Biochemical Genetics, Vol. 27, Nos. 7/8, 1989

Mitochondrial DNA Polymorphism in Native
Philippine Cattle Based on Restriction Endonuclease
Cleavage Patterns
Tomomasa Watanabe, 1'2 Joseph S. Masangkay, 3 Shigeharu Wakana, 4Naruya Saitou, 5 and Takeshi Tomita 6
Received 24 Oct. 1988--Final 4 May 1989

An analysis of patterns of cleavage of mtDNA by restriction endonucleases
was performed for nine individuals from the Philippine population of native
cattle. MtDNA polymorphisms were detected in the restriction patterns
generated by the following six enzymes, B a m H I , BglII, EcoRV, HindlII,
PstL and S c a L The restriction patterns showing polymorphisms were distributed nonrandomly among the nine individuals examined from the Philippine
population of native cattle, indicating the existence of two separate types of
mtDNA. These two types of mtDNA are very different from each other, at thelevel of subspecies. Since the native Philippine cattle are considered torepresent an admixture of European and Indian cattle, the two types of
mtDNA must be derived from the mtDNAs of both varieties. The polymorphic sites in mtDNA have been located on a restriction map, and thenucleotide substitutions at some of the sites have also been estimated.
KEY WORDS: cattle; mitochondrial D N A ; restriction endonuclease; Philippines.

INTRODUCTION
M i t o c h o n d r i a l D N A ( m t D N A ) p o l y m o r p h i s m s based on an analysis ofrestriction endonuclease digests have allowed us to clarify the genetic relationt Department of Biochemistry, Institute for Developmental Research, Aichi Prefecture Colony,
Kamiyacho, Kasugai, Aichi 480-03, Japan.
2 To whom correspondence should be addressed.
3 College of Veterinary Medicine, University of the Philippines, Los Banos, Laguna 3720,
Philippines.
4 Central Institute for Experimental Animals, Nogawa, Miyamaeku, Kawasaki 211, Japan.
5 Laboratory of Anthropology, Faculty of Science, Tokyo University, Hongo, Tokyo 113, Japan.
6 Laboratory of Animal Genetics, Faculty of Agriculture, Nagoya University, Furocho, Chikusa,
Nagoya 464, Japan.
431
0006-2928/89/0800-0431506.00/0© 1989PlenumPublishingCorporation

432

Watanabe, Masangkay, Wakana, Saitou, and Tomita

ships between various species, since mtDNA is maternally inherited and theevolutionary rate of nucleotide substitution is very rapid (Brown et al., 1979,
1982). We are investigating polymorphism in mtDNA of domestic animals,
such as the pig, cow, and chicken (Watanabe et al., 1985a, b, c, 1986; Wakana
et al., 1986). In the case of the cow, there are two major groups in the world,
namely, European cattle (humpless cattle) and Indian cattle (humped or
Zebu cattle). In a previous report (Watanabe et al., 1985c), the restriction
patterns of mtDNA in cattle were examined in the three breeds maintained inJapan: Japanese Black, Japanese Shorthorn, and Holstein. However, few
polymorphisms were detectable. The three breeds in Japan are all Europeantype cattle. Therefore, in the present study we examined the mtDNA from
native cattle in the Philippines which are thought to represent an admixture ofEuropean and Indian cattle.

MATERIALS AND METHODS

Chemicals. Fifteen restriction endonucleases, BamHI, BgllI, DraI, EcoRI,
EcoRV, HindlII, KpnI, PstI, PvulI, Sail, SacI, ScaI, StuI, XbaI, and XhoI,
were purchased from Toyobo Biochemicals. DNaseI was purchased fromWorthington Biochemical Co. Agarose was obtained from Nakarai Chemical
Ltd., and ethidium bromide from Katayama Chemical Ltd. Other reagentswere commercial preparations of the highest purity available.

Preparation of mtDNA, Restriction Endonuclease Digestion, and Gel
Electrophoresis. Mitochondrial DNA was prepared from kidneys of Philippine native cattle which were obtained from a slaughterhouse in the suburbs ofManila. The cattle were randomly chosen from the population so that theirmaternal relatedness is not known. The procedures for the preparation of
mtDNA, restriction endonuclease digestion, and agarose gel electrophoresis
have been described previously (Watanabe et al., 1985a, c).

Mapping of Cleavage Sites on mtDNA and Analysis of Nucleotide
Substitutions. Molecular weights of restriction fragments of mtDNA were
determined by comparison of their mobilities relative to those of fragments of~-DNA digested with HindlII and used as standards. Double-digestion
methods were used for the mapping of cleavage sites on the mtDNA molecule.
Determinations of the sites of cleavage by the restriction enzymes and of
nucleotide substitutions were made using a personal computer (NEC, PC8201), comparing them to the complete sequence of Anderson et al. (1982).
The A restriction pattern for all enzymes except one in this study matches thatinferred from the published sequence; for MspI pattern B is derived from the
known sequence.

Mitochondrial DNA Polymorphism in Cattle

433

RESULTS
Analysis of patterns of cleavage of mtDNA by restriction endonucleases was
performed for nine individual Philippine native cows. Fifteen restriction
enzymes which each recognize six base pairs were used in this study. MtDNA
polymorphisms were detected in the restriction patterns generated by the six
enzymes, BamHI, BgllI, EcoRV, HindlII, PstI, and ScaI, but no difference
was observed in the patterns generated by the other nine enzymes. In the
restriction patterns of enzymes in which no differences were detected, thenumbers of cleavage sites and the sizes of fragments were the same aspreviously reported (Watanabe et al., 1985c); there were seven fragments
detected for DraI, which is the only enzyme not used in the earlier report. In
total, 36 restriction sites by nine enzymes revealing no variation wererecognized. Figure 1 shows some restriction patterns that reveal polymorphisms, and Table I shows the number of cleavage sites of restriction enzymes

AB

H_~dm

~ill

Pstl

A C A

ABA

A B

Origin---)-23.1- 4./~

1

- Z3
- Z0

+

kb

Fig. 1. Comparison of patterns of cleavage by restriction endonucleases of mtDNA from
Philippine native cattle after electrophoresis on 0.9% agarose gels.

434

Watanabe, Masangkay, Wakana, Saitou, and Tomita

Table I. N u m b e r of Sites of Cleavage by Various Restriction Endonucleases, Demonstrating
Polymorphism in Bovine m t D N A , and the Size of Fragments
Restriction
endonucleaseType

N u m b e r ofcleavage sites

A
B

3
4

11.2
9.8

3.2
3.2

BgllI

A
B

2
1

9.7
16.3

6.6

EcoRV + Sail a

A
B

1
2

16.3
11.1

5.2

A
B
C

3
4
2

10.2
10.2
14.6

4.5
2.4
1.7

PstI

A
B

2
1

9.4
16.3

7.0

ScaI

A

9

B

9

4.2
1.3
6.0
1.2

2.6
1.2
2.6
0.5

BamHI

HindlII

Length offragments (kb)
1.9
1.9

1.4

1.7
2.0

1.7

2.6
0.5
2.6
0.2

1.9
0.2
1.7
0.2

1.8
1.3

aThere is a SalI restriction site common to A and B types of m t D N A .

that allow polymorphisms to be recognized and the length of relevant
fragments. No EcoRV cleavage site was detected in the A type of mtDNA,
but in the B type there was a single EcoRV restriction site. The B type ofHindlII restriction pattern was not found in this study, even though it was
found previously in Japanese Shorthorn and Holstein cows. The A and C typesof HindlII restriction pattern were observed in the Philippine native cattle.
The C type pattern contains only two bands of 14.6 and 1.7 kb, while the A
type pattern contains three bands, because of the presence of one more
restriction site in the fragment of 14.6 kb than is present in the C type. Thisadditional site is responsible for generation of the fragments of 10.2 and 4.5
kb. The B type mtDNA generates four bands, since fragments of 2.4 and 2.0
kb result from cleavage at yet one more site in the 4.5 kb fragment of the A
type mtDNA. On the basis of the differences in cleavage sites, the A type ofHindlII restriction pattern can be considered to be intermediate between the
B and the C types. In the ScaI restriction patterns, the A and B types eachgenerate nine fragments, but two of the cleavage sites are different from eachother. In the restriction patterns generated by BamHI, BgllI, and PstI, asingle cleavage site differs between each of the A and B types.
Altogether, polymorphisms were detected at seven restriction sites, andthe different patterns of restriction were distributed characteristically in thenine individual Philippine native cattle examined. Five cows were A type forMitochondriai DNA Polymorphism in Cattle

435

BamHI, BglII, EcoRV, HindlII, PstI, and ScaI and are designated Philippines-l, and this combination corresponds to that observed in the case ofJapanese- 1. Four of the native, Philippine cows gave patterns that were B, B,
B, C, B, and B type, respectively, and these cows are designated Philippines-2.
Since the Philippine native cattle are considered to represent an admixture ofEuropean and Indian cattle, the mtDNA in Philippines-1 must be derived
from the European cattle's type of mtDNA, and the Philippines-2 mtDNA
must be derived from the Indian cattle's type of mtDNA. Furthermore,combining the data in this study with those from a previous report, we cansummarize the restriction types, as shown in Table II. In the mtDNA of thethree Japanese breeds, three combinations were seen, A, A, and A (Japanese1A), A, A, and B (Japanese-lB), and B, B, and B (Japanese-2), with respect
to digestions with HindlII, TaqI, and MspI, respectively. TaqI and MspI are
enzymes that recognize 4 base pairs. On the basis of these results and therestriction map (Fig. 2), the genetic distance between types of mtDNA
detected in the three breeds in Japan and in the Philippine native cattle can be
estimated from the numbers of restriction sites observed. First, the distancewas calculated by the methods of Nei and Li (1979) for the cases in which
enzymes that recognize only 6 base pairs were used. These were 0.0015 _0.0015 between Philippines-1 and Japanese-2, 0.0095 _+ 0.0040 between
Philippines-1 and Philippines-2, and 0.0111 _+0.0043 between Japanese-2 andPhilippines-2. Second, the distance was calculated by the methods of Nei andTajima (1983) for the cases in which enzymes that recognize both 4 and 6
base-pairs were used. These were 0.0011 _+0.0011 between Japanese-lA andJapanese-lB, 0.0032 ___ 0.0019 between Japanese-lA and Japanese-2, and
0.0021 _+ 0.0015 between Japanese-lB and Japanese-2. Based on thesegenetic distances, phylogenetic trees of mtDNA types were constructed by
UPGMA (Fig. 3). The genetic distances estimated by the two procedures are
consistent. Philippines-2 is very distant from the other groups, to the extent
that it can be considered to be a subspecies.
Table II. Types of Restriction Polymorphisms in m t D N A A m o n g Three Japanese Breeds andPhilippine Native Cattle
Line

BamHI

BgllI

EcoRV

HindIII

PstI

ScaI

TaqI"

MspI °

Japanese-lA
Japanese- 1B
Japanese-2
Philippines- 1
Philippines-2

A
A
A
A
B

A
A
A
A
B

A
A
A
A
B

A
A
B
A
C

A
A
A
A
B

A
A
A
A
B

A
A
B
b
--

A
B
B
__
--

"TaqI and MspI, which were used in the previous study (Watanabe et al., 1985c), are enzymes
that recognize 4 base pairs.
bNot determined.

Watanabe, Masangkay, Wakana, Saitou, and Tomita

436
5

0

S
I

I

10

I

I
S
II
E

S
I

S
I ,

S
rl

S
I

EV

E

15

I

I

16.3 kb

I

H S~
I= I
Bg

SHS
Iiii
E

S (H)
i I
I
P

S
iI
B

H
,I t
E Eg

B

SS
I l l ,I P h i l i p p i n e s - 1
PB

H SI
I I

SHS
Jill

S
I I ,

,

J

I

i

SSS
III

E

P

B

E Bg

B

B

i

,I P h i l i p p i n e s - 2
B

Fig. 2. Maps of cleavage by restriction endonucleases of Philippines-i- and Philippines-2-type
mtDNA. Asterisks indicate the polymorphic sites detected in Philippine native cattle, and thepolymorphic site detected previously in two Japanese breeds is in parentheses. B, BamHI; Bg,
BgllI; E, EcoRI; Ev, EcoRV; H, HindlII; S, ScaI; SI, SalI; P, PstI.

Japanese-lA, B
Philippines- 1

A
IJapanese- 2
Philippines-2
Genetic-, -'d istance(/o)OJapanese-lA

'

0.1

.

0.2

.

0.4

.

.

0.6

.

.

0.8

1.0

1.2

B

Japanese-lB
Japanese- 2
Fig. 3. Dendrogram of three Japanese breeds and Philippine native cattle, based on genetic
distances. The distance in A is calculated by the methods of Nei and Li (1979) for the cases inwhich only 6 base pairs recognizing enzymes were used, and the distance in B is estimated by themethods of Nei and Tajima (1983) for the cases that both 4- and 6-base-pair enzymes wereused. The scale is the same in A and B.

M~tochondrial DNA Polymorphism in Cattle

437

The complete nucleotide sequence of bovine mtDNA has already beenreported by Anderson et al. (1982). Therefore, the restriction map, including
the polymorphic sites, is easily drawn. Figure 2 shows the restriction map ofboth the Philippines-1 and the Philippines-2 types of mtDNA, and if a single
nucleotide substitution has occurred between the two types of mtDNA, thepositions of seven polymorphic sites can be estimated on the nucleotide
sequence of mtDNA, by computer analyses, to be those shown. The sevenvariable sites in Philippines-2 relative to the completely reported sequence areas follows, listed according to enzyme, gene, location of site start, and, for thethree site gains, the inferred base change and its location: (1) EcoRV,tRNA va~, 1389, A -~ C, 1395; (2) BglII, COI, 6115; (3) BamHI COIII, 9600,
G -~ A, 9602; (4) ScaI, ND4, 11067; (5) HindIII, ND5, 12174; (6) ScaI
Cytb, 15605, G -~ A, 15608; and (7) PstI, 15738, tRNA Pr°. The mutations at
9602 and 15605 are third-position silent changes.
DISCUSSIONIn Philippine native cattle, the presence of two types of mtDNA has clearly
been demonstrated in this study. These two types of mtDNA are very different
from each other. In comparison with the mouse mtDNA sequence divergenceamong subspecies (Yonekawa et al., 1982; Ferris et al., 1983), it has been
considered that the sequence divergence, somewhat over 1%, between Philippines-1 and Philippines-2 is at the level of subspecies. No shifting type of
mtDNA, showing intermediate characteristics, was observed. This resultmeans that repeated nucleotide substitutions have not occurred in thePhilippine native cattle and that two groups of cattle of different maternal
origin were admixed in that country. From investigations of polymorphisms inthe beta chain of hemoglobin, Namikawa (1981) suggested that the Philippine native cattle developed from an admixture between European and Indian
types of cattle. One set of mtDNA restriction patterns examined in this study
must, thus, be derived from European cattle and another from Indian cattle.
In future, it will be necessary to examine the restriction patterns of mtDNA
from authentic Indian cattle.
In domestic animals, the mtDNA polymorphisms offer important information for the identification of their origins. The situation with respect topolymorphism of cow mtDNA is very similar to that for pig mtDNA.
Watanabe et al. (1985a, 1986) have indicated from studies of mtDNA
polymorphism that domestic pigs must be derived from two different maternal
origins, namely, European and Asian wild boars, and that one breed, theLarge White, must have arisen as an admixture of European and Asian pigs.
Mitochondrial DNA polymorphisms of the chicken, Gallus gallus domesticus
are quite different from those of the pig and cow (Wakana et al., 1986). The

438

Watanabe, Masangkay, Wakana, Saitou, and Tomita

cleavage patterns generated by 11 restriction enzymes, each of which recognizes 6 base pairs, were identical in all the 16 lines of domestic fowl examined,
including the red j u n g l e fowl, G a l l u s g a l l u s gallus. O n l y one v a r i a n t was
found, in a line of W h i t e Leghorns, in the p a t t e r n of digestion by M s p I , which
recognizes 4 base pairs. T h e domestic fowl seems to have a less divergent
population of m t D N A s t h a n the pig and the cow.

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