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两种虾脊兰属植物DNA提取方法的优化.docx

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两种虾脊兰属植物DNA提取方法的优化 Abstract Orchidaceaeisoneofthelargestfamiliesoffloweringplants,withahigheconomicandornamentalvalue.However,DNAextractionfromorchids,especiallyfromthegenusPaphiopedilum,remainsachallengingtaskduetothepresenceofsecondarymetabolitesandothercompoundsthatcaninterferewiththeDNAisolationprocess.Inthisstudy,weoptimizedtwoDNAextractionmethodsfortwospeciesofthegenusPaphiopedilum(PaphiopedilumdianthumandPaphiopedilummicranthum),usingdifferentconditionsoflysisbufferandextractionprotocol.Theresultsshowedthatbothmethodsyieldedhigh-qualityandquantityDNA,suitablefordownstreammolecularapplications.ThisstudyprovidesausefulreferenceforDNAextractionfromPaphiopedilumspecies. Introduction Orchidsareperennialherbsorlianascommonlyfoundintropicalandsubtropicalregionsaroundtheworld.Duetotheiruniqueanddiversefloralmorphology,orchidshavebeenvaluedfortheirbeautyandornamentalpurposessinceancienttimes.Withtherapiddevelopmentofmodernbiotechnologyandtheincreasingdemandforsustainableagriculture,orchidbreedingandbiotechnologyresearchhavebecomeimportantfieldsofinterestinrecentyears(Chenetal.,2012;Chughetal.,2009).Geneticdiversityanalysis,genemapping,andmolecularmarkerassistedbreedingareamongthemolecularapplicationsthatrequirehigh-qualityDNAextraction(Ghoshetal.,2014).However,DNAisolationfromorchidsisoftenchallenging,especiallyforthegenusPaphiopedilum,whichcontainssecondarymetabolitesthatinterferewithDNAisolation(Jhaetal.,2008).Thus,optimizingDNAextractionmethodsforPaphiopedilumspeciesisessentialformolecularbiologyresearchandbiotechnologicalapplications. Materialsandmethods PlantmaterialandDNAextraction Inthisstudy,twospeciesofPaphiopedilum,namelyPaphiopedilumdianthumandPaphiopedilummicranthum,wereselectedforDNAextraction.Freshleaveswerecollectedfrommatureplantsgrowninthegreenhouse.TwoDNAextractionmethodswereoptimized,includingtheCTABandSDSmethods. CTABmethod TheCTABmethodwasperformedaccordingtotheprotocoldescribedbyDoyle&Doyle(1990)withsomemodifications.Briefly,approximately100mgoff