山東主栽果樹AFLP指紋圖譜的構建.pdf

1、萊陽農學院碩士學位論文山東主栽果樹AFLP指紋圖譜的構建EstablishmentofAFLPFingerprintingOilFruitTreesMainlyCultivatedinShandong研究生姓名導師姓名專業(yè)研究方向瑚海騰祝軍教授果樹學生物技術AbstractFifty—threevarietiesbelongtofourkindoffruittreesmainlycultivatedinShandongincluding

2、apple(MaluspumilaMill)，pear(聊nL)，peach(PpelsicaBatch)andjujube(ZiziphusjujubeMill)，wereusedinthisexperimentTheDNAfingerprintingofthesefiftythreevarietieswereestablishedbyusingAFLPtechnologyTheidentificationofthesefiftyth

3、reevarieties，andthehereditaryrelationsanalysiswerealsomadebasedontheirDNAfingerprintingThegenomicDNAoftheseexperimentmaterialswereextractedbymodifiedCTABmethodThecomponentsofmodifiedisolationbufferare：3％CTAB14mol／LNaCl，2

4、0mmol／LEDTA，100mmol／LTriSHCI(pH80)，2％p—MercaptoethanalThegenomicDNAwaspurifiedbyphenol／chloroform／isoamylalcohol(twice)，andtheRNAiseliminatedfromthesolutionwithRNasefinallythegenomicDNAwaspurifiedbychloroform／isoamylalco

5、hol(once)TheextractedgenomicDNAoffruittreeswassuitableforAFLPanalysisAftersystematicallystudyingonthemainfactorsincludingquantitiesofenzymereactiontime，linkagetimeandSOon，anAFLPsilver—staininganal37sissystemforfruittrees

6、genomicDNAwasestablished：ThepurifiedfruitgenomicDNA200500ngNEBBuffer25pL，BSAO259L，EcoRI(20U／gL)25I_tL，MseI(10U／uL)O5pkEcoR，adapter(5pMol／gL)1OlaL，MseIadapter(50pMol／gL)1OL，andaddedddH20to509L，andincubatedat37。CovernightT

7、helinkedproductswereusedasforpre—anlplificationThepreamplifiedproductsw￡sdiluted20times、“thddH20andusedasforselectiveamplificationAttheendoftheselectiveamplification，thefruittreessamplesweredenaturedby79LLoadingbufferand

8、heatedup95℃for5min，thencooledoniceimmediatelyThePCRreactionproductswereanalyzedon60％denaturalacrylamide／bisacrylimadegelsbysilverstainingEightpairsofprimerswereselectedamongfortypairstOamplifyfor53varietiesandpreliminari