Int J Legal Med (2003) 117 : 218–225
DOI 10.1007/s00414-003-0379-2

O R I G I N A L A RT I C L E

Sayaka Maruyama · Kiyoshi Minaguchi · Naruya Saitou

Sequence polymorphisms of the mitochondrial DNA control region
and phylogenetic analysis of mtDNA lineages in the Japanese population

Received: 22 February 2002 / Accepted: 3 April 2003 / Published online: 5 July 2003
© Springer-Verlag 2003

Abstract Sequence polymorphisms of the hypervariablemitochondrial DNA (mtDNA) regions HVI and HVII, andcoding region polymorphisms were investigated in 211 unrelated individuals from the Japanese population. Sequence comparison of the HVI and HVII regions led tothe identification of 169 mitochondrial haplotypes definedby 147 variable positions. Among them 145 types were observed in only 1 individual; the other 24 types were sharedby 2 or more individuals. The gene diversity was estimatedat 0.9961, and the probability of two randomly selectedindividuals from the population having identical mtDNA
types was 0.86%. We also established phylogenetic haplogroups in the Japanese population based on the coding
and control region polymorphisms and compared the haplotypes with those in other Japanese, Korean and Chinese
populations. As a result, three new subhaplogroups, G4a,G4b, and N9b, and several haplotypes specific for the Japanese and Korean populations were identified. The present database can be used not only for personal identification but also as an aid for geographic or phenotype (race)
estimation in forensic casework in Japan.
Electronic Supplementary Material Supplementary material is available for this article if you access the article at
http://dx.doi.org/10.1007/s00414-003-0379-2
Keywords Mitochondrial DNA · Polymorphism ·Hypervariable regions · Coding region · Phylogeny

S. Maruyama (✉) · K. Minaguchi
Department of Forensic Odontology, Tokyo Dental College,
1-2-2 Masago Mihama-ku, 261-0011 Chiba City, Japan
Tel.: +81-43-2703786, Fax: +81-42-2703788,
e-mail: smaruyam@tdc.ac.jp
N. Saitou
Laboratory of Evolutionary Genetics,
National Institute of Genetics,
Mishima-shi, 411-8540 Shizuoka-ken, Japan

Introduction
Knowledge of the frequencies with which certain mitochondrial DNA sequences occur in a given population isof crucial importance for the application of mitochondrial
markers to forensic studies, because mtDNA sequencetypes are strongly correlated with geographic origin or
phenotype (race) (Forster et al. 2002). Recently, classifications of mtDNA lineages in east Asian populations using control region and coding region information havebeen developed (Yao et al. 2002; Kivisild et al. 2002) anda more reliable phylogenetic tree for mtDNA differentiation has been presented. These studies provide very useful
information for estimating geographic origin or phenotype
from biological evidence by DNA analysis.
With the increase in the number of foreign workers inJapan, the need to estimate geographic origin in forensic
casework is growing. However, there is a limit to distinguishing closely related ethnic groups using morphological information alone.
In this study, to further our understanding of mtDNA
polymorphisms in the Japanese population and to approach the possibility of geographic or phenotype estimation from individual biological evidence using mtDNA
polymorphism data, we have carried out a population studyof a large number of Japanese individuals that examined
the sequence variations in the HVI and HVII region andcoding region information specific for mtDNA haplogroups.
We further inferred individual HVI and HVII sequence
data sets from closely related populations such as otherJapanese, Koreans, and Chinese, and examined differencesamong these populations.

Materials and methods
Samples
Genomic DNA was extracted from blood samples of 211 healthy
unrelated Japanese individuals. Informed consent was obtained
from the blood donors. The names of the prefectures corresponding to the birthplaces and the numbers of individuals used in this219
Each amplification was performed in a 30 µl mixture containing
10 ng of genomic DNA, 10 mM Tris-HCl at pH 8.3, 50 mM KCl,
2.5 mM MgCl2, 0.02% gelatin, 200 µM dNTP, 400 nM of eachprimer, and 1.5 U of AmpliTaq Gold (Perkin-Elmer Cetus). PCR
amplification was done by 2-step or 3-step methods. PCR primers
and PCR conditions used are shown in Table 1. Amplicons using
the primer pairs L16093 and H16416 for HVI and L29 and H389
for HVII were purified for sequencing by one of the following
three methods:
1. Elution from silver-stained 8% acrylamide gels using the “crush
and soak” method (Sambrook et al. 1989), followed by re-amplification by PCR and purification using Centricon 100 (Amicon).
2. Purification using the QIAquick gel extraction kit (Qiagen) or
the StrataPrepTM DNA gel extraction kit (Stratagene) according to the manufacturer’s instructions.
3. Purification using QIAquick PCR purification kit (Qiagen) according to the manufacturer’s instructions.

Fig. 1 Geographic location and the number (in parenthesis) of thepresent Japanese samples. Abbreviations for prefectures in Table
S1 are HO Hokkaido, AM Aomori, AT Akita, YGA Yamagata, MG
Miyagi, FS Fukushima, TG Tochigi, IR Ibaraki, NG Niigata, GM
Gunma, ST Saitama, TK Tokyo, CB Chiba, KN Kanagawa, NN
Nagano, SO Shizuoka, AC Aichi, TY Toyama, IK Ishikawa, GF
Gifu, ME Mie, KT Kyoto, NR Nara, WK Wakayama, HG Hyogo,
OY Okayama, HS Hiroshima, TT Tottori, YGU Yamaguchi, KG
Kagawa, EH Ehime, FO Fukuoka, OI Ohita, and KM Kumamoto

Amplicons of the complete control region using the primer pairs
L15978 and H601 were purified with QIAquick PCR purification
kit (Qiagen). PCR for sequencing was performed using Dye Terminator or BigDyeTM Terminator cycle sequencing ready reaction
kits with AmpliTaq DNA Polymerase FS (Perkin Elmer Cetus).
Excess dye was removed using Centri-Sep Spin columns (Princeton) or DyeEx TM spin kits (Qiagen). Sequence analysis was performed on an ABI 373A DNA sequencer (Applied Biosystems) oran ABI 310 DNA sequencer (Applied Biosystems).
DNA sequences of partial fragments in 120 samples were determined from both strands using original primer pairs, and primers
used for sequencing complete 1,193 bp fragments in 91 samples
were the original PCR primer pairs, H16420, L16381, H194, andL155 (Table 1). For samples with length heteroplasmy in HVI and
HVII regions, primers L16194 and L317 were used for additionalsequencing. When ambiguous electrophoretograms for 20–30 nucleotides near the primers were obtained in the 29–389 fragments,
complete 1,193 bp fragments were further amplified and primer
L16381 was used for sequencing. While performing phylogenetic
analysis, the haplogroup motif and private mutations were checked
and reconfirmed by sequencing additional PCR products, even if
there was little doubt of their accuracy in comparison with the sequences for other samples in the same haplogroup and the databasefor neighbors (Bandelt et al. 2001).
Typing of other polymorphisms

study are shown in Fig. 1. Leukocyte preparations from blood weredigested with proteinase K (Sigma) at 55°C overnight, followed bytreatment with RNAse at 55°C for 2 h. DNA was extracted withphenol/chloroform, precipitated with ethanol, and resuspended in
TE buffer (10 mM Tris-HCl, 1 mM EDTA at pH 7.6) (Sambrook et
al. 1989).
PCR amplification and sequencing of control regions
We determined the nucleotide sequences of 211 samples from16024 to 16398 for HVI and from 49 to 369 for HVII. We useddifferent sized PCR products to determine the sequences of HVI
and HVII. Among the 211 samples, the nucleotide sequences of
120 samples and 91 samples were determined using the different
PCR products. In 120 samples, 3 partial segments (185 bp, 324 bp,
and 361 bp) of the control region were amplified using the primer
pairs L15978 and H16162, and L16093 and H16416 for HVI and
L29 and H389 for HVII, respectively (Table 1). In 91 samples, thecomplete non-coding control region (1,150 bp) was amplified using the primer pair L15978 and H601. Smaller sized products are
more suitable for use in actual forensic cases, however to expand
the database, amplification of the whole control region minimizes
the chances of making errors in the experiments, because one kindof template can be used to determine the sequences of both regions, and sequencing regions can be overlapped depending on theprimers (Bandelt et al. 2001).

We examined the nucleotide variation between np 10305 and 10420
by SSCP (single strand conformation polymorphism) analysis of
all of the samples (Table 1). SSCP analysis was performed in 17%
polyacrylamide gels as described by Fujita and Kiyama (1995) except that the gel contained 5% glycerine, the gel and reservoir
buffer were 0.475×TBE and 1×TBE, respectively, and a 16×36 cm
gel with 0.4 cm thickness was used. Electrophoresis was performed at 55 V/cm constant voltage at 15°C. All the products werevisualized by silver staining. When a different pattern was obtained, the PCR product was sequenced to confirm the mutation.
The intergenic COII/tRNALys 9 bp deletion (Horai et al. 1996),
and 3010 and 5178 mutations (Umetsu et al. 2001) were also determined from all of the samples as described in those studies.
Other coding region polymorphisms specific for each lineage wereselected by referring to the data of Yao et al. (2002) and Kivisild etal. (2002). Typing for these polymorphisms was performed by
SSCP analysis after restriction enzyme digestion, or by direct sequencing. PCR primers, annealing temperatures, and PCR methods used for SSCP analysis are shown in Table 1. SSCP analysis
and sequencing identified unexpected mutations in addition to the
expected ones. Some of these unexpected mutations were novel
ones for the new haplogroup. When the characteristics of sequences
did not match those of any known subhaplogroups, we further extended sequencing to find coding region mutations for the newsubhaplogroup. Primers to sequence other regions than thoseshown in Table 1 are available from the authors on request.

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Table 1 PCR primers, annealing temperature, and conditions for sequencing and SSCP analysis
Primers

Annealing
temperature and
PCR methoda

Polymorphic
sites or region

Detection
methodL15978/H16162
5’-CACCATTAGCACCCAAAGCTA-3’
5’-TATGTACTACAGGTGGTCAAG-3’
L16093/H16416
5’-CACCATTAGCACCCAAAGCT-3’5’-TTCACGGAGGATGGTGGT-3’
L029/H389
5’-CTCACGGGAGCTCTCCATGC-3’
5’-CTGGTTAGGCTGGTGTTAGG-3’
L15978/H601c
5’-CACCATTAGCACCCAAAGCTA-3’5’-CTTTGAGGAGGTAAGCTACATA-3’
L550/H698
5’-AACCAAACCCCAAAGACAC-3’
5’-GCATGTGTAATCTTACTAAGA-3’
L3143/H 3294
5’-TAAGGCCTACTTCACAAAGC-3’
5’-AGGAATTGAACCTCTGACTGT-3’
L3143/H3419
5’-TAAGGCCTACTTCACAAAGC-3’
5’-TTGGGGCCTTTGCGTAGTT-3’
L4688/H4856
5’-TATCCTCTTCAACAATATACTC-3’
5’-AAGCAGGCCGGATGTCAG-3’
L5109/H5253
5’-ACCGCATTCCTACTACTCA-3’
5’-GCAAAAAAGCCGGTTAGCG-3’
L8211/H8310
5’-TCGTCCTAGAATTAATTCCC-3’
5’-AGTTAGCTTTACAGTGGGCT-3’
L9803/H9972
5’-AGCCACAGGCTTCCACGGA-3’5’-TGGAGACATACAGAAATAGTC-3’
L10285/H10441
5’-CATGAGCCCTACAAACAACT-3’5’-GAGTCGAAATCATTCGTTTTG-3’
L10285/H10682
5’-ACATAGAAAAATCCACCCCTTACG-3’5’-TGCTTCGCAGGCGGCAAAGACTA-3’

58, 2-step

HVI

Sequencing

52, 2-step

HVI

Sequencing

58, 2-step

HVII

Sequencing

58, 3-step

16024-576

Sequencing

54, 2-step

663

SSCP

58, 2-step

3172.1, 3206

SSCP

58, 2-step

3172.1, 3206, 3338, 3394

NciI-SSCPb

56, 2-step

4715, 4761, 4793, 4833

SSCP

56, 2-step

5231, (5147), (5178)d

SSCP

58, 2-step

9-bp Deletion

8% Native gel

58, 2-step9824, 9923

SSCP

58, 2-step

10310, 10321, 10345, 10373
10362,10397,10398,10400
10410
10589, 10607, 10609, 10646(10654)dSSCP58, 2-stepaThe 2-step PCR was 95°C for 10 min, followed by 35 cycles ofdenaturation at 95°C for 50 s and annealing and extension at an appropriate temperature for 105 s. After the 35th cycle, a final extension step was performed at annealing and extension temperaturefor 10 min. The 3-step PCR was 95°C for 10 min, followed by 35
cycles of denaturation at 95°C for 1 min, annealing at 58°C for
1 min. and extension at 72°C for 3 min. After the 35th cycle, a final
extension step was performed at 72°C for 10 min.
bSSCP gel electrophoresis after digestion with restriction enzyme.

Data analysis
The mtDNAs were assigned to the haplogroups according to theclassification proposed by Yao et al. (2002) and Kivisild et al.
(2002). Using their data and those obtained in this study, we classified the published mtDNA sequence data of other Japanese, Korean, and Chinese populations by motif search and near matchingin HVI and HVII region, as described in those studies, to compare
the frequency distributions of these haplogroups.

SpeI-SSCPb

cThe following primers were also used for sequencing in addition
to the PCR primers; L16194: 5’-ATGCTTACAAGCAAGTACAG-3’, H16420: 5’-TGATTTCACGGAGGAT-3’, L16381:
5’-TCAGATAGGGGTCCCTTGA-3’, H194: 5’-GTATGTTCGCCTGTAATATT-3’, and L155: 5’-TATTTATCGCACCTACGTTC-3’, L317: 5’-CTTCTGGCCACAGCACTTAA-3’.
dMigration of samples with mutation sites in parentheses differedslightly from the samples with no mutations. To detect such polymorphisms, careful inspection is required.

Results and discussion
Determination of sequences
We studied possible influences on sequencing profiles thatmight depend on the purification procedure of the PCR
products as described in the Materials and Methods section: re-amplification from the acrylamide gel (method 1),
purification from agarose gel (method 2), or direct purifi-

221

cation from the amplification mixture (method 3). Ambiguous electrophoretograms for 20–30 nucleotides near
the primers were sometimes obtained because of highbackground noise, especially in cases of direct purificationfrom the amplification mixture (method 3). Almost all of
the samples purified from agarose gels and all of the samples purified by re-amplification from acrylamide gelsgave satisfactory results with no background. When wecompared the electrophoretograms from the same samples
after purification from agarose gel and direct purificationfrom the amplification mixture, the former gave better
electrophoretograms with no background in the first 20–30
nucleotides. Because we used Centri-Sep to remove theremaining dye in all samples, ambiguous results for the
background in the first 20–30 nucleotides should be attributable to the purity of the original PCR products. Similarly,
sequencing of the PCR products of the complete 1,150 bp
control region using original PCR primers sometimes gave
electrophoretograms with high background in the first
10–30 nucleotides; however, semi-nested PCR sequencing
using internal primers almost always gave satisfactory results with no background in the first 10–30 nucleotides.
Length heteroplasmy
In HVI, the transition of the T to a C at position 16189
leads to the formation of a C-stretch, always associated
Table 2 Distributions of the sequence variations in the region
from 16180 to 16193 in HVI and from 303 to 315 in HVII
RegionSequence variations

Number observed

HVI

AAAA
AAAA
AAAA
AAAA
AAAA
AAAA
AAAA
AAAA
AAAA
AAAA
AAAA
AAAC
AAAC
AAAC
AACC
AACC
AACC

Anderson sequence
128
2
3
9
4
1
3
5
5
4
9
14
1
6
14
3

HVII

CCCCCCC---TCCCCCCCCC------TCCCCCC
CCCCCC----TCCCCCC
CCCCCCTC--TCCCCCC
CCCCCCC---TCCCCCC
CCCCCCCC--TCCCCCC
CCCCCCCCC-TCCCCCC
CCCCCCCCCCTCCCCCC
CCCC------CCCCCCC

CCCCCTCCCCCCCCCTCCCCCTCCCTCCCCTCCCCTCCCCCCCTCTCCCCCCCTCCCCCCCCCCCTCCCTCCCCCTCCTCCCCCCCTCCCCC
CCCCCCCCCCCCCCCCCCCCC
CCCCCCCCCCCCCCCCCCCCC
CCCCCCCCCCCC
CCCCCCCCCCC
CCCCCCCCCCCCCCCCCCC--

Anderson sequence
1
5
1
76
86
39
1
2

with pronounced heteroplasmy and was observed in 27%of the individuals from the Japanese population. The number of bases in the C-stretch was determined from theL-strand by the following rule: when the height of theC peak suddenly declined and the height of the corresponding background peak attained approximately 50%of the height of the C peak, the preceding C peak was considered to be the final peak of the C-stretch. The results ofthe sequence variations and their distributions in the region from 16180 to 16193 are shown in Table 2. Amongthe 54 samples having a C-stretch without T in the sequence, 85% had 2 or 3 As preceding the homopolymeric
C-tract (16180–16183). When the number of As was 4,
the C repeat was 10 or 11 bp. When the number of As was
3, the length of the C repeat was mostly 12 or 11 bp.
When the number of As was 2, the most common typehad 12 Cs, and the second most frequent type had 13 Cs.
Thus the overall length of the A+C repeat sequence wasusually 14 or 15 bp. It appears that the length of this repeat is under some selective constraint, as proposed by
Bendall and Sykes (1995), Howell and Smejkal (2000),
and Malik et al. (2002). Although we determined thenumber of Cs in the C-stretches from 16184 to 16193 using the abovementioned rule, the total number of bases in
the C-stretch was fixed as 10 for the actual comparison ofindividual sequence variations and for haplogrouping, as
has been done in other similar reports.
In HVII a heteroplasmic situation often occurs with an
insertion of Cs between positions 303 and 309. In thiscase, the length of the C-stretch was determined from theH-strands, regarding the highest T peak as the base at position 302. The patterns of sequences from 300 to 315 in
the Japanese population are shown in Table 2. A pronounced heteroplasmic situation was observed in the majority of the samples with more than 9 Cs between positions 303 and 309, in 76% of the samples with 8 Cs, andin 1 sample with 7 Cs. In the samples with 8 Cs without
pronounced heteroplasmy, low peaks corresponding toother groups of length variants were usually observed. Although these samples might actually be heteroplasmic,
this could also be an artifact that arises during the PCR.
Thus we could not clearly discriminate all the samples
with and without length heteroplasmy in this region. In
two samples (nos.25 and 191 in Table S1), the transitionof the T to a C at position 310 leads to the formation of a
C-stretch associated with a high level of length heteroplasmy. Both samples showed additional high peaks corresponding to the T peak at position 302 in the H-strand.
Judging only from electrophoretograms, the major haplotype may have 11 Cs, but haplotypes with 10 and 12 Cs
can also be present in high proportions in both samples.
Because these two haplotypes belong to different haplogroups, this mutation may have occurred independently.
It appears that the total number of Cs in this region may
also be under some selective constraint, similar to the
HVI C-stretch.

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Population data
We have determined the nucleotide sequences of the HVI
and HVII control regions from 211 persons who were unrelated in their maternal lineages (Table S1). In 375 nucleotides in HVI (16024–16398), 93 (24.8%) sites were variable and in 321 nucleotides in HVII (49–369), 52 (16%)
sites were variable. A total of 112 and 96 different haplotypes were observed in HVI and HVII, respectively, inwhich 24 haplotypes of HVI and 29 haplotypes of HVII
were shared by more than 1 individual. The most common
haplotypes of HVI and HVII were found in 22 (10.4%)
and 25 (11.8%) individuals, respectively. Among the combinations of HVI and HVII, 169 haplotypes were identified: 145 types were unique, and the most common type wasobserved in 9 individuals, 1 type in 5 individuals, 2 types
in 4 individuals, 4 types in 3 individuals, and 16 types in
2 individuals. Gene diversity calculated from our data using the equation h=n(1–Σxi2)/(n–1), (where n is sample size
and xi is the frequency of i-th mtDNA type) (Tajima 1989)
was 0.9765 for HVI, 0.9685 for HVII, and 0.9961 for bothregions combined. The probability of two randomly selectedindividuals from a population having identical mtDNA
types (P=Σxi2) was 2.8% for HVI, 3.6% for HVII, and
0.86% for both regions combined.

Fig. 2 SSCP gel electrophoresis of the PCR products, 10285–
10441. Ten types were observed. Each lane shows the type possessing the following mutations. Lane 1 10397, 10398, 10400,
lane 2 10345, 10398, 10400, lane 3 10398, 10400,10410, lane 410398, 10400, lane 5 10321, 10398, 10400, lane 6 no mutation,lane 7 10310, lane 8 10398, lane 9 10362, 10398, 10400, 10410,
lane 10 10373, 10389, 10400

Phylogenetic studies
Phylogenetic differentiation of mtDNA in the Japanese
population was investigated using sequence variation in
HVI and HVII, and specific coding region mutations. We
referred to the classification tree for the east Asian population proposed by Yao et al. (2002) and Kivisild et al.
(2002). Because mutations between 10305 and 10420
provide useful information for analyzing haplogroups in
Asian populations, we used SSCP analysis to detect thosemutations. SSCP electrophoresis of the PCR products
10285–10441, identified 10 different types in 211 individuals (Fig. 2). Each type had characteristic sequence differences, which are informative for defining the macrohaplogroups, M and N, and some of the other subhaplogroups.
Since it was not difficult to discriminate each SSCP band
pattern, this method is not time-consuming when analyzing large numbers of samples. In addition, the small PCR
product size is suitable for analyzing degraded DNA samples in forensic cases. Analysis of 3,010 and 5,178 mutations was also useful in determining haplogroups in theJapanese population, because haplogroup D was very common in the population, and some samples in the haplogroup G were difficult to discriminate from haplogroup D
by only control region sequences.
The sequence variation of HVI and HVII and those atother polymorphic sites are shown in Table S1. All of thepresent samples could be placed in macrohaplogroups M
or N. The present data suggest new subhaplogroups, G4a,G4b, and N9b. Altogether, we distinguished 33 haplogroups; 1 sample with a G* haplotype was not specified further.

In this study 12 individuals (5.7%) shared the 1622316325-16362-150 motif (1 lacked 150) and 4793 and4833 mutations. Representative samples shared additionalsites for the haplogroup G but lacked sites specific forother M haplogroups, including G2. Thus we designatedthis haplogroup as G4a. The control region motif of G4a
has also been found in other Japanese (Seo et al. 1998;
Imaizumi et al. 2002) and Korean samples (Lee et al.
1997; Pfeiffer et al. 1998), and in one Taiwanese Han(Tsai et al. 2001), but it was not found in Chinese Han
samples (Yao et al. 2002). Two individuals (0.9%) sharedthe 16184-16214-16362-200 motif and 7867 and 15323
mutations in addition to specific sites for the haplogroup
G except 4833, but lacked specific sites for other M haplogroups including G2. Because these individuals shared
the mutation 7867 with those in haplogroup G4a, we tentatively designated this haplogroup as G4b. The control
region motif of G4b was also found in one Korean sample(Pfeiffer et al. 1998). Another sample also shared coding
region mutations at positions specific for haplogroup G,
suggesting that this sequence belongs to a new subhaplogroup. However, because a similar sequence was foundin only one sample from Korea (# P226 of Lee et al. 1997)
whose coding region information had not been obtained,
we indicated this haplogroup as G*. Although 4 individuals did not share characteristic sites other than simple motifs of recurrent sites, 16189-16223 in the control region,
they shared 5147 and 10607 mutations in addition to a
specific site for haplogroup N. Because they lacked 14178
and 5231 mutations specific for N9a and Y, we designatedthis haplogroup as N9b. A similar haplotype was also found223

in four Japanese individuals reported by Imaizumi et al.
(2002), and one Korean (Lee et al. 1997), but was not foundin Chinese (Tsai et al. 2001; Yao et al. 2002). Amongthese new haplogroups, G4a was also found in Ainu, andN9b-like sequences were also found in Ainu and Ryukyuans (Horai et al. 1996).
Other findings in this study were as follows:
1. M10 haplotypes found in this study shared the 3172.1
mutation in addition to the 10646 mutation. This site ispossibly a specific mutation of this haplogroup.
2. The 16093-16188.1-16193.1-16362-16390-146-150-152182-217 motif was found in 4 individuals in haplogroup D5. Although this haplotype seems to belong to
a new haplogroup, it was found only in the samples
from Chiba prefecture. A similar HVI motif has beenfound in one sample from Japanese (Yoshii et al. 1995),
but so far has not yet been found in other Japanese(Seo et al. 1998; Imaizumi et al. 2002), Korean (Lee etal. 1997; Pfeiffer et al. 1998), or Chinese Han populations (Tsai et al. 2001; Yao et al. 2002; Kivisild et al.
2002).
3. Most of the D4a haplotypes observed in this study
shared the 10410 mutation, suggesting the presence ofa subclade of D4a in the Japanese population, as indicated by Yao et al. (2002).
4. In the D4 haplotype 4 individuals shared the 1622416245-16292-19362 motif, which also occurred in 5 out
of 502 Japanese individuals in other studies (Yoshii etal. 1995; Seo et al. 1998; Nishimaki et al. 1999; Imaizumi et al. 2002; Koyama et al. 2002), in 2 Koreans(Pfeiffer et al. 1998), in 1 Han Chinese (Yao et al. 2002),
and in 1 central Asian (Comas et al.1998). In the D4
haplotype 6 individuals shared the 16245-16362-191.1194-199-207 motif which also occurred in 3 Japaneseand 1 Japanese individual in other studies (Seo et al.
1998; Imaizumi et al. 2002), and in 3 Koreans (Lee etal. 1997; Pfeiffer et al. 1998), but not in Chinese Hans
(Tsai et al. 2001; Yao et al. 2002). This motif seems todefine subclades of D4, although no coding region information is available at the moment.
5. Most of the individuals in the B4b1 group shared the16284-199-202-207 motif in addition to the haplogroup specific motif 16136-16189-16217. This motifis also frequent in other Japanese populations (Seo etal. 1998; Imaizumi et al. 2002), but occurred in only
one Korean (Lee et al. 1997; Pfeiffer et al. 1998), in
one Chinese (Yao et al. 2002), and in two Korean individuals with similar sequences (Lee et al. 1997; Pfeiffer et al. 1998). Thus this haplotype tends to be relatively restricted to the Japanese population.
6. All of the F1b haplotypes observed in this study sharedthe 16129-16232-16249-16311-152 motif in addition
to the haplogroup-specific motif 16189-16304-249del.
The complete motif was common to almost all of theother reported Japanese populations. Identical or very
similar motifs were found in Koreans (Lee et al. 1997;
Pfeiffer et al. 1998) and in one Chinese (Yao et al. 2002).
The sequence in three Chinese individuals (Yao et al.
2002) was slightly different from that of the Japanese;thus the complete motif tends to be restricted to theJapanese or Korean population.
7. One individual bore 12705C suggesting R status andan 16189-16217 motif suggesting B4 but did not bearthe 9 bp deletion. Similar HVI motifs were found in one
Korean (Lee et al. 1997) and one Japanese (Imaizumi
et al. 2002), and slightly different motifs were found in
one Ainu, two Ryukyuans (Horai et al. 1996), and one
Korean (Lee et al. 1997). Because we had not yet specified the haplogroup using coding region information,
we tentatively classified this sample as R*.
Haplogroup frequencies were compared with those of otherJapanese, Korean, and Chinese populations (Table 3). The
sequences of FO19 in D4 (16129, 16223, 16294, 16362,
16152, 16195, 16309.1C) and of TK24 in D4a (16129,
16223, 16362, 16152, 16195) were both very similar, anddiscrimination of each haplogroup was very difficult only
by near matching with the previously established sequences in the control region. Thus D4 and D4a werecombined as D4* for comparison. From the study of Seo
et al. (1998) 11 samples were excluded from comparisons,as well as 2 samples of Imaizumi et al. (2002), 18 samplesof Lee et al. (1997), 2 samples of Pfeiffer et al. (1998),
and 3 samples of Tsai et al. (2001) because we could not
assign them to proper haplogroups. In the comparison under these circumstances, the most common haplogroup in
the Japanese population was D4*, which was found in
33–36% of the population. The Korean population had
slightly lower frequencies than the Japanese, and Chinese
had frequencies less than half of these values (12–14%).
The haplogroup with the second highest frequency in theJapanese population was M7a, which included 6–14% of
the population. The frequency of M7a in the Korean population was approximately 3%, and this haplogroup so far
has not been observed in Chinese. The M7b1 haplotype,which is of Chinese origin, was not found in this study.
The frequency of macrohaplogroup M in the Japanese
population was the highest (70–74%) among the three
population studied, that in the Korean population wasslightly lower (65–71%), and that in the Chinese population was the lowest (52–56%). Other features observed in
the Japanese and Korean population were a restricted distribution of A5, in addition to the haplogroups G4a andN9b, as mentioned above.
Consequently, Japanese, Koreans and Chinese have
similar haplogroups, but the distribution of frequencies issomewhat different. Several haplogroups appear to be restricted to the Japanese and Korean populations. Moreover, other haplotypes which might be restricted to Japanand Korea were also found. These results suggest that determination of clades of the mtDNA phylogeny may help
us to estimate geographic origin or phenotype (race) in
forensic cases. Although we estimated most of the haplogroups from the information in the control region, there
were sequences that were difficult or ambiguous to assign
to specific haplogroups only by control region information. It is time-consuming to examine coding region information to determine phylogenetic clades. However, whenmuch more data are compiled, mtDNA haplogrouping

224
Table 3 Comparison of the estimated frequencies of mtDNA haplogroups in the Jaoanese, Korean and Chinese populations
Haplogroup

M7a1*
M7b1
M7b2
M7b
M7c1a
M7c1b
M7c1c
M7c1
M7c
M7
M8a
C
CZ
Z
M9*
M10
M
G2
G2a
G4a
G4b
G*
D5a
D5
D4b
D4*
D
A5
A
N9a
N9b
N*
Y
Y1
B4a
B4b1
B4*
B5a
B5b
B5
B
R9a
F1a
F1b
F1c
F1
F2a
F2
F
R+R10+R*
T1
HV

Estimated frequency (%)
Japanese-1(n=211)

Japanese-2(n=89)

Japanese-3(n=160)

Korean-1(n=288)

Korean-2(n=58)

10.0

13.5

6.3

3.4

2.8

5.6

5.6

2.8
0.7
2.8

0.5

1.1

0.6

1.0
2.1

5.2

5.2

0.7
0.6
0.5
0.9
1.4
0.9
1.4
0.5
2.8
5.7
0.9
0.5
0.5
4.7
0.5
35.5
5.2
3.8
1.9
1.9

1.1
2.2

1.1
2.2
1.1
1.1
1.1
4.5

1.1
3.4
33.7
1.1
3.4
2.2

0.6
1.3

3.1
1.3
1.9
0.6
4.4
3.1

1.9
3.1
3.8
32.5
0.6
5.0
1.9
3.8
2.5

0.5
0.5
6.6
0.5
0.9
1.9
0.5

2.2
3.4
1.1
1.1
3.4

0.9
4.3

3.4
3.4

0.6
1.3
1.3
3.8
1.9

1.7
3.5

1.7
8.6

3.4
3.4
1.7

27.9

2.8
6.9
3.5
2.1
3.1
1.4
2.1
1.0
4.2
1.4
0.3

1.0
0.3

0.6

1.7
0.3
0.7
1.4
0.7

Japanese-1 present study
Japanese-2 Seo et al. (1998)
Japanese-3 Imaizumi et al. (2002)
Korean-1 Lee et al. (1997)
Korean-2 Pfeiffer et al. (1998)
Taiwanese Tsai et al. (2000)
Chinese Yao et al. (2002)
M7a1 and M7a, M9 and M9a, D4 and D4a, N and N9, and B4 and B4cwere combined and shown as M7a*, M9*, D4*, N*, and B4*, respectively.
For comparison with the database, 9 samples from Seo et al. (1998) (Nos.

5.2

0.7
1.0
1.4
31.9

1.9
4.4

2.2
0.5

1.0
1.0
1.7
0.7
2.1
3.1
4.2
0.7

Taiwanese(n=152)Chinese(n=263)

2.6
0.7
3.9
0.7

3.4
1.5
0.8
0.8

1.3
1.3
1.3
1.3
3.9
2.6
0.7
1.3
1.3
2.6
2.6
2.6
1.3
1.3

1.5
1.1
4.9
3.0
1.9
2.7
1.9
4.2
0.4
3.0

2.6
5.3
0.7
12.5
1.3

3.8
2.7
0.8
14.2
0.4

1.7
3.4
1.7

6.6
2.6

7.2
4.6

1.7

1.3

0.4
1.1

3.4

1.7
5.2
3.4
3.4

3.4
1.7
1.7

7.9
3.3
2.0
1.3
0.7
1.3
0.7
2.0
5.9
2.0
0.7
0.7
1.3
2.0
2.0

4.6
3.4
2.3
2.3
1.5
0.4
1.5
1.1
5.7
3.0
1.1
1.9
2.3
0.8
1.1
0.4
0.4

24, 25, 31, 46, 48, 59, 63, 72, 89), 1 sample from Imaizumi et al. (2002)(JP003), and 3 samples from Lee et al. (1997) (P29, P33, H117) were excluded, because they seemed to be artefactual recombinations. Transitions
16242C/T-16248C/T and the transversion 16239C/G in the database of Seo
et al. (1998) were omitted from variable positions (Bandelt et al. 2001), because we could not find any such mutations in our samples. The 11 samples and 2 samples of Koreans which bore very similar sequences to thoseof D4 and G3 but lacked the 16362 mutation were regarded as D4 and G3
types, respectively, 7 samples with the motif 16223-16362-150 in Koreans
were regarded as D, althrough these sequences were similar to those of D5.

225

will become more useful in forensic science. We are also
confident that making efforts to minimize errors in the
database of the control region will increase the usefulnessof mtDNA polymorphism in any research fields such as
anthropology, forensic science, and medical genetics.
Acknowledgements This study was supported by the Grant-in
Aid for Scientific Research (B) (14370701) from the Ministry of
Education, Science, Sports and Culture of Japan.

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