分子标记辅助选择ppt课件.ppt

第十七章分子标记辅助选择第一节分子标记辅助选择的基本原理第二节质量性状的标记辅助选择第三节数量性状的标记辅助选择第四节分子标记辅助选择的挑战与发展策略,1,传统的育种主要是根据植株的表现型进行选择，而环境条件、基因间互作、基因型与环境互作等多种因素都会影响表型选择效率。育种者在长期的实践中不断探索运用遗传标记来提高育种的选择效率与育种预见性。遗传标记包括形态学标记、细胞学标记、生化标记与分子标记。,2,以DNA多态性为基础的分子标记，目前已在作物遗传图谱构建、重要农艺性状基因的标记定位、种质资源的遗传多样性分析与品种指纹图谱及纯度鉴定等方面得到广泛应用，尤其是分子标记辅助选择（molecularmarker-assistedselection，MAS）育种更受到人们的重视。,3,1、概念：通过基因定位找到与目标基因紧密连锁的分子标记后，可通过该分子标记间接地对目标性状进行选择。此法称分子标记辅助选择(MolecularAssistantSelection,MAS)。MAS是育种中的一个诱人领域,将给传统的育种研究带来革命性的变化。MAS主要应用在有利基因的转移和基因的累加等方面。,第一节分子标记辅助选择的基本原理,TesterM(2)selectionindependentofenvironments;(3)selectionwithoutlaboriousfieldworkorintensivelaboratorywork;(4)selectionatanearlierbreedingstage;(5)selectionformultiplegenesand/ormultipletraits;(6)wholegenomeselection.,7,Usefulifconventionalscreeningmethodsarelaborious,costly,orenvironmentallydependentSelectionsfordiseaseandinsectresistancecanbemadeintheabsenceofthepathogenorpestGreatestpotentialadvantageoverphenotypicselectionfortraitswithlowpenetranceorlowheritabilityMayreducepopulationsizesneededforphenotypicselectionMaypermitselectionofindividualplantsMayspeedupthebreedingprocessMaybeeffectiveforearlygenerationtestingSelectionsattheseedlingstagecanbeagreatadvantageincropswithalonggenerationtimeReducenumberofgenerationsinabackcrossingprogrambyselectingforrecoveryoftherecurrentparentgenomeaswellasgenesofinterestfromthedonorparent,2、MostsuitableforMAS,8,PyramidgenesforasingletraitthatcouldnototherwisebedistinguishedatthephenotypiclevelAccumulatingmultiplequantitativetraitloci(QTL)fordiseaseresistancemayprovideahigherlevelofresistanceand/ormoredurableresistancetochangesinthepathogenpopulationMASmaypromotedeploymentoffewerresistancegenesGenesformultipletraitsofinterestmayalsobecombinedinonecultivarwithrelativeefficiencyMarkertechnologiesprovidethepotentialtounderstandtheunderlyingcausesofepistasisandGXE,whichcouldgreatlyimproveselectionefficiency,9,QualitativetraitsandquantitativetraitswithhighheritabilityaremoreamenabletoMASthanquantitativetraitswithlowheritability,whichisgenerallythecaseforphenotypicselectionaswell.Epistasis(oreffectofgeneticbackground)andgenotypebyenvironmentinteractions(GXE)canconfoundprogressfromMASjustastheydoinconventionalselectionschemes.EffortstoimprovetheprecisionofQTLestimationthroughincreasedreplicationandmultilocationaltestingwillalsoincreasetheefficiencyofphenotypicselection,therebyreducingthegainsthatmaybeattainedthroughMAS.Catch22:Ifphenotypesarepoorindicatorsofgenotypes,youcannotmapQTLforuseinMASIfphenotypicdataaregood,youdontneedMAS,ParadoxofMAS,10,1)Howtightlyitislinkedtogenescontrollingimportanttraits.2)Therelativeimportanceofthosegenesindeterminingthephenotype.3)TheconsistencyoflinkagedisequilibriumbetweenthemarkerandQTL4)ThefrequencyoftheQTL(MASwillbemorebeneficialwhentheQTLisinlowfrequency),3.Theutilityofamarkerdependson,11,12,Requirementsforwide-scaleapplicationofMAS,ValidationofQTLinbreedingmaterialsMultiplemarkersinvicinityofQTLdesirableSimple,quick,inexpensiveprotocolsfortissuesampling,DNAextraction,genotyping,anddatacollectionEfficientdatatracking,management,andintegrationwithphenotypicdataDecisionsupporttoolsforbreedersoptimaldesignofselectionstrategiesaccurateselectionofgenotypes,13,大多数情况下，质量性状无需借助于分子标记，但采用分子标记辅助选择可提高选择效率：表现型测定难度大或费用太高；表现型只能在个体发育后期才能测量，而育种实践中希望在早期选择；除目标性状外，还需对遗传背景进行选择；质量-数量性状的选择。,第二节质量性状的标记辅助选择,14,1、标记辅助选择的基本方法1）前景选择（foregroundselection）对目标基因的选择单标记可靠性：取决于标记与目标基因间连锁的紧密程度。如，标记座位M/m与目标基因座位Q/q连锁，重组率为r，则在F2根据标记基因型M/M获得目标基因型Q/Q的概率为p=（1-r）2。选择正确率随重组率增加而迅速降低。,15,如要求正确率在90%以上，则标记与目标基因间的重组率必须5%。,16,如要求选到1株目标基因型的概率为P，则必须选择带有目标基因型M/M的植株的最少数目为：n=log（1-P）2/log（1-r）2即使重组率高达0.3，也只要选择7株具有基因型M/M的植株，就有99%的把握能保证其中有1株为目标基因型；而如果不用标记辅助选择（相当于标记与目标基因间无连锁，即r=0.5），则至少需要16株。,17,双边标记可靠性将大大提高。如标记M1/m1和M2/m2各位于目标基因座Q/q的一侧，与目标基因间的重组率分别为r1和r2，则：F1的基因型为M1QM2/m1qm2F1产生的标记基因型为M1M2的配子有两种类型，包含目标等位基因的M1QM2和包含非目标等位基因的双交换M1qM2，由于双交换发生频率很低，因此，在后代中通过同时跟踪M1和M2来选择目标等位基因Q，正确率必然很高。,18,在无干扰时，F2代通过选择标记基因型M1M2/M1M2而获得目标基因型Q/Q的概率为：p=（1-r1）2（1-r2）2/（1-r1）2（1-r2）+r1r2在两标记间的图距固定的情况下，r1=r2时p最小（即目标基因位于两标记间的中点）。在实际情况下，单交换间一般总是存在相互干扰的，双交换的概率更小，因此双标记选择的正确率比理论期望值更高。,19,20,2）背景选择（backgroundselection）对除了目标基因之外的其他部分（遗传背景）的选择在F2，根据两个相邻标记，可推测出它们之间的染色体区段的来源和组成。参考完整的分子遗传图谱，检测所有标记基因型，最后绘制出图示基因型（graphicgenotype）。,21,1,2,2,2,2,2,2,2,2,2,2,2,1,2,2,2,2,3,2,1,1,2,2,2,2,2,2,2,2,2,2,1,2,2,2,2,3,3,3,3,1,2,3,3,3,3,2,2,1,1,1,A品种B品种的F2代一个个体的图示基因型，共5对染色体，白色表示来自A品种的区段，黑色表示来自B品种的区段，红色表示发生了交换的区段；竖杠表示标记所在位置。,2,2,22,2、标记辅助选择在育种上的应用1）多基因聚合（genepyramiding）将分散在不同品种中的有用基因聚合到一个基因组中,23,Agene-pyramidingschemecumulatingsixtargetgenes.（Servin等，2004）,24,25,3个抗稻瘟病基因在染色体上的位置,例子1：通过分子标记辅助选择方法聚合水稻抗稻瘟病基因（Zhengelal.1995）,26,C101LACC101A51,C101LACC101PKT,F1,F1,F2150株,F2150株,用相邻标记进行选择,Pi-1,Pi-2,Pi-1,Pi-4,10个Pi-1和Pi-2纯合的植株,10个Pi-1和Pi-4纯合的植株,F2150株,标记辅助选择,F1,3个抗病基因纯合的植株,三个近等基因系,27,例2：N.Huangetal（1997）Pyramidingofbacterialblightresistancegenesinrice:marker-assistedselectionusingRFLPandPCR,DNAmarker-assistedselectionwasusedtopyramidfourbacterialblightresistancegenes,Xa-4,xa-5,xa-13andXa-21.Breedinglineswithtwo,threeandfourresistancegenesweredevelopedandtestedforresistancetothebacterialblightpathogen(Xanthomonasoryzaepv.oryzae).Thepyramidlinesshowedawiderspectrumandahigherlevelofresistancethanlineswithonlyasinglegene.,28,近等基因系,接种株数,标记分析株数,29,30,31,32,2）分子标记辅助回交育种（genetransfer，MAB）将供体亲本中的有用基因转移或渗入到受体亲本的遗传背景中，从而达到改良受体亲本个别性状的目的。在每一代都需通过对供体等位基因的选择使目标基因座位保持杂合，同时提高目标座位以外的其他染色体区域的受体基因组（Recipientgenomecontent，RGC）比例。,33,Marker-assistedbackcrossing,Foregroundselection：markerscanbeusedincombinationwithortoreplacescreeningforthetargetgeneorQTL.Recombinantselection:markerscanbeusedtoselectBCprogenywiththetargetgeneandrecombinationeventsbetweenthetargetlocusandlinkedflankingmarkers.Backgroundselection:MarkersunlinkedtothetargetlocuscanbeusedtoselectBCprogenywiththegreatestproportionofrecurrentparentgenome.,34,(1)前景选择（正选择）保证从每一回交世代选出的作为下一轮回交亲本的个体都包含目的基因。通过标记基因型的选择来控制目的座位基因型，可通过TCR值来评定效果。,35,TargetControlRate(TCR)：对任一回交后代个体，当其标记基因型为杂合的“供/受”时，其目标座位基因型也为“供/受”的概率。换句话说，一个个体在标记为期望基因型，而目标座位不是期望基因型的概率为（100-TCR）。,TargetControlRate=TCR(%)=Pr(TD/TR)|(MD/MR)100=Pr(TDMD/TRMR)/Pr(MD/MR)100TD和TR为目标座位上供体和受体的等位基因；MD和MR为标记座位上供体和受体的等位基因；Pr(X/Y)表示基因型X/Y的概率，|表示条件；,36,Itisclearlyseenthatcontrolofthetargetbyasinglemarkerisnotsatisfactoryinmostcases.Themarkermustbeascloseas1cMtothetargettokeeptheriskoflosingthetargetbelow5%overfiveBCgenerations.Evenwithasinglemarkerat1cM,theriskoflosingthetargetiscloseto10%inBC10.Forgreaterdistancesofasinglemarker,theriskbecomesrapidlytoohigh.,Targetcontrolledbysinglemarker（Hospital，2003）,单标记,37,当目标座位为两侧标记界定时，即使标记相距较远，效果也是很好的，如两侧标记分别相距10cM时，其控制效果与单标记距离在1cM的效果相近。很显然，这种结果的原因在于当有两侧标记时，要打破标记与目标的连锁需要进行双交换；而单标记情况下，只需要发生单交换。,Targetcontrolledbytwoflankingmarkers（Hospital，2003）,双标记,38,回交1回交2回交3回交6传统回交育种75.087.793.399.0标记辅助85.598.0100.0回交育种,回交育种中轮回基因组百分比,YoungandTanksley，1989，计算机模拟,(2)背景选择（负选择）加快遗传背景恢复成轮回亲本基因组的速度，以缩短育种年限.,39,指有利基因和不利基因间的连锁，使回交育种在导入有利基因的同时也带入了不利基因，常常造成性状改良后的新品种与原目标不一致.传统的回交育种中，无法鉴别目标基因附近所发生的遗传重组，只能靠碰巧来选择消除了连锁累赘的个体。大致需要100代才能基本消除连锁累赘的目的。,避免或减轻连锁累赘(linkagedrag)的问题。,40,用高密度的分子标记连锁图就有可能直接选择到在目标基因附近发生了遗传重组的个体。只要在BC1和BC2中进行标记辅助选择，即可得到含有目标基因的供体染色体片段长度不大于2cM的植株，从而只需两个回交世代就可达到基本消除连锁累赘的目的。,41,传统回交育种,标记辅助回交育种,年,F1,BC1,BC2,BC3,BC20,BC100,年,0.5,1,1.5,黑色代表供体基因组；白色代表轮回基因组（Tanksleyetal.1989）,0.5,1,1.5,2,10.5,50.5,42,Target-markerdistanceonthecarrierchromosomewas2cM.Eachnon-carrierchromosomewascontrolledbythreemarkers.,43,44,Example:MarkerassistedtransferofXa21:strategy,Minghui63IRBB21(Xa21),Minghui63F1,Minghui63BC1F1,Minghui63BC2F1,BC3F1,BC3F2(Xa21),SelectionforrecombinationoneithersideoftheXa21locus,SelectionforrecombinationontheothersideoftheXa21locus,SelectionforMinghui63background,Minghui63(Xa21),SelectionforXa21homozygotes,ChenShengZhangQifa,45,46,MH63(Xa21),MH63(CK),47,数量性状的遗传基础主效基因-微效基因多个微效基因作用数量性状的分子标记辅助选择主效基因的辅助选择微效基因的辅助选择全基因组选择,第三节数量性状的标记辅助选择,48,1.主效基因的分子标记选择,性状分解;性状数据获得;分子标记定位;利用近等基因系建立精细定位群体;精细定位和基因效应验证;分子标记的验证;分子标记辅助育种.,49,50,M.WissuwaM.YanoN.Ae,MappingofQTLsforphosphorus-deficiencytoleranceinrice(OryzasativaL.)TheorApplGenet(1998)97:777783,AsuccessstoryinMASonricePiefficiency,51,M.WissuwaJ.WegnerN.AeM.Yano,SubstitutionmappingofPup1:amajorQTLincreasingphosphorusuptakeofricefromaphosphorus-deficientsoil.TheorApplGenet(2002)105:890897,52,GraphicalgenotypesfortheKasalathsegmentonchromosome12of160selectedF3individualsofsixF2families,aswellasthemeansandstandarddeviationsfortillernumbersofgenotypicclasseswithinfamilies,53,Sequencecomparisonofphosphorusuptake1(Pup1)lociandflankingregionsinthreericevarieties.ANucmeralignmentoftheunmaskedKasalathsequenceassembledfromthreeBACcloneswiththecorrespondingsequencesingenotypesNipponbare(japonicareferencegenome)and93-11(indicareferencegenome)isillustrated.,SigridHeuer,etal.ComparativesequenceanalysesofthemajorquantitativetraitlocusPhosphorusuptake1(Pup1)revealacomplexgeneticstructure.PlantBiotechnologyJournal(2009)7,pp.456471,54,ChinJH,etal.,Developmentandapplicationofgene-basedmarkersforthemajorriceQTLPhosphorusuptake1.TheorApplGenet(2010)120:10731086,55,ChinJH,etal.,Developmentandapplicationofgene-basedmarkersforthemajorriceQTLPhosphorusuptake1.TheorApplGenet(2010)120:10731086,Pup1haplotypeof58ricegenotypes.ThePup1haplotypewasdeterminedbygenomicPCRusingsevenPup1gene-basedmarkers(K41K59)andtwocloselyflankingmarkers(RM28073,Ba76H14_7154).,56,Pup1haplotypesurvey.RiceaccessionsgenotypedwiththePup1markersweregroupedaccordingtotheirpreferredcroppingsystemsandvarietalgroup(indica,japonica,aus)(a).In(b)thesamegenotypesweregroupedaccordingtotheirclassificationasmodernortraditionalvarieties.,ChinJH,etal.,Developmentandapplicationofgene-basedmarkersforthemajorriceQTLPhosphorusuptake1.TheorApplGenet(2010)120:10731086,57,58,RootelongationandreductioninshootlengthunderPdeficiencyinhydroponicsculturesolution.AsetofcontrastingPup1near-isogeniclineswiththePup1locus(NILC443,NIL6-4,andNIL14-4)andsisterlineswithoutthePup1locus(NIL6-3,NIL14-6)weregrowninhydropnicssolutionwith0and100lMP,respectively,59,ChinJH,etal.,DevelopingRicewithHighYieldunderPhosphorusDeficiency:Pup1SequencetoApplication.PlantPhysiology,2011,Vol.156,pp.12021216,Pup1genemodelsandpositionsofmarkers.,60,Sequencecomparisonsofmarkeramplicons,61,PCRampliconsofPup1gene-specificmarkersinrepresentativericevarieties.,62,Pup1haplotypeindiversericegenotypes,63,Pup1coremarkers,64,FieldevaluationandselectionofIR64-Pup1andIR74-Pup1breedinglines,65,数量性状的标记辅助选择意义很大，但困难也很大，目前多局限在理论研究上，实际成功例子很少：（1）只有少数数量性状的全部QTL被精细定位出来，无法对数量性状进行全面的标记辅助选择；（2）育种过程中同时对许多目标QTL进行选择相当困难；（3）上位性效应对选择效果影响很大，常使选择达不到预期目的。,2.微效基因的辅助选择,66,1）基因型选择：即对每个目标QTL利用其两侧相邻标记或单个紧密连锁的标记进行选择，其原理和方法与质量性状相似。2）关键问题：Theuncertaintyofthetruetargetlocation，i.e.theexactlocationofthetargetisoftennotknown.,67,3）计算TCR值：假定QTL检测已经完成，QTL的期望（最可能）位置已经确定，同时已给出置信区间，而真实的目标是位于期望QTL附近的某一点，遵循一个均值为0、方差为置信区间长度的正态分布。对每一个可能的真实位置都计算TCR值，然后平均（HospitalandCharcosset，1997）。,68,4）不完全基因型选择，即对那些已定位的QTL进行基因型选择。在选择目标QTL的同时，同样也可以利用分子标记进行背景选择，加快遗传背景恢复成轮回亲本的速度。,69,分子标记在水稻产量遗传改良上的应用,通过分子技术在野生稻中，发现两个增产QTL，每个QTL具有比对照品种增产18%的效应（Xiao等，1995）。已将其中的一个QTL转移到栽培稻中，用这种携带野生稻增产QTL水稻配组的杂交稻，2003年在浏阳作双季晚稻百亩示范，亩产700公斤。,70,71,J23A/Q611,72,Shenetal.(2005)UsedBNL1521andBNL2961markersforQTLfs21selection,andBNL1122forQTLfs22selection.Thefiberstrengthoftheselectedindividualplantsshowedthegreatimprovement.,分子标记在棉花纤维强度遗传改良上的应用,73,NowitmaybefeasibletoaskhowbesttousemarkerstomakegeneticgainsdiscoveryofSNPsandotherhighdensitypolymorphicmarkershighthroughputgenotypinghasreducedcostscostsforphenotypingcontinuetoincreaseMaybepossibletoselectanarrayofmarkerswithoutestablishingsignificantassociationswithtraits,3全基因组选择(Genome-wideselection),74,Genome-wideselection,Inatrainingpopulation(bothgenotypicandphenotypicdataavailable),fitalargenumberofmarkersasrandomeffectsinalinearmodeltoestimateallgeneticeffectssimultaneouslyforaquantitativetrait.Theaimistocapturealloftheadditivegeneticvarianceduetoalleleswithbothlargeandsmalleffectsonthetrait.Inabreedingpopulation(onlygenotypicdataavailable),useestimatesofmarkereffectstopredictbreedingvaluesandselectindividualswiththebestGEBVs(genomicestimatedbreedingvalues).,75,Genome-wideselectionsimulationstudy,Simulationstudiesoftestcrossperformanceofdoublehaploidsinmaizeshowedgenome-wideselectionconsistentlymoreeffectivethanmarker-assistedrecurrentselection(MARS)GreatestadvantageforcomplextraitscontrolledbymanyQTLwithlowheritabilityMinimumnumberofmarkers=128to256Mostbeneficialforrecurrentselection(lesseffectiveforchoosingparentsofbreedingpopulationsorselectionofsingle-crosses),BernardoandYu,2007,76,GenomicSelectionMethods,RelativelynewapproachmanyquestionsremainCompositionoftrainingpopulationSelectioncriteriaChoiceofstatisticalmodelsLeastsquares(最小二乘法),Ridgeregression(回归),Bayesian,LassoSeereviewbyHeffneretal.,2009:Xu,2).thecostsoftissuecollectionandstorage;3).themethodofextractionofDNA/proteinforanalysis;4).theextenttowhichextractedDNAisusedforanumberofdifferentmarkers;5).theanalyticalmethodused;6).therateofthroughputoftheanalyticalsystem;7).thedegreetowhichtheanalysisutilizesmultiplexingandmulti-pooling;8).theeaseofscoringtheallelesaftertheanalysis;9).theunitcostsofthelaborforthedifferentoperations(Dreheretal.2003);10).thecapitalcostofthelaboratoryequipmentrequired;and11).theutilizationoftheequipmentrequired(thatis,theoverallthroughputofthelaboratory).,88,2.EconomicassessmentofMAS,Comparisonofcostsofmolecularmarkerwithphe