Molecular genetic analysis of ancient cattle bones excavated from archaeological sites in Jeju, Korea

Ancient cattle bones were excavated from archaeological sites in Jeju, Korea. We used molecular genetic techniques to identify the species and establish its relationship to extant cattle breeds. Ancient DNA was extracted from four sources: a humerus (Gonae site, A.D. 700-800), two fragments of radius, and a tooth (Kwakji site, A.D. 0-900). The mitochondrial DNA (mtDNA) D-loop regions were cloned, sequenced, and compared with previously reported sequences of various cattle breeds (9 Asian, 8 European, and 3 African). The results revealed that these bones were of the breed, Bos taurus, and a phylogenetic tree indicated that the four cattle bones formed a monophyletic group with Jeju native black cattle. However, the patterns of sequence variation and reports from archaeological sites suggest that a few wild cattle, with a different maternal lineage, may have existed on Jeju Island. Our results will contribute to further studies of the origin of Jeju native cattle and the possible existence of local wild cattle.     

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.     

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.     

Mitochondrial sequence variation in ancient horses from the Carpathian Basin and possible modern relatives

Movements of human populations leave their traces in the genetic makeup of the areas affected; the same applies to the horses that move with their owners This study is concerned with the mitochondrial control region genotypes of 31 archaeological horse remains, excavated from pre-conquest Avar and post-conquest Hungarian burial sites in the Carpathian Basin dating from the sixth to the tenth century. To investigate relationships to other ancient and recent breeds, modern Hucul and Akhal Teke samples were also collected, and mtDNA control region (CR) sequences from 76 breeds representing 921 individual specimens were combined with our sequence data. Phylogenetic relationships among horse mtDNA CR haplotypes were estimated using both genetic distance and the non-dichotomous network method. Both methods indicated a separation between horses of the Avars and the Hungarians. Our results show that the ethnic changes induced by the Hungarian Conquest were accompanied by a corresponding change in the stables of the Carpathian Basin.     

Phylogenetic relationships of Cheju horses to other horse breeds as determined by mtDNA D-loop sequence polymorphism

Historical records suggest that horses inhabiting the island of Cheju in Korea are descendants of Mongolian horses introduced in 1276. Other studies, however, suggest that horses may have been present on the island prior to the Mongolian introduction. To determine the origin of the Cheju horses we used a phylogenetic analysis of sequences of the mitochondrial DNA (mtDNA) D-loop region, including tRNA Pro and parts of tRNA thr and tRNA Phe sequences (1102-bp excluding the tandem repeat region). Maximum parsimony and neighbor-joining trees were constructed using sequences determined for seven Cheju, four Mongolian, one Przewalskii and two Chinese Yunnan horses, and published sequences for one Swedish and three Thoroughbred horses. Donkey mtDNA was used as an outgroup. We found that the mtDNA D-loop sequence varies considerably within Mongolian, Cheju and Thoroughbred horse breeds, and that Cheju horses clustered with Mongolian horses as well as with horses from other distantly related breeds. On the basis of these findings we propose that horses on Cheju Island are of mixed origin in their maternal lineage, and that horses may have existed and been traded on the island before the Mongolian introduction.     

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.     

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.     

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.     

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 DNA analysis of horses recovered from a frozen tomb (Berel site, Kazakhstan, 3rd Century BC)

Sequence polymorphism of the mitochondrial DNA D-loop was used to determine the genetic diversity of horses recovered from a Scythian princely tomb dating from the beginning of the 3rd century BC. Eight haplotypes were found among the 13 ancient horse samples tested. Phylogenetical analysis showed that these ancient horse's sequences, along with two Yakut ones, were distributed throughout the tree defined by modern horses' sequences and are closely related to them. No clear geographical affiliation of the specimens studied was thus determined. Our work, among others, supports the very ancient origin of the matrilines in 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.     

A pedigree-based study of mitochondrial D-loop DNA sequence variation among Arabian horses

Through DNA sequence comparisons of a mitochondrial D-loop hypervariable region, we investigated matrilineal diversity for Arabian horses in the United States. Sixty-two horses were tested. From published pedigrees they traced in the maternal line to 34 mares acquired primarily in the mid to late 19th century from nomadic Bedouin tribes. Compared with the reference sequence (GenBank X79547), these samples showed 27 haplotypes with altogether 31 base substitution sites within 397 bp of sequence. Based on examination of pedigrees from a random sampling of 200 horses in current studbooks of the Arabian Horse Registry of America, we estimated that this study defined the expected mtDNA haplotypes for at least 89% of Arabian horses registered in the US. The reliability of the studbook recorded maternal lineages of Arabian pedigrees was demonstrated by haplotype concordance among multiple samplings in 14 lines. Single base differences observed within two maternal lines were interpreted as representing alternative fixations of past heteroplasmy. The study also demonstrated the utility of mtDNA sequence studies to resolve historical maternity questions without access to biological material from the horses whose relationship was in question, provided that representatives of the relevant female lines were available for comparison. The data call into question the traditional assumption that Arabian horses of the same strain necessarily share a common maternal ancestry.     

Genome-wide analyses of the Jeju,Thoroughbred, and Jeju crossbred horse populations using the high density SNP array

The Jeju horse is an indigenous Korean horse breed that is currently registered with the Food and Agriculture Organization of the United Nations. However, there is severe lack of genomic studies on Jeju horse. This study was conducted to investigate genetic characteristics of horses including Jeju horse, Thoroughbred and Jeju crossbred (Jeju?×?Thoroughbred) populations. We compared the genomes of three horse populations using the Equine SNP70 Beadchip array. Short-range Linkage disequilibrium was the highest in Thoroughbred, whereas r² values were lowest in Jeju horse. Expected heterozygosity was the highest in Jeju crossbred (0.351), followed by the Thoroughbred (0.337) and Jeju horse (0.311). The level of inbreeding was slightly higher in Thoroughbred (??0.009) than in Jeju crossbred (??0.035) and Jeju horse (??0.038). F_ST value was the highest between Jeju horse and Thoroughbred (0.113), whereas Jeju crossbred and Thoroughbred showed the lowest value (0.031). The genetic relationship was further assessed by principal component analysis, suggesting that Jeju crossbred is more genetically similar to Thoroughbred than Jeju horse population. Additionally, we detected potential selection signatures, for example, in loci located on LCORL/NCAPG and PROP1 genes that are known to influence body. Genome-wide analyses of the three horse populations showed that all the breeds had somewhat a low level of inbreeding within each population. In the population structure analysis, we found that Jeju crossbred was genetically closer to Thoroughbred than Jeju horse. Furthermore, we identified several signatures of selection which might be associated with traits of interest. To our current knowledge, this study is the first genomic research, analyzing genetic relationships of Jeju horse, Thoroughbred and Jeju crossbred.     

Extensive mtDNA diversity in horses revealed by PCR–SSCP analysis

The hypervariable D-loop region of mitochondrial DNA (mtDNA) was amplified with the polymerase chain reaction using total horse DNA samples. Analysis of single strand conformation polymorphism (SSCP) of denatured amplification products was carried out by native polyacrylamide (8%) gel electrophoresis followed by silver staining. As many as 15 distinct SSCP variants were revealed when screening a total of 78 maternally unrelated horses representing five different breeds. All breeds showed a high degree of polymorphism and the estimated probability (PI_mt) that two maternally unrelated individuals have, by chance, identical SSCP variants varied between 0.14 and 0.30. We detected no heteroplasmy or deviations from strict and stable maternal inheritance when examining four maternal lineages, each represented by six to eight horses, separated by up to five generations from a common ancestral mare. The study establishes a simple screening method for detecting equine mtDNA types, which can be applied for tracing maternal genealogies and for association studies.     

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。      

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     

小口白甲鱼都柳江种群mtDNA D环的序列变异及遗传多样性

采用PCR结合DNA测序技术,测定分析了易危鱼类小口白甲鱼(Onychostoma lini)都柳江种群36个个体mtDNA D环约470bp序列的变异及遗传多样性。结果表明,在36个个体中,该序列的长度为469～475bp,其碱基组成为A+T的平均含量(68.4%)高于G+C(31.6%)。共检测到25个多态位点,其中转换19个、颠换6个。核苷酸多样性(π)为0.00575,平均核苷酸差异数(K)为2.695。36个个体分属5个单倍型,单倍型多样度(Hd)为0.260,单倍型间的平均遗传距离(P)为0.026。5个单倍型构建的UPGMA系统树聚为2个分支。目前小口白甲鱼都柳江种群mtDNA D环序列存在着较丰富的变异和遗传多样性。     

High microsatellite and mitochondrial diversity in Anatolian native horse breeds shows Anatolia as a genetic conduit between Europe and Asia

The horse has been a food source, but more importantly, it has been a means for transport. Its domestication was one of the crucial steps in the history of human civilization. Despite the archaeological and molecular studies carried out on the history of horse domestication, which would contribute to conservation of the breeds, the details of the domestication of horses still remain to be resolved. We employed 21 microsatellite loci and mitochondrial control region partial sequences to analyse genetic variability within and among four Anatolian native horse breeds, Ayvac?k Pony, Malakan Horse, H?n?s Horse and Canik Horse, as well as samples from indigenous horses of unknown breed ancestry. The aims of the study were twofold: first, to produce data from the prehistorically and historically important land bridge, Anatolia, in order to assess its role in horse domestication and second, to analyse the data from a conservation perspective to help the ministry improve conservation and management strategies regarding native horse breeds. Even though the microsatellite data revealed a high allelic diversity, 98% of the genetic variation partitioned within groups. Genetic structure did not correlate with a breed or geographic origin. High diversity was also detected in mtDNA control region sequence analysis. Frequencies of two haplogroups (HC and HF) revealed a cline between Asia and Europe, suggesting Anatolia as a probable connection route between the two continents. This first detailed genetic study on Anatolian horse breeds revealed high diversity among horse mtDNA haplogroups in Anatolia and suggested Anatolia’s role as a conduit between the two continents. The study also provides an important basis for conservation practices in Turkey.     

Molecular genetic analysis of remains of a 2,000-year-old human population in China-and its relevance for the origin of the modern Japanese population.

We extracted DNA from the human remains excavated from the Yixi site ( approximately 2,000 years before the present) in the Shandong peninsula of China and, through PCR amplification, determined nucleotide sequences of their mitochondrial D-loop regions. Nucleotide diversity of the ancient Yixi people was similar to those of modern populations. Modern humans in Asia and the circum-Pacific region are divided into six radiation groups, on the basis of the phylogenetic network constructed by means of 414 mtDNA types from 1, 298 individuals. We compared the ancient Yixi people with the modern Asian and the circum-Pacific populations, using two indices: frequency distribution of the radiation groups and genetic distances among populations. Both revealed that the closest genetic relatedness is between the ancient Yixi people and the modern Taiwan Han Chinese. The Yixi people show closer genetic affinity with Mongolians, mainland Japanese, and Koreans than with Ainu and Ryukyu Japanese and less genetic resemblance with Jomon people and Yayoi people, their predecessors and contemporaries, respectively, in ancient Japan.