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Fumihito A, Miyake T, Sumi S, Takada M, Ohno S, Kondo N. One subspecies of the red junglefowl (Gallus gallus gallus) suffices as the matriarchic ancestor of all domestic breeds. Proc Natl Acad Sci USA 91: 12505-12509

Authors:

Abstract

The noncoding control region of the mitochondrial DNA of various gallinaceous birds was studied with regard to its restriction fragment length polymorphism (RFLP) and sequences of the first 400 bases. Tandem duplication of the 60-base unit was established as a trait unique to the genus Gallus, which is shared neither by pheasants nor by quails. Unlike its close ally Gallus varius (green junglefowl), the red junglefowl Gallus gallus is a genetically very diverse species; the 7.0% sequence divergence was seen between those from Thailand (G. g. gallus and G. g. spadiceus) and the other from the Indonesian island of Java (G. g. Bankiva). Furthermore, the divergence increased to 27.83% if each transversion is regarded as an equivalent of 10 transitions. On the other hand, a mere 0.5-3.0% difference (all transitions) separated various domestic breeds of the chicken from two G. g. gallus of Thailand, thus indicating a single domestication event in the area inhabited by this subspecies of the red junglefowl as the origin of all domestic breeds. Only transitions separated six diverse domesticated breeds. Nevertheless, a 2.75% difference was seen between RFLP type I breeds (White Leghorn and Nagoya) and a RFLP type VIII breed (Ayam Pelung). The above data suggested that although the mitochondrion of RFLP type V was the main contributor to domestication, hens of other RFLP types also contributed to this event.
Proc.
Natl.
Acad.
Sci.
USA
Vol.
91,
pp.
12505-12509,
December
1994
Evolution
One
subspecies
of
the
red
junglefowl
(Gallus
gallus
gallus)
suffices
as
the
matriarchic ancestor
of
all
domestic
breeds
AKISHINONOMIYA
FuMIHITO*,
TETSUO
MIYAKEt,
SHIN-ICHIRO
SUMIt,
MASARU
TAKADAt,
SUSUMU
OHNO§,
AND
NORIo
KONDO*
*Yamashina
Institute
for
Ornithology,
115
Tsutsumine-aza,
Konoyama,
Abiko-shi,
Chiba
Prefecture
270-11,
Japan;
tWakunaga
Pharmaceutical
Company
Central
Laboratories,
1624
Shimokotachi,
Kodacho,
Takatagun,
Hiroshima
Prefecture
739-11,
Japan;
tThe
Research
Institute
of
Evolutionary
Biology,
2-4-28
Kamiyoga,
Setagaya-ku,
Tokyo
158,
Japan;
and
§Beckman
Research
Institute
of
the
City
of
Hope,
1450
East
Duarte
Road,
Duarte,
CA
91010-3000
Contributed
by
Susumu
Ohno,
September
6,
1994
ABSTRACT
The
noncoding
control
region
of
the
mitochon-
drial
DNA
of
various
gallinaceous
birds
was
studied
with
regard
to
its
restriction
fragment
length
polymorphism
(RFLP)
and
sequences
of
the
first
400
bases.
Tandem
duplication
of
the
60-base
unit
was
established
as
a
trait
unique
to
the
genus
Galus,
which
is
shared
neither
by
pheasants
nor
by
quails.
Unlike
its
close
ally
GaUus
varius
(green
junglefowl),
the
red
junglefowl
GaUus
gaUus
is
a
genetically
very
diverse
species;
the
7.0%
sequence
divergence
was
seen
between
those
from
Thai-
land
(G.
g.
gaUus
and
G.
g.
spadiceus)
and
the
other
from
the
Indonesian
island
of
Java
(G.
g.
Bankiva).
Furthermore,
the
divergence
increased
to
27.83%
if
each
transversion
is
regarded
as
an
equivalent
of
10
transitions.
On
the
other
hand,
a
mere
0.5-3.0%
difference
(all
transitions)
separated
various
domestic
breeds
of
the
chicken
from
two
G.
g.
gaius
of
Thailand,
thus
indicating
a
single
domestication
event
in
the
area
inhabited
by
this
subspecies
of
the
red
junglefowl
as
the
origin
of
all
domestic
breeds.
Only
transitions
separated
six
diverse
domesticated
breeds.
Nevertheless,
a
2.75%
difference
was
seen
between
RFLP
type
I
breeds
(White
Leghorn
and
Nagoya)
and
a
RFLP
type
VIII
breed
(Ayam
Pelung).
The
above
data
suggested
that
although
the
mitochondrion
of
RFLP
type
V
was
the
main
contributor
to
domestication,
hens
of
other
RFLP
types
also
contributed
to
this
event.
There
is
little
doubt
that
the
domestication
of
various
wild
animals
as
the
beasts
of
burden,
the
source
of
protein
and
fat,
and
the
instrument
of
war
and
recreation
played
many
pivotal
roles
in
the
cultural
evolution
of
mankind.
Of
special
interest
has
been
the
various
divine
rites
performed
in
association
with
various
domesticated
animals,
particularly
the
chicken.
For
documentation
of
so
recent
an
event
as
domestication,
nuclear
genes
with
their
low
mutation
rate
would
be
of
little
use.
On
the
contrary,
the
mitochondrial
genome
appears
particularly
suitable.
Its
high
mutation
rate
is
expected
to
remain
constant,
being
relatively
impervious
to
generation
time
differences
between
species.
It
may
be
recalled
that
an
organism
does
not
start
its
life
with
a
single
copy
but
with
hundreds
of
thousands
of
copies
of
the
mitochondrial
genome
harbored
by
the
egg
cytoplasm.
Accordingly,
generation
changes
do
not
constitute
significant
epochs
in
the
life
history
of
mitochondrial
DNA.
Furthermore,
the
extremely
useful
landmark
was
established
by
recent
studies
on
two
hyper-
variable
subregions
of
the
control
region
of
human
mitochon-
dria.
The
average
sequence
divergence
between
all
races
of
mankind
was
established
as
2.0%
and
the
rate
of
evolution
was
estimated
to
be
1%
sequence
divergence
per
71,000-
167,000
years
(1, 2).
It
follows
that
any
mitochondrial
se-
quence
divergence
substantially
above
2.0%
within
a
given
domesticated
species
creates
a
peculiar
paradox
of
either
domestication
occurring
before
the
emergence
of
mankind
or
at
least
domestication
occurring
within
the
African
cradle
before
the
exodus
of
certain
bands
to
the
Near
East
and
outward.
Indeed,
such
a
paradox
was
encountered
in
a
recent
study
on
the
mitochondrial
control
region
of
various
breeds
of
cattle.
Two
distinct
mitochondrial
lineages
separated
by
a
5.01%
sequence
difference
were
observed.
Furthermore,
this
dichotomy
did
not
follow
the
customary
Bos
taurus/Bos
indicus
split,
for
the
African
zebu
was
more
similar
to
European
taurine
breeds
than
to
Indian
zebu.
This
paradox
was
resolved
by
the
assumed
presence
of
two
subspecies
of
the
aurochs
(Bos
primigenius)
prior
to
the
emergence
of
humans
and
the
two
subsequent
independent
domestication
events
(3).
In
view
of
the
above
data,
we
have
decided
to
study
the
control
region
light
chain
(L
chain)
of
the
avian
mitochondria
on
various
gallinaceous
birds
with
regard
to
its
restriction
fragment
length
polymorphism
(RFLP)
as
well
as
sequences
of
the
first
400
bases
of
the
control
region.
In
human
studies,
64%
of
the
total
polymorphism
in
the
entire
control
region
was
found
among
the
first
400
bases
(4).
MATERIALS
AND
METHODS
Material.
Materials
used
for
the
present
study
are
summa-
rized
in
Fig.
1.
As
to
junglefowls,
10
of
the
red
junglefowl
(G.
g.
gallus
and
G.
g.
spadiceus)
were
gifts
from
the
Department
of
Forestry
of
the
Thai
government.
Five
specimens
of
G.
g.
bankiva
were
obtained
from
the
Indonesian
island
of
Java,
and
so
are
all
of
the
green
junglefowl
(Gallus
varius).
Four
additional
Thai
red
junglefowls
sampled
were
from
those
kept
in
the
Tama
Zoological
Garden
(Tokyo).
Of
various
domestic
breeds,
samples
from
all
those
clas-
sified
as
"occidental
breeds"
were
collected
at
the
Domestic
Fowl
Trust
of
England.
As
to
Asiatic
breeds,
those
starting
with
"ayam"
were
all
Indonesian
breeds
and
were
collected
there.
Others
were
either
collected
in
their
native
habitats
or
obtained
from
one
of
the
following
three
institutions
in
Japan:
Yamashima
Institute
for
Ornithology,
The
Research
Institute
of
Evolutionary
Biology,
or
Hiroshima
Animal
Husbandry
Experimental
Station.
Preparation
of
Cell
Lysate
and
Extraction
of
DNA.
At
least
5
,ul
of
peripheral
blood
was
blotted
on
a
small
piece
of
filter
paper
(approximately
5
x
5
mm)
and
kept
dried
during
transportation.
Blood
was
eluted
from
a
filter
paper
in
500
IlI
of
phosphate-buffered
saline.
After
centrifugation
at
5000
rpm
for
2
min,
cell
pellets
were
suspended
in
100,ul
of
10
mM
Tris-HCl
(pH
8.3)
buffer
containing
50
mM
KCl,
1.5
mM
MgCl2,
0.001%
gelatin,
0.45%
Nonidet
P-40,
0.45%
Tween
20,
and
200
gg
of
proteinase
K
per
ml.
The
suspension
was
incubated
for
30
min
at
60°C
and
was
heated
at
94°C
for
15
min
Abbreviation:
RFLP,
restriction
fragment
length
polymorphism.
12505
The
publication
costs
of
this
article
were
defrayed
in
part
by
page
charge
payment.
This
article
must
therefore
be
hereby
marked
"advertisement"
in
accordance
with
18
U.S.C.
§1734
solely
to
indicate
this
fact.
12506
Evolution:
Akishinonomiya
et
al.
FIG.
1.
Distribution
of
14
RFLP
types
among
121
individuals
of
G.
gallus
(red
junglefowls
and
domestic
breeds)
and
G.
varius
(green
junglefowls)
is
shown.
Mitochondrial
control
region
am-
plified
by
PCR
contained
six
polymor-
phic
sites
for
four
restriction
enzymes.
V,
Vsp
I;
A,
Alu
I;
Ms,
Mse
I;
Mb,
Mbo
II.
On
the
left,
each
RFLP
type
is
defined
as
cleavable
(+)
or
not
cleavable
(-)
at
each
of
the
six
sites.
RFLP
types
of
G.
gallus
are
numbered
in
Roman
numerals
I-VIII,
whereas
those
of
G.
varius
are
shown
as
A-F.
Nevertheless,
types
I
and
II
of
the
former
and
types
A
and
B
of
the
latter
are
related
(see
text).
With
regard
to
each
wild
species
and
subspecies
as
well
as
to
each
domestic
breed,
distribution
is
ex-
pressed
as
number
of
individuals
of
a
particular
RFLP
type
per
total
number
studied.
Aside
from
14
Thai
red
jungle-
fowls
(10
G.
g.
gallus
and
4
G.
g.
spadi-
ceus)
and
5
Indonesian
red
junglefowls
(G.
g.
bankiva)
and
30
green
junglefowls,
72
individuals
representing
26
diverse
domestic
breeds,
3
of
them
in
2
varieties
each
were
studied.
Although
all
domestic
breeds
are
ultimately
of
Asiatic
origin,
those
long
established
in
Europe
and
the
New
World
were
classified
as
occidental
in
contrast
to
those
that
stayed
in
Asia.
to
stop
the
reaction.
The
cell
lysate
was
then
extracted
twice
with
400
,l
of
phenol/chloroform/isoamyl
alcohol
(25:24:1)
and
total
DNA
was
recovered
from
ethanol
precipitation.
The
DNA
pellet
was
dissolved
in
200
,l
of
10
mM
Tris-HCl
(pH
7.5)
buffer
containing
1
mM
EDTA.
PCR.
Conserved
primer
pair
H1255
(5'-CATCTTG-
GCATCTTCAGTGCC-3')
and
L16750
(5'-AGGACTACG-
GCTTGAAAAGC-3')
was
used
to
amplify
the
control
region
for
RFLP
analysis.
L
and
H
refer
to
the
light
and
heavy
chains
and
the
number
designates
the
position
of
the
3'
end
of
the
primer
in
the
reference
sequence
(5).
Two
microliters
of
the
total
DNA
or
cell
lysates
was
subjected
to
35
amplification
cycles
by
using
Taq
(Thermus
aquaticus)
DNA
polymerase
(Takara
Shuzo,
Kyoto)
accord-
ing
to
the
manufacturer's
instructions,
with
denaturation
at
94°C
for
1
min,
annealing
at
55°C
for
1
min,
and
extension
at
72°C
for
2
min.
Detection
of
RFLP.
After
testing
36
restriction
enzymes,
the
following
four
were
chosen
as
suitable:
Alu
I
(recognition
sequence,
AGCT),
Mse
I
(TTAA),
MboII(TCTTC),
and
Vsp
I
(ATTAAT).
RFLPs
were
detected
on
either
1.5%
or
4.0%
agarose
gels
after
30
min
to
1
hr
of
electrophoresis
at
80
V.
Nucleotide
Sequencing.
Because
of
the
presence
of
an
EcoRI
site
within,
the
primer
H1254
(5'-ATGAATTCTTGGCATCT-
TCAGTGCCA-3')
was
used
instead
of
H1255
to
obtain
PCR
products
for
cloning.
The
base
sequence
of
another
primer
already
given,
L16775,
did
contain
a
HindIII
site.
When
the
above
H1254/L16775
pair
was
used
for
PCR
amplification,
3.0
mM
MgCl2
replaced
the
1.5
mM
concentration
recommended.
PCR
products
were
digested
with
EcoRI
and
HindIII
and
purified
by
agarose
gel
electrophoresis.
Ligation
of
the
DNA
segments
into
the
EcoRI/HindIII
site
of
the
cloning
vector
pUC118,
transformation
of
Escherichia
coli
JM109,
and
sin-
gle-strand
DNA
preparation
by
using
helper
phage
M13K07
were
performed
as
described
(6).
To
minimize
errors
intro-
duced
by
Taq
DNA
polymerase
during
PCR,
two
or
three
clones
obtained
from
each
sample
were
used
for
sequencing.
Sequencing
was
carried
out
with
the
BcaBEST
dideoxynu-
cleotide
sequencing
kit
(Takara
Shuzo)
using
fluorescein
iso-
thiocyanate
labeled
M13
forward
primer
(Shimazu,
Kyoto)
and
DNA
sequencer
DSQ1
(Shimazu).
OBSERVATIONS
RFLP
Within
the
1200-
to
1300-Base
Control
Region.
As
noted
in
Materials
and
Methods,
four
restriction
enzymes
recognized
specific
polymorphic
cleavage
sites
within
the
control
region,
thus
yielding
different
sized
fragments
readily
distinguishable
by
gel
electrophoresis.
These
four
restriction
enzymes
were
Vsp
I,
Alu
I,
Mse
I,
and
Mbo
II.
Inasmuch
as
the
last
two
enzymes
recognized
two
polymorphic
sites
each,
a
total
of
six
sites
were
involved
in
RFLP
(Fig.
1).
The
first
four
polymorphic
sites
are
identified
in
Fig.
2.
Of
the
potential
64
(26)
types
involving
six
sites,
eight
were
found
among
domestic
chickens
and
their
wild
ancestor,
red
junglefowls.
Six
addi-
tional
types
were
seen
among
more
distantly
related
green
junglefowls.
Thus,
14
of
the
64
potential
types
are
in
existence.
Fig.
1
shows
that
regardless
of
whether
they
belong
to
the
breeds
long
established
in
the
West
(Europe
and
North
America)
or
to
the
breeds
that
remained
in
Asia,
the
pre-
dominant
RFLP
type
among
domesticated
chickens
was
type
V,
closely
followed
by
type
I.
While
type
V
was
also
found
in
more
than
half
of
the
red
junglefowls
of
three
subspecies
sampled,
types
I,
II,
and
IV
have
not
thus
far
been
found
among
red
junglefowls.
Conversely,
type
VII
has
been
con-
fined
to
the
Thai
red
junglefowl
(G.
g.
spadiceus)
in
spite
of
TYP
RFLP
JUNGLEFOWLS
DOMESTIC
FOWLS
V
A
MS
MB
MS
MB
ASIATIC
OCCIDENTAL
NAGOYA
1/1
GIFU-JIDORI
2/2
-
-
- -
+
+
BLACK
SILKY
1/3
WHITE
LEGHORN
3/3
THAI
BANTAM
5/8
INDIAN
GAME
2/2
AYAM
KATAI
1/5
_.
BLACK
SILKY
1/3
III
+
_
_
+
+
THAI
RED
3/14
THAI
BANTAM
3/8
WHITE
LEGHORN
(HIROSHIMA
VAR.)1/2-
IV
+
_
_
+ +
+
TOHMARU
1/2
BLACK
SILKY
1/3
WHITE
LEGHORN
(HIROSHIMA
VAR.)1/2
BARRED
PLYMOUTH
ROCK
3/3
TOHMARU
1/2
WHITE
PLYMOUTH ROCK
1/1
WHITE
SILKY
1/1
RHODE
ISLAND
RED
1/1
DARK
BRAHMA
1/1
LIGHT
SUSSEX
2/2
HOUDAN
2/2
BUFF
COCHIN
2/2
THAI
RED
7/14
~~~FAYOMI
1/1
PARTRIDGE
COCHIN
2/2
V
+
+
-
+
+
+
THAI
RED
7/14
MALAY
GAME
2/2
SILVER
GREY
DORKING
2/2
INDONESIAN
RED
3/5
SUMATRA
GAME
2/2
JERSEY
BLACK
GIANT
2/2
AYAM
BANGKOK
2/2
BROWN
LE6HORN
1/1
AYAM
BEKISAR
2/4
LA
FRECHE
1/1
AYAM
CEMANI
1/2
ARAUCANA
1/1
AYAM KEDU
2/9
AYAM
PELUNG
1/5
THAI
RED
1/14
Vl
+
+
_
+
+
INDONESIAN
RED
1/5
AYAM
CEMANI
1/2
VIl
+
+
+
+
+
+
THAI
RED
3/14
AYAM
BEKISAR
2/4
VIII
+
+
_
+
+
NDONESIAN
RED
1/5
AYAM
KEDU
7/9
AYAM
PELUNG
4/5
A
-
+
4
GREEN
1/30
B
--- - - -
GREEN
1/30
GREEN
18/30
C
-
-
+
-
_
_
GREEN
3/30
(60-BASE-LONG
INSERTION)
D
-
-
+
-
+
_
GREEN
2/30
GREEN
3/30
E
+
-
+
_
_
GREEN
1/30
(120-BASE-LONG
INSERTION)
F
_
+-
_
_
_
_
GREEN
1/30
Proc.
Natl.
Acad.
Sci.
USA
91
(1994)
Proc.
Natl.
Acad.
Sci.
USA
91
(1994)
12507
RFLP
1)
AYAM
PEEUNG
#76
(VIII)
AATTTTATTTTTTAACCTAACTCCCCTACTAA6TGTACCCCCCCTTTCCCC,CA666GGGTATACTAT6CATAATCGT6CATACATTTATAT
94
2)
NA6OYA
#
1
(l)
AATMATTTTTTAACCTAACTCCCCTACTAA6T6TACCCCCCCMCCCC"A6666G6GTATACTAT6CATAATC6TGCATACAMATAT
94
3)
BARRED
PLYMOUTH
ROCK
#
1
(V)
AAMTATTTMAACCTAACTCCCCTACTAAGT6TACCCCCCCMCCCCCCCAG6GG666GTATACTAT6CATAATCGTGCATACAMATAT
94
4)
THAI
RED
JUNGLEFOWL
#11
(V)
AATTTTATTTMAACCTAACTCCCCTACTAA6TGTACCCCCCCMCCCCC
CAG666666GTATACTATGCATAATCGTGCATACATTTATAT
94
5)
THAI
RED
JUNGLEFOWL
#
3
(VII)
AATTTTATTTMAACCTAACTCCCCTACTAAGTGTACCCCCCCTTTCCCCCCCAGG6666GTATACTATGCATAATCGTGCATACATTTATAT
94
6)
INDN.
RED
JUNGLEFOWL
#15
(VIII)
AATTTTATTTTTTAACCTAACTCCCCTACTAA6T6TACCCCCCCTTTCCCC,A6G66GG6TATACTAT6CATAATCGT6CATACAMATAT
94
7)
GREEN
JUNGLEFOWL
#32
(C)
AATTATM
TTAACCCAACTCCCCTACTAA6TGTACCCCCCCMCCCCCA6GGG66GG6TATACTATGCATAATCGTGCATACATTTATAT
94
8)
GREEN
JUNGLEFOWL
#
2
(D)
AATMAMTTTAACCCAACTCCCCTACTAAGTGTACCCCCCCTTTCCCCAGG666GGGTATACTATGCATAATC6T6CATACATTTATAT
94
9)
GREEN
JUNGLEFOWL
#50
(E)
AATTMTATTTTTTAACCCAACTCCCCTACTAAGTGTACCCCCCCTTTCCCC,CCCAGGGGGGGTATACTATGCATAATCGTGCATACAMATAT
94
00)
JAPANESE
WUAIL
REf.
AAcAcT-'TTTMAACCTAACTCCCCTACTTA6TGTACCCCCCCMCCCC"AGGG666TATACTATGCATAATC6T6CATACAMATAT
93
1)
DMB
(VIII)
ACCACATATATTAT66TACC20TAATATATACTATATAT6TACTAAACCCATTATATGTATACG66C6TACTATATTCCACAMCTCCCAATGTCCATTCTATGCATG
206
2)
D0B
(1)
ACCACATATATTAT20TACC66TAATATATACTATATAT6TACTAAACCCATTATATGTATAC666CATTAACCTATATTCCACATCTCCCAATGTCCATTCTATGCATG
206
3)
0MB
{V)
A2ACATATATTAT06TACCG6TAATATATACTATATATGTACTAAACCCATTATATGTATAC6CArrScTATATTCCACATTTCTCCAATGTCCATTCTATGCATG
206
4)
T
RJF
(V)
ACCACATATATTAT66TACC62TA6TATATACTATATATGTACTAAACCCATTATATGTATAC6G6CAlTCTATATTCCACAMCTCCCAAT6TCCATTCTATGCATG
206
5)
T
RJF(VI
I)
ACCACATATATTAT66TACC20TAATATATACTATATATGTACTAAACCCATTATATGTATACGGG6T^ACTATATTCCACATCTCCCAATGTCCATTCTATGCATG
206
6)
1
RJFPVI11)
ACCACATATATTATVGTACCG2TAATATATACTATATATGTACTAAACCCATTATATGTATAC666cUTACATTCCTCAMCTCCAATGTCCTTCcATGCATG
206
7)
GJF
(C)
ACCACATATATTAT20TACC6GTAATATATACTATATAT6TACTAAACCCATTATATGTATACGGACATTAAcCTAcATTCCcCAMCTCCCcATGT;CATTCZAT6ATG
206
8)
GJF
(D)
ACCACATATATTAT2GTACCGGTAATATATACTATATATGTACTAAACCCATTATATGTATACGCACATTAACCTAZATTCCCCAMCTCCCZATGT;CATTCZATG;AT6
206
9)
GJF
(E)
ACCACATATAc2TATGTACC0GTAATATATACTATATATGTACTAAACCCATTATATGTATAC6GAC6AT
CTAcATTCCcCAMCTCCCcATGTACATTCcATGAATG
206
00)J
Au
20CCACATATA3TATGTACCGTAATATATATATATA6TACTAAACCCATTATATGTATAC3G6CATTA-CATATTGTCCcCATTTCTCCCcATGTACATT-AGTGCATG
203
ALUJ
'
1S
1)
DMB
(VIII)
ATCCAG6ACAT-AC-T;ATTCACCCTCCCCATAGAChZ
244
CCAAACCACTACCAAGTCACCTAAT6AATGTTGCAGGACATAAATCTCACTCTCATGCT
307
2)
DMB
(1)
ATCCAG6ACAT-AC-CCATT;ACCCTCCCCATAGACAG;T
244
CCAAACCACTA
CAAGCCACCT
AACTATGAATGTTACAGGACATAAATCTCACTCTCATGTT
307
3)
DRB
(V)
ATCCAG6ACAc-AC-TCATTCACCCTCCCCATAGAChI
244
CCAAACCACTACCAAGTCACC
ATGAATGGTTACAGGACATAAATCTCACTCTCATGTI
307
4)
T
RJF
(V)
ATCCAGGACAT-AC-TCATTCACCCTCCCCATA6AChSI
244
CCAAACCACTACCAA6TCACCTA
IT6jIAIT6TTCA66ACATAAATCTCACTCTCAT6CL
307
5)
T
RJFPVII)
ATCCAG6ACAT-AC-TCATTCACCCTCCC;ACAGAL:
244
CCAAACCACZACCAA6TCAz2AATATGAATG6TTACAGGACATAAATCTCACTCTCATGTL
307
6)
I
RJFPVIII)
ATCCAA6TCAT-TC-TTAGTCATATTCCCCATAAGCh
244
C;:AACCACTACCAAGACACCTAACTATGAATGGTTACA
GACATAAcTCT;ACTCTCATGCT
306
7)
GJF
(C)
ATCCA'G'CAT;ACA'TC-TC--C"ACCC;ATA;C'CACT
243
C;A;ACCACTAACAG6TCAC)ITCTAITGAATGGTTACAG6ACATACTTCTdAAACCAGTGCT
306
8)
GJF
(D)
ATCCA;GTCATTACATC-GTC--CTACCCCATATcTAACT
243
CTATACCACTAACAGGTCACLTTiATGAAT6TACAGGACATAccTCTAAT;CTCAT6GT
306
9)
GJF
(E)
ATCCAA;GCAT;ACGTC:GTC::C;ACCC;ATA;ZCA6CT
243
CrnACCACTAACAGGTCACTTAACTAT6AAT6GTTACA66ACATAccTCTAT'ACTAGTGCT
306
00)
J
au
CTCCAAGACAT-AAACCATAC-GTTCACCTAGTAATAG'A'-
240
----------------------------------------------------------
-j
oin
rVTDA
rnv
7)
GJF
(C)
CTACCCCTAACAGGTCACCTAACTAT6AAT
GTTACAG6ACATACTTCTAATACcAGT6CT
367
9)
GJF
(E)
CTACCCCcAACAGGTCACCTAACTAT6AATG6TTACAG6ACATACcTCTAATT6TAGTGCT
367
13RD
EXTRA
COP
7)
GJF
(C)
---------
9)
GJF
(E)
CTACCCCCAACAG6TCACCTAACTATGAATGGTTACAGGACATACCTCTAATATTAGTGC
1)
DMB
(VIII)
CcTCCCCC-AACAAGTCACC-TAACTATGAATGGTTACAGGACATACAMAACTACCATGTT
368
-CTAACCCAm
GA
MGCTCG-CCGTATCAG
399
2)
DMB
(1)
CTcCCCCC-AACAAGTCACC-TAACTATGAATGGTTACAGGACATACAMAACTACCATGTT
368
-CTAACCCATTMGGTTATGCTCG-CCGTATCAG
399
3)
DMB
(V)
CTT.CCC-AACAAGTCACC-TAACTATGAATGGTTACAGGACATACAM
AACTACCATGTT
368
-CTAACCCAMGGTTATG
TCG-CCGTATCAG
399
4)
T
RJF(V)
-LICCCCC-AACAAGTCACC-cAACTATGAATGGTTACAGGACATACAMAACTACCATGTT
368
-CTAACCCAT6GGATGCTCGTCCGTATCAG
400
5)
T
RJF(VI
I).
C
CCCT-AcCAAGTCACC-TAACTATGAATG6TTCAGGACATATAMAACTACCATGTT
368
-CTAACCCATTTGGTTATGCTCG-CCGTATCAG
399
6)
RJF(VIII)CcTCCCCC-AACAAGTCACC-TAACTAT6AATGGTTACAGGACATACATTTAACTACCATG;T
367
TCTAACCC
TTTGGTTATGCTCGT--GTATCAG
398
7)
GJF
(C)
CTACCCC;-AACA6GTCACC-TAACTATGAATGGTTACA66ACATACATcTAACTACCATG6T
430
-CTAACC-,A
=CTCGT--GTAcCAG
456
8)
GJF
(D)
CTACCCC;-AACA6GTCACC-TAACTATGAATGGTTACAGGACATACATTTAACTACCATG;T
367
-CTAACCCATGGTTATGCTCGT-CGTAcCAG
398
9)
GJF
(E)
CTACCCCC-AACAsGTCACC-TAACTATGAATGGTTACAGGACATACATCTAACTACCATGAT
489
-CTAACC-AMTGGTTATG-TCGT--GTA-CAG
516
10)
J
Au
CTT;CCAC;AACA6GACACCATAACTAT6AATGGTT0CAGGACATA
cTTA-CTA-AATAcT
302
TAGciCCC
AGACGTTATGCT^G-C0TAcCAG
334
the
sampling
of
>27
diverse
domestic
breeds.
Of
particular
interest
was
type
VIII.
Among
domesticated
chickens,
this
type
was
seen
only
in
those
breeds
originated
in
Indonesia.
At
the
same
time,
1
of
the
19
red
junglefowls
exhibiting
RFLP
type
VIII
was
also
of
Javanese
origin.
The
above
data
appeared
to
have
suggested
the
multiple
sites
of
domestica-
tion-i.e.,
Indonesian
breeds
starting
from
the
independent
regional
domestication
of
G.
gallus
bankiva.
In
the
past,
various
population
studies
utilizing
isozyme
as
well
as
blood
group
polymorphism
suggested
such
multiple
and
indepen-
dent
sites
of
domestication
(8).
The
green
junglefowl
(G.
varius)
manifested
its
own
poly-
morphism
composed
of
six
allelic
forms,
here
designated
as
types
A,
B,
C,
D,
E,
and
F.
However,
Fig.
1
shows
that
while
the
second
Mbo
II
site
in
all
individuals
of
G.
gallus
was
cleavable
by
the
enzyme,
the
corresponding
site
in
all
30 G.
varius
was
not.
If
one
excludes
this
second
Mbo
II
site
from
consideration
as
reflecting
a
pair
of
species-specific
traits
separating
G.
gallus
from
G.
varius,
type
I
of
the
former
now
becomes
the
same
as
type
A
of
the
latter
and
the
same
applies
to
type
II
and
type
B.
The
above
suggests
that
RFLP
observed
in
G.
gallus
and
G.
varius
has
been
a
very
ancient
polymor-
phism
antedating
the
separation
of
G.
gallus
from
G.
varius.
FIG.
2.
L-chain
sequences
of
the
first
400
bases of
the
mitochondrial
control
region
from
nine
gallinaceous
birds
rep-
resenting
two
wild
species (G.
gallus
and
G.
varius)
and
three
domestic
breeds
are
aligned
using
the
published
Japanese
quail
(C.
coturnixjaponica)
sequence
as
the
reference
(7).
These
nine
individuals
and
their
RFLP
types
are
identified
on
the
left.
At
each
polymorphic
site,
the
majority
base
is
shown
in
a
large
capital
letter,
whereas
a
minority
base(s)
is
shown
in
small
capital
letters
marked
by
asterisks.
CCC
base
triplets
underlined
in
the
first
section
were
bases
missed
in
two
previous
publications
(5,
7).
Four
poly-
morphic
positions
within
three
potential
restriction
sites
are
so
indicated,
Vsp
I
in
the
second
section,
Alu
I
and
Mse
I
in
the
third
section,
and
Mbo
II
in
the
bottom
section.
At
these
sites,
cleavable
se-
quences
are
underlined.
The
invariant
14-base
sequence
in
the
center
of
each
60-base
unit
is
underlined
and
so
is
the
invariant
10-base
unit
residing
near
the
end
(last
section).
Tandem
Duplication
of
60-Base
Unit
Within
the
Control
Region
as
the
Genus-Specific
Trait
of
Galus.
Before
compar-
ison
of
base
sequences
with
regard
to
the
first
400
bases of
the
control
region
L
chain,
tandem
duplication
of
one
60-base
unit
in
members
of
the
genus
Gallus
as
a
genus-specific
trait
should
be
noted.
The
control
region
base
sequence
of
White
Leghorn
chickens
(5)
and
that
of
the
Japanese
quail
Coturnix
coturnixjaponica
(7)
have
been
published,
and
it
was
shown
that
the
control
region
of
the
latter
was
41
bases
shorter
than
that
of
the
former.
Our
own
sequencing
of
type
I
White
Leghorn
and
Japanese
quail
produced
only
one
discrepancy
from
the
published
sequences
noted
above.
The
base
triplet
CCC
underlined
in
the
first
section
of
Fig.
2
was
missing
from
the
published
sequences
of
both
White
Leghorn
(5)
and
Japanese
quail
(7).
As
shown
in
the
fifth
section
of
Fig.
2
(marked
"Original"),
the
60-base
unit
containing
the
invariant
tetradecamer
AAC-
TATGAATGGTT
in
its
center
is
present
as
a
single
unit
in
the
quail,
whereas
tandem
duplication
of
this
unit
was
observed
in
all
11
G.
gallus
as
well
as
all
four
G.
varius
individuals.
In
Fig
2,
a
copy
of
the
original
located
immediately
upstream
is
marked
"lst
copy."
As
shall
be
reported
separately,
we
found
this
duplication
to
be
present
also
in
the
third
and
fourth
members
of
the
genus
Gallus:
the
grey
junglefowl
c
Evolution:
Akishinonomiya
et
al.
12508
Evolution:
Akishinonomiya
et
al.
(Gallus
sonnerati)
and
Lafayette's
junglefowl
(Gal
ettei).
Yet,
their
closest
relatives,
various
pheasai
genus
Phasianus,
were
quail-like,
having
this
60-ba
a
solitary
state.
Among
members
of
the
family
Pha
pheasants
are
far
more
closely
related
to
the
chic
quails
are,
as
evidenced
by
the
fact
that
pheasant
hybrids
are
fully
viable,
albeit
sterile,
whereas
oi
2.0%
of
the
incubated
eggs
produce
live
chicken-
quail
hybrids
(9).
Yet,
the
sequence
comparison
bel
original
and
its
first
copy
on
every
one
of
the
15
s
individuals
of
G.
gallus
and
G.
varius
indicated
average
difference
was
20%.
Interestingly,
the
difference
between
originals
of
the
Japanese
qua
Gallus
was
25%.
It
would
thus
appear
that
separati
pheasant
lineage
from
the
chicken
lineage
occurred
soon
after
that
of
the
quail
lineage
from
the
e
pheasant-chicken
lineage.
Tandem
repeats
within
ti
region
of
mitochondrial
DNA
have
previously
been
in
two
papers:
79-base
tandem
repeats
in
three
subs
the
masked
shrew
(Sorex
sinreus)
(10)
and
10-bas
repeats
in
canine
mitochondrial
DNA
(11).
Once
duplication
started,
further
duplication
wc
been
inevitable
(12).
Indeed,
one
extra
copy
of
th(
unit
was
found
in
three
green
junglefowls
of
RFL]
while
two
extra
copies
were
found
in
one
green
junj
RFLP
type
E
(Figs.
1
and
2).
One
each
of
these
in
with
one
and
two
extra
copies
was
sequenced.
)
quence
comparison
was
made
between
the
original
junglefowl
nos.
32
and
50
and
their
own
"secc
copies,"
the
uniform
sequence
difference
of
13
noted.
The
above
data
revealed
that
the
initial
furti
cation
that
produced
a
second
extra
copy
from
th
was
a
rather
ancient
affair,
probably
antedating
the
s
of
G.
varius.
Indeed,
the
presence
of
the
second
e:
was
also
noted
in
certain
individuals
of
G.
sonnera
as
G.
layfayettei.
This
shall
be
reported
separal
generation
of
the
"third
extra
copy"
by
green
jungl
50,
on
the
other
hand,
was
a
very
recent
event,
for
i
only
by
a
single
base
substitution
from
the
second
e.
of
the
same
individual
(Fig.
2).
Sequence
Differences
Between
G.
varius
and
G.
g
Affinity
of
All
the
Domestic
Breeds
to
Thai
Red
Jung]
10.542
(51.60%)
18.302
(94.5%)
20
15 10
g.
gallus).
Of
the
four
G.
varius
individuals
sequenced,
two
(nos.
6
and
32)
were
of
the
same
RFLP
type
C.
In
spite
of
the
fact
that
the
latter
was
endowed
with
the
second
extra
copy,
these
two
demonstrated
the
least
sequence
divergence
of
1.50%.
Furthermore,
all
the
substitutions
were
transitions
(Fig.
3).
In
view
of
the
considerable
antiquity
of
the
second
extra
copy
already
discussed,
this
probably
means
the
recent
loss
of
the
second
extra
copy
by
the
lineage
represented
by
no.
6.
The
difference
between
these
two
RFLP
type
C
individuals
and
no.
2
of
RFLP
type
D
increased
to
2.25%,
while
a
3.20%
sequence
difference
separated
no.
50
of
RFLP
type
E
from
the
rest.
Furthermore,
these
differences
included
a
few
transversions
(Figs.
2
and
3).
In
contrast
to
the
green
junglefowl,
which
is
a
local
species
confined
to
the
Indonesian
Islands,
the
red
junglefowl
(G.
gallus)
inhabits
a
very
large
area:
the
Asian
mainland
stretch-
ing
from
northeastern
India
in
the
west
to
the
western
coast
of
China
to
the
east.
In
addition,
its
range
includes
various
Indonesian
Islands
where
it
is
sympatric
with
G.
varius
as
well
as
Hainan
Island
in
the
South
China
Sea.
It
is
no
surprise
that
G.
gallus
has
often
been
subdivided
into
five
subspecies
(13).
As
shown
in
Fig.
2,
when
dealing
with
different
subspecies,
the
same
RFLP
type
was
no
indication
of
genetic
similarity.
Both
red
junglefowl
no.
15
and
the
domestic
breed
ayam
pelung
no.
76
typed
as
RFLP
type
VIII
and
they
were
from
the
same
Indonesia
island.
Yet,
5.75%
sequence
divergence
separated
the
two.
Furthermore,
9
of
the
23
substitutions
were
transversions
(Fig.
2).
The
above
clearly
excluded
the
involvement
of
G.
gallus
bankiva
in
the
domestication
event.
In
sharp
contrast,
all
three
Thai
red
junglefowls
(two G.
g.
gallus
and
one
G.
g.
spadiceus)
were
very
close
to
all
breeds
of
domestic
chicken.
The
closest
affinity
of
only
0.5%
(one
each
of
transition
and
deletion)
difference
was
seen
between
Thai
red
junglefowl
no.
11
of
RFLP
type
V
and
a
member
of
the
Indonesian
breed,
ayam
cemani,
of
the
same
RFLP
type
(Fig.
4).
Of
three
subspecies
of
the
red
junglefowl,
G.
g.
gallus
(Thai
nos.
8
and
11)
was
far
more
closely
related
to
G.
g.
spadiceus
(Thai
no.
3)
from
the
adjacent
area
than
to
G.
g.
bankiva
from
Java
(Indonesian
no.
15).
Nevertheless,
a
transversion
was
involved
in
a
difference
between
the
first
two
and
RFLP
type
VII
was
unique
to
G.
g.
spadiceus.
1.25%
(1.25X)
as
* *
a
*
i
86
5
4
2
O
F
THAI
RED
JUNGLEFOWL
#
11
(V)
L
THAI
RED
JUNGLEFOWL
#
8
(V)
J
THAI
RED
JUNGLEFOWL
#
3
(VIl)}
INDONESIAN
RED
JUNGLEFOWL
#
15
(V
GREEN
JUNGLEFOWL
#
6
(C)
GREEN
JUNGLEFOWL
#
32
(C)
(60-BASE-LONG
INSERTION)
GREEN
JUNGLEFOWL
#
2
(D)
GREEN
JUNGLEFOWL
#
50
(E)
i-
n
(120-BASE-LONG
INSERTION)
JAPANESE
QUAIL
} d
PERCENT
SEQUENCE
DIFFERENCE
FIG.
3.
Dendrogram
based
on
sequence
divergence
with
regard
to
the
first
400
bases
of
the
mitochondrial
control
region
of
four
G.
varius
and
two
G.
g.
gallus
and
one
each
of
G.
g.
spadiceus
and
G.
g.
bankiva.
Japanese
quail
(7)
was
chosen
as
the
outgroup.
Sequence
difference
is
shown
as
percentage
at
each
branch
point.
Often
larger
percentages
in
parentheses
are
derived
by
regarding
each
transversion
as
an
equivalent
of
10
transitions.
Proc.
Natl.
Acad.
Sci.
USA
91
(1994)
Proc.
Natl.
Acad.
Sci.
USA
91
(1994)
12509
167
171
210
217
220
221
225
243
246 254 256
261
265
281
282 306 309
310 315
317
327
342
391' 394'
THAI
RED
JUNGLEFOWL
#
8
(V)
T
C C
T
T
C
C
C
C T
C
T
C
G
T
C
T
TC
A
T
G
-
-
THAI
RED
JUNGLEFOWL
#11
(V)
T T
C
T
T C
C
C
C T
C
T
C
G
C C
T T
CA
C
A
T
-
AYAM
CEMANI
(V)
T T
C
T T
C
C C
C
T
C
T
C
G
C
C
T
T C
A
T
A
-
-
BARRED
PLYMOUTH
ROCK
(v)
T T
C C
T
CC
C
C
T
C
T
C A C
T
T
T C
A
T
A
-
-
WHITE
LEGHORN(HIROSHIMA
VAR.)
(V)
T
T
C
C
T
C
C
C
T
T
C
T
C A C
T
T T
C
A
T
A
-
T
WHITE
LEGHORN(HIROSHIMA
VAR.)
(IV)
T
T
C
C
T
C
C
T
C
T
C
T
C A C
T T T
CA
T
A
-
T
THAI
RED
JUNGLEFOWL
#
3
(VII)
T
C C
T T
C
C C
C
C C
T
T
A C
T T T
T
C
T
G
-
-
AYAM
PELUNG
(VIII)
T T
C
T
T T
C C
C
T
C
T
C
G C C C
T
C
A
T
A
-
-
WHITE
LEGHORN
(I)
C
T
T T T
C
T
T
C
T
T
C
C A C