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($    (Y2$ Y!  C<< c g "  zoB.` hp x (#XB (#XXXzoXSXXX@<<<<x@    1  A  2 `*Times New RomanTT3|x<6X9`("Courier NewTTd6X@DQ@<6X9`("Courier NewTTXXx6X@DQX@\  `*Times New RomanTTC\  P6QP2 `*Times New RomanTTXX P7XP(>Y$HP LaserJet 4,,,,,,0uv(+00U Y!  n=<dd=B.` hp x (#XBXSXXXXXS  _ResistancetosoybeansuddendeathsyndromeandrootcolonizationbyFusariumsolanif. % sp.glycine. innearisogeneiclines.  l G V.N.Njiti,T.W.Doubler,R.J.Suttner,L.E.Gray,P.T.Gibson,andD.A.Lightfoot*   T.W.Doubler,currentaddressICISeeds,Slater,IO,V.N.Njiti,R.J.Suttner,P.T.GibsonandD.A.Lightfoot.Dept.ofPlantandSoilScience,MolecularScienceProgram,SouthernIllinoisUniv.,Carbondale,IL62091;andL.E.Gray,USDA,ARS,Dept.ofCropScience,UniversityofIllinois,Urbana,IL61801.Authorsnamesarenecessarytoreportfactuallyontheavailabledata;however,theUSDAneitherguaranteesnorwarrantsthestandardoftheproduct,andtheuseofthenamebyUSDAimpliesnoapprovaloftheproducttotheexclusionofothersthatmayalsobesuitable.Correspondingauthor.EmailGA4082@SIU.EDU.򀀀 2!  Abbreviations:C96,Coralocationin1996;DI,diseaseincidence;DS,diseaseseverity;DX,diseaseindex;IF,infectionfrequency;IS,infectionseverity;QTL,quantitativetraitloci;NIL,nearisogeneicline;RIL,recombinantinbredline;R95,Ridgwaylocationin1995;R96,Ridgwaylocationin1996;SDS,suddendeathsyndrome.U95,Ullinlocationin1995;VR94,VillaRidgelocationin1994;  ,p$   򀀀ABSTRACT Onegenomicregioninsoybean(Glycinemax(L.)Merr.)cultivar'Essex'andthreeincultivar'Forrest'underliefieldresistanceoftheirRILprogenytosuddendeathsyndrome(SDS)leafscorch.RootinfectionbyFusariumsolani(Mart.)Sacc.f.sp.glycineprecedestheleafscorchcausedbySDS.ForrestshowsratereducingresistancetobothrootcolonizationbyF.solaniandleafscorchwhereasEssexdoesnot.OurobjectivewastodeterminewhethergenomicregionsthatunderlieresistancetotheSDSleafscorchalsocausedresistancetorootcolonizationbyF.solaniusingnearisogeneiclines(NILs).TheNILswerederivedfromindividualplantsselectedfromwithinonerecombinantinbredline(ExF34).ExF34washeterogenouswithinregionsoflinkagegroupC2andG,eachofwhichencompassedquantitativetraitloci(QTL)forresistancetoSDS.UsingDNAmarkers,fourgenotypicclassescouldbeidentified.TheQTLeffectswerecomparedwithtwoSDSdiseaseparameters,leafscorchmeasuredasSDSdiseaseindex(DX)attheR6.5growthstageandrootcolonizationbyF.solanimeasuredasinfectionseverity(IS)attheR5.5andR8growthstages.TheForrestalleleofthegenomicregiononlinkagegroupGwasconsistentlyassociatedwithdecreasedDX(P<0.05to0.0004)andIS(P<0.05to0.0017).TheEssexalleleofthegenomicregiononlinkagegroupC2causedadecreaseinDX(P>0.05)andISinthesusceptiblepairofthefourNILclasesatR5.5butnotR8.Therefore,theQTLonlinkagegroupGandC2conferseparatecomponentsofresistancetoSDSthataresuitableforselectioninaresistancegenepyramid.ǀManyimportanttraitsinagricultureshowcontinuousvariationininheritancestudies.Phenotypicvariationiscausedbythesegregationofindependentpolygenesofsmalleffect(Pattersonetal.,1990).Thesepolygenescanactadditivelyonasinglecomponentofatraitorindependentlyondistinctcomponentsofasingletrait.Polygenescanbedetectedandmappedwithin1020cMintervalsasquantitativetraitloci,inintercrossderivedpopulations,particularlywithDNAmarkers(Darvasietal.,1995).Localizationto10cMintervalsissufficientforsomeaspectsoftheirstudyandmanipulation.̀IdentificationofthecomponentsofcomplextraitscanbedifficulttoachieveinF2orRILpopulationsbecauseofthesegregationofseveralpolygenessimultaneouslyaffectingthesametrait(Hnetkovskyetal.,1996;Mansuretal.,1996).However,analysisofnearisogeniclinescanallowtheaccuratedeterminationoftheeffectofsegregationofspecificgenomicregionsoncomplextraitsandcomponentsofcomplextraitsinahomogeneousgeneticbackground.Suchisogeniclinescanbedevelopedbyanumberofmethodsincluding;substitutionmappinginBC2F1lines(Pattersonetal.,1990);mappinginnearisogeniclines(Bentolilaetal.,1991);mappinginadvancedintercrosslines(Darvasietal.,1995);mappinginrecombinantbackcrossinbredlines(EshedandZamir,1995);andmappinginheterogeneousrecombinantinbredderivednearisogeniclinepopulations(Haleyetal.,1994).EachoftheserelatedmethodsinvolvesthestudyofapolymorphicgenomicregioncontainingaQTLwithin1040cMintervalusingmolecularmarkers.Analysisoftheeffectofallelesofthegenomicregiononphenotypeandcomponentsofthephenotypearethenmadeinanotherwisehomogeneousgeneticbackground.̀Severeyieldlossesoccurinsoybean(Glycinemax(L.)Merr.)duetosuddendeathsyndrome(Wratheretal.,1995;1996).SDSmaybecausedbythefungusFusariumsolani(Mart.)Sacc.f.sp.phaseoli(ODonnellandGray,1995;Rupe,1989;Royetal.,1989;Achenbachetal.,1996).PathologydataareequivocalonwhetherF.solanif.sp.phaseoliiscongenicwithF.solanif.sp.glycinesform.nov.(Gray,1991;Roy,1997;Gray,unpublished)butgeneticevidenceindicatesverycloserelatednessofthefungiwithDNAsequencevariationlessthanthatexpectedforseparatespeciesoffungi(ODonnellandGray,1995,Achenbachetal.,1996).TheSDSassociatedF.solaniTypeB(Roy,1997)isrelatedtoneitherF.solanif.sp.phaseolinorf.sp.glycinesandisnotassociatedwithpathogenicity(Achenbachetal.,1996)orhostplantresistanceinsouthernIllinois(Njitietal.,1997)...WhilstF.solanif.sp.glycine(hereafterF.solani)infectsandcolonizesonlytherootsandthecrown,thediseaseisoftenrecognizedandquantifiedbyitsfoliarscorch(Gibsonetal.,1994).FieldresistancetoSDSinadaptedsoybeansisbroad,incompleteandquantitative(Gibsonetal.,1994;Hnetkovskyetal.,1996;Njitietal.,1996,Njitietal.,1997).ResistancetothefoliarscorchofSDSappearstobepartlyderivedfromaForrestalleleofaregionoflinkagegroupGandanEssexalleleofaregionoflinkagegroupC2(Changetal.,1996).Forrest,butnotEssex,hasbeenshowntobepartiallyresistanttocolonizationofthetaprootbyF.solani(Njitietal.,1997).However,EssexissomewhatmoreresistanttothedevelopmentoffoliarSDSsymptomsthanothersusceptiblecultivars(Gibsonetal.,1994;Njitietal.,1997).̀Inthisreport,wefirstaimedtoshowifgenomicregionsthatunderlayresistancetoSDSintheprimaryRILpopulationcouldalsocontrolresistancetoSDSinasecondary,butderived,NILpopulationindifferentenvironments.Second,weaimedtodeterminewhethertheEssexalleleoftheC2genomicregionandtheForrestalleleoftheGgenomicregionreduceSDSfoliarsymptomsbyreducingrootcolonizationbyF.solaniorbyincreasingresistancetofoliarscorchdevelopment. @..MATERIALSANDMETHODS PlantMaterial ̀Thecrossof'Essex'(SmithandCamper,1973)by'Forrest'(HartwigandEpps,1973)(ExF)wasmadeandanF5derivedpopulationof100RILsgenerated.DuringthestudiesdescribedhereintheRILswereadvancedtotheF5:13generationfromneverlessthan300plantspergeneration.EssexissusceptibletoSDS,whileForrestisresistanttoSDS(Gibsonetal.,1994;Hnetkovskyetal.,1996).ThepotentialfornaturalselectionforresistancetoSDSwasminimizedbyallinbreedingbeingcarriedoutinfieldswithnohistoryofSDSsymptoms...ResidualheterogeneitywithinRILs,theoretically6.25%,wasabout8%asdetectedbycodominantmarkersattheF5:9(Hnetkovskyetal.,1996;Changetal.,1996).FortheRFLPmarkerBng122DonlinkagegroupG,sixRILsthatwereheterogeneousweredetected.OneoftheseRILs,ExF34wasalsoheterogeneousforK455DonlinkagegroupC2.ThereforeExF34washeterogeneousforthemajorQTLforresistancetoSDSonlinkagegroupsGandC2(Changetal.,1996).TheExF34RILwashomogeneousfortheForrestderivedresistancealleleforthethirdmajorQTLforSDSresistancefoundonlinkagegroupN(Hnetkovskyetal.,1996).TheRILwashomogeneousfortheEssexsusceptibilityalleleforthefourthmajorQTLforSDSresistance(QTL2G)alsofoundonlinkagegroupG(Changetal.,1996).Therefore,theExF34RILandderivedNILswerepredictedtobemoderatelySDSsusceptibleandsegregateforpartialSDSresistancepossiblyshowingbigenicinheritance.ModeratetohighSDSscoresimprovetheabilityofdistinguishingsignificantcultivardifferencesbyfoliarsymptomrating(Hnetkovskyetal.,1996)butarenotnecessaryfordistinguishingcultivarsbyrootinfectionrating(Njitietal.,1997)...TheExF34RILwasusedtoextractNILpopulationsof40individualsattheF5:9generationbyseedtorowdescent.TheNILswereexpectedtocontrastfortheheterogeneosregionsencompassingthetwoQTLforSDSresistancebutotherwisebealmostcompletelyhomogeneous.SublinepopulationsfromtheF5:9:11toF5:9:13wereusedtotestforresistancetosoybeanSDSfoliarsymptomsandsoybeanrootinfectionbyF.solaniinthefield. AssaysofResistancetoSDS. ..AllfieldsfortheseassayswereselectedbasedonahistoryofuniformSDSleafsymptomsandalllocationswereinsouthernIllinois.Theexperimentswereconductedinthreephases.Thefirstphasein1994and1995wasdesignedtoaccuratelydetermineSDSleafsymptoms,thesecondandthirdphasesin1996weredesignedtodeterminetheextentofrootcolonizationbyF.solaniandaddtotheSDSleafsymptomdata.Thefirstphaseinvolvedall40NILS,EssexandForrestinarandomizedcompleteblockdesignintworowplotswithtworeplications.TheexperimentwasconductedinthreesouthernIllinoislocationswhereSDSleafsymptomsweresevere(Essexdiseaseindex(DX)>12)ormoderate(EssexDX>3;Table1),atVillaRidgein1994(VR94),Ridgwayin1995(R95)andUllinin1995(U95).Intheseandthefieldexperimentsdescribedbelowrowswere0.75mwideand3.0mlong,withabout17plants/m.̀ThesecondphasewasatonelocationwhereSDSleafsymptomshadbeensevereinpreviousyears.TwentysixsoybeanNILs,EssexandForrestwereplantedatRidgwayin1996(R96)inarandomizedcompleteblockdesignintworowplotswithtworeplications.The26NILswereselectedforconsistentlyhighorlowDXscoresduring1994and1995.RootcolonizationbyF.solaniatR96wasassayedintaprootscollectedatharvestmaturity(R8,seedescriptionbelow).̀Inthethirdphase,fourNILsrepresentingthefourgenotypicclassesdetectedbyDNAmarkers(Table1)werestudiedmorecloselyforresistancetorootcolonizationbyF.solani.ThefourNILswereselectedbasedonthepresenceorabsenceoftheSDSresistanceQTLonlinkagegroupGandlinkagegroupC2.TheNILsselectedincluded:346(G+,C2+),345(G+,C2),3429(G,C2+),and3425(G,C2).In1996theNILswereplantedatRidgwayandCora(C96),Illinois,inarandomizedcompleteblockdesignexperimentsusingsixrowplotswiththreereplications.TheoutertworowswereusedfordestructivesamplingandthemiddletworowswereusedforplotevaluationofSDSdiseaseincidenceandseverity.Theinterveningrowspreventeddisturbancebyrootsamplingoftherowsusedforleafsymptoms...Thesoiltypeswere:Bonniesiltloam,finesiltymixed,acid,mesicTypicFluvaquentsatRidgway(R95andR96wereneighboringfieldswiththesamesoiltype);BelknapSiltLoam,coarsesilty,mixed,acid,mesicTypicFluquaventsatVillaRidge;Pattonsiltyclayloam,finesiltymixed,mesic,TypicHaplaquollsatUllin;andGorhamsiltclayloam,finesilty,mixed,mesicFluvaquenticHaplaquollsatCora.Experimentswereplantedbetween15Mayand15Juneeachyear. SDSDiseaseScoring ̀ThemethodsforSDSleafsymptomscoringhavebeendescribedindetail(Matthewsetal.,1991;Hnetkovskyetal.,1996,Njitietal.,1996).Fromtheonsetofdisease,plotswereratedweeklyfordiseaseincidence(DI;0100%),diseaseseverity(DS;19),andthereproductivestage(R;R0R8;Fehretal.,1971).ThelastscorebeforeandthefirstscoreafterR6(fullpod)wereusedtostandardizeDIandDStotheR6stage.Thediseaseindex(DX;0100)wascalculatedasDI*DS/9. RootcolonizationbyF.solani. ..RootsamplesforthefourselectedNILsandparentswerecollectedattheR5.5(midpodfill)developmentalstage(Fehretal.,1971,Gibsonetal.,1994)fromRidgwayandCorain1996.AnothersetofrootsampleswascollectedattheR8(harvestmaturity)stagefor26NILsfromRidgway.FollowingNjitietal.,(1996)fiveplantsperplot(10or15plantspergenotypeperlocation)wererandomlyharvested,recoveringatleast15cmofthetaproot.Rootsfromsampledplantsweretransportedonicetothelaboratorywheretheywerestoredat4oC(1to7d)untiltheywereprocessedforF.solaniisolationandquantification...BasedonthefindingbyRupe(1989)thattheepidermaltissueofthetaproothadthehighestfrequencyofF.solanirecovery,theisolationinthisstudywaslimitedtothetaproot.Arestrictivemedium(Njitietal.,1997)thatlimitedfungalgrowthandrestrictedbacterialgrowthwasused.PlantsamplesweretakenattheR5.5andR8toaidinprocessingsamplesinatimelymanner.WehaveshowndifferencesinISbetweengenotypesencompassedthesegrowthstages.FromtheR5.5samples(Essex,ForrestandfourNILs),fifteen1cmrootsegmentsperplantwereculturedfromeachof15plants.Thisinvolved450rootsegmentsperNILandparentor2,700segmentsintotal.FromtheR8samples(EssexForrestand26NILs)five1cmrootsegmentsperplantwereculturedfromeachof10plants.Thiswas50rootsegmentsperNIL,about350pergenotypicclassor1,300NILsegmentsand100parentsegmentsintotal.Segmentswereplacedonthemediumandtheplateswereincubatedatroomtemperaturefor14days.Purecoloniesofallslowgrowingfungifromeachsegmentweretransferredontofreshmedium,consistingof24g/lofPDAandagar,10ml/lof10%NP10and0.064g/loftetracycline.Theplateswereallowedtoincubateatroomtemperaturefor14days...ThepercentageofplantsyieldingblueF.solanifromatleastonesegmentwasdeterminedasinfectionfrequency(IF).ThepercentageofsegmentsyieldingblueF.solanifromallsampledplantsofeachplotweredeterminedasinfectionseverity(IS).ThesegmentsyieldingothertypesofF.solani(typeB)werealsonoted. DNAMarkers ̀BacterialstrainscontainingclonedsoybeanPstIgenomicDNAinsertswereobtainedfromDr.R.Shoemaker,USDAARS,Ames,Iowa(ShoemakerandSpecht,1995).PolymorphiclociweredetectedandscreenedasdescribedbyChangetal.(1996).PolymorphicRFLPlociwerereferredtoafterCreganetal.(1995).Microsatellitemarkersweregeneratedandscoredby6%denaturingPAGEexactlyasdescribedbyAkkayaetal.(1995).ForRAPDmarkerstheamplificationreactionsweredoneafterWilliamsetal.(1990).DNAwasamplifiedasdescribedpreviously(Hnetkovskyetal.,1996;Changetal.,1996).NomenclatureoflinkagegroupsfollowedShoemakerandSpecht(1995)asmodifiedbyMansuretal.(1996). Delimitingthegenomicregionsunderlyingresistance ..Forlinemeancomparisons,thedataweresubjectedtoanalysisofvariance(ANOVA;SASInstituteInc.,Cary,NC),withmeanseparationbyLSD(GomezandGomez,1984).GraphswereconstructedbyQuattroProversion5.0(NovellInc.,Orem,Utah).TodeterminetheeffectofgenomicregionsassociatedwithresistancetoSDS,theNILswereclassifiedasEssextypeorForresttypeforeachpolymorphicDNAmarker.MarkerswerecomparedwithSDSdiseasescoresbyANOVA.TheprobabilityofassociationofeachmarkerwitheachtraitwasdeterminedandasignificantassociationwasdeclaredifP0.05(unlessnotedotherwiseinthetext)sincethedetectionoffalseassociationsisreducedinisogeniclines(LanderandBotstein,1989;Pattersonetal.,1990). @..RESULTSANDDISCUSSION ..SufficientSDSleafsymptomstodistinguishresistantandsusceptiblecultivarswereobtainedfromonlythreeofthefivelocationsplanted.SDSDXlinemeanswere7.6atV94,23.6atR95and5.1atU95.TheEssexcultivarDXmeanswere12.9atV94,34.9atR95and3.4atU95(Table1).SDSDXheritabilitieswere71%atV94,69%atR95and63%atU95.However,atR96andC96SDSDXlinemeansandEssexcultivarDXmeansweretoolow(<2%)todistinguishresistantandsusceptiblegenotypesorcalculateheritabilities.In1996,theabscenceofleafsymptomswasduetolateseasondroughtinterferingwithleafsymptomdevelopment.̀Incontrast,theR96andC96locationsprovideddataforF.solaniISthatcoulddistinguishbetweenresistantandsusceptiblecultivars.TheheritabilitiesofISwere70%atR96atR8,78%atR96atR5.5and76%atC96atR5.5.AtR96attheR8samplingdatetheISlinemean(n=4)was25.5%atR96andthemeanforjustEssexwas49.0%(Table1).AtC96attheR5.5samplingdatetheISlinemean(n=26)was37.2%andthemeanforjustEssexwasabout56%(Table2).Inapreviousstudy,themeanISforEssexatR8wasabout43%atR95andatU95(Njitietal.,1997).Therefore,infectionwasasseverein1996asatenvironmentsin1995wheresignificantSDSleafsymptomsoccured.ThedatareinforcetheconclusionofNjitietal.,(1997)thatSDSDXisanindirectandoftenineffectiveindicatorofboththeprogressionofinfectionandthedegreeofcultivarresistance. PolymorphismandLinkage. ̀Thepresentreportsummarizestheanalyseswith5polymorphicloci.TwolociwereidentifiedbyRFLPmarkers,twolocibyRAPDbandsandonelocusbyamicrosatellitemarker.Markersthatwerenotpolymorphicinthenearisogeniclinepopulationincludedthe74discretelocipreviouslymappedtotwentythreelinkagegroupsencompassing1,722to2,262cMofthesoybeangenome(Changetal.,1997).̀TheExF34derivednearisogeneiclinepopulationwasshowntobepolymorphicintworegions.Aheterogeneousregionofabout10cMencompassingSATT163(P.Cregan,personalcommunication),Bng122DandOI03512butnotextendingtoOG13490(Changetal.,1997;Prabhuetal.,1997)segregatedwithinthispopulation.Asecondheterogeneousregionofabout20cMencompassingK455D,OP13200andOO05250butnotextendingtoOO05450(Changetal.,1997)segregatedwithinthispopulation. GenomicregionsAssociatedWithSDSDiseaseIndexandInfectionSeverityintheNILPopulation.̀ TodetectgenomicregionsconditioningpartialresistancetoSDS,wetestedassociationsbetweenF5:9:13genotypicclassesandmeanSDSDXandSDSISphenotypes.ThechromosomalregiononlinkagegroupGidentifiedbyBng122D1andOI03512,previouslyshowntobestronglyassociated(P0.0001,R2=18%)withmeanDXintheRILpopulation(Changetal.,1996),wassignificantlyassociatedwithbothmeanDX(P=0.0004,R2=38%)andIS(P=0.0017,R2=47%)intheNILpopulation(Table1,Fig.1&Fig.2).InboththeRILandNILpopulationsForrestprovidedthebeneficialalleleforresistancetoSDS.HeritabilityofISatR96(R8)was70%andheritabilityofDXwasabove63%inallenvironmentssothepercentageofvariationexplainedbyQTL1Gwashigherthanthe3035%expectedfromthebigenicinheritancemodelandtraitheritabilities...However,thechromosomalregiononlinkagegroupC2thatwasshowntobestronglyassociatedwithmeanDI(P=0.0004,R2=14%)butonlymoderatelywithmeanDX(P0.001,R2=12%)intheRILpopulation(Changetal.,1996)wasnotassociatedwithmeanDX(P=0.06,R2=12%)orISatR96(P=0.52)intheNILpopulation...Although,theregionofC2didnothaveasignificantassociationwithSDSDXintheNILpopulation,Table1indicatesthatdiseasemeanswerereducedwhenevertheEssexallelewaspresentinthisregion.Inaddition,inboththeRILandNILpopulationsEssexprovidedthebeneficialalleleforresistancetoSDS.TheC2andGgenomicregionswereadditive(P<0.001)anddidnotinteractamongthe26NILsor100RILs(P>0.05)asjudgedbytwowayanova.Theyjointlyexplained21%ofthevariationinDXintheRILpopulationand50%ofthevariationinISandDXintheNILpopulation...ThelackofassociationintheC2regionwithDXwithintheNILpopulationascomparedtotheRILpopulationcouldbeareflectionofseveralphenomena.TheQTLeffectmayhavebeenexaggeratedintheRILpopulation.ThesmallsamplesizeintheNILpopulation(comparedtotheRILpopulation)andvariabilityofISandDXscoresmayreducetheQTLsignificance.Epistaticinteraction(s)affectingtheQTLmayhaveoccuredwithintheRILpopulationthatwereexcludedfromtheNILpopulation.ThesegregationoftheGchromosomalregion(thatstronglyaffectsDX)mayhaveincreasedthevariabilitywithinthepopulationandreducedthesignificanceofthereductioninDXcausedbytheQTLC2.TheR5.5studiescanexcludesomeofthesepossibilities. GenomicregionsAssociatedWithSDSDiseaseIndexandInfectionSeverityinthefourNILswithcontrastinggenotypes. ..TheQTLeffectswerecomparedwithtwoSDSdiseaseparameters,leafscorchmeasuredasSDSdiseaseindex(DX)androotcolonizationbyF.solanimeasuredasinfectionseverity(IS).ThefourNILsthatwereintensivelysampledattheR5.5growthstageshowedaconsistent(P0.05)effectoftheGchromosomalregionattwolocations.TheregionidentifiedonlinkagegroupGreportedpreviously(Changetal.,1996;1997)wassignificantlyassociatedwithlowmeanIS(<22.3%)andDX(<4.4)inbothisolinescontainingtheForrestallele.IncontrastIS(>40.6%)washighandDX(>12.1)wasmoderateinbothisolinescontainingtheEssexallele(Table2,Fig.1andFig.2).Therefore,ratereducingresistancetosoybeanrootinfectionbyF.solaniandthedevelopmentofleafsymptomswascontrolledbytheGregioninthetwoexperimentalphases...WhenconsideringtheQTLonlinkagegroupC2theresultsobtainedfromthefourNILssampledintensivelyattwolocationsattheR5.5wasnotconsistentwiththeNILpopulationdataattheR8growthstage.TheregiononlinkagegroupC2waseffectiveinsignificantlyreducingISandDXintheSDSsusceptibleisolinepair(Table2).ComparingisolinesExF3429andExF3425,ISwassignificantlylowerfortheNILcarryingtheEssexallelethantheNILcarryingtheForrestalleleinthisregioninthemeanoftwoenvironments(Table2)butnotindividualenvironments(Figure1).Inaddition,theISwasnotsignificantlyhigherin3429thaninthe345NILcontainingtheSDSresistantQTLonlinkagegroupGbutnotlinkagegroupC2...WhetherthedifferencebetweenthesecondphasepopulationstudyandthethirdphaseselectedNILstudyissignificantisunclearsincetheLSDswerelarge(Table2).IftheQTLwerecomplexlocispreadoverseveralcentimorgansandtheNILpopulationcontainedsomerecombinantsthedifferencemighthaveageneticbasis.InfactnorecombinantsweredetectedbetweenOI03512andBng122DbutthreebetweenK455,OP13200andOO05250(Doubleretal.,1997).TheeffectoftheGQTLdoesappeartomasktheeffectoftheC2QTLsincelines346and355donotsignificantlydifferforISorDX.However,fixationoftheGalleledidnotimprovetheassociationsforC2attheR8growthstageintheNILpopulation.TheregionofchromosomeC2mayreduceDXbyepistaticinteractionswithasecondlocuswithintheRILpopulationthatisexcludedfromtheNILpopulationbutnottheselectedisolines.However,nosignificant(P<0.005)interactinglocuswasidentifiedbyanyoftheexistingDNAmarkersintheRILpopulation(Changetal.,1997)asdeterminedbyEpistat(Chaseetal.,1997).Therefore,mostlikelythedifferencereflectsexperimentalvariability.WeconcludethatwhiletheregionsofGstronglyaffectsDXandIStheregionofC2affectsDXandIStoamuchlesserdegree.ItshouldbenotedthislocuscanalsoreduceSCNindexofparasitismwithinselectedextremepopulations(Chang,1995).TodeterminewethertheQTLonC2wasidentifiedasanSDSresistanceQTLinerrororasacomplexlocuswillrequireadditionalfieldstudies. ImplicationsforbreedingresistancetoSDS ̀WehaveshownthatthetwogenomicregionsunderlyingresistancetoSDScanbeisolatedinnearisogeniclines.WithintheselinestheeffectofthegenomicregionscanbecomparedandcomponentsoftheSDSresistancetraitdistinguished.TheregionofchromosomeGclearlyaffectsrootIS,theeffectonleafDXprobablyoccursduetothelowerpathogenload.Theregionmayconferresistancetoinfectionorcolonizationbyrecognitionofthepathogen.Ifso,thismayrepresentadefeatedrecognitionfactorthatisnolongerabletoconfercompletehypersensitiveresistance.Thelackofasexualstage(Achenbachetal.,1996),broadhostrange(Gray,1991)andprofligateproductionofphytotoxins(Jinetal.,1996)providecircumstantialevidencethatF.solanihasevolvedmechanismscapableofdefeatinghypersensitiveresistanceinmanyplantspecies.ResistancetoatranslocatedtoxinproducedbyF.solani(Jinetal.,1996)doesnotappeartounderlietheresistanceintheseNILs(G.Hartmanpers.comm.).However,F.solaniproducesmanyphytotoxinsthathavenotbeentestedagainstthisgenomicregion...IncontrasttogenomicregionG,thegenomicregionC2mayreduceDXmorethanIS,suggestingadifferentmechanismalthoughitshouldbenotedthatthesedifferencesweremarginallysignificantandnotconsistent.WhethertheactionofC2reflectsacomplexlocuswithseveralinteractinggenesforSDSresponseorwasidentifiedbyerrorintheRILpopulationisnotyetclear.ComplexQTLexist(Mansuretal.,1996;Chaseetal.,1997)andmayunderlietheadvantageofelitecultivarsselectedbysoybeanbreedersoverelitepopulationsselectedbyDNAmarkers.IneithercasetheresistancefromEssexongenomicregionC2canbecombinedwiththeresistancefromForrestongenomicregionGandmayproducesoybeancultivarswithimprovedSDSresistance(Hnetkovskyetal.,1996;Schmidtetal.,1997).Identificationofotherlociwithdistinctmechanismsofresistanceandtheconstructionofnovelgenepyramidsshouldbepossiblebythemethodwehavedemonstrated. 򀀀Acknowledgments ParticularthankstoM.SchmidtandJ.KleinforexcellentmanagementofthefieldprograminsouthernIllinois.ThankstoalltheworkersontheSDSfieldteamatSouthernIllinoisUniversityatCarbondalefrom19941996.WethankDr.O.MyersJr.forcriticalreadingofthemanuscript.WealsowanttothankPerryCreganforSATT163andearlyreleaseofothermarkers.ThisworkwassupportedinpartbygrantsfromtheIllinoisSoybeanProgramOperatingBoardNos.93191323and94201433,andNorthCentralSoybeanProgramOperatingBoardNos.9520431and9520432.  ," p x (#,0.. ..REFERENCES D:X` hp x 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   \R'3+ \ H  X  &Xd&MCHHP X`h!$p&( +x-/MXXXO<6X9`("Courier NewTTOTable1.AssociationsbetweenthegenomicregionsoflinkagegroupsC2andGwithSDSdiseaseindex(DX)andinfectionseverity(IS)byF.solaniintheExF34NILpopulationandparents.򀀀̀DNALinkagePercentAllelicMeansParentMeansPopulationMeanTrait(MarkernGroup'PR2󀀀EssexForrestEssexForrestNILs򀀀V94DXOI0351240..G0.0014219.5+1.05.9+1.012.9+0.30.6+0.37.6+1.1R95DXOI0351240..G0.00063731.2+2.816.9+2.334.9+2.71.7+0.223.6+2.2MCHhHp x(08"$&@)+-MU95DXOI0351240..G0.0009358.7+1.72.0+0.83.4+1.20.0+0.03.4+1.1MeanDXOI03512120..G0.00043816.5+1.78.2+1.017.1+4.70.8+0.38.2+4.4C96ISOI0351226..G0.00174751.0+4.326.5+4.949.0+5.814.8+4.425.5+4.4V94DXK45540..C20.1276.1+0.78.0+0.9R95DXK45540..C20.10718.7+2.925.3+2.6U95DXK45540..C20.0692.9+0.96.1+1.3MeanDXK455120..C20.0699.1+1.413.1+1.5C96ISK45526..C20.52238.5+0.544.0+5.0򀀀'GandC2indicatetheregionoflinkagegroupsGandC2,namedafterShoemakerandSpecht(1995),thatcontainQTLforresistancetoSDS(Changetal.,1996).(DXattheR6.5growthstagewascalculatedfromthemeansofVR94,R95andU95.ISwasdeterminedinR96attheR8growthstage.    \R3'* \     &Xd&;18@ HPX ";Table2.Diseaseindexandinfectionseveritymeansforfournearisogeneiclines,EssexandForrestandpooledgenotypicclassesfrom26NILs.) h 8")򀀀..Disease..Infection..InfectionGenotype..index(DX)(..severity(IS)..frequency(IF)򀀀Selectedlines346(G+C2+)'..4.1(..15.3..70.0345(G+C2)..4.4..22.3..80.03429(GC2+)..12.1..40.6..96.63425(GC2)..20.5..60.2..95.0 LSD(0.05)..7.9..19.1..25.4 AlllinespooledwithinGenotypicclassesG+C2+..5.5..32.5..81.7G+C2..9.1..29.0..88.2GC2+..12.4..56.1..97.5GC2..15.0..57.1..93.3 LSD(0.05)..5.8..13.4..13.5 ParentsForrest(G+C2)..0.8..14.8..76.6Essex(GC2+)..17.1..49.0..100.0 LSD(0.05)..8.1..18.1..30.2 򀀀'G+indicateForrestalleleonlinkagegroupG;C2+indicateEssexalleleonlinkagegroupC2afterShoemakerandSpecht(1995).(DXwascalculatedfromthemeansofVR94,R95andU95.ISandIFweredeterminedinC96andR96attheR5.5growthstageforselectedlinesbutonlyatR96attheR8growthstageforalllinespooledwithingenotypicclasses.FigurelegendsFigure.1:F.solaniinfectionseverity(IS)atmidpodfill(R5.5)atRidgway1996(a)andatCora1996(b)amongfournearisogeneiclinesthatcontrastforSDSresistance,EssexandForrest.Barscarryingthesamecapitallettersarenotsignificantlydifferentfromeachother.GandC2indicatetheregionsoflinkagegroupsGandC2thatdifferintheseNILs.The+representthepresenceoftheresistancealleleattheregioncontainingaQTLforresistancetoSDS.TherepresentthepresenceofthesusceptibilityalleleattheregioncontainingaQTLforresistancetoSDS.ǀFig.2:MeanSDSdiseaseindex(DX)andF.solaniinfectionseverity(IS)atharvestmaturity(R8)forfourcontrastinggenotypeclassesamong26NILs.Barsofthesamediseaseparameterandcarryingthesameletterarenotsignificantlydifferent.GandC2indicatetheregionsoflinkagegroupsGandC2thatdifferintheseNILs.The+representthepresenceoftheresistancealleleattheregioncontainingaQTLforresistancetoSDS.TherepresentthepresenceofthesusceptibilityalleleattheregioncontainingaQTLforresistancetoSDS.̀..OldTable1.AssociationbetweenmarkersandtraitdataintheExF34derivednearisogeneicline(NIL)population.򀀀) hx08")Marker..K455D..OI03450n..36..28Linkagegroup..C2..G򀀀SDSdiseaseindex(DX)̀Probability..0.06..0.0004̀R2..0.09..0.38̀Means:Essex..9.11.4..16.51.7̀Forrest..13.11.5..8.21.3F.solaniinfectionseverity(IS)̀Probability..0.52..0.0017̀R2..0.02..0.47̀Means:Essex..38.56.4..51.04.3̀Forrest..44.05.0..26.54.9򀀀  April20,1997Dr.PerryB.Cregan,USDAARS,SoybeanandAlfalfaResearchLab.,Bldg.011,HH19,BARCWest,Beltsville,MD207052350.DearPerry,̀pleasefindenclosedfourcopiesofamanuscriptforCropScienceson:̀ ResistancetosoybeansuddendeathsyndromeandrootcolonizationbyFusariumsolanif.sp.glycine. innearisogeneiclines. byV.N.Njiti,T.W.Doubler,R.J.Suttner,L.E.Gray,P.T.Gibson,andD.A.Lightfoot*̀̀Aspartofthe"C7"experimentpleasesendoutforreview.Dr.StMartinorhisdelegatewithknowledgeofNjitietal.,1997wouldbeappropriate.̀Sincerely,DavidA.LightfootAssistantProfessor  PS.ThanksforthemicrosatellitesonGandD,theyarecriticaltoourwork.DoyouhaveanyonA2yetthatcouldhelpustohelpBenandLilawalktoRhg4?ThefinemappingpaperforTAGwhichyouwillcoauthorisbeingimprovedbyadditionofallthefinemapmarkers(4,SSRsand6AFLPs)thisshouldbecompletebyMay15.Iwillfaxyouafreshdraftatthattime.IfyouthinkouruseofSATT163intheabovepapermeritscoauthorshipofthismanuscriptpleaseforwardittoanothereditor.July14,1997Dr.PerryB.Cregan,USDAARS,SoybeanandAlfalfaResearchLab.,Bldg.011,HH19,BARCWest,Beltsville,MD207052350.DearPerry,̀pleasefindtherevisedcopyofamanuscriptC97781forCropScienceson:̀ ResistancetosoybeansuddendeathsyndromeandrootcolonizationbyFusariumsolanif.sp.glycine. innearisogeneiclines. byV.N.Njiti,T.W.Doubler,R.J.Suttner,L.E.Gray,P.T.Gibson,andD.A.Lightfoot*Ibroadlyagreedwithalltherefereescomments,exceptdroppingFigure1.Thefiguredoesnotduplicatetable2,ratherprovidesthereaderwithanindicationofenvironmenttoenvironmentvariabilityforIS.ThisisimportantsinceISisnotacommonlyusedmeasurebyCropScientists(althoughitshouldbe).̀Sincerely,DavidA.LightfootAssistantProfessorPS.ThefinemappingpaperforTAGwhichyouwillcoauthorisalmostreadytosubmit.Iwillfaxyouafreshdraftatthattime.Reviewer2Point1.Page12line227234.Ididnotspecifywhichallelewasbeneficial.Ithinkthisconfusedthisreviewer.Point2.Table1andlines199208.Ihaveattemptedtoclarifywhatwasdone.PreviouslydetailsfromtheMaterialsandMethodshadtoberecalledtounderstandthedata.Point3.Page18Line273.Ihaveclarifiedthetext.TherewerenorecombinantsbetweenOI03andBng122.ThatwastrueoftheotherNILpopulationwestudy.Point4.Markerscoresarenotshown,onlytheanalysis.Thisistypicalforpapersofthisgenre.Reviewer3Point1line38.Manyitis.Point2line79.asnoted.Point3line122.DXisdefined,parentincludedandexperimentaldesigncommonalitiesbroughtforwardassuggested.Point4.line123127.Notedparentsincludedandclarified.Point5.seepoint3Point6.seepoint3,5Point7.Fehr1971iscited.Point8.Line165,ournumberswerewrong,thankyou!Point9.R96datawereonlyforn=4,sotheycannotbeincludedwithvalidityifthereareanyundetectedheterogenousregionsthataffectSDS.ModifiedthetexttoremovethisambiguityPoint10.Line240241.ImeantDXexcludingIS.Thisisclarified.However,youmadeaninterestingobservationweoverlooked.Point11.Line267.AsyounotedtheeffectofC2wasonlysignificantinpooleddatafromR96andC96.Point12.Line268.C+GwassignificantlymoreSDSsusceptiblethanG+C+.Textwasmodified.point13.Line.Ihaveattemptedanexplanation.SincethetrendsareaspredictedandaspoolingdataincreasessignificanceofthosedifferenceswepredictedIstillbelieveC2isaQTLforSDSresistance.The1997datawillconfirmorrefutethatsupposition.Point14.IagreeFig2couldbedeleted.Butinpoints1113thedatainFigure1arecriticaltoevaluatingthedata.Isuggestacompromise.Point15.Tablesareclarifiedasnoted.