高级遗传育种

ImprovementofBacterialBlightResistanceofMinghui63,anEliteRestorerLineofHybridRice,byMolecularMarker-AssistedSelection,ShengChen,X.H.Lin,C.G.Xu,andQifaZhang,Minghui63isarestorerlinewidelyusedinhybridriceproductioninChina,however,thislinehasbecomeincreasinglysusceptibletobacterialblight(BB),resultinginarapiddeclineofitsuseinriceproduction.TheobjectiveofthisstudywastoimprovetheBBresistanceofMinghui63byintrogressingXa21,abroadspectrumBBresistancegene,intoMinghui63bymolecularmarker-assistedselection(MAC).,迄今为止，已经鉴定或命名了20多个白叶枯病抗性基因(Xa1至Xa23)，在这些基因中Xa21是经过充分鉴定已被公认的对白叶枯病生理小种抗性谱最广的显性抗性基因。该基因全抗国际上的白叶枯病的鉴别菌系和我国稻区的全部7个白叶枯病病原型，比其他白叶枯病抗性基因更具有育种价值。1995年由中国科学院遗传研究所植物生物技术实验室参加的国际合作克隆了Xa21基因，成为水稻基因组研究和植物抗病基因克隆的重大突破,从而开辟了水稻抗白叶枯病基因工程新领域。,MAS（Markerassistedselection),是利用与目标性状基因紧密连锁的遗传标记,对目标性状进行跟踪选择的一项育种技术。,EXPERIMENTALMATERIALS,1.IRBB21,anisogeniclineofIR24containingXa21.2.Minghui63,therestorerlineforanumberofelitehybridswidelygrowninChina,wastherecipientparenttobeimproved.3.Zhengshan97A,themalesterilelineforanumberofwidelygrownhybrids,wasusedtomakehybridswithMinghui63aswellastheimprovedversionofMinghui63.4.Zhenzhuai，acultivarhighlysusceptibletoalltheXooraces,wasusedasasusceptiblecheck.,Xoo简介,白叶枯病是水稻的主要病害之一，如在籼稻的主栽品种中多数品种全感白叶枯病的鉴别菌系，少数品种仅局限于对部分生理小种的抗性。国际水稻所对亚洲白叶枯病抗性基因在水稻产区的分布进行调查，在分析的3711个我国水稻品种中，带有已知白叶枯病抗性基因的品种只有7.33%。目前我国抗性品种携带的抗源主要是Xa4和Xa3基因，抗谱窄，抗性水平低。杂交稻对白叶枯病的抗性状况也不理想。1993年全国水稻育种协作组杂交稻新组合白叶枯病抗性鉴定表明，在44个组合中只有2个组合表现为中抗。由于缺乏对选用优良抗源的认识，新的杂交稻组合在白叶枯病抗性改良上进步并不大。因此迫切需要拓宽抗病基因源，选择抗谱广，抗性水平高的抗性基因，改良杂交水稻对白叶枯病的抗性。,EXPERIMENTALMETHODS,MAS（Markerassistedselection标记辅助育种)是利用与目标性状基因紧密连锁的遗传标记,对目标性状进行跟踪选择的一项育种技术。RFLP(RestrictionFragmentLengthPolymorphisma,限制性内切酶片段长度多态性)作为第一代分子生物学标记自问世以来已广泛运用于多门生物学科研究中，但它运用于植物抗性研究还只是近几年的事。RFLP能对植物的抗性基因进行定位和分离，利用RFLP技术，对于核基因组或叶绿体基因组、尤其是后者，若能提取纯净DNA，则可直接从酶切后的电泳图谱看出其多态性，利用这一方法可以测定种群内、种群间不同水平的物种在污染环境下抗性分化进化水平上的差异。RFLP可以用来测定多态性是由父本还是母本产生的，也可用来测定由多态性产生的突变类型究竟是由碱基突变或倒位、还是由缺失、插入造成的。,RFLPandPCRMarkersUsedforMAS,XooStrains,InoculumPreparation,Inoculation,andDiseaseScoring,Atotalof17Xoostrainswereusedinthisstudy(seeTable1forrepresentativeexamples,includingninestrains.Fordiseasescoring,thelengthoflongestlesionsoffiveundamagedleavesofeachindividualwasmeasured21dafterinoculation.Aplantwasclassifiedasresistantiftheaveragelesionlengthwasshorterthan3.0cm,moderatelyresistantifthelesionwas3.0to6.0cm,moderatelysusceptibleifthelesionwas6.0to9.0cm,andsusceptibleifthelesionwaslongerthan9.0cm.,Table1.Reactionsofsixlinerepresentativeexamplesofthe17Xanthomonasoryzacpv.oryzac(Xoo)strainsusedfortestingBBresistance,ToestablishaMASsystemforintrogressingXa21fromIRBB21toMinghui63.200individualsfromtheF2populationofacrossbetweenthesetwolineswereassayedindividuallywith11markers(seeFig.1)surroundingtheXa21genomicregion.Fourofthemarkers,281.21.C189(convertedfromRFLP),andAB9werePCRmarkers,andtheremainingwereRFLPmarkersselectedfromtwohighdensitymaps.AlocallinkagemapforthisgenomicregionwasconstructedwithMapmaker/Exp3.0withLOD3.0.Cosegregationwasalsotestedbetweenresistanceandthe,markerrepresentingtheXa21locusbyinoculatingthispopulationwithXoostrainPxo99.,TheexperimentalproceduresforRFLPassays,includingDNAisolation,digestion,electrophoresisandsouthernblothybridization,showthatAB9isadominantmarkerwithPCRfragmentfromthedonorparentIRBB21detectedinthebreedingpopulationandtheremainingthreemarkerarecodominant,Fig1.ThelinkagemapoftheXa21genomicregiononricechromosome11,MAS,ThelocallinkagemapofXa21genomicregiononchromo-some11,constructedonthebasisof200individualsoftheF2populationfromtheMinghui63/IRBB21cross,isshowninFig.1,fromwhichaMASsystemwasestablished.,TheCrossingandSelectionScheme,TheXa21genewasinrogressedintoMinghui63followingarecurrentbackcrossingprocedure,combinedwithtandemselectionusingmolecularmarkers.Theentireschemetookthreegenerationsofbackcrossesandonegenerationofselfingtocomplete.Inthisscheme,theprogenyofeachbackcrosswasfirstselectedforthepresenceoftheXa21genebymeansofbothPCRanddiseaseinoculation.TheXa21-containingindividualsintheBC1F2wereselectedforrecombinationbetweenXa21andtheothermarkerslocus.TheXa21containingplantsintheBc3F1wereassayedwithalargenumberofmolecularmarkerscoveringtheentirericegenometoidentifyindividualsthatwerehomozygousfortheMinghui63genotypesatallmarkerloci,excepttheXa21locus.Theselectedindividualswerethenself-fertilizedtoproduceindividualsthatwerehomozygousfortheXa21geneatthislocus,thuscompletingthebreedingprocedure.,Screeningof354RFLPprobesfromthetwopublishedhigh-densitymapsidentified158markersthatweretwopolymorphicbetweenMinghui63andIRBB21(Table2).Thesemarkersweredistributedquiteevenlyinthelinkagemapexceptthatthereweremoremarkersfromchromosome11thanfromtheotherchromosome.Onehundredtwenty-eightofthe158polymorphicmarkers,representingallthe12chromosomeswiththeintervallessthan30cM,wereusedfor“cleaningup”thegeneticbackgroundofselections.,Table2ScreeningRFLPmarkersthatwerepolymorphicbetweenMinghui63andIRBB21,Amongatotalof250plantsthatcarriedXa21inBC3F1,twoplantswerefoundtobehomozygousfortheMinghui63genotypesatallmarkerlociexcepttheRG103locusresidingintheXa21generegion.ThereweresixadditionalplantsthatwereheterozygousatonetofourmarkerlocibesidestheRG103locus.ThelesionlengthsoftheseeightplantswerenotsignificantlydifferentfromthatofIRBB21whentestedagainstPxo99,astrainbelongingtoXooRace6.(Table3),Table3.Themolecularmarkergenotypesandresistanceofeightselectedindividualsfrom250BC2F1resistantplants.Themolecularmarkersgenotypesarebasedon128RFLPlocithatwerepolymorphicbetweenMinghui63andIRBB21andevenlydistributedonthe12ricechromosomes.,AlltheremainingmarkerlociwerehomozygousfortheMinghui63genotypes.Averagelesionlengthof5inoculatedleavesat21dafterinoculationwithPxo99.,Thereeightplantswereself-pollinatedtoproduceBC3F2,fromwhichprogenieshomozygousfortheXa21allelewereobtain.ComparedwiththeoriginalMinghui63,theBBresistanceoftheseselectedlineswasgreatlyimprovedaccordingtotheresultsofinoculationswithatotalofXoostrains(seeTable4fortheresultfromsomeofthestrains).,Table4.ExamplesofBBresistanceofeightselectedlinesfromBC3F2,expressedasaveragelesionlengthSDwheninoculatedwithsixisolatesofthepathogen,cm,ComparisonoftheAgronomicPerformance,TheresultsfromtheWuhanplantinginthesummershowedthatyieldandyieldcomponenttraitswerealmostidenticalbetweentheoriginalShanyou63andShanyou63(Xa21)whentherewasnoartificialinoculation.Ontheotherhand,themeasurementsforyieldandtwooftheyieldcomponenttraits(grainsperpanicleandgrainweight)ofShanyou63(Xa21)appearedtobehigherthanShanyou63whenchallengedwiththedisease.,IntheHainantestwithoutartificialinoculation,Minghui63andMinghui63(Xa21)wereidenticalforalltheagronomictraitsexamined,aswereShanyou63andShanyou63(Xa21)(Table5).Underheavilydiseasedconditions(Table5),Minghui63(Xa21)showedsignificantlyhighergrainweightandspikeletfertilitythanMinghui63.ThedifferencesinyieldrelatedtraitswereevenmorepronouncedbetweenShanyou63andShanyou63(Xa21)(Table5).Yieldandallthreeyieldcomponenttraits(tillersperplant,grainperpanicle,andgrainweight)werehigherforShanyou63(Xa21)thanShanyou63.,Table5.AgronomicperformanceofMinghui63Shanyou,IBRR21andXa21containingversionsofMinghui63andShanyou63underfieldconditionswithorwithoutartificialinoculationtestedinthespringof1999inHainanIsland,Leastsignificantdifferencesat0.05and0.01probabilitylevels,respectively,DICUSSION,UsingMASandthreegenerationsofbackcrossesfollowedbyonegenerationofselfing,animprovedversionofMinghui63wereobtained,thebestrestorerlinewidelyusedinriceproductioninChina.,TheexperimentalshowedthatthisprovedversionofMinghui63contained