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.     

The Lusitano horse maternal lineage based on mitochondrial D-loop sequence variation

The analysis of mitochondrial D-loop sequences (408 bp) from 145 Lusitano founder mares yielded a total of 27 different haplotypes. The distribution of these mtDNA sequences was quite unequal, with the three most frequent ones representing 56.5% of all the Lusitano founder mares and 14 haplotypes (51.9%) being rare variants found only once in the sampling. Four main haplotype clusters were present in the Lusitano breed. The comparison of these sequences with other equine haplotypes shows that they fall in groups shared with other horse breeds. These data support the hypothesis of multiple domestication events in many distinct geographic areas over a broad time span. However, the analysis of 145 Lusitano, 55 Pura Raza Espanola and 18 Sorraia sequences indicates that half of the samples (50.9%) fall in one specific-cluster (A), which has previously been described as characteristic of the Iberian and Northern African horse breeds. The presence of a phylogeographic structure in cluster A associated with its star-like structure was interpreted as suggestive of a centre of horse domestication in the Iberia Peninsula.     

Origin and history of mitochondrial DNA lineages in domestic horses

Domestic horses represent a genetic paradox: although they have the greatest number of maternal lineages (mtDNA) of all domestic species, their paternal lineages are extremely homogeneous on the Y-chromosome. In order to address their huge mtDNA variation and the origin and history of maternal lineages in domestic horses, we analyzed 1961 partial d-loop sequences from 207 ancient remains and 1754 modern horses. The sample set ranged from Alaska and North East Siberia to the Iberian Peninsula and from the Late Pleistocene to modern times. We found a panmictic Late Pleistocene horse population ranging from Alaska to the Pyrenees. Later, during the Early Holocene and the Copper Age, more or less separated sub-populations are indicated for the Eurasian steppe region and Iberia. Our data suggest multiple domestications and introgressions of females especially during the Iron Age. Although all Eurasian regions contributed to the genetic pedigree of modern breeds, most haplotypes had their roots in Eastern Europe and Siberia. We found 87 ancient haplotypes (Pleistocene to Mediaeval Times); 56 of these haplotypes were also observed in domestic horses, although thus far only 39 haplotypes have been confirmed to survive in modern breeds. Thus, at least seventeen haplotypes of early domestic horses have become extinct during the last 5,500 years. It is concluded that the large diversity of mtDNA lineages is not a product of animal breeding but, in fact, represents ancestral variability.     

Microsatellite diversity, population subdivision and gene flow in the Lipizzan horse

Blood samples of 561 Lipizzan horses from subpopulations (studs) of seven European countries representing a large fraction of the breed's population were used to examine the genetic diversity, population subdivision and gene flow in the breed. DNA analysis based on 18 microsatellite loci revealed that genetic diversity (observed heterozygosity = 0.663, gene diversity = 0.675 and the mean number of alleles = 7.056) in the Lipizzan horse is similar to other horse breeds as well as to other domestic animal species. The genetic differentiation between Lipizzan horses from different studs, although moderate, was apparent (pairwise F(ST) coefficients ranged from 0.021 to 0.080). Complementary findings explaining the genetic relationship among studs were revealed by genetic distance and principal component analysis. One genetic cluster consisted of the subpopulations of Austria, Italy and Slovenia, which represent the classical pool of Lipizzan horse breeding. A second cluster was formed by the Croatian, Hungarian and Slovakian subpopulations. The Romanian subpopulation formed a separate unit. The largest genetic differentiation was found between the Romanian and Italian subpopulation. Genetic results are consistent with the known breeding history of the Lipizzan horse. Correct stud assignment was obtained for 80.9% and 92.1% of Lipizzan horses depending on the inclusion or exclusion of migrant horses, respectively. The results of the present study will be useful for the development of breeding strategies, which consider classical horse breeding as well as recent achievements of population and conservation genetics.     

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 DNA D‐loop sequence variation in maternal lineages of Iranian native horses

To understand the origin and genetic diversity of Iranian native horses, mitochondrial DNA (mtDNA) D‐loop sequences were generated for 95 horses from five breeds sampled in eight geographical locations in Iran. Sequence analysis of a 247‐bp segment revealed a total of 27 haplotypes with 38 polymorphic sites. Twelve of 19 mtDNA haplogroups were identified in the samples. The most common haplotypes were found within haplogroup X2. Within‐population haplotype and nucleotide diversities of the five breeds ranged from 0.838 ± 0.056 to 0.974 ± 0.022 and 0.011 ± 0.002 to 0.021 ± 0.001 respectively, indicating a relatively high genetic diversity in Iranian horses. The identification of several ancient sequences common between the breeds suggests that the lineage of the majority of Iranian horse breeds is old and obviously originated from a vast number of mares. We found in all native Iranian horse breeds lineages of the haplogroups D and K, which is concordant with the previous findings of Asian origins of these haplogroups. The presence of haplotypes E and K in our study also is consistent with a geographical west–east direction of increasing frequency of these haplotypes and a genetic fusion in Iranian horse breeds.     

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.     

Characterization of cytochrome b diversity in Chinese domestic horses

Previous mitochondrial DNA (mtDNA) D‐loop and microsatellite studies have shown that Chinese horses have multiple maternal origins and high genetic diversity. To better characterize maternal genetic origins and diversity of Chinese domestic horses, we conducted a comprehensive analysis of 407 complete 1140 bp sequences of the horse mitochondrially encoded cytochrome b (CYTB) gene, including 323 horses from 13 Chinese indigenous breeds and 84 reference sequences from GenBank. A total of 114 haplotypes were identified, of which 73 appeared among the 13 Chinese horse breeds. The high mitochondrially encoded cytochrome b haplotypic diversity suggests multiple maternal origins in Chinese horses.     

An Overview of Ten Italian Horse Breeds through Mitochondrial DNA

BackgroundThe climatic and cultural diversity of the Italian Peninsula triggered, over time, the development of a great variety of horse breeds, whose origin and history are still unclear. To clarify this issue, analyses on phenotypic traits and genealogical data were recently coupled with molecular screening.MethodologyTo provide a comprehensive overview of the horse genetic variability in Italy, we produced and phylogenetically analyzed 407 mitochondrial DNA (mtDNA) control-region sequences from ten of the most important Italian riding horse and pony breeds: Bardigiano, Esperia, Giara, Lipizzan, Maremmano, Monterufolino, Murgese, Sarcidano, Sardinian Anglo-Arab, and Tolfetano. A collection of 36 Arabian horses was also evaluated to assess the genetic consequences of their common use for the improvement of some local breeds.ConclusionsIn Italian horses, all previously described domestic mtDNA haplogroups were detected as well as a high haplotype diversity. These findings indicate that the ancestral local mares harbored an extensive genetic diversity. Moreover, the limited haplotype sharing (11%) with the Arabian horse reveals that its impact on the autochthonous mitochondrial gene pools during the final establishment of pure breeds was marginal, if any. The only significant signs of genetic structure and differentiation were detected in the geographically most isolated contexts (i.e. Monterufolino and Sardinian breeds). Such a geographic effect was also confirmed in a wider breed setting, where the Italian pool stands in an intermediate position together with most of the other Mediterranean stocks. However, some notable exceptions and peculiar genetic proximities lend genetic support to historical theories about the origin of specific Italian breeds.     

Genetic analysis of Sicilian autochthonous horse breeds using nuclear and mitochondrial DNA markers

Genetic diversity and relationship among 3 Sicilian horse breeds were investigated using 16 microsatellite markers and a 397-bp length mitochondrial D-loop sequence. The analysis of autosomal DNA was performed on 191 horses (80 Siciliano [SIC], 61 Sanfratellano [SAN], and 50 Sicilian Oriental Purebred [SOP]). SIC and SAN breeds were notably higher in genetic variability than the SOP. Genetic distances and cluster analysis showed a close relationship between SIC and SAN breeds, as expected according to the breeds' history. Sequencing of hypervariable mitochondrial DNA region was performed on a subset of 60 mares (20 for each breed). Overall, 20 haplotypes with 31 polymorphic sites were identified: A higher haplotype diversity was detected in SIC and SAN breeds, with 13 and 11 haplotypes respectively, whereas only one haplotype was found in SOP. These were compared with 118 sequences from GenBank. BLAST showed that 17 of the 20 haplotypes had been reported previously in other breeds. One haplotype, found in SIC, traces back to a Bronze Age archaeological site (Inner Mongolia). The 3 Sicilian breeds are now rare, and 2 of them are officially endangered. Our results represent a valuable tool for management strategies as well as for conservation purposes.     

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.     

Genetic analysis of seven Italian horse breeds based on mitochondrial DNA D‐loop variation

To understand the origin and genetic diversity of Italian horses, mitochondrial DNA D‐loop sequences were generated for 163 horses from seven breeds. Sequence analysis of a 480‐bp segment revealed a total of 84 haplotypes with 57 polymorphic sites, indicating multiple maternal origins and high genetic diversity. Comparison of the haplotypes with the equine mtDNA haplotype/haplogroup nomenclature showed a haplogroup distribution in the Italian breeds more similar to that found in the Middle East breeds than in the European breeds, probably due to the economic and cultural relationship with the Middle East in the past centuries.     

A new view on dam lines in Polish Arabian horses based on mtDNA analysis

Polish Arabian horses are one of the oldest and the most important Arab populations in the world. The Polish Arabian Stud Book and the Genealogical Charts by Skorkowski are the main sources of information on the ancestors of Polish Arabs. Both publications were viewed as credible sources of information until the 1990s when the data regarding one of the dam lines was questioned. The aim of the current study was to check the accuracy of the pedigree data of Polish dam lines using mtDNA analysis. The analyses of a 458 bp mtDNA D-loop fragment from representatives of 15 Polish Arabian dam lines revealed 14 distinct haplotypes. The results were inconsistent with pedigree data in the case of two lines. A detailed analysis of the historical sources was performed to explain these discrepancies. Our study revealed that representatives of different lines shared the same haplotypes. We also noted a genetic identity between some lines founded by Polish mares of unknown origin and lines established by desert-bred mares.     

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.     

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.     

A novel maternal lineage revealed in sheep (Ovis aries)

It is generally believed that domestic sheep have two maternal lineages (haplotypes A and B), based on mitochondrial DNA analysis. In the present study, we provide evidence that a novel maternal lineage (haplotype C) is exhibited in Chinese native sheep. To verify this finding, 231 samples were collected from six Chinese local breeds, which cover the vast geographical region of sheep inhabitation in China. For comparison, 50 samples were collected from two Western breeds collected in China. Mitochondrial DNA was screened by PCR single-strand conformational polymorphism (SSCP), leading to the identification of novel band patterns in ND2 and ND4 genes in the Chinese breeds. Interestingly, mutations at the two loci were in strong linkage disequilibrium. Direct sequencing of the DNA fragments revealed a non-synonymous substitution in ND2. Furthermore, two synonymous mutations were identified by comparisons of the novel type (haplotype C) and the established types (haplotypes A and B). The entire mitochondrial control region for 55 samples was then sequenced to construct a phylogenetic tree and median joining network. Both the tree and network demonstrated a topology of three groups, which is in consistent with the SSCP analysis. Unlike Western breeds, Chinese breeds are composed mainly of haplotypes A and B, but with a small fraction of haplotype C. According to Fu's test and mismatch distribution, haplotype C has not been subject to a recent population expansion. Based on these results, we propose a novel origin for Chinese sheep.     

Mitochondrial DNA Cleavage Patterns Distinguish Independent Origin of Chinese Domestic Geese and Western Domestic Geese

It has generally been assumed, based on morphology, that Chinese domestic goose breeds were derived from the swan goose (Anser cygnoides) and that European and American breeds were derived from the graylag goose (Anser anser). To test the validity of this assumption, we investigated the mtDNA cleavage patterns of 16 Chinese breeds and 2 European breeds as well as hybrids produced between a Chinese breed and a European breed. After 224 mtDNAs, isolated from the Chinese and European breeds, were digested by 19 restriction endonucleases, variations of the cleavage patterns were observed for four enzymes (EcoRV, HaeII, HincII, and KpnI). All Chinese breeds and their maternal hybrids except the Yili breed showed an identical haplotype, named haplotype I or the Chinese haplotype; the European breeds and the Yili breed showed another haplotype, named haplotype II or the western haplotype. None of the haplotype found in the Chinese type was detectable in the western type and vice versa. The two haplotypes were found to differ from each other at 8.0% of the sites surveyed and with a 0.72% sequence divergence. Using 2% substitution per million years calibrated from the genera Anser and Branta, the two domestic geese haplotypes were estimated to have diverged approximately 360,000 years ago, well outside the 3000-6000 years in domestic history. Our findings provide the first molecular genetic evidence to support the dual origin assumption of domestic geese in the world. Meanwhile, the four mtDNA restriction fragment length polymorphisms can be used as maternal genetic markers to distinguish the two types of domestic geese.     

Phylogenetic relationships of Argentinean Creole horses and other South American and Spanish breeds inferred from mitochondrial DNA sequences

South American horses constitute a direct remnant of the Iberian horses brought to the New World by the Spanish conquerors. The source of the original horses was Spain, and it is generally assumed that the animals belonged to the Andalusian, Spanish Celtic, Barb or Arabian breeds. In order to establish the relationship between Argentinean and Spanish horses, a portion of the mitochondrial D-loop of 104 animals belonging to nine South American and Spanish breeds was analysed using SSCP and DNA sequencing. The variability found both within and between breeds was very high. There were 61 polymorphic positions, representing 16% of the total sequence obtained. The mean divergence between a pair of sequences was 2.8%. Argentinean Creole horses shared two haplotypes with the Peruvian Paso from Argentina, and the commonest haplotype of the Creole horses is identical to one of the Andalusian horses. Even when there was substantial subdivision between breeds with highly significant Wright's Fixation Index (FST), the parsimony and distance-based phylogenetic analyses failed to show monophyletic groups and there was no clear relationship in the trees between the South American and any of the other horses analysed. Although this result could be interpreted as mixed ancestry of the South American breeds with respect to the Spanish breeds, it is probably indicating the retention of very ancient maternal lineages in the breeds analysed.     

Microsatellite diversity, pedigree relatedness and the contributions of founder lineages to thoroughbred horses

The thoroughbred (TB) horse is one of the oldest breeds of domestic animals, with pedigree records spanning three centuries. Because the population is essentially closed, there is concern about loss of genetic variation. Here we report two parallel analyses. In the first, genetic variation in the current population is measured using data from 13 microsatellite loci in 211 horses with relationships calculated based on allele sharing. In the second analysis, pedigree information is used to calculate genetic relationships between animals based on shared ancestry. These two measures of relationship are compared and shown to be closely related. Together, they provide an estimate of the amount of genetic variation which existed in founder animals. This study confirms the narrow genetic base of the breed and provides comprehensive analysis of contributions of founder animals. Seventy-eight percent of alleles in the current population are derived from 30 founders, 27 of these male. Ten founder females account for 72% of maternal lineages, while one founder stallion is responsible for 95% of paternal lineages.     

Genetic Diversity and Systematic Evolution of Chinese Domestic Ducks Along the Yangtze-Huai River

To investigate the population structure and systematic evolution of the domestic duck in China, we sequenced the 667 bp mitochondrial DNA (mtDNA) D-loop control region of 106 ducks from nine breeds along the Yangtze-Huai River. Of the total analyzed sites, 34 (5.1%) were polymorphic due to transitions, transversions, insertions, and deletions. Nucleotide content was 25.6% A, 33.3% C, 15.2% G, and 25.9% T. In total, 31 haplotypes were identified in the target region; of these, the major haplotype was A7, and nine haplotypes were shared by the tested ducks. The haplotype diversity (Hd) and average nucleotide diversity (Pi) were 0.798% and 0.28%, respectively. Hd was highest in the Jingjiang shelduck, followed by the Youxian and Enshi shelducks, and it was lowest in the Wendeng black duck. Nucleotide diversity (Dxy) among the nine breeds ranged from 0.139 to 0.433%, and the Kimura 2-parameter distances were 0.0013-0.0044. Molecular variance indicated that a very high proportion of the insignificant genetic variance was attributable to variations within breeds. Phylogenetic analysis of 31 haplotypes revealed only one distinct maternal lineage in the tested ducks, and no evidence was found of a contribution of the Anas zonorhyncha group B haplotype to the maternal origin of Chinese domestic duck breeds along the Yangtze-Huai.