genetic stability analysis of diapause-induced

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DOI:10.1111/j.1570-7458.2010.00978.xGeneticstabilityanalysisofdiapause-inducedmultivoltinesilkwormBombyxmorigermplasmusingintersimplesequencerepeatmarkersR.Saravanakumar,K.M.Ponnuvel*&S.M.H.QadriBiotechnologyLaboratory,CentralSericulturalGermplasmResourcesCentre,PBNo44,ThallyRoad,Hosur635109,TamilNadu,IndiaAccepted:20January2010Keywords:Lepidoptera,Bombycidae,diapauseinduction,molecularmarkers,polymerasechainreaction,primers,phylogeneticrelationship,PCR,ISSR-PCRAbstractConservationofsilkworm[BombyxmoriL.(Lepidoptera:Bombycidae)]germplasmaidsinpreven-tionofgeneticerosionandraceextinction.Multivoltinesilkwormgermplasmmaintenanceinvolvesrearingfivegenerationsperyearandthisrequiresexpenditure,manpower,andinfrastructure,inadditiontofrequentexposureofthesilkwormstodiseases,pests,andpredators.Tominimizetheseproblems,analternativeprotocolwasdevelopedbywayofinductionofeggdiapauseinthemultivol-tinesilkwormsthroughrearingofthefourthandfifthinstarsatlowtemperature(18–20°C)underregulatedphotoperiod(L6:D18).Thismethodallowedinductionofeggdiapauseintheselectedracesatlevelsrangingfrom26to94%andthediapause-inducedeggsweremaintainedundercoldpreser-vationconditions.Thestabilityofgenotypiccharacterswasconfirmedinthediapause-inducedsilkwormbatchesaftertheseventhgeneration,bycomparisonwithcontrolbatchesofsilkwormusingintersimplesequencerepeat-polymerasechainreaction(ISSR-PCR).AnalysisofNei’sgeneticdis-tanceforthedifferentracesindicatednosignificantvariationbetweencontrolanddiapause-inducedraces.Thisrevealsthatalltheselectedracesmaintainedgeneticstabilityevenaftertheseventhgenera-tionatthephenotypicandmolecularlevel.Hence,itcanbeconcludedthatinductionofeggdiapauseisanappropriatealternativemethodtopreservemultivoltineracesforlongerperiodsoftime.tionofsilkwormgermplasmresourcesrepresentingracesIntroductionofvariousgeographicaloriginsthatalsorevealdiverseBombyxmoriL.(Lepidoptera:Bombycidae)isadomesti-morphological,reproductive,biochemical,growth,andcatedsilkwormandinviewofitslonghistoryofdomesti-yieldparameters.Eggsofmultivoltinesilkwormracesarecation,manynaturallyevolvedstrainshavebeenpreservedfor30–35daysat5°C,facilitatingfiverearingmaintained.Conservationofsilkwormgermplasmisadif-cyclesperyear(Kumaresanetal.,2004).Ontheotherficultandtechnicallydemandingactivityrequiringskilledhand,eggsofbivoltineracesarepreservedfor10monthslabour,causingmaintenanceoflargecollectionsofsilk-incoldstorageandonlyonerearingisconductedperyear.wormracestobeaverycostlyaffair.Furthermore,conser-Bivoltineracesundergohibernation(diapause),whichvationofmultivoltinesilkwormgermplasmusingthehelpstowithstandlowtemperatureforlongerperiodsoftraditionalpracticeofcontinuousrearingisriskyasitnottime,whereasmultivoltineraceslackhibernationandcan-onlyinvitesdiseasesandpestsbutalsoleadstogeneticero-notbestoredforlongerperiodsoftime.Generally,dia-sion(Radhakrishnanetal.,2003).TheCentralSericulturalpauseisgeneticallycontrolledaswellasinfluencedbyGermplasmResourcesCentre(CSGRC)hasalargecollec-environmentalfactors,suchastemperatureandphoto-period.Nagayama&Yamamoto(1987)developedapro-tocoltoinduceeggdiapauseinmultivoltinesilkwormsby*Correspondenceandpresentaddress:K.M.Ponnuvel,SeriBiotechregulatingphotoperiodandrearingatroomtemperature.ResearchLaboratory,CentralSilkBoard,SarjapurRoad,Carmelram–Post,Kodathi,Bangalore560035,India.E-mail:kmpvel@yahoo.Kogure(1933)foundthatlightexertsthesameeffectascomhightemperatureanddarknessasthatoflowtemperature.Ó2010TheAuthorsEntomologiaExperimentalisetApplicata135:170–176,2010170JournalcompilationÓ2010TheNetherlandsEntomologicalSociety GeneticstabilityinBombyxmori171Illuminationfor15hperdayisnecessaryfortheeggslationdiscriminationandgeneticvariationindiamond-(duringincubation)toproducemothsthatlay100%dia-backmoth,Plutellaxylostella(L.)populations(Rouxetal.,pauseeggs.Thetropicalmultivoltinesilkwormexhibitsa2007)andtheISSR-PCRtechniquehasbeenfoundsuit-facultativetypeofdiapausewhichisreversible.Kobayashiabletostudyintra-andinter-specificvariationinnoctuidetal.(1986)andNagayama&Yamamoto(1987)reportedmoths(Luqueetal.,2002).thatanintermediatetemperatureregime(20–23°C)dur-Currently,DNA-basedmarkersarebeingincreasinglyingthelatefourthandfifthinstarsandpupalstagepro-usedtomonitorthegeneticstabilityofgermplasmduringmotestheproductionofdiapauseeggsintheprogenyoflong-termconservation.Manymicro-propagatedplantletsseveralmultivoltineracesofB.mori.Similarmethodswereofbanana,MusaacuminateL.,thatweredevelopedfromadoptedtoinducediapauseinselectedmultivoltinesilk-axillaryshootbudexplantsover10yearsoldwerescreenedwormracesmaintainedatCSGRC,Hosur.ItwasobservedforgeneticstabilityusingRAPDandISSRmarkersthatdiapausegetsreversedwhentheoriginalenvironmen-(Lakshmananetal.,2007).Thesemarkersarereliableastalconditionsarerestored.theyaretissueandenvironmentindependent(Ranietal.,Genomeanalysisofthesilkwormhasbeenperformed1995).Itiswellknownthatmoleculardataaremorereli-byseveralgroupsofresearcherswiththeintentionofchar-ablethanphenotypicobservationsforevaluatingvaria-acterizinggeneticdiversitybyDNAfingerprintingusingtionsandmonitoringgeneticstability(Leroyetal.,2000).intersimplesequencerepeat-polymerasechainreactionISSRamplificationisavaluablemethodfordetermining(ISSR-PCR)(Reddyetal.,1999a),heterologousminisatel-geneticvariabilityamongsilkwormgenotypes.Inthisdoc-liteprobe,Bkm(Sharmaetal.,1998),microsatellitelociument,wereporttheresultsofourinvestigationsonthe(Reddyetal.,1999b)aswellasrandomamplifiedpoly-useoftheISSRtechniquetoevaluategeneticstabilityofmorphicDNA(RAPDs)(Nagaraja&Nagaraju,1995).diapause-inducedsilkwormracescomparedwithcontrolNagarajuetal.(2001)comparedtheutilityofmultilocussilkwormraces.restrictionfragmentlengthpolymorphism(RFLPs)andthreePCR-basedtechniquesviz.,RAPD,ISSR-PCR,andMaterialsandmethodssimplesequencerepeats(SSRs),inthegeneticcharacter-izationof13silkwormstrains.Chatterjee&MohandasSelectionofgeneticmaterial(2003)usedtheISSRtechniquetofindalinkbetweenpro-Ninemultivoltinesilkwormraces,viz.,PureMysore,RongductivitytraitsandmolecularmarkersinsomeselectedDaizo,GNM,PMX,WAI-1,WAI-4,MW13,Daizo,andsilkwormraces.MU303representingvariousgeographicallocalitiesinMolecularmarkersareknowntohavemanyadvantagesIndiaandChina(Table1)andsusceptibleto45daysofovermorphologicalandbiochemicalmarkersastheyarecoldpreservationwereselectedforthestudy(Kumaresanmorestableandindependentofenvironmentalinfluencesetal.,2004).(Berbatzky&Tanksley,1989;Gepts,1993).ISSRmarkersareusefulindetectinggeneticpolymorphismamongclo-EggdiapauseinductionprotocolselyrelatedplantsoranimalsbygeneratingalargenumberInitially,eggswereincubatedat25°C,80%r.h.,andofmarkersthattargetmultiplemicrosatellitelocidistrib-L16:D8photoperiod.Afterhatching,thesilkwormswereutedacrossthegenome.Furthermore,theyaresimplertorearedasperstandardrearingmethodofKrishnaswamiusethantheSSRtechnique,aspriorknowledgeofthe(1978)uptothethirdinstar.Afterthethirdmoult,300targetsequenceflankingtherepeatisnotrequiredlarvaewererearedatlowtemperature(18–20°C)and(Zietkiewiczetal.,1994).TheuseofISSRinphylogenyregulatedphotoperiod(L6:D18)uptococoonharvestanalysesandinpopulationgeneticshasbeendocumented(Nagayama&Yamamoto,1987).Afteremergence,mothsinawidevarietyoforganisms(Deshpandeetal.,2001),werematedfor4hat25°Candallowedtolayeggs.AfterparticularlyingeneticdiversityanalysisamongB.mori48h,eggswereobtainedfromeachraceandpercentagesstrains(Reddyetal.,1999b;Chatterjee&Pradeep,2003;ofdiapauseinductionwererecorded.Thediapause-Veluetal.,2008).inducedeggswerepreservedoveraperiodof3months(atIntersimplesequencerepeatsarepresumablynon-cod-25°Cfor10days,20°Cfor4days,15°Cfor4days,5°Cingsequencesandaredispersedthroughoutthegenome.for50days,2.5°Cfor20days,and15°Cfor3days)andISSRmarkersareadvantageousinviewofproductionof6months(at25°Cfor20days,20°Cfor4days,15°Cmanyfragments,reproducibility,andlowcosts.Thus,for4days,5°Cfor90days,2.5°Cfor60days,and15°CISSRcouldbeanunbiasedtoolforevaluatingchangesinfor3days)atvarioustemperatures(Vemanandareddydiversityinagronomicallyimportantcrops(Morenoetal.,etal.,2004).Aftercompletionofthe3and6monthspres-1998).ISSRmarkershavebeenusedasatoolforpopu-ervationschedule,theeggswerereleasedandrearingwas 172Saravanakumaretal.Table1Diapauseinduction(mean%%diapauseGeographical±SE)intheseventhgenerationofnineRaceinductioncoordinatesGeographicaloriginmultivoltinesilkwormracesPureMysore26.4±7.212°15¢N,76°37¢EIndia(KarnatakaState)RongDaizo93.7±2.530°16¢N,120°7¢EChinaGNM83.3±3.130°16¢N,120°7¢EChinaPMX34.1±4.712°15¢N,76°37¢EIndia(KarnatakaState)WAI-138.7±5.420°53¢N,74°46¢EIndia(MaharastraState)WAI-472.4±3.220°53¢N,74°46¢EIndia(MaharastraState)MW1392.0±1.112°15¢N,76°37¢EIndia(KarnatakaState)Daizo92.9±1.730°16¢N,120°7¢EChinaMU30333.1±2.912°15¢N,76°37¢EIndia(KarnatakaState)conducted.Thisprocesswascontinueduptosevengener-amplifiedDNAfragmentswerescoredaspresent(=1)orationsunderthesameconditionsoftemperature,humid-absent(=0).Toanalysethegeneticstabilityinthepopu-ity,andphotoperiodasdescribedabove.lation,variousparametersbasedonthebandingpatternswereassessed,suchaspercentagepolymorphism(P),GenomicDNAextractiondiversityamongpopulations(DST),andgeneticdistanceMothswerecollectedfromtheseventhgenerationdia-betweendiapause-inducedandcontrolsamplesofsilk-pause-inducedbatchandcorrespondingcontrolbatchofworm,usingTFPGA1.3software(Miller,1997).Bootstrapeachracetoanalysegeneticstability.GenomicDNAwasanalysiswasconductedusing1000replicates.extractedfrom10individualmothsfollowingthephe-nol:chloroform:isoamylalcoholextractionmethod(SuzukiResultsetal.,1972).TheextractedDNAwasdried,dissolvedin)1DiapauseinductionresponseTEbufferanddilutedto10ngllthroughserialdilutionandquantifiedona0.8%(wtvol)agarosegelagainstEggdiapausewasinducedinallselectedmultivoltinesilk-)1uncutkDNA(10ngll)asastandard.wormracesunderlowtemperatureandshortdayphoto-period(L6:D18).MeandiapauseinductionvariedfromPCRamplificationwithISSRprimers26.4%(PureMysore)to93.7%(RongDaizo)(Table1).FifteenISSRprimersoriginallydevelopedbytheUniver-Allracesrespondedtothediapauseinductiontreatment;sityofBritishColumbia(Abot,2001)wereusedfortheindividualsofRongDaizo,Daizo,andMW13raceslaidstudy.PCRamplificationwasperformedinaPTC200onlydiapauseeggs,whereasthoseofGNMracelaidamix-thermalcycler(MJResearch,Watertown,MA,USA)usingtureofdiapauseandnon-diapauseeggs.Mostnon-dia-20llreactionmixturecontaining30ngDNA,2.0ll10·pauseeggswerelaidbyindividualsfromPureMysore,PCRbuffer(MBIfermentas,Burlington,ON,Canada),PMX,andMU303races.Thediapause-inducedeggswere0.2mMofdNTP,2.5mMMgCl2,0.15lMprimer,andpreservedundercoldpreservationconditionsforaperiod1.0UofTaqDNApolymerase.ThePCRschedulewasof3monthsandsuccessiverearingwasconductedupto94°Cfor2min,followedby35cyclesof94°Cfor30s,sevengenerations.50°Cfor30s,72°Cfor2minandafinalextensionof10minat72°C.StabilityanalysiswithISSRprimersThePCRproductswereresolvedona1.5%(wtvol)Thediapause-inducedsilkwormracesweresubjectedtoaagarosegelandelectrophoresiswascarriedoutatacon-geneticanalysistesttoconfirmtheoriginalgenotypes.Thestantvoltageof80Vfor2h.Theexperimentwasrepeatedgeneticfidelityofdiapause-inducedsilkwormafterseventhreetimesandthebandsthatappearedconsistentlyinallgenerationswascomparedwithcontrolsilkwormindivid-threegelswerescoredandusedforanalysis.WeakbandsualsusingISSRprimers.Atotalof15ISSRprimerswerewerenotconsideredforanalysis.Inthisexperiment,ISSRused(Table2).Itwasfoundthatdifferenceinpolymor-patternsincontrolanddiapause-inducedbatcheswerephism(P)existedbetweencontrolanddiapause-inducedcompared.races.Atotalof192scorablebandswererecorded,ofwhich178bandswerepolymorphic,showing92.7%poly-Statisticalanalysismorphismamongthecontrolanddiapause-inducedraces.DatawereanalysedusingNei’scoefficientofgeneticdis-Amongthe15primers,UBC812,813,841,844,and873tance(Nei,1972,1978)forclusteranalysis.Consistentyielded100%polymorphicbandsindifferentraces, GeneticstabilityinBombyxmori173Table2ListofISSRprimersusedandtheirpolymorphismlevelinsilkwormSizerangeTotalNo.polymorphicPrimer5¢–3¢primersequence(bp)no.bandsbands%polymorphismUBC807AGAGAGAGAGAGAGAGT400–3000171694.1UBC808AGAGAGAGAGAGAGAGC350–3000181794.4UBC809AGAGAGAGAGAGAGAGG300–2500151493.3UBC811GAGAGAGAGAGAGAGAC600–3000131292.3UBC812GAGAGAGAGAGAGAGAA400–25001717100UBC813CTCTCTCTCTCTCTCTT700–30001212100UBC822TCTCTCTCTCTCTCTCA400–15007457.1UBC827ACACACACACACACACG300–2250181688.9UBC841GAGAGAGAGAGAGAGAYC300–11001313100UBC844CTCTCTCTCTCTCTCTCRC400–20001010100UBC855ACACACACACACACACYT400–2000141392.9UBC861ACCACCACCACCACCACC500–22509777.8UBC864ATGATGATGATGATGATG450–2500161487.5UBC873GACAGACAGACAGACA300–25001313100whereasprimerUBC822showedonly57.1%polymor-Withrespecttodiversity(DST)analysis,amongthephism.However,allthesebandswereconsideredforthenineraces,thelargestdistance(0.732)wasobservedanalysisofgeneticstabilitybetweencontrolanddiapause-betweenDaizoandWAI-1.Similarly,geneticdistanceinducedraces.SomeoftheprimersrevealednodifferencesbetweenMW13andRongDaizowaslarge(0.706).Theinthebandingpatternbetweencontrolanddiapause-smallestdistance(0.296)wasobservedbetweenWAI-1inducedsamples(Figure1A,B).andWAI-4bothoriginatingfromthesameprovinceinFigure1ISSRprofilesof10individualseachoftreated(diapause-induced)andcontrolracesof(A)MW13and(B)WAI-1,amplifiedusingprimerUBC-812andresolvedina1.5%agarosegel.M=sizeruler,DNAladder(FermentosLifeSciences,Hanover,MD,USA). 174Saravanakumaretal.Table3Nei’sgeneticdistancebetweenninepopulationsofmultivoltinesilkwormracesRacesPMRDGNMPMXWAI-1WAI-4MW13DaizoMU303PureMysore(PM)–RongDaizo(RD)0.445–GNM0.5840.539–PMX0.3400.5390.445–WAI-10.4250.5960.6550.496–WAI-40.5500.6430.6070.5390.296–MW130.5170.7060.5730.5070.6070.561–Daizo0.4750.4150.5500.5730.7320.6800.528–MU3030.6190.6680.5610.4350.5500.5280.5390.655–Figure2Unrootedtreeobtainedfromthegeneticdistancevaluesof10individualseachof18populationsforcontrol(C)anddiapause-induced(DI)races.India(Table3).Twomajorgroupswerefound,whichoftheindividualswithinrace.TheresultsclearlyindicateclusteredatD=0.575.Onegroupcomprisedsixthatgeneticstabilitywasmaintainedevenaftersevensilkwormraces,viz.,PM,PMX,WAI-1,WAI-4,MU303,generations.andMW13.Theothermajorgroupclusteredat0.380,includingDaizo,RongDaizoandGNM.ThisinformationDiscussionclearlyindicatesthatthegeneticdistancewashigherbetweenindigenousandexoticracesthanbetweenindige-Itisawellknownfactthatinductionandterminationofnousorbetweenexoticraces.diapausearecontrolledbyhormonessecretedbyendo-Comparisonbetweendiapause-inducedandcontrolcrineglandsandmediatedbyenvironmentalstimulisamplesrevealedanunrootedtreebasedongeneticdis-(Wigglesworth,1970).Undernormalconditions,thetancewhichdifferentiatedthediapause-inducedfromthesubesophagealganglion(SG)synthesizesthediapausecontrolraces.Thegeneticdistance(0.024–0.08)washormonethatinduceslayingofdiapauseeggsinbivoltineobservedbetweendiapause-inducedandcontrolsamples.silkworms,whereasinmultivoltinesilkwormstheSGTherangevariedfrom0.024inWAI-Itomaximumofsecretesverylittlediapausehormoneandthereforethey0.08inGNM(Figure2),andthisindicatesheterozygositylaynon-diapauseeggs(Yamashita&Yaginuma,1991).Yu GeneticstabilityinBombyxmori175etal.(1993)statedthatinthecaseofmultivoltinesilk-Referencesworms,tolerancetolowtemperatureisaracialcharacteris-ticanddiapauseinductionisadaptiveinnature.AbotP(2001)Individualandpopulationvariationininverte-Inthisstudy,weobservedthatRongDaizorecordedbratesrevealedbyInter-simpleSequenceRepeats(ISSRs).highestdiapauseinduction(93.7%),followedbyMW13JournalofInsectScience1:1–3.(92.9%)andGNM(83.3%),whereasPureMysoreBerbatzkyR&TanksleySD(1989)Restrictionfragmentsasrecordedthelowest(26.4%).Maximuminductionofmolecularmarkersforgermplasmevaluationandutilization.TheuseofPlantGeneticResources(ed.byAHDBrown,OHdiapauseinRongDaizoandGNMmaybeduetotheirFrankel,DRMarshel&JTWilliams),pp.353–362.CambridgeoriginfromthetemperatezoneofChina.InthecaseofUniversityPress,NewYork,NY,USA.MW13with92.0%diapause,oneoftheparentswasNB7,ChatterjeeSN&MohandasTP(2003)IdentificationofISSRabivoltinerace.Shimizuetal.(2005)reportedinductionmarkersassociatedwithproductivitytraitsinsilkworm,Bom-ofdiapauseinmultivoltinesilkwormracesintherangebyxmoriL.Genome46:438–447.of70–92%underconditionsoflowtemperatureandChatterjeeSN&PradeepAR(2003)Molecularmarker(RAPD)regulatedphotoperiod.associatedwithgrowth,yieldandoriginofthesilkworm,Thediapause-inducedeggsweremaintainedunder3-BombyxmoriL.inIndia.RussianJournalofGenetics39:1365–and6-monthpreservationschedules.Thehatching1377.percentageofeggsrecordedonrearingafter3andDeshpandeAU,ApteGS,BahulikarRA,LaguMD,KulkarniBG6monthseggpreservationdidnotdiffersignificantlyfrometal.(2001)GeneticdiversityacrossnaturalpopulationsofthreemontaneplantspeciesfromtheWesternGhats,Indiathecontrol.ISSRmarkersrevealeddifferentiationofninerevealedbyintersimplesequencerepeats.MolecularEcologymultivoltinesilkwormracesbasedonhighnumber(192)10:2397–2408.ofscorablebandswith92.7%polymorphism.Hence,theseGeptsP(1993)Theuseofmolecularandbiochemicalmark-primersmaybeconsideredastoolstodifferentiateersincropevolutionstudies.EvolutionaryBiology27:silkwormraces.However,nodifferencesinthebanding51–94.patternsofdiapause-inducedandcontrolbatcheswereJayaswalKP(1996)ImpactofDiapauseonSilkProductioninobserved.TheresultsobtainedareinaccordancewiththeBombyxmoriL.PhDDissertation,UniversityofCalcutta,Cal-principlethatthenumberofindividualsusedtoestimatecutta,India.averageheterozygositycanbeverysmallifalargenumberKobayashiJ,EbinumaH&KobayashiM(1986)Effectoftemper-oflociisstudied(Nei,1978).PhylogeneticreconstructionsatureonthediapauseeggproductioninthetropicalraceoftheweremadebasedonISSRdatausingUPGMA.silkworm,Bombyxmori.JournalofSericulturalScienceofJapan55:343–348.ThegeneticdistanceanalysisrevealedsmalldistancesKogureM(1933)Theinfluenceoflightandtemperatureoncer-betweentheracescollectedfromthesameregionandlargetaincharactersofthesilkwormBombyxmori.Journalofthedistancesbetweenindigenousandexoticraces.DiapauseDepartmentofAgriculture,KyushuImperialUniversity4:inductiondidnotaltergeneticstability;evenafterseven1–93.generationsbandingpatternsoftheselectedracesweresta-KrishnaswamiS(1978)NewTechnologyofSilkwormRearing.ble.Jayaswal(1996)reportedthattheweightofthesilkBulletinNo.2.CentralSericulturalResearchandTrainingshellishighlyheritable,race-specific,andstable,evenInstitute,CentralSilkBoard,Mysore,India.underfluctuatingenvironmentalconditions.Earlierstud-KumaresanPK,ThangaveluK&SinhaRK(2004)StudiesoniesconductedbySaravanakumar&Ponnuvel(2007)alsolong-termpreservationofeggsofIndiantropicalmultivoltineindicatednosignificantvariationinphenotypiccharacterssilkworm(BombyxmoriL.)geneticresources.Internationalofdiapause-inducedandcontrolsilkwormsaftersevenJournalofIndustrialEntomology9:79–87.LakshmananV,VenkataramareddySR&BhagyalakshmiNgenerations.Diapauseinductioninrelatedracesdidnot(2007)Molecularanalysisofgeneticstabilityinlong-termaltertheirgeneticstabilityevenaftersevengenerations.micropropagatedshootsofbananausingRAPDandISSRThus,itisquitepossibletoreducethenumberofcropmarkers.ElectronicJournalofBiotechnology10:106–113.cyclesinmultivoltineraces,providingawayforsuccessfulLeroyXJ,LeonK,CharlesG&BranchardM(2000)Cauliflowerconservationofmultivoltinesilkwormgermplasms.somaticembryogenesisandanalysisofregenerantstabilitybyISSRs.PlantCellReports19:1102–1107.LuqueC,LegalL,StaudterH,GersC&WinkM(2002)ISSRAcknowledgements(InterSimpleSequenceRepeats)asgeneticmarkersinNoc-Thisresearchworkwassupportedbygrantsfromthetuids(Lepidoptera).Hereditas136:251–253.DepartmentofBiotechnology,MinistryofScienceandMillerMP(1997)ToolsforPopulationGeneticAnalysisTechnology,GovernmentofIndia(BTPR4503PBD(TFPGA)Version1.3.DepartmentofBiologicalSciences,NorthernArizonaUniversity,Flagstaff,AZ,USA.190992003dated26thMarch2004). 176Saravanakumaretal.MorenoS,MartinJP&OrtizJM(1998)IntersimplesequencetureanalysisofPlutellaxylostellapopulationsnativetodiffer-repeatsPCRforcharacterizationofcloselyrelatedgrapevineentgeographicalareas.MolecularPhylogeneticsandEvolutiongermplasm.Euphytica101:117–125.43:240–250.NagarajaGM&NagarajuJ(1995)GenomefingerprintingoftheSaravanakumarR&PonnuvelKM(2007)Eggdiapauseinduc-silkwormBombyxmoriusingrandomarbitraryprimers.Elec-tioninmultivoltinesilkwormBombyxmoriforlong-termtrophoresis16:1633–1638.germplasmpreservation.InternationalJournalofIndustrialNagarajuJ,ReddyKD,NagarajaGM&SethuramanBN(2001)Entomology15:1–7.ComparisonofmultilocusRFLPsandPCR-basedmarkersys-SharmaA,NiphadkarMP,KathirvelP&NagarajuJ(1998)DNAtemsforgeneticanalysisofthesilkwormBombyxmori.Hered-fingerprintvariabilitywithinandamongthesilkwormBombyxity86:588–597.morivarietiesandestimationoftheirgeneticrelatednessusingNagayamaK&YamamotoT(1987)EffectoflowtemperatureBkm-derivedprobe.JournalofHeredity90:315–319.during4thand5thlarvalandpupalstagesonthediapauseinShimizuK,HirokawaM,TatematsuK&KosegawaE(2005)severalmultivoltinesilkwormstrains.JournalofSericulturalEffectiveinductionofeggdiapauseinmultivoltinesilkwormScienceofJapan56:247–248.racesbylowtemperatureandshort-photoperiodrearingcon-NeiM(1972)Geneticdistancebetweenpopulations.Americandition.TheJournalofSericulturalScienceofJapan74:1–7.Naturalist106:283–292.SuzukiY,GageLP&DonaldBD(1972)ThegenesforsilkfibroinNeiM(1978)EstimationofaverageheterozygosityandgeneticinBombyxmori.JournalofMolecularBiology70:637–649.distancefromasmallnumberofindividuals.Genetics89:583–VeluD,PonnuvelKM,MuthulakshmiM,SinhaRK&Qadri590.SMH(2008)Analysisofgeneticrelationshipinmutantsilk-RadhakrishnanS,KumaresanP,SinhaRK&ThangaveluKwormstrainsofBombyxmoriusingInterSimpleSequence(2003)Inductionofdiapauseintheeggsofmultivoltinesilk-Repeat(ISSR)markers.JournalofGeneticsandGenomics35:worm(Bombyxmori)germplasmforlong-termpreservation.291–297.ProceedingsofNationalSeminar,SSTL,Bangalore,pp.155–VemanandareddyG,VeeraiahTM&SamsonMV(2004)Silk-158.Director,SSTL,Centralsilkboard,India.wormseedpreservationschedulesforbivoltines–newdimen-RaniV,ParidaA&RainaSN(1995)Randomamplifiedpolymor-sions.IndianJournalofSericulture43:25–34.phicDNA(RAPD)markersforgeneticanalysisinmicroprop-WigglesworthVB(1970)InsectHormones.OliverandBoyd,agatedplantsofPopulusdeltoidsMarsh.PlantcellReports14:Edinburgh,UK.459–462.YamashitaO&YaginumaT(1991)Silkwormeggsatlowtemper-ReddyKD,NagarajuJ&AbrahamEG(1999a)Geneticatures:implicationsforsericulture.InsectsatLowTempera-characterizationofthesilkwormBombyxmoribysimpleture(ed.byRELee&DLDenlinger),pp.424–445.Chapmansequencerepeat(SSR)anchoredPCR.Heredity83:681–andHall,NewYork,NY,USA.687.YuSJ,ChenJS&HsiehFK(1993)StudiesonthepreservationofReddyKD,AbrahamEG&NagarajuJ(1999b)Microsatelliteineggsofthemultivoltinesilkworm,BombyxmoriL.Chinesethesilkworm,Bombyxmori:abundance,polymorphismandJournalofEntomology13:141–150.straincharacterization.Genome42:1057–1065.ZietkiewiczE,RafalskiA&LabudaD(1994)Genomefinger-RouxO,GevreyM,ArvanitakisL,GersC,BordatD&LegalLprintingbysimplesequencerepeat(SSR)-anchoredpolymer-(2007)ISSR-PCR:toolfordiscriminationandgeneticstruc-asechainreactionamplification.Genomics20:176–183.