breeds. Bos taurus cattle generally have poor adaptation
to tropical environments, but high growth potential in
stress-free conditions, while Bos indicus have the reverse
characteristics. The formation of kind of this composite has
been successfully achieved, e.g. Brangus, Braford, Simbrah,
Charbray and Belmont Red (Hammond et al., 1992).
rapidly to selection than purebreeds because of a greater
selection intensity permitted by retained heterosis in
reproduction rate, and increased additive genetic variance
associated with differences in gene frequencies between
the parental breeds (Barker, 1989; Dickerson, 1973).
The beef cattle selection experiment carried out by
Agriculture Western Australia at Wokalup research
station involved a comparison of additive genetic
variation and response to selection between a composite
multibreed (approximately comprised of ¼ Brahman,
Charolais and Friesian, and 1/8 Angus and Hereford) and a
purebred Hereford population. Quantitative genetic
analyses by Meyer et al. (1993; 1994) and Meyer (1995)
showed the composite breed to have consistently high
phenotypic and genetic variation for a range of growth
and mature size traits.
The aims of this part of the study were therefore to:
1. Investigate polymorphism in the growth hormone
gene and mitochondrial DNA in the composite and
purebred Hereford herds from the Wokalup selection
experiment;
2. Compare genetic diversity in the growth hormone
gene and mitochondrial DNA in the composite and
purebred Hereford herds.
This project was part of a big project with the final
objective of finding gene markers for production traits in
beef cattle.
MATERIAL AND METHODS
Cattle
A total of 194 Hereford and 235 composite breed
cattle from Wokalup Research Station were used in this
study. They were part of a selection experiment
described in detail by Meyer et al. (1993) and
maintained at Agriculture Western Australia’s Wokalup

© 1999 Jurusan Biologi FMIPA UNS Surakarta

 SUTARNO and LYMBERY – Genetics Variation
Research Station. Most of the cattle used in this study
were purebred Herefords or a composite breed
comprising approximately 1/4 Brahman, Charolais and
Friesian, and 1/8 Angus and Hereford. Both of the
populations were closed in 1977, and selection lines
established from 300 cows and 12 bulls. Approximately
60 cows and 10 bulls were replaced each year, with
replacements selected on pre-weaning growth rate. Control
lines were regenerated from frozen embryos and semen from
1990 to 1995.
Calving occurred in April-May each year. Calves
were weighed at birth and monthly thereafter until
weaning in December-January. Milk production was
estimated for all lactating females each year by the
weigh-suckle-weigh method (Meyer et al., (1993).
Estimated breeding values (EBVs) for birth weight, the
calf component of 200-day growth rate and the maternal
component of 200-day growth rate were obtained for all
animals in the herd each year. Approximately 60 cows
and 10 bulls were replaced each year, with replacement
selection based initially on pre-weaning growth, and
later on an index of increased breeding values for the
direct and maternal components of pre-weaning growth,
and reduced breeding values for birth weight. Estimated
breeding values (EBVs) were provided by Breedplan, the
within-herd genetic evaluation system of the Australian
National Beef Recording Scheme. In 1989 and 1990
frozen embryos from the foundation populations were
implanted to generate control lines for each breed type.
DNA Extraction
Genomic DNA and mitochondrial DNA were
extracted from leucocytes or whole blood using the
method as described in detail by Sutarno (1998).
PCR-RFLP Analysis
Conditions for PCR-RFLP analysis of the growth
hormone gene loci 1 and 2 (GH-L1 and GH-L2) and
mitochondrial DNA (D-loop and ND-5) are as described
by Sutarno (1998).
1 2
223bp
171bp
52bp
Figure 1. Example of gel photographs showing growth hormone gene polymorphism detected by PCR-RFLP using, 1. AluI in locus I (GH-L1)
and 2. MspI in locus 2 (GH-L2).
Data Analysis
Growth Hormone Gene
The locus 1 and 2 fragments were analyzed using the
“infinite alleles” model of the computer program
GENESTRUT (Constantine, Hobbs & Lymbery, 1994)
treating each fragment as a separate locus with two
alternative alleles. Genetic diversity was described by
the mean number of alleles per locus (A), mean observed
heterozygosity (Ho) and mean expected unbiased
heterozygosity (He; Nei 1978), with variance calculated
by the method of Nei (1978). Differences between mean
expected heterozygosities were tested using the method
of Archie (Archie, 1985).
For each locus, genotypic frequencies expected under
Hardy Weinberg equilibrium were calculated from
allelic frequencies using Levene’s correction (Levene,
1949) for small sample size. Deviation of observed from
expected frequencies were tested by X2, and the extent of
deviation expressed by Wright’s fixation index, with the
approximate variance of Brown (1970). Associations
between the loci were examined with Burrow’s
composite measure of linkage disequilibrium ( AB),
tested for significance by X2 as outlined by Weir (1990)
Mitochondrial DNA
Genetic diversity at the mtDNA loci was analyzed
using the “infinite sites” model in the program
RESTSITE (Miller, 1991). The nucleotide diversity, or
average number of nucleotide substitutions per site
within breeds (d) was estimated over all loci by the
method of Nei and Li (1979), with standard errors
calculated by jackknifing (Nei & Miller, 1990). Allelic
frequencies within each breed were calculated separately
for each locus / restriction site combination. Associations
between restriction site polymorphisms at the same or
different loci were tested by comparing observed
haplotype frequencies with those expected from allelic
frequencies, using a X2 test.
600bp 600bp
329bp
224bp
100bp 105bp
100bp
10

RESULTS
Linkage Disequilibrium
Growth Hormone Gene As expected, there were significant associations
The results of the PCR-RFLP in both regions of growth between the two growth hormone loci in both breeds
hormone gene (GH-L1 and GH-L2) were presented in Figure 1. (Table 4).

Table 4. Estimates of Burrows composite measure of linkage

Genetic Diversity disequilibrium ( AB + SE) for the two growth hormone loci in
Standard measures of genetic diversity at the growth composite and Hereford breeds of cattle.
hormone loci between composite and Hereford are shown
Breed P
in Table 1. Differences in expected heterozygosity and AB

nucleotide diversity between breeds were not significant. Composite 13.68 + 0.161 <0.001
There were no differences in genetic diversity between Hereford 5.39 + 0.008 <0.05
sexes in either breed (data not shown).

Table 1. Standard measures of genetic diversity (+ standard error) over

both loci of the growth hormone gene for composite and Hereford
cattle.
Mitochondrial DNA

Breed N Observed No of Unbiased Genetic Diversity

heterozygosity alleles expected
heterozigosity
Standard measures of genetic diversity at the
Composite 211 .371 + .275 2.00 + .000 .425 + .060 mitochondrial D-loop and ND-5 between composite and
Hereford are shown in Table 5. There were no
Hereford 165 .256 + .131 2.00 + .000 .261 + .143
differences in genetic diversity between sexes in either
breed (data not shown).
Allelic Frequencies
Table 5. Genetic diversity over mitochondrial D-loop and ND-5 loci
Allelic frequencies at the growth hormone loci in for composite and Hereford breeds. d is the nucleotide diversity, or
composite and Hereford breeds are shown in Table 2. average number of nucleotide substitutions per site (+ standard error)
There were no significant differences in allelic within breeds.
frequencies between sexes in either breed for any locus
(data not shown). Breeds N d (Nei and Li)
Composite 235 0.039185 + 0.015663
Table 2. Allelic frequencies at both loci of the growth hormone gene
for composite and Hereford breeds. Hereford 194 0.050723 + 0.019578

Breed N GH-L1 GH-L2

L V MspI (+) MspI (-)
Allelic Frequencies
Composite 211 .589 .411 .757 .243 Allelic frequencies at the mitochondrial D-loop and
Hereford 165 .765 .235 .915 .085 ND-5 loci are shown in Table 6. There were no
significant differences in allelic frequencies between
Genotypic Frequencies
sexes in either breed for any locus (data not shown).
Table 3 shows the observed and expected
heterozygosities at each growth hormone locus in each Linkage Disequilibrium
breed. Fixation indices were generally positive, Out of 10 possible mtDNA haplotypes, only 3 were
indicating fewer heterozygotes then expected, but this observed in the composite breed and 3 in the Hereford.
was significant only in the composites at locus 2. Over The most common haplotypes were significantly over-
both breeds, Fis was not significantly different from 0 for represented in both breeds (Table 7), indicating
either locus. extensive associations within and between loci.
Table 3. Heterozygosities observed (Ho) and expected (He) under
Hardy-Weinberg equilibrium, and fixation indices (Fi with variances in Table 7. Frequency of mtDNA haplotypes observed and expected
parentheses) at the two growth hormone loci in Hereford and (from products of allelic frequencies) in composite and Hereford.
composite breeds. Fis is the weighted mean of values across breeds.
Breed Haplotypes Expected Observed P
Locus Breed Fis Frequency Frequency
Composite Hereford Composite AAAAA 74.9 159 <0.005
GH-L1 Ho 0.428 0.360 ABBBA 7.3 75 <0.005
He 0.485 0.360
F 0.118 0.002 .069 ABBBB 0.04 1 <0.005
(0.005) (0.006)

GH-L2 Ho 0.303 0.145 Hereford AAAAA 32.6 110 <0.005

He 0.369 0.156 ABBBA 14.5 69 <0.005
F 0.178* 0.066 (0.009) .098*
(0.006) BBBBA 1.2 15 <0.005
 SUTARNO and LYMBERY – Genetics Variation
Table 6. Allelic frequencies at mitochondrial D-loop and ND-5 for composite and Hereford breeds. At each locus the common allele is designated
as A, and the less common allele B.
Breed D-loop
TaqI PstI SspI
A B A B A B
Composite .685 .315 .685 .315 .685 .315
Hereford .567 .433 .567 .433 .567 .433
DISCUSSION
Extensive genetic diversity was found in both breeds
of cattle in the growth hormone gene and mitochondrial
DNA. As expected, restriction site variation in the two
growth hormone regions or in the regions of
mitochondrial DNA surveyed was not independent,
confirming restricted recombination between sites.
Populations of livestock often show reduced
heterozygosity across all nuclear loci as a consequence
of inbreeding arising from small population sizes and
artificial selection. This was not evident in the present
study. Heterozygote deficiency was found only at GH-
L2 in the composite breed. This may have resulted from
selection against heterozygotes, perhaps as a
consequence of previous artificial selection policies, but
more data are needed to confirm this hypothesis.
Composite breeds are expected to be genetically
more diverse than purebreeds, because of recombination
of the loci from different breeds which takes place
during breed formation. Greater diversity at the
individual locus level in composite breeds is expected to
provide greater genetic variance in quantitative traits and
more rapid response to selection. Empirical tests of this
theory are rare and have invariably involved estimation
of quantitative genetic parameters in composite and
purebred populations, rather than comparisons of allelic
or nucleotide diversity at individual loci. Howe & James
(1973) found a greater realized heritability for
sternopleural bristle number in composite, than in
parental lines of Drosophila melanogaster. Liu et al.
(1991) found greater genetic variance for growth traits in
a composite, than in purebred beef cattle population.
Meyer et al. (1993; 1994), working with the same
populations studied here, found greater phenotypic and
genetic variance for growth and size traits among
composite than among purebred Hereford.
Although there was a trend for greater expected
heterozygosity at the growth hormone locus in the
composite than in the Hereford population, this trend
was not statistically significant. For mitochondrial DNA,
diversity was actually greater (although not significantly
so) in the Herefords. Simulation studies conducted by
Gunawan & James (1986) and Mohd-Yusuff &
Dickerson (1991) indicate that additive genetic variation
may or may not be increased in composite populations,
depending on the degree of dominance, diversity in gene
frequency among parental lines, and the mean gene
frequencies in the composite. Mohd-Yusuff & Dickerson
(1991) suggested that favourable dominant alleles at
QTLs should be fixed at equilibrium, so that genetic
ND-5
ApaI AvaII HindIII SpeI
A B A B A B A B
.995 .005 1.000 .000 .685 .315 .685 .315
1.000 .000 .923 .077 .567 .433 .567 .433
variance for the trait in composite populations will only
exceed that in their purebred parental populations when
dominance is partial or when frequencies of dominant
and overdominant alleles are less than expected at
equilibrium. The theoretical point is whether the growth
hormone and mitochondrial DNA loci studied here are
actually QTLs. This will be addressed in the next
publications.
ACKNOWLEDGEMENT
I am grateful to Patrick Donnelly and the staff at
Wokalup Research Station, Agriculture Western
Australia, especially Phil Moyle, Bob Jones and Richard
Seaward for the phenotypic data and sample collections.
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