doi:10.1111/j.1365-2052.2011.02285.

High microsatellite and mitochondrial diversity in Anatolian native horse breeds shows Anatolia as a genetic conduit between Europe and Asia
E. Koban*, M. Denizci*,1, O. Aslan*,1, D. Aktoprakligil*, S. Aksu*, M. Bower, B. K. Balcioglu*, A. Ozdemir Bahadir*, R. Bilgin, B. Erdag*, H. Bagis*, and S. Arat*
*TUBITAK Marmara Research Center, Genetic Engineering and Biotechnology Institute, 41470 Gebze, Kocaeli, Turkey. Glyn Daniel Laboratory for Archaeogenetics, McDonald Institute for Archaeological Research, University of Cambridge, Cambridge, UK. Bogazici University, Institute of Environmental Sciences, Bebek 34342, Istanbul, Turkey. Adiyaman University, Medical Faculty, 02040 Adiyaman, Turkey.

Summary

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, Ayvack Pony, Malakan Horse, Hns Horse and Canik Horse, as well as samples from indigenous horses of unknown breed ancestry. The aims of the study were twofold: rst, 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 rst detailed genetic study on Anatolian horse breeds revealed high diversity among horse mtDNA haplogroups in Anatolia and suggested Anatolias role as a conduit between the two continents. The study also provides an important basis for conservation practices in Turkey. Keywords Anatolia, domestication, Equus, genetic diversity, horse, microsatellite, mitochondrial DNA.

Introduction
Although biodiversity and conservation studies are traditionally associated with wildlife species (Frankham et al. 2002), one research focus is increasingly turning towards conservation of the worlds domestic animal species as well.
Address for correspondence S. Arat, TUBITAK Marmara Research Center, Genetic Engineering and Biotechnology Institute, 41470 Gebze, Kocaeli, Turkey. E-mails: sezen.arat@mam.gov.tr, sezenarat@yahoo.com
1

The contributions of these two authors are equal.

Accepted for publication 4 July 2011

In 2007, the FAO (2007a) reported that, of the 7616 breeds of domesticated animals and birds, 20% were at risk of extinction. The importance of conserving domestic animal diversity was recognized with the rst Global Plan of Action for Animal Genetic Resources being declared in Interlaken (37 September 2007) and adopted by 109 countries (FAO 2007b), including Turkey. The loss of genetic and phenotypic diversity, both within breeds and in the number of breeds, is dramatically increasing owing to socioeconomic and environmental changes in the world. For conservation actions to be effective, it is essential that wild ancestors be identied and the complexity surrounding the domestication histories of our core domestic animals be unravelled (Bruford et al. 2003). 401

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Koban et al. Compared to the other domestic species, except for donkeys, the domestication of the horse came relatively late (BejaPereira et al. 2004). The precise timeline is a point of discussion but has been suggested to be around 6000 BP (Clutton-Brock 1999). Archaeological ndings (Baker et al. 2002; Zaitseva et al. 2005; Outram et al. 2009) and mitochondrial DNA studies on both living and ancient samples from Eurasian horse populations have revealed seven maternal lin` eages (HAHG) and a complex domestication history (Vila et al. 2001; Jansen et al. 2002; Wallner et al. 2003; Lindgren et al. 2004; McGahern et al. 2006; Lei et al. 2009). Anatolia is one of the places in the world from which data are missing in the current databases. The Anatolian peninsula, which contains a part of the Fertile Crescent, forms a land bridge between Asia and Europe and has been important in the course of the domestication of several species, as revealed by genetic and archaeological data from domestic and/or wild species (Zohary & Hopf 2000; Naderi et al. 2008; Zeder 2008; Rezaei et al. 2010). These ndings suggest that genetic analysis of Anatolian horse breeds may shed some light on the domestication history of the horse as well as on the diversication of present horse breeds. Similar to the horse populations throughout the world that have been declared at risk (FAO 2007a; Thirstrup et al. 2008), there has been a sharp decline in the number of horses in Turkey owing to technological advancement and increasing mechanization. Moreover, the native breeds have been replaced by valuable imported breeds, like Thoroughbred or Arabian horses, for racing and economic purposes. Hence, urgent genetic characterization studies from Anatolia are critical, as this decline in numbers will inevitably lead to a loss of important genetic information, including preserved patterns that are the result of the domestication process as well as important characteristics such as disease resistance and environmental adaptation. To prepare action plans and set up sustainable conservation and management strategies for horse breeds, the rst step is to dene the present breeds and evaluate both phenotypic and genotypic characteristics. For this reason, the Turkish Ministry of Agriculture and Rural Affairs has supported the national TURKHAYGEN-1 project (http://www.turkhaygen. gov.tr), the aims of which include the genotyping of domestic animal species, including Anatolian horse breeds. This study is the rst molecular characterization of four Anatolian horse breeds carried out within the context of the TURKHAYGEN-1 project. The breeds studied were Ayvack Pony (A.PONY) from north-west Anatolia, Hns Horse (HKK) from East Anatolia, Malakan Horse (MLK) from north and north-east Anatolia, and Canik Horse (CNK) from North Anatolia. Although they all previously had been used for heavy tasks, currently they are bred as a hobby or for racing. However, A.PONY is still used in elds for olive harvesting. Please see Appendix S1 for information on the breeds. The results reported are based on 21 microsatellite loci and partial mtDNA D-loop sequence diversity of these four Anatolian native horse breeds. We compared these with other native horses from Turkey, which cannot be assigned to any known breed based on phenotypic characters alone (i.e. grade horses; Pavia & Posnikoff 2005). This study is the rst study on genetic diversity within and among Anatolian horses. The objectives of the study were (i) to produce data from prehistorically and historically important Anatolia, because understanding the global domestication process for horses requires a combined data analysis that also includes Anatolian horses and (ii) to analyse the data from Anatolian breeds in the context of conservation studies carried out by the Ministry of Agriculture and Rural Affairs in order to contribute conservation and management strategies for native Anatolian horses.

Materials and methods

Sampling
Turkey is climatically divided into seven regions bordered by geographic barriers. Each native domestic breed is adapted to one of these geographic regions. Therefore, extensive sampling was carried out covering many distant villages in each of the nine different provinces (Fig. 1) in order to capture as much genetic diversity as possible for each breed. For the MLK breed only, villages in three provinces were, visited as they all had this breed (41 samples from Kars, 12 dr). samples from Ardahan, and 11 samples from Ig Distinctive phenotypes of Anatolian horse breeds were diagnosed using old textbooks (e.g. Batu 1938) and the FAO Domestic Animal Diversity Information System (DAD-IS: http://dad.fao.org) and through the help of animal scientists who evaluated the individuals sampled on the basis of phenotypic characters. Only the samples that could easily be assigned phenotypically to their respective breeds were kept in their respective breed population. The others, so-called Anatolian native grade horses that were not assigned to a breed based on phenotypic characters, were sampled (N = 94) for comparison with breed-assigned individuals. For population analysis purposes, native grade horses sampled in the same geographic region, including Erzurum r (AGR), Kars (KRS), Ardahan (ARD), Ig dr (IGD) (ERZ), Ag and Van (VAN), were assigned to an East Anatolian group (EAH), and samples collected from Kayseri (KYS) were assigned to a Central Anatolian group (CAH) (Fig. 1). The details about the breeds (names, regions and sample sizes) are given in Table 1, which also includes the results of the estimated summary statistics based on microsatellite data.

Genotyping and sequencing

Blood samples from 237 individuals from four morphologically distinct horse breeds, A.PONY, HKK, MLK and CNK, were collected into vacuum tubes containing K3EDTA. DNA from the blood samples was isolated using a standard

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High genetic diversity found in Anatolian horses

403

Europe

The Caucasus The Balkans The Black Sea

8
Figure 1 The centre of the provinces visited for sampling of the horse populations. The numbers reect samples collected from 10 to 15 villages from each of these provinces for the following breeds: (1) HKK and ERZ, (2) MLK and KRS, (3) KYS, (4) CNK, (5) AGR, (6) MLK and IGD, (7) MLK and ARD, (8) A.PONY and (9) VAN. The seven geographic regions of Turkey are shown on the upper right corner.

Anatolia 3

2 1 5

7 6 9

The Mediterranean

The Middle East 0 135 km

Table 1 Sample names, acronyms and the sample sizes (N) of the populations and, based on the microsatellite data, the estimated parameters of observed heterozygosity (HO), non-biased expected heterozygosity (HE) per population, mean number of alleles (MNA) per population, average number of alleles after Bonferroni correction (allelic richness) and population FIS estimates. Population name Ayvack Pony Hns Horse Malakan Horse Canik Horse r Ag dr Ig Ardahan Kars Erzurum Van Kayseri Group of geographic origin EAH EAH EAH EAH EAH EAH CAH Allelic richness 6.94 6.65 6.71 6.74 6.75

Acronym A.PONY HKK MLK CNK AGR IGD ARD KRS ERZ VAN KYS

N 49 60 64 64 14 15 4 17 17 15 12

HO 0.79 0.79 0.75 0.80 0.78

HE 0.79 0.80 0.79 0.80 0.80

MNA 9.00 8.81 8.43 9.62 9.62

FIS 0.001 0.016 0.081 0.002 0.025 (ns) (ns) (***) (ns) (*)

0.77

0.79

6.95

6.95

0.029 (ns)

ns, not signicant; EAH, East Anatolian group. *P < 0.05, **P < 0.01, ***P < 0.001.

phenolchloroform extraction protocol (Sambrook et al. 1989). All individuals (N = 331) were genotyped at 21 microsatellite loci (Table S1) in three multiplex PCRs. The master mix components, the reaction conditions for all three multiplex panels, and the run conditions are provided in Appendix S2. All the PCR products were run on a Beckman Coulter CEQ8800 Genetic Analysis System for allelic readings. For size calling, only the size standard was taken as a reference. Randomly chosen samples were PCR-amplied at least twice to check the repeatability of the allele size calling. The mtDNA partial D-loop amplication products (details are given in Appendix S2) were puried using an Agarose Gel Extraction Kit (Roche) according to the manufacturers instructions. Sequencing was carried out using the Genome Lab Dye Terminator Cycle Sequencing with Quick Start Kit

(Beckman Coulter) and a Beckman Coulter CEQ8800 Genetic Analysis System (details are given in the Appendix S2). Once results were obtained, the sequence quality was checked using the sequence analysis software from the Beckman Coulter CEQ8800 Genetic Analysis System. Proofreading of the sequences and checking the chromatograms and contig construction were carried out using BIOEDIT software (Ibis Biosciences). Only sequences with good contig alignments (N = 285) were used in the data analysis. Sequences were deposited in GenBank (accession numbers HM483870HM484172). The sequences were 301 bp long between the positions 15 492 and 15 792 of the reference sequence X79547 (Xu & Arnason 1994). For haplogroup analysis, sequences were truncated to 247 base pairs (between 15 494 and 15 740 bp of the reference sequence

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Koban et al. X79547; Xu & Arnason 1994) in order to be comparable with the previously published data (as cited by McGahern et al. 2006; Lei et al. 2009).

Statistical analysis
Microsatellite data The data were rst analysed using FREENA (Chapuis & Estoup 2007) for the presence and frequency of null alleles, and selected samples were then re-genotyped on the basis of these results. There were few signicant deviations from normal distributions of the expected allele frequencies found in the nal check with FREENA, but this deviation was found in less than 5% of the total number of alleles. Therefore, all the data were included in the further statistical analysis. GENETIX v4.05.2 (Belkhir et al. 19962004) was employed to estimate variation within samples by gene diversity (HE), observed heterozygosity (HO) and Wrights FIS (Wright 1931) using Weir & Cockerhams (1984) approach. The data set was permutated 1000 times over loci to test the signicance of estimated FIS values. Factorial correspondence analysis (FCA) was also performed with this software. In addition, ARLEQUIN v3.5 (Excofer et al. 2005) was used to estimate HW deviations. Pairwise genetic differences were measured by Wrights FST, and the signicance of differences was tested using a permutation approach. Allelic variation and allelic richness were estimated using FSTAT (Goudet 1995). Partitioning of molecular variation within and among the breeds was assessed by AMOVA using ARLEQUIN v3.5. Identication of genetic clusters and assignment of individuals to these clusters were made using STRUCTURE v2.0 (Pritchard et al. 2000). The number of clusters, K, was tested from K = 1 to 10 (run 20 times for each K value) using different burn-in periods and Markov chain Monte Carlo (MCMC) repetitions ranging from 30 000 to 300 000 in order to check whether there was a bias from burn-in period and MCMC repetition choices due to convergence; the most likely K value (K = 3) was estimated as described in Evanno et al. (2005). A neighbor-joining (NJ) tree was constructed based on pairwise DA genetic distances (Takezaki & Nei 1996) using the POPULATIONS (v 1.0) software (Langella 1999).

Figure 2 A median-joining network of Anatolian horses compared with Eurasian horse populations (N = 1662). Purple pie slices represent Anatolian horses. Clades are labelled according to Jansen et al. (2002). Nodes are proportional to the frequency of haplotypes.

(N = 1662, Fig. 2) using NETWORK (Bandelt et al. 1999). For this network analysis, the star contraction algorithm was applied and haplotypes with a frequency of two or greater were used (i.e. 1-active method). Hypervariable regions were down-weighted, and the parameters were set according to Jansen et al. (2002).

Results
Population differentiation within and among samples
Microsatellite data revealed high genetic variation within populations with 623 alleles/locus, an average of 0.65 0.86 HO/locus and an average of 0.660.88 HE/locus (see Table S2). There were few private alleles, all with low frequencies (between 0.0061 and 0.0417). The chi-square test did not reveal any signicant deviation (v2 = 0.01968; P > 0.05) from HardyWeinberg expectations. Among the populations, the average HO/population was between 0.75 and 0.80, and the average HE/population was about 0.80. The allelic richness estimated using the Bonferroni correction resulted in lower (6.656.95) than observed (6.95 9.62) allelic averages. The population FIS estimates were not signicant except for MLK and EAH. The high number of observed alleles across loci did not reveal a distinct differentiation between populations as reected in the AMOVA analysis results; 98% of the total genetic variation was contained within groups. The estimated K = 3 (based on the method of Evanno et al. 2005) in the structure analysis suggested three distinct groups. However, each group comprised a mixture of breeds, not in concordance with geographic origin or breed of origin. Furthermore, the assignment tests resulted in more than 50% of the individuals within the same population being assigned to a different population. The parallel results revealed by FCA showed one central cluster including all

Sequence data Diversity measures and demographic statistics comprising mismatch distributions, Fus FS and Tajimas D were estimated using DNASP v5.0 (Librado & Rozas 2009). Mitochondrial haplotypes were assigned with reference to previously published data (Jansen et al. 2002; McGahern et al. 2006; Lei et al. 2009). The Anatolian data were compared with the mtDNA D-loop sequences available in the public record (Lei et al. 2009), and median-joining networks were constructed in order to understand the relationship between Anatolian and Eurasian horses

2011 The Authors, Animal Genetics 2011 Stichting International Foundation for Animal Genetics, 43, 401409

High genetic diversity found in Anatolian horses the individuals (four breeds and grade horses) except for some samples, most of which belonged to A.PONY (Fig. 3). Moreover, the phylogenetic tree based on microsatellite genotypes did not have a well-resolved topology with high bootstrap support (Fig. 4). The only lineage that had branch support greater than 60% included CNK, HKK and EAH, and its internal branch (between HKK and EAH) had a support of greater than 70%.

405

mtDNA diversity within Anatolian samples

One hundred haplotypes were observed in 285 horse samples based on 301-bp sequences resulting from 58 variable sites, of which 57 were polymorphic and one was an insertion compared to the reference sequence (see haplotype 53, i.e. H_53; all the haplotypes are provided in Table S3). Among the polymorphic sites, 12 were singletons and 45 were parsimony informative sites. Nucleotide diversity (p) across all individuals was estimated at 0.026, and the haplotype diversity (Hd) was estimated at 0.98. The average number of nucleotide differences (k, pairwise mismatches) between the sequence pairs was 7.02. The only indel, compared to the reference sequence (position 40i, as given in Table S3), was an insertion detected in two A.PONY samples. The sequencing of these two individuals was repeated in order to conrm this indel. There were three more sites (see sequence positions 18, 40 and 157 in SM4) where indels, as deletions, were present in comparison with the reference sequence. The lowest Hd was estimated for A.PONY (0.957), and the highest Hd was
Figure 4 Neighbor-joining tree constructed based on DA genetics distances estimated from microsatellite genotypic data.

estimated for HKK (0.979). The highest p (0.026) and the highest average pairwise differences (k = 7.863) were also estimated for A.PONY (details are given in Table 2). The estimates of neutrality tests among overall samples and for each population based on Tajimas D and Fus Fs statistics did not reveal a signicant result (P > 0.10), which argues against the possibility of a recent population expansion. AMOVA showed that the total genetic variation was partitioned within groups (98%), which is a nding similar to the AMOVA analysis based on microsatellite data of the same individuals.

Haplogroup distribution and network analysis

The variation observed at the microsatellite loci within individuals was also observed in the mtDNA control region

120 110 100 90 80 70 60 50 40 30 20 10 0 10 20 30 40 50 60 70 80 90 8000 6000 4000 2000 0 2000 4000 Axe 3 (1.40 %)

Axe 4 (1.34 %)

HKK CNK MLK A.PONY EAH CAH

Axe 3 (1.22 %) 6000 8000

Figure 3 Factorial correspondence analysis of Canik (CNK), Malakan (MLK), Hns (HKK) and Ayvack Pony (A.PONY) horses, and East Anatolian (EAH) and Central Anatolian (CAH) horses of unknown breed ancestry. The circle encloses the A.PONY samples differing from the rest of the samples that were included in a mixed single cluster. 2011 The Authors, Animal Genetics 2011 Stichting International Foundation for Animal Genetics, 43, 401409

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Koban et al.
Table 2 The summary statistics of the variation detected among the populations based on the mtDNA D-loop sequences. The columns show the number of sequences (N), the number of mutations as compared to the reference sequence (variable sites and indels), number of haplotypes observed (NHPT), haplotype diversity (Hd), nucleotide diversity (p), number of pairwise differences (k) and the haplogroup frequencies (HA ) HG) detected among the populations. Mutations (variable+indel) 32 41 42 34 38 (31 (41 (42 (34 (38 + + + + + 1) 0) 0) 0) 0)

Population A.PONY HKK MLK CNK EAH

N 48 60 58 55 64

NHPT 21 40 33 30 39

Hd 0.957 0.979 0.970 0.968 0.973

p 0.026 0.024 0.025 0.021 0.024

k 7.863 7.324 7.397 6.265 7.233

HA (%) 27.0 41.9 51.3 40.5 53.1

HB (%) 0.0 2.3 7.7 5.4 2.0

HC (%) 27.0 23.3 15.4 27.0 10.2

HD (%) 32.4 16.3 12.8 5.4 20.4

HE (%) 0.0 0.0 0.0 2.7 0.0

HF (%) 13.5 16.3 7.7 18.9 14.3

HG (%) 0.0 0.0 5.1 0.0 0.0

EAH, East Anatolian group.

sequences. Each population contained individuals with different haplotypes, and haplotype frequencies varied according to breed and sampling location (Table 2). The reconstructed network revealed few novel haplotypes with respect to the reference samples used in the analysis ` et al. 2001; Jansen et al. 2002; McGahern et al. 2006; (Vila Lei et al. 2009). However, the novel haplotypes observed in the Anatolian samples were not divergent; they were only different from the most common haplotypes of each haplogroup by a few base pairs and had low frequencies. The most common haplotypes observed in other populations from the rest of the world were also the most common haplotypes observed in the Anatolian populations. The frequency of Anatolian samples contained in haplogroups HC1 and HC2 was relatively high. The HC1 clade is considered distinctive for Northern European ponies (Jansen et al. 2002). However, this clade did not contain exclusively haplotypes of A.PONY horses within the Anatolian samples. Among the 22 Anatolian individuals present in this clade, four belonged to the A.PONY breed and 18 belonged to the other Anatolian populations. Similarly, HC2 contained individuals from different Anatolian breeds.

Discussion
The level of allelic variation (623/locus and 6.959.62/ population) and heterozygosity (0.8/population and 0.66 0.88/locus) in Anatolian horse breeds at the microsatellite loci analysed was found to be considerably greater than that reported from a wide range of breeds. For example, Bjrnstad & Red (2002) reported 319 alleles/locus and 0.310.78 heterozygosity/locus heterozygosity estimates in their study, which included native Norwegian breeds (Fjord Horse, Nordland/Lyngen Horse, Dle Horse, Coldblooded Trotter) as well as the Icelandic horse, Shetland Pony, Standardbred Trotter and Thoroughbred breeds. In addition, Aberlee et al. (2004) found 3.436.70 alleles/ population and 0.520.74 heterozygosity/population estimates among 12 breeds, namely Przewalskis Horse, Icelandic horse, Sorraia horse, Exmoor Pony, Hanoverian Warmblood, Arabian, the South German Coldblood, Rhen-

ish German Draught Horse, Mecklenburg Coldblood, Saxon Thuringa Coldblood, Black Forest Horse and Schleswig Draught Horse. Moreover, estimates of 614 alleles/locus, 5.177.92 alleles/population and 0.610.81 heterozygosity/locus were reported by Marletta et al. (2006) among 11 breeds: Jaca Navarra, Asturcon, Caballo Gallego, Losina, Pottoka, Menorquina, Mallorquina, Sanfratellano, Sicilian Oriental Purebred, Sicilian Indigenous and Spanish Thoroughbred horses. Despite the high allelic diversity observed in the present study, the FCA and structure and assignment analysis did not reveal strong population differentiation as seen, for example, in Thoroughbred horses, Arabian horses or Spanish horses (Can on et al. 2000). During domestication and subsequent breed development, as certain traits are selected for, alleles can be expected either to be lost from the gene pool or to increase in frequency. Selective breeding affects heterozygosity and leads to differentiation. Based on both the oral history about the controlled breeding practices during the Ottoman times and the breeding practices to raise distinctive horses for troopers of the army in the early times of the Turkish Republic, we were expecting to be able to genetically distinguish between the breeds Anatolia once had. However, high allelic diversity and low differentiation were observed in Anatolian horse breeds of today (Fig. 3). This may reect a lack of selective breeding in Anatolia in the last few decades. Alternatively, the high genetic diversity found in both Anatolian horse breeds and grade horses may reect a diverse founder population or introgression from non-native or commercial horses. Detecting no deviations from Hardy Weinberg expectations may also suggest a high mixture of Anatolian breeds and no population structuring. The NJ tree constructed based on microsatellite data (Fig. 4) had only one lineage, which included CNK, HKK and EAH samples, with a high bootstrap value (>60%). CNK is known to have introgression from Caucasus in the last millennium. In addition, HKK is known to have introgression from Turkish Arabian horses. EAH horses are East Anatolian horses of mixed origin. HKK is also found in East Anatolia. This higher bootstrap support in the tree may be reecting some common origins for these three horse

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High genetic diversity found in Anatolian horses populations. The only deviation within populations (based on FIS estimates) was observed in MLK horses, which are known to have experienced a sharp population decline in the last two decades and whose samples were collected from marginal rural areas of three distant provinces having little or no connection with the others. This pattern is repeated in EAH horses, which comprised samples collected from six distinct geographical areas. So, the signicant FIS estimates might be suggesting some population substructuring (Wahlund effect). However, MLK horses are known to have mixed origins, which might also have led to this deviation in the FIS estimate. The results revealed some minor differentiation between the A.PONY, which is additionally distinguishable owing to its phenotypic features and the purpose for which it is used (i.e. the collection of olives in Ayvack region), and the other breeds analysed (Fig. 3). This genetic differentiation reects a phenotypic selection process, which has resulted in a unique morphology, in particular the low body stature of this breed. However, owing to a decrease in the number of olive farms and increasing mechanization in olive elds, the number of A.PONY is in decline, and there is an increasing tendency to breed these individuals with taller horses. ` et al. 2001; All previously reported haplogroups (Vila McGahern et al. 2006; Lei et al. 2009) were detected among Anatolian horses (Fig. 2). The haplogroups had high haplotype diversities (0.960.98; Table 2). Moreover, the average pairwise difference (k = 6.37.9; Table 2) was high compared to that found in domestic livestock species. The high mtDNA genetic variation can indicate the broad genetic base of founder mares of Anatolian native horse breeds (Kavar et al. 2002) and/or repeated recruitment of wild horses into the breeding stock after domestication or introgression from commercial breeds into the native breeding pool. Geographically distant and phenotypically different Anatolian populations shared common haplotypes, and each population contained more than one haplotype. The dened haplogroups and the haplotypes in the literature are of diverse origin from Asia to America. Finding all of the worlds mitochondrial haplogroups present in a relatively small region, i.e. Anatolia, compared to Eurasia, as well as the high diversity revealed by the microsatellite analysis, suggests that Anatolia has captured the full range of Eurasian horse diversity. If this is a reection of levels of ancient genetic diversity, this would indicate that Anatolia might have played an important role in the process of horse domestication, as has been suggested for other domestic species (Loftus et al. 1999; Bruford et al. 2003; Naderi et al. 2008; Zeder 2008). However, it is probable that there were prehistoric and historic events that profoundly affected diversity. For example, this high diversity might be reecting the role of Anatolia as a passageway between Asia and Europe. Therefore, high diversity found in mtDNA sequences should be interpreted with care, as different events may result in similar ndings. Among the mtDNA haplogroups HAHG, HE was found only in CNK and HG was found only in MLK. HB was present in all populations except for A.PONY (Table 2). The comparative results show that HF is present in Asian populations with high frequency but in European populations with low frequency (McGahern et al. 2006; Lei et al. 2009). Anatolia, being between Europe and Asia, had an intermediate frequency for HF in its populations (Table 2). HF might have been introduced to the domestic horse gene pool in Asia and might have reached Europe through Anatolia. Horse population movements in Eurasia from east to west and vice versa are known to have occurred throughout history, the most well known of these via the Silk Road. Another cline was observed for HC, which had low frequencies in Central and East Asian populations, especially in Far Eastern populations (McGahern et al. 2006; Lei et al. 2009) and Turkey (Table 2). The frequency of HC is high in European populations. Again, horse population movements, including via the Silk Road, might have played a role in introducing HC from Europe to Asia through Turkey. These data could be valuable when understanding the origin of the breeds and/or haplogroups. Anatolia has played an important role in the domestication of the sheep, goat and pig (Clutton-Brock 1999; Zeder 2008). This rst detailed study of Anatolian horse samples revealed high genetic diversity based on both microsatellite markers and mtDNA control region sequences. Moreover, the sampling trips of the study were very valuable, as there is no registry and inventory programme on horse numbers and breeds in Turkey. According to both ofcial and unofcial sources (the records of the Ministry and books on horses), there were more than eight horse breeds in Anatolia. However, except for an old textbook (Batu 1938), no detailed study on breed characteristics could be found. The sampling trips of this study revealed for the rst time the sharp decrease in breed diversity and population sizes. The current study provides basic genetic diversity information on native breeds. The allelic diversity at the microsatellite loci as well as the mtDNA diversity can be used in starting breeding and management programmes. Assignment and structure test results can be employed in choosing which individuals to keep from each breed. The native breeds were once distinctive and were used in the farm elds (A.PONY and MLK), local ambling races (CNK) and jereed games (HKK), contributing to the local economy. Before losing the diversity present in Anatolian horse breeds, both the ancient horse remains from Anatolia and modern samples from other native breeds should be analysed. The results from the present and future studies should be interpreted with care to inform conservation, management and breeding strategies. Setting up proper management and breeding strategies for horses would help to increase the value of the local breeds and thus help local economies. Every new technique and accumulated information from different techniques may reveal different aspects to consider

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Koban et al. when studying the domestication process and would help us to understand the whole picture of horse domestication. Therefore, the present and future studies can contribute further to understanding the evolutionary history of horse domestication and breed diversication worldwide.
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Acknowledgements
We thank the staff of the Ministry of Agriculture and Rural Affairs (veterinarians, veterinary technicians and zootechnicians) working at directories in the provinces visited during sampling for their invaluable help in collection of the samples. We also would like to extend deep thanks to the rul owners who allowed us to sample their horses; to Ertug Gu (the former president of the Association on the lec Improvement of Anatolian Horses) for his valuable contri_ butions and help in phenotypic evaluation; to Prof. M. Ihsan Soysal (Namik Kemal University, Turkey), who helped in sampling and phenotypic evaluation; and to our special technician Gazi Turgut for invaluable help in the laboratory and to Aydn Bahar for his technical support. We are indebted to the three anonymous reviewers and the journals technical editors for their critical reading and for helping us improve the manuscript. This study was funded by The Scientic and Technological Research Council of Turkey (project no: KAMAG-106G005).

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Supporting information
Additional supporting information may be found in the online version of this article. Appendix S1 Anatolian breed Information. Appendix S2 Supplementary methods. Table S1 The loci analysed in the present study, their chromosome numbers, PCR multiplex sets, primer sequences, expected size ranges and citations. Table S2 Summary of the number of observed alleles, observed heterozygosity (HOBS) and non-biased expected heterozygosity (HNB) in each locus across all samples. Table S3 The mutations detected among the 302 bp region of mtDNA D-loop sequences of 285 anatolian samples. As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer-reviewed and may be re-organized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing les) should be addressed to the authors.

2011 The Authors, Animal Genetics 2011 Stichting International Foundation for Animal Genetics, 43, 401409