Gene 316 (2003) 111 – 118
www.elsevier.com/locate/gene

The diversity of bovine MHC class II DRB3 genes in Japanese Black,
Japanese Shorthorn, Jersey and Holstein cattle in Japan
S. Takeshima a, N. Saitou b, M. Morita c, H. Inoko d, Y. Aida a,*a

Retrovirus Research Unit, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan
Division of Population Genetics, National Institute of Genetics, Mishima 411-8540, JapancLivestock Improvement Association of Japan, 316 Kanamarumachi, Maebashi 371-0121, Japand
Department of Immunotransplantation, Tokai University School of Medicine, Seibodai, Isehara 259-1100, Japanb

Received 16 December 2002; received in revised form 13 May 2003; accepted 6 June 2003
Received by T. Gojobori

Abstract
We sequenced exon 2 of the major histocompatibility complex (MHC) class II DRB3 gene from 471 individuals in four different Japanese
populations of cattle (201 Japanese Black, 101 Holstein, 100 Japanese Shorthorn, and 69 Jersey cattle) using a new method for sequencebased typing (SBT). We identified the 34 previously reported alleles and four novel alleles. These alleles were 80.0 – 100.0% identical at thenucleotide level and 77.9 – 100.0% identical at the amino acid level to the bovine MHC (BoLA)-DRB3 cDNA clone NR1. Among the 38
alleles, eight alleles were found in only one breed in this study. However, these alleles did not form specific clusters on a phylogenetic tree of236-base pairs (bp) nucleotide sequences. Furthermore, these breeds exhibited similar variations with respect to average frequencies ofnucleotides and amino acids, as well as synonymous and non-synonymous substitutions, in all pairwise comparisons of the alleles found in
this study. By contrast, analysis of the frequencies of the various BoLA-DRB3 alleles in each breed indicated that DRB3*1101 was the mostfrequent allele in Holstein cattle (16.8%), DRB3*4501 was the most frequent allele in Jersey cattle (18.1%), DRB3*1201 was the mostfrequent allele in Japanese Shorthorn cattle (16.0%) and DRB3*1001 was the most frequent allele in Japanese Black cattle (17.4%),
indicating that the frequencies of alleles were differed in each breed. In addition, a population tree based on the frequency of BoLA-DRB3
alleles in each breed suggested that Holstein and Japanese Black cattle were the most closely related, and that Jersey cattle were moredifferent from both these breeds than Japanese Shorthorns.
D 2003 Elsevier B.V. All rights reserved.
Keywords: BoLA-DRB3 allele; Sequence-based typing; Allele frequency; Population tree; Cattle breed; Major histocompatibility complex

1. Introduction
The major histocompatibility complex (MHC) class I andclass II molecules exhibit an extraordinarily high degree ofgenetic polymorphism compared to many vertebrate species(Kennedy et al., 2002; Otting et al., 2000; Seddon andEllegren, 2002; Imanishi et al., 1992; Yuhki and O’Brien,

Abbreviations: MHC, major histocompatibility complex; SBT, sequence based typing; BoLA, bovine MHC; bp, base pair(s); PCR,
polymerase chain reaction; SSP, sequence specific primer; TAE, Tris
acetate EDTA; HLA, human MHC; DLA, dog MHC.
* Corresponding author. Tel.: +81-48-462-4408; fax: +81-48-4624399.
E-mail address: aida@postman.riken.go.jp (Y. Aida).
0378-1119/$ - see front matter D 2003 Elsevier B.V. All rights reserved.
doi:10.1016/S0378-1119(03)00744-3

1997). The MHC polymorphism occurs predominantly atresidues involved in peptide binding (Brown et al., 1993),
and there is compelling evidence that the polymorphism ismaintained by some form of balanced selection (Hughes andNei, 1989). The essential role of MHC molecules in the
immunological recognition of foreign peptide antigens
implies that the cause of such selection is related to theeffects of MHC polymorphism on host defenses against
pathogens.
The MHC variability in natural populations is of greatinterest to evolutionary biologists because of the typicallyhigh levels of polymorphism. Consequently, representative
species of several mammalian orders, including Artiodactyla, Carnivora, Cetacea, Primates, and Rodentia have beencharacterized with respect to MHC allelic diversity. Some

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S. Takeshima et al. / Gene 316 (2003) 111–118

species exhibit extremely high levels of MHC polymorphism (Mikko and Andersson, 1995; Yuhki and O’Brien,1997), whereas, others are characterized by very low levels(Ellegren et al., 1993). Moreover, even in species that arecharacterized by high levels of MHC polymorphism, some
populations are monomorphic or oligomorphic. The transspecies polymorphism hypothesis seems to provide a
universal explanation for differences in levels of MHC
variability and other features of the MHC in natural
populations (Klein et al., 1993). Variability within andamong populations of a certain species is directly related tothe interplay between effective population size, time ofdivergence, and the intensity of selective pressure at aparticular locus. The gene frequencies of various humanpopulations were determined for human MHC (HLA)
genetic polymorphism (Imanishi et al., 1992) in order toassist in understanding the origins of given populations.
Similarly, many studies have indicates that differences
exist between breeds of cattle and dogs with regard to
frequencies of MHC class II alleles. In cattle, interpretations of polymerase chain reaction – restriction fragment
length polymorphism (PCR-RFLP) and sequencing studies
indicate that there are significant differences in allelicfrequencies of BoLA-DRB3.2 in Jersey (Gilliespie et al.,1999), Holstein (Dietz et al., 1997), Argentine creole(Giovambattista et al., 2001), Japanese Shorthorn (Takeshima et al., 2002), and Brazilian dairy Gir cattle (da Mota
et al., 2002). Moreover, Kennedy et al. (2002) genotyped
dog MHC (DLA) class II alleles of over 80 breeds indomestic dogs by polymerase chain reaction-sequencespecific oligonucleotide probe (PCR-SSOP) and showed
considerable variation in the allelic frequencies among
breeds. Thus, the frequencies of alleles of MHC genes indifferent populations allow the differentiation and reconstruction of genetic distances among populations, providing a molecular basis for determination of the possible
common origin of populations.
Around the second century A.D., cattle migrated from
North China via the Korean peninsula to Japan. This
movement of cattle was accompanied by the introductionof rice cultivation. Both genetic (Namikawa, 1980) andmorphological (Ogawa et al., 1989) studies have demonstrated that native Japanese cattle belong to the species Bos
taurus and are representative of the ‘‘Turano –Mongolia’’
type (Felius, 1995). By contrast, at the end of the 19th
century, several European breeds of cattle were introduced
into Japan for enhancement of native breeds (Obata et al.,1996). Japanese Black, which is the main breed of beef
cattle in Japan, has been less influenced by European breeds
than other breeds in Japan and other countries. Japanese
Shorthorns are derived from crosses between Shorthorn
cattle and Nanbu cattle and are raised predominantly inthe northern part of Japan. Jersey cattle were first bred on asmall island and this breed has existed in effective genetic
isolation, because of strict breeding practices, for about 200
years (French et al., 1996).

There is one predominant class II DRB locus in cattle,
namely, bovine MHC (BoLA)-DRB3 (Lewin, 1996), andthis locus is also the most polymorphic class II locus incattle. Sixty-four BoLA-DRB3.2 alleles in exon 2 (http://
www.projects.roslin.ac.uk/bola/drb3pcr.html; Gilliespie etal., 1999) were distinguished by the PCR-RFLP method
described by van Eijk et al. (1992). However, the utilityof such typing is limited in the case of alleles that are
split into several further alleles at the nucleotide level.
Indeed, 89 alleles have been published in various breedsof cattle by sequencing of cloned genomic DNA, cDNAor cloned products of PCR (Ammer et al., 1992; Aida etal., 1995; Russell et al., 1997, 2000; Maillard et al.,1999, 2001; Takeshima et al., 2001, 2002). Recently, wedeveloped a more precise method for sequence-based
typing (SBT), which allows the identification of specific
BoLA-DRB3 alleles in large numbers of animals (Takeshima et al., 2001). The present study, using our SBT
methods, was designed to determine the nucleotide
sequences of exon 2 of the BoLA-DRB3 alleles of a totalof 471 individuals belonging to four distinct breeds ofcattle, namely, Japanese Black, Japanese Shorthorn, Holstein and Jersey. The frequencies of alleles and thephylogenetic relationships among BoLA-DRB3 alleles are
discussed.

2. Materials and methods
2.1. Animals and extraction of DNA
Samples from a total of 471 individuals, obtained fromthe Livestock Improvement Association of Japan, were
examined for the distribution of BoLA-DRB3 alleles: 201
head of Japanese Black cattle; 100 head of Japanese
Shorthorn cattle; 101 head of Holstein cattle; and 69 headof Jersey cattle. High-molecular-weight DNA was preparedfrom whole blood with 10% sodium dodecyl sulfate (SDS)
and a mixture of phenol and chloroform (Takeshima et al.,2001).
2.2. Typing by PCR with sequence-specific primers (PCRSSP) and nucleotide sequencing
To avoid preferential allele amplification in heterozygous individuals that would presumably occur becauseof mismatches in the regions of primer annealing or
secondary structure, the first round of amplification by
PCR with the locus-specific primers ERB3N and HL031
and subsequent amplification by PCR-SSP with distinct,
group-specific 3Vprimers (Sp1 through Sp8) and a locusspecific 5V primer, DRB3B, were performed as described
previously. In addition, we used the locus-specific 3V
primer DRB3ALL, which allowed amplification of thealleles in all previously identified groups (Takeshima
et al., 2001).

S. Takeshima et al. / Gene 316 (2003) 111–118

After PCR-SSP, amplified DNAs in 5 Al of the reaction
mixture were fractionated by electrophoresis on a 2%
agarose/Tris acetate EDTA (TAE) gel. The gel was stainedwith ethidium bromide and bands of DNA were visualized
under UV light.
DNA sequencing of each product of PCR-SSP was
performed by the dideoxy chain termination method witha BigDyek Terminator Cycle Sequencing Ready Reaction
Kit and an automated sequencer ABI PRISMk 310 Genetic
Analyzer (Applied Biosystems, Foster City, CA) or with the
CEQk 2000 DNA analysis system (Beckman Coulter,
Fullerton, CA).
2.3. Sequence analysis
General sequence analysis was performed using theLasergene program (DNASTAR, Madison, WI). Nucleotide
sequences were compared with sequences in the GenEMBL
database using the BLAST algorithm. New alleles werenamed by the BoLA nomenclature committee.
2.4. Statistical analysis
Allele frequencies ( f ) were obtained by direct counting.
The observed frequencies of heterozygotes (H observed), given
in Table 2, were obtained directly by dividing the number ofheterozygous individuals by the total number of individuals.
The expected frequencies of heterozygote (H expected) were
those expected from the Hardy– Weinberg equilibrium, as
calculated using Arlequin ver. 2.000 (Schneider et al.,2000).
2.5. Calculation of genetic distances and construction of
population tree
Pairwise genetic distances were estimated on the bases ofKimura’s two-parameter model. The gene tree was constructed from a distance matrix that was based on NeighborJoining method of Saitou and Nei (1987). To test thesignificance of the branches, 1000 bootstrap replicate calculations were performed. An analysis of breed diversity,
based on allele frequency, was performed using the com-

Fig. 1. Neighbor-Joining tree constructed from the 236-bp nucleotide
sequences of the h1 domain coding-regions of BoLA-DRB3 alleles derived
from four distinct breeds of cattle; Holstein (H); Japanese Shorthorn (S);Japanese Black (B); and Jersey (J). The tree is based on Kimura’s twoparameter distances. Numbers are bootstrap percentages that support eachnode. Bootstrapping was performed with 1000 replicates to assess thereliability of individual branches. Alleles that were only found in one breedin this study are shown in boldface. New alleles are underlined. Letters inparentheses indicate the breeds in which the each allele was found. The
nucleotide sequences reported in this paper have been submitted to theInternational Nucleotide Sequence Database and have been assigned
accession numbers AB033384 through AB033402, AB033404, AB033405,
AB048732 through 048735, AB048814 through 048816, AB049442,
AB053167, AB060152, AB060153 and Z82996.

113

puter program gd5.exe. This program generates estimates of
Nei’s (1972) genetic distances and the population tree wasconstructed by the UPGMA method. Pairwise comparisonsof nucleotide and amino acid substitutions between alleles
were conducted according to the number of differences. The
numbers of non-synonymous and synonymous nucleotide
substitutions per site were estimated for each pair as

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S. Takeshima et al. / Gene 316 (2003) 111–118

Fig. 2. Alignment of the predicted amino acid sequences of the h1 domains encoded by four new BoLA-DRB3 alleles (accession numbers are AB053167 for
BoLA-DRB3*0504, AB033396 for BoLA-DRB3*1302, Z82996 for BoLA-DRB3*3701 and AB033404 for BoLA-DRB3*4002) derived from 471 animals thatbelonged to four distinct cattle breeds, Holstein, Japanese Shorthorn, Japanese Black and Jersey. The numbering refers to positions of amino acids in the mature
protein. Amino acid residues identical to those encoded by the BoLA-DRB3 cDNA clone NR-1 are indicated by dots (Aida et al., 1995). Homology scores also
refer to this cDNA clone.

described by Nei and Gojobori (1986) using the Jukes –Cantor’s formula. Analyses of phylogeny and molecular
evolution were performed using MEGA version 2.1 (Kumar
et al., 2001).

3. Results and discussion
3.1. Identification and characterization of BoLA-DRB3
alleles obtained from four distinct breeds of cattle
The genotypes of 471 individuals from four different
Japanese populations of cattle were determined for exon 2of the BoLA-DRB3 allele by PCR-SBT typing (Fig. 1). The
34 previously reported alleles and four novel alleles wereidentified: 20 previously reported alleles were obtained from
100 Japanese Shorthorns; 14, including one new allele, from69 Jersey cattle; 18 from 101 Holsteins; and 24, including
three new alleles, from 201 Japanese Blacks.
These 38 DRB3 alleles were 80.0– 100.0% identical at
the nucleotide level and 77.9 –100% identical at the aminoacid level to the BoLA-DRB3 cDNA clone NR1 (Aida et al.,1995). Nucleotide sequences and deduced amino acidsequences of four new alleles are shown in Fig. 2. Thesefour new alleles were assigned allele names by the BoLA
nomenclature committee (http://www.projects.roslin.ac.uk/
bola/bolahome.html). One new allele was designated
DRB3*1302 and differed from DRB3*1301 at positions 30
and 37. Another new allele was designated DRB3*0504 anddiffered from BoLA-DRB3*0501 at position 86. The third

Table 1Frequencies of amino acid and nucleotide substitutions in pairwise comparisons of BoLA-DRB3 allelesa
Comparisonb

HO vs. HO (N = 153)
JS vs. JS (N = 190)
JE vs. JE (N = 91)
JB vs. JB (N = 276)
BoLA-DRB3 vs.
-DRB3 (N = 4371)
Bovine TNF a vs.
TNF a (N = 21)aAmino acid substitutions

Nucleotide substitutions

Non-synonymous substitutionsc

Synonymous substitutionsc

Mean

Range

Mean

Range

Mean

Range

Mean

Range

0.18
0.17
0.18
0.18
0.17

0.04 – 0.27
0.03 – 0.26
0.06 – 0.24
0.01 – 0.27
0.00 – 0.29

0.08
0.09
0.08
0.09
0.08

0.02 – 0.13
0.05 – 0.18
0.03 – 0.12
0.01 – 0.13
0.00 – 0.14

0.11
0.11
0.11
0.11
0.11

0.04 – 0.17
0.02 – 0.17
0.05 – 0.16
0.01 – 0.19
0.00 – 0.20

0.04
0.05
0.05
0.05
0.04

0 – 0.12
0 – 0.12
0 – 0.09
0 – 0.12
0 – 0.15

0.011

0 – 0.024

0.007

0.002 – 0.014

0.005

0 – 0.011

0.13

0 – 0.033

The analysis involved codons 9 through 86 of h1 domain.
N, Number of pairwise comparisons of BoLA-DRB3 alleles from four distinct breeds of cattle, namely, Holstein (HO); Japanese Shorthorn (JS); Jersey(JE); and Japanese Black (JB); four new alleles found in this study and 89 BoLA-DRB3 alleles had been previously published and 236 HLA-DRB1 alleles(http://www.authonynolan.org.uk/HIG/index.html).
cRates of non-synonymous and synonymous substitutions were calculated by method of Nei and Gojobori (1986).
b

S. Takeshima et al. / Gene 316 (2003) 111–118

new allele, DRB3*4002, differed from DRB3*4001 at
positions 12, 26, 30 and 38. The remaining new allele,
DRB3*3701, differed from DRB3*2501 at positions 34, 39,
57, 59 and 60.
3.2. A Neighbor-Joining tree of BoLA-DRB3 alleles
obtained from the four distinct breeds of cattle
We generated a Neighbor-Joining tree using the 236-basepairs (bp) nucleotide sequences of the various h1 domains
found in the four different breeds (Fig. 1). According to thistree, there were no significant differences among the allelesof all four breeds, including the four new alleles and theeight alleles that were not found in other breeds. The lowbootstrap values indicated that there were no significant
branches on this tree. Thus, a Neighbor-Joining tree indicates that BoLA-DRB3 sequences isolated from one breed
were shared by other breeds.
3.3. Frequencies of amino acid and nucleotide substitutions
for all pairwise comparisons of BoLA-DRB3 alleles
obtained from the four distinct breeds of cattle
We examined the evolution of polymorphism of the
BoLA-DRB3 gene by comparing the amino acid and nucleotide substitutions for all pairs of alleles in the four different
breeds, namely, the 20 alleles in Japanese Shorthorns, the 14
alleles in Jersey cattle, the 18 alleles in Holsteins and the 24
alleles in Japanese Blacks (Table 1). The cattle populations
each showed similar mean numbers of amino acid substitutions per site (average 0.17– 0.18; range 0.01 –0.27). Likewise, all pairwise comparisons within the 4 new alleles
found in this study and the 89 DRB3 alleles previously
published averaged 0.17 (range 0.00 –0.29). This result was
similar to the mean of amino acid substitutions in pairwise
comparisons of the 14 BoLA-DRB3 alleles analyzed by
Sigurdardottir et al. (1991). In such a comparison ofpolymorphic positions exclusively, there were no significant
differences in terms of the mean and the range for the aminoacid substitutions in the four different breeds.
Next, we calculated rates of synonymous and non-synonymous nucleotide substitution rates in all pairwise comparisons of all alleles found in the four different breeds(Table 1). We also calculated these values for the gene forbovine TNF-a as a control. There were no significant
differences in terms of the mean and ranges for silent
substitutions in the four different breeds. Moreover, values
for non-synonymous substitutions were greater than thosefor synonymous substitutions in BoLA-DRB3, suggestingthat there is selection for genetic diversity in BoLA-DRB3
exon 2. These findings showed the same tendency as
previous results by Sigurdardottir et al. (1991). By contrast,in the gene for TNF-a, values for synonymous substitutions
were greater than those for non-synonymous substitutions.
Thus, all pairwise comparisons of the alleles found in thisstudy indicate that there is no significant difference in the115

levels of polymorphism of BoLA-DRB3 among the four
different breeds. Therefore, these results may suggest thatsome alleles have been maintained intact since the divergence of different breeds. Indeed, the phylogenetic tree
constructed from nucleotide sequences of DRB3 shows that
there is no typical clade for the four different breeds (Fig. 1).
3.4. Distribution of BoLA-DRB3 alleles in the four breeds ofcattleTo differentiate between the allelic variations in the four
distinct breeds, we determined the frequencies of BoLATable 2
Frequencies of BoLA-DRB3 alleles and heterozygosities (H) for the cattle
breeds studied
DRB3
alleleHolsteina(N = 101)(allele no.
202)

Japanese
Shorthorna(N = 100)(allele no.
200)

Jerseya(N = 69)(allele no.
138)

Japanese
Blacka(N = 201)(allele no.
402)

0101
0201
0301
0501
0502
0503
0504
0601
0701
0801
0901
0902
1001
1101
1103
1201
1301
1302
14011
1501
1601
1701
1801
20012
2002
2006
2201
2502
2601
2703
2710
2801
3202
3401
3701
4002
4401
4501
H observed
H expected

11.4
5.5
0.5
–
–
–
–
4.5
1.0
–
1.0
5.9
5.9
16.8b
–
14.4
–
–
5.0
13.4
3.5
1.0
–
1.0
–
–
0.5
–
–
8.4
–
–
0.5
–
–
–
–
–
92.1
90.1 F 0.7

7.5
7.5
14.5
6.5
–
0.5
–
–
0.5
12.0
1.0
4.5
–
8.0
–
16.0b
1.0
–
5.0
–
–
–
3.5
3.5
3.5
–
–
–
0.5
1.0
0.5
–
–
3.0
–
–
–
–
92.0
91.2 F 0.7

–
16.7
2.2
–
–
–
–
–
6.5
5.1
–
–
–
2.2
–
0.7
–
–
–
–
2.9
–
–
–
–
14.5
–
11.6
–
0.7
–
1.4
–
–
10.1
–
7.2
18.1b
91.3
88.7 F 1.0

3.5
4.5
–
0.5
3.0
2.5
0.5
–
4.7
1.0
1.7
7.2
17.4b
11.0
1.2
8.5
–
5.0
2.0
7.7
12.9
–
–
1.2
–
–
–
–
–
1.7
–
–
–
0.2
–
0.8
1.2
–
90.5
91.4 F 0.5abN, number of typed unrelated individuals. H, heterozygosity rate.
The most frequent alleles in each breed are given in boldface.

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S. Takeshima et al. / Gene 316 (2003) 111–118

DRB3 alleles in each breed and compared them with thosein other populations (Table 2). All breeds examinedexhibited extremely high diversity with respect to DRB3,
with heterozygosity between 90.5% and 94.1%, and thesevalues were close to the expected heterozygosity of 88.7–
91.4%. The DRB3*1101 allele was the most frequent in
Holsteins (16.8%) and was detected at moderate frequencies
in Japanese Shorthorns (8.0%) and Japanese Blacks(11.0%). Among 24 alleles, DRB3*1001 was the mostfrequent allele in Japanese Blacks (17.4%); it was infrequentin Holsteins (5.9%) and absent in Jersey cattle and Japanese
Shorthorns. In addition, three alleles (DRB3*0504, *1302
and *4002) were found only in Japanese Blacks, were also
new alleles. In the 100 Japanese Shorthorns examined,
DRB3*1201 and DRB3*0301 were the most common andsecond most common alleles with frequencies of 16.0% and14.5%, respectively. Within the 69 Jersey cattle, theDRB3*4501 and DRB3*0201 alleles were the most common, with frequencies of 18.1% and 16.7%, respectively,
and DRB*4501 was not found at all in the other breeds.
Similarly, three alleles (DRB3*2006 and *2502) were also
detected only in Jersey cattle, as indicated in Fig. 1. In
addition, DRB3*3701 was found only in Jersey cattle, wasalso new alleles (Fig. 1).
The frequency of alleles we determined in this study
generally agrees with other published research. The majorityof the more abundantly (with gene frequencies higher than5%) detected alleles in Holsteins (alleles 0101, 0201, 0902,
1001, 1101, 1201, 14011, 1501, 2703) and in Jerseys (0201,
0701, 0801, 2006, 2502, 3701, 4401, 4501) in this study arein concordance with the data previously published in otherpopulation of these breeds (e.g. Dietz et al., 1997; Sharif etal., 1998; Gilliespie et al., 1999). However, similar small
differences in gene frequencies were observed among theJapanese local populations and other populations of Holsteins and Jerseys.
Our investigation clearly suggests that the remarkably
dissimilar patterns of distribution of BoLA-DRB3 alleles inthe different breeds might be the result of differential
selection after the separation of the major population groupsof cattle. Moreover, it appears that BoLA-DRB3 sequences
isolated from one breed are shared by other breeds, suggesting that these allelic lineages might be derived from common ancestral alleles that existed prior to the divergence of
these breeds.
Comparison of the frequencies of BoLA-DRB3 alleles inbreeds shows that four different breeds exhibit similarlyhigh levels of heterozygosities. These levels are similar to
those reported for Holstein and Argentinean Creole cattle(Giovambattista et al., 2001). Moreover, all pairwise comparisons of the BoLA-DRB3 alleles suggest that there is no
significant difference in the levels of polymorphism of
BoLA-DRB3 alleles among the four different breeds andvalues for non-synonymous substitutions were greater thanthose for synonymous substitutions in BoLA-DRB3 in all
four different breeds. These results provide evidence for the
existence of over-dominant selection in the evolution of
BoLA-DRB3, as in the case of the HLA-DRB1 (Hughes andNei, 1989).
The degrees of genetic variation at the DRB1 loci inhuman and dog were measured by their heterozygosity. It
appeared that there are significant differences in heterozygosities at the HLA-DRB1 among various human populations, although values of heterozygosities of these
populations tend to be similar to those of cattle (Mizuki etal., 1997). By contrast, the level of heterozygosities at DLADRB1 was very low: 59.5%, and lower than those of cattle(Kennedy et al., 2002).

Fig. 3. Rooted UPGMA tree (A) constructed from Nei’s genetic distances (B), which were based on the allele frequencies of alleles of the BoLA-DRB3 gene in
the four populations studied.

S. Takeshima et al. / Gene 316 (2003) 111–118

3.5. Population tree based on frequencies of BoLA-DRB3
alleles
Putative evolutionary relationships among different populations can be determined from the genetic distances
derived from frequencies of alleles (Mizuki et al., 1997).
We constructed a population tree that was based on thefrequencies of BoLA-DRB3 alleles using the UPGMA
method (Fig. 3A). The smallest genetic distances for
DRB3 alleles were those between Holsteins and JapaneseBlacks (0.2802) (Fig. 3B). The genetic distance between
Jersey cattle and the other three breeds was large and Jerseys
were clustered on a different branch from the other three
breeds. Collectively, the results suggest that the Holsteinand Japanese Black breeds were the closest to each other,with Jersey being farther from these two breeds thanJapanese Shorthorn.
The evolution of cattle breeds has been described on thebasis of variations in mitochondrial DNA using the genetic
distance based on nucleotide substitution to construct theevolutionary tree. Although such analysis showed thatbreeds of B. taurus and Bos indicus can be clearly differentiated (Loftus et al., 1994), differences in the evolution of
the various breeds were less clear. Allelic distributions of
the microsatellite markers also have been analyzed with theconstruction of a population tree (MacHugh et al., 1997).
On this tree, Simmental and Holstein were closest, andJersey was farther from these breeds than Hereford. In thepresent study, we demonstrated the genetic contribution offour major breeds in Japan by constructing a population treeof four cattle breeds using their allele frequency distributions. Ours is the first tree to use the frequency of BoLA
alleles. The introduction of foreign breeds of cattle (Brown
Swiss, Shorthorn, Devon, Ayrshire, Simmental and Holstein) for crossbreeding with native cattle in Japan was
encouraged by the Japanese government after the Meiji
restoration in 1867. Moreover, part of the Holstein breedin Japan are crosses between Japanese native cattle andHolstein breed cattle. Our tree suggests that the JapaneseBlack has a great genetic contribution from Holstein cattle
which breed was used to upgrade the native Japanese cattle.
By contrast, although the Jersey breed was also the European breed, its genetic contribution was very limited withrespect to the Japanese Black and Japanese Shorthorn
breeds. It is suggested that the Japanese breeds were moresimilar to each other than the diversity of European breeds
with respect to MHC distribution. Our present finding that
Holstein and Japanese Black breeds may be more closely
related to each other than to Jerseys supports the results of
microsatellite DNA studies by MacHugh et al. (1997).
Cattle are grouped into two species, B. taurus and B.
indicus, and BoLA-DRB3 sequences are shared by the twogroups. Japanese Black, Holstein, Japanese Shorthorn, andJersey cattle analyzed in this study belong to B. taurus.
Other studies describe that B. indicus has a quite different
frequency compared with B. taurus and have a lot of alleles

117

that are not found in B. taurus (Mikko and Andersson, 1995;
Maillard et al., 1999; da Mota et al., 2002). These results
indicate that many more alleles remain to be discovered.
Therefore, further sequencing data from B. indicus areneeded to characterize the variability of bovine MHC.

Acknowledgements
This work was supported by Grants-in-Aid for Scientific
Research (A) and (B) from the Japan Society for the
Promotion of Science (JSPS), and by a special grant for thepromotion of research from RIKEN.

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