三疣梭子蟹微卫星文库的构建及多态性位点筛选

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1、●◆上海海洋大学硕士学位论文MicrosatellitelibraryConstructionandpolymorphiclociscreeningfortheswimmingcrab，PortunustrituberculatusABSTRACTmswimmingcrab(Portunustrituberculatus)isanimportantfisheryresource，widelydistributedincoastalareasofChina．11圮swimmingcrabwasregarededasoneofthemarineaquaculturespeciesfrom198

2、1．Ithasbecomeoneofthemainaquaculturespeciesduetoitsconsiderableeconomicvalue，highediblenutritionalvalueandfastgrowingspeed．Becauseofvariousreasons，theresourcesofPtrituberculatusdroppedsharply，defenseabilitydeclinedandnaturalgermplasmresourcesdegradedseriouslyinrecentyears．Inordertorestoreandimprov

3、ethequalityoftheparentcrabandcrabseed，keepthehealthydevelopmentofaquaculture，avoiddecrementofwildgermplasmresources，andrealizesustainableutilizationofthenaturalresources，researchonthegeneticgermplasmresourcesofP．trituberculatusisneededurgently．Therefore，developingasetofeffectiveandreasonablemolecu

4、largeneticmarkersisnecessaryforgeneticgermplasmresourcesconservationresearchforthisspecies．MicrosatelliteDNAlibraryoftheswimmingcrabWasestablishedusingthemagneticbeadhybridizationenrichmentmethods．T0talgenomicDNAWasextractedanddigested、析tllrestrictionenzymeSau3AI．Fragmentsinrangeof200--1000bpwerea

5、garose-gelpurifiedandligated、析tllspecialadaptor,andthecombinationsWashybridized、Ⅳitllbiotin—labelledmicrosatelliteprobe(CAh2，(GA)i2，(TGC)s．Byusingthehighbindingaffinityofbiotintostreptavidin—coatedmagneticbeads，single-strandedDNAcontainingtheselectedmicrosatellitesequenceswerecaptured．ThetargetedD

6、NAWasused鹪thetemplateforPCRamplificationusingtheadapter嬲primers；andtheproductswerethencloneddirectly,screenedbybacteriumliquidPCRmethodsubsequently．Ofthe60positiveclones，42microsatellitelociwereacquired，whichwerefurthercategorizedintoperfect(73．8％)，imperfect(21．4％)，andcompound(4．8％)types．Amongthem

7、icrosatelliterepeats，10,--30repeatsweremostabundant．III●上海海洋大学硕士学位论文ByusingPremierPrimer5．0，wedesigned31pairsofmicrosatelliteprimers，amongwhich16pairscanamplifyexpectedproduct．These16pairsofprimerswerefurtherused