Download Identification of genetic polymorphisms in bovine mitochondrial

Published December 4, 2014
Identification of genetic polymorphisms in bovine
mitochondrial deoxyribonucleic acid1
E. Kim,*2 H. S. Cheong,†2 J. S. Bae,‡ J. Chun,‡ T. J. Park,‡ K. Lee,*
Y. Yun,* and H. D. Shin†‡3
*Department of Veterinary Medicine, Graduate School, Cheju National University, Cheju, Korea, 153-801;
†Department of Genetic Epidemiology, SNP Genetics Inc., WooLim Lion’s Valley, 371-28, Gasan-Dong,
Geumcheon-Gu, Seoul, Korea, 153-801; and ‡Department of Life Science, Sogang University,
1 Shinsu-dong, Mapo-gu, Seoul, Korea, 121-742
ABSTRACT: In this study, the intent was to identify genetic polymorphisms of mitochondrial (mt) DNA
in Korean cattle (Bos taurus coreana) and to analyze
the genetic relationship between Korean cattle and
other breeds. Whole mtDNA genomes (16,338 bp) of
26 animals (16 Korean cattle and 10 Holsteins) were
directly sequenced. Multiple alignments, including 26
whole-mtDNA sequences obtained by direct sequencing
and 10 mtDNA sequences from a public database (National Center for Biotechnology Information), revealed
393 mtDNA polymorphisms (382 SNP, 3 heteroplas-
mies, and 8 insertion-deletion polymorphisms). Estimated gene diversity of mtDNA was 0.00198 among
these 36 animals. Phylogenic analysis with mtDNA
polymorphisms revealed a distinct genetic difference
between Bos taurus (Korean, Japanese Black, Holstein,
and Fleckvieh breeds) and Bos indicus (Nellore and
Zwergzebu breeds). The genetic information regarding
mtDNA polymorphisms identified in this study would
be useful for further investigation of mtDNA in other
breeds.
Key words: Bos taurus coreana, direct sequencing, Korean cattle, mitochondrial deoxyribonucleic acid,
phylogenic analysis, polymorphism
©2010 American Society of Animal Science. All rights reserved.
INTRODUCTION
More than 800 breeds of cattle are widely distributed
throughout the world. The cattle species are thought
to have originated from the aurochs, Bos primigenius,
which has since been divided into humpless taurine
(Bos taurus) and humped zebu (Bos indicus) through
domestication (Epstein, 1971; Epstein and Mason,
1984; MacHugh et al., 1997). Bos taurus includes Korean native, Japanese Black, Holstein, Jersey, Angus,
Limousin, Fleckvieh, and other cattle breeds. They are
typical breeds of Europe, northeastern Asia, and parts
of Africa, and are adapted to cooler climates. Bos indicus includes Gir, Guzerat, Kankrej, Indo-Brazilian,
Brahman, Nelore, Zwergzebu, and other cattle breeds.
1
This research was supported by the Bio research and development program through the Korea Science and Engineering Foundation (Daejeon), funded by the Ministry of Education, Science and
Technology (Seoul; M10741670000-07N4167-00000).
2
E. Kim and H. S. Cheong contributed equally to this work.
3
Corresponding author: [email protected]
Received June 21, 2009.
Accepted April 13, 2010.
J. Anim. Sci. 2010. 88:2551–2555
doi:10.2527/jas.2009-2235
They are more common in Africa and South Asia and
are adapted to more tropical environments than other
domestic cattle.
After the completion of human mitochondrial (mt)
DNA sequencing (Anderson et al., 1981), the complete
mt genomes were sequenced for the following additional
organisms: common chimpanzees (Horai et al., 1995;
Arnason et al., 1996), mice (Bibb et al., 1981), fin
whales (Arnason et al., 1991), blue whales, and horses
(Arnason and Gullberg, 1993).
In addition, the complete 16,338-nucleotide DNA sequence of the bovine mt genome was determined (Anderson et al., 1982). The gene arrangement was found
to be identical to that of humans. It contains 12S and
16S rRNA, 22 tRNA, 13 protein components of the
inner mt membrane, and a variable noncoding control
region or displacement loop (D-loop), which regulates
transcription and replication of the coding DNA strand
of the mtDNA (Anderson et al., 1981; Clayton, 1991).
Several studies have reported that genetic diversity
exists in bovine mtDNA (Bradley et al., 1996; Hiendleder et al., 2008; Zhang et al., 2009). Among them,
Hiendleder et al. (2008) reported 237 polymorphisms in
a comparison of 2 complete bovine mtDNA sequences
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Kim et al.
Table 1. Sequence information used in this study1
Breed
Bos taurus
Korean
Holstein
Japanese Black
Fleckvieh
Bos indicus
Nellore
Zwergzebu
Total
No. of sequences
1
16
10
7
1
1
1
Genome information
Source
AY526085
Direct sequencing
Direct sequencing
AB074962- AB074968
AF492351
NCBI
This study
This study
NCBI
NCBI
AY126697
AF492350
36
NCBI
NCBI
1
The complete mitochondrial DNA of 16 Korean and 10 Holstein cattle were sequenced directly in this
study. For multiple-alignment and phylogenic analysis, other sequences, including those of Japanese Black (B.
taurus), Fleckvieh (B. taurus), Nellore (B. indicus), and Zwergzebu (B. indicus) cattle, were also used. NCBI
= National Center for Biotechnology Information.
from the Simmental breed (B. taurus) and Dwarf Zebu
breed (B. indicus). To date, however, large-scale identification of polymorphisms in a large number of whole
mtDNA has not been performed. In this study, it was
our intent to identify genetic polymorphisms of whole
mtDNA by direct sequencing of 16 Korean and 10 Holstein-Friesian cattle. In addition, phylogenic analysis
using mtDNA variants was performed.
MATERIALS AND METHODS
All the experimental procedures conducted in this
study were approved by the Animal Ethics Committees
of the National Agricultural Cooperative Federation
(Seoul) and the National Livestock Research Institute
(Suwon) of Korea.
Animals
The animal subjects used in this study included 16
Korean cattle (also called Hanwoo), which were bred
at the Hanwoo Improvement Center, National Agricultural Cooperative Federation, and 10 Holstein-Friesian
cattle, which were bred at the National Livestock Research Institute.
Preparation of DNA and Direct Sequencing
of Complete mtDNA
Total DNA (genomic and mtDNA) was extracted from blood samples (5 mL of whole blood cells in
EDTA tubes) using a commercial DNA extraction kit
(QIAamp DNA Blood Midi Kit, Qiagen, Valencia, CA).
A total of 43 primer sets were designed to amplify complete mtDNA sequences (Supplemental Table 1; http://
jas.fass.org/content/vol88/issue8/). Polymerase chain
reaction was performed in a solution of 30 µL, which
consisted of sterile distilled water, 10 ng of template
DNA, 1.25 pmol of each primer, 250 µM deoxynucleotide 5′-triphosphate, 1× buffer, and 0.15 units of Taq
polymerase under touchdown PCR conditions (Don et
al., 1991), using a thermal cycler (Applied Biosystems,
Foster City, CA). Products from PCR were gel-purified
using the CleanUp Kit (a 96-well PCR purification
plate, Millipore, Billerica, MA). After performing Big
Dye sequencing, capillary electrophoresis was carried
out using a 3730 DNA Analyzer following the protocol
of the manufacturer (Applied Biosystems).
Data Analysis
The mtDNA of Korean native cattle (GenBank accession number AY526085) was used as a reference sequence. To compare their complete mtDNA sequence
with that of other breeds, sequences from B. taurus (7
Japanese Black, 1 Fleckvieh) and B. indicus (1 Nellore,
1 Zwergzebu) were obtained from the National Center
for Biotechnology Information (http://www.ncbi.nlm.
nih.gov/sites/entrez; Table 1). Multiple alignment of
mtDNA sequencing was performed to discover sequence
variants using SeqMan software (DNASTAR, Madison,
WI).
Alignment of sequences was achieved using the ClustalW package (Thompson et al., 1994). Sites representing a gap in the aligned sequences were included in
the analysis. Multiple-alignment parameters are as
follows: DNA transition weight = 0.5, weight matrix
= IUB/BESTFIT, gap open penalty = 15, gap extension penalty = 6.66, and DNA divergent sequences =
30. Phylogenies were constructed using the neighborjoining method (Saitou and Nei, 1987) incorporated
in PHYLIP (version 3.69; Felsenstein, 1993). In the
neighbor-joining analysis, 1,000 bootstrap iterations
were performed. Genetic distance was calculated using
ClustalDist in the ClustalW package (Thompson et al.,
1994).
Gene diversity was estimated for B. taurus (Korean,
Japanese Black, Holstein, Fleckvieh) and B. indicus
(Nellore and Zwergzebu) as n[(n − 1)Σxixjdij]−1, where
n is the number of DNA sequences examined; xi and
xj are the population frequencies of the ith and jth
DNA sequence, respectively; and dij is the proportion
of nucleotides that differ between the ith and jth DNA
sequence (Jorde et al., 2000).
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Bovine mitochondrial deoxyribonucleic acid polymorphisms
Table 2. Genes and number of polymorphisms in 36 cattle, including Bos taurus and
Bos indicus
Gene
Full name
D-loop
12S rRNA
16S rRNA
tRNA-Leu
tRNA-Met
tRNA-Ala
tRNA-Asn
tRNA-Cys
tRNA-Asp
tRNA-Gly
tRNA-Ser
tRNA-Glu
tRNA-Thr
tRNA-Pro
ND1
ND2
ND3
ND4
ND4L
ND5
ND6
COX1
COX2
COX3
ATP6
ATP8
CYTB
Intergenic
L-strand
Total
Displacement loop
12S ribosomal RNA
16S ribosomal RNA
Transfer RNA leucine
Transfer RNA methionine
Transfer RNA alanine
Transfer RNA asparagine
Transfer RNA cysteine
Transfer RNA aspartate
Transfer RNA glycine
Transfer RNA serine
Transfer RNA glutamate
Transfer RNA threonine
Transfer RNA proline
NADH dehydrogenase subunit 1
NADH dehydrogenase subunit 2
NADH dehydrogenase subunit 3
NADH dehydrogenase subunit 4
NADH dehydrogenase subunit 4L
NADH dehydrogenase subunit 5
NADH dehydrogenase subunit 6
Cytochrome C oxidase subunit 1
Cytochrome C oxidase subunit 2
Cytochrome C oxidase subunit 3
ATP synthase subunit 6
ATP synthase subunit 8
Cytochrome b
—
—
—
RESULTS
Complete mtDNA sequences were obtained by direct sequencing of 16 Korean cattle and l0 Holsteins.
A total of 393 mtDNA polymorphisms among 36 cattle, including B. taurus and B. indicus, were identified
in the whole-mtDNA genome. Information regarding
these polymorphisms is listed in Supplemental Table
2 (http://jas.fass.org/content/vol88/issue8/), including Single Nucleotide Polymorphism Database submitter SNP accession numbers, the gene regions, minor
allele frequency, and nucleotide change. The numbers
of mtDNA polymorphisms in each gene region were as
follows: 77 in the D-loop, 10 in 12S rRNA, 28 in 16S
rRNA, 152 in NADH dehydrogenase subunits 1 to 6, 5
in NADH dehydrogenase subunit 4L, 21 in tRNA, 55
in cytochrome C oxidase subunits 1 to 3, 8 in ATP8,
11 in ATP6, 24 in cytochrome b, 1 in the L-strand
replication origin site, and 1 in the intergenic region
(Table 2). The number of polymorphisms with alleles
found only in B. taurus and B. indicus were 137 and
209, respectively (Supplemental Table 2). Among the
393 mtDNA polymorphisms, most of the nucleotide
changes (91.4%) were transitions (A to G: 37.7%; C to
T: 53.7%; Table 3). In this study, only 3 heteroplasmic
loci were observed. However, many more heteroplasmies
may exist that could not be detected because of smaller
copy numbers than the detection limit of the technol-
No. of polymorphisms
77
10
28
2
1
1
1
2
5
1
1
1
3
3
28
19
10
30
5
49
16
28
13
14
11
8
24
1
1
393
ogy used in this study (direct DNA sequencing). Gene
diversity of the mtDNA genome among the 36 cattle in
this study was calculated as 0.00198 (Table 4).
To analyze the genetic relationship among breeds,
phylogenic analyses using representative mtDNA sequences of each breed were performed. The representative sequences (the most common allele represented in
the sequence used) of Korean, Japanese Black, and Holstein cattle were exactly matched. Therefore, we used 4
sequences, 1 representative sequence for Korean, Japanese Black, and Holstein cattle, and 3 individual sequences of Fleckvieh, Nellore, and Zwergzebu cattle, for
Table 3. Types of nucleotide substitutions in mitochondrial DNA polymorphisms
Nucleotide
substitution
C/T
A/G
A/C
G/T
C/G
A/T
Others1
Total
Number
Percentage
211
148
10
2
7
4
11
393
53.7
37.7
2.5
0.5
1.8
1.0
2.8
100
1
Others include insertion-deletion polymorphisms and heteroplasmies.
2554
Kim et al.
Figure 1. Unrooted neighbor-joining tree constructed from Korean cattle (KC)/Japanese Black (JB)/Holstein (HS), Fleckvieh, Nellore, and
Zwergzebu sequences. The numbers at the branches are confidence values based on the bootstrap method of Felsenstein (1993).
phylogenetic and genetic distance analyses. As expected, B. taurus and B. indicus showed a distinct genetic
difference from each other (Figure 1 and Supplemental
Table 3; http://jas.fass.org/content/vol88/issue8/).
DISCUSSION
Bovine mtDNA, like that of other higher animals, is
double-stranded, circular, and lacking in introns, with
a highly conserved size and gene content. It encodes 22
tRNA, 2 rRNA, and 13 subunits of the oxidative phosphorylation pathway (Anderson et al., 1982; Wolstenholme, 1992; Shadel and Clayton, 1997). Mitochondrial
DNA has a unique mode of inheritance (maternally inherited), and has a greater mutation rate than nucleic
DNA (Ballard and Whitlock, 2004; Haag-Liautard et
al., 2008), which is due to the mutagenic properties
of reactive oxygen species (the harmful by-products of
oxidative phosphorylation), an error-prone polymerase,
and limited mtDNA repair (Bogenhagen, 1999).
In this study, 393 polymorphisms including 3 heteroplasmic loci were detected from a 16-kb genome among
36 animals. Although polymorphisms were distributed
throughout the gene region, a large portion of polymorphisms was found in the D-loop (77 polymorphisms/400
bp), which is known as the noncoding and hypervariable region. This greater polymorphic rate in the Dloop region has been reported in humans and bovines
(Aquadro and Greenberg, 1983; Hiendleder et al., 2008).
Similar to our results, Hiendleder et al. (2008) found 49
polymorphisms in the D-loop region among 237 bovine
mtDNA variants. The frequency of polymorphisms was
0.024% (393 polymorphisms/16,338 bp), which was
much greater than the frequency of autosomes [8.94 ×
10−4, estimated from a Celera (Rockville, MD)-publicly
funded Human Genome Project comparison].
Table 4. Gene diversity estimates of whole-mitochonchrial DNA sequences among cattle populations
Breed
All
Bos taurus Korean
Japanese Black
Holstein
Fleckvieh
Bos indicus Nellore/Zwergzebu
Number
Gene diversity
36
34
16
7
10
1
2
0.00198
0.00116
0.00063
0.00028
0.00034
—
0.00086
To identify the origin and genetic diversity of cattle
in northeastern Asia, mt analyses have been performed
on Japanese, Mongolian, and Korean cattle (Mannen et
al., 2004), and these showed that Japanese and Korean
cattle contained only B. taurus haplotypes. The Korean
and Japanese samples were composed of haplogroups
that were also found throughout Europe, the Near East,
and especially East Asia. In this study, the representative sequences of 3 B. taurus breeds (Korean, Japanese
Black, and Holstein cattle) were exactly matched. Although our study was not based on haplotype analysis,
we also showed that 2 East Asia breeds (Korean and
Japanese cattle) and a Europe breed (Holsteins) were
closely related.
Because our study was performed with a limited sample size (n = 36) and we used only 2 sequences for B.
indicus (Zwergzebu and Nellore), the genetic diversity
estimates and phylogenetic analysis might be fragile.
However, this study is the first large-scale study identifying polymorphisms by using a reasonable number
of complete bovine mtDNA sequences. The genetictechnical information in this study would be useful for
further investigation.
In summary, we identified 393 mtDNA polymorphisms in 36 animals. Gene diversity and phylogenetic
analyses showed a close genetic background of 3 B.
taurus breeds (Korean, Japanese Black, and Holstein
cattle) and also revealed a distinct genetic difference
between B. taurus and B. indicus.
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