การวิเคราะห์ พีซีอาร์ อาร์เอฟแอลพี ของไมโตคอนเดรียดีเอ็นเอ ...digital_collect.lib.buu.ac.th/journal/Science/v15n2/87-98.pdf ·

87

การวิเคราะห์พีซีอาร์อาร์เอฟแอลพีของไมโตคอนเดรียดีเอ็นเอเพื่อบ่งชี้ความแตกต่างของประชากรปลาหมอ

(Anabas testudineus)ในประเทศไทย

PCR-RFLPAnalysisofMitochondrialDNAtoDifferentiatePopulationsofClimbingPerch

(Anabas testudineus)inThailand

SyarifHidayat1,2 and Wansuk Senanan1*1DepartmentofAquaticScience,FacultyofScience,BuraphaUniversity.

2DepartmentofAquaculture,FacultyofFisheries,BrawijayaUniversity,Malang-Indonesia.

บทคัดย่อ

การเพาะเลี้ยงปลาหมอ(Anabas testudineus)ได้รับความสนใจในหลายๆประเทศเนื่องจากเป็นปลาที่มีการบริโภคอย่าง

แพร่หลาย เป็นแหล่งโปรตีนที่มีราคาถูก และเพิ่มความสำคัญทางเศรษฐกิจในระดับนานาชาติ กรมประมงของประเทศไทยจึงมี

ความสนใจที่จะคัดเลือกปลาชนิดนี้ให้มีลักษณะที่ดีขึ้นสำหรับการเพาะเลี้ยงสัตว์น้ำ อย่างไรก็ตามยังขาดข้อมูลเบื้องต้นถึงระดับ

ความหลากหลายทางพนัธกุรรมและความแตกตา่งทางพนัธกุรรมของแหลง่ปลาทีใ่ชใ้นการปรบัปรงุพนัธ์ุการศกึษานีจ้งึใชเ้ทคนคิพซีอีาร์

อาร์เอฟแอลพี (polymerase chain reaction restriction fragment length polymorphism; PCR-RFLP) ของบริเวณดีลูป

ของไมโตคอนเดรียในการบ่งชี้ความแตกต่างระหว่างประชากรธรรมชาติ (6ประชากร)และประชากรโรงเพาะฟัก (2ประชากร)

ของปลาหมอ (Anabas testudineus) (n = 21-29) การวิเคราะห์ความแปรปรวนแสดงความหลากหลายทางพันธุกรรมของ

ตัวอย่างจากโรงเพาะฟักที่น้อยกว่าตัวอย่างจากธรรมชาติอย่างมีนัยสำคัญ (P = 0.01; ค่าเฉลี่ยความหลากหลายแฮพโพลไทป์ =

0.52และ0.10ในตัวอย่างจากธรรมชาติและจากโรงเพาะฟักตามลำดับ)อย่างไรก็ตามไม่พบความสัมพันธ์ระหว่างการปรากฏ

รูปแบบของแฮพโพลไทป์ กับแหล่งของตัวอย่าง การศึกษานี้ยังพบความแตกต่างกลุ่มตัวอย่างในเกือบทุกกลุ่ม โดยการวิเคราะห์

ความแปรปรวนทางพันธุกรรม (Analysis of Molecular Variance; AMOVA) พบ 57.83% ของความแปรปรวนเกิดจาก

ความต่างระหว่างประชากรนอกจากนี้ค่าFSTระหว่างคู่ตัวอย่างยังบ่งชี้ความแตกต่างระหว่างเกือบทุกคู่การทดสอบยกเว้นสองคู่

(ระหว่างตัวอย่างจากโรงเพาะฟัก 2 ตัวอย่าง และ ระหว่าง ชลบุรี, CH และ ราชบุรี, RT) แผนภูมิแสดงความสัมพันธ์ทาง

วิวัฒนาการ (Neighbor-Joining dendrogram) ที่สร้างจากค่าระยะห่างทางพันธุกรรมNei’s genetic distance ยังบ่งชี้ความ

แตกต่างระหว่างตัวอย่างจากจังหวัดสกลนคร(SN)กับตัวอย่างอื่นๆแต่ไม่ได้แสดงความสัมพันธ์อย่างชัดเจนระหว่างตัวอย่างที่เหลือ

(นครปฐม (NP), RT, CH และนครศรีธรรมราช (NS)) ซึ่งเป็นตัวแทนของแม่น้ำสายหลักในประเทศไทย ดังนั้นเทคนิคพีซีอาร์

อาร์เอฟแอลพีของบริเวณดีลูป จึงเป็นเทคนิคที่มีประสิทธิภาพในการประเมินความหลากหลายทางพันธุกรรมภายในและระหว่าง

ประชากรปลาหมอได้ อย่างไรก็ตาม ระดับความหลากหลายทางพันธุกรรมดังกล่าวอาจยังไม่เพียงพอในการแสดงความสัมพันธ์ทาง

พนัธกุรรมระหวา่งกลุม่ตวัอยา่งบางกลุม่ได้ขอ้มลูจากการศกึษานีส้ามารถใชป้ระกอบการตดัสนิใจในการจดัการระดบัความหลากหลาย

ทางพันธุกรรมที่มีอยู่ในปัจจุบันของประชากรโรงเพาะฟัก และช่วยในการออกแบบประชากรตั้งต้นในการคัดเลือกปรับปรุงพันธ์ุของ

ปลาชนิดนี้ได้

คำสำคัญ:พีซีอาร์อาร์เอฟแอลพีบริเวณดีลูปไมโตคอนเดรียดีเอ็นเอAnabastestudineusประเทศไทย

--------------------------------------------------

Correspondingauthor.E-mail:[email protected]

SyarifHidayatandWansukSenanan/BuraphaSci.J.15(2010)2:87-98

88

Abstract

Climbingperch(Anabas testudineus)isacommontropicalfresh-andbrackishwaterfishspeciesinAsia.Its

aquaculturehasreceivedattentioninmanyAsiancountriesbothasanemergingeconomicspeciesandaffordable

proteinsource.TheThaiDepartmentofFisheries,therefore,hasbeeninterestedinaselectivebreedingprogram

forthisspecies.However,thelackofgeneticdatapreventssuchsystematicbreedingprogram.Weperformed

polymerasechainreactionrestrictionfragmentlengthpolymorphism(PCR-RFLP)analysisofmitochondrialD-loop

regiontodifferentiatesixwildandtwohatcherypopulationsofAnabas testudineusinThailand(n=21-29).

Analysisofvarianceindicatedthathatcherypopulationshadlowergeneticdiversitythanwildpopulations(P =

0.01;averagehaplotypediversity=0.52and0.10inthewildandhatcherysamples,respectively).However,there

wasnoclearrelationshipbetweenthepresenceofhaplotypesandgeographiclocations.Almostallpopulations

weregeneticallydistinct.AnalysisofMolecularVariance(AMOVA)indicated57.83%amongpopulationgenetic

variation. Pairwise FSTvaluesshowedsignificantdivergenceinalmostallpopulationpairs,exceptfortwopairs

(twohatcherysamples;andChonburi,CHandRatchaburi,RT).Neighbor-JoiningdendrogrambasedonNei’s

geneticdistancesuggestedhighdivergencebetweenSakhonNakhon,SNandtheremainingsamples,butitdid

notresolvetherelationshipsamongNakhonPathom(NP),RT,CHandNakhonSiThammarat(NS)representing

majorriversystemsinThailand.ThePCR-RFLPofthemitochondrialD-loopregionwasaneffectivetechnique

toevaluategeneticdiversitywithinandamongpopulations.However,thelevelofpolymorphismmaynotbe

adequatetoresolvetherelationshipsamongsomepopulations.Theresultscanaidthemanagementofexisting

geneticvariationwithinhatcheriesandthedevelopmentofabasepopulationforselectivebreeding.

Keywords:PCR-RFLP,D-loopregion,mitochondrialDNA,Anabastestudineus,Thailand

SyarifHidayatandWansukSenanan/วารสารวิทยาศาสตร์บูรพา.15(2553)2:87-98

89

Introduction

Climbingperch(Anabas testudineus)isacommon

tropicalfresh-andbrackishwaterfishspeciesinAsia.

ItsaquaculturehasreceivedattentioninmanyAsian

countriesbothasanemergingeconomicspeciesand

affordableproteinsource.ProductionofA. testudineus

inThailandincreasedfrom7,700tonsin2001to16,200

tonsin2005(ThailandDepartmentofFisheries,2006).

Withgrowingnumberoffarms(550farms(recordedin

2005and1,024farmsrecordedin2007)inThailand,there

areinterestsindevelopingdomesticatedbroodstockof

thisspecies(Dr.SangaLeesanga,ChumphonFisheries

TestandResearchCenter,Personalcommunication).

An initial step for a breeding program is to

developabasepopulation.Acriticalrequirementfor

aneffectivebreedingprogramistoknowwhetherthe

speciesexistsasasinglegeneticunit(homogeneous)

or as series of relatively genetically distinct groups

(heterogeneous)(Beaumont&Hoare,2003).Ferguson

(1994)suggestedthatacommercialbreedingprogram

shouldstartfromabasepopulationwithawide-range

genetic base. Thiswill ensure for long-term genetic

responses(Hayeset al.,2006).Todevelopsuchbase

population,geneticdataofthesourcepopulationswill

be critical. The genetic datawill help us determine

whetherpotentialsourcesforthebasepopulationare

geneticallydistinct.Inaddition,thesedatacanaidthe

decisionstoconserveoriginalgenepoolforfutureap-

plication(Tinniet al.,2007).

MitochondrialDNA(mtDNA)geneticmarkerhas

been proven successful in differentiating populations

ofseveralfishspeciessuchasswordfish(Chowet al.,

1997),sockeyesalmon(Brykovet al.,2003),Dollyvarden

(Oleiniket al.,2004),andfreshwatergoby(Takahashi

&Ohara,2003).MtDNAcanbemoresensitive than

allozymeornuclearDNAtodetectgeneticdifferences

betweenpopulationsthatarerecentlydiverged(Parker

et al.,1998).Ithassmallereffectivepopulationsize(Liu

&Cordess,2004)andhigherrateofnucleotidesubstitu-

tionsthanallozymesornuclearDNA(Brownet al.,1979).

Inaddition,mtDNAisappropriateforpopulationgenetic

studybecausemitochondrialgenomeissmall,maternally

inherited, and only small amount of sample needed

foranalysis.TheD-looporcontrolregion(non-coding

region)ofmtDNAisgenerallymorevariablethanthe

coding region due to its lower functional constrains and

lackofselectionpressure(Liu&Cordess,2004).Inthis

study,weevaluatedgeneticvariationofA. testudineus

from different geographic areas using PCR-RFLP of

mtDNAD-loopregion.Theinformationobtainedfrom

thestudycanhelpdesignmanagementstrategiesto

maximizegeneticvariationinhatcherypopulationsas

wellastohelpidentifypopulationswithuniquegenetic

identityforconservation.

Materialsandmethods Wecollected fin clips of adultA. testudineus

fromsixwildandtwohatcherypopulations(Table1).

Sampleswerecollectedbetweenyears2007to2008.

The2007sampleswerecollectedbythestaffofthe

ChumphonFisheriesTestandResearchCenter.Wild

samplescamefromNakhonSiTammarat(NS),Uttaradit

(U),SakhonNakhon(SN),Chonburi(CH),Ratchaburi

(RT),andNakhonPathom(NP),whilehatcherysamples

were from Chumphon Fisheries Test and Research

Center(CU1)andaprivatefarminChumphon(CU2).

Sampleswerepreservedin95%ethanol.

DNAwasextractedfollowingaprotocolmodified

fromAljanabiandMartinez(1997).PCRamplification

wasperformedusingprimerMTA-01(5’-AAGCCAGGA

TTCTAAATTAAA-3’)andMTA-02(5’-TCTTCAGTG

TTATGCTTTGA-3’)designedfromavailablesequence

of mitochondrial D-loop region of A. testudineus in

the online database, Genbank (accession number

EF179144).A 10µl PCR reaction contains 1XPCR

buffer,0.4mMMgCl2,0.2mMofdNTP,0.2µMofeach

primers,10-100ngofDNAtemplate,and0.2unitof


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Taqpolymerase (Vivantis).PCRwasperformed ina

thermalcycler (TGradient)witha temperatureprofile

consistingof40cyclesofdenaturationat93oCfor1

minute,annealingat49oCfor1minute30secondsand

extensionat72oCfor1minute.Thecycleendedwith1

cycleof72oCfor10minutes.PCRamplifiedabout903

basepairs(bp)fragmentoftheD-loopregion.

Weanalyzedrestrictionpatternsofsevenrestric-

tionenzymes;ApoI,AvaII,EcoRII,HaeII,HindII,AflII,and

BccI (Table2).Weanalyzed thepotential restriction

sitesusingacompleteD-loopsequencefromGenBank

(EF179144)andsixindividualsfromNS,CHandNP.

Digestionreactionwasperformedusing1µgofPCR

productfor1Unitofrestrictionenzyme.Digestionwas

donein10µlofreactionvolumeandincubatedforat

leastsixhouratthespecifiedincubationtemperature.

DNAfragmentscleavedbytherestrictionenzymewere

electrophorezedon1.5%agarosegelandstainedwith

Table 1. DescriptionsofA. testudineussamplesusedinthisstudy.

No LocationSample

code

Sample

numberRiversystem

Geographic

region

Collection

year

1

2

3

4

5

6

7

8

Uttaradit

NakhonPathom

SakhonNakhon

Ratchaburi

Chonburi

NakhonSiTammarat

ChumphonFisheriesTest

andResearchCenter

PrivatefarminChumphon

U

NP

SN

RT

CH

NS

CU1

CU2

21

23

22

26

29

21

28

21

ChaoPhraya

ChaoPhraya

Mekong

Maeklong-Petchaburi

Eastern

Peninsular

-

-

Northern

Central

Northeast

Central

Eastern

Southern

-

-

2007

2008

2007

2007

2008

2007

2007

2007

Table 2. Specificityofrestrictionenzymesusedinthisstudyandthepotentialfragmentsizeafterrestriction

(generatedusingacompletesequenceofA. testudineusD-loopregionfromGenbank,EF179144,and

apartialsequenceobtainedinourstudy).

Enzyme Recognitionsites Incubationtemperature(oC) Potentialrestrictioncuts(basepairs)

ApoI

AvaII

EcoRII

HaeII

HindII

AflII

BccI

RAATTY

GGWCC

CCWGG

RGCGCY

GTYRAC

CTTAAG

CCATC(N)4

50

37

37

37

37

37

37

71,217,620

409,499

8,143,160,220,377

392,516

81,167,660

655,114*

86,95,239,488

*UsingresultfromtheDNAsequencingresultinthepreliminarystudy(769bp)

A=Adenosine;G=Guanine;T=Thymidine;C=Cytosine;R=GorA;Y=TorC;W=AorT;

N=Anybase.


91

ethidium bromide. The electrophoretic migration of

restrictedDNA fragments (haplotype)onagelwere

comparedagainst50bpDNAladder.TheDNAfragments

werevisualizedunderUVlight(VILBERLOURMATETX-

40M).Eachdistincthaplotypepatternwasassignedwith

aletter.Eachindividualfishwasassignedbymultiple

lettersrepresentingitscompositehaplotype.

We evaluated two levels of genetic variation

(withinandamongpopulations).Forwithinpopulation

geneticvariation,weestimatedhaplotypediversity(h)andnucleotidediversity(π)usingDAimplementedin

REAP (McElroy et al., 1992). Homogeneity test was

performed using MONTE implemented in REAP to

determine whether the haplotype frequencies differ

significantlyamongpopulations.Totestwhetherwithin-

populationgeneticvariationdiffersamongpopulations,

weperformedanalysisofvariance(ANOVA)ofhaplotype

numberandhaplotypediversityusingSPSS11(SPSS

Inc.).Toexaminewhether thepresenceofparticular

haplotypescorrelatewithgeographiclocationsofthe

samples,weconstructedaNeighbor-Joiningdendrogram

basedonNei’sgeneticdistanceofcompositehaplotypes

usingPHYLIPversion3.68(Felsenstein,1989).

Toevaluatepopulationgeneticdivergence,two

approacheswereused:(1)analysisofmolecularvari-

ance (AMOVA),and (2)geneticdistanceandcluster

analysis.AMOVAtestwascalculatedusingARLEQUIN

version3.11(Excoffieret al.,2005).Apairwisecompari-

sonamongpopulations(FST)andExacttestswerealso

performedusingARLEQUIN.ANei’sgeneticdistance

matrix(ahundredbootstrapreplicates)wasobtained

usingMTDIS (Danzmann,1998).Thedistancematrix

thenusedtoconstructaNeighbor-Joiningdendrogram

usingPHYLIPversion3.68(Felsenstein,1989).

Results and discussionsWithinpopulationgeneticvariation

Restrictionanalysisyieldedfifteencompositehap-

lotypeswiththenumberofhaplotypeineachsample

rangingfromone(CU2)tosix(NPandSN)(Table3).

SNhadaveryuniquesetofcompositehaplotypes

to other populations. This observation may indicate

thatSNsampleisgeneticallydistinctfromtheothers,

althoughsmallnumberofsamplescouldalsoexplain

why there were no shared haplotypes between SN

andotherpopulations.Amongallhaplotypes,h2 was themostcommonhaplotypewhichpresentinalmost

allsamplesexceptNSandSN.CHandNPshared

thehighestnumberofhaplotypewiththreehaplotypes

presentinbothsamples.MonteCarlosimulationtest

(1000simulations)forhaplotypefrequencyheterogene-

ityamongpopulationsshowedsignificantdifferences

inhaplotypedistributionamongallpopulations(χ2 =

98.53,P<0.001).Clusteranalysisbasedonthelevel

divergenceofmtDNAhaplotypesdidnotrevealstrong

geographicalassociation(Hidayat,2010).

Bothhaplotypediversityandnucleotidediversity

suggestedasimilargeneticdiversitypatternwithNP

havinghighesthaplotypediversity(0.78)andnucleotide

diversity (0.028) and CU2 having lowest haplotype

diversity (Table 3). Nucleotide diversity values were

muchlowerthanhaplotypediversityineverysample.

Most samples had relatively low nucleotide diversity

(0.000-0.028). The level of MtDNA diversity of the

examined A. testudineus populations was similar to

otherfreshwaterfishes.Thehaplotypediversityvalues

(h)observedintheA. testudineuswildpopulations(h =0.39-0.78;average=0.52)(Table3)arewithinthe

rangesobservedinotherfreshwaterspecies,suchas

anAmazoniaspecies,Brycon opalinus,wildpopulations

(h=0.00-0.89;average=0.60;Hilsdorfet al.,2002);andGoldfish,Carassius auratus gibelio,(h=0.00-0.84;average=0.55;Brykovet al.,2002).Thevaluesobserved

inthisstudyarehigherthanthoseobservedinami-

gratoryfishspeciessuchasbrowntrout,Salmo trutta,

(h=0.00-0.54;average=0.30;Weisset al.,2000).

Althoughtheaveragevalueofhaplotypediversity

foundinthisstudywaslowerthanthoseobservedin


92

severalmarinefishes,somepopulationshadreasonably

highlevelofgeneticdiversity.Bothsamplesfromthe

ChaoPhrayariversystem(UandNP)showedhigher

geneticdiversitythantheotherriversystems(average

h=0.66and0.45forsamplesfromtheChaoPhraya

and other river systems, respectively). This result is

similartotheobservationinanotherspecieswithsimilar

lifehistorytoA. testudineus.Na-Nakornet al.(2004)

detected higher level of genetic diversity ofClarias

macrocephalusintheChaoPhrayariversystemcom-

paredtotheMekong,South-east,andPeninsularriver

systems;theauthorssuggestedthattheChaoPhraya

riversystemmightprovideagoodbreedinggroundwith

littlegeographicalbarriers.Thus,thesefishspecieswere

abletomaintainalargepopulationsizeencountering

the effects of genetic drift.

TheabilityofA. testudineus to ‘walk’ and sur-

vive on land for an extended period of timemight

increasethechanceforgeneflowbetweenneighboring

populations.Geneticcontributionfromdistinctgenetic

sources will increase genetic variation of the receiving

populations.Sokhenget al.(1999)reportedthehistory

oflateralmigrationofthisspeciesfromtheMekong

mainstreamorotherpermanentwaterbodiestoflooded

areasduringthefloodseason.

Table 3.Compositehaplotypes,haplotypefrequencies,haplotypediversity(h±SE),andnucleotidediversity(π) of the studied A. testudineuspopulations.

Composite

haplotypes

Samplinglocations

CH NP NS SN U CU1 CU2 RT

(n=29) (n=23) (n=21) (n=22) (n=21) (n=28) (n=21) (n=26)

h1 BBBBBBB 21(0.72) 4(0.17) - - - - - 13(0.50)

h2 BBBBBBC 7(0.24) 8(0.34) - - 12(0.57) 25(0.89) 21(1.00) 13(0.50)

h3 BBBBBBA - - - - 8(0.38) 3(0.11) - -

h4 BBBBABC - - - - 1(0.04) - - -

h5 BBBBAAB - - - 1(0.04) - - - -

h6 CBBBBBB - 1(0.04) 16(0.76) - - - - -

h7 CBBBBBC 1(0.03) 3(0.13) 4(0.19) - - - - -

h8 CBBBBAB - - - 2(0.09) - - - -

h9 CBBBBAC - - - 16(0.72) - - - -

h10 CBBBABB - - 1(0.04) - - - - -

h11 CBBAAAB - - - 1(0.04) - - - -

h12 CBBAAAC - - - 1(0.04) - - - -

h13 CABBBAC - - - 1(0.04) - - - -

h14 DBBBBBB - 1(0.04) - - - - - -

h15 DBBBBBC - 6(0.26) - - - - - -

Haplotypediversity

(h±SE)0.42±0.06 0.78±0.03 0.39±0.08 0.47±0.09 0.54±0.04 0.20±0.07 0.00±0.00 0.51±0.01

Nucleotidediversity

(π)0.004 0.028 0.000 0.000 0.001 0.000 0.000 0.000


93

ANOVA suggested that wild populations had

highergeneticvariationcomparedtohatcherypopula-

tions(averageh=0.52and0.10,respectively;P=0.01).Thefindingsaretypicalforseveralspecies.Sekinoet al.

(2002)foundthatwildpopulationsofJapaneseFlounder

Paralichthys olivaceushadhighermtDNAvariation(aver-

age h=0.99)thanhatcherypopulations(h=0.69-0.79;average=0.76).StudyofHemigrammocypris rasborella

inJapanbyWatanabeet al.(2009)alsoshowedhigher

mtDNAvariationinthewildpopulations(h=0.27-0.79;average=0.51)comparedtothehatcherypopulations

(h=0.00-0.79;average=0.39).Possiblereasonsforthe reduction in genetic variation in these hatchery

populationsincludebottleneckeffectsandinbreeding.

In A. testudineus hatchery populations, some

aquaculturepractices,suchasselectivebreeding,using

unequalnumbersofmaleandfemalebreeders,andus-

ingsmallnumberoffoundersmayleadtothereduction

ofgeneticvariation.Theindividualsfrombothhatchery

populationshaveundergoneatleastfourgenerations

of domestication and some selection for body size

andgrowth(Dr.SangaLeesanga,ChumphonFisheries

TestandResearchCenter,personalcommunication).A

breedingschemeusedattheChumphonFisheriesTest

andResearchCenter(CU1sample)isagroupspawning

of100-200individualswitha1:1maletofemaleratio

(Dr. Sanga Leesanga,Chumphon Fisheries Test and

ResearchCenter, personal communication). However,

massspawningmightallowfordifferentialbroodstock

contributionwhichwillreducegeneticvariation(Frost

et al.,2006).EventhoughthebroodstockattheChum-

phonFisheriesTestandResearchCenterwasderived

fromthebroodstockattheprivatefarm,thelevelof

geneticdiversityinCU1samplewasslightlyhigherthan

thatobservedintheCU2sample.Thisobservationmay

reflectthedifferenceinpracticesbetweenthesetwo

hatcheries(e.g.,theintensityofselection,sexratio,and

numberofbreeders).

Lowlevelofgeneticdiversityinhatcherypopula-

tionsmightalsoreflectaconsequenceofdomestication

selection.Thistypeofselectionusuallyfavorsindividuals

(orgenotypes)withtheabilitytosurviveincaptivity.It

mayinadvertentlyreducegeneticvariationinthesuc-

cessive generations.

AmongPopulationGeneticVariation Allanalysesindicatedgeneticdifferencesamong

populations.AMOVAindicatedthat57.83%oftheob-

servedgeneticvariationwasaresultofamongpopula-

tion genetic variation. Pairwise FST values ranged from

0.06 (CU1-CU2) to0.87 (NS-CU2).PairwiseFST also

indicated significantgeneticdifferences in almost all

Table 4. Pairwise FSTcomparisonsamongpopulations.*and**indicatedsignificantlevelsatP<0.05and0.002

afterBonferronicorrection,respectively.

CH NP NS SN U CU1 CU2 RT

CH

NP 0.33**

NS 0.56** 0.48**

SN 0.72** 0.53** 0.65**

U 0.48** 0.23** 0.75** 0.70**

CU1 0.61** 0.27** 0.84** 0.76** 0.15*

CU2 0.66** 0.29** 0.87** 0.77** 0.31** 0.06

RT 0.06 0.19** 0.60** 0.68** 0.27** 0.39** 0.45**


94

samplepairs(Table4),exceptfortheCH-RT,CU1-CU2,

andU-CU1(0.002<P<0.05).Neighbor-Joiningdendrogram

basedonNei’sgeneticdistancealsosuggestedgenetic

divergenceamongsamples,exceptforCU1andCU2

(Figure1).However,itcouldnotresolvetherelation-

shipsamongNP,RT,CHandNS.

The examined A. testudineus populations ex-

hibitedapatternofgeneticvariationtypicaltomany

freshwaterfishspecies,suchasOpsariichthys bidens

and Xiphophorus cortezi (among population genetic

variation=92.21%and95.03%,respectively)(Perdices

et al.,2005;Rodriguezet al.,2007).However,astudy

on Hypseleotris compressa populations in Australia

didnotshowthispatternofgeneticdiversity(among

populationgeneticvariation=1.4%);theauthorssus-

pectedthatthisobservationmaybeduetohistorical

connectivity between the populations (McGlashan &

Hughes,2001).

RFLPofmtDNAusedinthisstudyrevealeda

higherlevelofpopulationdifferentiationcomparedto

nuclearDNA.TheRAPDstudyofA. testudineus detected

17%amongpopulationsgeneticvariation(Unpublished

data;anidenticalsetofpopulationsexceptforCHand

NP).Thisdifferencemaybeduetohighervariabilityand

sensitivitytogeneticdriftofD-loopmtDNAcompared

tonuclearDNA.

Most populations examined were genetically

distinct.Amongwildpopulationpairs,onlytheCH-RT

pairdidnotshowsignificantFSTvalue(FST=0.06;P>

0.05)(Table4).Amongthegeneticallydistinctpopula-

tions,highestFSTvalue inwildpopulationpairswas

betweenNSandU(0.75;P<0.002)whilethelowest

valuewasfoundbetweenNPandRT(0.19;P<0.002).

Geographicaldistanceprobablyexplainedthepattern

ofthisgeneticdifferentiation.TheinsignificantFST value

betweenCHandRTmightreflecthistoricalconnectivity

18

Figure 1. A consensus genetic dendrogram based on Nei’s genetic distance. Numbers on each node

represent numbers of the same node generated over 100 bootstrap replicates. Figure 1.AconsensusgeneticdendrogrambasedonNei’sgeneticdistance.Numbersoneachnoderepresent

numbersofthesamenodegeneratedover100bootstrapreplicates.

´


95

of the rivers (Maeklong-Petchaburi andEastern river

systems)oritmightbecoincident.Therehasbeenno

recordofthefirstspeculation.Furthermore,NPwhichis

apopulationlocatedbetweenthetwopopulationswas

geneticallydistinctfrombothpopulations.Thesecond

speculationispossibleasgeneticdriftasarandom

processthatcanleadtoasimilargeneticoutcomein

independentpopulations,especiallyifthesepopulations

are small.

The low FSTvaluebetweentheCU1andCU2

samples(FST=0.06,P>0.05)reflectedthehistorical

exchange between these two populations. The CU1

samplewasderived from theCU2whichhadbeen

domesticatedforsomegenerations(Dr.SangaLeesanga,

Director of Chumphon Fisheries Test and Research

Center;Personalcommunication).Fromthehaplotype

foundinthisstudy(Table3)andthegeneticdendro-

gram(Figure1),somefoundersfortheCU2sample

mayhavecamefromthecentralpartofThailand.

Geneticdendrogramgeneratedinthisstudysug-

gestedthreemajorclusters(Figure1).SNwashighly

divergedfromothersamples.Thisfindingcoincideswith

thepresenceofuniquehaplotypesfoundinSN.The

dendrogramalsodifferentiatedUfromthe remaining

wildsamples(bootstrapvalue=84%).However,the

dendrogramdidnot resolve the relationshipsamong

somepopulationslocatedindifferentriversystems(NP,

RT,NS,andCHrepresentingChaoPhraya,Maeklong-

Petchaburi, Peninsular and eastern river systems

respectively).Anextensivestockingprogramalloverthe

countryusingbreedersfromtheChoaPhrayaRiverbasin

mayalsoexplainthisobservation(Dr.SangaLeesanga,

ChumphonFisheriesTestandResearchCenter,Personal

communication). Incontrast to thestrongpopulation

structureofthisspeciesdetectedbyallozymes(Sekino

&Hara,2000),thelevelofpolymorphismofRFLPof

mtDNAD-loopmaynotbeadequatetoresolvethese

relationships.Also,smallsamplesizesusedinthestudy

maynotallowthedetectionofallavailablepolymorphism

ineachpopulation.

Recommendationsforbroodstockmanagement

Afteronlyafewgenerationsofdomestication,

the levelofgeneticvariation inhatcherypopulations

wasquitelow.Thehatcheriesmayneedtoconsider

changingsomeaquaculturepracticestoincreasege-

neticvariationandprolongthelossofgeneticvariation.

Thesepracticesmayincludeusinga1:1maletofemale

ratio,andavoidusingagroupspawningforbroodstock

development.

BreedingschemeusedattheChumphonFisheries

TestandResearchCenterisalready1:1maletofemale

ratio.However,inagroupspawning,thecontributionof

parentsislikelyunknown.Groupspawningalsotends

toskewfamilyrepresentationinthenextgenerations

(Frostet al.,2006). Itwouldbe interesting toasses

thecontributionoftheparentsineachspawningusing

parentageassignmentofhypervariablemarkerssuch

asmicrosatelliteDNA.

Decliningingeneticvariationinhatcherypopu-

lationscouldalsodue to lownumberof individuals

inafoundingpopulation.Kapuscinski&Miller(2007)

recommendedusing60to500individuals.Althoughthe

ChumphonFisheriesTestandResearchCenterhave

beenusing100-200individualstostartapopulation,

usingdomesticatedstockasasourcepopulationmay

haverestrictedtheavailablegeneticdiversity.To in-

creasegeneticvariation,thesehatcheriesmayneedto

introduce new genetic material from the wild or other

geneticallydistincthatcherystocks.Studycomparing

naturalandhatcherypopulationsofBetta splendens in

ThailandbyMeejuiet al.(2005)foundthatobserved

heterozygosityinhatcherypopulationswashigherthan

thewild(0.081-0.125forthehatcherypopulationsand

0.065innaturalpopulation)asaresultofroutinestock

exchangebetweenhatcheries.

Conclusions We detected high haplotype diversity in the

samples that located in the Chao Phraya river


96

systemcomparedtootherriversystems.Bothhatch-

erysampleshadlowerhaplotypediversitycompared

to thewildsamples.Significantdifferences found in

almostallpopulationpairsofA. testudineusexamined

with57.83%ofvariationwasduetovariationamong

population.However,clusteranalysisdidnotshowed

strongpopulationstructuringbasedonthegeographic

locations of the rivers.

RFLPofmtDNAisaneffectivetoolforevaluating

geneticvariationofpopulations.Fromthisstudy,despite

thelownumberofsamples,mtDNAmarkershowedhigh

valueofgeneticvariationinNPandU.Itsuccessfully

revealedgeneticdifferencesamongalmostallpopulation

pairs,anditevensuccessfullydifferentiatedCHfrom

NPalthoughtheywere locatedclosetoeachother.

However,restrictionmappinginthispartofDNAmight

underrepresenttheavailablelevelofgeneticvariation,

especiallywhengeneticchangesoccurinnucleotides

notspecifictorestrictionenzyme.Thisrestrictedlevel

of variationmay have limited our ability to provide

strong population structure among samples. Genetic

markerswithhigherresolutionsuchassequencingof

mtDNAD-loopregionmayprovideadditionalinsights

ongeneticstructureofthesepopulations.

Acknowledgements WethankDr.SangaLeesanga(DirectorofChum-

phonFisheriesTestandResearchCenter)forvaluable

information related to the samples and the staff of

ChumphonFisheriesTestandResearchCenterforthe

assistanceincollectingthe2007samples.TheFaculty

ofScience,BuraphaUniversityprovidedpartialfunding

for this research.

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