基于mdna控制区序列的长江中下游白头鹤种群遗传结构分析

基于mdna控制区序列的长江中下游白头鹤种群遗传结构分析

u2004范围遗传因素是这一因素的特征。从遗传因素的角度来看，这是期望的。第一阶段的目标预算反映在哪里（冯卓等人，2002）。第二面可渗透性特征的分类（zhangetal.，2002）。TheHoodedCrane(Grusmonacha)isalargemigratorywadingbird,designatedasvulnerableintheIUCNRedList(IUCN,2011),andasaFirst-classNationaProtectedWildAnimalspeciesinChina.Duringthepasttwodecades,duetohabitatloss,hunting,pollution,pesticidesandhumanactivities,thepopulationoftheHoodedCranehasundergoneaprecipitousdecline(BirdLifeInternational,2001).ThecraneswinterinJapan,SouthKoreaandChina(BirdLifeInternational,2001).ThelakesandcoastalintertidalzonesinthemiddleandlowerreachesoftheYangtzeRiveraremajorwinteringareasforthesebirds.DelanyandDerek(2006)estimatethatthereareabout1000–1500individualsofthisspecieswinteringinChina.ThemainwinteringsitesareShengjinLake(about300)(Xuetal.,2006)andCaiziLake(276)(Zhang,2006a)inAnhui,PoyangLake(255)inJiangxi(JiandZeng,2006)andChongmingDongtaninShanghai(about130)(Zhang,2006b).However,morerecently,duetohumandisturbances,lakewetlandshavebecomeseriouslydegradedandhabitatsforthewinteringpopulationareconstantlylosing(WangandYan,2002).Thestatusofthepopulationhasbecomeofmajorconcern.Inordertoimproveconservationofwinteringpopulations,itisnecessarytohaveamorethoroughknowledgeaboutthestatusofboththeirhabitatandgeneticbackground.Non-invasivesamplingisanimportantapproachforstudyingconservationgeneticsofendangeredwildlife(Lietal.,2001).Faecalsamplingisoneofthesimplestnon-invasivesamplingmethodswithminimalimpactonanimals(Wang,2001).Atpresent,faecalDNAanalysishasbeensuccessfullyappliedinstudiesofOtistarda(Brodericketal.,2003;Idaghdouretal.,2003),Tetraourogallus(Regnautetal.,2006a,2006b;Jancobetal.,2010),Ardeotisnigriceps(Ishtiaqetal.,2011),Grusjanponensis(Honmaetal.,2011),G.monacha(DaiandZhou,2011;Honmaetal.,2011),G.vipio(Honmaetal.,2011),aswellasotherthreatenedorendangeredbirdspecies.Inthisstudy,usingfecesassourcesofDNAsamples,weanalyzedthegeneticdiversityofHoodedCranepopulationswinteringinthemajorwetlandsinthemiddleandlowerreachesoftheYangtzeRiverbasedonthemtDNAD-loopsequences.Theobjectivesofthisstudyweretounderstandthegeneticdiversityandstructureofthesepopulationsandtoaccumulatebasicdataconcerningtheconservationgeneticsofthewinteringpopulations.杏仁核分子身份证colectedSampleswerecollectedfromfourwinteringHoodedCranepopulations(Fig.1).Beforefaecalsampleswerecollected,theforagingareasandovernightroostsofthecraneswereobservedwithbinocularsortelescopes.Theminimumdistanceamongindividualbirdsobservedwasabout1–2m.Freshfaecalsampleswerecollectedimmediatelyafterthecranesflewawaytotheirforaginggroundorthenextmorningattheovernightroosts.Inordertoavoidthateachsamplecamefromthesamebird,wecollectedsamplesateachsamplinglocationonlyonce,withaminimumdistancebetweensamplesofatleast2m.WeuseddisposablePEglovestocollectsamples,whichwereplacedinto50mLcentrifugetubesandthen2to3timesitsvolumeofabsoluteethylethanolwasaddedtopreservethesamples.Atotalof221faecalsampleswerecollected,consistingof84samplesfromShengjinLake(SJ),61fromCaiziLake(CZ),30fromPoyangLake(PY)and46fromChongmingDongtan(DT).Inaddition,ninedownfeathersamplesfromwinteringcranesinShengjinLakeandfourmusclesamplesfromdeadbirdsfoundintheShengjinLakeNationalNatureReserveduring2008–2011werecollected.Thefeatherandmusclesampleswereplacedat-20°C.DNAextraction,PCRamplificationandsequencingDNAwasextractedfromfaecalsamplesusingtheimprovedmethodofGuanidineThiocyanateandGlassMilk(Reedetal.,1997;DaiandZhou,2011).AfterDNAwasextracted,70%ethanolwasaddedtowashit2–3times.Afterairdrying,50µLofddH2Owasadded.DNAfromeachfaecalsamplewasextractedrepeatedlyfourtimes,afterwhichtheextractedDNAwaspooledandpurified(Zhaoetal.,2005;Regnautetal.,2006b).DNAextractionfromfeatherandmusclesampleswascarriedoutusingtheconventionalbutimprovedProtease-Phenol/Chloroformmethod(Sambrooketal.,1989;Zanetal.,2008).LC16004(5′-GAGCCCTAGAAAACAAAATA-3′),LC16575(5′-ACAAAAGAAACCCCCAAACTCA-3′)andHC01342(5′-AAGAATTCTGCGGATACTTGCATGT-3′)werechosenasprimersfortheamplificationofDNAD-loopsequencesofmitochondriaofHoodedCranes(Hasegawaetal.,1999).ThePCRmix(50µL)comprisedof3-5µLofanextract,0.2µMofeachprimer,0.4mg/mLofBSAand1×EasyTaqPCRSuperMix(Trans).Cyclingparametersconsistedofaninitialdenaturationat94°Cfor3minfollowedby35amplificationcyclesof45sat94°C,1minat58°Cand1minat72°Cwithaterminalcycleof10minat72°C(Hasegawaetal.,1999).Theamplifiedproductsweredetectedwithagelimagerafterelectrophoresisfor30minat120Vwith1.5%EB-agarosegel.Finally,atwo-waysequencingwasperformedonthepurifiedPCRproducts(SangonBiotech(Shanghai)Co.,Ltd.).离子束诱变alinmartMtDNAD-loopsequencesoftwoHoodedCraneswinteringinJapanwereobtainedfromGenBank(AB017625andAB023813).TheSeqManIIprogramfromtheDNASTARsoftwarepackage(Burland,2000)wasusedtomatchthecoupledsequencesofonesampleandcomparedthemwiththesequencesfromGenBankinordertoconfirmthetargetsequences.Atotalof72mtDNAD-loopsequenceswereobtainedforgeneticanalysis.ThesequenceswerealignedusingtheClustalWinMEGA4.0software(Tamuraetal.,2007);haplotypeswereidentifiedbyDAMBE(XiaandXie,2001).WehadsubmittedthedatafromthesehaplotypestoGenBankandreceivedtheaccessionnumbers(JQ015266-JQ015288).AstherepresentativesoftheJapanesewinteringpopulation(JA),thetwosequencesobtainedfromGenBankwereanalyzed.Haplotype(h)andnucleotidediversities(π)werecalculatedusingDnasp5.10software(Rozasetal.,2003).Geneticdifferencesbetweenpopulations(FST)wereobtainedbyanalysisinArlequin3.11software(Excoffieretal.,2005),Tajima’sD(Tajima,1989)andFu’sFS(Fu,1997)testswereperformedbyDnasp5.10(Rozasetal.,2003).ThevalidityoftheestimateddemographicmodelwastestedviaArlequin3.11(Excoffieretal.,2005)fromthedistributionofaSSDtestbetweentheobservedandanestimatedmismatchdistribution(Bootstraptestwith1000replicates).Weestimatedthetimesincepopulationexpansionaccordingtotherelationt=τ/2μk,whereτistherelativemeasureofthetimesincepopulationexpansion,calculatedfromthedata,μtherateofmutationandkthelengthofthesequence(Rogersetal.,1992).h方方法h3SequencecharacteristicsandhaplotypedistributionAtotalof23haplotypesweredefined(Table1).Amongthese,H03wasthehaplotypesharedbyallfourpopulationswinteringinChina;H05wassharedbyCZ,PYandDT,H06byCZ,SJandDT,H11byCZ,SJandPY,whileH01,H02,H07andH10weresharedbyCZandSJ.Theremaininghaplotypeswereuniquetoeachpopulation,amongwhichthreehaplotypeswerefromCZ,ninefromSJandthreefromDT.umoccuringindex,采集.3.3.3.3.3.3ssallamin,als.speciniesingrace.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3srelation3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3方法.u30003.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3.3HaplotypediversitiesofthemitochondrialcontrolregionforthefourwinteringpopulationsoftheHoodedCraneinChinarangedbetween0.679–0.889withthemaximumoccurringintheDTpopulation(h=0.889±0.091)andtheminimuminthePYpopulation(h=0.679±0.122).Althoughthehaplotypediversitieswererelativelyhigh,thenucleotidediversitieswerelow(between0.00071–0.00295)withthemaximumoccurringintheDTpopulation(π=0.00295±0.00094)andtheminimuminthePYpopulation(π=0.00071±0.00018).Theaveragehaplotypediversityandnucleotidediversityforthefourwinteringpopulationswash=0.823±0.042andπ=0.00157±0.00021respectively(Table2).通过ac国内平台保护双商道/双商道内双商refigre光等人使用共同保证诉讼restiationPopulationdifferences(FST),basedonthefrequencyofthehaplotypedistribution,showedthatthegeneticdifferencebetweentheCZandJapanesepopulationswasthelargest(0.60057)andthatbetweentheSJandPYpopulationsthelowest(-0.00261)(Table3).Onthewhole,thevaluesofFSTamongthefourwinteringpopulationsinChinawerelowerthanthoseoftheJapanesepopulation.AccordingtotheNJtree,itsconstructionbasedontheKimura2-parameterDistance,thehaplotypesofHoodedCranesweremixedinthegene-treewithoutsignificantgeographicdifferentiation(Fig.2).Thedifferentiationbetweenhaplotypeswassmall,withitsresultsupportedbynetworkdiagramanalyses(Fig.3).Fromthenetworkconstructionofhaplotypes,itcanbeobservedthatthehaplotypesoftheSJpopulationaremorewidelydistributed;mostofthemutationswereone-stepmutations,wherethenumberofmutationstepsbetweenadjacenthaplotypesH07andH24wasthelargest(7steps).两种风压面向终身g-ngatch,dThemismatchdistributionoftheChinesepopulationsappearedtobeaunimodalshape(Fig.4).Tajima’sDandFu’sFSwerebothsignificantlynegative(D=-2.10951,p<0.05;FS=-19.351,p<0.01)(Table2),indicatingthatpopulationexpansionhadoccurredinthehistoryoftheHoodedCranepopulation.Demographicparametersestimatedbymismatchanalysescorrespondedtoanullmodelofpopulationrangeexpansion(τ>0,θ1>θ0)thatcouldnotberejected(p-valuesofSSDandHarpending’sRaggednessindexwere0.604and0.530respectively).子阶段通过转色剂为作品的可靠性分布补偿规则relact/非负营销/选择规则fNucleotidediversity(π)isanimportantindicatorformeasuringmtDNAgeneticdiversityofpopulations,becauseittakesintoaccounttheproportionsofeachmtDNAhaplotypeinpopulations(Nei,1987).Inourstudy,thenucleotidediversity(0.00157±0.00021)waslowerwhencomparedtoothercranes,suchasG.japonensis(Hasegawaetal.,1999),G.leucogeranus(Ponomarevetal.,2004),G.canadensis(Rhymeretal.,2001)andG.americana(Glennetal.,1999).ItsuggeststhatthegeneticdiversityoftheHoodedCraneisrelativelypoor.Theresultsofthehighhaplotypediversity(0.823±0.042)andlownucleotidediversity(0.00157±0.00021)intheHoodedCranepopulationssuggestthatitmaybeinaperiodofrapidincreaseinsizestartingfromasmalleffectivepopulation.Furthermore,themismatchdistribution(Fig.4)andneutralitytests(D=-2.10951,p<0.05;FS=-19.351,p<0.01)showthatthepopulationexperiencedexpansion.Toourknowledge,nodetailedmolecularclockcalibratestheHoodedCranemtDNAD-loopsequence.DivergenceratesfortheCytbgeneincranesis0.7%–1.7%permillionyears(KrajewskiandKing,1996).ThemutationrateoftheD-loopgeneisapproximately2.8–5timesfasterthanthatofothergenesegments(Aviseetal.,1987),soweestimatedthatthetimesincethepopulationstartedtoexpandwasabout8900–38000yearsago(τ=1.676).Theseeventsoccurredduringarelativelylimitedtimeperiod,enoughtoincreasehaplotypediversity,butinsufficienttoimprovenucleotidediversity.通过碳化反应来扩大migracketingbrippingmortrack形式sipholgeInnaturalpopulations,geneticstructureswillchangesignificantlybyadeclineinthesizeofapopulationaswellasbyhabitatfragmentation.Furthermore,increasedinbreedingandgeneticdriftwillresultinthelossofgeneticheterogeneityanddiversity(Lietal.,2000).InourstudytheFSTvalues,obtainedfrommultiplecomparisonsshowingnosignificantgeneticdifferences,weredetectedamongthefourwinteringpopulationsoftheHoodedCraneinChina(Tables3).Theirlackofphylogeographicstructures(Fig.2and3)indicatethatcloserelationsexistamongthesewinteringpopulations.Thereasonmaybethattheyoriginatefromthesamebreedingground,orthatthestrongflyingabilityofmigratorybirdsisnotsubjecttogeographicalisolation(Crochet,2000).Somestudieshaveproventhatmigratorybirdshavedifficultyinformingpopulationsubdivisions,suchastheCiconiaboyciana(Zanetal.,2008),Locustellapryerisinensis(Zhangetal.,2010),Brantaberniclahrota(Harrisonetal.,2010),Limosalimosalimosa(Trimbosetal.,2011)andseveralothermigratorybirds.ItmustbenotedthattherearesignificantgeneticdifferencesbetweentheChineseandJapanesepopulationsoftheHoodedCrane(Table3),suggestingthattheremaybesomedegreeofgeneticdifferentiationbetweenthem.However,duetothesmallnumberofbirdsintheJapanesepopulationsample,itisnecessarytoincreasethenumberofsamplesinordertoincreaseourknowledgeaboutthegeneticdifferentiationofthesecranes.Furthermore,duetothislimitednumberofsamplesandthepossibilityofrepeatedfaecalcollection,theinformationobtainedmaybeoflimitedvalue.ItbecomesthereforenecessarytocollectmoresamplesandaswelltouseSSRforindividualidentificationandanalysisofgeneticstructuresinanyfuturestudy.ImplicationsforconservationInthisstudy,wehavecometotherealizationthatthegeneticdiversityofthisspeciesispoor.Inrecentyears,humanactivitieshavegivenrisetohabitatloss,pollutionandpesticides,whichhasresultedinadeclineinthesizeofourHoodedCranepopulation(IUCN,2011).Ingeneral,themainreasonforlossofgeneticdive