Journal of Heredity 2003:94(5):374–380
DOI: 10.1093/jhered/esg079

Ó 2003 The American Genetic Association

Microsatellite Variation in Japanese andAsian Horses and Their Phylogenetic
Relationship Using a European Horse
Outgroup
T. TOZAKI, N. TAKEZAKI, T. HASEGAWA, N. ISHIDA, M. KUROSAWA, M. TOMITA, N. SAITOU,
AND H. MUKOYAMA
From the Department of Molecular Genetics, Laboratory of Racing Chemistry, 1731-2 Tsurutamachi, Utsunomiya,Tochigi 320-0851, Japan (Tozaki and Kurosawa); Max-Planck Institut für Biologie, Abteilung Immungenetik, Corrensstrasse 42,
Tuebingen 72076, Germany (Takezaki); Equine Research Institute, Japan Racing Association, 321-4 Tokami, Utsunomiya,Tochigi 320-0856, Japan (Hasegawa and Ishida); Department of Physiological Chemistry, School of Pharmaceutical
Sciences, Showa University, 1-5-8 Hatanodai, Shinagawa, Tokyo 142-8555, Japan (Tozaki and Tomita); Division ofPopulation Genetics, National Institute of Genetics, 1111 Yata, Mishima, Shizuoka 411-8540, Japan (Saitou); andDepartment of Animal Science, Faculty of Applied Life Science, Nippon Veterinary and Animal Science University, 1-7-1
Kyonacho, Musashino, Tokyo 180-8602, Japan (Mukoyama).
Address correspondence to Teruaki Tozaki, Department of Molecular Genetics, Laboratory of Racing Chemistry, 1731-2
Tsurutamachi, Utsunomiya, Tochigi 320-0851, Japan, or e-mail: ttozaki@nyc.odn.ne.jp (ttozaki@lrc.or.jp).

Abstract
The genetic relationships of seven Japanese and four mainland-Asian horse populations, as well as two European horse
populations, were estimated using data for 20 microsatellite loci. Mongolian horses showed the highest average
heterozygosities (0.75–0.77) in all populations. Phylogenetic analysis showed the existence of three distinct clusters
supported by high bootstrap values: the European cluster (Anglo-Arab and thoroughbreds), the Hokkaido-Kiso cluster, andthe Mongolian cluster. The relationships of these clusters were consistent with their geographical distributions. Basing ourassumptions on the phylogenetic tree and the genetic variation of horse populations, we suggest that Japanese horses
originated from Mongolian horses migrating through the Korean Peninsula. The genetic relationship of Japanese horses
corresponded to their geographical distribution. Microsatellite polymorphism data were shown to be useful for estimating
the genetic relationships between Japanese horses and Asian horses.

In the domestic livestock species of Japan, the populations of
native horses (Equus caballus) have been insulated from otherhorse populations and relatively free of artificial selection.
Currently there are eight local populations of Japanesehorses: Hokkaido, Kiso, Misaki, Noma, Taishu, Tokara,
Miyako, and Yonaguni. The phylogenetic relationships among
these Japanese horse populations is not well elucidated.
Horses native to Japan have been kept in their local areas
to avoid extinction, although there was previously somegene flow between the Japanese horse populations. The
population sizes of these Japanese horses range from 35
to 100 horses, except for the native horses from
Hokkaido, which include more than 2,000 horses (Nozawa
1992).
Ishida et al. (1995) conducted a phylogenetic study ofthoroughbreds, Japanese (Hokkaido) horses, Mongolian374

horses, and Przewalskii’s wild horses using the mitochondrial
DNA (mtDNA) D-loop region. The study suggested that the
Asian horses were similar to each other and distinct fromthoroughbreds.
Hayashida (1958) classified Japanese horses into twogroups according to body size and location. Medium-size andsmall-size horses are 130–140 cm and 110–120 cm in withersheight, respectively. The medium-size horses—Hokkaido,
Kiso, and Misaki horses—live mainly on the three mainislands of Japan (Hokkaido, Honshu, and Kyushu). The smallsize horses—Noma, Taishu, Tokara, Miyako, and Yonaguni
horses—live on islets (Shikoku, Tsushima, Tokara, Miyako,
and Yonaguni) south and west of the main islands. Based on
this classification, Hayashida (1958) proposed a two-wave
migration hypothesis for the evolution of Japanese horse
populations.

Tozaki et al.  Phylogenetic Relationship of Japanese Horses

Figure 1. The location of seven Japanese horses, one Korean horse, and three Mongolian horses.

However, a recent study using the protein polymorphisms of 22 loci from 2,415 horses belonging to 34
horse populations in Japan and Asia (Nozawa et al. 1998)
was inconsistent with this two-wave migration. The
phylogenetic relationships among Japanese horses could
not correspond to the geographical distribution in Japan(Nozawa et al. 1998). The difference between thephylogenetic relationship and the geographical distribution
might be a consequence of a small population size,
a bottleneck effect, and low polymorphism for protein loci.
The 22 loci examined had monomorphism or two to threealleles in Japanese horses and were less informative for thedetermination of a phylogenetic relationship, althoughuseful for Asian horses.
Recently many microsatellites were isolated from horse
genome DNA (Tozaki et al. 2000a,b,c, 2001b). Microsatellites, in particular the (CA/TG)n repeats, which wereconstructed with tandemly repeated DNA sequences consisting of poly(dC-dA)/(dG-dT) repeats, have been found to
be common in all eukaryotic genomes, occurring once forevery 30–60 kb (Litt and Luty 1989; Stallings et al. 1991;
Weber and May 1989). As microsatellites are also frequently
polymorphic and evolutionarily conserved in the eukaryotic
genome, they provide useful markers for comparative studies
of genetic variation, parentage testing, and studies of geneflow (Bjørnstad and Røed 2001; Cunningham et al. 2001;
Tozaki et al. 2001a) and have recently been the markers ofchoice for analyses of population structure in wild anddomesticated species (Bowcock et al. 1994; Kim et al. 2001;
Takahashi et al. 1998).

The genetic relationships of many horse populations in
Europe have been investigated recently using microsatellites(Canon et al. 2000; Vila et al. 2001). However, the genetic
relationships of horse populations in Japan have not been
investigated using microsatellites.
In the present study, we analyzed 20 microsatellite loci,which were originally characterized in thoroughbred horses
to infer the phylogenetic relationships among Japaneseand Asian horses. In addition, we compared the levelsof polymorphism between Japanese and Asian horses.

Materials and Methods
Animals
Sampling localities are shown in Figure 1. We collected blood
samples from 60 Mongolian horses: Dzaamar (n ¼ 22),
Bajandzargalan (n ¼ 18), and Garshar (n ¼ 20). In addition,
we collected blood samples from a total of 135 Japanesehorses: Hokkaido (n ¼ 24), Kiso (n ¼ 21), Misaki (n ¼ 16),
Noma (n ¼ 16), Taishu (n ¼ 10), Tokara (n ¼ 24), andYonaguni (n ¼ 24). We also collected blood samples from
Korean horses (n ¼ 21), thoroughbred horses (n ¼ 25), andAnglo-Arab horses (n ¼ 18). No horses were injured in any
way during the collecting of samples. Genomic DNAs wereprepared from whole blood samples. Genomic DNAs fromthoroughbred horses were extracted using the MagExtractor
System MFX-2000 (Toyobo, Osaka, Japan) according to the
manufacturer’s protocols. Following digestion with proteinase K (2 mg/ml) in extraction buffer (10 mM Tris, 10375

Journal of Heredity 2003:94(5)mM EDTA, 0.1 M NaCl, 2% SDS), genomic DNAs from
Asian horses were extracted by phenol/chloroform, precipitated with ethanol, washed, and resuspended in a sterile
TE buffer (10 mM Tris-HCl, 0.1 mM EDTA, pH 8.0).
Microsatellites
In this study we used the following 20 microsatellite loci:
TKY2 (Sakagami et al. 1995); TKY3 (Tozaki et al. 1995);
HTG3, HTG4, HTG5, HTG6 (Ellegren et al. 1992); HTG7,
HTG10, HTG15 (Marklund et al. 1994); HMS1, HMS2,
HMS3, HMS5, HMS7, HMS8 (Guérin et al. 1994); VHL20(van Haeringen et al. 1994); and AHT4, ASB1, ASB2, and
ASB3 (Swinburne et al. 2000).
Genotyping
Forward primers of all the microsatellites were labeled with
Cy-5 (Amersham Biosciences, Piscataway, NJ). Each polymerase chain reaction (PCR) amplification was performed ina total volume of 20 ll of the following mixture: 20–50 ng ofequine genomic DNA, 5 pmol of a Cy-5-labeled forward
primer, 5 pmol of reverse primer, 200 lM of dNTP, 2 ll of
103 reaction buffer; and 0.1 U of rTaq polymerase (Takara
Bio, Shiga, Japan). PCR amplification entailed initial denaturation (948C, 4 min); 30 cycles of 1 min each at 948C, 55–
608C, and 728C; and then 60 min at 728C for final extension
in a GeneAmp PCR System 9600 (Applied Biosystems,
Foster City, CA). The final extension step at 728C for 60 min
promoted a complete nontemplated 39-nucleotide addition.
The amplified fragments were analyzed through electrophoresis with an ALF express DNA sequencer (Amersham
Biosciences) using an 8% polyacrylamide gel containing 7 M
urea (Biomate, Tokyo, Japan). Finally, the raw data obtained
were converted to dinucleotide repeat polymorphic band
patterns using a Fragment Manager (Amersham Biosciences).

most loci displayed polymorphism between individuals.
Allele frequency distributions and heterozygosities of 20
microsatellites showed unique features for each population.
Although the other populations were polymorphic, with 2 to
10 alleles at all loci, the Misaki and Tokara populations weremonomorphic at three loci (HTG7, HMS3, and HMS8)and six loci (HTG5, HTG6, HTG7, HMS8, ASB1, and
ASB2), respectively; these loci also showed relatively lesspolymorphism than the other loci when the other horse
populations were analyzed.
A total of 130 alleles were detected in this study and theaverage number of alleles was 4.6 (Table 1). The observed
number of alleles and average heterozygosities for all
populations are shown in Tables 1 and 2. The average
heterozygosity was 0.62, ranging from 0.34 in the Tokara
population of Japanese horses to 0.77 in the Bajandzargalan
population of Mongolian horses. Four populations ofJapanese horses (Misaki, Noma, Tokara, and Yonaguni)
showed lower heterozygosity than the average value. Similar
to the heterozygosity data, the observed number of alleles
ranged from 42 in the Tokara population to 125 in the
Dzaamar population, and the average allele number was 4.6.
The average allele numbers of the aforementioned four
populations were lower than the average value. In particular,
low heterozygosities and a small number of alleles wereobserved in the Tokara and Misaki populations. Conversely,
high heterozygosities and a large number of alleles wereobserved in three populations of Mongolian horses. Three
populations of Mongolian horses had more alleles than theother populations at 17 loci. The average heterozygosity forJapanese horses was less than for any other population.
Furthermore, the number of alleles per locus also tended to
be fewer among Japanese horses.

Genetic Distances and Population Relationships
Estimation of Genetic Distances and the Constructionof a Phylogenetic Tree
Alleles were designated according to PCR product size andallele frequencies were estimated by direct counting. Genetic
differences among populations were estimated by calculating the DA distance (Nei 1983) and (dl)2 distance (Goldstein
et al. 1995). A neighbor-joining (NJ) tree was constructedfrom the distances (Saitou and Nei 1987), and 1,000
bootstrap replicates were generated.

Results
Heterozygosities and the Number of Alleles
Polymerase chain reaction amplification of 20 (CA/TG)nmicrosatellites, which were mainly isolated from thethoroughbred horse genome, was successful with minor
modifications of the original condition for the 13 horse
populations studied. Allele frequencies and heterozygosities
were calculated. (The allele frequencies are available from thesenior author upon request.) Despite possible inbreeding,376

Estimates of the genetic distances among the 13 populations
based on the allele frequency data on 20 microsatellites arepresented in Table 3. The DA values always range between
0 and 1 and are nonlinearly related to the number of genesubstitutions. Mean genetic distances ranged from 0.057(Bajandzargalan to Garshar populations), 0.065 (Bajandzargalan to Garshar populations), and 0.075 (Bajandzargalan toGarshar populations) to 0.491 (Tokara to thoroughbred
populations) and 0.481 (Misaki to Noma populations), while
the (dl)2 distance ranged from 0.057 (Bajandzargalan toGarshar populations), 0.065 (Bajandzargalan to Garshar
populations), and 0.075 (Bajandzargalan to Garshar populations) to 0.491 (Tokara to thoroughbred populations) and
0.481 (Misaki to Noma populations). Considering all distancemeasures, the three populations of Mongolian horses wereclosest.
DA and (dl)2 distance matrices were used to build
phylogenetic trees with the NJ method. The NJ method forconstructing evolutionary trees from distance data has beenreported to be one of the most effective methods (Saitou andNei 1987). Figures 2 and 3 show the phylogenetic tree

Population
5
5
3
2
2
2
5
6
5
6
6
5
3
4.2

5
3
3
4
2
5
4
5
6
6
6
4
4
4.4

6
4
3
3
1
2
3
7
7
6
5
6
4
4.4

5
2
2
3
1
5
4
5
8
7
6
4
5
4.4

Population

4
4
1
4
1
3
4
5
4
3
3
2
2
3.1

5
6
4
2
2
5
6
10
9
7
6
6
6
5.7

4
4
2
3
2
4
2
5
6
7
6
3
3
3.9

4
4
2
4
4
5
3
5
6
5
6
4
3
4.2

8
6
3
6
2
5
4
6
6
7
5
5
5
5.2

5
5
1
4
3
5
4
7
6
8
7
5
5
5.0

2
3
4
3
2
3
2
3
3
3
3
3
4
2.9

5
5
5
4
4
4
4
5
6
8
7
6
5
5.2

5
4
1
4
1
4
3
4
5
5
5
3
4
3.7

7
7
4
3
3
5
5
8
9
7
10
4
4
5.9

4
5
4
4
1
3
4
5
5
5
5
3
3
3.9

7
9
7
6
1
6
4
10
9
6
9
6
8
6.8

5
6
2
4
4
4
3
5
7
6
6
5
4
4.7

7
6
4
5
2
5
5
8
8
8
8
8
4
6.0

5.1
4.8
3.0
3.7
2.1
4.1
3.8
5.9
6.3
6.0
6.0
4.5
4.2
4.6

0.58
0.73
0.52
0.07
0.29
0.21
0.61
0.14
0.79
0.66
0.66
0.56
0.64
0.50

0.49
0.51
0.34
0.53
0.48
0.53
0.66
0.82
0.80
0.75
0.76
0.71
0.58
0.61

0.51
0.48
0.19
0.67
0.41
0.78
0.69
0.42
0.54
0.79
0.70
0.56
0.56
0.56

0.60
0.57
0.55
0.66
0.00
0.11
0.58
0.64
0.83
0.77
0.70
0.48
0.12
0.51

0.54
0.47
0.48
0.46
0.00
0.54
0.60
0.66
0.67
0.79
0.76
0.65
0.61
0.56

0.72
0.73
0.00
0.70
0.00
0.61
0.39
0.81
0.67
0.77
0.74
0.55
0.69
0.57

0.66
0.82
0.77
0.37
0.45
0.82
0.64
0.84
0.85
0.81
0.75
0.85
0.83
0.73

0.27
0.34
0.53
0.59
0.51
0.63
0.20
0.56
0.41
0.56
0.68
0.60
0.63
0.52

0.63
0.46
0.46
0.73
0.67
0.78
0.24
0.57
0.72
0.73
0.77
0.69
0.71
0.63

0.73
0.85
0.53
0.81
0.23
0.85
0.58
0.85
0.81
0.81
0.80
0.45
0.52
0.68

0.67
0.73
0.00
0.67
0.55
0.86
0.54
0.82
0.83
0.78
0.76
0.72
0.76
0.67

0.41
0.51
0.68
0.57
0.51
0.72
0.49
0.62
0.68
0.59
0.61
0.70
0.64
0.60

0.78
0.70
0.76
0.64
0.64
0.80
0.70
0.71
0.81
0.85
0.86
0.81
0.80
0.76

0.74
0.75
0.00
0.72
0.00
0.38
0.62
0.74
0.72
0.75
0.72
0.68
0.62
0.57

0.84
0.71
0.30
0.43
0.57
0.71
0.62
0.87
0.84
0.80
0.89
0.72
0.76
0.70

0.67
0.73
0.58
0.71
0.00
0.66
0.59
0.61
0.70
0.77
0.75
0.66
0.60
0.62

0.86
0.84
0.80
0.72
0.00
0.88
0.69
0.88
0.87
0.84
0.80
0.88
0.85
0.76

0.34
0.78
0.18
0.72
0.54
0.53
0.49
0.77
0.82
0.80
0.71
0.71
0.48
0.61

0.80
0.81
0.30
0.76
0.43
0.82
0.72
0.81
0.84
0.86
0.81
0.80
0.77
0.73

0.63
0.66
0.43
0.61
0.34
0.64
0.54
0.69
0.75
0.77
0.75
0.66
0.63
0.62

TKY2 TKY3 HTG3 HTG4 HTG5 HTG6 HTG7 HTG10 HTG15 HMS1 HMS2 HMS3 HMS5 HMS7 HMS8 AHT4 ASB1 ASB2 ASB3 VHL20 Average

Heterozygosity for 20 microsatellites in each population

Hokkaido
0.68
Kiso
0.66
Misaki
0.62
Noma
0.70
Tokara0.45
Taishu0.52
Yonaguni
0.12
KoreanCheju
0.72
Mongolian Dzaamar
0.81
Bajandzargalan 0.84
Garshar
0.82
European Anglo-Arab
0.41
Thoroughbred 0.40
Average
0.60

Japanese

Table 2.

3
4
2
2
2
3
3
3
4
4
4
5
4
3.3

TKY2 TKY3 HTG3 HTG4 HTG5 HTG6 HTG7 HTG10 HTG15 HMS1 HMS2 HMS3 HMS5 HMS7 HMS8 AHT4 ASB1 ASB2 ASB3 VHL20 Average

The number of alleles and average alleles of 20 microsatellites

Hokkaido
6
Kiso
3
Misaki3
Noma3
Tokara2
Taishu3
Yonaguni
3
KoreanCheju
6
Mongolian Dzaamar
6
Bajandzargalan 6
Garshar
6
European Anglo-Arab3
Thoroughbred 3
Average
4.1

Japanese

Table 1.

Tozaki et al.  Phylogenetic Relationship of Japanese Horses

377

378

0.195

0.185
1.554
1.710
0.162
0.251
1.561
1.912

1.850
2.115

0.339
0.306
0.442
0.384
0.491
0.290
0.376
0.197
0.224
0.219
0.220
0.082
0.301
0.296
0.434
0.327
0.435
0.262
0.405
0.171
0.187
0.197
0.202
0.180
0.193
0.376
0.264
0.376
0.209
0.274
0.128
0.075
0.152
0.180
0.374
0.250
0.382
0.173
0.278
0.110

0.174
0.197
0.374
0.239
0.371
0.164
0.246
0.110
0.065
0.057

Thoroughbred
Anglo-Arab
GarsharBajandzargalan
Dzaamar

0.343
0.347
0.335
1.271
1.429
0.852
0.728
0.698
0.748
1.438
1.605

1.225
0.888
0.621
0.695
0.982
1.759
2.318
5.555
4.644
5.012
4.194
4.165
4.821
5.670
5.653

3.414
2.473
1.748
1.692
1.438
1.502
1.445
3.148
3.309
3.270
6.630
3.604
2.802
2.079
2.664
2.637
3.172
3.006
2.598

5.293
2.674
5.559
1.169
1.794
1.394
0.885
1.104
1.063
2.329
2.830

0.172
0.213
0.341
0.199
0.375
0.182
0.274
0.314
0.320
0.445
0.332
0.385
0.309
0.243
0.281
0.421
0.274
0.458
0.266
0.351
0.481
0.408
0.459

0.491
4.410
1.967
5.151
0.800
1.409
0.776
0.454
0.623
0.425
2.273
2.803

0.175

0.426
0.448
0.472
0.405

KoreanHokkaido

Kiso

Misaki

Noma

Yonaguni
TaishuTokaraHokkaodo
Kiso
Misaki
Noma
TokaraTaishuYonaguni
KoreanDzaamarBajandzargalan
GarsharAnglo-Arab
Thoroughbred

Table 3.

Genetic distances among all pairs of populations of Japanese, Asian, and European horse populations calculated using DA (above diagonal) and (dl)2 (below diagonal)

Journal of Heredity 2003:94(5)Figure 2. Neighbor-joining dendrogram (Saitou and Nei
1987) of Japanese, Mongolian, and Korean horse varieties
based on DA (Nei 1983). The numbers represent the percentage
of bootstrap values from 1,000 replications of resampled loci.
European horses are positioned as an outgroup. The linear
scale relates the branch lengths to DA units.

obtained from DA and (dl)2 distance values using the NJ
method. However, these trees showed some differencesfrom each other. In order to test the reliability of the tree
topography, dendrograms from 1,000 replications ofresample loci were constructed using both distances. Highbootstrap values were obtained when using the DA distance,
which indicated the high confidence of the obtained trees.
The DA distance clustered together both European populations with a bootstrap value of 99%, and Mongolian
populations with bootstrap values of 89% and 92%.
Hokkaido and Kiso horses clustered with a bootstrap valueof 91%.

Discussion
The task of resolving the genetic relationships among
Japanese horses is a difficult one, because each population
size of Japanese horses is small and Japanese horses had
substantial gene flow between the populations (Nozawa
1992). Recently, many microsatellites were isolated from thehorse genome (Tozaki et al. 2000a, 2000b, 2000c, 2001b),
and the microsatellites showed multiple alleles as well as highheterozygosity among European horse breeds such as
thoroughbreds. Microsatellites may be a useful approach forresolving the relationships of Japanese horse populations.
In this study we analyzed the allele frequencies andgenotype distributions of Japanese horses to investigate
a phylogenetic relationship among the populations. In
addition, we analyzed the allele frequencies and genotype
distributions among Mongolian and Korean horses toconsider the ancestral populations for Japanese horse
populations.
Twenty microsatellites, which were isolated from thethoroughbred genome, showed high heterozygosity andTozaki et al.  Phylogenetic Relationship of Japanese Horses

Figure 3. Neighbor-joining dendrogram (Saitou and Nei
1987) of Japanese, Mongolian, and Korean horse varieties basedon (dl)2 (Goldstein et al. 1983). The numbers represent thepercentage of bootstrap values from 1,000 replications ofresampled loci. European horses are positioned as an outgroup.
The linear scale relates the branch lengths to (dl)2 units.

were used for analyzing allele frequencies of Asian horse
populations, Anglo-Arab breeds, and thoroughbreds. The
average heterozygosity within populations analyzed in thisstudy was 0.62 (range 0.34–0.77). Nozawa et al. (1998)
investigated genetic variation among Asian horses using 22
blood protein polymorphisms; this study showed an average
heterozygosity between 0.0584 and 0.1257. The microsatellites tested in our study were therefore effective tools inanalyzing the genetic population structure of Asian horses.
The average heterozygosity within each population ofMongolian horses ranged from 0.75 to 0.77. Mongolian
horses had the highest values in all populations, demonstrating that Mongolian horses have retained the largest
amount of genetic variation of all the populations studied.
In contrast, the average heterozygosity within each
population of Japanese horses ranged between 0.34 and
0.66. In addition, Mongolian horses possessed all the alleles
found in Japanese horses. These results are attributed to thefact that Mongolian horses are descended from the ancestral
populations of Japanese horses. This assumption is supported by the historical fact that native horses on the Asian
continent were frequently transported to Japan through the
Korean peninsula.
The Tokara population showed the lowest heterozygosity
in this study. Furthermore, the Tokara population showed
the lowest value of protein polymorphisms (Nozawa et al.
1998). According to the literature, the population size of theTokara group has decreased to low numbers in the past,
but has recently increased due to breeding. Thus the main
reason for this low-level genetic variation is most likely
the population bottleneck.
The phylogenetic reconstructions using DA and (dl)2
gave similar clustering results. However, the different
clustering was not unexpected since the bootstrap values inboth trees were low. The DA tree showed much higher values
than the (dl)2 tree. This difference can be explained by thefact that (dl)2 is based on the assumption that microsatellites
follow a stepwise mutation model, while DA is based on theinfinite allele model. All the populations in Japan were
derived from one original horse population, which wastransported about 2,000 years ago through the Korean
peninsula. Furthermore, these populations localized to theparticular areas in Japan mentioned in this article. After
localization, the populations were affected by the gene flowof each other. Thus allele frequencies of Japanese horses
might not be the effect of a stepwise mutation of microsatellites, indicating that the DA tree might be suitable forthis study. In addition, Takezaki and Nei (1996) studied
the efficiencies of several genetic distance measures, such as
DA (Nei 1983), DC (Cavalli-Sforza and Edwards 1967), DSW(Shriver et al. 1995), and (dl)2 (Goldstein et al. 1995), whenapplied to microsatellites. Their computer simulation
showed that the DA and DC distances are most efficient in
obtaining the correct tree topology (Takezaki and Nei 1996).
In this study, our phylogenetic tree showed several
clusters, including the European cluster (Anglo-Arab,thoroughbreds), the Hokkaido-Kiso cluster, and the Mongolian cluster, because of high bootstrap values. The
relationship of the clusters to each other corresponds totheir geographical distributions. In particular, the Hokkaido
and Kiso populations clustered with a high bootstrap value.
This relationship corresponds to the historical fact that the
Hokkaido population was made up of horses transported
from Honshu, Japan’s mainland, for the purpose of
agriculture. The Kiso population is geographically locatedin the area nearest to the Hokkaido population in Japan.
Although Misaki and Tokara populations have long branch
lengths and low bootstrap values, these populations wereclustered into one group close to the Yonaguni population.
The long branch lengths for the Tokara and Misaki
populations in the tree might be caused by a bottleneck
effect. The Misaki, Tokara, and Yonaguni populations werealso close to the Noma population genetically. Furthermore,
the genetic relationship of the Taishu population was close to
the Mongolian, Korean, Hokkaido-Kiso groups, and the four
populations. The genetic relationships constructed in thisstudy corresponded to geographical distribution in Japan.
Furthermore, the correspondence between the genetic
relationship and the geographical distribution supports the
hypothesis that Japanese horses are descended from
Mongolian horses through the Korean peninsula and havespread all over Japan, because there were no horses in Japan
about 2,000 years ago, according to the literature.
In conclusion, we analyzed the genetic variation andphylogenetic relationship of Asian and Japanese horses using
microsatellites, and we support the view that Japanese horses
originated from Mongolian horses through the Korean
Peninsula. The genetic relationship of Japanese horses toeach other corresponds to their geographical distribution.
Our results also show that several horse populations in Japan
have low genetic variation. Because almost all Japanesehorses are in small populations, breeders should monitor

379

Journal of Heredity 2003:94(5)genetic variation to protect these horse populations. Genetic
marker data, as described here, could be an effective tool.
In the future, an investigation of the pedigree of
populations and the genetic variations in those populations
will be necessary. As our data show, microsatellites area useful tool for studying the genetic relationships amongclosely related horse breeds. Since the microsatellites in thisstudy are highly polymorphic, they can also be applied forparentage testing. Thus we believe that using microsatellites
is important in the evaluation of genetic variations.

Acknowledgments
We thank Dr. M. Sakagami for coordinating this project and for helpful
discussion. We are grateful to Drs. N. Oguri, T. Oyunsuren, and Y. Lim forcollecting blood samples of Asian and Japanese horses. We are also gratefulto the staff of the blood-typing section of the Laboratory of Racing
Chemistry for collecting blood samples of Japanese horses.

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Received August 29, 2002
Accepted May 31, 2003
Corresponding Editor: Oliver A. Ryder