Microsatellite Analysis
of Genetic Diversity
in Wild and Farmed Emus
(Dromaius novaehollandiae)
E. L. Hammond, A. J. Lymbery,
G. B. Martin, D. Groth, and
J. D. Wetherall
The emu (Dromaius novaehollandiae) occupies most regions of the Australian
continent and in recent times has been
farmed for meat, oil, and leather. Very little
is known about the genetic structure of
natural or farmed populations of these
birds. We report a preliminary study of
genetic variation in emus undertaken by
typing birds from five farms and two natural
populations at five polymorphic microsatellite loci. Genetic diversity was high for all
populations and there was little evidence of
inbreeding, with most populations conforming to Hardy–Weinberg equilibrium for most
loci. Significant heterozygote deficiencies
at one locus in a number of populations
were detected and may indicate the presence of null alleles. Comparisons of allele
frequencies showed little evidence of genetic differentiation either among farmed
populations or between farmed and natural
populations.
The emu (Dromaius novaehollandiae) is
a well-known member of the ratite family
376 The Journal of Heredity 2002:93(5)
Table 1. Mean number of alleles per locus and total gene diversity (H) in samples of emus from seven
populations
Population
Source
Sample size
Mean alleles/locus
Total gene diversity
Mt. Gibson
Harvey
York
Toodyay
Thailand
Wild A
Wild B
Farmed
Farmed
Farmed
Farmed
Farmed
Wild—west of fence
Wild—east of fence
20
19
15
19
9
11
14
12
11
10
11
6
8
11
0.89
0.87
0.82
0.87
0.79
0.80
0.87
All farmed populations, except Thailand, were in Western Australia. The two natural populations were also
from Western Australia and separated by a vermin-proof fence.
of flightless birds and is widely dispersed
across the Australian continent. There is
very little morphological variation among
emu populations across Australia and
there are no data on their genetic
variability. Several studies have used
mitochondrial sequence analysis to examine the phylogenetic relationships
among ratite birds (Baker et al. 1995;
Freitag and Robinson 1993; Haddrath and
Baker 2001), but no studies to date have
examined ratite population structure
using microsatellite analysis.
The emergence of an emu industry in
the 1980s, aimed at commercial production of meat, oil, and leather, has heightened interest in characterizing emu
pedigrees and improving their genetic
management. Within Western Australia,
many farms have birds that are descendants of emus from the founding stock of
the first emu farm established in Wiluna
in 1976. However, poor written records of
male:female ratios, overall stock numbers, and the source of breeding stock
make it difficult to estimate the level of
relatedness among domesticated stock.
The number of emus at each farm
appears variable over time, and culls
and severe reductions in stock size can
follow large breeding seasons. Estimation
of expected levels of genetic variation
among farmed stock is further confounded by a lack of knowledge of existing
genetic variation in wild populations.
More information would be very helpful
in defining pedigrees, assessing the levels
of inbreeding, and (eventually) in defining
markers for desirable heritable traits.
We have previously published the first
panel of five microsatellite markers isolated in this species (Taylor et al. 1999)
and here we report a preliminary assessment of genetic variation in emus sampled from farms and from the wild in
Western Australia. Data from wild emus
were used to assess whether isolating
mechanisms are operating in natural
populations of these birds. Any factor
that limits gene flow among populations
can act as an isolating mechanism, including the long stretches of verminproof fences that have been erected in
an effort to control emu movements.
Data from farmed emus were used to
determine if farmed populations were
less genetically diverse than wild populations and to assess the genetic relationships among different farmed
populations. The results described here
represent the first molecular genetic
characterization of emu populations.
Materials and Methods
Sampling
Blood was sampled from populations of
wild and farmed emus from various
locations in Western Australia and from
one farmed population in Thailand
(Table 1). When sampling birds from
commercial farms, the farmer was usually able to advise which birds were
unrelated, and samples were not knowingly taken from related birds. Farmed
emus were restrained and calmed, usually by holding the bird under the wings,
while 200 ll of blood was taken by
syringe from the jugular vein. The blood
was immediately mixed with 1 ml 70%
ethanol and stored at 48C pending DNA
extraction by standard phenol-chloroform procedures (Maniatis et al. 1982).
Wild emus panic when restrained, and
previous attempts to capture them have
stressed the birds to such an extent that
they have seriously injured themselves.
The use of drugs, either in water sources
or as a dart, has also been largely
unsuccessful. We therefore obtained
a scientific research permit to kill a number of wild birds; we employed the
services of professional marksmen to
ensure a clean shot. Groups of emus—consisting of a number of smaller birds of
approximately the same height and one
full-sized bird—were considered a family
unit, and only one member was sampled.
From seven distinct populations, 107
individual animals were sampled. These
were identified as either wild or farmed,
together with the location from which
they were obtained (Table 1). Most of the
emus on Western Australian farms sampled in this study originated, directly or
indirectly, from the Wiluna emu farm that
was established in 1976 with 100 breeding pairs caught from the surrounding
area. The Mount Gibson and Toodyay
farms supplemented their stock with
more wild birds. The Toodyay farm
contained some birds that were descendants of emus taken from the Ben Covens
and Sand Plains areas around Toodyay.
The Thailand farm was established in the
1990s with emus imported from the
United States, but the origin of these
emus is unknown. Wild emus were
sampled from two areas in Western
Australia, to the west and to the east of
a vermin-proof fence that was erected in
1900 and subsequently extended and
maintained to control emu movements
into agricultural areas.
Microsatellite Typing
Loci were amplified by polymerase chain
reaction (PCR) in 10 ll reaction volumes,
using 50 ng DNA, 4 pmol forward primer,
end-labeled with an IRD 800 infrared dye
(Li-Cor Inc., Lincoln, NE), 1 pmol unlabeled
forward primer, and 5 pmol unlabeled
reverse primer, 200 lM dNTPs, 0.1 U
Amplitaq Gold (Perkin-Elmer, USA), 0.1 M
Tris-HCl pH 8.3, 0.5 M KCl, and 1.5 mM
MgCl2 (Taylor et al. 1999). A summary of
the primer pairs and properties of the final
panel of five microsatellite loci is given in
Taylor et al. (1999). Gel electrophoresis
was used for genotyping, as described
previously (Taylor et al. 1999). All loci
generated light stutter bands typical of
dinucleotide microsatellite loci, but these
did not preclude successful identification
of the main DNA fragments used to assign
genotypes.
Analysis
Genotypic and allelic frequencies were
calculated for all populations at all microsatellite loci. Genetic diversity within
populations was described by the mean
number of alleles per locus and the mean
expected unbiased heterozygosity or total gene diversity (H; Nei 1978). Genotypic
frequencies expected under Hardy–Weinberg equilibrium were calculated from
allelic frequencies using Levene’s (1949)
Brief Communications 377
Table 2.
Allele frequencies at each locus for each population
Locus
Alleles
Total
Emu5
Population
Mt. Gibson
Harvey
York
Toodyay
Thai
Wild B
Wild A
1
0.000
0.056
0.000
0.000
0.000
0.000
0.000
2
0.200
0.167
0.036
0.062
0.125
0.143
0.000
3
0.175
0.194
0.036
0.156
0.375
0.179
0.167
4
0.300
0.250
0.179
0.406
0.062
0.250
0.389
5
0.075
0.139
0.536
0.125
0.375
0.107
0.222
6
0.000
0.111
0.000
0.156
0.000
0.107
0.000
7
0.000
0.000
0.000
0.062
0.000
0.000
0.000
8
0.150
0.028
0.036
0.031
0.000
0.143
0.222
9
0.025
0.028
0.143
0.000
0.000
0.036
0.000
10
0.000
0.000
0.000
0.000
0.062
0.000
0.000
11
0.075
0.028
0.036
0.000
0.000
0.036
0.000
Emu63
Population
Mt. Gibson
Harvey
York
Toodyay
Thai
Wild B
Wild A
1
0.029
0.000
0.000
0.000
0.000
0.000
0.000
2
0.000
0.000
0.000
0.000
0.000
0.037
0.000
3
0.029
0.029
0.067
0.038
0.000
0.148
0.000
4
0.000
0.000
0.000
0.000
0.000
0.000
0.045
5
0.000
0.000
0.133
0.038
0.000
0.037
0.000
6
0.176
0.059
0.100
0.077
0.375
0.074
0.045
7
0.029
0.000
0.100
0.077
0.000
0.000
0.091
8
0.029
0.088
0.000
0.000
0.000
0.000
0.000
9
0.000
0.000
0.000
0.000
0.000
0.000
0.091
10
0.000
0.029
0.000
0.038
0.250
0.037
0.045
11
0.029
0.088
0.033
0.000
0.000
0.000
0.091
12
0.000
0.088
0.033
0.000
0.000
0.074
0.045
13
0.029
0.000
0.067
0.000
0.000
0.037
0.000
14
0.029
0.088
0.100
0.000
0.125
0.111
0.045
15
0.029
0.029
0.033
0.077
0.000
0.000
0.136
Population
Mt. Gibson
Harvey
York
Toodyay
Thai
Wild B
Wild A
16
0.147
0.029
0.000
0.000
0.000
0.000
0.091
17
0.059
0.059
0.033
0.038
0.000
0.037
0.045
18
0.029
0.000
0.033
0.038
0.000
0.000
0.000
19
0.000
0.059
0.000
0.000
0.000
0.000
0.182
20
0.000
0.118
0.033
0.000
0.000
0.074
0.000
21
0.029
0.000
0.067
0.000
0.000
0.037
0.000
22
0.029
0.029
0.000
0.077
0.000
0.037
0.000
23
0.029
0.000
0.067
0.038
0.000
0.000
0.000
24
0.029
0.059
0.000
0.038
0.250
0.000
0.000
25
0.000
0.000
0.000
0.077
0.000
0.037
0.000
26
0.029
0.000
0.000
0.038
0.000
0.037
0.000
27
0.029
0.000
0.033
0.038
0.000
0.037
0.000
28
0.059
0.029
0.000
0.038
0.000
0.000
0.000
29
0.000
0.000
0.000
0.038
0.000
0.000
0.000
30
0.029
0.029
0.000
0.000
0.000
0.000
0.000
Population
Mt. Gibson
Harvey
York
Toodyay
Thai
Wild B
Wild A
32
0.000
0.000
0.033
0.038
0.000
0.000
0.000
33
0.000
0.000
0.000
0.000
0.000
0.000
0.045
34
0.029
0.000
0.000
0.038
0.000
0.074
0.000
35
0.000
0.000
0.000
0.038
0.000
0.037
0.000
36
0.000
0.000
0.000
0.038
0.000
0.000
0.000
37
0.029
0.029
0.000
0.038
0.000
0.037
0.000
38
0.029
0.000
0.000
0.000
0.000
0.000
0.000
Emu50
Population
Mt. Gibson
Harvey
York
Toodyay
Thai
Wild B
Wild A
1
0.050
0.115
0.115
0.026
0.000
0.000
0.000
2
0.025
0.038
0.000
0.000
0.000
0.077
0.105
3
0.025
0.000
0.115
0.000
0.000
0.077
0.105
4
0.050
0.154
0.077
0.079
0.056
0.115
0.105
5
0.075
0.077
0.038
0.105
0.167
0.000
0.053
6
0.000
0.077
0.000
0.105
0.333
0.038
0.053
7
0.000
0.000
0.000
0.000
0.222
0.000
0.105
8
0.100
0.154
0.115
0.211
0.111
0.192
0.000
9
0.125
0.077
0.077
0.105
0.000
0.154
0.211
10
0.075
0.000
0.077
0.132
0.056
0.038
0.053
11
0.050
0.038
0.038
0.026
0.000
0.077
0.000
12
0.175
0.154
0.115
0.026
0.000
0.115
0.105
13
0.050
0.077
0.115
0.079
0.000
0.038
0.105
14
0.175
0.000
0.115
0.079
0.000
0.000
0.000
15
0.000
0.000
0.000
0.000
0.000
0.038
0.000
16
0.025
0.000
0.000
0.000
0.056
0.000
0.000
17
0.000
0.038
0.000
0.026
0.000
0.038
0.000
40
26
26
38
18
26
19
Emu33
Population
Mt. Gibson
Harvey
York
Toodyay
Thai
Wild B
Wild A
1
0.000
0.000
0.000
0.000
0.000
0.000
0.050
2
0.000
0.000
0.000
0.028
0.000
0.036
0.000
3
0.079
0.000
0.107
0.111
0.000
0.000
0.050
4
0.237
0.062
0.179
0.167
0.167
0.214
0.400
5
0.211
0.219
0.250
0.222
0.278
0.107
0.100
6
0.079
0.031
0.036
0.111
0.000
0.071
0.000
7
0.079
0.062
0.036
0.028
0.000
0.036
0.000
8
0.026
0.000
0.000
0.028
0.000
0.000
0.000
9
0.000
0.125
0.036
0.028
0.056
0.036
0.000
10
0.105
0.000
0.071
0.028
0.000
0.000
0.000
11
0.053
0.125
0.143
0.111
0.000
0.107
0.400
12
0.000
0.031
0.000
0.028
0.000
0.036
0.000
13
0.000
0.000
0.000
0.000
0.056
0.036
0.000
14
0.053
0.031
0.036
0.056
0.056
0.000
0.000
15
0.053
0.094
0.036
0.028
0.222
0.179
0.000
16
0.026
0.156
0.071
0.028
0.167
0.107
0.000
17
0.000
0.062
0.000
0.000
0.000
0.036
0.000
38
32
28
36
18
28
20
Emu18
Population
Mt. Gibson
Harvey
York
Toodyay
Thai
Wild B
Wild A
1
0.222
0.167
0.160
0.133
0.056
0.107
0.100
2
0.074
0.000
0.000
0.100
0.000
0.036
0.000
3
0.370
0.500
0.560
0.433
0.500
0.536
0.550
4
0.074
0.056
0.040
0.133
0.056
0.107
0.050
5
0.074
0.056
0.000
0.000
0.000
0.000
0.000
6
0.074
0.056
0.000
0.000
0.000
0.000
0.000
7
0.000
0.000
0.040
0.000
0.111
0.000
0.000
8
0.000
0.000
0.120
0.000
0.000
0.000
0.000
9
0.000
0.000
0.040
0.000
0.000
0.000
0.000
10
0.000
0.000
0.000
0.000
0.000
0.000
0.100
11
0.037
0.111
0.040
0.000
0.111
0.000
0.050
12
0.074
0.000
0.000
0.200
0.111
0.179
0.150
13
0.000
0.056
0.000
0.000
0.056
0.036
0.000
correction for small sample size. Deviations of observed from expected frequencies were tested by the exact test of Guo
and Thompson (1992). The extent of
deviation from Hardy–Weinberg equilibrium within populations was expressed for
each locus by Wright’s fixation index (F),
and across all loci for each population, Fvalues were summarized by the weighted
378 The Journal of Heredity 2002:93(5)
40
36
28
32
16
28
18
31
0.000
0.059
0.033
0.000
0.000
0.000
0.000
34
34
30
26
16
27
22
mean, FIS. Burrow’s composite measure of
linkage disequilibrium (AB) was estimated for all pairwise combinations of loci in
all populations and tested for significance
as outlined by Weir (1990).
Genetic differentiation among populations was assessed by an analysis of molecular variance (AMOVA; Excoffier et al.
1992). Distances among all multilocus
27
18
25
30
18
28
20
genotypes were estimated by counting
the number of different alleles (Michalakis and Excoffier 1996) and the total
variation among genotypes partitioned
into that due to differences within populations, that due to differences among
populations in Australia, and that due to
differences among populations in Australia and Thailand. A nonparametric
Table 3. Heterozygosities observed (Ho) and expected (He) under Hardy–Weinberg equilibrium, and fixation indices (F) at five microsatellite loci within seven
populations of emus
Population
Locus
Emu5
Ho
He
F
Emu63
Ho
He
F
Emu50
Ho
He
F
Emu33
Ho
He
F
Emu18
Ho
He
F
FIS
Mt. Gibson (n 5 20)
Harvey (n 5 19)
0.50
0.82
0.4006*
0.58
0.85
0.2943*
0.88
0.94
0.0769
0.94
0.96
0.0192
0.95
0.93
ÿ0.0418
0.92
0.92
0.0000
0.95
0.88
ÿ0.0746
1.00
0.87
ÿ0.1579
0.0430
York (n 5 15)
Toodyay (n 5 19)
Thailand (n 5 9)
0.44
0.79
0.4517*
0.28
0.76
0.6782*
0.92
0.98
0.0728
0.50
0.77
0.3636
0.70
0.69
0.0385
1.00
0.96
ÿ0.0435
1.00
0.94
ÿ0.0722
0.95
0.91
ÿ0.0452
0.89
0.84
ÿ0.0667
0.60
0.68
ÿ0.0667
1.00
0.92
ÿ0.0871
0.81
0.90
0.1014
0.93
0.89
ÿ0.0464
0.94
0.90
ÿ0.0490
0.89
0.86
ÿ0.0407
1.00
0.76
0.0000
0.93
0.91
ÿ0.0242
0.78
0.74
ÿ0.0566
0.0772
0.67
0.64
ÿ0.0476
0.0324
0.80
0.75
ÿ0.0667
0.0727
0.91
0.94
0.1290
ÿ0.0539
0.78
0.68
ÿ0.1626
ÿ0.0097
0.71
0.68
ÿ0.0526
0.6
0.96
0.3808*
0.67
0.74
0.1111
0.2018*
Wild A (n 5 11)
1.00
0.92
ÿ0.3333
Wild B (n 5 14)
0.64
0.87
0.2687*
FIS is the weighted mean of F-values across loci for each population. N 5 sample size.
* Significant at the 5% level, with the Bonferroni correction.
permutation approach (Excoffier et al.
1992) was used to test the significance of
the variance component estimates.
Results and Discussion
A total of 96 alleles were detected in the
107 individual emus typed. As shown in
Table 2, the allele frequency distributions for the five microsatellite loci were
similar between the groups of emus, and
indicate that the groups sampled are not
differentiated. The distributions of the
alleles are similar to those seen with
microsatellite loci in humans and many
other species, displaying a large number
of alleles and high levels of heterozygosity. Locus emu63 was exceptionally
polymorphic and is clearly the most informative for population characterization
and individual identification, including
parentage assignment (Taylor et al.
2000). Over all populations, the mean
number of alleles detected per locus was
19, although the actual number of observable alleles at each locus ranged
from 13 at locus emu18 to 38 at locus
emu63. Some alleles were detected in just
one population. There was a direct relationship between the number of samples taken from a source and the number
of alleles detected, indicating that increased sample size is required to provide more accurate knowledge of alleles
and their frequency distribution. There
was no evidence of associations between
alleles at different microsatellite loci, and
none of the 10 pairs of loci showed
significant linkage disequilibrium as measured by (AB) (data not shown).
Values for total genetic diversity at all
loci are compared among populations in
Table 1. Given the sample size limitations
of the study, these values should be
viewed as minimum estimates, as further
sampling may reveal even higher levels of
genetic diversity. In general, the farmed
Thai and natural Wild A populations were
less genetically diverse than the other
farmed or natural populations, but again
further sampling is required to determine
whether these data accurately reflect the
total genetic diversity of the source.
For most loci, in most populations, the
genotypic frequencies did not deviate
significantly from Hardy–Weinberg equilibrium (Table 3). The exception was
locus emu5, where there were significant
heterozygote deficiencies in five of the
seven populations. Such locus-specific
effects cannot be accounted for by
genome-wide phenomena such as inbreeding or a Wahlund effect. Null alleles
(alleles that fail to amplify because of
large increases in the size of the product
or mutations at flanking primer sites)
could explain the heterozygote deficiencies, because heterozygotes for the null
allele will appear as homozygotes for the
amplified allele.
Although farmed populations of emus
tended to have higher fixation indices
than natural populations, the general
concordance between the expected and
observed heterozygosity values suggests
that, for the most part, the populations
are not inbred (Table 3). The mean FIS
value over loci was significantly greater
than zero for the Thai population, but
this was principally due to the relatively
greater value at locus emu5, and does not
seem to indicate a general heterozygote
deficiency in this population. There is
therefore little evidence for extensive
inbreeding in farmed emus. This suggests, first, that initial selection of emus
for breeding stock sampled a large proportion of the genetic diversity present in
wild populations, and second, that there
has been little restriction of the gene
pool in farmed populations since they
were founded. The pair bonding of males
and females (Blache et al. 2000) has
prevented emu farms from implementing
systematic programs of genetic improvement that would tend to increase inbreeding and reduce genetic diversity
(MacHugh et al. 1997; Norris et al. 1999).
When total genetic variation was partitioned within and among populations by
AMOVA, most genetic variation (94%)
was found within populations, with only
3% found between emus in different
countries (Thailand and Australia) and
3% between different populations from
within Australia. The general lack of
genetic differentiation among different
farmed populations, or between farmed
and natural populations, may be attributable to the relatively short time the
populations have been separated and
Brief Communications 379
the absence of selective breeding in
captivity. However, given the large number of alleles detected at each locus, relatively small changes in genetic structuring
may not be detectable with the limited
number of samples analyzed in this study.
We did not detect any genetic differences between emus sampled from wild
populations that are separated by vermin-proof fences. The fences may be
effective in preventing emu movement,
but they were erected only 100 years ago,
and this may be insufficient time for
genetic differences to develop. The data
suggest that no isolating mechanisms
exist, so the morphological homogeneity
seen in the emu (O’Brien 1990) appears
to be a true indicator of genetic uniformity in the species. However, most of the
wild emus in this study originated from
regions within 1000 km of Perth, a limitation in sampling that must be taken into
account. Samples from populations located in Eastern Australia or from the far
north of Western Australia were not
available for this study and are needed
to permit better estimates of genetic
diversity in this widely disseminated,
apparently homogeneous species.
From the Department of Biomedical Sciences, Curtin
University, Perth 6000, Australia (Hammond, Groth,
and Wetherall); Division of Veterinary and Biomedical Science, Murdoch University, Perth 6150, Australia (Lymbery); and School of Animal Biology,
University of Western Australia, Crawley 6009,
Australia (Martin). This work was carried out at
Curtin University and the University of Western
Australia. Our thanks to Gemma Graham and Dominique Blache at the Animal Science Group, University of Western Australia, for assistance with sample
collection. We are also grateful to Professor Stephen
J. Davies for his contribution to sample collection
and specific knowledge of emus. This work was
partly supported by the Rural Industries Research &
Development Corporation and the Australian Research Council. Address correspondence to Emma
Hammond, Centre for Clinical Immunology and Biomedical Statistics, 2nd floor, North Block, Royal
Perth Hospital, Perth, Australia, 6000, or e-mail:
[email protected].
Ó 2002 The American Genetic Association
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Received September 11, 2001
Accepted August 8, 2002
Corresponding Editor: Susan J. Lamont