development of molecular markers for authentication_chang ho_thesis

-- 2.0

l , , , , .

:

l , , .

l .

.

(Legal Code) .

Disclaimer

. .

. .

. , .

http://creativecommons.org/licenses/by-nc-nd/2.0/kr/legalcodehttp://creativecommons.org/licenses/by-nc-nd/2.0/kr/

- I -

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

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

- III -

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)

- I -

Development of Molecular Markers for Authentication of

Korean Ginseng and Japanese Ginseng

Chang-HoAhn

DepartmentofForestry

GraduateSchool,KangwonNationalUniversity

Abstract

PanaxginsengC.A.Meyer,commonlyknownasKoreanginseng,isa

perennialherbnativetoKoreaandChina.Itsrootsarehighlyprizedfor

various medicinalproperties.The internationaltrade of ginseng is

increasingyearly.Duetothisfact,disguisedChineseginsengintoKorean

ginseng became a problem in world and also in Korean market.

Therefore,an efficientmethod is required to distinguish between P.

ginsengandotherPanaxspeciesataDNA level.Themolecularmarkers

wereaccepted asmoreconfidentmethodsthan themorphologicaland

chemicalanalysis.MolecularauthenticationsofdifferentPanaxspecies

werereportedinsomeextentusingRAPD,PCR-RFLP,genesequences.

However,molecularmarkerto authenticate the Korean ginseng from

otherforeignginsengisremainedtobeclear.Amplifiedfragmentlength

polymorphism (AFLP)analysis hastheadvantageofdetecting length

- II -

differencesassmallas1bp,andisabletodetectmoreindependentloci

forpolymorphism than othercurrentlyavailablePCR-basedtechniques.

Microsatellites are highly polymorphic,codominantmarkers thathave

provenusefulforavarietyofpurposesincludingfinger-printing.

Inthisstudy,wedevelopedageneticmarkersasafast,efficientand

easymethodsfortheauthenticationamongdifferentPanaxspeciesby

usingtheAFLPandmicrosatelliteanalysistechnique.AFLPanalysiswas

carriedouttoauthenticatetheP.japonicusamongP.ginseng andP.

quinquefolius.A clearspecies-specificAFLPbandsforP.japonicuswas

generated.AfterisolationandsequencingoftheAFLPfragment,aDNA

sequence(293bp)wasobtainedandnamedJG14.Oligonucleotideprimer

(23mer)wasdesignedforamplifying191bpofthesequenceofJG14.

PCRanalysisrevealedaclearamplifiedbandforP.japonicusbutnotin

3otherPanaxspecies(P.ginseng,P.quinquefoliusandP.notoginseng).

Thissequencecharacterized amplified regions(SCAR)markerwillbe

usedforrapidauthenticationofP.japonicusamongotherrelatedPanax

species.This is the first report of species-specific SCAR marker

developmentinP.japonicus.

Ontheotherhands,microsatellitefragments(252clones)wereisolated

from genomic library. Then sequence of these fragments was

determinated.Baseuponsequenceinformation,38microsatelliteprimers

were obtained. Among the primers tested, 6 highly polymorphic

microsatellitemarkers(calledSSRmarker)suchasPG409,PG450,PG491,

andPG582wereselectedforanalysisofginseng.SSRanalysisrevealed

- III -

thatthesemarkerscouldbeappliedtodistinguishKoreanginsengfrom

Chineseginseng.

Conclusively,thesekindsofmarkerwillofferanefficientapproachto

authenticateKoreanginsengfrom chineseginsengandJapaneseginseng.

- IV -

ABSTRACT

LIST OFTABLES

LIST OFFIGURES

I

INTRODUCTION 1

Chapter1.DevelopmentofspeciesspecificAFLP-derived

SCARmarkerforauthenticationofPanaxjaponicusC.A.

Meyer 4

Abstract

1.1Introduction

1.2MaterialsandMethods

1.2.1PlantmaterialsandextractionofgenomicDNA

1.2.2GenomicDNAdigestionandadapterligation

1.2.3AFLPanalysis

1.2.4Cloning,DNAsequencingandSCARprimerdesign

1.2.5PCRanalysis

1.3ResultsandDiscussion

1.3.1AFLPanalysis

1.3.2IsolationofAFLPfragmentforSCARmarkerdesign

1.3.3PCRamplificationusingSCARprimers

4

5

6

6

7

7

8

9

10

10

11

11

Chapter2.Developmentofnew microsatellitemarkersfor

Panaxginseng20

Abstract

2.1Introduction



2.2.2PrimerdesignandPCRamplification

20

21

22

22

23

CONTENTS

- V -

2.2.3Electrophoresisandsilverstaining


2.3.1Primerdesign

2.3.2AuthenticationofKoreanginsengfrom Chineseginseng

2.3.3AnalysisinYangyang-gun(A.D.T.C.)samples

23

24

24

24

25

Literaturecited 37

- VI -

LISTOFTABLE

Table1.1Dataofcollectedsamplesofvariouskindsofginsengrootsfor

AFLP13

Table1.2OligonucleotideadaptersandprimersusedforAFLPanalysis

14

Table2.1SamplesofvariousginsengforMicrosatelliteanalysis27

Table 2.2 Yangyang-gun (A.D.T.C.)samples ofvarious ginseng for

Microsatelliteanalysis28

Table2.3Microsatelliteprimers,nucleotidesequences,coremotifsand

numberofrepeatsandexpectedproductsizeforSSR29

- VII -

LISTOFFIGURE

Fig.1.1AFLP analysisoftheP.japonicus,P.quinquefolius,and P.

ginsengusingprimercombinationMseI+GTT/EcoRI+TA15

Fig.1.2SequenceofthespeciesspecificDNA fragmentsofP.japonicus.

16

Fig.1.3PCRanalysisusingJG14primers17

Fig.1.4PCRanalysisfrom variouskindsofsamplesusingJG14primers

18

Fig.1.5PCRamplificationofSCAR fragmentindifferentsamplesofP.

japonicus19

Fig.2.1GeneticdiversityofSSR analysisusingPG409primerbetween

Korean-andChineseginseng30







Fig.2.5GeneticdiversityofSSR analysisusingPG491primeramong

theYangyang-gunsamplesprovidedfrom A.D.T.C.andChinese

ginseng34

Fig.2.6GeneticdiversityofSSR analysisusingPG582primeramong

theYangyang-gunsamplesprovidedfrom A.D.T.C.andChinese

ginseng35

- 1 -

INTRODUCTION

Panax(Araliaceae)isagenusofabout12speciesdistributedinthe

worldandeachofthem hasbeenusedastraditionalmedicines.Panax

ginseng, commonly known as Korean and Asian ginseng. P.

quinquefolium is an another important ginseng species (American

ginseng),whichisdistributedinNorthAmerica.P.japonicusisavailable

inJapanandSouthernareaofChina.

Panaxspecieshavebeenusedastonicsforanti-stress,anti-fatigue,and

anti-aging purposes.Ginseng productshavebecomevery popularand

haveattracted worldwideconsumption.However,Orientalginseng and

Americanginsengareknowntohavedifferentpropertiesandmedicinal

values.The red form ofOrientalginseng,produced by steaming,is

"warm"andknowntoreplenishthe"vitalenergy",whereasAmerican

ginsengis"cool"andismainlyusedforreducingthe"internalheat"and

promotingthesecretionofbodyfluids.

In addition,ginsenosides (ginseng saponins)are known to be the

bioactive components ofginseng.According to the difference in the

aglyconeinthesesaponins,ginsenosidesareclassifiedintothreetypes:

the20(S)-protopanaxadioltype(e.g.,ginsenosidesRb1,Rc,Rb2,andRd),

the20(S)-protopanaxadioltype(e.g.,ginsenosidesRg1,Rf,andRe),and

the oleanolic acid type (e.g.,ginsenosides Ro) (Chan etal.,2000).

GinsenosidesRg1,Re,Ro,Rb1,Rc,Rb2,and Rd arepresentin both

Oriental and American ginsengs in different proportions, whereas

ginsenosideRfcanonlybefoundinOrientalginseng(Chuangetal.,

1995).Incontrast,Americanginsengcontains24-(R)-pseudoginsenoside

F11,anocotilloltypetriterpenewhichisabsentinOrientalginseng(Dou

etal.,1998;Chenetal1981).

AmongPanaxspecies,KoreanP.ginseng isthebest-knownproduct

- 2 -

with high medicinalvalues in the world.However,in the ginseng

marketsworldwide,Americanginsengusuallycommandsamuchhigher

price than the sun-dried Orientalginseng.Since the roots ofthese

ginsengs are similar in appearance and many commercialginseng

productsareinform ofpowderorshreddedslices,identificationofthe

originsoftheginsengproductsisnotaneasytask.Duetothisfact,

disguisedChineseandAmericanginsengintoKoreanginsengbecamea

problem inworldandalsoinKoreanmarket.InKorea,wild-grownand

wild-cultivatedKoreanginsengissoldwithhighpricesandthedisguise

ofChinesewildginsengintoKoreanginsengisseriousproblem inKorea,

whichmayberesultedinthedepressionofginsengmarket(Kim etal.,

2005).

Therefore,itisveryimportanttoauthenticatetheKoreanginsengfrom

Chinese ginseng and from otherdifferentginseng species.Thereare

somereportsonthemorphologicalandchemicalauthenticationofKorean

ginsengbetweenotherforeignP.ginsenganddifferentginsengspecies.

TherearesomedifferencesbetweenP.ginsengandP.quinquefoliusin

morphological characters and saponins (Chung et al.,1995,1998).

However,itisvery difficulttodifferentiatethecharactersofginseng

withinsamespeciesalthoughtheywerecultivatedindifferentcountry.

Themolecularmarkerswereacceptedasmoreconfidentmethodsasthe

morphologicalandchemicalanalysis.Molecularauthenticationsofdifferent

PanaxspecieswerereportedinsomeextentusingRAPD (Shim etal.,

2003;Um etal.,2001),PCR-RFLP (Nganetal.,1999;Fushimietal.,

1997),genesequences(Komatsyetal.,2001;Shuetal.,1997).However,

molecularmarkerto authenticate the Korean P.ginseng from other

foreignP.ginsengisremainedtobeclear.

Amplifiedfragmentlengthpolymorphism (AFLP)isaPCR basedtool

usedin geneticsresearch,DNA fingerprinting,and in thepracticeof

- 3 -

geneticengineering.AFLP-PCRisahighlysensitivemethodfordetecting

polymorphismsinplantspecies.

Microsatellites,alsoknown asSSRs(simplesequencerepeats)area

smallarray oftandemly arranged 1-6 bases spread throughoutthe

genomes.MicrosatellitesasDNA markersareadvantageousovermany

other markers as they are highly polymorphic, highly abundant,

co-dominant inheritance,analytically simple and readily transferable.

MicrosatellitesarereportedtobemorevariablethanRFLPsorRAPDs,

andhavebeenwidelyutilizedinplantgenomicstudies.

Inthepresentstudy,thecombinationofthesemarkersdeterminatedby

twotechniqueswillofferan efficientapproachtoauthenticateKorean

ginsengfrom foreignginseng.

- 4 -

Chapter 1. Development of species specific

AFLP-derivedSCAR markerforauthenticationof

PanaxjaponicusC.A.Meyer

Abstract

Panaxjaponicusisanimportantmedicinalplant.Theaim ofthisstudy

was to develop species-specific molecular markers for P.japonicus.

Amplifiedfragmentlengthpolymorphism (AFLP)wascomparedamongP.

japonicus,P.ginsengandP.quinquefolius.Aclearspecies-specificAFLP

markerforP.japonicuswasgenerated.Afterisolationandsequencingof

theAFLPfragment,aDNA sequence(293bp)wasobtainedandnamed

JG14.Oligonucleotideprimer(23mer)wasdesignedforamplifying191bp

ofthesequenceofJG14.PCRanalysisrevealedaclearamplifiedbandfor

P.japonicus but not in 3 other Panax species (P.ginseng, P.

quinquefoliusandP.notoginseng).Thissequencecharacterizedamplified

regions (SCAR)markerwillbe used forrapid authentication ofP.

japonicusamongotherrelatedPanaxspecies.Thisisthefirstreportof

species-specificSCARmarkerdevelopmentinP.japonicus.

- 5 -

1.1Introduction

Panaxisagenuscomprisingseveralspeciesofslow-growingperennial

medicinalplants,inthefamilyAraliaceae.Theygrow intheNorthern

HemisphereineasternAsia(Korea,northernChina,easternSiberiaand

Japan)andNorthAmerica,typicallyincoolerclimates.P.japonicusC.A.

Meyer(Japanese name;Chikusetsuninjin)is distributed in Japan and

south-western China,and has a long rhizome,being morphologically

similartobamboorhizome.IthasbeenreportedthattherhizomesofP.

japonicus have an antiulcer action and have been used to treat

fibrinolysis(Yamaharaetal.,1987;Matsudaetal.,1989),antiobesity(Han

etal.,2005),and suppression ofapoptosis(Hosono-Nishiyama etal.,

2006)andtosmoothcoughsandreducephlegm (ChangandBut,1986)

Themain activecomponentin P.japonicusisocotillol-typesaponins

(Zouetal.,2002).

Theidentification ofspecies-specificDNA markersofPanaxspecies

wouldbeofgreatimportancetodiscriminatethespecies.Analysisof

well-characterizedcompounds,ginsenosides,isthemostpopularmethod

foridentifyingthePanaxspeciesandqualitycontrolofginsengproducts

(Chanetal.,2000).TheprofilesofginsenosidesinrootsofPanaxspecies

are very similar and affected significantly by growth and storage

conditions,and harvesttimes.Genetictoolsareconsidered toprovide

more standardized and reliable methods for authentication of plant

materialsattheDNA level.UsingPanaxspecies,themethodsdeveloped

previously includelow-CotDNA fingerprinting (Hoand Leung,2002),

randomlyamplifiedpolymorphicDNA(RAPD)(Baietal.,1997;Shaw and

But,1995)orarbitrarily primed polymerase chain reaction (AP-PCR)

(Cheungetal.,1994),PCR-random fragmentlengthpolymorphism (RFLP)

(Fushimietal.,1997;Ngan etal.,1999),amplified fragmentlength

- 6 -

polymorphism (AFLP)(Kim etal.,2005)andmicrosatellitemarkers(Hon

etal.,2003)andinternaltranscribedspacers(ITS)sequencesofribosomal

DNA (WenandZimmer,1996),andmicrochipelectrophoresis(Qinetal.,

2005).

Paran and Michelmore (1993) developed a technique known as

sequence-characterized amplified regions(SCAR).SCAR markershave

beenderivedfrom RAPD andAFLPmarkersandhaveprovenusefulin

identifyingtheplantsattheintraorinter-specificlevel.SCARasaPCR

basedgeneticmarkerisagenomicDNA fragmentthatisidentifiedby

PCR amplificationusingapairofspecificoligonucleotideprimers(Paran

and Michelmore,1993).Theconversion ofsuch markersinto SCARs

based on the markersequence information significantly improves the

reproducibility and reliability ofPCR assays (Paran and Michelmore,

1993).SCAR markers are highly advantageous for quick and easy

assessment(ParanandMichelmore,1993).Wangetal.(2001)developeda

SCAR markerfrom RAPD fragmentstoauthenticateP.ginsengandP.

quinquefoliusspecies.

In thisstudy,aspecies-specificSCAR marker(named JG14)forP.

japonicuswasobtainedfrom AFLP fragments.PCR amplificationusing

JG14 specific primers clearly demonstrated the specific band for P.

japonicusbutnotforP.ginseng,P.quinquefoliusandP.notoginseng



Seeds of P.ginseng were collected from Kangwon-do (Hambaek

mountain),Kyungi-do(Ansungginsengcultivatedfield)ofSouthKorea,

JilinProvince(ChangBaimountain)ofChinaandPrimorye(Ussuriysk)

- 7 -

in Russia(Table1.1).SeedsofP.quinquefoliuswerecollected from

Wisconsin(fieldcultivatedginsengandwild-simulatingginseng)inthe

UnitedStates.SeedsofP.japonicuswerecollectedfrom Tochigi(Nikko

NationalPark) and Nagano (two samples from different plants in

Nyuugasayama),andHokkaido(NopporoForestPark)inJapan.Seedsof

P.notoginsengwerecollectedfrom Yunnanprovince(twofieldcultivated

plants)ofsouthwestern China.Dehisced seeds moisture-chilled for6

monthsweresowedinsoil.Aftertwomonthsofculture,rootsofthe

plantswereusedtostudytheAFLPanalysis.TotalgenomicDNA was

extractedfrom ginsengrootswithaDNeasyPlantMinikit(Qiagene,

Germany) using the procedure specified by the manufacturer.Two

hundredmilligramsofsampleswerefrozeninliquidnitrogen,groundinto

powder,and then the procedure ofthe manufacturerfollowed.DNA

concentrationwasdeterminedbyabsorbanceat260nm.

1.2.2GenomicDNAdigestionandadapterligation

TheAFLPprocedureusedwasthatdescribedbyVosetal.(1995).To

obtain the restriction fragments, 500 ng of genomic DNA was

double-digested with 5 units ofEcoRI(TaKaRa,Japan)and MseI

(TaKaRa,Japan)for12hat37.Afteradditionofethanol,DNA was

precipitated by centrifuge at14000 rpm for 30 min.The ends of

double-digested DNA fragments were ligated with EcoRIand MseI

adaptersfor12hat14 (Table1.2).Afterligation,a10-folddiluted

DNA solutionwasusedforpre-amplification.

1.2.3AFLPanalysis

Each20 PCRmixturecontained5 DNA,2 dNTP(0.2mM),

- 8 -

0.5 DNA primers(Table1.2),and2 TaqDNA polymerase(EX

Taq,TaKaRa,Japan).AmplificationwasperformedinaDNA thermal

cycler(AppliedBiosystems9800,FosterCity,CA,U.S.A.)for20cycles.

Theinitialcyclewas2minat94.Subsequentcycleswere30sat9

4,1minat60,and1minat72,followedby10minat96 for

thelastcycles.Pre-amplificationPCRproductswerediluted50-foldwith

waterand usedforselectiveamplification.Theamplification mixture

(20,finalvolume)contained5 pre-amplificationmixture,2.0

10buffer,2 dNTP,0.5 DNA primer(E1-5andM1-2ofTable

1),and2 TaqDNA polymerase(EX Taq,TaKaRa,Japan).After30

sat94,30sat65,1minat72 forthefirstcycle,followedbya

loweringoftemperature(1)inthenext12cycles,thenat56 forthe

remaining23cycles;extension for1minat72.Amplifiedsamples

were loaded onto a 6% denaturing polyacrylamide gel (5.75% Long

Ranger,BMA U.S.A.,7M urea,1TBE)andelectrophoresedfor4hat

15mA.Thegelwasfixedin10%aceticacidandsilverstained.

1.2.4Cloning,DNAsequencingandSCARprimerdesign

Amplifiedspecificbandswereexcisedfrom AFLPgelwitharazorblade

andtheDNA extractedusingtheQIA quickGelExtractionkitaccording

to the manufacturer's instructions (Qiagen, Valencia, CA, U.S.A.).

Ligation ofthePCR productwascarried outwith apGEM-T Easy

vector (Promega) according to manufacturer's instructions and then

re-amplified alongsidetheoriginalAFLP reactionstoensurethatthe

correctbandshadbeencloned.Thepurifiedligationreactionwasdiluted

with50 water,anda5 aliquotwassubsequentlymixedwith40

competentcellsandplacedonicefor1handthenplacedintoa

cuvetteand electroporated(GenePulser;BioRad,Hercules,CA,U.S.A.).

- 9 -

One milliliter of LB medium was added immediately following

electroporationandincubatedfor1hat37 withshaking,afterwhicha

200 aliquotwasplated ontoLB agarplatescontaining ampicillin,

IPTG and X-galand incubated overnightat37.Up to six white

coloniesfrom eachtransformationreactionwerestreakedontoLBplates

toproducesinglecolonies.

Plasmid DNA wasextracted from overnightculturesoftransformed

bacterialcellsusingtheminiprepprocedure(SambrookandRussell,2001)

,andsampleswerediluted1:50insterilewater.Ten-microliteraliquots

ofthedilutedminiprepweremixedwith6 sterilewater10buffer

(Roche)and2 EcoR1restrictionenzyme(Roche)andthereactions

incubatedat37 for1h.Theentirereaction,alongwith5 ofa

1-kbladder(Gibco-BRL,Gaithersburg,MD,U.S.A.),waselectrophoresed

ona1% horizontalagarosegelandstainedwithethidium bromide.Two

sequencingreactionsweresetupusing1 ofplasmidDNA combined

with 4 ofM13 forward and reverse primer,respectively.These

reactionswerebroughtuptoatotalvolumeof18 withsterilewater.

DNAsequencingwasperformedusingpUC/M13primersonanautomated

sequencer(AppliedBiosystems9700).Theforwardandreverseprimers

weredesignedusingprimerselect(DNAStar).

1.2.5PCRanalysis

ThedesignedSCARprimerpairs(oneforwardandonereverseprimer)

wereusedtotestthefourPanaxspecies(P.ginseng,P.quinquefolius,

P. notoginseng and P. japonicus). Testing was done to ensure

amplificationofthebandwiththeexactmolecularweightanddetermine

optimalannealingtemperature.Twodifferentannealingtemperatures(5

8,62)werescreenedtodeterminetheoptimalannealingtemperature.

- 10 -

ThePCR reactiontoamplifytheSCAR markerconsistedof32cycles,

eachoneconsistingofa30sstepat94,a1minstepat58 or62,

anda1minat72.PCRproductswererunona1.5% (w/v)agarose

gelandstainedinethidium bromideasstatedabove.Thepresenceor

absence oftheSCAR band was visually scored and compared with

samplesofeachspecies.


1.3.1AFLPanalysis

AFLP isbasedonselectivePCR amplificationofrestrictionfragments

from adigestoftotalgenomicDNA using PCR.TheAFLP analysis

procedureismoretime-consumingthanRAPD (ParanandMichelmore,

1993).However,amajoradvantageofAFLPmarkersistheircapacityto

revealmanypolymorphicbandsinonelanecomparedtoRAPD markers

(ParanandMichelmore,1993).TheAFLPprimersshowninTable1were

screenedforpolymorphism amongPanaxspecies.ThenumberofAFLP

bandsgenerated from Panax samplesranged from 65to 73in each

primercombination.Everyprimershoweddistinctpolymorphicfragments.

EcoRI+TA/MseI+GTT primercombinationshowedabout15polymorphic

AFLPbandsinP.japonicuscomparedtothoseofP.quinquefoliusand

P.ginseng.Polymorphism wasgreaterinP.japonicuscomparedtoP.

quinquefoliusandP.ginseng.Thisconfirmsthatthedistinctpolymorphic

bandsbytheAFLPtechniqueamongthedifferentPanaxspeciescanbe

used as a molecularmarkerto authenticate the Panax species.We

previouslydescribedclearpolymorphicbandsbetweenSouthKoreanP.

ginsengandChineseP.ginsengbyAFLPanalysisthatcouldbeapplied

toauthenticateKoreanP.ginsengfrom ChineseP.ginseng(Kim etal.,

- 11 -

2005).

1.3.2IsolationofAFLPfragmentforSCARmarkerdesign

AnAFLPfragment(JG14,arrow markedinFig.1.1)forP.japonicus

wasuniqueinP.japonicusandnotconservedintheotherspecies.The

JG14fragmentwasclonedandsequenced.A 293bpDNA fragmentwas

obtained. Forward and reverse oligonucleotide primers (23 mer)

(underlinedinFig.1.2)weredesignedfrom 192bpofthesequenceof

JG14toamplifythisAFLPfragment.Theprimersequenceswere:JG14-

F:5-GGAATGGCATGCATAGATATGGA-3andJG14-R:5-GTGTG

CATGTCATTACCCCGAAA-3. BLAST results revealed that the

sequenceshadnohomologywithknownplantnucleotidesequencesat

sequence-similaritylevels.

1.3.3PCRamplificationusingSCARprimers

Among the four kinds of Panax species tested,P.ginseng,P.

quinquefolius,P.japonicusandP.notoginseng,theJG14SCAR marker

primersisolatedfrom theAFLPfragmentfrom P.japonicusshoweda

clearband(191-bp)byPCRforP.japonicusbutnobandinP.ginseng,

P.quinquefolius,andP.notoginseng(Fig.1.3).PCR analysisusingthe

JG14SCAR markerprimersusing varioussamplesofP.ginseng,P.

quinquefoliusandP.notoginsengcollectedatdifferentplacesshowedno

PCRbandexceptforsamplesfrom P.japonicus(Fig.1.4).Theoptimized

protocolisdescribedasfollows.Eachreactionconsistedof2 genomic

DNA,10.3 sterilewater,2.5 10PCR buffer(12 mM MgCl2

added),2 ofboththeforwardandreverseprimers,and0.2 Taq

polymerase.The optimized amplification program requires an initial

- 12 -

denaturationof5minat94;32cyclesof30sat94,1minstepat

62 and1minat72;afinalextensionof5minat72 (Fig.1.3B).

Lowering theannealing temperatureto58 wasalsoshoweddistinct

PCRproductinP.japonicus(Fig.1.3A).

PCR analysis ofdifferentsamples ofP.japonicus collected from

differentplacesinJapanindicatedthattheJG14SCAR fragmentwas

conserved in allsamplesofP.japonicusdistributed throughoutJapan

(Fig.1.5).TheSCAR primerswereused to amplify P.ginseng,P.

quinquefolius,P.notoginsengandP.japonicus.

Speciesauthentication ofP.japonicusamong theotherthreePanax

specieswasclearlydemonstratedusingthisJG14specificprimer.This

report is the first describing SCAR marker development for the

authenticationofP.japonicus.SCARmarkershavebeendevelopedfrom

RAPD fragmentsfortheauthenticationofP.quinquefolius(Wangetal.,

2001).

Inconclusion,aspecies-specificSCAR markerforP.japonicuswas

obtainedfrom AFLPfragments.PCR amplificationJG14specificprimers

clearlydemonstratedtheuniquebandonlyinP.japonicusandnotinP.

ginseng,P.quinquefoliusandP.notoginseng.TheSCARmarkersforP.

japonicuswillbeusedforrapidauthenticationofthisspecies.

- 13 -

Table 1.1. Samples of various ginseng roots for AFLP analysis

No. Samples Scientific name Collecion site

1Cultivated ginseng

in Korea

Panax ginseng Hambaek mountain

Kangwon-do

2Ansung

Kyungi-do

3Cultivated ginseng

in China

Panax ginseng Chang Bai mountain

Jinlin

4Cultivated ginseng

in Russia

Panax ginseng Ussuriysk

Primorye

5Cultivated ginseng

in U.S.A.

Panax quinquefolius Wisconsin

U.S.A.

6Cultivated ginseng

in Japan

Panax japonicus Nikko National Park

Tochigi

7Nyuugasayama

Nagano

8Nopporo Forest Park

Hokkaido

9Cultivated ginseng

in China

Panax notoginseng Yunnan

Southwestern China

- 14 -

Table1.2.OligonucleotideadaptersandprimersusedforAFLPanalysis

Adapter (5' to 3') Primer sequences (5' to 3')

EcoRI-adapter E0: GAC TGC GTA CCA ATT C Forward: E1: GAC TGC GTA CCA ATT CAT

CTCGTAGACTGCGTACC E2: GAC TGC GTA CCA ATT CAC

Reverse: E3: GAC TGC GTA CCA ATT CTA AATTGGTACGCAGTCTAC E4: GAC TGC GTA CCA ATT CTG

E5: GAC TGC GTA CCA ATT CACA

MseI-adapter Forward: M0: GAT GAG TCC TGA GTA A

GACGATGAGTCCTGAG M1: GAT GAG TCC TGA GTA ACT G

Reverse: M2: GAT GAG TCC TGA GTA AGT T TACTCAGGACTCAT

- 15 -

Fig.1.1.AFLP analysisoftheP.japonicus,P.quinquefolius,andP.

ginseng using primercombination MseI+GTT/EcoRI+TA.Arrowheads

indicatespecificbandforgenesequencing.

- 16 -

Fig.1.2.SequenceofthespeciesspecificDNA fragmentsofP.japonicus.

Totallength ofthe fragmentis 293 bp.Designed primers forPCR

analysisareunderlinedandnamedJG14.

- 17 -

Fig.1.3.PCRanalysisusingJG14primers.A:PCRamplificationofSCAR

fragmentinP.japonicus.M isthe100-bpmarker.PgisP.ginsengfrom

Kangwon-do(Hambaekmountain).PqisP.quinquefoliusfrom Wisconsin

(wild-simulatingginseng)inU.S.A.PnisP.notoginsengfrom Yunnan

provinceofsouthwesternChina.PjisP.japonicusfrom Tochigi(Nikko

NationalPark)in Japan.Black arrowhead indicatesexpectedamplified

band(191-bp)byJG14primers.

- 18 -

Fig.1.4.PCRanalysisfrom variouskindsofsamplesusingJG14primers.

M isthe100-bpmarker.SamplesofP.ginsengwascollectedfrom (Pg1)

Kangwondo (Hambaek mountain),(Pg2)Kyungi-do (Ansung ginseng

cultivated field) of South Korea,(Pg3) Jilin Province (Chang Bai

mountain)ofChinaand(Pg4)Primorye(Ussuriysk)inRussia.Samples

ofP.quinquefoliuswerecollected from cultivated ginseng (Pq1)and

wild-simulating ginseng (Pq2)in Wisconsin (U.S.A.).Samples ofP.

japonicuswerecollectedfrom (Pj1)Tochigi(NikkoNationalPark),(Pj2)

Nagano(Nyuugasayama).SamplesofP.notoginsengwerecollectedfrom

(Pn1andPn2)YunnanprovinceofsouthwesternChina.

- 19 -

Fig.1.5.PCRamplificationofSCARfragmentindifferentsamplesofP.

japonicus.SamplesofP.japonicuswerecollectedfrom NikkoNational

Park(Pj1)andNagano(Pj2,Pj3),andNopporoForestParkofHokkaid

(Pj4) in Japan.Black arrowhead indicates expected amplified band

(191-bp)byJG14primers.M isthe100-bpmarker.

- 20 -

Chapter 2. Development of new microsatellite

markersforPanaxginseng

Abstract

Microsatellites,also called simplesequencerepeats (SSRs),arevery

usefulmolecular genetic markers commonly used in plant breeding,

speciesidentificationandlinkageanalysis.Becausetheyareco-dominant,

multiallelic,easilyscoredandhighlypolymorphic.Inthepresentstudy,

we constructed a microsatellite-enriched genomic library of Panax

ginseng.Tri-ntrepeatunitswerethemostabundant(57.9%),followedby

di-nt repeats (27.8%) and tetra-nt repeats (14.3%).After sequence

analysison 992randomly pickedpositivecolonies,126(12.7%)ofthe

colonieswerefound tocontain microsatellitesequences,and 6primer

pairs were designed.By polymorphism assessment,4 primer(PG409,

PG450,PG491,andPG582)testedwereshown tobepolymorphicand

Korean ginseng wasclearly distinguished from chineseginseng.This

studyrepresentsthefirstreportofthebulkisolationofmicrosatellitesby

screening a microsatellite-enriched genomic library in P.ginseging.

These microsatellite markers provide powerfultools to authenticate

Koreanginsengfrom Chineseginseng.

- 21 -

2.1Introduction

Panaxginseng,commonly known asKorean orAsian ginseng,isa

perennialherbnativetoKoreaandChinaandhasbeenusedasanherbal

remedy ineastern Asiaforthousandsofyears.TherootsofKorean

ginsengarehighlyprizedforseveralmedicinalproperties.Traditionally,

thecrophasbeenharvestedfrom thewild,however,intherecentyears

increasing demand in the world markethad led to its uncontrolled

harvestingthatresultedinthevirtualextinctionoftheplantinitsnative

habit.Asaresult,ginsengisbeingdevelopedasahorticulturalcropnow

days.Furthermore,thedisguiseofChineseandAmericanginsenginto

Koreanginseng hadbecameaproblem in recentyearsin Koreaand

abroad.Therefore,anefficientmethodisrequiredtodistinguishbetween

P.ginsengandotherPanaxspeciesataDNAlevel.

Themolecularmarkerswereacceptedasmoreconfidentmethodsasthe

morphologicalandchemicalanalysis.Molecularauthenticationofdifferent

Panax species werereported in some extentusing random amplified

polymorphism (RAPD)(Kim andSohn,2005;Mihalovetal.,2000;Um et

al.,2001),restrictionfragmentlengthpolymorphisms(RFLPs)(Fushimiet

al.,1997;Ngan etal.,1999),amplifiedfragmentlength polymorphisms

(AFLPs)(Kim etal.,2005;Choietal.,2008),amplificationofminisatellite

regionDNA (DAMD)(Haetal.,2002).However,molecularmarkerto

authenticatetheKoreanginsengfrom otherforeignginsengisremained

tobeclear.

Microsatellites,also referred to as shorttandem repeats (STRs)or

(SSRs),arepolymorphiclocipresentinnuclearandorganellarDNA that

consistofrepeatingunitsof1-6basepairsinlength(Turnpennyand

Ellard,2005).Theyhavebeenwidelyusedforpositionalcloningofgenes

ofinterest,and construction ofgenome-wide physicalmap,and to

- 22 -

improve understanding ofgenome evolution (Bonierbale etal.,1988;

Kurataetal.,1997;Martinetal.,1993;Tanksleyetal.,1996).Despite

theirimpotance,thedevelopmentofmicrosatellitemarkersystemsfor

manyplantshasbeenlimitedbecauseitisnecessarytoisolate,clone,

sequenceand characterizemicrosatellitelociin mostspeciesthatare

being examined forthe firsttime.The relatively low frequency of

microsatellitesinplantgenomescomparedtohumanoranimalgenomes

may presentan additionaltechnicalproblem (Powelletal.,1996).To

increase the efficiency of microsatellite marker development, novel

approachestoenrichSSRshavebeenproposedandsuccessfullyapplied

inmanyplants(Edwardsetal.,1996;Hamiltonetal.,1999).Therefore,it

maybeusefultosearchformicrosatellitesinpublishedDNAdatabases.

Inthepresentstudy,wedevelopedasetofmicrosatellitemarkersfrom

KoreanputativewildP.ginsengversusChineseP.ginseng.


2.2.1.PlantmaterialsandextractionofgenomicDNA

ThePanax ginseng samplesused wereeitherfresh ordried roots.

SamplesofputativewildP.ginsengsampleswereobtainedasleaffrom

Punggi-eup,Inje-gun,Hwacheon-gunandcultivatedP.ginsengsamples

were purchased in China (Table 2.1).Furthermore,10 samples of

mountain cultivated P.ginseng were provided from Yangyang-gun

(AgriculturalDevelopment and Technology Center) (Table 2.2).The

samples were ground into fine powders after freezing with liquid

nitrogen.GenomicDNA wasthenextractedusingaDNeasyplantDNA

isolationkit(Qiagen,Germany).

- 23 -

2.2.2.PrimerdesignandPCRamplification

Theflankingsequencesoftherepeatmotifswereusedtodesignspecific

primers.Base upon database,various microsatellites were obtained.

Amongthem,6primerswereselectedforanalysis(Table2.3).

PCRreactionswereperformedin25 volumemadeinDNAfreewater

containing10-20ngofgenomicDNA,0.5pmolesofeachprimer(Table

2.3),0.2mM ofeachdNTP,2mM MgCl2 and0.5unitofTaqDNA

polymerase(EX Taq,TaKaRa,Japan).ReactionswererunonaDNA

thermalcycler (Applied Biosystems 9800,Foster City,CA,U.S.A.).

Cyclingconditionswere94 for5min;followedby35cyclesof94 for

30s,appropriateannealingtemperature58 for30s,72 for1minand

finalextensionstepat72 for5min.

2.2.3.Electrophoresisandsilverstaining

Microsatellite PCR amplified products were electrophoresed in 6%

denaturingpolyacrylamidegelcontaining7M ureain1TBEbufferata

constantvoltageof1000-1500Vfor2-3h.

Afterelectrophoresis,carefullyseparatetheplatesusingaplasticwedge.

The gelshould beattached strongly to theshortglassplate.After

placing thegelplateina10% aqueousaceticacid,thesolution was

shakenlightlyuntilloadingdyedisappeared.Afterthreewashingsofgel

platewithtwominuteintervalsusingultrapurewaterandallmoistures

wereremoved.Afterabout90minutesofdyeinginasolutionof2Lof

ultrapurewater,3 of37% formaldehydeand2gofsilvernitrate,gel

platewasgainwashedwithultrapurewater.A mixedsolutionof2Lof

ultrapurewater,60gofsodium carbonate,3 of37% formaldehydeand

400 ofsodium thiosulfate(10mg/)thathadbeencooledinadvance

- 24 -

wasseparatedintotwodevelopingsolutionsof1L.Gelplatewasplaced

insidethefirstdevelopingsolution.OncethefirsttemplateDNA beganto

appear,theplatewasplacedinthesecondsolutionanddevelopeduntil

thebandappeared.Developedgelplatewasprocessedinafixsolution

for2-3minutesandwashed2-3timesusingultrapurewater.Locationof

bandwasinterpretedaftercompletelydryingthegelplate.


2.3.1Primerdesign

Theflankingsequencesoftherepeatmotifswereusedtodesignspecific

primers.Among the38SSR primers,six microsatelliteprimerswere

analysedtoauthenticatetheKoreanginsengfrom Chineseginseng.From

six primers,fourKoreanginsengspecificSSR markerswereobtained,

which was clearly distinguished the Korean ginseng from Chinese

ginseng.

2.3.2AuthenticationofKoreanginsengfrom Chineseginseng

SSR analysisrevealedthattherearecleardifferencesinbandpatterns

ofKoreanginsengandChineseginseng.DependedupontheSSRmarkers

(PG409,PG450,PG491andPG582)tested,specificbandsforKoreanor

Chineseginsengweregeneratedprimers(Figs.2.1-2.4).

Amongthefourprimerstested,PG409primerwereclearlydemonstrated

themoleculardifferencesbetweenKoreanginsengandChineseginseng

(Fig2.1).However,amongtheKoreanginsengsamples,thesamplefrom

Inje-gunwasexceptionallydifferentfrom othersamples(Fig2.1,arrow).

Moreover, thebandpatterninonesample(China3)ofChineseginseng

- 25 -

wassameintheKoreanginsenginall4primers(PG409,PG450,PG491

andPG582).ThisresultindicatesthatsomeamountofKoreanginseng

seedsarecultivatedinChina,whichsamplescanmakenew problem to

distinguishtheChineseginsengbymolecularanalysis.

Also,incaseofPG409andPG450,specialbandsforInje-gunginseng

werefound(Figs.2.1,2.2).ThespecialbandsinInje-gunginsengmight

indicatethenew wildvarityofKoreanmountainginseng.In caseof

PG491,thebandpatternwasgenerallysimilarbetweentheKoreanand

Chinese ginsengs,butthere were darkerand specific bands in the

Chineseginseng (Fig 2.3).Todistinguish theKorean ginseng among

Chineseginseng,itseemsthatanalysisofbandpatternsusingseveral

primersisimportanttoclearlydistinguishtheKoreanginsengs.

IncaseofPG582,thebandpatternwasdifferentbetweentheKorean

and Chineseginsengs,and therewasspecific bands fortheChinese

ginseng(Fig.2.4).

ThefourSSR primers(PG409,PG450,PG491and PG582)canbe

developedasthemarkertodistinguishtheKoreanginsengfrom Chinese

ginseng.However,itisnotappropriatetodistinguishtheKoreanginseng

usingsingleprimerset.EspeciallyinPG450,italsoseemsinappropriate

for the marker because ofminute difference between Korean- and

Chineseginseng.Therefore,theseveralprimersshouldbeusedasthe

markertodistinguishtheKoreanandChineseginsengs.

Formoresecureandclearidentificationofreproductivity,itisnecessary

tousemorediversesamplesinadditiontothoseusedinthisstudy,and

todevelopmorenew SSRmarkers.

2.3.3AnalysisinYangyang-gun(A.D.T.C.)samples

Differentbanding patterns were compared in 10 randomly selected

- 26 -

ginseng from Yangyang-gun (provided from A.D.T.C.)using primer

PG491andPG582.

In caseofPG491,theband pattern revealed thatthere werethree

specificbandsin theChineseginseng although generalband patterns

weresimilar(Fig2.5).Especiallythesamplefrom Myeonokchi-rishowed

thesametendencyofbandpatternasthatoftheChineseginseng.

Finally,inthebandpatterngeneratedbyPG582,Myeonokchi-risample

also showed the same band pattern to the Chinese ginseng.This

indicatesthatKorean ginseng iscultivated in Chinaand imported to

Korea.

Inconclusion,4SSR primer(PG409,PG450,PG491,andPG582)were

showntodistinguishtheKoreanginsengamongtheChineseginseng.

- 27 -

Table 2.1. Samples of various ginseng for Microsatellite analysis

No. Samples Scientific name Collection site

1Wild ginseng

in Korea

Panax ginseng Punggi-eup,

Gyeongsang-do

2Hwacheon-gun 1,

Kangwon-do

3Samcheok-si,

Kangwon-do

4Inje-gun,

Kangwon-do

5Hwacheon-gun 2,

Kangwon-do

6Hwacheon-gun 3,

Kangwon-do

7Hwacheon-gun 4,

Kangwon-do

8Hwacheon-gun 5,

Kangwon-do

9Cultivated ginseng

in China

Panax ginseng Chang bai mountain 1,

Jinlin

10Chang bai mountain 2,

Jinlin

11Chang bai mountain 3,

Jinlin

- 28 -

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

Microsatellite analysis

No. Samples Scientific name Collection site

1

Cultivated ginseng

in Korea

Panax ginseng

Songhyun-ri

2Myeonokchi-ri

3Beopsuchi-ri

4Songhyun-ri

5Wol-ri

6Myeonokchi-ri

7Songhyun-ri

8Cultivated ginseng

in China

Panax ginseng Hunchun

9Cultivated ginseng

in Korea

Panax ginseng Beopsuchi-ri

10Jukjeongja-ri

- 29 -

Table2.3.Microsatelliteprimers,nucleotidesequences,coremotifsand

numberofrepeatsandexpectedproductsizeforSSR

SSR

designationSequence(5'3')

Repeatmotif

andno.of

repeats

Expected

size(bp)

PG22 ATCAAGTTGGAAATCAGGTGGG (TC)22107

GTGTCTATGCAAGTTGCGGCTGPG281 AAACTCTCTTCTAGTCTTCTTGCC (CTAT)12

201GTAGGTATACATACATGTACGGAA

PG409 GATCAATCAGAAACAAAGAAAGCT (TGA)7195

GCTGCTCTTTCTGGGTATGCTTPG450 GATCTTCTGGATGATTTCGACAT (GA)13

172TTGCCCACCCCTTTCTCCACC

PG491 AGGGAGTACGGAAGGATGGAAG (TC)14211

GCTCAGTGTTTACAGACACAATT

PG582 GAATTCATGCTCGGACTCAGTC (GGA)7298

CCACTATTCTCCATCTCTCACC

- 30 -

Fig.2.1.GeneticdiversityofSSR analysisusingPG409primerbetween

Korean-andChineseginseng.Thefigureshowstheallelicprofileofa

microsatellitelocusin P.ginseng specieson 6% polyacrylamidegels

silver-stained. Polymorphism was detected clearly in Korean- and

Chineseginseng.Arrow ()indicatesexpectedamplifiedChineseginseng

specificbands.Arrow heads( )indicateChineseginsengspecificbands

indicatesexpectedamplifiedKoreanginsengspecificbands.

- 31 -




silver-stained.Polymorphism wasdetectedbetweenKorean-andChinese

ginseng.Arrow ()indicatesexpectedamplifiedChineseginsengspecific

bands.Allow heads( )indicatesexpectedamplifiedInje-gunginseng

specificbands.

- 32 -





ginseng.Arrow ()indicatesexpectedamplifiedChineseginsengdarker

andspecificbands.

- 33 -





ginseng.Arrow ()indicatesexpectedamplifiedChineseginsengspecific

bands.

- 34 -

Fig.2.5.GeneticdiversityofSSRanalysisusingPG491primeramongthe

Yangyang-gun samples produced from A.D.T.C.and Chinese ginseng.

ThefigureshowstheallelicprofileofamicrosatellitelocusinP.ginseng

species on 6% polyacrylamide gels silver-stained.Polymorphism was

detected in samplesofYangyang-gun.Arrow ()indicatesexpected

amplifiedChineseginsengdarkerandspecificbands.Arrow heads( )

indicateMyeonokchi-riginsengspecificbands.

- 35 -

Fig.2.6.GeneticdiversityofSSRanalysisusingPG582primeramongthe

Yangyang-gun samples produced from A.D.T.C.and Chinese ginseng.

ThefigureshowstheallelicprofileofamicrosatellitelocusinP.ginseng

species on 6% polyacrylamide gels silver-stained.Polymorphism was

detected in samplesofYangyang-gun.Arrow ()indicatesexpected

amplifiedChineseginsengdarkerandspecificbands.Arrow heads( )

indicateMyeonokchi-riginsengspecificbands.

- 36 -

Literaturecited

Bai,D.P.,Brandle,J.,andReeleder,R.(1997)GeneticdiversityinNorth

Americanginseng(PanaxquinquefoliusL.)growninOntariodetectedby

RAPDanalysis.Genome,40,111-115.

Bonierbale,M.W.,Plasisted,R.L.,andTanksley,S.D.(1988)RFLP

mapsbasedonacommonsetofclonesrevealmodesofchromosomal

evolutioninpotatoandtomato.Genetics120,1095-1103.

Chan,T.W.,But,P.P.H.,Cheng,S.W.,Kwok,I.M.,Lau,F.W.,and

Xu,H.X.(2000)Differentiation and authentication ofPanaxginseng,

Panaxquinquefolius,andginseng productsby using HPLC-MS.Anal.

Chem.,72,1281-1287.

Chang,H.,andBut,P.(1986)PharmacologyandapplicationofChinese

materialmedica.Vol.1,WorldScientificPublishing,Singapore,1986,p.

17.

Chen,S.E.,Staba,E.J.,Taniyasu,S.,Kasai,R.,andTanaka,O.(1981)

Furtherstudyondammarane-saponinsofleavesandstemsofAmerican

ginseng,Panaxquinquefolium.PlantaMed.42,406-409.

Cheung,K.S.,Kwan,H.S.,But,P.P.H.,andShaw,P.C.(1994)

PharmacognosticalidentificationofAmericanandOrientalginsengroots

by genomic fingerprinting using arbitrarily primed polymerase chain

reaction(AP-PCR).J.Ethnopharmacol.42,67-69.

Chuang,W.C.,Wu,H.K.,Sheu,S.J.,Chiou,S.H.,Chang,H.C.,and

- 37 -

Chen,Y.P.(1995)A comparative study on commercialsamples of

ginsengradix.PlantaMed.61,459-465.

Chung,Y.Y.,Lee,M.G.,Chung,C.M.,andJo,J.S.(1998)Comparison

ofplantgrowth and morphologicalcharacteristics among the Korean

ginseng,theAmericanginsengandtheBambooginseng.J.GinsengRes.

22,147-153.

Chung,Y.Y.,Chung,C.M.,Ko,S.R.,Choi,K.T.(1995)Comparisonof

agronomiccharacteristicsandchemicalcomponentofPanaxginseng C.

A.Meyer and Panax quinquefolium L.Korean J.Ginseng Sci.19,

160-164.

Choi,Y.E.,Ahn,C.H.,Kim,B.B.,andYoon,R.S.(2008)Development

ofspecies specific AFLP-derived SCAR markerforauthentication of

PanaxjaponicusC.A.Meyer.Biol.Pharm.Bull.31,135-138

Dou,D.Q.,Hou,W.B.,Chen,Y.J.(1998)Studiesonthecharacteristic.

constituentsofChineseginsengandAmericanginseng.PlantaMed.64,

585-586.

Edwards,K.J.,J.H.Baker,A.Daly,C.Jonesand A.Karp (1996)

Microsatellite libraries enriched forseveralmicrosatellite sequences in

plants.Biotechniques20,758-760.

Fushimi,H.,Komatsu,K.,Isobe,M.,andNamba,T.(1997)Applicationof

PCR-RFLPandMASA analyseson18SribosomalRNA genesequence

forthe identification ofthree ginseng drugs.Biol.Pharm.Bull.20,

765-769.

- 38 -

Ha,W.Y.,Shaw,P.C.,Liu,J.,Yau,F.C.,and Wang,J.(2002)

AuthenticationofPanaxginsengandPanaxquinquefoliususingamplified

fragment length polymorphism (AFLP) and directed amplification of

minisatelliteregionDNA(DAMD).J.Agric.FoodChem.50,1871-1875.

Hamilton,M.B.,Pincus,E.L.,Di-Fiore,A.andFleischer,R.C.(1999)

UniversallinkerandligationproceduresforconstructionofgenomicDNA

librariesenrichedformicrosatellites.Biotechniques27,500-507.

Han,L.K.,Zheng,Y.N.,Yoshikawa,M.,Okuda,H.,andKimura,Y.

(2005)Anti-obesity effects ofchikusetsusaponins isolated from Panax

japonicusrhizomes.BMCComplement.Altern.Med.5,9.

Ho,I.S.H.,andLeung,F.C.(2002)Isolationandcharacterizationof

repetitiveDNA sequencesfrom Panaxginseng.Mol.Genet.Genomics

266,951-961.

Hon,C.C.,Chow,Y.C.,Zeng,F.Y.,andLeung,F.C.C.(2003)Genetic

authentication ofginseng and othertraditionalChinesemedicine.Acta

Pharmacol.Sin.24,841-846.

Hosono-Nishiyama,K.,Matsumoto,T.,Kiyohara,H.,Nishizawa,A.,

Atsumi,T.,andYamadaH.(2006)SuppressionofFas-mediatedapoptosis

ofkeratinocytecellsby chikusetsusaponinsisolated from therootsof

Panaxjaponicus.PlantaMed.72,193-198.

Kim,B.B.,Jeong,J.H.,Jung,S.J.,Yun,D.W.,Yoon,E.S.,andChoi,

Y.E.(2005)AuthenticationofKoreanPanaxginsengfrom chinesePanax

- 39 -

ginsengandPanaxquinquefoliusbyAFLPanalysis.J.PlantBiotechnol.

7,81-86.

Kim,J.J.,Jo,B.H.,Lee,K.L.,Yoon,E.S.,Ryu,G.H.,andChung,K.

W.(2007)Identificationofnew microsatellitemarkersinPanaxginseng.

Mol.Cells24,60-68.

Kim,S.M.,andSohn,J.K.(2005)Identificationofaricegene(Bph1)

conferringresistancetobrownplanthopper(NilaparvatalugensStal)using

STSmarker.Mol.Cells20,30-34.

Komatsu,K.,Zhu,S.,Fushimi,H.,Qui,T.K.,Cai,S.,andKadota,S.

(2001)Phylogeneticanalysisbasedon18S rRNA geneandmatK gene

sequencesofPanaxvietnamensisandfiverelatedspecies.PlantaMed.67,

461-465.

Kurata,N.,Umehara,Y.,Tanoue,H.,and Sasaki,T.(1997)Physical

mapping ofthe rice genome with YAC clones.PlantMol.Biol.35,

101-113.

Martin,G.B.,Brommonschenkel,S.H.,Chunwongse,J.,Frary,A.,Ganal,

M.W.,etal.(1993)Map-based cloning ofa protein kinase gene

conferringdiseaseresistanceintomato.Science262,1432-1436.

Matsuda,H.,Samukawa,K.,Fukuda,S.,Shiomoto,H.,Tung,C.N.,and

Kubo,M.(1989)StudiesofPanaxjaponicusfibrinolysis.PlantaMed.55,

18-21.

Mihalov,J.J.,Marderosian,A.D.,and Pierce,J.C.(2000) DNA

- 40 -

identificationofcommercialginseng samples.J.Agric.FoodChem.48,

3744-3752.

Ngan,F.,Shaw,P.,But,P.P.H.,and Wang,J.(1999)Molecular

authenticationofPanaxspecies.Phytochemistry50,787-791.

Paran,I.,and Michelmore,R. W. (1993) Development of reliable

PCR-basedmarkerslinkedtodownymildew resistancegenesinlettuce.

Theor.Appl.Gen.85,985-993.

Powell,W.W.,Machery,G.C.,Provan,J.(1996)Polymorphism revealed

bysimplesequencerepeats.TrendsGenet.1,215-222.

Qin,J.,Leung,F.C.,Fung,Y.,Zhu,D.,and Lin,B.(2005)Rapid

authentication ofginseng speciesusing microchip electrophoresiswith

laser-inducedfluorescencedetection.Anal.Bioanal.Chem.381,812-819.

Sambrook,J.,andRussell,D.W.(2001)"MolecularCloning:ALaboratory

Manual,"ColdSpringHarborLaboratoryPress,New York.

Shaw,P.C.,andBut,P.P.H.(1995)AuthenticationofPanaxspecies

andtheiradulterantsbyrandom-primedpolymerasechainreaction.Planta

Med.61,466-469.

Shim,Y.H.,Choi,J.H.,Park,C.D.,Lim,C.J.,Cho,J.H.,andKim,H.

J.(2003)MoleculardifferentiationoPanaxspeciesbyRAPD analysis.

Arch.Pharm.Res.26,601-605.

Shu,Z.,Hirotoshi,F.,Shaoqing,C.,andKatsuko,K.(2003)Phylogenetic

- 41 -

relationshipinthegenusPanax:inferredfrom chloroplasttrnK geneand

nuclear18SrRNAgenesequences.PlantaMed.69,647-653.

Tanksley,S.D.,Grandillo,S.,Fulton,T.M.,Zamir,D.,Eshed,Y.,etal.

(1996)AdvancedbackcrossQTL analysisinacrossbetweenanelite

processinglineoftomatoanditswildrelativeL.pimpinellifolium.Theor.

Appl.Genet.92,213-224.

Turnpenny,P.and Ellard,S.(2005) Emery's Elements of Medical

Genetics,12th.ed.Elsevier,London

Um,J.Y.,Chung,H.S.,Kim,M.S.,Na,H.J.,Kwon,H.J.,etal.(2001)

MolecularauthenticationofPanaxginsengspeciesbyRAPDanalysisand

PCR-RFLP.Biol.Pharm.Bull.24,872-875.

Vos,P.,Hogers,R.,Bleeker,M.,Reijans,M.,VandeLee,T.,Hornes,M.,

Frijters,A.,Pot,J.,Peleman,J.,Kuiper,M.,andZabeau,M.(1995)AFLP:

a new technique for DNA fingerprinting.Nucleic Acids Res.23,

4407-4414.

Wang,J.,Ha,W.Y.,Ngan,F.N.,But,P.P.H.,andShaw,P.C.(2001)

Applicationofsequencecharacterizedamplifiedregion(SCAR)analysisto

authenticatePanaxspeciesandtheiradulterants.PlantaMed.67,781-783.

Wen,J.,andZimmer,E.A.(1996)PhylogenyandbiogeographyofPanax

L.(theginseng genus,araliaceae):inferencesfrom ITS sequencesof

nuclearribosomalDNA.Mol.Phylogenet.Evol.6,167-177.

Yamahara,J.,Kubomura,Y.,Miki,K.,andFujimura,H.(1987)Anti-ulcer

- 42 -

actionofPanaxjaponicusrhizome.J.Ethnopharmacol.19,95-101.

Yun,T.K.(2001)BriefintroductionofPanaxginsengC.A.Meyer.J.

KoreanMed.Sci.16,3-5.

Zou, K., Zhu, S., Tohda, C., Cai, S., and Komatsu, K. (2002)

Dammarane-typetriterpenesaponinsfrom Panaxjaponicus.Nat.Prod.65,

346-351.

- 43 -

(PanaxginsengC.A,Meyer) (Araliaceae)

,

. 12

.

(Panaxginseng) ,,

.

, .

.

.

, ,,,,

AFLP microsatellite

.

- 44 -

AFLP primer JG14 PCR

, .

microsatellite 4 priemr , PG409

. primer

marker

marker

.

- 45 -

2

. ,

.

.

.

,

.

, ,

.

.

, Dr.Anbu,Dr.Harada,

, .

,Dr.

Faisal, , , , ,

.

, ,

, , , , ,

, , , , ,

, , , ,

, , .

- 46 -

, , , , ,

, , , ,

.

,, ,

, ,,

.

.

,

,

.

INTRODUCTIONChapter 1. Development of species specific AFLP-derived SCAR marker for authentication of Panax japonicus C. A. MeyerAbstract1.1 Introduction1.2 Materials and Methods1.2.1 Plant materials and extraction of genomic DNA1.2.2 Genomic DNA digestion and adapter ligation1.2.3 AFLP analysis1.2.4 Cloning, DNA sequencing and SCAR primer design1.2.5 PCR analysis

1.3 Results and Discussion1.3.1 AFLP analysis1.3.2 Isolation of AFLP fragment for SCAR marker design1.3.3 PCR amplification using SCAR primers

Chapter 2. Development of new microsatellite markers for Panax ginsengAbstract2.1 Introduction2.2 Materials and Methods2.2.1 Plant materials and extraction of genomic DNA2.2.2 Primer design and PCR amplification2.2.3 Electrophoresis and silver staining

2.3 Results and Discussion2.3.1 Primer design2.3.2 Authentication of Korean ginseng from Chinese ginseng2.3.3 Analysis in Yangyang-gun (A.D.T.C.) samples

Literature cited

12INTRODUCTION 1Chapter 1. Development of species specific AFLP-derived SCAR marker for authentication of Panax japonicus C. A. Meyer 4 Abstract 4 1.1 Introduction 5 1.2 Materials and Methods 6 1.2.1 Plant materials and extraction of genomic DNA 6 1.2.2 Genomic DNA digestion and adapter ligation 7 1.2.3 AFLP analysis 7 1.2.4 Cloning, DNA sequencing and SCAR primer design 8 1.2.5 PCR analysis 9 1.3 Results and Discussion 10 1.3.1 AFLP analysis 10 1.3.2 Isolation of AFLP fragment for SCAR marker design 11 1.3.3 PCR amplification using SCAR primers 11Chapter 2. Development of new microsatellite markers for Panax ginseng 20 Abstract 20 2.1 Introduction 21 2.2 Materials and Methods 22 2.2.1 Plant materials and extraction of genomic DNA 22 2.2.2 Primer design and PCR amplification 23 2.2.3 Electrophoresis and silver staining 23 2.3 Results and Discussion 24 2.3.1 Primer design 24 2.3.2 Authentication of Korean ginseng from Chinese ginseng 24 2.3.3 Analysis in Yangyang-gun (A.D.T.C.) samples 25Literature cited 36

development of molecular markers for authentication_chang ho_thesis

Documents