Genetic diversity of Cheju horses (Equus caballus) determined by using mitochondrial DNA D-loop polymorphism

We used sequence polymorphism of the mitochondrial DNA D-loop (968 bp excluding the tandem repeat region) to determine genetic diversity of horses inhabiting Cheju (a southern island of Korea). Seventeen haplotypes with frequencies from 1.5 to 21.5% were found among 65 Cheju horse samples. Genetic diversity (h) of the 17 haplotypes was calculated to be 0.91, indicating that the extant Cheju horse population consists of diverse genetic groups in their maternal lineage. Phylogenetic analysis showed that 17 types of Cheju (D-loop sequences determined), 5 Mongolian, 6 Arabian, 3 Belgian, 2 Tsushima, 2 Yunnan, 1 Przewalskii, and 3 Thoroughbred horses (published sequences for the latter seven breeds) showed that Cheju horses were distributed into many different clusters in the tree. Four Mongolian horses clustered with separate Cheju horse groups, showing that some Cheju horses are clearly of Mongolian origin. The analysis of partial sequences (284 bp) of the D-loop of 109 horses showed that Thoroughbred, Mongolian, Lipizzan, and Arabian breeds are as diverse as Cheju horses. Our data together with others' suggest that most horse breeds tested with reasonably sufficient numbers of samples are diverse in their maternal lineages and also are not uniquely different from each other.     

Genetic characterization of horse bone excavated from the Kwakji archaeological site,Jeju, Korea

We determined the nucleotide sequences of the hypervariable D-loop region of mitochondrial DNA (mtDNA) from horse bone (humerus, A.D. 700 to A.D. 800) that was excavated from the Kwakji archaeological site, Jeju, Korea. We compared them with ones from extant horses. We designed three pairs of oligonucleotide primers from the tRNA-Thr and tRNA-Phe gene regions of mtDNA that are highly conserved among many other animal species. We cloned 232, 336, and 644 bp from the horse bone in order to determine the mtDNA D-loop sequence. The sequence was 1,124 bp long; the middle contained 19 tandem repeats of an 8-bp sequence (TGTGCACC) that is specific to equines. The mtDNA D-loop region contained each base (total number, percentage of total) as follows: A (317, 28.20%), C (336, 29.89%), G (169, 15.04%), and T (302, 26.87%). This sequence, like those of other horse populations, was AT rich. Sequence divergence was the lowest (1.71%) between the ancient horse bone and that of the Thoroughbred horse 1. The neighbor-joining and strict consensus tree of three of the most parsimonious trees also suggested that the ancient bone was considerably unrelated to native Jeju horses. The molecular phylogenetic characteristics of the horse bone that was excavated from the Kwakji archaeological site (Jeju, Korea) showed that some horse breeds may have existed on Jeju Island, Korea before Mongolian horses were introduced. The horse bone that was excavated from the Kwakji archaeological site may aid future research on the origin and ancestry of native Jeju horses.     

Molecular phylogeny of Indian horse breeds with special reference to Manipuri pony based on mitochondrial D-loop

Manipuri pony is the geographically distant breed of horse from the five recognized horse breeds found in the Indian subcontinent. The phylogenetic relationship of Manipuri pony with the other breeds is unknown. The diversity in the mitochondrial (mt) DNA D-loop region is employed as an important tool to understand the origin and genetic diversification of domestic horses and to examine genetic relationships among breeds around the world. This study was carried out to understand the maternal lineages of Manipuri pony using the 247 bp region of the mtDNA D-loop. The dataset comprised of eleven numbers of self developed sequences of Manipuri pony, 59 and 35 number of retrieved sequences of Indian horse breeds and other worldwide breeds respectively. A total of 35 haplotypes was identified with a high level of genetic diversity in the Indian breeds. A total of seven major mtDNA haplogroups (A–G) was identified in the Indian horse breeds that indicated the abundance of mtDNA diversity and multiple origins of maternal lineages in them. The majority of the studied sequences of Indian breeds (33.3 %) were grouped into haplogroup D and least (3.9 %) in haplogroup E. The Manipuri breed showed the least F_ST distance (0.03866) with the most diverged Indian breeds and with Thoroughbred horse among the worldwide. This study indicated a close association between Manipuri pony and Thoroughbred.     

Mitochondrial D-loop sequence variation among Italian horse breeds

The genetic variability of the mitochondrial D-loop DNA sequence in seven horse breeds bred in Italy (Giara, Haflinger, Italian trotter, Lipizzan, Maremmano, Thoroughbred and Sarcidano) was analysed. Five unrelated horses were chosen in each breed and twenty-two haplotypes were identified. The sequences obtained were aligned and compared with a reference sequence and with 27 mtDNA D-loop sequences selected in the GenBank database, representing Spanish, Portuguese, North African, wild horses and an Equus asinus sequence as the outgroup. Kimura two-parameter distances were calculated and a cluster analysis using the Neighbour-joining method was performed to obtain phylogenetic trees among breeds bred in Italy and among Italian and foreign breeds. The cluster analysis indicates that all the breeds but Giara are divided in the two trees, and no clear relationships were revealed between Italian populations and the other breeds. These results could be interpreted as showing the mixed origin of breeds bred in Italy and probably indicate the presence of many ancient maternal lineages with high diversity in mtDNA sequences.     

Multiple maternal origins of native modern and ancient horse populations in China

To obtain more knowledge of the origin and genetic diversity of domestic horses in China, this study provides a comprehensive analysis of mitochondrial DNA (mtDNA) D-loop sequence diversity from nine horse breeds in China in conjunction with ancient DNA data and evidence from archaeological and historical records. A 247-bp mitochondrial D-loop sequence from 182 modern samples revealed a total of 70 haplotypes with a high level of genetic diversity. Seven major mtDNA haplogroups (A–G) and 16 clusters were identified for the 182 Chinese modern horses. In the present study, nine 247-bp mitochondrial D-loop sequences of ancient remains of Bronze Age horse from the Chifeng region of Inner Mongolia in China ( c. 4000–2000a bp ) were used to explore the origin and diversity of Chinese modern horses and the phylogenetic relationship between ancient and modern horses. The nine ancient horses carried seven haplotypes with rich genetic diversity, which were clustered together with modern individuals among haplogroups A, E and F. Modern domestic horse and ancient horse data support the multiple origins of domestic horses in China. This study supports the argument that multiple successful events of horse domestication, including separate introductions of wild mares into the domestic herds, may have occurred in antiquity, and that China cannot be excluded from these events. Indeed, the association of Far Eastern mtDNA types to haplogroup F was highly significant using Fisher's exact test of independence ( P  = 0.00002), lending support for Chinese domestication of this haplogroup. High diversity and all seven mtDNA haplogroups (A–G) with 16 clusters also suggest that further work is necessary to shed more light on horse domestication in China.     

中国蒙古马与国外纯血马mtDNA D-Loop高变区序列比较

比较分析了4匹中国蒙古马和4匹国外纯血马的线立体DNA（mtDNA）D-Loop高变区400bp核苷酸序列的变异情况。结果发现,4匹中国蒙古马mtDNA D-Loop高变区的平均核苷酸变异率为3.69%,而纯血马的为4.00%,其核苷酸变异类型均包括转换、颠换和缺失3种形式,其中以转换最为常见。核苷酸变异基因座多,并且存在长度变异,不同变异在个体之间差异也很大,因此说明中国蒙古马和国外纯血马的mtDNA D-Loop高变区都具有丰富的多态性。Abstract：Mitochondrial DNA D-Loop varied region 400bp sequence variations in 4 Chinese Mongolian horses and 4 External Thoroughbred horses were analyzed in this experiment. The results showed that the average nucleotide mutational rate of mtDNA D-Loop varied region in 4 Chinese Mongolian horses was 3.69%,while External Thoroughbred horses were 4.00%. Three types of mutations including transition,transversion and deletion were all found in the investigated mtDNA D-Loop regions,of which transition was the most frequent. Nucleotide mutational loci were abundant,length mutations were found and great differences were all observed among the 8 horses. It showed there existed much polymorphism in the mitochondrial DNA D-Loop varied region of Chinese Mongolian horses and External Thoroughbred horses.     

Phylogenetic relationships of the Hucul horse from Romania inferred from mitochondrial D-loop variation

The existence of the Hucul horse on Romanian territory has been documented from the very distant past; today Hucul is a unique breed that is part of the FAO Program for the Preservation of Animal Genetic Resources. We compared Hucul with several primitive European and Asiatic breeds in order to elucidate the origin of these horses. We analyzed a 683-bp mitochondrial DNA (mtDNA) D-loop fragment in a population of Hucul horses and compared the polymorphic sites with sequences from other primitive breeds, including Exmoor, Icelandic Pony, Sorraia, Przewalski Horse, Mongolian Wild Horse, Konik, and Shetland Pony, as well as with Arabian, Akhal Teke and Caspian Pony. The sequences were truncated to 247 bp to accommodate short sequence data for the other species. Eighty horses were analyzed; 35 polymorphic sites representing 33 haplotypes were observed. The mean percentage of polymorphic sites was 14.2% for this mtDNA fragment. A neighbor-joining phylogenetic tree was constructed based on Kimura two-parameter distances and the Network 3.111 software was used for phylogenetic analysis. The Hucul horse was classified separately from all other primitive breeds. It is possible that the Hucul horse is not part of the pony class, as it segregated apart from all primitive pony breeds. We found multiple origins in the maternal lineage of domestic horse breeds and demonstrated the uniqueness of the Hucul breed; its origins remain unclear.     

Genetic diversity and relationships among native Japanese horse breeds,the Japanese Thoroughbred and horses outside of Japan using genome‐wide SNP data

Eight horse breeds—Hokkaido, Kiso, Misaki, Noma, Taishu, Tokara, Miyako and Yonaguni—are native to Japan. Although Japanese native breeds are believed to have originated from ancient Mongolian horses imported from the Korean Peninsula, the phylogenetic relationships among these breeds are not well elucidated. In the present study, we compared genetic diversity among 32 international horse breeds previously evaluated by the Equine Genetic Diversity Consortium, the eight Japanese native breeds and Japanese Thoroughbreds using genome‐wide SNP genotype data. The proportion of polymorphic loci and expected heterozygosity showed that the native Japanese breeds, with the exception of the Hokkaido, have relatively low diversity compared to the other breeds sampled. Phylogenetic and cluster analyses demonstrated relationships among the breeds that largely reflect their geographic distribution in Japan. Based on these data, we suggest that Japanese horses originated from Mongolian horses migrating through the Korean Peninsula. The Japanese Thoroughbreds were distinct from the native breeds, and although they maintain similar overall diversity as Thoroughbreds from outside Japan, they also show evidence of uniqueness relative to the other Thoroughbred samples. This is the first study to place the eight native Japanese breeds and Japanese Thoroughbred in context with an international sample of diverse breeds.     

中国17个黄牛品种mtDNA变异特征与多态性分析

中国黄牛品种资源丰富,尚有28个地方固有品种.为了进一步深入了解这些宝贵遗传资源,本研究通过mtDNA变异特征与多态性分析揭示这些来自中国不同地域地方黄牛的母系起源与分子系统学特征.在17个品种84个体的mtDNA D-loop全序列中,一共检测到了102个核苷酸替代突变位.由此定义的53个单倍型被类聚为2个明显的单倍群：普通牛和瘤牛.mtDNA D-loop全序列变异的第一个特征是转换发生的频率远高于颠换;第二个特征是缺失与替代突变共存;第三个特征是缺失突变率比较高.所有D-loop全序列的核苷酸多样性和单倍型多样性分别为0.026 78±0.000 50和0.919±0.027.普通牛D-loop单倍型在北方牛种群中占有优势(80%～100%),而瘤牛单倍型在南方牛种群中占有优势(42.9%～100%),2种不同单倍型在中原牛种群中的分布也存在差异.2种不同单倍型在中国不同地域17个黄牛品种中的差异性分布揭示出了瘤牛mtDNA基因在中国黄牛中自南而北、由高到低的流动模式,这种基因流动模式的形成可能是由历史事件、地理隔离以及气候环境差异等造成的.     

Genetic relationship between Mongolian and Norwegian horses?

Human populations of Central Asian origin have contributed genetic material to northern European populations. It is likely that migrating humans carried livestock to ensure food and ease transportation. Thus, eastern genes could also have dispersed to northern European livestock populations. Using microsatellite data, we here report that the essentially different genetic distances DA and (deltamu)2 and their corresponding phylogenetic trees show close associations between the Mongolian native horse and northern European horse breeds. The genetic distances between the northern European breeds and Standardbred/Thoroughbred, representing a southern-derived source of horses, were notably larger. We suggest that contribution of genetic material from eastern horses to northern European populations is likely to have occurred.     

A genetic analysis of the Italian Salernitano horse

Salernitano (SAL) is an ancient Italian horse breed developed over the course of the ages together with Napoletano and, during the 20th century, by crossing with Thoroughbred horse lines. Excellent in hurdle jumping, this breed is currently facing a concrete risk of extinction due to the lack of appropriate management strategies. This research is the first SAL genetic characterization that aims to set up the basic knowledge for a conservation plan. A representative sample of 61 SALs was analyzed by means of a set of 16 microsatellites markers (short tandem repeats (STRs)). The sequence of hypervariable D-loop mtDNA region was also performed on a subset of 24 mares in order to study the maternal diversity and obtain a complete picture of the internal genetic variation. All the molecular data were analyzed together with those obtained from three Sicilian horse breeds investigated in a previous research (Siciliano, Sanfratellano and Sicilian Oriental Purebred). STRs markers revealed a moderate level of genetic diversity in SAL (alleles/locus 5.1, He 0.67) and confirmed the hunch of genetic erosion. Autosomal variability highlighted a very light deficit of homozygotes (FIS=−0.067). Experimental D-loop sequences were compared by multiple alignments with those retrieved from biological databases and revealed two unreported haplotypes. The phylogenetic network, which was built on mtDNA sequences, included various cosmopolitan and European horses and showed SAL haplotypes distributed among different mtDNA lineages.     

Molecular Analysis Using Mitochondrial DNA and Microsatellites to Infer the Formation Process of Japanese Native Horse Populations

To assess the genetic diversity of Japanese native horse populations, we examined seven such populations using mitochondrial DNA (mtDNA) and microsatellite analyses. Four reference populations of Mongolian horses and European breeds were employed as other equids. In the mtDNA analysis, the control region (D-loop) of 411 bp was sequenced, and 12 haplotypes with 33 variable sites were identified in the Japanese native horses. The phylogenetic tree constructed by haplogrouping and using worldwide geographic references indicated that the haplotypes of the Japanese native horses were derived from six equid clusters. Compared with the foreign populations, the Japanese native populations showed lower within-population diversity and higher between-population differentiation. Microsatellite analysis, using 27 markers, found an average number of alleles per locus of 9.6 in 318 native and foreign horses. In most native populations, the within-population diversity was lower than that observed in foreign populations. The genetic distance matrix based on allelic frequency indicated that several native populations had notably high between-population differentiation. The molecular coancestry-based genetic distance matrix revealed that the European populations were differentiated from the Japanese and Mongolian populations, and no clear groups could be identified among the Japanese native horse populations. The genetic distance matrices had few correlations with the geographic distribution of the Japanese native populations. Based on the results of both mtDNA and microsatellite analyses, it could be speculated that each native population was formed by the founder populations derived from Mongolian horses. The genetic construction of each population appears to have been derived from independent breeding in each local area since the time of population fission, and this was accompanied by drastic genetic drift in recent times. This information will help to elucidate the ancestry of Japanese native horses. An erratum to this article can be found at     

Mitochondrial D-loop sequence variation among the 16 maternal lines of the Lipizzan horse breed

Mitochondrial DNA from 49 Lipizzan horses representing 16 maternal lines from the original stud at Lipica was used for SSCP analysis and DNA sequencing. The SSCP analysis of the 444 bp long fragment of the D-loop region extending from the tRNA(Pro) gene to the central conserved sequence block revealed three distinct groups of SSCP patterns. Both ends of the D-loop region (378 bp and 310 bp), which are considered as the most variable regions within the mammalian mitochondrial DNA, were sequenced. According to 49 polymorphic sites identified within the both parts of the D-loop region, the 16 maternal lines were grouped into 13 distinct mitochondrial haplotypes. The minimal difference between two different haplotype DNA sequences was one nucleotide and the maximal 24 nucleotides. The inheritance of mitochondrial haplotypes was stable and no sequence variation potentially attributable to mutation within maternal line was observed. Considerable DNA sequence similarity of Lipizzan mitochondrial haplotypes with the haplotypes from other breeds was observed. Phylogenetic analysis of the sequence data revealed a dendrogram with three separated branches, supporting the historical data about the multiple origin of the Lipizzan breed.     

Phylogenetic relationships of Northeast Asian cattle to other cattle populations determined using mitochondrial DNA D-loop sequence polymorphism

Phylogenetic relationships of Northeast Asian cattle to various other cattle breeds including Bos taurus, Bos indicus, and Bison bison were assessed using mtDNA D-loop sequences. A neighbor-joining tree was constructed using sequences determined for 4 Cheju Black, 4 Cheju Yellow, 4 Korean Yellow cattle (Bos taurus), and 2 American Brahman cattle (Bos indicus), and also published sequences for 31 Japanese Black cattle, 45 European breed cattle, 6 African zebus, 2 African taurines, and 6 Indian zebus. Five American bisons (Bison bison) were used as an outgroup. The neighbor-joining tree showed that American bisons and Indian zebus are clearly separate from other cattle breeds, respectively, and African cattle clustered together, although with a low bootstrap probability (<50%). Results indicate that cattle in Northeast Asia, Europe, and Africa are closely related to each other–suggesting their recent divergence, but are separate from Indian zebus.     

贵州黄牛mtDNA D-loop 遗传多样性研究

对贵州4个地方黄牛品种共计82个个体的线粒体DNA D-loop区全序列910 bp进行分析,检测到31种单倍型,其核苷酸多态位点65个,约占所测核苷酸总长的7.14%,其中有62个转换,2个颠换,1个转换/颠换共存。贵州4个黄牛品种mtDNA D-loop区核苷酸多样度（π值）为2.16%～2.61%,单倍型多样度（H）为0.695～0.909,表明贵州黄牛mtDNA遗传多样性比较丰富。根据单倍型构建了贵州4个黄牛品种的NJ分子系统树。聚类表明,贵州黄牛有普通牛和瘤牛2大母系起源,其影响较为均一。并探讨了用核苷酸多样度π值的大小来衡量黄牛群体遗传分化程度的可行性。      

Sequence variation and structural conservation in the D-loop region and flanking genes of mitochondrial DNA from Japanese pond frogs

The nucleotide sequences of the D-loop region and its flanking genes of the mitochondrial DNA (mtDNA) from Japanese pond frogs were determined by the methods of PCR, cloning, and sequencing. The frogs belonged to two species, one subspecies, and one local race. The gene arrangements adjacent to the D-loop region were analyzed. The frogs shared a unique mitochondrial gene order that was found in Rana catesbeiana; i.e., cyt b--D-loop region--tRNA(Leu(CUN))--tRNA(Thr)--tRNA(Pro)--tRNA(Phe)--12S rRNA. The arrangements of the three tRNA genes of these frogs were different from those of X. laevis, a species which has the same overall structure as in mammals. Highly repetitive sequences with repeat units (16-bp or 17-bp sequence specific for each taxon) were found in the D-loop region. The length of repetitive sequences varied from 0.6 kbp to 1.2 kbp, and caused the extensive size variation in mtDNA. Several short sequence elements such as putative TAS, OH, CSB-1, and CSB-2 were found in the D-loop region of these frogs. The sequences of these short regulatory elements were conserved in R. catesbeiana, X. laevis, and also in human. The comparison of sequence divergences of the D-loop region and its adjacent genes among various taxa revealed that the rates of nucleotide substitutions depend on genes. The nucleotide sequences of the 3'-side segment of the D-loop region were the most variable among taxa, whereas those of the tRNA and 12S rRNA genes were the most conservative.     

Phylogenetic relationships of Asian and European pig breeds determined by mitochondrial DNA D-loop sequence polymorphism

Phylogenetic relationships among Asian and European pig breeds were assessed using 1036 bp of mitochondrial DNA (mtDNA) D-loop sequences. An unweighted pair-group method with arithmetic mean (UPGMA) tree was constructed on the basis of maximum likelihood distances using sequences determined for three Cheju (Korea), 11 Chinese, one Westran (Australian feral origin) and two European pigs (Berkshire and Welsh), and also published sequences for four Japanese (including two Wild Boars), one Yucatan miniature, five European (including Large White, Landrace, Duroc, Swedish and Wild Boar) and two Meishan pigs. The Colombian collared peccary (Tayassu tajacu) sequence was also determined and used as an outgroup. The maximum parsimony with heuristic search method was used to determine bootstrap support values. Asian-type pigs clustered together (bootstrap support 33%), but were separate from European-type pigs that also clustered together (93%). The Westran pig, derived from the feral descendants of pigs inhabiting Kangaroo Island of South Australia, clustered with Asian pigs, demonstrating Asian origin of their mitochondria. Berkshire and Large White clustered with Asian pigs, indicating that Asian pigs were involved in the development of these breeds. Our findings clearly demonstrate that pigs indigenous to China, Korea and Japan are only recently diverged from each other and distinctly different from European-type pigs. European pig breeds consist of pigs with mitochondria of Asian and non-Asian type, some of which were formed from closely related maternal ancestors, if not from a single ancestor.     

SSCP analysis of pig mitochondrial DNA D-loop region polymorphism

The sequence polymorphism that occurs in the mitochondrial DNA (mtDNA) displacement (D)-loop region is useful as a cytoplasmic DNA marker. We cloned the mtDNA D-loop regions of five breeds of pig by polymerase chain reaction (PCR) and determined their sequences. The sequence diversities in D-loop regions among five breeds of pig were located in the starting area of heavy-strand replication. From these sequences, we designed primers for PCR-mediated single-strand conformation polymorphism (PCR-SSCP) analysis that amplified the most polymorphic 227 bp fragment of the D-loop region. The results of PCR-SSCP analysis clearly showed that four types of polymorphism (A to D) are found in Landrace (A), Large White (A, B), Duroc (A), Göttingen miniature pig (B) and Meishan (C, D). The same polymorphisms were also detected from each porcine embryo by this method. Our results show that PCR-SSCP analysis is useful in detecting polymorphisms in the D-loop region of pigs and pig embryos.     

Phylogeography of Varicorhinus barbatulus (Cyprinidae) in Taiwan based on nucleotide variation of mtDNA and allozymes

The phylogeographical patterns and population genetic structures of Varicorhinus barbatulus in Taiwan were investigated based on genetic diversity of 34 allozyme loci and nucleotide sequences of 3' end of the cytochrome b gene, tRNA genes, D-loop control region, and the 5' end of the 12S rRNA of mtDNA. Allozyme and mtDNA analyses revealing evident geographical structuring suggest limited gene flow between populations (F(ST)=0.511 and 0.791, respectively). Low genetic variability within populations (P=5.56%; He=0.018) based on allozymes and significantly negative Tajima's D statistics based on mtDNA suggest that most populations in Taiwan may have originated from a small number of founders followed by demographic expansion. The gene genealogy of mtDNA identified six lineages corresponding to major drainages that were separated by the geological barriers due to vicariant events. A minimum spanning network based on nucleotide substitutions reflects divergence from populations of the Miao-li Plateau to northern and southern regions of the island. In contrast to a previous hypothesis that suggests an early invasion to eastern part of Taiwan prior to the lifting of central mountain range some one million years ago, the mtDNA genealogy and molecular dating reveal very recent colonization of the eastern population. Nested clade analyses revealing significant associations between genetic structure and geographical division identify past fragmentation and range expansion as major phylogeographical events that shaped the geographical distribution of this species in Taiwan.     

Mitochondrial DNA sequences of various species of the genus Equus with special reference to the phylogenetic relationship between Przewalskii's wild horse and domestic horse

The noncoding region between tRNA^Pro and the large conserved sequence block is the most variable region in the mammalian mitochondrial DNA D-loop region. This variable region (ca. 270 bp) of four species of Equus, including Mongolian and Japanese native domestic horses as well as Przewalskii's (or Mongolian) wild horse, were sequenced. These data were compared with our recently published Thoroughbred horse mitochondrial DNA sequences. The evolutionary rate of this region among the four species of Equus was estimated to be 2–4 × 10^–8 per site per year. Phylogenetic trees of Equus species demonstrate that Przewalskii's wild horse is within the genetic variation among the domestic horse. This suggests that the chromosome number change (probably increase) of the Przewalskii's wild horse occurred rather recently.Correspondence to: N. Ishida