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(Legal Code) .
Disclaimer

Dissertation for the Degree

of Master

Development of Molecular Markers for Authentication

of Korean Ginseng and Japanese Ginseng

by
Chang-Ho Ahn
Department of Forestry

Graduate School
Kangwon National University

February, 2009

- I -

Under the Guidance of

Professor Yong-Eui Choi

Development of Molecular Markers for Authentication

of Korean Ginseng and Japanese Ginseng
A DISSERTATION
Submitted to the Graduate School of
Kangwon National University in Partial
Fulfillment of the Requirements
for the Degree of

Master of Chang-Ho Ahn

by
Chang-Ho Ahn
Department of Forestry
February, 2009

Approved by Committee of the Graduate School of

Kangwon National University in Partial Fulfillment of
the Requirements for the Degree of
Master of Agriculture

Chang-Ho Ahn

December, 2008

Thesis Committee ;
(Signature)
(Signature)
(Signature)

- III -

Development of Molecular Markers for Authentication of

Korean Ginseng and Japanese Ginseng
Chang-HoAhn
Depa
r
t
mentofFor
es
t
r
y
Gr
a
duat
eSc
hool
,Ka
ngwonNa
t
i
onalUni
ver
s
i
t
y

Abst
r
act

Panaxgi
ns
eng C.
A.Me
ye
r
,commonl
y known asKor
e
an gi
ns
e
ng,i
sa
pe
r
e
nni
alhe
r
b nat
i
vet
oKor
e
aandChi
na.I
t
sr
oot
sar
ehi
ghl
y pr
i
z
e
df
or
var
i
ous me
di
c
i
nal pr
ope
r
t
i
e
s
. The i
nt
e
r
nat
i
onal t
r
ade of gi
nse
ng i
s
i
nc
r
e
asi
ng year
l
y.Duet
ot
hi
sf
ac
t
,di
s
gui
s
e
dChi
nes
egi
ns
engi
nt
oKor
e
an
gi
nse
ng be
came a pr
obl
em i
n wor
l
d and al
s
o i
n Kor
e
an mar
ke
t
.
The
r
e
f
or
e
, an e
f
f
i
ci
e
nt me
t
hod i
sr
e
qui
r
e
dt
o di
s
t
i
ngui
sh be
t
we
e
n P.
gi
ns
eng andot
he
rPanaxspe
ci
e
sataDNA l
e
vel
.Themol
e
cul
armar
ker
s
we
r
e acc
e
pt
e
d as mor
ec
onf
i
de
ntme
t
hods t
han t
he mor
phol
ogi
caland
c
hemi
calanal
ys
i
s
.Mol
e
cul
ar aut
he
nt
i
c
at
i
ons of di
f
f
e
r
ent Panax s
pe
c
i
e
s
we
r
er
e
por
t
e
di
n some e
xt
e
ntus
i
ng RAPD,PCR-RFLP,ge
nese
que
nc
e
s
.
Howe
ve
r
,mol
ec
ul
ar mar
ke
rt
o aut
he
nt
i
c
at
et
he Kor
e
an gi
nse
ng f
r
om
ot
herf
or
e
i
gn gi
nse
ng i
sr
emai
ne
dt
o bec
l
e
ar
.Ampl
i
f
i
e
df
r
agme
ntl
engt
h
pol
ymor
phi
sm (
AFLP) anal
ys
i
s has t
he advant
age of det
e
ct
i
ng l
e
ngt
h

- I -

di
f
f
er
e
nce
sassmal
las1bp,andi
sabl
et
odet
e
ctmor
ei
nde
pe
nde
ntl
oc
i
f
or pol
ymor
phi
s
m t
han ot
her c
ur
r
e
nt
l
y avai
l
abl
e PCR-bas
e
dt
e
c
hni
que
s
.
Mi
c
r
osat
e
l
l
i
t
e
s ar
e hi
ghl
y pol
ymor
phi
c
,c
odomi
nant mar
ke
r
st
hat have
pr
ove
nuse
f
ulf
oravar
i
et
yofpur
pos
e
si
nc
l
udi
ngf
i
nger
-pr
i
nt
i
ng.
I
nt
hi
ss
t
udy,we de
vel
ope
d a ge
net
i
c mar
ke
r
s as a f
as
t
,ef
f
i
c
i
e
ntand
e
as
y me
t
hods f
or t
he aut
he
nt
i
c
at
i
on among di
f
f
e
r
e
ntPanax s
pe
c
i
e
s by
us
i
ngt
heAFLP andmi
c
r
osat
e
l
l
i
t
eanal
ysi
st
e
c
hni
que
.AFLP anal
ys
i
swas
c
ar
r
i
ed outt
o aut
he
nt
i
c
at
et
he P.j
aponi
c
us among P.gi
ns
eng and P.
qui
nquef
ol
i
us
.A cl
ears
pe
c
i
e
s-spe
ci
f
i
cAFLP bandsf
orP.j
aponi
c
uswas
gene
r
at
e
d.Af
t
e
ri
s
ol
at
i
on and se
quenc
i
ng oft
heAFLP f
r
agment
,aDNA
s
eque
nce(
293bp)wasobt
ai
ne
d and name
dJ
G14.Ol
i
gonuc
l
e
ot
i
depr
i
me
r
(
23 me
r
)was de
s
i
gne
df
orampl
i
f
yi
ng 191 bp oft
he se
quenc
e ofJ
G14.
PCR anal
ys
i
sr
e
ve
al
e
d ac
l
earampl
i
f
i
edband f
orP.j
aponi
c
usbutnoti
n
3ot
he
rPanaxs
pe
c
i
e
s(
P.gi
ns
eng,P.qui
nquef
ol
i
usandP.not
ogi
ns
eng)
.
Thi
s se
que
nc
ec
har
act
e
r
i
z
e
d ampl
i
f
i
e
dr
e
gi
ons (
SCAR) mar
ke
r wi
l
lbe
us
e
df
orr
api
d aut
he
nt
i
c
at
i
on ofP.j
aponi
c
usamong ot
he
rr
e
l
at
e
d Panax
s
pe
ci
es
. Thi
s i
s t
he f
i
r
s
tr
e
por
t of s
pe
c
i
e
s-spec
i
f
i
c SCAR mar
ke
r
de
vel
opme
nti
nP.j
aponi
c
us
.
Ont
heot
he
rhands,mi
c
r
os
at
e
l
l
i
t
ef
r
agment
s(
252c
l
one
s)we
r
ei
sol
at
e
d
f
r
om

genomi
c l
i
br
ar
y. The
n s
eque
nce of t
he
s
e f
r
agme
nt
s was

de
t
e
r
mi
nat
e
d.Bas
e upon se
que
nc
ei
nf
or
mat
i
on,38 mi
c
r
osat
e
l
l
i
t
e pr
i
me
r
s
we
r
e obt
ai
ne
d. Among t
he pr
i
me
r
s t
es
t
ed, 6 hi
ghl
y pol
ymor
phi
c
mi
c
r
osat
e
l
l
i
t
emar
ke
r
s(
c
al
l
e
dSSR mar
ke
r
)s
uc
hasPG409,PG450,PG491,
and PG582 wer
ese
l
e
c
t
e
df
oranal
ys
i
sofgi
nse
ng.SSR anal
ys
i
sr
e
ve
al
ed

- II -

t
hatt
he
semar
ker
s coul
d beappl
i
e
dt
o di
s
t
i
ngui
sh Kor
e
an gi
ns
e
ng f
r
om
Chi
ne
s
egi
ns
e
ng.
Co
nc
l
us
i
ve
l
y,t
he
s
e ki
nds ofmar
ke
rwi
l
lof
f
e
ran e
f
f
i
c
i
e
ntappr
oac
ht
o
aut
he
nt
i
c
at
eKor
e
angi
nse
ngf
r
om c
hi
nes
egi
nse
ngandJ
apane
s
egi
ns
e
ng.

- III -

CONTENTS
ABSTRACT

I
LI
ST OF TABLES

LI
ST OF FI
GURES

I
NTRODUCTI
ON

1
Chapt
er1.Devel
opmentofspeci
esspeci
f
i
cAFLP-der
i
ved
SCAR mar
kerf
oraut
hent
i
cat
i
on ofPanaxj
aponi
cusC.A.
Meyer

4
Abs
t
r
ac
t

4
1.
1I
nt
r
oduc
t
i
on

5
1.
2Mat
e
r
i
al
sandMet
hods

6
1.
2.
1Pl
antmat
e
r
i
al
sande
xt
r
ac
t
i
onofge
nomi
cDNA

6
1.
2.
2Ge
nomi
cDNA di
ge
s
t
i
onandadapt
e
rl
i
gat
i
on

1.
2.
3AFLP anal
ys
i
s

7
1.
2.
4Cl
oni
ng,DNA se
que
nc
i
ngandSCAR pr
i
merde
si
gn

8
1.
2.
5PCR anal
ys
i
s

9
1.
3Re
s
ul
t
sandDi
sc
us
si
on

10
1.
3.
1AFLP anal
ys
i
s

10
1.
3.
2I
s
ol
at
i
onofAFLP f
r
agme
ntf
orSCAR mar
ke
rde
si
gn

11
1.
3.
3PCR ampl
i
f
i
c
at
i
onusi
ngSCAR pr
i
mer
s

11
Chapt
er2.Devel
opmentofnew mi
cr
osat
el
l
i
t
emar
ker
sf
or
Panaxgi
nseng

20
Abs
t
r
ac
t

20
2.
1I
nt
r
oduc
t
i
on

21
2.
2Mat
e
r
i
al
sandMet
hods

22
2.
2.
1Pl
antmat
e
r
i
al
sande
xt
r
ac
t
i
onofge
nomi
cDNA

22
2.
2.
2Pr
i
merde
si
gnandPCR ampl
i
f
i
c
at
i
on

23

- IV -

2.
2.
3El
ec
t
r
ophor
es
i
sands
i
l
ve
rst
ai
ni
ng

23
2.
3Re
s
ul
t
sandDi
sc
us
si
on

24
2.
3.
1Pr
i
merde
si
gn

24
2.
3.
2Aut
he
nt
i
c
at
i
onofKor
eangi
ns
e
ngf
r
om Chi
ne
s
egi
ns
e
ng
24
2.
3.
3Anal
ys
i
si
nYangyang-gun(
A.
D.
T.
C.
)s
ampl
e
s

25
Li
t
er
at
ur
eci
t
ed

37

- V -

LI
ST OF TABLE
Tabl
e1.
1Dat
aofcol
l
e
c
t
e
ds
ampl
e
sofvar
i
ouski
ndsofgi
ns
eng r
oot
sf
or
AFLP

13
Tabl
e 1.
2 Ol
i
gonuc
l
e
ot
i
de adapt
er
s and pr
i
mer
s us
e
df
orAFLP anal
ys
i
s

14
Tabl
e2.
1Sampl
e
sofvar
i
ousgi
nse
ngf
orMi
c
r
os
at
e
l
l
i
t
eanal
ysi
s

27
Tabl
e 2.
2 Yangyang-gun (
A.
D.
T.
C.
)s
ampl
e
s of var
i
ous gi
ns
e
ng f
or
Mi
c
r
osat
e
l
l
i
t
eanal
ysi
s

28
Tabl
e 2.
3 Mi
c
r
os
at
el
l
i
t
e pr
i
me
r
s,nuc
l
e
ot
i
de se
quenc
e
s,cor
e mot
i
f
s and
numbe
rofr
epe
at
sande
xpe
c
t
e
dpr
oductsi
z
ef
orSSR

29

- VI -

LI
ST OF FI
GURE
Fi
g.1.
1 AFLP anal
ys
i
s of t
he P.j
aponi
c
us
,P.qui
nquef
ol
i
us
,and P.
gi
ns
eng usi
ngpr
i
me
rc
ombi
nat
i
onMs
eI
+GTT/
Ec
oRI
+TA

15
Fi
g.1.
2Se
que
nceoft
hes
pe
c
i
e
ss
pe
ci
f
i
cDNA f
r
agme
nt
sofP.j
aponi
c
us
.

16
Fi
g.1.
3PCR anal
ys
i
sus
i
ngJ
G14pr
i
me
r
s

17
Fi
g.1.
4PCR anal
ysi
sf
r
om var
i
ouski
ndsofs
ampl
e
susi
ng J
G14pr
i
me
r
s

18
Fi
g.1.
5PCR ampl
i
f
i
cat
i
on ofSCAR f
r
agmenti
n di
f
f
e
r
e
nts
ampl
e
sofP.
j
aponi
c
us

19
Fi
g.2.
1 Ge
ne
t
i
c di
ve
r
s
i
t
y ofSSR anal
ysi
s usi
ng PG409 pr
i
me
rbe
t
we
e
n
Kor
e
an-andChi
ne
s
egi
ns
e
ng

30
Fi
g.2.
2 Ge
ne
t
i
c di
ve
r
s
i
t
y ofSSR anal
ysi
s usi
ng PG450 pr
i
me
rbe
t
we
e
n
Kor
e
an-andChi
ne
s
egi
ns
e
ng

31
Fi
g.2.
3 Ge
ne
t
i
c di
ve
r
s
i
t
y ofSSR anal
ysi
s usi
ng PG491 pr
i
me
rbe
t
we
e
n
Kor
e
an-andChi
ne
s
egi
ns
e
ng

32
Fi
g.2.
4 Ge
ne
t
i
c di
ve
r
s
i
t
y ofSSR anal
ysi
s usi
ng PG582 pr
i
me
rbe
t
we
e
n
Kor
e
an-andChi
ne
s
egi
ns
e
ng

33
Fi
g.2.
5 Ge
net
i
c di
ve
r
s
i
t
y ofSSR anal
ysi
s us
i
ng PG491 pr
i
mer among
t
heYangyang-gun s
ampl
e
s pr
ovi
de
df
r
om A.
D.
T.
C.and Chi
ne
s
e
gi
nse
ng

34
Fi
g.2.
6 Ge
net
i
c di
ve
r
s
i
t
y ofSSR anal
ysi
s us
i
ng PG582 pr
i
mer among
t
heYangyang-gun s
ampl
e
s pr
ovi
de
df
r
om A.
D.
T.
C.and Chi
ne
s
e
gi
nse
ng

35

- VII -

I
NTRODUCTI
ON
Panax (
Ar
al
i
ac
eae
)i
s a ge
nus ofabout12 spe
ci
e
s di
st
r
i
but
e
di
nt
he
wor
l
d and e
ac
h oft
he
m has be
e
n use
d as t
r
adi
t
i
onalme
di
c
i
ne
s.Panax
gi
ns
eng, c
ommonl
y known as Kor
ean and As
i
an gi
nse
ng. P.
qui
nquef
ol
i
um i
s an anot
her i
mpor
t
ant gi
ns
e
ng s
pe
c
i
e
s (
Ame
r
i
c
an
gi
nse
ng)
,whi
c
hi
sdi
s
t
r
i
but
e
di
nNor
t
hAme
r
i
c
a.P.j
aponi
c
usi
savai
l
abl
e
i
nJ
apanandSout
he
r
nar
e
aofChi
na.
Panaxspec
i
e
shavebee
nus
edast
oni
c
sf
orant
i
-s
t
r
e
s
s,ant
i
-f
at
i
gue
,and
ant
i
-agi
ng pur
pos
e
s.Gi
nse
ng pr
oduc
t
s have be
c
ome ver
y popul
ar and
have at
t
r
ac
t
e
d wor
l
dwi
de c
ons
umpt
i
on.Howe
ve
r
,Or
i
ent
algi
nse
ng and
Ame
r
i
c
an gi
ns
e
ng ar
e known t
o have di
f
f
e
r
e
ntpr
ope
r
t
i
e
s and me
di
c
i
nal
val
ues
.The r
ed f
or
m of Or
i
e
nt
algi
nse
ng,pr
oduc
e
d by st
e
ami
ng,i
s
"
war
m" and known t
or
e
pl
e
ni
sh t
he "
vi
t
ale
ner
gy"
,whe
r
e
as Ame
r
i
can
gi
nse
ng i
s"
c
ool
"andi
smai
nl
y use
df
orr
e
duc
i
ng t
he"
i
nt
e
r
nalhe
at
"and
pr
omot
i
ngt
hes
ec
r
e
t
i
onofbodyf
l
ui
ds.
I
n addi
t
i
on, gi
nse
nos
i
de
s (
gi
ns
e
ng s
aponi
ns) ar
e known t
o be t
he
bi
oac
t
i
ve c
ompone
nt
s of gi
nse
ng. Ac
c
or
di
ng t
ot
he di
f
f
e
r
e
nc
ei
n t
he
agl
ycone i
nt
he
se saponi
ns
,gi
ns
enos
i
de
s ar
e cl
as
si
f
i
e
di
nt
ot
hr
e
et
ype
s:
t
he20(
S)
-pr
ot
opanaxadi
olt
ype(
e
.
g.
,gi
ns
e
nos
i
desRb1,Rc
,Rb2,and Rd)
,
t
he 20(
S)
-pr
ot
opanaxadi
olt
ype (
e
.
g.
,gi
ns
e
nos
i
des Rg1,Rf
,and Re)
,and
t
he ol
e
anol
i
c ac
i
d t
ype (
e
.
g.
, gi
nse
nos
i
de
s Ro) (
Chan et al
.
, 2000)
.
Gi
nse
nos
i
de
s Rg1,Re
,Ro,Rb1,Rc,Rb2,and Rd ar
e pr
e
se
nt i
n bot
h
Or
i
e
nt
al and Ame
r
i
c
an gi
ns
e
ngs i
n di
f
f
e
r
ent pr
opor
t
i
ons
, whe
r
e
as
gi
nse
nos
i
de Rfc
an onl
y be f
ound i
n Or
i
e
nt
algi
ns
eng (
Chuang etal
.
,
1995)
.I
nc
ont
r
as
t
,Ame
r
i
c
an gi
ns
e
ng c
ont
ai
ns 24-(
R)
-ps
e
udogi
ns
e
nos
i
de
F11,an ocot
i
l
l
olt
ypet
r
i
t
e
r
pe
newhi
c
hi
sabs
e
nti
n Or
i
e
nt
algi
ns
eng (
Dou
etal
.
,1998;Che
netal1981)
.
Among Panax spec
i
e
s
,Kor
ean P.gi
ns
eng i
st
he be
st
-known pr
oduc
t

- 1 -

wi
t
h hi
gh me
di
ci
nal val
ue
s i
n t
he wor
l
d. Howe
ve
r
,i
n t
he gi
nse
ng
mar
ke
t
s wor
l
dwi
de
,Amer
i
c
an gi
nse
ng us
ual
l
yc
ommands a muc
h hi
ghe
r
pr
i
c
et
han t
he sun-dr
i
e
d Or
i
e
nt
al gi
ns
e
ng.Si
nc
et
he r
oot
s of t
he
s
e
gi
nse
ngs ar
e s
i
mi
l
ar i
n appe
ar
ance and many comme
r
c
i
al gi
ns
e
ng
pr
oduct
s ar
ei
nf
or
m ofpowde
rors
hr
e
dde
ds
l
i
c
e
s
,i
de
nt
i
f
i
c
at
i
on oft
he
or
i
gi
ns oft
he gi
ns
e
ng pr
oduc
t
si
s notan e
as
yt
as
k.Due t
ot
hi
sf
ac
t
,
di
s
gui
s
e
d Chi
nes
e and Ame
r
i
c
an gi
ns
e
ng i
nt
o Kor
e
an gi
ns
e
ng bec
ame a
pr
obl
em i
n wor
l
d and al
s
oi
n Kor
e
an mar
ke
t
.I
n Kor
e
a,wi
l
d-gr
own and
wi
l
d-cul
t
i
va
t
e
d Kor
e
an gi
nse
ng i
ss
ol
d wi
t
h hi
gh pr
i
ce
sandt
hedi
sgui
s
e
ofChi
ne
s
ewi
l
dgi
nse
ngi
nt
oKor
eangi
ns
engi
sse
r
i
ouspr
obl
e
mi
nKor
e
a,
whi
c
h may ber
e
s
ul
t
e
di
nt
hede
pr
es
s
i
on ofgi
ns
e
ng mar
ke
t(
Ki
m etal
.
,
2005)
.
The
r
e
f
or
e
,i
ti
sve
r
yi
mpor
t
antt
oaut
he
nt
i
cat
et
heKor
e
an gi
nse
ng f
r
om
Chi
ne
s
e gi
nse
ng and f
r
om ot
he
r di
f
f
e
r
e
nt gi
ns
eng s
pe
c
i
e
s.The
r
e ar
e
s
omer
e
por
t
son t
hemor
phol
ogi
c
aland che
mi
calaut
he
nt
i
c
at
i
on ofKor
e
an
gi
nse
ng be
t
we
e
n ot
herf
or
e
i
gn P.gi
ns
eng and di
f
f
er
e
ntgi
ns
e
ng s
pe
c
i
e
s.
The
r
ear
es
omedi
f
f
e
r
e
nc
e
s be
t
wee
n P.gi
ns
eng and P.qui
nquef
ol
i
us i
n
mor
phol
ogi
cal c
har
ac
t
er
s and saponi
ns (
Chung et al
.
, 1995, 1998)
.
Howe
ve
r
,i
ti
s ve
r
y di
f
f
i
cul
tt
o di
f
f
er
e
nt
i
at
et
he char
ac
t
e
r
s ofgi
nse
ng
wi
t
hi
nsamespe
ci
e
sal
t
hought
he
ywer
ec
ul
t
i
vat
e
di
ndi
f
f
e
r
e
ntc
ount
r
y.
Themol
e
cul
armar
ke
r
swe
r
eacc
e
pt
e
dasmor
ec
onf
i
de
ntme
t
hodsast
he
mor
phol
ogi
calandc
he
mi
c
alanal
ys
i
s
.Mol
e
c
ul
araut
he
nt
i
c
at
i
onsofdi
f
f
e
r
e
nt
Panax spe
ci
e
s we
r
er
e
por
t
e
di
ns
ome e
xt
e
ntus
i
ng RAPD (
Shi
m etal
.
,
2003;Um etal
.
,2001)
,PCR-RFLP (
Ngan etal
.
,199
9;Fushi
mietal
.
,
1997)
,ge
nes
e
que
nce
s(
Komat
s
y etal
.
,2001;Shu etal
.
,1997)
.Howe
ve
r
,
mol
ec
ul
ar mar
ke
rt
o aut
he
nt
i
c
at
et
he Kor
e
an P.gi
ns
eng f
r
om ot
he
r
f
or
e
i
gnP.gi
ns
eng i
sr
e
mai
ne
dt
obec
l
e
ar
.
Ampl
i
f
i
e
df
r
agme
ntl
e
ngt
h pol
ymor
phi
sm (
AFLP)i
s a PCR bas
e
dt
ool
use
di
n ge
ne
t
i
c
sr
e
s
e
ar
c
h,DNA f
i
nge
r
pr
i
nt
i
ng,and i
nt
he pr
act
i
c
e of

- 2 -

gene
t
i
ce
ngi
ne
e
r
i
ng.AFLP-PCR i
sahi
ghl
ys
e
nsi
t
i
veme
t
hodf
ordet
e
ct
i
ng
pol
ymor
phi
s
msi
npl
antspec
i
e
s
.
Mi
c
r
osat
e
l
l
i
t
e
s
,al
s
o known as SSRs (
s
i
mpl
es
eque
nce r
e
pe
at
s
) ar
ea
s
mal
l ar
r
ay of t
ande
ml
y ar
r
ange
d 1-6 bas
es spr
e
ad t
hr
oughout t
he
genomes
.Mi
c
r
os
at
e
l
l
i
t
e
s as DNA mar
ke
r
s ar
e advant
age
ous ove
rmany
ot
her mar
ke
r
s as t
hey ar
e hi
ghl
y pol
ymor
phi
c
, hi
ghl
y abundant
,
c
o-domi
nant i
nhe
r
i
t
anc
e, anal
yt
i
c
al
l
y si
mpl
e and r
e
adi
l
y t
r
ans
f
e
r
abl
e
.
Mi
c
r
osat
e
l
l
i
t
e
sar
er
e
por
t
e
dt
o be mor
evar
i
abl
et
han RFLPs orRAPDs,
andhavebee
nwi
de
l
yut
i
l
i
z
e
di
npl
antge
nomi
cst
udi
es
.
I
nt
hepr
e
s
e
ntst
udy,t
hec
ombi
nat
i
onoft
he
semar
ke
r
sde
t
e
r
mi
nat
e
dby
t
wo t
ec
hni
ques wi
l
lof
f
er an e
f
f
i
c
i
e
ntappr
oach t
o aut
he
nt
i
c
at
e Kor
e
an
gi
nse
ngf
r
om f
or
e
i
gngi
ns
e
ng.

- 3 -

Chapt
er 1. Devel
opment of speci
es speci
f
i
c
AFLP-der
i
ved SCAR mar
ker f
or aut
hent
i
cat
i
on of
Panaxj
aponi
cusC.A.Meyer
Abst
r
act
Panaxj
aponi
c
usi
san i
mpor
t
antme
di
ci
nalpl
ant
.Theai
m oft
hi
ss
t
udy
was t
o deve
l
op spec
i
e
s
-s
pe
c
i
f
i
c mol
e
c
ul
ar mar
ke
r
sf
or P. j
aponi
c
us
.
Ampl
i
f
i
e
df
r
agmentl
e
ngt
hpol
ymor
phi
s
m(
AFLP)wasc
ompar
e
damongP.
j
aponi
c
us
,P.gi
ns
eng andP.qui
nquef
ol
i
us
.A c
l
e
arspe
ci
e
s
-s
pe
c
i
f
i
cAFLP
mar
ke
rf
orP.j
aponi
c
uswasge
ne
r
at
e
d.Af
t
e
ri
s
ol
at
i
on ands
e
que
nc
i
ng of
t
heAFLP f
r
agment
,aDNA s
e
que
nc
e(
293bp)wasobt
ai
ne
d and na
me
d
J
G14.Ol
i
gonuc
l
e
ot
i
depr
i
me
r(
23me
r
)wasde
s
i
gne
df
orampl
i
f
yi
ng19
1bp
oft
hes
e
que
nc
eofJ
G14.PCR anal
ys
i
sr
e
ve
al
edac
l
e
arampl
i
f
i
e
dbandf
or
P. j
aponi
c
us but not i
n 3 ot
he
r Panax s
pe
c
i
e
s (
P. gi
ns
eng, P.
qui
nquef
ol
i
usand P.not
ogi
ns
eng)
.Thi
ss
e
que
ncec
har
a
ct
e
r
i
z
e
d ampl
i
f
i
e
d
r
egi
ons (
SCAR) mar
ke
r wi
l
l be use
d f
or r
api
d aut
he
nt
i
cat
i
on of P.
j
aponi
c
usamong ot
he
rr
e
l
at
e
d Panax s
pe
c
i
e
s.Thi
si
st
hef
i
r
s
tr
e
por
tof
s
pe
ci
es
-s
pe
c
i
f
i
cSCAR mar
ke
rde
ve
l
opme
nti
nP.j
aponi
c
us
.

- 4 -

1.
1I
nt
r
oduct
i
on
Panax i
sage
nusc
ompr
i
s
i
ng s
e
ve
r
als
pe
ci
esofs
l
ow-gr
owi
ng per
e
nni
al
me
di
c
i
nalpl
ant
s
,i
nt
he f
ami
l
y Ar
al
i
ace
ae.The
y gr
ow i
nt
he Nor
t
he
r
n
He
mi
s
phe
r
ei
ne
as
t
e
r
n As
i
a(
Kor
e
a,nor
t
he
r
n Chi
na,e
as
t
e
r
n Si
be
r
i
a and
J
apan)andNor
t
hAme
r
i
ca,t
ypi
cal
l
yi
nc
ool
e
rcl
i
mat
e
s.P.j
aponi
c
usC.A.
Me
ye
r(
J
apane
se name
; Chi
kus
e
t
s
uni
nj
i
n) i
s di
s
t
r
i
but
e
d i
n J
apan and
s
out
h-we
s
t
e
r
n Chi
na,and has a l
ong r
hi
z
ome,be
i
ng mor
phol
ogi
c
al
l
y
s
i
mi
l
art
obamboor
hi
z
ome
.I
thasbe
e
nr
e
por
t
e
dt
hatt
her
hi
z
ome
sofP.
j
aponi
c
us have an ant
i
ul
c
e
r act
i
on and have be
e
n us
ed t
o t
r
eat
f
i
br
i
nol
ys
i
s(
Yamahar
aetal
.
,1987;Mat
s
udaetal
.
,1989)
,ant
i
obes
i
t
y(
Han
et al
.
,2005)
,and suppr
es
s
i
on of apopt
os
i
s(
Hos
ono-Ni
s
hi
yama et al
.
,
2006)and t
os
moot
hc
oughs and r
e
duce phl
e
gm (
Chang and But
,1986)
The mai
n act
i
ve compone
nt i
n P.j
aponi
c
us i
s ocot
i
l
l
ol
-t
ype s
aponi
ns
(
Zouetal
.
,2002)
.
The i
de
nt
i
f
i
cat
i
on of spec
i
e
s
-s
pe
c
i
f
i
c DNA mar
ke
r
s of Panax s
pe
c
i
e
s
woul
d be ofgr
e
ati
mpor
t
ance t
o di
s
cr
i
mi
nat
et
he spe
ci
e
s
.Anal
ys
i
s of
we
l
l
-char
ac
t
e
r
i
z
e
dc
ompounds
,gi
ns
e
nosi
de
s
,i
st
he mos
tpopul
arme
t
hod
f
ori
de
nt
i
f
yi
ng t
hePanaxspe
ci
e
sand qual
i
t
yc
ont
r
olofgi
ns
e
ng pr
oduct
s
(
Chanetal
.
,2000)
.Thepr
of
i
l
e
sofgi
ns
e
nos
i
desi
nr
oot
sofPanaxs
pe
c
i
e
s
ar
e ve
r
y s
i
mi
l
ar and af
f
e
c
t
e
d s
i
gni
f
i
c
ant
l
y by gr
owt
h and s
t
or
age
c
ondi
t
i
ons
,and har
ve
s
tt
i
me
s.Ge
net
i
ct
ool
s ar
ec
onsi
de
r
e
dt
o pr
ovi
de
mor
e s
t
andar
di
z
e
d and r
e
l
i
abl
e me
t
hods f
or aut
he
nt
i
c
at
i
on of pl
ant
mat
e
r
i
al
satt
heDNA l
eve
l
.Us
i
ng Panaxs
pe
c
i
e
s,t
heme
t
hodsde
ve
l
ope
d
pr
e
vi
ous
l
yi
nc
l
ude l
ow-Cot DNA f
i
nge
r
pr
i
nt
i
ng (
Ho and Le
ung,2002)
,
r
andoml
yampl
i
f
i
e
dpol
ymor
phi
cDNA (
RAPD)(
Baietal
.
,1997;Shaw and
But
,1995) or ar
bi
t
r
ar
i
l
y pr
i
me
d pol
yme
r
as
ec
hai
n r
e
act
i
on (
AP-PCR)
(
Cheungetal
.
,1994)
,PCR-r
andom f
r
agme
ntl
e
ngt
hpol
ymor
phi
sm (
RFLP)
(
Fus
hi
mi et al
.
, 1997; Ngan et al
.
,1999)
,ampl
i
f
i
e
d f
r
agme
nt l
e
ngt
h

- 5 -

pol
ymor
phi
s
m (
AFLP)(
Ki
m e
tal
.
,2005)andmi
c
r
os
at
e
l
l
i
t
emar
ke
r
s(
Hon
etal
.
,2003)andi
nt
e
r
nalt
r
ansc
r
i
be
dspac
e
r
s(
I
TS)s
e
que
nc
e
sofr
i
bosomal
DNA (
We
n and Zi
mme
r
,1996)
,and mi
c
r
ochi
pe
l
e
c
t
r
ophor
e
s
i
s(
Qi
n etal
.
,
2005)
.
Par
an and Mi
che
l
mor
e (
1993) de
ve
l
ope
d a t
e
chni
que known as
s
eque
nce
-c
har
act
e
r
i
z
e
d ampl
i
f
i
e
dr
e
gi
ons (
SCAR)
.SCAR mar
ke
r
s have
be
e
n der
i
ve
df
r
om RAPD and AFLP mar
ker
sand havepr
ove
n us
e
f
uli
n
i
de
nt
i
f
yi
ng t
hepl
ant
satt
hei
nt
r
aori
nt
e
r
-s
pe
c
i
f
i
cl
e
ve
l
.SCAR asaPCR
base
d ge
ne
t
i
c mar
ke
ri
s a ge
nomi
c DNA f
r
agme
ntt
hati
si
de
nt
i
f
i
ed by
PCR ampl
i
f
i
c
at
i
on usi
ng a pai
rofspe
ci
f
i
col
i
gonuc
l
e
ot
i
depr
i
me
r
s(
Par
an
and Mi
c
hel
mor
e
,1993)
.The c
onve
r
s
i
on of s
uc
h mar
ke
r
si
nt
o SCARs
base
d on t
he mar
ke
rs
e
que
nc
ei
nf
or
mat
i
on s
i
gni
f
i
c
ant
l
y i
mpr
ove
st
he
r
epr
oduc
i
bi
l
i
t
y and r
e
l
i
abi
l
i
t
y of PCR as
says (
Par
an and Mi
che
l
mor
e
,
1993)
. SCAR mar
ke
r
s ar
e hi
ghl
y advant
age
ous f
or qui
c
k and e
asy
as
s
e
ss
me
nt(
Par
anandMi
c
he
l
mor
e
,1993)
.Wangetal
.(
2001)de
ve
l
ope
da
SCAR mar
ke
rf
r
om RAPD f
r
agme
nt
st
o aut
he
nt
i
cat
eP.gi
ns
eng and P.
qui
nquef
ol
i
uss
pe
c
i
e
s.
I
nt
hi
s st
udy,a spec
i
e
s
-s
pe
c
i
f
i
c SCAR mar
ke
r(
name
dJ
G14) f
or P.
j
aponi
c
us was obt
ai
ned f
r
om AFLP f
r
agme
nt
s
.PCR ampl
i
f
i
c
at
i
on us
i
ng
J
G14 s
pe
c
i
f
i
c pr
i
me
r
s cl
ear
l
y de
mons
t
r
at
e
d t
he s
pe
ci
f
i
c band f
or P.
j
aponi
c
usbutnotf
orP.gi
ns
eng,P.qui
nquef
ol
i
usandP.not
ogi
ns
eng

1.
2Mat
er
i
al
sandMet
hods
1.
2.
1Pl
antmat
e
r
i
al
sande
xt
r
act
i
onofge
nomi
cDNA
See
ds of P. gi
ns
eng we
r
e c
ol
l
e
c
t
e
d f
r
om Kangwon-do (
Hambae
k
mount
ai
n)
,Kyungi
-do (
Ans
ung gi
ns
eng c
ul
t
i
vat
e
df
i
el
d)ofSout
h Kor
e
a,
J
i
l
i
n Pr
ovi
nc
e(
Chang Baimount
ai
n)ofChi
na and Pr
i
mor
ye (
Us
sur
i
ys
k)

- 6 -

i
n Rus
si
a(
Tabl
e 1.
1)
.Se
eds of P.qui
nquef
ol
i
us we
r
e col
l
e
c
t
e
df
r
om
Wi
s
c
onsi
n(
f
i
e
l
dc
ul
t
i
vat
ed gi
ns
e
ng and wi
l
d-s
i
mul
at
i
ng gi
nse
ng)i
nt
he
Uni
t
e
d St
at
es
.See
dsofP.j
aponi
c
uswe
r
ec
ol
l
ec
t
ed f
r
om Toc
hi
gi(
Ni
kko
Nat
i
onal Par
k) and Nagano (
t
wo s
ampl
e
s f
r
om di
f
f
e
r
e
nt pl
ant
s i
n
Nyuugas
ayama)
,and Hokkai
do(
Noppor
oFor
es
tPar
k)i
nJ
apan.See
dsof
P.not
ogi
ns
eng we
r
ec
ol
l
e
c
t
e
df
r
om Yunnanpr
ovi
nc
e(
t
wof
i
e
l
dc
ul
t
i
vat
e
d
pl
ant
s
) of s
out
hwe
s
t
e
r
n Chi
na.De
hi
sc
e
d s
e
e
ds moi
s
t
ur
e
-c
hi
l
l
e
df
or 6
mont
hs we
r
es
owe
di
n soi
l
.Af
t
e
rt
wo mont
hs ofcul
t
ur
e,r
oot
s oft
he
pl
ant
swe
r
eus
ed t
os
t
udy t
heAFLP anal
ysi
s.Tot
alge
nomi
cDNA was
e
xt
r
ac
t
e
df
r
om gi
ns
e
ng r
oot
s wi
t
h a DNeas
y Pl
antMi
niki
t(
Qi
age
ne
,
Ge
r
many) us
i
ng t
he pr
oce
dur
e s
pe
ci
f
i
e
d by t
he manuf
ac
t
ur
e
r
. Two
hundr
e
dmi
l
l
i
gr
amsofs
ampl
e
swe
r
ef
r
oz
e
ni
nl
i
qui
dni
t
r
oge
n,gr
oundi
nt
o
powde
r
, and t
he
n t
he pr
oc
e
dur
e of t
he manuf
ac
t
ur
e
rf
ol
l
owe
d. DNA
c
once
nt
r
at
i
onwasde
t
er
mi
ne
dbyabs
or
banc
eat260nm.
1.
2.
2Ge
nomi
cDNA di
ge
st
i
onandadapt
e
rl
i
gat
i
on
TheAFLP pr
oce
dur
euse
d wast
hatde
s
cr
i
be
d by Vosetal
.(
1995)
.To
obt
ai
n t
he r
e
st
r
i
ct
i
on f
r
agme
nt
s
, 500 ng of ge
nomi
c DNA was
doubl
e
-di
ge
s
t
e
d wi
t
h 5 uni
t
s of Ec
oRI (
TaKaRa, J
apan) and Ms
eI
(
TaKaRa,J
apan)f
or12 h at37
.Af
t
e
raddi
t
i
on ofet
hanol
,DNA was
pr
e
ci
pi
t
at
ed by c
e
nt
r
i
f
uge at 14000 r
pm f
or 30 mi
n. The e
nds of
doubl
e
-di
ge
s
t
e
d DNA f
r
agme
nt
s we
r
el
i
gat
e
d wi
t
h Ec
oRI and Ms
eI
ada
pt
er
sf
or12 h at14
(
Tabl
e 1.
2)
.Af
t
e
rl
i
gat
i
on,a 10-f
ol
d di
l
ut
ed
DNA s
ol
ut
i
onwasuse
df
orpr
e
-ampl
i
f
i
c
at
i
on.
1.
2.
3AFLP anal
ys
i
s
Each20 PCR mi
xt
ur
econt
ai
ne
d5 DNA,2 dNTP (
0.
2mM)
,

- 7 -

0.
5 DNA pr
i
mer
s(
Tabl
e1.
2)
,
and 2 Taq DNA pol
yme
r
as
e(
EX
Taq,TaKaRa,J
apan)
.
Ampl
i
f
i
ca
t
i
on was pe
r
f
or
me
di
n a DNA t
he
r
mal
c
yc
l
e
r(
Appl
i
e
dBi
os
ys
t
ems9800,Fos
t
e
rCi
t
y,CA,U.
S.
A.
)f
or20c
yc
l
e
s
.

Thei
ni
t
i
alc
yc
l
ewas2mi
n at94
.Subs
e
que
ntc
yc
l
e
swer
e30sat9
4
,1mi
n at60,and 1mi
n at72,f
ol
l
owe
dby 10mi
n at96 f
or
t
hel
astcycl
es
.Pr
e
-ampl
i
f
i
c
at
i
onPCR pr
oduct
swe
r
edi
l
ut
e
d50-f
ol
dwi
t
h
wat
e
r and use
d f
or s
e
l
e
ct
i
ve ampl
i
f
i
cat
i
on.The ampl
i
f
i
c
at
i
on mi
xt
ur
e
(
20,
f
i
nalvol
ume)c
ont
ai
ne
d5
pr
e
-a
mpl
i
f
i
c
at
i
onmi
xt
ur
e,
2.
0
10buf
f
e
r
,2 dNTP,0.
5 DNA pr
i
me
r(
E1-5andM1-2ofTabl
e
1)
,and2 Taq DNA pol
yme
r
as
e(
EX Taq,
TaKaRa,J
apan)
.Af
t
e
r30
sat
94
,30sat65
,1mi
nat
72 f
ort
hef
i
r
s
tc
yc
l
e
,f
ol
l
owe
dbya
l
owe
r
i
ng oft
e
mper
at
ur
e(
1
)i
nt
hene
xt12cycl
es
,t
he
nat56 f
ort
he
r
emai
ni
ng 23 cycl
es
;e
xt
e
ns
i
on f
or 1 mi
n at72
.Ampl
i
f
i
e
d sampl
e
s
we
r
el
oade
d ont
o a 6% de
nat
ur
i
ng pol
yac
r
yl
ami
de ge
l
(
5.
75% Long
Range
r
,BMA U.
S.
A.
,7M ur
e
a,1TBE)ande
l
e
c
t
r
ophor
e
s
e
df
or4hat

15mA.Thege
lwasf
i
xe
di
n10%ace
t
i
cac
i
dandsi
l
ve
rs
t
ai
ne
d.
1.
2.
4Cl
oni
ng,DNA s
e
que
nc
i
ngandSCAR pr
i
me
rde
s
i
gn

Ampl
i
f
i
e
ds
pe
c
i
f
i
cbandswe
r
ee
xci
s
e
df
r
om AFLP ge
lwi
t
har
az
orbl
ade
andt
heDNA e
xt
r
act
e
dus
i
ng t
heQI
A qui
ckGe
lExt
r
ac
t
i
on ki
tac
cor
di
ng
t
o t
he manuf
ac
t
ur
e
r
'
s i
nst
r
uc
t
i
ons (
Qi
age
n, Val
e
nc
i
a, CA, U.
S.
A.
)
.
Li
gat
i
on of t
he PCR pr
oduc
t was c
ar
r
i
e
d out wi
t
h a pGEM-T Easy
ve
ct
or (
Pr
ome
ga) ac
c
or
di
ng t
o manuf
ac
t
ur
e
r
'
s i
ns
t
r
uc
t
i
ons and t
he
n
r
e-ampl
i
f
i
e
d al
ongs
i
de t
he or
i
gi
nalAFLP r
e
act
i
ons t
oe
ns
ur
et
hat t
he
c
or
r
e
c
tbandshad be
e
nc
l
one
d.Thepur
i
f
i
e
dl
i
gat
i
on r
e
ac
t
i
on wasdi
l
ut
e
d
wi
t
h 50 wat
er
,anda5 a
l
i
quotwass
ubse
quent
l
y mi
xe
d wi
t
h 40
c
ompe
t
e
ntc
el
l
s and pl
ac
e
d on i
ce f
or 1 h and t
he
n pl
ac
e
di
nt
oa
c
uve
t
t
e and e
l
e
c
t
r
opor
at
e
d(
Gene
Pul
se
r
;Bi
oRad,He
r
c
ul
e
s,CA,U.
S.
A.
)
.

- 8 -

One mi
l
l
i
l
i
t
e
r of LB me
di
um

was adde
d i
mme
di
at
e
l
y f
ol
l
owi
ng

e
l
e
c
t
r
opor
at
i
on andi
nc
ubat
e
df
or1hat37 wi
t
hs
haki
ng,af
t
erwhi
c
ha
200 al
i
quot was pl
at
e
d ont
o LB agar pl
at
e
s cont
ai
ni
ng ampi
c
i
l
l
i
n,
I
PTG and X-gal and i
ncubat
e
d ove
r
ni
ght at 37
.Up t
os
i
x whi
t
e
c
ol
oni
e
sf
r
om e
ac
ht
r
ans
f
or
mat
i
on r
e
ac
t
i
on we
r
es
t
r
e
ake
d ont
o LB pl
at
e
s
t
opr
oduc
es
i
ngl
ec
ol
oni
e
s.
Pl
as
mi
d DNA was e
xt
r
ac
t
e
df
r
om ove
r
ni
ght cul
t
ur
e
s of t
r
ansf
or
med
ba
ct
e
r
i
alc
e
l
l
susi
ng t
hemi
ni
pr
e
ppr
oc
e
dur
e(
Sambr
ookandRus
s
el
l
,2001)
,ands
ampl
e
swe
r
edi
l
ut
e
d1:50i
nst
e
r
i
l
ewat
er
.Te
n-mi
cr
ol
i
t
e
ral
i
quot
s
oft
he di
l
ut
ed mi
ni
pr
ep we
r
e mi
xe
d wi
t
h 6 st
e
r
i
l
e wat
er10 buf
f
e
r

(
Roc
he
)and 2 Ec
oR1 r
es
t
r
i
c
t
i
on e
nz
yme (
Roc
he
)and t
he r
e
ac
t
i
ons
i
nc
ubat
ed at37
f
or1 h.The e
nt
i
r
er
e
ac
t
i
on,al
ong wi
t
h 5 ofa
1-kbl
adde
r(
Gi
bc
o-BRL,Gai
t
he
r
s
bur
g,MD,U.
S.
A.
)
,wase
l
e
c
t
r
ophor
e
s
e
d
on a1% hor
i
z
ont
alagar
os
ege
land s
t
ai
ne
d wi
t
he
t
hi
di
um br
omi
de
.Two
s
eque
nci
ng r
e
act
i
onswe
r
es
e
tupus
i
ng 1 ofpl
asmi
d DNA combi
ne
d
wi
t
h 4 of M13 f
or
war
d and r
e
ve
r
s
e pr
i
me
r
,r
es
pe
c
t
i
vel
y.The
s
e
r
eac
t
i
onswer
ebr
oughtupt
oat
ot
alvol
umeof18 wi
t
hst
e
r
i
l
ewat
e
r
.

DNA se
que
nc
i
ngwaspe
r
f
or
me
dus
i
ngpUC/
M13pr
i
me
r
sonanaut
omat
e
d
s
eque
nce
r(
Appl
i
e
d Bi
os
ys
t
e
ms 97
00)
.The f
or
war
d and r
e
ve
r
se pr
i
me
r
s
we
r
ede
s
i
gne
dusi
ngpr
i
me
rs
el
e
ct(
DNASt
ar
)
.
1.
2.
5PCR a
nal
ys
i
s

Thede
s
i
gne
d SCAR pr
i
me
rpai
r
s(
onef
or
war
d and oner
e
ve
r
sepr
i
me
r
)
we
r
e us
e
dt
ot
es
tt
hef
ourPanax s
pe
c
i
e
s(
P.gi
ns
eng,P.qui
nquef
ol
i
us
,
P. not
ogi
ns
eng and P. j
aponi
c
us
)
. Te
s
t
i
ng was done t
o e
nsur
e
ampl
i
f
i
c
at
i
on oft
heband wi
t
ht
hee
xactmol
e
c
ul
arwe
i
ghtand det
e
r
mi
ne
opt
i
malanne
al
i
ng t
e
mper
at
ur
e.Two di
f
f
er
e
ntanneal
i
ng t
e
mpe
r
at
ur
e
s(
5
8
,62)we
r
es
cr
e
e
nedt
odet
e
r
mi
net
heopt
i
malanne
al
i
ng t
empe
r
at
ur
e
.

- 9 -

The PCR r
e
ac
t
i
on t
o ampl
i
f
yt
he SCAR mar
ke
rc
ons
i
s
t
e
d of32 c
yc
l
e
s,
e
ac
honecons
i
s
t
i
ngofa30sst
e
pat94,a1mi
ns
t
e
pat58
or62,
and a 1 mi
n at72
.PCR pr
oduc
t
swe
r
er
un on a 1.
5% (
w/
v)agar
ose
geland s
t
a
i
ned i
ne
t
hi
di
um br
omi
de as s
t
at
ed above.The pr
e
s
e
nce or
abse
nc
e of t
he SCAR band was vi
sual
l
ys
c
or
e
d and c
ompar
ed wi
t
h
s
ampl
e
sofe
ac
hs
pe
ci
es
.

1.
3Resul
t
sandDi
scussi
on
1.
3.
1AFLP anal
ys
i
s
AFLP i
s base
d on s
e
l
e
c
t
i
ve PCR ampl
i
f
i
c
at
i
on ofr
es
t
r
i
c
t
i
on f
r
agme
nt
s
f
r
om a di
ge
s
t oft
ot
algenomi
c DNA us
i
ng PCR.The AFLP anal
ysi
s
pr
oc
e
dur
ei
s mor
et
i
me
-c
ons
umi
ng t
han RAPD (
Par
an and Mi
che
l
mor
e
,
1993)
.Howe
ve
r
,amaj
oradvant
ageofAFLP mar
ke
r
si
st
he
i
rcapaci
t
yt
o
r
eve
almany pol
ymor
phi
cbandsi
n onel
anecompar
e
dt
oRAPD mar
ke
r
s
(
Par
anandMi
c
he
l
mor
e
,1993)
.TheAFLP pr
i
me
r
sshowni
nTabl
e1we
r
e
s
cr
e
e
ne
df
orpol
ymor
phi
s
m among Panax s
pe
c
i
e
s
.The numberofAFLP
bands gene
r
at
e
df
r
om Panax s
ampl
e
sr
ange
df
r
om 65 t
o 73 i
ne
ac
h
pr
i
me
rcombi
nat
i
on.Eve
r
y pr
i
me
rs
howe
ddi
s
t
i
nc
tpol
ymor
phi
cf
r
agme
nt
s
.
Ec
oRI
+TA/
Ms
eI
+GTT pr
i
me
rc
ombi
nat
i
on showe
d about15 pol
ymor
phi
c
AFLP bands i
n P.j
aponi
c
uscompar
e
dt
ot
hose ofP.qui
nquef
ol
i
usand
P.gi
ns
eng.Pol
ymor
phi
s
m was gr
e
at
e
ri
n P.j
aponi
c
us c
ompar
ed t
o P.
qui
nquef
ol
i
usand P.gi
ns
eng.Thi
sc
onf
i
r
mst
hatt
hedi
st
i
nctpol
ymor
phi
c
bandsby t
heAFLP t
e
c
hni
queamong t
hedi
f
f
e
r
entPanaxspe
ci
e
sc
an be
use
d as a mo
l
e
cul
ar mar
ke
rt
o aut
he
nt
i
c
at
et
he Panax s
pe
ci
es
. We
pr
e
vi
ous
l
y de
sc
r
i
be
d cl
earpol
ymor
phi
c bands be
t
we
en Sout
h Kor
e
an P.
gi
ns
eng and Chi
ne
seP.gi
ns
eng by AFLP a
nal
ys
i
st
hatc
oul
dbeappl
i
e
d
t
o aut
he
nt
i
cat
eKor
e
an P.gi
ns
eng f
r
om Chi
nes
eP.gi
ns
eng (
Ki
m etal
.
,

- 10 -

2005)
.
1.
3.
2I
sol
at
i
onofAFLP f
r
agme
ntf
orSCAR mar
ke
rde
s
i
gn
An AFLP f
r
agme
nt(
J
G14,ar
r
ow mar
ked i
n Fi
g.1.
1)f
orP.j
aponi
c
us
wasuni
quei
n P.j
aponi
c
usand notc
ons
e
r
ve
di
nt
heot
herspec
i
e
s
.The
J
G14f
r
agme
ntwasc
l
one
dand s
e
que
nc
ed.A 293bpDNA f
r
agme
ntwas
obt
ai
ne
d. For
war
d and r
e
ve
r
se ol
i
gonuc
l
e
ot
i
de pr
i
me
r
s (
23 me
r
)
(
under
l
i
ne
di
n Fi
g.1.
2)we
r
e de
s
i
gne
df
r
om 192 bp oft
he s
e
que
nc
e of
J
G14t
oampl
i
f
yt
hi
sAFLP f
r
agme
nt
.Thepr
i
me
rs
e
que
nc
eswer
e
:J
G14F :5-GGAATGGCATGCATAGATATGGA-3andJ
G14-R :5-GTGTG
CATGTCATTACCCCGAAA-3. BLAST

r
e
sul
t
s r
e
ve
al
e
d t
hat t
he

s
eque
nce
s had no homol
ogy wi
t
h known pl
antnuc
l
e
ot
i
de se
quenc
e
s at
s
eque
nce
-s
i
mi
l
ar
i
t
yl
e
ve
l
s
.
1.
3.
3PCR a
mpl
i
f
i
c
at
i
onus
i
ngSCAR pr
i
me
r
s
Among t
he f
our ki
nds of Panax s
pe
ci
es t
e
s
t
e
d, P. gi
ns
eng, P.
qui
nquef
ol
i
us
,P.j
aponi
c
us and P.not
ogi
ns
eng,t
he J
G14 SCAR mar
ke
r
pr
i
me
r
si
s
ol
at
ed f
r
om t
he AFLP f
r
agme
ntf
r
om P.j
aponi
c
us s
howe
da
c
l
e
arband(
191-bp)by PCR f
orP.j
aponi
c
usbutnobandi
nP.gi
ns
eng,
P.qui
nquef
ol
i
us
,and P.not
ogi
ns
eng (
Fi
g.1.
3)
.PCR anal
ys
i
s usi
ng t
he
J
G14 SCAR mar
ke
r pr
i
me
r
s us
i
ng var
i
ous sampl
e
s of P.gi
ns
eng,P.
qui
nquef
ol
i
usand P.not
ogi
ns
eng col
l
e
c
t
e
d atdi
f
f
e
r
e
ntpl
ac
e
sshowed no
PCR bande
xc
e
ptf
ors
ampl
e
sf
r
om P.j
aponi
c
us(
Fi
g.1.
4)
.Theopt
i
mi
z
e
d
pr
ot
ocoli
sde
s
c
r
i
bedasf
ol
l
ows
.Eac
hr
e
ac
t
i
oncons
i
s
t
e
dof2 ge
nomi
c
DNA,10.
3 st
e
r
i
l
e wat
e
r
,2.
5 10PCR buf
f
e
r(
12 mM MgCl
2
adde
d)
,2 ofbot
ht
hef
or
war
d and r
e
ve
r
sepr
i
me
r
s,and 0.
2 Taq
pol
yme
r
as
e
. The opt
i
mi
z
e
d ampl
i
f
i
cat
i
on pr
ogr
am r
e
qui
r
e
s an i
ni
t
i
al

- 11 -

de
nat
ur
at
i
on of5mi
n at94;32c
yc
l
e
sof30sat94,1 mi
ns
t
e
p at
62
and 1 mi
n at72;af
i
nale
xt
e
ns
i
on of5mi
n at72 (
Fi
g.1.
3B)
.
Lowe
r
i
ng t
he anne
al
i
ng t
empe
r
at
ur
et
o 58
was al
s
os
howe
d di
s
t
i
nc
t
PCR pr
oducti
nP.j
aponi
c
us(
Fi
g.1.
3A)
.
PCR anal
ys
i
s of di
f
f
e
r
e
nt s
ampl
e
s of P. j
aponi
c
us c
ol
l
e
c
t
e
d f
r
om
di
f
f
er
e
ntpl
ac
e
si
nJ
apan i
ndi
c
at
ed t
hatt
he J
G14 SCAR f
r
agme
ntwas
c
onse
r
ve
di
n al
ls
ampl
e
s of P.j
aponi
c
us di
s
t
r
i
but
e
dt
hr
oughoutJ
apan
(
Fi
g.1.
5)
.The SCAR pr
i
me
r
s we
r
e us
e
dt
o ampl
i
f
y P.gi
ns
eng,P.
qui
nquef
ol
i
us
,P.not
ogi
ns
eng andP.j
aponi
c
us
.
Spe
ci
es aut
hent
i
c
at
i
on ofP.j
aponi
c
us among t
he ot
he
rt
hr
ee Panax
s
pe
ci
es was c
l
e
ar
l
y de
mons
t
r
at
e
d usi
ng t
hi
sJ
G14 s
pe
c
i
f
i
c pr
i
mer
.Thi
s
r
epor
t i
s t
he f
i
r
st de
s
cr
i
bi
ng SCAR mar
ker de
ve
l
opme
nt f
or t
he
aut
he
nt
i
c
at
i
on ofP.j
aponi
c
us
.SCAR mar
ke
r
shavebe
e
n de
vel
ope
df
r
om
RAPD f
r
agme
nt
sf
ort
heaut
hent
i
c
at
i
on ofP.qui
nquef
ol
i
us(
Wang etal
.
,
2001)
.
I
nc
onc
l
usi
on,a spec
i
e
s
-s
pe
c
i
f
i
c SCAR mar
ke
rf
or P.j
aponi
c
us was
obt
ai
ne
df
r
om AFLP f
r
agme
nt
s
.PCR ampl
i
f
i
cat
i
on J
G14 s
pe
c
i
f
i
c pr
i
me
r
s
c
l
e
ar
l
y de
monst
r
at
edt
heuni
queband onl
yi
n P.j
aponi
c
usand noti
n P.
gi
ns
eng,P.qui
nquef
ol
i
usandP.not
ogi
ns
eng.TheSCAR mar
ke
r
sf
orP.
j
aponi
c
uswi
l
lbeus
e
df
orr
api
daut
he
nt
i
c
at
i
onoft
hi
ss
pe
ci
es
.

- 12 -

Table 1.1. Samples of various ginseng roots for AFLP analysis

No.
1

Samples
Scientific name
Panax
ginseng
Cultivated ginseng
in Korea

Kangwon-do
Ansung

2
3
4
5
6

Kyungi-do
Cultivated ginseng Panax ginseng
in China

Chang Bai mountain

Jinlin

Cultivated ginseng Panax ginseng

in Russia

Ussuriysk
Primorye

Cultivated ginseng Panax quinquefolius Wisconsin

in U.S.A.

U.S.A.

Cultivated ginseng Panax japonicus

in Japan

Nikko National Park

Tochigi
Nyuugasayama

Nagano
Nopporo Forest Park

8
9

Collecion site
Hambaek mountain

Hokkaido
Cultivated ginseng Panax notoginseng
in China

Yunnan
Southwestern China

- 13 -

Tabl
e1.
2.Ol
i
gonuc
l
e
ot
i
deadapt
e
r
sandpr
i
me
r
sus
e
df
orAFLP anal
ys
i
s
Adapter (5' to 3')

Primer sequences (5' to 3')

EcoRI-adapter
Forward:
CTCGTAGACTGCGTACC
Reverse:
AATTGGTACGCAGTCTAC

E0:
E1:
E2:
E3:
E4:
E5:

MseI-adapter
Forward:
GACGATGAGTCCTGAG
Reverse:
TACTCAGGACTCAT

GAC
GAC
GAC
GAC
GAC
GAC

TGC
TGC
TGC
TGC
TGC
TGC

GTA
GTA
GTA
GTA
GTA
GTA

CCA
CCA
CCA
CCA
CCA
CCA

ATT
ATT
ATT
ATT
ATT
ATT

C
CAT
CAC
CTA
CTG
CACA

M0: GAT GAG TCC TGA GTA A

M1: GAT GAG TCC TGA GTA ACT G
M2: GAT GAG TCC TGA GTA AGT T

- 14 -

Fi
g.1.
1.AFLP anal
ys
i
s oft
he P.j
aponi
c
us
,P.qui
nquef
ol
i
us
,and P.
gi
ns
eng us
i
ng pr
i
me
rc
ombi
nat
i
on Ms
eI
+GTT/
Ec
oRI
+TA. Ar
r
owheads
i
ndi
c
at
es
pe
c
i
f
i
cbandf
orge
nes
eque
nci
ng.

- 15 -

Fi
g.1.
2.Se
que
nc
eoft
hespec
i
e
sspec
i
f
i
cDNA f
r
agme
nt
sofP.j
aponi
c
us
.
Tot
all
e
ngt
h of t
he f
r
agme
nt i
s 293 bp.De
si
gne
d pr
i
me
r
sf
or PCR
anal
ys
i
sar
eunde
r
l
i
ne
dandnamedJ
G14.

- 16 -

Fi
g.1.
3.PCR anal
ys
i
susi
ngJ
G14pr
i
me
r
s.A:PCR ampl
i
f
i
c
at
i
onofSCAR
f
r
agme
nti
nP.j
aponi
c
us
.M i
st
he100-bpmar
ke
r
.Pgi
sP.gi
ns
eng f
r
om
Kangwon-do(
Hambae
kmount
ai
n)
.Pqi
sP.qui
nquef
ol
i
usf
r
om Wi
s
c
onsi
n
(
wi
l
d-si
mul
at
i
ng gi
nse
ng)i
n U.
S.
A.Pn i
s P.not
ogi
ns
eng f
r
om Yunnan
pr
ovi
nc
eofsout
hwe
st
e
r
n Chi
na.Pji
sP.j
aponi
c
usf
r
om Tochi
gi(
Ni
kko
Nat
i
onalPar
k) i
nJ
apan.Bl
ack ar
r
owhe
ad i
ndi
c
at
e
se
xpec
t
ed ampl
i
f
i
ed
ba
nd(
191-bp)byJ
G14pr
i
me
r
s
.

- 17 -

Fi
g.1.
4.PCR anal
ys
i
sf
r
om var
i
ouski
ndsofs
ampl
e
sus
i
ngJ
G14pr
i
me
r
s.
M i
st
he100-bpmar
ke
r
.Sampl
esofP.gi
ns
eng wascol
l
e
c
t
e
df
r
om (
Pg1)
Kangwondo (
Hambae
k mount
ai
n)
,(
Pg2) Kyungi
-do (
Ans
ung gi
ns
e
ng
c
ul
t
i
vat
e
d f
i
el
d) of Sout
h Kor
e
a, (
Pg3) J
i
l
i
n Pr
ovi
nc
e (
Chang Bai
mount
ai
n)ofChi
na and (
Pg4)Pr
i
mor
ye (
Uss
ur
i
ys
k)i
n Russ
i
a.Sampl
e
s
of P.qui
nquef
ol
i
us wer
ec
ol
l
e
ct
e
df
r
om c
ul
t
i
vat
ed gi
nse
ng (
Pq1) and
wi
l
d-si
mul
at
i
ng gi
ns
e
ng (
Pq2) i
n Wi
sc
ons
i
n (
U.
S.
A.
)
. Sampl
es of P.
j
aponi
c
uswe
r
ec
ol
l
e
ct
e
df
r
om (
Pj
1)Toc
hi
gi(
Ni
kko Nat
i
onalPar
k)
,(
Pj
2)
Nagano(
Nyuugas
ayama)
.Sampl
e
sofP.not
ogi
ns
eng we
r
ecol
l
e
c
t
e
df
r
om
(
Pn1andPn2)Yunnanpr
ovi
nceofsout
hwe
st
e
r
nChi
na.

- 18 -

Fi
g.1.
5.PCR ampl
i
f
i
cat
i
on ofSCAR f
r
agme
nti
n di
f
f
e
r
e
ntsampl
e
sofP.
j
aponi
c
us
.Sampl
e
s ofP.j
aponi
c
us we
r
ec
ol
l
ec
t
ed f
r
om Ni
kko Nat
i
onal
Par
k(
Pj
1)and Nagano (
Pj
2,Pj
3)
,and Noppor
o For
e
stPar
k ofHokkai
d
(
Pj
4) i
n J
apan. Bl
ac
k ar
r
owhe
ad i
ndi
cat
e
s expe
c
t
e
d ampl
i
f
i
ed band
(
191-bp)byJ
G14pr
i
me
r
s
.M i
st
he100-bpmar
ke
r
.

- 19 -

Chapt
er 2. Devel
opment of new

mi
cr
osat
el
l
i
t
e

mar
ker
sf
orPanaxgi
nseng
Abst
r
act
Mi
c
r
osat
e
l
l
i
t
e
s
,al
s
oc
al
l
e
ds
i
mpl
e se
quenc
er
e
pe
at
s(
SSRs
)
,ar
e ve
r
y
use
f
ul mol
e
cul
ar gene
t
i
c mar
ke
r
s commonl
y use
d i
n pl
ant br
e
e
di
ng,
s
pe
ci
esi
de
nt
i
f
i
c
at
i
on andl
i
nkageanal
ys
i
s
.Be
c
aus
et
he
y ar
ec
o-domi
nant
,
mul
t
i
al
l
e
l
i
c
,e
as
i
l
ys
c
or
e
d and hi
ghl
y pol
ymor
phi
c
.I
nt
he pr
e
se
nts
t
udy,
we c
ons
t
r
uct
e
d a mi
c
r
osat
e
l
l
i
t
e
-enr
i
c
he
d ge
nomi
c l
i
br
ar
y of Panax
gi
ns
eng.Tr
i
-ntr
e
pe
atuni
t
swe
r
et
hemos
tabundant(
57.
9%)
,f
ol
l
owe
dby
di
-nt r
e
peat
s (
27.
8%) and t
e
t
r
a-nt r
e
peat
s (
14.
3%)
. Af
t
e
r s
e
que
nc
e
anal
ys
i
s on 992 r
andoml
y pi
c
ke
d posi
t
i
ve c
ol
oni
e
s,126 (
12.
7%) oft
he
c
ol
oni
e
s we
r
ef
ound t
oc
ont
ai
n mi
c
r
osat
e
l
l
i
t
es
e
que
nc
es
,and 6 pr
i
me
r
pai
r
s we
r
e de
s
i
gned.By pol
ymor
phi
sm as
se
s
s
me
nt
,4 pr
i
me
r(
PG409,
PG450,PG491,and PG582) t
e
st
e
d wer
e shown t
o be pol
ymor
phi
c and
Kor
e
an gi
ns
eng was c
l
e
ar
l
y di
s
t
i
ngui
s
he
df
r
om chi
ne
s
e gi
ns
e
ng.Thi
s
s
t
udy r
epr
e
s
e
nt
st
hef
i
r
s
tr
e
por
toft
hebul
ki
sol
at
i
onofmi
c
r
os
at
el
l
i
t
e
sby
s
cr
e
e
ni
ng a mi
c
r
osat
e
l
l
i
t
e-e
nr
i
c
he
d ge
nomi
c l
i
br
ar
y i
n P. gi
ns
egi
ng.
The
s
e mi
c
r
osat
e
l
l
i
t
e mar
ke
r
s pr
ovi
de powe
r
f
ul t
ool
s t
o aut
he
nt
i
c
at
e
Kor
e
angi
ns
engf
r
om Chi
ne
segi
nse
ng.

- 20 -

2.
1I
nt
r
oduct
i
on
Panax gi
ns
eng,commonl
y known as Kor
e
an or As
i
an gi
nse
ng,i
sa
pe
r
e
nni
alhe
r
bnat
i
vet
oKor
e
aandChi
naandhasbe
e
nus
e
dasanhe
r
bal
r
emedy i
ne
as
t
e
r
n Asi
af
or t
hous
ands ofyear
s.The r
oot
s ofKor
ean
gi
nse
ng ar
e hi
ghl
y pr
i
z
e
df
orse
ve
r
alme
di
c
i
nalpr
oper
t
i
e
s.Tr
adi
t
i
onal
l
y,
t
hec
r
ophasbe
en har
ve
s
t
e
df
r
om t
hewi
l
d,howe
ve
r
,i
nt
her
e
ce
ntyear
s
i
nc
r
e
asi
ng de
mand i
n t
he wor
l
d mar
ke
t had l
e
d t
o i
t
s unc
ont
r
ol
l
ed
har
ve
s
t
i
ng t
hatr
es
ul
t
e
di
nt
hevi
r
t
uale
xt
i
nct
i
onoft
hepl
anti
ni
t
snat
i
ve
habi
t
.Asar
es
ul
t
,gi
nse
ngi
sbe
i
ng de
ve
l
ope
dasahor
t
i
cul
t
ur
alcr
opnow
days.Fur
t
he
r
mor
e
,t
he di
sgui
s
e ofChi
nes
e and Amer
i
c
an gi
ns
e
ng i
nt
o
Kor
e
an gi
nse
ng had be
c
ame a pr
obl
e
m i
nr
e
c
ent year
si
n Kor
ea and
abr
oad.The
r
e
f
or
e
,an e
f
f
i
c
i
e
ntme
t
hod i
sr
e
qui
r
e
dt
o di
st
i
ngui
s
h bet
we
e
n
P.gi
ns
eng andot
he
rPanaxs
pe
ci
esataDNA l
e
ve
l
.
Themol
e
c
ul
armar
ke
r
swer
eacc
e
pt
e
dasmor
ec
onf
i
de
ntme
t
hodsast
he
mor
phol
ogi
c
aland c
he
mi
c
alanal
ysi
s.Mol
e
c
ul
araut
he
nt
i
cat
i
on ofdi
f
f
er
e
nt
Panax s
pe
c
i
e
s we
r
er
e
por
t
e
di
n some e
xt
e
nt us
i
ng r
andom ampl
i
f
i
e
d
pol
ymor
phi
sm (
RAPD)(
Ki
m andSohn,2005;Mi
hal
ov etal
.
,2000;Um et
al
.
,2001)
,r
es
t
r
i
c
t
i
on f
r
agme
ntl
e
ngt
h pol
ymor
phi
s
ms(
RFLPs
)(
Fus
hi
miet
al
.
,1997;Ngan etal
.
,1999)
,ampl
i
f
i
ed f
r
agme
ntl
e
ngt
h pol
ymor
phi
s
ms
(
AFLPs
)(
Ki
me
tal
.
,2005;Choietal
.
,2008)
,ampl
i
f
i
c
at
i
onofmi
ni
sat
e
l
l
i
t
e
r
egi
on DNA (
DAMD)(
Ha etal
.
,200
2)
.Howe
ve
r
,mol
e
c
ul
ar mar
ke
rt
o
aut
he
nt
i
c
at
et
he Kor
e
an gi
nse
ng f
r
om ot
he
rf
or
e
i
gn gi
nse
ng i
sr
e
mai
ne
d
t
obecl
ear
.
Mi
c
r
osat
e
l
l
i
t
e
s
,al
s
or
e
f
e
r
r
ed t
o as s
hor
tt
ande
m r
e
pe
at
s(
STRs
) or
(
SSRs
)
,ar
epol
ymor
phi
cl
ocipr
e
s
e
nti
n nuc
l
e
arand or
gane
l
l
arDNA t
hat
c
onsi
s
tofr
e
pe
at
i
ng uni
t
s of1-6 bas
e pai
r
si
nl
e
ngt
h(
Tur
npe
nny and
El
l
ar
d,2005)
.The
y havebe
e
nwi
de
l
y us
e
df
orposi
t
i
onalc
l
oni
ng ofge
ne
s
of i
nt
er
e
s
t
, and cons
t
r
uc
t
i
on of ge
nome
-wi
de phys
i
c
al map, and t
o

- 21 -

i
mpr
ove under
s
t
andi
ng of ge
nome e
vol
ut
i
on (
Boni
e
r
bal
e et al
.
, 1988;
Kur
at
a etal
.
,1997;Mar
t
i
n etal
.
,1993;Tanks
l
e
y etal
.
,1996)
.De
s
pi
t
e
t
he
i
ri
mpot
anc
e
,t
he de
ve
l
opme
nt of mi
cr
os
at
e
l
l
i
t
e mar
ke
rs
ys
t
e
ms f
or
many pl
ant
s has be
e
nl
i
mi
t
ed be
c
aus
ei
ti
s nec
e
s
sar
yt
oi
s
ol
at
e,c
l
one
,
s
eque
nce and char
ac
t
e
r
i
z
e mi
c
r
os
at
e
l
l
i
t
el
ocii
n mos
ts
pe
c
i
e
st
hat ar
e
be
i
ng e
xami
ne
d f
or t
he f
i
r
st t
i
me
. The r
e
l
at
i
ve
l
y l
ow f
r
eque
ncy of
mi
c
r
osat
e
l
l
i
t
es i
n pl
antge
nome
s compar
e
dt
o human orani
malge
nome
s
may pr
e
se
nt an addi
t
i
onalt
e
c
hni
calpr
obl
em (
Powe
l
let al
.
,1996)
.To
i
nc
r
e
ase t
he e
f
f
i
c
i
e
ncy of mi
c
r
osat
e
l
l
i
t
e mar
ke
r de
ve
l
opme
nt
, nove
l
appr
oac
he
st
oe
nr
i
c
h SSRs have be
e
n pr
opos
e
d and suc
c
es
s
f
ul
l
y appl
i
e
d
i
nmanypl
ant
s(
Edwar
dsetal
.
,1996;Hami
l
t
onetal
.
,1999)
.The
r
e
f
or
e
,i
t
maybeuse
f
ult
os
e
ar
c
hf
ormi
c
r
osat
e
l
l
i
t
esi
npubl
i
s
hedDNA dat
abas
e
s.
I
nt
hepr
es
e
nts
t
udy,wede
ve
l
oped as
e
tofmi
c
r
osat
e
l
l
i
t
emar
ke
r
sf
r
om
Kor
e
anput
at
i
vewi
l
dP.gi
ns
eng ve
r
s
usChi
nes
eP.gi
ns
eng.

2.
2Mat
er
i
al
sandMet
hods
2.
2.
1.Pl
antmat
e
r
i
al
sandext
r
act
i
onofge
nomi
cDNA
The Panax gi
ns
eng sampl
es us
ed we
r
e ei
t
he
rf
r
es
h or dr
i
ed r
oot
s.
Sampl
e
sofput
at
i
vewi
l
dP.gi
ns
eng sampl
eswe
r
eobt
ai
nedasl
eaff
r
om
Punggi
-e
up,I
nj
e
-gun,Hwac
he
on-gun and c
ul
t
i
vat
e
d P.gi
ns
eng s
ampl
e
s
we
r
e pur
chase
d i
n Chi
na (
Tabl
e 2.
1)
. Fur
t
he
r
mor
e
, 10 s
ampl
e
s of
mount
ai
n c
ul
t
i
vat
e
d P. gi
ns
eng we
r
e pr
ovi
de
d f
r
om Yangyang-gun
(
Agr
i
c
ul
t
ur
al De
ve
l
opme
nt and Te
chnol
ogy Ce
nt
er
)(
Tabl
e 2.
2)
. The
s
ampl
e
s wer
e gr
ound i
nt
o f
i
ne powder
s af
t
e
r f
r
e
e
z
i
ng wi
t
h l
i
qui
d
ni
t
r
oge
n.Ge
nomi
cDNA wast
he
ne
xt
r
ac
t
ed usi
ng a DNe
asy pl
antDNA
i
s
ol
at
i
onki
t(
Qi
age
n,Ger
many)
.

- 22 -

2.
2.
2.Pr
i
me
rde
s
i
gnandPCR ampl
i
f
i
c
at
i
on
Thef
l
anki
ngs
eque
nce
soft
her
e
pe
atmot
i
f
swe
r
eus
e
dt
ode
s
i
gns
pe
c
i
f
i
c
pr
i
me
r
s. Bas
e upon dat
abase
, var
i
ous mi
c
r
os
at
e
l
l
i
t
e
s we
r
e obt
ai
ne
d.
Amongt
hem,6pr
i
me
r
swe
r
ese
l
e
c
t
e
df
oranal
ysi
s(
Tabl
e2.
3)
.
PCR r
e
act
i
onswe
r
epe
r
f
or
me
di
n25 vol
umemadei
nDNA f
r
e
ewat
e
r
c
ont
ai
ni
ng 10-20ng ofge
nomi
cDNA,0.
5pmol
esofe
ac
h pr
i
mer(
Tabl
e
2.
3)
,0.
2 mM ofeac
h dNTP ,2 mM MgCl
nd 0.
5 uni
tofTaq DNA
2 a
pol
yme
r
as
e(
EX Taq,TaKaRa,J
apan)
.Re
ac
t
i
ons wer
er
un on a DNA
t
he
r
mal c
yc
l
e
r(
Appl
i
e
d Bi
os
ys
t
e
ms 9800, Fos
t
e
r Ci
t
y, CA, U.
S.
A.
)
.
Cycl
i
ngc
ondi
t
i
onswe
r
e94
f
or5mi
n;f
ol
l
owe
dby35c
yc
l
e
sof94 f
or
30s,appr
opr
i
at
eanne
al
i
ngt
e
mpe
r
at
ur
e58
f
or30s,72 f
or1mi
nand
f
i
nale
xt
e
nsi
ons
t
epat72
f
or5mi
n.
2.
2.
3.El
e
ct
r
ophor
e
si
sands
i
l
vers
t
ai
ni
ng
Mi
c
r
osat
e
l
l
i
t
e PCR ampl
i
f
i
e
d pr
oduc
t
s we
r
e e
l
ec
t
r
ophor
es
e
d i
n 6%
de
nat
ur
i
ng pol
yacr
yl
ami
dege
lc
ont
ai
ni
ng 7M ur
e
ai
n 1TBE buf
f
e
rata
c
onst
antvol
t
ageof1000-1500V f
or2-3h.
Af
t
e
re
l
e
c
t
r
ophor
e
s
i
s
,c
ar
e
f
ul
l
ys
epar
at
et
hepl
at
e
susi
ng apl
as
t
i
cwedge
.
The ge
ls
houl
d be at
t
ache
ds
t
r
ongl
y t
ot
he s
hor
t gl
as
s pl
at
e
.Af
t
e
r
pl
ac
i
ng t
he ge
lpl
at
ei
n a 10% aqueous ac
e
t
i
c ac
i
d,t
he s
ol
ut
i
on was
s
hake
nl
i
ght
l
y unt
i
ll
oadi
ng dyedi
s
appe
ar
e
d.Af
t
e
rt
hr
e
ewas
hi
ngsofge
l
pl
at
e wi
t
ht
wo mi
nut
ei
nt
e
r
val
s usi
ng ul
t
r
apur
e wat
e
rand al
lmoi
s
t
ur
e
s
we
r
er
emove
d.Af
t
e
rabout90mi
nut
e
sofdye
i
ng i
n as
ol
ut
i
on of2L of
ul
t
r
apur
ewat
er
,3 of37% f
or
mal
de
hydeand 2g ofsi
l
ve
rni
t
r
at
e,ge
l
pl
at
ewasgai
n washe
dwi
t
hul
t
r
a
pur
ewat
e
r
.A mi
xe
ds
ol
ut
i
on of2L of
ul
t
r
apur
ewat
e
r
,60gofsodi
um car
bonat
e,3 of37% f
or
mal
de
hydeand
400 ofs
odi
um t
hi
osul
f
at
e(
10mg/
)t
hathad be
e
nc
ool
e
di
n advanc
e

- 23 -

wass
e
par
at
e
di
nt
ot
wode
ve
l
opi
ng s
ol
ut
i
onsof1L.Gelpl
at
ewaspl
ac
e
d
i
ns
i
det
hef
i
r
stde
ve
l
opi
ngsol
ut
i
on.Onc
et
hef
i
r
s
tt
e
mpl
at
eDNA begant
o
appe
ar
,t
he pl
at
e was pl
ac
e
di
nt
he se
c
ond s
ol
ut
i
on and de
ve
l
ope
d unt
i
l
t
he band appe
ar
e
d.De
ve
l
oped gelpl
at
e was pr
oc
es
s
e
di
naf
i
xs
ol
ut
i
on
f
or2-3mi
nut
e
sandwashe
d2-3t
i
mesusi
ngul
t
r
apur
ewat
e
r
.Locat
i
onof
bandwasi
nt
e
r
pr
e
t
e
daf
t
e
rc
ompl
e
t
e
l
ydr
yi
ngt
hege
lpl
at
e
.

2.
3Resul
t
sandDi
scussi
on
2.
3.
1Pr
i
me
rde
s
i
gn
Thef
l
anki
ngs
eque
nce
soft
her
e
pe
atmot
i
f
swe
r
eus
e
dt
ode
s
i
gns
pe
c
i
f
i
c
pr
i
me
r
s.Among t
he 38 SSR pr
i
mer
s
,s
i
x mi
cr
os
at
e
l
l
i
t
e pr
i
me
r
s we
r
e
anal
ys
e
dt
oaut
hent
i
c
at
et
heKor
e
an gi
ns
e
ng f
r
om Chi
ne
s
egi
ns
e
ng.Fr
om
s
i
x pr
i
me
r
s,f
our Kor
ean gi
ns
eng s
pe
c
i
f
i
c SSR mar
ke
r
s wer
e obt
ai
ne
d,
whi
c
h was c
l
e
ar
l
y di
s
t
i
ngui
s
hed t
he Kor
e
an gi
ns
eng f
r
om Chi
ne
se
gi
nse
ng.
2.
3.
2Aut
he
nt
i
cat
i
onofKor
e
angi
ns
engf
r
om Chi
ne
segi
nse
ng
SSR anal
ys
i
sr
e
ve
al
e
dt
hatt
he
r
ear
ecl
eardi
f
f
e
r
e
nc
e
si
n band pat
t
er
ns
ofKor
e
angi
ns
e
ng andChi
ne
s
egi
nse
ng.De
pende
dupont
heSSR mar
ke
r
s
(
PG409,PG450,PG491 and PG582)t
e
s
t
e
d,s
pe
ci
f
i
c bands f
orKor
e
an or
Chi
ne
s
egi
ns
e
ng we
r
ege
ne
r
at
edpr
i
mer
s(
Fi
gs
.2.
1-2.
4)
.
Among t
hef
ourpr
i
me
r
st
e
st
e
d,PG409pr
i
me
rwe
r
ec
l
e
ar
l
y de
mons
t
r
at
e
d
t
he mol
e
cul
ardi
f
f
e
r
e
nc
e
s bet
we
e
n Kor
e
an gi
ns
e
ng and Chi
nes
e gi
ns
e
ng
(
Fi
g 2.
1)
.Howe
ve
r
,among t
heKor
e
angi
ns
e
ng sampl
es
,t
hes
ampl
ef
r
om
I
nj
e
-gun wase
xc
e
pt
i
onal
l
y di
f
f
e
r
e
ntf
r
om ot
hersampl
es(
Fi
g 2.
1,ar
r
ow)
.
Mor
e
ove
r
,t
heband pat
t
e
r
ni
n onesampl
e(
Chi
na3)ofChi
ne
s
egi
ns
e
ng

- 24 -

wassamei
nt
heKor
e
an gi
nse
ng i
n al
l4pr
i
mer
s(
PG409,PG450,PG491
and PG582)
.Thi
sr
es
ul
ti
ndi
c
at
e
st
hatsome amountofKor
e
an gi
ns
e
ng
s
ee
ds ar
ec
ul
t
i
vat
e
di
n Chi
na,whi
ch sampl
e
scan makenew pr
obl
e
m t
o
di
s
t
i
ngui
sht
heChi
ne
s
egi
ns
e
ngbymol
e
c
ul
aranal
ysi
s.
Al
so,i
ncaseofPG409andPG450,s
pe
c
i
albandsf
orI
nj
e
-gungi
ns
e
ng
we
r
ef
ound (
Fi
gs.2.
1,2.
2)
.Thes
pe
c
i
albandsi
nI
nj
e
-gun gi
ns
eng mi
ght
i
ndi
c
at
et
he ne
w wi
l
d var
i
t
y ofKor
e
an mount
ai
n gi
nse
ng.I
nc
ase of
PG491,t
he band pat
t
e
r
n was ge
ne
r
al
l
y si
mi
l
arbe
t
we
en t
he Kor
e
an and
Chi
ne
s
e gi
ns
e
ngs
, but t
her
e wer
e dar
ke
r and s
pe
c
i
f
i
c bands i
n t
he
Chi
ne
s
e gi
ns
e
ng (
Fi
g 2.
3)
.To di
s
t
i
ngui
s
ht
he Kor
e
an gi
ns
e
ng among
Chi
ne
s
e gi
ns
e
ng,i
ts
e
e
ms t
hatanal
ys
i
s ofband pat
t
er
ns us
i
ng se
ve
r
al
pr
i
me
r
si
si
mpor
t
antt
ocl
e
ar
l
ydi
s
t
i
ngui
s
ht
heKor
e
angi
ns
e
ngs
.
I
nc
as
e ofPG582,t
he band pat
t
er
n was di
f
f
e
r
e
ntbe
t
we
e
nt
he Kor
e
an
and Chi
nes
e gi
nse
ngs,and t
he
r
e was s
pe
c
i
f
i
c bands f
or t
he Chi
ne
s
e
gi
nse
ng(
Fi
g.2.
4)
.
The f
our SSR pr
i
me
r
s(
PG409,PG450,PG491 and PG582) c
an be
de
vel
ope
d ast
hemar
ke
rt
odi
s
t
i
ngui
sh t
heKor
e
an gi
ns
eng f
r
om Chi
ne
s
e
gi
nse
ng.Howeve
r
,i
ti
snotappr
opr
i
at
et
odi
s
t
i
ngui
s
ht
heKor
e
angi
ns
e
ng
usi
ng si
ngl
epr
i
me
rse
t
.Es
pe
ci
al
l
yi
n PG450,i
tal
so s
ee
msi
nappr
opr
i
at
e
f
or t
he mar
ke
r bec
aus
e of mi
nut
e di
f
f
e
r
e
nc
e be
t
we
e
n Kor
e
an- and
Chi
ne
s
e gi
ns
e
ng.The
r
e
f
or
e
,t
he s
eve
r
alpr
i
me
r
s shoul
d be us
e
d as t
he
mar
ke
rt
odi
st
i
ngui
s
ht
heKor
eanandChi
ne
s
egi
ns
e
ngs
.
Formor
es
e
c
ur
eandcl
eari
de
nt
i
f
i
c
at
i
onofr
e
pr
oduc
t
i
vi
t
y,i
ti
sne
c
e
ss
ar
y
t
ousemor
edi
ve
r
ses
ampl
e
si
n addi
t
i
on t
ot
hos
eus
e
di
nt
hi
sst
udy,and
t
ode
ve
l
opmor
enew SSR mar
ke
r
s.
2.
3.
3Anal
ys
i
si
nYangyang-gun(
A.
D.
T.
C.
)s
ampl
e
s
Di
f
f
e
r
e
nt bandi
ng pat
t
e
r
ns we
r
e c
ompar
e
d i
n 10 r
andoml
y se
l
ec
t
ed

- 25 -

gi
nse
ng f
r
om Yangyang-gun (
pr
ovi
ded f
r
om A.
D.
T.
C.
) us
i
ng pr
i
me
r
PG491andPG582.
I
nc
as
e of PG491,t
he band pat
t
er
nr
e
veal
e
dt
hat t
he
r
e we
r
et
hr
e
e
s
pe
ci
f
i
c bands i
nt
he Chi
ne
s
e gi
nse
ng al
t
hough ge
ne
r
alband pat
t
e
r
ns
we
r
es
i
mi
l
ar(
Fi
g 2.
5)
.Espe
ci
al
l
yt
hes
ampl
ef
r
om Mye
onokc
hi
-r
is
howe
d
t
hes
amet
e
nde
nc
yofbandpat
t
e
r
nast
hatoft
heChi
ne
s
egi
ns
e
ng.
Fi
nal
l
y,i
nt
heband pat
t
e
r
n ge
ner
at
e
d by PG582,Myeonokchi
-r
isampl
e
al
so showe
d t
he s
ame band pat
t
e
r
n t
o t
he Chi
nes
e gi
nse
ng. Thi
s
i
ndi
c
at
es t
hat Kor
e
an gi
nse
ng i
s cul
t
i
vat
e
di
n Chi
na and i
mpor
t
e
dt
o
Kor
e
a.
I
nc
onc
l
us
i
on,4 SSR pr
i
me
r(
PG409,PG450,PG491,and PG582)we
r
e
s
hownt
odi
s
t
i
ngui
s
ht
heKor
e
angi
ns
e
ngamongt
heChi
ne
s
egi
ns
e
ng.

- 26 -

Table 2.1. Samples of various ginseng for Microsatellite analysis

No.
1

Samples
Wild ginseng

Scientific name
Panax ginseng

in Korea

Gyeongsang-do
Hwacheon-gun 1,

Kangwon-do
Samcheok-si,

Kangwon-do
Inje-gun,

Kangwon-do
Hwacheon-gun 2,

Kangwon-do
Hwacheon-gun 3,

Kangwon-do
Hwacheon-gun 4,

Kangwon-do
Hwacheon-gun 5,

8
9

Collection site
Punggi-eup,

Kangwon-do
Cultivated ginseng

Panax ginseng

in China

Chang bai mountain 1,

Jinlin
Chang bai mountain 2,

10

Jinlin
Chang bai mountain 3,

11

Jinlin

- 27 -

Table 2.2. Yangyang-gun (A.D.T.C.) sample of various ginseng for

Microsatellite analysis
No.

Samples

Scientific name

Myeonokchi-ri

Beopsuchi-ri

3
4

Cultivated ginseng

Panax ginseng

in Korea

Myeonokchi-ri

Songhyun-ri

Songhyun-ri
Wol-ri

Collection site
Songhyun-ri

Cultivated ginseng

Panax ginseng

Hunchun

in China
Cultivated ginseng

Panax ginseng

Beopsuchi-ri

in Korea
Jukjeongja-ri

10

- 28 -

Tabl
e 2.
3.Mi
c
r
osat
e
l
l
i
t
e pr
i
me
r
s
,nuc
l
e
ot
i
de s
eque
nce
s
,cor
e mot
i
f
s and
numbe
rofr
epe
at
sande
xpe
c
t
e
dpr
oductsi
z
ef
orSSR
Re
peatmot
i
f

SSR
de
s
i
gnat
i
on
PG22
PG281
PG409
PG450
PG491
PG582

Seque
nc
e(
5'
3'
)

ATCAAGTTGGAAATCAGGTGGG
GTGTCTATGCAAGTTGCGGCTG
AAACTCTCTTCTAGTCTTCTTGCC
GTAGGTATACATACATGTACGGAA
GATCAATCAGAAACAAAGAAAGCT
GCTGCTCTTTCTGGGTATGCTT
GATCTTCTGGATGATTTCGACAT
TTGCCCACCCCTTTCTCCACC
AGGGAGTACGGAAGGATGGAAG
GCTCAGTGTTTACAGACACAATT
GAATTCATGCTCGGACTCAGTC
CCACTATTCTCCATCTCTCACC

- 29 -

andno.of
r
e
pe
at
s
(
TC)
2
2
(
CTAT)
1
2
(
TGA)
7
(
GA)
13
(
TC)
1
4
(
GGA)
7

Expe
c
t
e
d
s
i
z
e(
bp)
107
201
195
172
211
298

Fi
g.2.
1.Ge
ne
t
i
c di
ve
r
si
t
y ofSSR anal
ysi
sus
i
ng PG409 pr
i
me
rbet
we
e
n
Kor
e
an- and Chi
ne
s
e gi
ns
e
ng.The f
i
gur
es
hows t
he al
l
e
l
i
c pr
of
i
l
e ofa
mi
c
r
osat
e
l
l
i
t
el
ocus i
n P.gi
ns
eng s
pe
c
i
e
s on 6% pol
yacr
yl
ami
de ge
l
s
s
i
l
ve
r
-s
t
ai
ned. Pol
ymor
phi
s
m was de
t
ec
t
ed c
l
e
ar
l
y i
n Kor
ean- and
Chi
ne
s
egi
ns
eng.Ar
r
ow (
)i
ndi
c
at
e
se
xpe
ct
e
dampl
i
f
i
e
dChi
ne
segi
ns
e
ng
s
pe
ci
f
i
cbands
.Ar
r
ow he
ads( )i
ndi
cat
eChi
ne
s
egi
ns
e
ng s
pe
ci
f
i
cbands
i
ndi
c
at
ese
xpe
ct
e
dampl
i
f
i
e
dKor
e
angi
ns
e
ngs
pe
c
i
f
i
cbands
.

- 30 -

Fi
g.2.
2.Ge
ne
t
i
c di
ve
r
si
t
y ofSSR anal
ysi
sus
i
ng PG450 pr
i
me
rbet
we
e
n
Kor
e
an- and Chi
ne
s
e gi
ns
e
ng.The f
i
gur
es
hows t
he al
l
e
l
i
c pr
of
i
l
e ofa
mi
c
r
osat
e
l
l
i
t
el
ocus i
n P.gi
ns
eng s
pe
c
i
e
s on 6% pol
yacr
yl
ami
de ge
l
s
s
i
l
ve
r
-s
t
ai
ned.Pol
ymor
phi
sm wasde
t
ec
t
ed be
t
wee
n Kor
e
an- and Chi
ne
se
gi
nse
ng.Ar
r
ow (
)i
ndi
c
at
e
se
xpec
t
edampl
i
f
i
e
dChi
ne
s
egi
ns
eng s
pe
c
i
f
i
c
bands
.Al
l
ow he
ads ( ) i
ndi
c
at
es e
xpe
c
t
e
d ampl
i
f
i
e
dI
nj
e-gun gi
ns
e
ng
s
pe
ci
f
i
cbands
.

- 31 -

Fi
g.2.
3.Ge
ne
t
i
c di
ve
r
si
t
y ofSSR anal
ysi
sus
i
ng PG491 pr
i
me
rbet
we
e
n
Kor
e
an- and Chi
ne
s
e gi
ns
e
ng.The f
i
gur
es
hows t
he al
l
e
l
i
c pr
of
i
l
e ofa
mi
c
r
osat
e
l
l
i
t
el
ocus i
n P.gi
ns
eng s
pe
c
i
e
s on 6% pol
yacr
yl
ami
de ge
l
s
s
i
l
ve
r
-s
t
ai
ned.Pol
ymor
phi
sm wasde
t
ec
t
ed be
t
wee
n Kor
e
an- and Chi
ne
se
gi
nse
ng.Ar
r
ow (
)i
ndi
c
at
e
se
xpe
ct
e
d ampl
i
f
i
e
d Chi
ne
segi
ns
e
ng dar
ke
r
ands
pe
c
i
f
i
cbands
.

- 32 -

Fi
g.2.
4.Ge
ne
t
i
c di
ve
r
si
t
y ofSSR anal
ysi
sus
i
ng PG582 pr
i
me
rbet
we
e
n
Kor
e
an- and Chi
ne
s
e gi
ns
e
ng.The f
i
gur
es
hows t
he al
l
e
l
i
c pr
of
i
l
e ofa
mi
c
r
osat
e
l
l
i
t
el
ocus i
n P.gi
ns
eng s
pe
c
i
e
s on 6% pol
yacr
yl
ami
de ge
l
s
s
i
l
ve
r
-s
t
ai
ned.Pol
ymor
phi
sm wasde
t
ec
t
ed be
t
wee
n Kor
e
an- and Chi
ne
se
gi
nse
ng.Ar
r
ow (
)i
ndi
c
at
e
se
xpec
t
edampl
i
f
i
e
dChi
ne
s
egi
ns
eng s
pe
c
i
f
i
c
bands
.

- 33 -

Fi
g.2.
5.Ge
ne
t
i
cdi
ve
r
s
i
t
yofSSR anal
ysi
susi
ngPG491pr
i
me
ramongt
he
Yangyang-gun sampl
e
s pr
oduc
e
df
r
om A.
D.
T.
C.and Chi
ne
s
e gi
nse
ng.
Thef
i
gur
es
howst
heal
l
e
l
i
cpr
of
i
l
eofami
c
r
osat
e
l
l
i
t
el
ocusi
nP.gi
ns
eng
s
pe
ci
es on 6% pol
yac
r
yl
ami
de ge
l
s si
l
ve
r
-st
ai
ne
d. Pol
ymor
phi
sm was
de
t
e
c
t
e
di
ns
ampl
e
s of Yangyang-gun.Ar
r
ow (
) i
ndi
cat
e
se
xpec
t
ed
ampl
i
f
i
e
d Chi
ne
se gi
nse
ng dar
kerand spe
ci
f
i
c bands.Ar
r
ow he
ads ( )
i
ndi
c
at
eMye
onokc
hi
-r
igi
nse
ngspec
i
f
i
cbands.

- 34 -

Fi
g.2.
6.Ge
ne
t
i
cdi
ve
r
s
i
t
yofSSR anal
ysi
susi
ngPG582pr
i
me
ramongt
he
Yangyang-gun sampl
e
s pr
oduc
e
df
r
om A.
D.
T.
C.and Chi
ne
s
e gi
nse
ng.
Thef
i
gur
es
howst
heal
l
e
l
i
cpr
of
i
l
eofami
c
r
osat
e
l
l
i
t
el
ocusi
nP.gi
ns
eng
s
pe
ci
es on 6% pol
yac
r
yl
ami
de ge
l
s si
l
ve
r
-st
ai
ne
d. Pol
ymor
phi
sm was
de
t
e
c
t
e
di
ns
ampl
e
s of Yangyang-gun.Ar
r
ow (
) i
ndi
cat
e
se
xpec
t
ed
ampl
i
f
i
e
d Chi
ne
se gi
nse
ng dar
kerand spe
ci
f
i
c bands.Ar
r
ow he
ads ( )
i
ndi
c
at
eMye
onokc
hi
-r
igi
nse
ngspec
i
f
i
cbands.

- 35 -

Li
t
er
at
ur
eci
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oot
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(
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