小叶锦鸡儿SSR引物开发及遗传多样性研究

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1、兰州大学硕士学位论文小叶锦鸡儿SSR引物开发及遗传多样性研究姓名：韩永增申请学位级别：硕士专业：畜牧学、草业科学指导教师：高洪文20100501兰州大学2010届硕士学位论文小叶锦鸡儿SSR引物开发及遗传多样性研究AbstractCaraganamicrophyllaI．am．isperennialforageshrubwhichisdistributedmostlyinNortheasternChina，InnerMongolia，Hebei，Shanxi，Shaanxi，Shandongetc．alsoinMongoliaandSiberia．Becauseit’Sdroughtresi

2、stance；coldresistanceandresistanttosoilimpoverishment，itbecomesfavorableforageshrubandecologicalprotectionpioneerplantinNortheasternandNorthwesternChina．Thusithasgreatpotentialfordevelopmentandutilization．TenpopulationsofCaraganamicrophyllaLam．，whichcainefromcentralandeasternInnerMongolia，wereusedi

3、ntheexperiment．ThematerialswereprovidedbyInstituteofAnimalSciences，ChineseAcademyofAgriculturalSciences．Theworkmainlyincluded：1)todevelopthepolymorphicSSRprimersforCaraganamicrophyllaLain．usingISSR-PCRtechnology．2)tOestablishandoptimizeSSR-PCRsystemandFiltratetheSSRprimersusingtheoptimizedSSR-PCRsy

4、stem．3)toanalyzethegeneticdiversityusingSSRmarkers．11leresultswereasfollowed．1．FoutythreepairsofSSRprimersweredesignedbasedonthefourISSRprimers，SI：(AG)10，S2：(AC)10，S3：(GA)10，S4：(CA)10．AndtheaverageSuccessratewas11．17％．Ourresultsalsoindicatedthattherewerelargenumbersof似G)nfragmentsandlessof(AC)nfrag

5、mentsinthegenomeofCaraganamicrophyllaLain．ComparedtothetraditionalmethodofSSRmarkerdevelopment，thistechniqueWasmoreefficient．Itmustbenotedthattherearetwicesequencingandthreetimesprimersynthesis．2．OrthogonaldesignwasappliedtooptimizetheSSR-PCRsystemonCaraganamicrophyllaI_am．，whichemployedfourlevelsa

6、ndfivefactors(DNA,M92+，dNTP，primers，andTaqDNApolymerase，respectively)．TheoptimumSSRreactionincluded40ngtemplateDNA，3mmol／LM矿+，300gmol／LdNTP,o．6#mol／Lprimer,1UTaqDNApolymerase，lxBufferwithtotal25／zLreactionvolumes．ThePCRprocedurewasallinitialstepof94℃forlOmin，followedby10cyclesof94℃for45s，65℃forlmin

7、(decreasing1℃everycycle)，72"Cforlmin；30cyclesof94"(2for45s，55℃forlmin，72℃for1rain，andafinalextensionat72℃for10min，4℃hold．3．ChoosingthedevelopedSSRprimersbytheoptimizedsystem，37pairs(86．05％)hadtargetfragment