BioSMART ISSN: 1411-321X

Volume 1, Nomor 1 April 1999

Halaman: 1-8

Genetic Diversity within and between Breeds of Beef Cattle in

Western Australia and Bali: 1. Mitochondrial DNA

SUTARNO
Jurusan Biologi FMIPA UNS Surakarta

ABSTRACT

Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP), a PCR based technique, is now
extensively used in genetic marker analysis. This technique was used in this study to genotype 8 different breeds of cattle from
Western Australia and Bali cattle (Bos javanicus) from Indonesia, at the D-loop and ND-5 regions of mitochondrial DNA.
Polymorphisms were found in the D-loop region using 10 restriction enzymes, and in ND-5 using 6 restriction enzymes. The
genetic diversity analysis indicated that there was little evidence of differences in genetic diversity among the cattle breeds
examined, the only major discrepancies were the absence of mtDNA variation among Belgian Blue and Murray Grey cattle. In
contrast to the growth hormone, the UPGMA phenogram constructed based on the mitochondrial DNA polymorphisms could
clearly separate between Bos javanicus from other breeds.

Key words: PCR-RFLP, cattle, mtDNA, genetic diversity

INTRODUCTION well as in population genetic studies. Avise (1994)

Mitochondrial DNA is a circular doubled
stranded DNA, 16.338kb long in cattle (Anderson
et al., 1982). Mammalian mitochondria carry
multiple copies of mtDNA which are inherited
maternally (Hutchison et al., 1974; Wallace, 1993),
since inheritance is predominantly through the egg
cytoplasm (Wallace, 1993).
Information on genetic diversity and genetic
relationships among cattle breeds may be very
useful in cattle breeding programs. Genetic
diversity is the basis for livestock breeding (Buis,
Oldenbroek & Vanderwerf, 1994), because it is
used as a starting point for the improvement of
breeds by artificial selection. Understanding the
extent and pattern of genetic variability among
breeds may help in the development of more
rational breeding programs and is a prerequisite to
the informed conservation of genetic resources
(Kidd et al., 1974).
Advanced techniques of molecular biology have
provided the opportunity to study genetic diversity
within and among breeds at the single gene level
(e.g. Baker & Manwell, 1991; Loftus et al., 1994b;
MacHugh et al., 1994; Moazamigoudarzi et al.,
1997; Ricketts & Vandenplas, 1992). Many DNA
markers, either of genomic DNA or cytoplasmic
DNA, have been generated recently by utilizing
molecular techniques. Candidate QTLs, such as the
growth hormone gene, BoLA gene and casein gene
have been extensively studied. Mitochondrial
DNA, a cytoplasmically inherited DNA, has
become an area of interest for studying the
maternal inheritance of many traits in livestock, as
has extensively reviewed the advantages of using
mitochondrial DNA, a cytoplasmically inherited
DNA, as a genetic marker in population studies.
Recently, Loftus et al. (1994a) used mtDNA
polymorphisms to study the phylogeny of different
breeds of cattle from Europe, Asia and Africa.
The aims of this part of the study were therefore to:
1. Investigate polymorphisms in the mitochondrial
DNA of different breeds of cattle;
2. Compare the genetic diversity at these genomic
regions in different breeds of cattle.
This preliminary experiment was part of the
large experiment utilizing large groups of cattle
with final objection of finding genetic markers for
production traits in beef cattle.
MATERIALS AND METHODS
Experimental Animals
Blood samples were taken from eight breeds of
cattle, comprising 18 Simmental, 13 Limousins, 16
Charolais, 25 Friesian, 12 Hereford, 6 Angus, 6
Belgian Blue and 13 Murray Grey, and also 2 Bali
cattle (Bos javanicus) for use in genetic diversity
studies. Samples were taken on farms, or in
abattoirs, and for most breeds cattle from a number
of unrelated herds were sampled.
DNA Extraction
Mitochondrial DNA was extracted from
leucocytes or whole blood using a Proteinase
K/SDS digestion followed by a phenol/chloroform
extraction, or the Wizard Genomic DNA Extraction
Kit (Promega).

© 1999 Jurusan Biologi FMIPA UNS Surakarta

2 BioSMART Vol. 1, No. 1, April 1999, hal. 1-8
PCR Amplification of Mitochondrial D-loop and
ND-5
The non-coding D-loop region and part of the
gene coding for NADH dehydrogenase sub-unit 5
(ND-5) were amplified by PCR, using primers D-L
/ D-R and ND-L / ND-R (Suzuki, Kemp & Teale,
1993) described below:
D-loop Primers:
D-L : 5’-TAG TGC TAATACCAACGGCC-3
D-R : 5’-AGGCAT TTTCAG TGCCTTGC-3'
ND-5 Primers:
ND-L : 5'-ATCCGTTGGTCTTAGGAACC-3'
ND-R : 5'-TTGCGGTTACAAGGATGAGC-3'
The D-loop primers yielded a PCR product of
1142 bp between positions 15601 and 404,
representing the whole D-loop and flanking
sequence at both ends, while the ND-5 primers
resulted in a PCR product of 453 bp between
positions 12058 and 12510 (Figure 1).
DL
D-loop
B
Cyt
6
ND
-R
ND
ND5
Bovine mt DNA
16338 bp
ND-L
ND4
ND
4L
ND
3
CO
III
ATPase 6&8
Figure 1. Schematic diagram of the location of the primers
(DL, DR, ND-L and ND-R) used for the
amplification of D-loop and ND-5 regions of bovine
mitochondrial DNA.
The D-loop and ND-5 amplifications were
performed under the same conditions, except the
annealing temperatures were slightly higher for D-
loop amplification. Dyna Wax (Finnzymes Oy) was
used instead of paraffin oil in all reactions to
produce a “hot start” amplification. All amplification
reactions were performed in a 50 ml reaction mix
consisting of 200 ng of template DNA, 0.15 mM
each of the oligonucleotide primers, 200 mM each
dNTPs, 2 mM MgCl2, 10x buffer and 1.5 units Taq
DNA polymerase (Biotech, Australia). Negative
controls (lacking template DNA) were included in
all reactions, and produced no products. PCR
amplification conditions for the mitochondrial D-
loop and ND-5 were as previously described
(Sutarno and Lymbery, 1997).
RFLP Analysis
Amplified D-loop fragments were digested
with 10 enzymes (AvaII, SspI, PstI, TaqI, HpaII,
BstXI, Bam HI, ApaI, DraI, NsiI), and amplified
ND-5 fragments were digested with 6 enzymes
(SpeI, HindIII, EcoRI, BstXI, SnaBI, DraI).
Aliquots of 7 ml (amplified growth hormone
loci) of DNA was put in a sterile tube. Master mix
containing enzyme, buffer and water was added to
the tube containing amplified DNA and incubated
as directed by the manufacturer. Digested samples
were then electrophoresed on 1-2% agarose gels
(Promega) in TAE buffer (40mM Tris-HCl; 20mM
Acetate; 2mM EDTA, pH adjusted to 7.9).
Electrophoresis was performed using horizontal
gels, in electrophoretic cells (Bio-Rad, Richmond,
USA) for 90 minutes at 55 volts. Ethidium bromide
was included in the gel at a final concentration of
0.12 mg/ml. After electrophoresis, DNA was
visualized under UV-illumination and
photographed using Polaroid type 57 film with a
red filter.
12
S Data Analysis
RN
A Genetic diversity at the mtDNA loci was
DR
analyzed using the “infinite sites” model in the
16S
program RESTSITE (Miller, 1991). The nucleotide
R
NA
diversity, or average number of nucleotide
substitutions per site within breeds (d) was
ND 1
estimated over all loci by the method of Nei and Li
ND 2
(1979), with standard errors calculated by
jackknifing (Nei & Miller, 1990). Allelic
I
CO
frequencies within each breed were calculated
II
CO separately for each locus/restriction site
combination. Genetic distances between breeds
were calculated using the equation of Jukes-Cantor
(1969), and these distance matrices were used to
construct phenograms of relationships between
breeds using UPGMA.
RESULTS
Restriction Site Polymorphisms in Mitochondrial
D-loop and ND-5
Products resulting from amplification of the
mitochondrial D-loop (1142bp) by PCR using
primers D-L and D-R, and the ND-5 (453bp)
region, using primers ND-L and ND-R, are shown
in Figure 2 and 3
Across all breeds except Murray Grey and
Belgian Blue, polymorphisms were detected in
mitochondrial D-loop and ND-5 fragments. Figure
3.9 and 3.10 showed photographs of mitochondrial
D-loop and ND-5 polymorphisms detected by
PCR-RFLP using SspI and HindIII.
 SUTARNO - Genetic Diversity 1. Mitochondrial DNA 3

100bp marker
V011

V012

V013

V519

V520
V015
1142bp
600bp

100bp

Figure 2. Photograph of an ethidium bromide stained agarose gel showing the specificity of the PCR products (1142 bp)
representing the whole mitochondrial D-loop and flanking sequence at both ends amplified using primers D-L and
D-R. Lane 1= J97 (Simmental), 2= W003 (Limousin), 3= L34D (Charolais), 4= DF1 (Friesian), 5= TBB1 (Belgian
Blue), 6= Her1 (Hereford).

100bp marker
V011

V012

V013

V015
V519

V520

600bp
453bp

100bp

Figure 3. Photograph of an ethidium bromide stained agarose gel showing the specificity of the PCR products (453 bp) of
mitochondrial ND-5 between positions 12058 and 12510, amplified using primers ND-L and ND-R. Lane 1= Sim3
(Simmental), 2= K85 (Limousin), 3= L33 (Charolais), 4= DF2 (Friesian), 5= Bambi2 (Murray Grey), 6= BL2 (Bali
cattle).
100bp marker
V003
V006
V021
V022
V023
V024
V013
V015
V016
V542
V551
V543
V564
uncut

850bp
600bp 660bp
290bp
100bp 110bp

Figure 4. An example of the gel photographs of an ethidium bromide stained agarose gel showing growth mitochondrial D-loop
polymorphism detected by PCR-RFLP using SspI.
4 BioSMART Vol. 1, No. 1, April 1999, hal. 1-8

The PCR products of the D-loop were digested Table 1. Restriction sites for the 10 restriction enzymes in the
by the 10 enzymes listed in Table 1. About 78 - 1142-bp PCR product of mitochondrial D-loop.
Enzymes Alleles Number of Fragment size (kb)
102 bp were screened by these enzymes sites
representing 6.8% to 9.3 % of the D-loop region, HpaII A 3 0.60, 0.23, 0.19, 0.02
and the cleavage patterns of those enzymes are TaqI A 2 0.70, 0.41, 0.03
shown in Table 2. B 1 0.73, 0.41
Five polymorphic sites in this region were PstI A 1 1.00, 0.14
B 0 1.14
detected with the enzymes TaqI, PstI, SspI, ApaI DraI A 2 0.85, 0.18, 0.11
and AvaII. These polymorphic sites are due to the SspI A 1 0.85, 0.29
addition of an SspI site, absence of a TaqI site, B 2 0.66, 0.29, 0.15
absence of a PstI site, and addition of ApaI and BamHI A 1 0.60, 0.54
ApaI A 1 0.63, 0.51
AvaII restriction sites. Eight haplotypes were found B 2 0.63, 0.46, 0.05
with these 5 polymorphic sites, and restriction BstXI A 1 0.67, 0.47
maps of these haplotypes are shown in Figure 5. AvaII A 1 0.70, 0.44
B 2 0.66, 0.44, 0.04
NsiI A 1 0.71, 0.43

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1
1
A B C D E J F AG H I B A D

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1
2
A * C D E J F AG H I B A D

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1
3
A * * D E J F AG H I B A D

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1
4
A * * D E J F AG H I B A E* D

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1
5
A * * D E J F AG H I B A E* D G*

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1
6
A * * D E J F AG H I B A E* D
I*
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1
7
A * C D E J F AG H I B A E* D

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1
8
A B * D E J F AG H I B A D
I*

Figure 5. Restriction maps of mitochondrial D-loop haplotypes. The enzymes are: HpaII (A), TaqI (B), PstI (C), DraI (D), SspI
(E), BamHI (F), ApaI (G), BstXI (H), AvaII (I) and NsiI (J). The restriction sites are according to the sequence of
Anderson et al. (1982). Polymorphic sites are indicated by *. The eight haplotypes coded by the alleles in Table 3.5 are:
1. AAAAA; 2. ABAAA; 3. ABBAA; 4. ABBBA; 5. BBBBA; 6. ABBBB; 7. ABABA; and 8. AABAB.
 SUTARNO - Genetic Diversity 1. Mitochondrial DNA 5

The PCR products of mitochondrial ND-5 were The standard measures of genetic diversity at
digested by 6 enzymes, corresponding to 42bp or mitochondrial D-loop and ND-5 over all breeds are
9.3 % of the region, and the cleavage patterns of shown in Table 3., and allelic frequencies in Table
the ND-5 region are shown in Table 3.6. The 4.
cutting sites of all enzymes were as predicted by
the previously published sequence (Anderson et al., Table 3. Genetic diversity for 8 breeds of Bos taurus
1982). Two polymorphic sites in this region were and Bali cattle (Bos javanicus). d is the nucleotide
detected with SpeI and HindIII enzymes. Two diversity, or average number of nucleotide substitutions
haplotypes were found at the ND-5 locus and the per site (+ standard error) within breeds over all
restriction enzyme/ locus combinations.
restriction maps of these haplotypes are shown in
Figure 6.
Breeds N d (Nei – Li)
Simmental 18 0.022218 + 0.007650
Table 2. Restriction sites for the 6 restriction enzymes Limousin 13 0.036073 + 0.014667
in the 453-bp PCR product of mitochondrial ND-5. Charolais 16 0.044106 + 0.018512
Friesian 25 0.055590 + 0.021303
Enzymes Alleles Number of Fragment Belgian Blue 6 0.000000 + 0.000000
sites size (kb) Hereford 12 0.053076 + 0.020582
BstXI A 1 0.42, 0.04 Angus 6 0.046118 + 0.019518
SpeI A 1 0.38, 0.08 Murray Grey 13 0.000000 + 0.000000
B 0 0.46 Bali cattle 2 0.121956 + 0.080763
HindIII A 1 0.33, 0.13
B 0 0.46 Genetic Distances Between Breeds
SnaBI A 1 0.29, 0.17
DraI A 1 0.30, 0.16 The matrix of Jukes-Cantor genetic distances
EcoRI A 1 0.36, 0.10 calculated from the mtDNA (D-loop and ND-5)
restriction site data is shown in Table 5.

0 0.1 0.2 0.3 0.4

1
A B C D E F

0 0.1 0.2 0.3 0.4

2
* *
A D E F

Figure 6. Restriction maps of ND-5. The enzymes are: BstXI (A), SpeI (B), Hind III (C), SnaBI(D) DraI (E) and EcoRI (F). The
restriction sites are according to the sequence of Anderson et al. (1982). Polymorphic sites are indicated by *. The two
haplotypes coded by the alleles in Table 3.6 are: 1. (AAAAAA); and 2. (ABAABA).

Table 4. Allelic frequencies for 8 breeds of Bos taurus and Bali cattle (Bos javanicus) based on PCR-RFLP analysis in the
mitochondrial D-loop and ND-5. For each polymorphic restriction site the common allele is coded A, and the uncommon allele is
coded B.

Breed D-loop ND-5

TaqI PstI SspI ApaI AvaII HindIII SpeI
A B A B A B A B A B A B A B
Simmental 0.94 0.06 0.83 0.17 0.83 0.17 0.94 0.06 1.00 0.00 0.83 0.17 0.78 0.22
Limousin 0.77 0.23 0.77 0.23 0.77 0.23 1.00 0.00 1.00 0.00 0.69 0.31 0.69 0.31
Charolais 0.69 0.31 0.69 0.31 0.69 0.31 1.00 0.00 1.00 0.00 0.69 0.31 0.69 0.31
Friesian 0.68 0.32 0.56 0.44 0.56 0.44 1.00 0.00 0.96 0.04 0.56 0.44 0.56 0.44
Belgian Blue 1.00 0.00 1.00 0.00 1.00 0.00 1.00 0.00 1.00 0.00 1.00 0.00 1.00 0.00
Hereford 0.67 0.33 0.67 0.33 0.67 0.33 1.00 0.00 1.00 0.00 0.67 0.33 0.67 0.33
Angus 0.67 0.33 0.67 0.33 0.67 0.33 0.83 0.17 1.00 0.00 0.67 0.33 0.67 0.33
Murray Grey 1.00 0.00 1.00 0.00 1.00 0.00 1.00 0.00 1.00 0.00 1.00 0.00 1.00 0.00
Bali cattle 0.50 0.50 0.50 0.50 0.50 0.50 1.00 0.00 1.00 0.00 0.50 0.50 0.50 0.50
6 BioSMART Vol. 1, No. 1, April 1999, hal. 1-8

Table 5. Matrix of Jukes-Cantor distances (above diagonal) calculated from mtDNA between breeds of cattle.

Simmental Limousin Charolais Friesian B.Blue Hereford Angus M.Grey Bos javanicus
Simmental - 0.0011 0.0010 0.0083 0.0006 0.0021 0.0020 0.0006 0.0217
Limousin - 0.0025 0.0026 0.0025 0.0020 0.0051 0.0025 0.0286
Charolais - 0.0003 0.0065 0.0040 0.0053 0.0065 0.0317
Friesian - 0.0159 0.0016 0.0012 0.0015 0.0382
B. Blue - 0.0088 0.0027 0.0000 0.0132
Hereford - 0.0059 0.0088 0.0341
Angus - 0.0027 0.0330
M. Grey - 0.0132
B.javanicus -

The UPGMA phenograms of relationships between breeds calculated from the distance matrix of
Jukes-Cantor distances from mtDNA are shown in Fig. 7.

Simmental 1

Belgian Blue 5

Murray Grey 8

Limousin 2

Angus 7

Charolais 3

Friesian 4

Hereford 6

Bos Javanicus 9

Figure 7. UPGMA phenograms of relationships between breeds, calculated from the distance matrix of Jukes-Cantor distances
from mtDNA.

DISCUSSION DNA level, can also be used as a check on the level

Genetic variation within breeds is important and
its study has become a subject of interest in
livestock species, as it has many applications in
animal breeding and genetics. Archibald (1983)
listed applications such as the identification of
animals and parentage testing, gene mapping and
identifying markers for performance traits. Since
all phenotypic characters are influenced by the
genetic information carried by DNA, DNA
variation may be correlated with variation in
performance traits. This idea is the basis for marker
assisted selection (MAS), which has aroused much
interest in recent years (Schwerin et al., 1995;
Soller, 1994). Genetic variation, measured at the
of genetic variation in quantitative traits maintained
within breeds. This will be extremely valuable in
maintaining pools of genetic diversity, which
artificial selection often acts to reduce (Archibald,
1983).
As demonstrated in this study, recent
developments in molecular techniques have
resulted in an abundance of data on genetic
polymorphisms from DNA analysis. These data
will provide us with a better understanding of the
nature of genetic variation within and between
cattle breeds. PCR-RFLP analysis can detect the
same type of polymorphisms as traditional RFLP
analysis, but without the need for Southern blotting
(Cushwa & Medrano, 1996), thus decreasing the
 SUTARNO - Genetic Diversity 1. Mitochondrial DNA
time taken and increasing sensitivity (Weber &
May, 1989). This method is therefore very useful
for the study of genetic variation.
Polymorphisms were found in the mitochondrial
D-loop region with ApaI, PstI, and TaqI, and in the
mitochondrial ND-5 region using HindIII,
confirming previous reports (Suzuki et al., 1993;
Watanabe et al., 1985b). New polymorphisms were
found in the D-loop by PCR-RFLP analysis using
AvaII and SspI, and in the ND-5 region using SpeI
(Sutarno & Lymbery, 1997). Compared to the
standard bovine sequence of mitochondrial DNA
(Anderson et al., 1982), these variant sequences
have an extra AvaII and SspI site in the D-loop and
have lost an SpeI site in mitochondrial ND-5.
Sutarno and Lymbery (1997) suggested that the
most likely scenarios for restriction site gains in the
D-loop are a T to C transition at position 16273 to
create a new AvaII site, and a C to T transition at
position 245 to create a new SspI site. This was
confirmed with the sequence data (data not shown)
There was little evidence of differences in
genetic diversity among the cattle breeds examined
in this study based on mitochondrial DNA (Table
4). The only major discrepancies were the absence
of mtDNA variation among Belgian Blue and
Murray Grey cattle. This may indicate a restriction
of dam lines in these breeds in Western Australia,
but may simply be an artifact of the small sample
sizes in this study.
For conservation purposes, study of the genetic
variation between livestock breeds is very
important. Adaptation to their different
environmental challenges may have resulted in a
unique combination of alleles specific to certain
breeds, and this would be difficult to recreate (Hall
& Bradley, 1995). Breeds which are very different
to others may need to be conserved, since the genes
and gene combinations that they carry may be
useful to agriculture in the future.
I found substantial genetic divergence between
Bos taurus and Bos javanicus (Bali cattle) in
mtDNA (Figure 6), although not in the growth
hormone gene (shown in the next series). A
previous study (Baker & Manwell, 1991) has also
suggested the genetic distinctness of Bali cattle. It
has long been demonstrated that mtDNA evolves
more rapidly than the nuclear genome (Brown,
1980). The rapid evolution of mtDNA has recently
made this cytoplasmically inherited DNA a
favoured source of information for population and
evolutionary studies (Bhat, Mishra & Bhat, 1990;
Loftus et al., 1994a; Suzuki et al., 1993; Watanabe
et al., 1985b).
Bali cattle (Bos javanicus), more popularly
called Banteng, have been domesticated largely in
7
Indonesia, especially in Java, Bali and Borneo. The
wild type of Banteng currently occurs in Burma,
Thailand, Kampuchea and Indonesia (Baker &
Manwell, 1991). Cattle of this type superficially
resemble Zebu, as they possess a hump, but the
bone structure of the head is quite different.
Copland (1996) suggested that Bali cattle are more
similar to ancestral cattle than other modern types.
According to the assessment done by AWCSG
(Asian Wild Cattle Specialist Group) in 1995, Bos
javanicus has been categorized as endangered, due
to disease, hunting, hybridization or trade (Heinen
& Srikosamatara, 1996). Indeed, the introduction of
modern cattle to Indonesia in the last few decades
has partly caused a reduction in the diversity of
Bali cattle. This is unfortunate because they are
considered an original species with several
economic advantages such as high fertility rate,
adaptability and carcass percentage.
Another important application of genetic
variation between breeds is to predict the crosses
between breeds that will produce crossbreed
offspring with maximum heterosis. Much more
attention has been paid in recent years to the
utilization of heterosis in beef cattle and other
livestock species. However, because there are so
many breeds that could be used for crossbreeding,
it is impossible to experimentally cross and
compare all breeds. The ability to predict the
magnitude of heterosis effects between breeds is
therefore crucially needed (Goddard & Ahmed,
1982). Since previous reports have indicated that
heterosis does not increase in any simple way with
increasing performance differences between
parental populations (Robertson & Reeve, 1955),
and relationships between heterosis and marker
gene distance have been found (Ehiobu, Goddard
& Taylor, 1990; Goddard & Ahmed, 1982; Graml
& Pirchner, 1984), the study of genetic variation at
the DNA level in parental populations may increase
the accuracy of predicting heterosis in
crossbreeding.
Previous studies using blood group loci (Graml
& Pirchner, 1984), allozymes (Ehiobu et al., 1990;
Goddard & Ahmed, 1982), minisatellites (Gavora
et al., 1996) and RAPDs (Helms et al., 1997) to
measure genetic distance, have found only
moderate associations with heterosis. Graml and
Pirchner (1984) and Helms et al. (1997) suggested
that markers more closely related to performance
genes may improve the predictability of heterosis.
Whether or not, the mtDNA polymorphisms
associated with production traits in beef cattle, it
remains to be tested experimentally.
8 BioSMART Vol. 1, No. 1, April 1999, hal. 1-8
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