Biochemical Genetics, Vol. 24, Nos. 5/6, 1986

Pig Mitochondrial DNA: Polymorphism, Restriction
Map Orientation, and Sequence Data
Tomomasa Watanabe, 1 Yukimasa Hayashi, 2 Jun Kimura, 3 Yukio Yasuda, 4
Naruya Saitou, 5 Takeshi Tomita, 6 and Nobuaki Ogasawara I

Received 25 Sept. 1985~-Final17 Jan. 1986

Restriction endonuclease cleavage patterns of mitochondrial DNA (mtDNA)
in pigs were analyzed using 18 enzymes which recognize six nucleotides and 1
four-nucleotide-recognizing enzyme. Pigs including Taiwan native breeds
and miniature strains maintained in Japan were examined in this study;fourcommercial breeds of pigs and Japanese wild boars have been investigatedearlier [Watanabe, T., et al. (1985). Biochem. Genet. 23:105]. mtDNA
polymorphisms were observed in the cleavage patterns of five restriction
enzymes, B g l l I , EcoRV, S c a L S t u L and T a q L The results support theprevious hypothesis that pigs must be derived from two different maternal
origins, European and Asian wild boars, and that a breed, Large White,arises from both European and Asian pigs. Two HindlII cleavage fragments
were cloned into the HindllI site of M13mplO and were partially sequenced
by the dideoxynucleotide-chain termination method. Furthermore, D r a I and
S t u I cleavage sites were newly determined on the restriction endonucleasemap. On the basis of these results, the restriction endonuclease cleavage mapof pig mtDNA was rewritten. Comparing sequence data of pig mtDNA at 237
positions with those of cow, human, mouse, and rat mtDNA, the sequence

Department of Biochemistry, Institute for Developmental Research, Aichi Prefecture Colony,
Kamiyacho, Kasugai, Aichi 480-03, Japan.
2 Department of Genetics, Institute for Developmental Research, Aichi Prefecture Colony,
Kamiyacho, Kasugai, Aicbi 480-03, Japan.
3 Medical Research Institute, Nippon Well-Being Foundation, Ikki-machi, Aizuwakamatsu,
Fukushima 965, Japan.
4 Central Institute for Experimental Animals, Nogawa, Miyamaeku, Kawasaki 211, Japan.
5 Center for Demographic and Population Genetics, University of Texas, Health Science Center,
Houston, Texas 77225.
6 Laboratory of Animal Genetics, Faculty of Agriculture, Nagoya University, Furocbo, Chikusa
Nagoya 464, Japan.
385
0006-2928/86/0600-0385505.00/0 © 1986 Plenum Publishing Corporation

386

Watanabe et al.

difference, silent and replacement changes, and transitions and transversions
among mammalian species were estimated. The relationships among them
are discussed.

KEYWORDS:pigs; mitochondrial DNA; restriction endonuclease;East Asia.
INTRODUCTION
Since animal mitochondrial DNA (mtDNA) is maternally inherited and theevolutionary rate of nucleotide substitution is very rapid (Brown et al., 1979,
1982), mtDNA polymorphism based on restriction endonuclease analysis has
allowed us to clarify genetic relationships of various species. We are investigating mtDNA polymorphism of domestic animals and fowls (Watanabe etal., 1985a, b, c; Wakana et al., 1986). In pigs, polymorphism of mtDNA has
been reported previously (Watanabe et al., 1985a) and suggests that two typesof mtDNAs, of European and Asian wild boar origins, exist in pigs. This paper
describes the further investigation of mtDNA polymorphism in four Taiwan
native breeds and three miniature strains maintained in Japan, the restriction
endonuclease cleavage map orientation, and the sequence data.
MATERIALS AND METHODS
Animals. Four breeds of Taiwan native pigs were introduced into Japan in1982 and are maintained at the Medical Research Institute, Nippon WellBeing Foundation. These breeds are Taoyaun, Meinung, Short-Ear, andLanyu. The former two breeds originated from native pigs of South China,
and the latter two from pigs of Southeastern Asia.
Three strains of miniature pigs are maintained in Japan. The Ohmini
strain was originally developed from the small pigs of Manchuria in the
Republic of China and are maintained at the Nippon Research Laboratory for
Domestic Animals. The G~ttingen miniature strain was introduced into theCentral Institute for Experimental Animals from Germany. The GSttingenstrain was developed from a cross of Homel miniatures with a small type ofVietnamese pig (Haring et al., 1965), and the unpigmented gene wasintroduced by crossing with the Landrace breed. The Pitman-Moore strain
was maintained at the Nippon Institute for Biological Science, originallydeveloped from feral hogs from the southern United States.
Chemicals. Eighteen restriction endonucleases were purchased from
Nippon Gene Co. or Takara Shuzo Co. In addition, DraI was obtained fromPharmacia. [o~-3/P]dCTP, the M13mpl0 cloning kit, and the sequencing kit
were purchased from Amersham. Alkaline phosphatase was from Boerhinger-

Pig Mitochondrial DNA Polymorphism

387

Mannheim Co. Other reagents were commercial preparations of the highest
purity available,

MtDNA Preparations, Restriction Endonuclease Digestion, and Gel
Electrophoresis. mtDNA was prepared from kidneys of pigs. The procedures
for mtDNA preparation, restriction endonuclease digestion, agarose gelelectrophoresis, and polyacrylamide gel electrophoresis have been described
previously (Watanabe et al., 1985a, c).
DNA Cloning and Sequencing. Pig mtDNA was digested with HindIII
and was ligated with an alkaline phosphatase-treated vector, M13mpl0(Groneborn and Messing, 1978; Messing et al., 1981). The ligation productwas used to transform Escherichia coli JM105, in order to prepare asingle-strand template. The sequence of a HindIII-digested fragment wasdetermined by dideoxynucleotide-chain termination method using a flanking
universal primer and [a-32p]dCTP (Sanger et al., 1977, 1980).
RESULTS AND DISCUSSION
Eighteen restriction endonucleases which recognize six base pairs were used inthis study, mtDNA polymorphisms were detected in the cleavage patterns of
4 restriction enzymes, BgllI, EcoRV, ScaI, and StuI, but no difference wasobserved in the patterns of the other 14 restriction enzymes, BamHI, BbeI,
DraI, EcoRI, HindlII, KpnI, MluI, PstI, PvulI, SacI, Sail, Sinai, XbaI, andXhoI, among pigs examined in this study and in the previous one (Watanabe
et al., 1985a). As shown in Table I, mtDNA from all four breeds of Taiwan
Table I. Types of Restriction Endonuclease Cleavage Patterns in Four Native Breeds of Taiwan
Pigs and Three Miniature Strains Maintained in Japan
Restriction pattern
BreedsNo.
examined

BgllI

EcoRV

ScaI

StuI

Taiwan native breeds
Taoyaun
Meinung
Short-ear
Lanyu

1
2
1
1

B
B
B
B

A
A
A
A

B
B
B
B

B
B
B
B

Miniature strains
maintained in Japan
Ohmini
GSttingen
Pitman-Moore

2
2
4

B
B
A

A
A
A

B
B
A

B
B
A

388

Watanabe et al,native pigs showed the cleavage types of B in BgllI, A in EcoRV, B in ScaI,and B in StuI. The cleavage types of mtDNA from two strains of miniature
pigs, Ohmini and G6ttingen, were also B-A-B-B in BglII-EeoRV-ScaIStuI, respectively. On the other hand, a miniature pig of the Pitman-Moore
strain showed the cleavage type of mtDNA, A-A-A-A. DraI was first used inthis study, when seven fragments were observed in the cleavage patterns andthe molecular weights were estimated as 6.54, 2.54, 2.29, 1.96, 1.52, 1.23, and0.39 kb. Therefore, the percentage sequence divergence among pigs showing
different cleavage patterns was recalculated with equations 10 and 16 of thereport by Nei and Li (1979). An assumption of this method is that acleavage-site difference is due to the substitution of one base pair. The
percentage sequence divergence of mtDNA between A - A - A - A and BA-B-B was 1.75, and that between A - A - A - A and A - B - A - A was 0.17.
From these results, the relationships among various breeds of pigs andJapanese wild boars based on the restriction patterns are diagrammed in Fig.
1. The restriction patterns of Landrace, Hampshire, Duroc, Pitman-Moore
and Large White ! are classified as the European type. The patterns of Large
White II, Ohmini, Gfittingen, Taiwan native breeds, and Japanese wild boars
are grouped as the Asian type. The time of divergence between both Europeanand Asian types was calculated to be about 0.8-0.9 x 10 6 years. The
hypothesis that the pigs examined in our studies are from different maternal
origins, European and Asian wild boars, and that the breed, Large White,arises from both European and Asian pigs is strongly supported.

% Hampshire%0
Landrace
Iio

DurocI ( ) Durocll
0.17
//o

Pitman-Moore

0% Large White [I 1.75
Japanese
Large WhitelI = wi|d boarsoII 0
Taiwan0"~ G6tt ingen o naiive breeds
Taoyaun Meinung,o//Ohmini

Short--ea~and Lar~uJ

Fig. 1. The relationships among several breeds of pigs andJapanese wild boars based on the restriction endonucleasecleavagepatterns of mtDNA. The numbers indicatethe estimated percentagesequencedifferencesamongthem.

Pig MitochondrialDNA Polymorphism

389

Since restriction enzymes recognizing four base pairs cut far more often
than six-base enzymes, the use of four-base enzymes could easily detect
nucleotide sequence difference. An example employing TaqI is shown in Fig.
2 and Table 1I. mtDNAs of pigs between European and Asian groups wereclearly different from each other in the Taql cleavage pattern, but mtDNAs
belonging in the same group had similar restriction patterns. Bottleneck
effects must occur in each groups of pigs as well as in inbred laboratory micereported by Ferris et al. (1982), although it is not clear when the effectoccurred, either prior to or contemporary with domestication of the pig.
On the basis of blood groups and protein polymorphism, Tanaka et al.

12345678910-.-)

Fig. 2, Polyacrylamide gel electrophoresis of Taql digests of pig
mtDNA. (1) Duroc II; (2) Large White I; (3) Large White II;(4) Landrace; (5) Duroc I; (6) Duroc I; (7) Large White II; (8)Japanese wild boar; (9) Ohmini; (10) Ohmini. Samples 1, 2, 4,
5, and 6 are A types, and samples 3, 7, 8, 9, and 10 are B types.
Arrows indicate the different fragments between A and B
types.

390

Watanabe et al.

Table II. Typesof RestrictionEndonucleaseCleavagePatterns Digested by TaqI andComparison with Thoseof BgllI, EcoRV, ScaI, and StuIi

SampleNo.

Breeds

TaqI

BgllI

EcoRV

ScaI

StuI

1
2
3
4
5
6
7
8
9
10

Duroc II
Large White I
Large White II
Landrace
Duroc I
Duroc I
Large White !I
Japanesewild boar
OhminiOhmini

A
A
B
A
A
A
B
B
B
B

A
A
B
A
A
A
B
B
B
B

B
A
A
A
A
A
A
A
A
A

A
A
B
A
A
A
B
B
B
B

A
A
B
A
A
A
B
B
B
B

(1983) concluded that the European and Asian pig populations are distantly
related; Taiwan native breeds and Ohmini miniature pigs are classified in t h e
Asian group. The G~ttingen miniature breed is also close to the Asian group(Oishi et al., 1980), but the Pitman-Moore miniature breed is close to theEuropean group (Oishi and Tomita, 1976). These results correspond well withthe genetic similarity based on the m t D N A cleavage patterns. Kurosawa et al.
(1983) have reported from immunogenetic studies that East-West geographical clines of some antigen frequencies are clearly observed in wild boars on
the Eurasian continent (Buschman, 1965; Wiatroszka, 1970; Tikhonov et al.,1972) and that wild boars in Japan are closer to Far Eastern than Middle
Asian Transcausian and European boars. Wild boars on the Eurasiancontinent need to be investigated from the view of m t D N A polymorphism,
because the study could suggest some ideas about the process of domesticationand evolution of pigs.
As the restriction endonuclease map of pig m t D N A was only provisionally drawn in the previous report, position 0 was not identified in the D-loop
region, unlike human, mouse, and bovine mtDNAs. Although the complete
nucleotide sequences of human, mouse, and bovine mtDNAs have alreadybeen determined (Anderson et al., 1981, 1982; Bibb et al., 1981), no sequence
data have been reported yet for pig mtDNA. Sequence data could make aprecise restriction endonuclease map orientation by means of the homology
test with human, mouse, and bovine mtDNAs. Pig m t D N A has four H i n d l I I
cleavage sites, so that four cleavage fragments, of 9.2, 4.3, 2.5, and 0.5 kb, areobtained. The two H i n d l I I cleavage fragments of 4.3 and 2.5 kb from
Pitman-Moore were cloned into the H i n d I I I site of M 1 3 m p l 0 and weresequenced by the dideoxynucleotide-chain termination method. A clone with a
4.3-kb fragment inserted has the nucleotide sequence of the terminal as shownPig Mitochondrial DNA Polymorphism

391

~ o

>

~_
<

~

<

~.

~i~-

.--II--

~

~

~

L

~

>-.~

~z

<
~-.~

~_~
<

~

392

Watanabe et al.

~J

<~

..=

<i:

<~

<£

~E

<<

o

>~<

< : ¢..~

~_<~
<<

<~

~

?=~ - o
<~
<

~>-~
~_

<~

o_

c.~

I-----lid

EPig Mitochondrial DNA Polymorphism

393

in Fig. 3. The nucleotide sequence of the terminal of the other clone including
a 2.5-kb fragment is presented in Fig. 4. The sequences of both fragments
were identified to correspond to a part of the ATPase subunit by analysis ofalignment with the sequence data of other mammals. With these data we haveassigned position 0 of pig mtDNA in the D-loop region in Fig. 5. Furthermore,
Dral and StuI cleavage sites on the map were newly determined in this study.
On the basis of these results, the restriction endonuclease map of pig mtDNA
was oriented as shown in Fig. 5. Up to here, polymorphic sites were notdetermined to be located in any genes. The A type has three BgllI cleavage
sites in regions 12 S RNA, Coil, and URF5, but the B type has only one site in
the Coil region. The A type in EcoRV has an EcoRV cleavage site in URF5,
but the B type has two sites in the 16 S RNA and URF5 regions. The
polymorphic sites for StuI must be located in URF4L and Cytb; a cleavage

I :~Sd'reJo '
l'eJ,

~o7
~os

.s!H

Pig m t D N A
16.35kb

ire

Glo

Met

- Hindn'I, X b a l- EcoRI

f'stttl

"StoZ
to

~"r"

Fig. 5. The restriction endonuclease cleavage map of pig mtDNA. Position 0 must be in the
D-loop region and ATPase 6 is located between position 8.43 and position 8.92. Restriction
endonucleases in parentheses show the different polymorphic sites among pigs.

394

Watanabe et aL

Table III. Sequence Differences Among Mammalian Mitochondrial DNAs at 237 Positions ~

Pig
Cow
Human
Mouse
Rat

Pig

Cow

Human

Mouse

Rat

-19.8
25.7
19.4
21.5

47
-25.3
25.3
24.9

61
60
-26.2
24.1

46
60
62
-17.3

51
59
57
41
--

°The number of sequence differences between any two mtDNAs appears in the upper right-handsection of the matrix, and the percentage sequence difference is given in the lower left-handsection,site of the A type is in the URF4L region and not in the Cytb region and thatof the B type is in the Cytb region and not in the URF4L region.
As the mtDNA sequence of pig mtDNA is available also in the rat (Pepeet al., 1983), the sequence differences among five species of mammalian
mtDNAs were compared at 237 positions (Table III). It would be reasonable
that the number of differences between the mouse and the rat, which are bothrodents, is the least. Of interest is that the number of differences between the
pig and the cow is almost the same as that between the pig and the mouse andbetween the pig and the rat. Pigs and cows might be distantly related,
although both species are artidactyl, and the pig is considered to be closer thanthe cow to rodents. As shown in Table IV, the number of nucleotide
substitutions was counted after dividing them into silent and replacementchanges and transitions and transversions. With respect to the number of
replacements and transversions, the differences between the pig and the cow
are indeed smaller than between any other pairs except the mouse and the rat.
Table I V . The Number of Nucleotide Substitutions Among Mammalian Mitochondrial
D N A s at 237 Positions
Species compared

SilentReplacement

Transition

Transversion

Pig-cow
Pig-human
Pig-mouse
Pig-rat
Cow-human
Cow-mouse
Cow-ratHuman-mouse
Human-rat
Mouse-rat

25
24
21
21
26
30
27
22
21
23

22
37
25
30
34
30
32
40
36
18

24
32
19
22
32
22
29
27
27
23

23
29
27
29
28
38
30
35
30
18

Pig Mitoehondrial DNA Polymorphism

395

More sequence data for pig mtDNA will be necessary to discuss therelationships among mammalian mtDNAs in detail.
ACKNOWLEDGMENTS

We would like to express our thanks to Dr. R. Semba of the Institute forDevelopmental Research for his useful discussions and his kind guidance on
how to analyze the sequence data with a hand-type personal computer.
We also thank Mr. H. Ohmi of the Nippon Research Laboratory for
Domestic Animals and Drs. M. Mitsui and M. Mizutani of the Nippon
Institute for Biological Science for making the animals available for thisstudy.
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