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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 2551 2552 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). 2553 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. 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