PART1. MOLECULAR GENETICS AND THE STUDY OF

MoleculargeneticanalysisofhybridisationSCIENCEFORCONSERVATION:105GeoffreyK.ChambersandElizabethS.MacAvoyPublishedbyDepartmentofConservationP.O.Box10-420Wellington,NewZealandScienceforConservationpresentstheresultsofinvestigationsbyDOCstaff,andbycontractedscienceprovidersoutsidetheDepartmentofConservation.Publicationsinthisseriesareinternallyandexternallypeerreviewed.©February1999,DepartmentofConservationISSN1173–2946ISBN0–478–21799–4ThispublicationoriginatedfromworkdoneunderDepartmentofConservationInvestigationno.2135and2240,carriedoutbyGeoffreyK.ChambersandElizabethS.MacAvoy,InstituteforMolecularSystematics,SchoolofBiologicalSciences,VictoriaUniversityofWellington,POBox600,Wellington.ItwasapprovedforpublicationbytheDirector,Science&ResearchUnit,ScienceTechnologyandInformationServices,DepartmentofConservation,Wellington.CataloguinginPublicationChambers,GeoffreyK.Moleculargeneticanalysisofhybridisation/GeoffreyK.ChambersandElizabethS.MacAvoy.Wellington,N.Z.:Dept.ofConservation,1999.1v.;30cm.(Scienceforconservation,1173-2946;105.)Includesbibliographicalreferences.ISBN04782179941.Hybridization.I.MacAvoy,ElizabethS.II.Title.III.Series:Scienceforconservation(Wellington,N.Z.);105.575.13220zbn99-011120CONTENTSPART1.MOLECULARGENETICSANDTHESTUDYOFHYBRIDISATIONINNATURALPOPULATIONS5GeoffreyK.ChambersandElizabethS.MacAvoyAbstract51.Background52.Generalconcepts63.Surveyofgenetictargetsandanalyticalmethods6Molecularcytogenetics6Allozymeelectrophoresis7Proteinsequencing7Restrictionfragmentlengthpolymorphism(RFLP)7DNAfingerprinting8PCRandDNAsequencing8Amplifiedfragmentlengthpolymorphism(AmFLP)8SSCP,TGGE,DGGEandheteroduplexanalysis9RandomlyamplifiedpolymorphicDNA(RAPD)9Microsatelliteanalysis94.Moleculargeneticanalysisofhybridisation:Twocasestudies105.Concludingremarks126.Acknowledgements127.References13PART2.MITOCHONDRIALDNASEQUENCEFROMBLACKANDPIEDSTILTS15ElizabethS.MacAvoyandGeoffreyK.ChambersAbstract151.Introduction152.Background153.Objectives164.Materialsandmethods164.1Materials164.2Methods17DNAsequencing17Restrictionenzymedigestion174.3Selectedgenetictargets18(1)Cytotchromeb18(2)Controlregion18(3)LongPCRtarget184.4Searchforrestrictionenzymemarkers184.5Phylogeneticanalysis205.Results205.1DNAextraction205.2Cytochromebsequences205.3Restrictionenzymemarkersforcytochromebhaplotypes215.4Controlregionsequences225.5Restrictionenzymemarkersforcontrolregionhaplotypes245.6Phylogeneticanalysis255.7Samplenumberingintissuecollections256.Discussion267.Conclusions288.Recommendations289.Acknowledgements2910.References295Part1.MoleculargeneticsandthestudyofhybridisationinnaturalpopulationsGeoffreyK.ChambersandElizabethS.MacAvoyAbstractTherangeofmoleculartoolsinpresentuseforthestudyofpopulationgeneticsinclude:molecularcytogenetics,allozymeelectrophoresis,proteinsequencing,restrictionfragmentlengthpolymorphism(RFLP),DNAfingerprinting,PCRandDNAsequencing,amplifiedfragmentlengthpolymorphism(AmFLP),SSCP,TGGE,DGGEandheteroduplexanalysis,randomlyamplifiedpolymorphicDNA(RAPD),andmicrosatelliteanalysis.Underlyingprinciples,advantages,anddisadvantagesoftheseleadingandmostversatiletechnologiesareexaminedinthecontextoftwocasestudies.Thissurveywillintroduceconservationbiologistsandwildlifemanagerstothemostcommonlyusedtechniquesandhelpthemtoselectthemostcost-effectivemoleculargenetictoolsforconservationstudy.1.BackgroundRecentadvancesinthedevelopmentofmoleculargenetictechniqueshaveprovidedconservationbiologistswitharapidlyincreasingdiversityofpowerfullaboratorytools.Asaresult,wildlifemanagersarenowfacedwithanalmostbewilderingrangeofalternativesfromwhichtochoose.Makingsuchdecisionsisdifficultsincethemethodswhicharecurrentlyavailabledifferconsiderablywithrespecttoversatility,cost,samplingrequirementsanddevelopmenttimeforeachnewspecies.Inparticular,theadventofthepolymerasechainreaction(PCR),whichmakesitpossibletoobtainhighlydiscriminatinganalysesfromrelativelysmallbiologicalsampleshasspawnedaverywiderangeofmoreorlessreliableanalyticalsystemswithinthelasttenyears.Thepurposeofthisarticleistooutlinetheleadingcharacteristicsofthemostcommonlyusedtechniques,asaguidetotheselectionofthemostcost-effectivesystem.ThishasbeendoneinthecontextoftwospeciallyselectedcasestudiesandbyreferringtootherworkcarriedoutpreviouslyinNewZealand.62.GeneralconceptsAspopulationsdivergemutationsoccurinDNAandindividualsbegintoaccumulategeneticchangeswhicheventuallycometocharacteriseeachpopulation.Thesechangesarerecordedintheirgenomesandmayalsobeexpressedingeneproducts(RNAandprotein).Thosechangeswithfunctionalconsequences,maygiverisetoobservablemorphologicalandbehaviouralcharacteristics.Itfollows,therefore,thatappropriatelydiscriminatingmolecularanalysesofgenesandgeneproductscanprovideverysensitivetoolswithwhichtomeasurepopulationdifferentiation,evenintheabsenceofmoreovertcharacters.Italsofollowsthattherateofmolecularevolutionofthegenetictargetisofcriticalimportancewhenselectingboththetargetitselfandthemethodofanalysis.Rapidlyevolvingmarkers,suchassomerepetitiveDNAsequences,willprovideg