第五章第一节图位克隆基本原理

第五章、植物基因克隆5.1植物基因克隆的基本原理5.2苹果抗黒猩病基因的克隆5.3水稻主效基因的克隆5.4水稻米香基因的克隆5.5数量性状基因克隆5.6玉米主效QTL克隆和利用5.1植物基因克隆的基本原理Strategiesforgenecloning1)Map-basedgenecloning2)Transposontagging3)Candidategeneapproach5.1.1Map-basedgenecloningGeneisolationwithoutpriorknowledgeofthegeneproductRequirements:PopulationofindividualswithgeneticdifferencesinthetraitofinterestGeneresponsiblecanbemappedtoachromosomalpositionadjacenttomolecularmarkers(e.g.RFLP,microsatmarkers)FirststepistoidentifymolecularmarkersthatareclosetothegeneofinterestInitiallysmallpopulationused(60-150)ThensaturatetheregionwithothermarkersMuchlargerpopulation(300-1000)Usemarkersbasedoncomparativemappinginfoe.g.Arabidopsis-BrassicacolinearitytohelpfindcanolagenesLookingforonesthatrarelyshowrecombination5.1.1Map-basedgenecloning-continue目标基因定位RRTargetgeneM1M2P1F1P2BC1×BC1-1BC1-100BC1-99BC1-98BC1-2M1M2目标基因精细定位RRRRRRRRRRTargetgeneM1M2Map-basedgenecloning

-ChromosomewalkingNext,screenalargeinsertgenomiclibrary(e.g.BAC)withthemarkerstoisolateclonesthatcontainthemarkerInvolvescreatingnewmarkersBasedonsequencesattheendsoftheselectedclonesScreenthelibrarywiththenewmarkersGoalistofindasetofclonesthatcovertheregionharboringthelocusofinterest.CreateacontigofBACswhichareanchoredbetweentheclosestmarkersflankingthelocusofinterestIfyouareluckysomebodyelsehasalreadywalkedbyandcreatedacontigwhichcanbeusedMap-basedgenecloning

-ChromosomewalkingMap-basedgenecloning

-ChromosomewalkingIdentifyingthetargetgene:YACsorBACscancontainseveraldifferentgenesMakeacDNAlibraryfromtissuethatshouldbeexpressingthegeneofinterestidentifyYACorBACstocDNAs&identifythosethatmatchtheregionrepresentedbytheYACorBACsIfmorethan1cDNAmatchesusehigh-resolutionmappingandlookfortheonethatco-segregateswiththegeneofinterestMap-basedgenecloning

-chromosomelanding

Chromosomewalkingishamperedinlargegenomes: LargeamountsofDNAbeingtraversedHighfrequencyofrepetitiveDNAChromosomelandingIdealsituationwheretheflankingmolecularmarkersidentifiedinthefirstmappingstepleadstothedirectisolationofgenomicfragment(s)thatcontainthegeneofinterest

Map-basedgenecloning

-chromosomelandingForchromosomelandingtobesuccessful,themarkersmustliewithinaphysicaldistancefromthegenethatislessthantheaverageinsertsizeofthegenomiclibrarybeingscreenedFacilitatedbytheavailabilityofhigh-densitymolecularlinkagemapsAndhigh-densitycomparativemapsfromcloselyrelatedspeciesMap-basedgenecloning

-chromosomelandingHigh-volumemarkertechnologiesAllowsthousandsofpotentiallypolymorphicDNAsegmentstobegeneratedRAPDs,AFLPBSADevelopmentofNILs(nearisogeniclines)UseofYACsorBACslargerinsertsTanksleyetal.1995.Chromosomelanding:aparadigmformap-bas