Genetics: Early Online, published on July 9, 2014 as 10.1534/genetics.114.167486
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UnravelingGenomicComplexityataQuantitativeDiseaseResistanceLocusinMaize
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TiffanyM.Jamann*,JesseA.Poland§,JudithM.Kolkman†,LaurieG.Smith‡,RebeccaJ.
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Nelson*†
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*Dept.ofPlantPathologyandPlant‐MicrobeBiology,CornellUniversity,Ithaca,NY14853
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§
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Agronomy,KansasStateUniversity,Manhattan,KS66506
USDA‐ARS,HardWinterWheatGeneticsResearchUnit,Manhattan,KS66502,Dept.of
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†
Dept.ofPlantBreeding,CornellUniversity,Ithaca,NY14853
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‡
SectionofCellandDevelopmentalBiology,DivisionofBiologicalSciences,Universityof
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CaliforniaatSanDiego,LaJolla,CA92093
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Copyright 2014.
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Shorttitle:Structuralvariationandpan1indiseaseresistance
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Keywords:quantitativediseaseresistance,northernleafblight,Setosphaeriaturcica,
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multiplediseaseresistance,Stewart’swilt
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Correspondingauthor:
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RebeccaNelson
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303GPlantScienceBuilding
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236TowerRoad
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Ithaca,NY,14853
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607‐254‐7475
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rjn7@cornell.edu
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ABSTRACT
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Multiplediseaseresistancehasimportantimplicationsforplantfitness,giventhe
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selectionpressurethatmanypathogensexertdirectlyonnaturalplantpopulationsand
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indirectlyviavarietyimprovementprogramsoncropplants.Evidenceofalocus
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conditioningresistancetomultiplepathogenswasfoundinbin1.06ofthemaizegenome
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withtheallelefrominbredline‘Tx303’conditioningquantitativeresistancetonorthern
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leafblight(NLB)andqualitativeresistancetoStewart’swilt.Todissectthegeneticbasisof
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resistanceinthisregionandtorefinecandidategenehypotheses,wemappedresistanceto
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thetwodiseases.Bothresistancephenotypeswerelocalizedtooverlappingregions,with
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theStewart’swiltintervalrefinedtoa95.9‐kbsegmentcontainingthreegenes,andthe
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NLBintervaltoa3.60‐Mbsegmentcontaining117genes.Regionsoftheintrogression
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showedlittletonorecombination,suggestingstructuraldifferencesbetweentheinbred
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lines‘Tx303’and‘B73’,theparentsofthefine‐mappingpopulation.Weexaminedcopy
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numbervariationacrosstheregionusingnext‐generationsequencingdataandfoundlarge
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variationinreaddepthin‘Tx303’acrosstheregionrelativetothereferencegenomeof
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inbredline‘B73’.Inthefine‐mappingregion,associationmappingforNLBimplicated
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candidategenes,includingaputativezincfingerandpan1.Wetestedmutantallelesand
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foundthatpan1isasusceptibilitygeneforNLBandStewart’swilt.Ourdatastrongly
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suggestthatstructuralvariationplaysanimportantroleinresistanceconditionedbythis
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region,andpan1,ageneconditioningsusceptibilityforNLB,mayunderlietheQTL.
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INTRODUCTION
Thegenesandlocithatinfluencehost‐pathogeninteractionsvaryinalleleeffects,
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specificities,andlinkagerelationships.Whilediseaseresistancecanbeconditionedby
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singlegeneswithlargeeffect(Bent1996;JonesandDangl2006),theemergingmodelof
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resistanceformanyplantdiseasesiscomplexinnature,withmanygenesandloci
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functioninginconcertandeachcontributingasmallproportionofthetotalphenotypic
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variation(Kumpetal.2011;Polandetal.2011;Cooketal.2012b).Eachlocushasaunique
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profile,withsomelocicontributingbroad‐spectrumprotectionagainstdiversepathogen
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speciesandstrains.Investigatingtheseintricaciesofferstheopportunitytounderstandthe
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diversewaysinwhichplantsdefendthemselvesagainstmicrobialassault.
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Correlatedresponsestomultiplediseaseshavebeenobservedinvarious
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germplasmpanels,implyingthattherearelociandgenesthatconditionbroad‐spectrum
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resistance(Rossietal.2006;Gurungetal.2009;Wisseretal.2011).Atthechromosomal
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segmentlevel,diseaseandinsectresistancelocico‐localizeinanon‐randomfashion
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(McMullenandSimcox1995;Williams2003;Wisseretal.2005)andlocihavebeen
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identifiedthatconferresistancetodiversepathogenisolatesandtaxa(Zwonitzeretal.
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2010;Chungetal.2011;Belcheretal.2012).Thereisevidencetosuggestthatgene
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clusterscanconferresistancetomorethanonedisease.Aclusterofgermin‐likeproteins
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confersresistancetoriceblastandsheathblightofrice(Manosalvaetal.2009).Similarly,
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resistancegenehomologs,whichareknowntoco‐localizewithbroad‐spectrumdisease
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resistanceloci,canclusterinthegenomeandcontributeadiversityofspecificities(Lopex
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etal.2003;Ramalingametal.2003).Pleiotropyremainsuncommoninmaize,and
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correlatedresponsesmaybeduetolinkageorpopulationstructure(Wallaceetal.2014),
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althoughinsomecases,individualgeneshavebeenshowntoconditionMDR.Forexample,
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theputativeABCtransporterLr34ofwheatprovidesprotectionagainstleafrust,stripe
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rust,andpowderymildew(Krattingeretal.2009).Patternrecognitionreceptorsareable
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todetectmolecularpatternsfromdiverseorganismstoconferdiseaseresistance(Zipfel
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andRathjen2008).
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Whileinsomecasessinglegenesorallelescommonacrossdiversegermplasm
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conferdiseaseresistance,increasingly,theroleofstructuralvariationinplantsisbeing
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exploredanditseffectsonphenotypicvariationrecognized(Spingeretal.2009;Chiaetal.
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2012;McHaleetal.2012).Asquantitativetraitloci(QTL)aresubjectedtofine‐mapping,
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somelocifractionateintomanyQTL,eachconditionedbyoneormoregenes(Steinmetzet
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al2002;StuderandDoebley2011;Johnsonetal.2012).Insomecases,thealleleeffect
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conditionedbyeachQTLissmallenoughthattheindividuallocuscannotbeidentifiedin
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isolation(Polandetal.2011;Buckleretal.2009).Inothercases,singleresistanceloci,such
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asRhg1,areconditionedbymultiplegenespresentinvaryingcopynumbersindifferent
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lines(Cooketal.2012a;Maronetal.2013).Whole‐genomestudieshaveinfactsuggested
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thatstructuralvariationisgenerallyassociatedwithdiseaseresistance:structural
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variationinplantsco‐localizeswithresistancenucleotide‐bindingproteins,receptor‐like
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proteins,anddiseaseresistanceQTL(Laietal.2012;McHaleetal.2012;Xuetal.2012).
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Theconventionalapproachofgeneticisolationandtransgeniccomplementation
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remainsthegoldstandardfordemonstratingthefunctionofagene.Thisapproach,
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however,isprovinginadequatefordealingwiththecomplexityunderlyingsomeloci,
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particularlyforstructuralvariation.Strongevidencefortheimportanceofcopynumber
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variationinexplainingtraitvariation(Cooketal.2012a;Maronetal.2013)andthe
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emergingmodelofplantdefensewithmanylocieachcontributingasmalleffectcombineto
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challengethisparadigm(Kumpetal.2011;Polandetal.2011;Cooketal.2012b).Thereisa
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needforanewapproachthatcantakeadvantageofwholegenomeanalyses,address
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presence/absencevariation,andexaminelociwithsmalleffects.Thisstudyrepresents
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suchanapproachandprovidesinsightsintoageneticallycomplexlocusaffectingdiverse
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traits.
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Inmaize,chromosomalbin1.06hasbeenidentifiedasakeylocusforstabilizing
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yieldunderadverseconditions,includingbothbioticandabioticstress(Landietal.2002;
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Tuberosaetal.2002;Landietal.2010).Inadditionaltoplantarchitecturaltraitsandyield
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underabioticstress,resistancetomanydiseaseshasbeenlocalizedtobin1.06,including
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northernleafblight(NLB),Stewart’swilt,southernleafblight(SLB),commonrust,grey
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leafspot(GLS),andearandstalkrotcausedbymultiplefungi(Wisseretal.2006;Chunget
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al2010b;Zwonitzeretal.2010).InaQTLstudyoftherecombinantinbredline(RIL)
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populationKi14xB73evaluatedforthreefoliarfungaldiseases,NLB,GLS,andSLB,a33
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Mbregionspanningbins1.05and1.06wastheonlylocusidentifiedthatconferred
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resistancetoallthreediseases(Zwonitzeretal.2010).AnumberofQTLstudiesforNLB
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resistanceinmaizehaveidentifiedQTLatbin1.06,ranginginphysicalsizefromthreeto
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30Mb(Freymarketal.1993;Welzetal.1999;Wisseretal.2006;Chungetal.2010b;Van
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Esbroecketal.2010;Chungetal.2011;Polandetal.2011).Additionally,bin1.06harbors
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thedominantStewart’swiltresistancegeneSw1(Mingetal.1999).
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BothNLB,causedbythefungusSetosphaeriaturcica,andStewart’swilt,causedby
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thebacteriumPantoeastewartii,arefoliar,hemibiotrophicdiseasesimportanttomaize
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production.Bothpathogensspreadthroughthevasculartissue,causingwiltedlesionsby
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pluggingxylemvessels(JenningsandUllstrup1957;Roperetal.2011).Theimportanceof
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geneslocalizedtomaizebin1.06inresistancetobothNLBandStewart’swilthasbeen
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describedinmultiplemappingpopulations.UsingapopulationofTx303xB73
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introgressionlines(Szalmaetal.2007),Chungetal.(2010)showedthattheNLBresistance
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QTLat1.06protectsagainstfungalpenetration.
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resolutionmappingpopulationsatthislocusandevaluatedNLBandStewart’swilt
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resistanceusingasetofTx303xB73near‐isogeniclines(NILs)(Szalmaetal.2007;Chung
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etal.2010b).Fine‐mappingallowedustodissectthelinkagerelationshipbetweenthe
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major‐effectStewart’swiltQTLandtheminor‐effectNLBQTLandtoidentifycandidate
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genes.Usingassociationmapping,wefurtherrefinedthelistofcandidategenesforNLB
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resistanceandusingmutantsconfirmedaroleforthereceptor‐likekinase,pan1,inplant
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defense.Furthermore,multiplelinesofevidenceindicatedalackofgenomicstabilityatthe
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region,includingreducedrecombinationacrossportionsofthefine‐mappingregioninthe
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NILpopulationandindicatorsofcopynumbervariation.
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Toexplorethegenomiccomplexityofthisimportantregion,weconstructedhigh
MATERIALSANDMETHODS
PlantMaterials:NILsusedforfine‐mappingwerederivedfromtheTBBC3(Tx303
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xB73Backcross3)population,asetofchromosomalsegmentsubstitutionlineswith
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Tx303introgressionsinaB73background(Szalmaetal.2007;Chungetal.2010b).Chung
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etal.(2010)identifiedfamiliesTBBC3‐38andTBBC3‐39,bothwithintrogressionsin1.06,
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assignificantlymoreresistantthanB73.Selectedfamiliesdevelopedfromtheselineswere
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chosenforfine‐mapping:TBBC3‐38_19E,TBBC3‐38_15G,andTBBC3‐38_17A(Chungetal.
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2010b).ThedetailsofpopulationdevelopmentandevaluationareshowninFigureS1.
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Briefly,apopulationwasdevelopedbycrossingTBBC3‐38_19EtoB73.IntheF2generation,
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435individualswerescreenedforrecombinantswithflankingmarkerssnp_01_0042
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(180,394,924AGP_V2)andsnp_01_0005(195,557,990bpAGP_V2).Atotalof113
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recombinantplantswereidentified,butseedwasavailablefromonly100plants.Seedfrom
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15heterozygousF2individualswasadvancedtotheF3generationtoscreenforadditional
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recombinants.Apopulationof4,080F3seedswasplantedand2,929plantswerescreened
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withflankingmarkerssnp_01_0042(180,394,924AGP_V2)andsnp_01_0005(195,557,990
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bpAGP_V2).PositionsarebasedonB73genomesequencereleaseAGP_V2(Schnableetal.
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2009).IndividualF3plants(n=874)wereidentifiedasrecombinantsfromtheF3
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population.Recombinantplantswereself‐pollinatedandhomozygousrecombinants
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identified.Homozygousrecombinantswereincreasedandevaluatedfordiseaseresistance.
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Subsequently,apopulationof1,546F3plantswasscreenedfromsnp_01_0059
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(184,633,349bpAGP_V2)tosnp_01_0083(189,352,206bpAGP_V2),yieldinganadditional
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156recombinants.Homozygousplantswereidentified,self‐pollinatedandscreenedfor
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NLBandStewart’swilt(FigureS1).
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Diseasetrials
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Northernleafblight:NLBtrialswerecarriedoutattheCornellUniversityRobert
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MusgraveResearchFarminAurora,NY.Inthefine‐mappingpopulations,194,80,and146
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homozygousrecombinantswerescreenedforNLBin2010,2011,and2012,respectively.
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Linescarryingmutationsinthegenespan1andpan2weretestedinAurora,NYin2011,
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2012,and2013,andinIthaca,NYin2011.PlantswereinoculatedwithS.turcicaisolate
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StNY001(race1)usingpreviouslydescribedinoculationprocedures(Chungetal.2010a).
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Briefly,culturesofthefungusweregrownonlactosecaseinagarforthreetofourweeks
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priortoinoculationorsorghumculturing.Asporesuspensionwaspreparedbyflooding
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thecultureswith5mLsterilizeddistilledwaterandconidiaweredislodgedusingaglass
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rod.Thesporesuspensionwasfilteredthroughtwolayersofcheeseclothandadjustedtoa
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concentrationof4x103sporespermlusingahaemocytometer.Thesporesuspension
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containedafinalconcentrationof0.02%Tween20.Sorghumseedcultureswereprepared
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bysoaking900mLofsorghumgrainsin600mLdistilledwaterovernightinaone‐gallon
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clearmilkjugandautoclavedtwicefor25minutes.OnemLofunfilteredsporesuspension
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wasthenintroducedtoeachjug,whichwasthenculturedatroomtemperatureforabout
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threeweeksbeforefieldinoculationswereconducted.Jugswereshakendailytoprevent
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cakingandprovideuniforminfestation.Forfieldinoculations,0.50mLsporesuspension
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and~1.25mLsorghumgrainscolonizedbyS.turcicawereplacedintothewhorlofeach
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plantatthefive‐tosix‐leafstage.
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Diseasedleafarea(DLA)wasratedonaperrowbasisthreetimesafterfloweringat
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anintervalofseventotendaysusingapercentagescaleof0‐100withincrementsofone,
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where0indicatesaplantwithnodiseaseand100indicatesacompletelydiseasedplant.
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Areaunderthediseaseprogresscurve(AUDPC)wascalculatedasdescribedpreviously
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(Chungetal.2010a).
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Stewart’swilt:Stewart’swilttrialswereconductedattheMusgraveResearchFarm
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inAurora,NYin2010,2011,and2012forfine‐mappingand2012and2013forpan1and
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pan2mutants.Resultsfrom2013werenotincludedintheanalysisduetoflooding.Plants
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wereinoculatedwithPantoeastewartiistrainPsNY003,originallycollectedinNYin1991,
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atthefive‐tosix‐leafstage,withinoculumpreparedandamodifiedpinprickmethodused
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forinoculationsaspreviouslydescribed(Chungetal.2010a).DLAwasratedonaperrow
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basisattwotofourweeksafterinoculationonarowbasisusingapercentagescaleof0to
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100,with0beingnodiseaseand100beingcompletelydiseased.
SNPMarkerDevelopment:Themaizediversityprojectdatabase
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http://www.panzea.org(Canaranetal.2008)wasusedtolocatepolymorphismsbetween
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thetwoinbredlinesusinganumberofdatasetsincludingmarkersfromtheNAMgenetic
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map(McMullenetal.2009),HapMapV1(Goreetal.2009),andHapMapV2(Chiaetal.
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2012).Singlenucleotidepolymorphisms(SNPs)polymorphicbetweenB73andTx303
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werechosenforthisstudy.SNPmarkersusedforthefine‐mappingstudyareshownin
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TableS1.Inaddition,anIlluminaMaizeSNP50Beadchipassay(Illumina)wasconductedon
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pooledDNAfromfamiliesTBBC3‐38_05FandTBBC3‐38_19EattheDavidH.Murdock
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ResearchInstitute,(Kannapolis,NC,USA)whichincludedgenotypicinformationfor52,686
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SNPs.
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DNAExtractions:BothSigmaExNAmp(Sigma‐Aldrich)andCTABDNAextractions
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wereused.ExNAmpDNAextractionswereusedtoidentifyrecombinantsandhomozygous
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recombinants.CTABextractionswereusedforgenotypeconfirmationandbreakpoint
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analysis.ToperformExNAmpextractions,onemm2ofplanttissuewascollectedina0.2‐
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mLPCRtubeandthetubeswereplacedonice.EightμLofextractionbufferwasaddedto
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eachtube,andtubeswereincubatedat95°Cfortenminutes.Followingtheincubation,8μL
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ofdilutionbufferwasadded.TheresultingDNAwasdiluted1:100withwaterforKASPar
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(LGCGenomics,Hoddesdon,Herfordshire,UK)genotyping.CTABDNAextractionswere
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performedusingabout0.1mgoffreshtissueasdescribedpreviously(Doyleetal.1987;
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Chungetal.2010a).
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Allele‐specificPCR:WhenusingExNAmp‐extractedDNA,10μLof1:100diluted
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DNAwasdrieddownina384‐wellKASParplate(LGCGenomics)anda4‐μLreaction
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performed.ForreactionsusingCTABextractedDNA,DNAwasquantifiedand
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approximately5ng/μLofDNAwasusedperreaction.Reactionconditionswereasfollows:
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1xKASParreactionmix(LGCGenomics),0.4mMMgCl2,0.41mMcommonreverseprimer,
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and0.165μMofeachallele‐specificprimer.Standardoligonucleotideswereobtainedfrom
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IDT(Coralville,IA,USA).PCRthermocyclingparameterswereasfollows:94°Cfor15
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minutes,20cyclesof94°Cfor10seconds,57°Cfor5seconds,and72°Cfor10seconds,
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followedby26cyclesof94°Cfor10seconds,57°Cfor20seconds,and72°Cfor40seconds.
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ResultswerereadusinganAppliedBiosystems7900HT(LifeTechnologies)andanalyzed
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usingSDSv2.1(LifeTechnologies).
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Experimentaldesignandstatisticalanalysis:Arandomizedincompleteblock
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designwasusedforallfine‐mappingfieldexperiments,withthreereplicationsforNLB
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trialsandtwoforStewart’swilttrials.NILscarryingB73andTx303allelesacrossthe
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regionwereincludedineachblockaschecklines.Tworowswereplantedaroundtheedge
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oftheexperimenttoreducebordereffects.Forthebreakpointanalysis,bestlinear
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unbiasedpredictors(BLUPs)werecalculatedusingthe‘lmer’commandinthelme4
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packageinRversion2.14(RDevelopmentCoreTeam2013)whereline,year,replication
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withinyear,andblocknestedwithinreplicationwerefittedasrandomfactorsinamixed‐
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effectsmodelforNLB.Similarly,Stewart’swiltBLUPswerecalculatedusingthe‘lmer’
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commandincludinglineandyearasrandomeffects.Fine‐mappingstatisticalanalyseswere
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completedinRversion2.14(RDevelopmentCoreTeam2013)usingR/qtl(Bromanetal.
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2003).First,individualswithfewerthan11genotypedmarkersandmarkerswithfewer
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than250individualsgenotypedwereremovedfromtheanalysis.Ageneticmapwasthen
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constructedusingtheKosambimappingfunction(Kosambi1943).Singlemarker
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regressionwasconducted(Patersonetal.1990;Kumpetal.2010)usingthe‘scanone’
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functioninR/qtl.Confidenceintervalswerecalculatedonthebasisofa95%Bayescredible
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intervalusingthefunction‘bayesint’inR/qtl(Bromanetal.2003).Genotypesand
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phenotypescanbefoundinFileS1.
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Recombinationratediversity:Recombinationrateswerecalculatedusing
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genotyping‐by‐sequencingSNPsontheNAMsub‐populations,usingphasedandfully
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imputedat1‐cMresolutiongenotypes(AllZea_GBSv2.3)
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(http://panzea.org/lit/data_sets.html).GeneticmapswereconstructedusingR/qtlwith
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the“est.map”functioninR(Bromanetal.2003;RDevelopmentCoreTeam2013).
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Readdepthvariation:Illuminasingleandpaired‐endsequencingreadsaveraging
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~4xcoverageofthegenomegeneratedaspartoftheHapMapV1andV2projects(NCBI
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accessionSRA051245)wereusedforthereaddepthvariationanalysis(Chiaetal.2012).
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AlignedsequencingreadsforB73andTx303(Chiaetal.2012)weredownloadedfrom
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iPlant(Goffetal2011).SAMtoolswasusedtocountthenumberofreadsateachnucleotide
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location(Lietal.2009).APerlscriptwaswrittentodividetheintervalinto11binsofequal
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size(327,181bp)andtocompilethenumberofreadsperbin.
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Candidategeneidentification:AllB73RefGenV2(AGP_V2)filteredgenesbetween
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thetwoflankingmarkerssnp_01_0047at185,737,089bp(AGP_V2)andsnp_01_0082at
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189,336,643bp(AGP_V2)ofthenarrowedNLBfine‐mappingintervalwereconsideredas
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candidategenes.SNPsfromgenome‐widenestedassociationmappingwithabootstrap
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posteriorprobability(BPP)>0.01thatfellwithinthenarrowedfine‐mappingintervalwere
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consideredfurtherascandidates(Polandetal.2011;Chiaetal.2012).
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Associationanalysisofthe282‐linemaizediversitypanel:Associationmapping
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wasconductedfortheNLBfine‐mappinginterval.BLUPsthatincludeddesignfactors,
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floweringtime,andpopulationstructurewereusedforassociationanalysis(Wisseretal.
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2011).Amixedlinearmodel(MLM)wasimplementedusingTASSELv4(Bradburyetal.
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2007).Themarkersassayedincluded47,445IlluminaMaizeSNP50SNPs(Cooketal.
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2012b)and425,035genotyping‐by‐sequencingSNPs(Romayetal.2013),filteredto
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removesiteswith>20%missingdata(Olukoluetal.2013).Thekinship(K)matrixwas
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constructedinTASSELusinga5,000‐SNPsubsetoftheIlluminaMaizeSNP50datasetthat
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hadnomissingdata(Olukoluetal.2013).Qvalueswerecalculatedusingqvaluepackage
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(Storey2002).AssociationswithafalsediscoveryrateofFDR<0.15werenoted.
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Mutantanalysis:Mutantsinthepan1andpan2geneswereevaluatedforNLBand
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Stewart’swiltreaction.Twomutantallelesofpan1wereevaluatedinaB73background:
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onemutantgeneratedbyethylmethanesulfonatemutagenesis(pan1‐EMS)andoneline
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withaMutator1(Mu1)transposoninpan1(GallagherandSmith2000;Cartwrightetal.
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2009).Twoethylmethanesulfonateallelesofpan2wereevaluatedinaB73background:
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pan2‐Oandpan2‐3(Cartwrightetal.2009).Allpan1andpan2mutantallelesexceptpan2‐O
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canbeconsiderednullallelesbasedonthenatureofthemutationsandanalysisofPAN
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proteinaccumulationinmutants,whereaspan2‐Oisamissenseallelethatmayencodea
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partiallyfunctionalprotein(Cartwrightetal.2009).pan1mutantlineswerebackcrossedto
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B733‐5Xandpan2mutantswerebackcrossedtoB733‐7X.Formutantanalysisacomplete
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blockdesignwasused,withfivereplicationsperlocationforpan1andpan2mutants.NLB
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resultswereanalyzedwithamixedlinearmodelinJMP9.0(SAS,Cary,NC,USA),with
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genotypeasafixedeffectandreplicationnestedwithinenvironment,andenvironmentas
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randomeffects.Stewart’swiltresultswereanalyzedwithgenotypeasafixedeffectand
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replicationasarandomeffect.
RT‐PCRanalysis:TissueforRNAextractionwascollectedfrommatureleaftissue
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of(BC4F3)BC1F5plantscarryingeithertheB73(qNLB1.06B73)orTx303allele
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(qNLB1.06Tx303)attheqNLB1.06locusduringthesummerof2011and2012.RNAwas
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extractedusinganRNeasykit(Qiagen)andfirstcDNAwaspreparedfromthisRNAusinga
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RETROscriptFirstStrandcDNASynthesiskit(LifeTechnologies).PCRwascarriedout
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usingthefollowingprimersforamplificationofpan1(5’‐TCGGGATGGAGCTGGAGGAG‐3’
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and5’‐TGGACAGACGCACGGACCAC‐3’)andactinasacontrol(5’‐
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TCAGCAGGTCTTCTCTTTCTT‐3’and5’‐TCCTTCATATTTCCTTCGTTC‐3’)withQ5HotStart
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TaqPolymerase(NewEnglandBiolabs).pan1andactinPCRproductswerequantifiedfrom
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gelimagesusingNIHImageJv.1.47g.
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RESULTS
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IdentificationofMDRinbin1.06:AnumberofQTLstudieshavelocalized
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resistancetoNLBtomaizebin1.06,withvaryingresolution(TableS2).Thesestudieshave
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consistentlyimplicatedtheregionspanningfrom180to205Mb.LinescarryingaTx303
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introgressionatthisintervalwerefoundtobeassociatedwithresistancetoNLBand
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Stewart’swilt(familiesTBBC3‐38andTBBC‐39oftheTBBC3population)(Chungetal.
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2010b).Basedongeneticbackgroundandseedavailability,TBBC3‐38_19E,TBBC3‐38_15A,
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andTBBC3‐38_17Gwereselectedforfine‐mapping.TheTx303introgressioninTBBC3‐38
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spansfromss196428597(172,877,033bp)toss196518155(196,244,799bp)(Figure1).
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Anintervalof15.16Mbspanningfromsnp_01_0042(180,394,890bpAGP_V2)to
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snp_01_0005(195,557,990bpAGP_V2)wastargetedforfine‐mapping,basedonNIL
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introgressionlocationsandpreviousQTLmappingstudies(TableS2andFigure1).
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Fine‐mappingofMDRat1.06:TwomarkersflankingtheqNLB1.06region,
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snp_01_0042andsnp_01_0005,wereusedtoscreen435F2and4,475F3plants.We
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identifiedatotalof1,130recombinantsspanningthe15Mbintervalofinterest.Plants
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wereself‐pollinatedandprogenywereassayedforhomozygous(fixed)recombinants.
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Recombinantplantswereselectedforphenotypicevaluationbasedonbreakpointanalysis
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andseedavailability.Fixedrecombinantswerescreenedfordiseasephenotypeina
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randomizedincompleteblockdesignwithtwocontrolNILs,onecarryingtheB73alleleand
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onecarryingtheTx303alleleintheregionofinterest.Fixedrecombinantswerescreened
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forNLB(n=194,80,and146in2010,2011,and2012,respectively)andStewart’swilt
312
(n=60,78,and140in2010,2011and2012,respectively)(Figure1).Anadditional17SNP
313
markerswereassayedonthepopulationtodeterminethephysicalpositionofbreakpoints
314
(TableS1).Fivewereremovedfromthebreakpointanalysisbecauseofmissinggenotypes
315
oralackofrecombinationwithneighboringmarkers.Theorderofthephysicalmap
316
matchedtheorderofthegeneticmap.Basedonthesedata,qNLB1.06wasnarrowedtoa
317
3.60‐Mbregionflankedbysnp_01_0047(185,737,089bpAGP_V2)andsnp_01_0082
318
(189,336,643bpAGP_V2)(Figure1).qSw1.06wasnarrowedtoa95.9‐kbintervalflanked
319
bysnp_01_0137(187,245,104bpAGP_V2)andsnp_01_0139(187,341,010bpAGP_V2)
15
320
(Figure1).Whileconfidenceintervalsdifferedforthediseases,breakpointanalysesfor
321
bothshowedsimilarprofiles.
322
Genomicintegrity:Weobservedalowfrequencyofrecombinationacrosspartof
323
thefine‐mappingregion.TodeterminewhetherthiswasananomalyonlyfoundintheNIL
324
fine‐mappingpopulation,weexaminedtherecombinationratesacrosstheRILpopulations
325
thatcomprisetheNAMpopulationbyexamininggeneticdistancesinNAMsub‐populations,
326
asshowninFigure2.Wefoundadepressedrecombinationrateacrossportionsofthe
327
targetintervalintheTx303xB73RILpopulation,confirmingalowrateofrecombination
328
foundinthecurrentstudybetweenB73andTx303inthisregion.Furthermore,mostNAM
329
founderlinesshowedlowrecombinationratesacrosspartsoftheregion.Hp301,however,
330
showedslightlyhigherlevelsofrecombination,suggestingstructuralsimilaritybetween
331
B73andHp301.Otherlines,suchasMs71andM37W,showedrepressedrecombinationin
332
otherregionsoftheinterval.
333
Becauselowrecombinationratesarehypothesizedtobeduetostructuralvariation
334
suchasinversions,indels,transposableelements,orpresence/absencevariation
335
(McMullenetal.2009),weexaminedIlluminareadsfromtheHapMapV2project(Chiaetal.
336
2012)toassessreaddepthvariationasaproxyforstructuralvariation.Wefoundvariation
337
inthenumberofTx303readsthatmappedtotheB73referencesequence,whilelittle
338
variationwasobservedinthenumberofB73readsthatmappedtothereferencesequence
339
(Figure3).TheregionwithlittletonorecombinationintheNAMsubpopulationsshoweda
340
reducednumberofreadsinTx303.Conversely,theregionwithsignificantCNVNAMGWAS
341
associationsat187Mbhadanelevatednumberofreadsthatmappedtothereference
342
genomeinthislocation,suggestingpossibleduplicationsandgenomeexpansion.
16
343
CandidategenesunderlyingqSw1.06Tx303:TheStewart’swiltfine‐mappingregion
344
wasnarrowedtoa95.9‐kbintervalthatcontainsthreegenesintheB73reference:a
345
putativezincfinger(GRMZM2G445684)andtwouncharacterizedgenes
346
(GRMZM2G445676,andAC213857.4_FG001).Thetwouncharacterizedgeneshaveno
347
knownhomologsandnoannotateddomains.ResistanceatthislocusisconferredbyTx303,
348
anditisthereforeplausiblethattheresistancegeneisabsentfromtheB73reference.
349
CandidategenesunderlyingqNLB1.06:Thefine‐mappingregionfallingwithinthe
350
Bayes95%confidenceintervalforNLBresistance,185.7Mbto189.3Mbofmaize
351
chromosome1,comprises117annotatedcodinggenes,30pseudogenes,and39
352
transposableelementsintheB73genomesequence(TableS3).Anumberofgeneswithin
353
thenarrowedfine‐mappingintervalarecrediblecandidatesbasedontheinvolvementof
354
thosegeneclassesinplantdefenseasdocumentedinthescientificliterature.Theseinclude
355
threeputativeleucinerichrepeat‐encodinggenes,threeputativeproteinkinases,two
356
putativewall‐associatedreceptorkinases,andoneputativelipoxygenasesharinghomology
357
withArabidopsisthalianaLOX2.Furthermore,genome‐widenestedassociationmapping
358
conductedbyChiaetal.(2012)identifiedthreesignificantassociationsclusteredwithinthe
359
40‐kbregionspanning187.23to187.27MbAGP_V1.Themostsignificanthitwasan
360
intergenic10kbreaddepthvariationwithabootstrapposteriorprobability(BPP)of44,
361
whichwas30kbupstreamofaputativeserine‐threonineproteinkinaseand129kb
362
downstreamofaputativezinc‐fingerencodinggene(GRMZM2G441903).Twoadditional
363
polymorphisms,oneintronicSNP(BPP=3)andoneintergeniccopynumbervariation
364
(BPP=1)werewithin40kbofthesignificantassociationwithBPP=44.
17
365
Associationmapping:Associationanalysisofthisregionusingthe282‐line
366
Goodmandiversitypanel(Flint‐Garciaetal.2005)evaluatedforNLB(Wisseretal.2011)
367
identifiedanassociationbetween185.7Mbto189.3Mb(Figure4).Forsignificant
368
intergenicassociations,adjacentgeneswereconsideredaswellasgenesimplicatedby
369
long‐distancelinkagedisequilibrium.TheSNPimplicatedbyassociationanalysiswas
370
locatedat188,018,070bp(AGP_V2)(p‐value=1.72x10‐4,q‐value=0.136)260bp
371
downstreamofaSpoUmethylase(GRMZM5G854901)and13.54Kbupstreamofan
372
uncharacterizedgenewithahelix‐loop‐helixDNA‐bindingdomain(GRMZM5G879527).
373
Thereceptor‐likekinasepan1(GRMZM5G836190)islocated37.555kbfromthesignificant
374
diversitypanelassociation,adistancecloseenoughtolinktheSNPtopan1bylong‐range
375
LD(Chiaetal.2012)ortheSNPcouldimplicateadownstreamelementregulatingthe
376
expressionofpan1.
377
pan1isasusceptibilitygeneforNLB:pan1(GRMZM5G836190atposition
378
187,978,007‐187,980,232onchromosome1)wasinitiallyacandidategenefordisease
379
resistanceat1.06basedonitslocationinsidethefine‐mappinginterval,itsproximitytoan
380
NLBassociationfromthediversitypanelanalysis,anditsidentityasareceptor‐likekinase
381
(Cartwrightetal.2009)(thisclassofproteinsisknowntodetectmicrobe‐associated
382
molecularpatterns;(Zipfel2008)).Phenotypicallysimilar,butunlinked,pan2mutants
383
(Zhangetal.2012)werealsoassessedtotestofthehypothesisthatthepangenesinfluence
384
thediseaseresponsethroughtheirknowneffectonstomatalmorphologyorrelated
385
pathways.Wetestedpan1andpan2mutantsforNLBandStewart’swiltinreplicated,
386
multi‐yeartrialsandfoundasignificantgenotypeeffect(p‐value<0.0001)forboth
387
diseases.Bothmutantswithnullallelesofpan1werefoundtobesignificantlymore
18
388
resistanttobothdiseasesthanB73,indicatingpan1isasusceptibilitygeneforNLBand
389
Stewart’swilt,whilepan2mutantsshowednosignificantdifferencefromB73(Figure5).
390
ForNLB,pan1‐Muhada41%decreaseinAUDPC,ascomparedwithB73,andwasgenerally
391
moreresistantthanpan1‐ems,whichhada27%decreaseinAUDPC,ascomparedtoB73.
392
ForStewart’swilt,pan1mutantswerenearlyimmune.
Theseresultssuggestthatpartialorcompletelossofpan1maycontributetothe
393
394
increasedresistancetoNLBseenforqNLB1.06Tx303comparedtoqNLB1.06B73.Weexamined
395
thegeneactionoftheQTL,andintwodifferentenvironmentsfoundthattheQTLactedina
396
dominantfashion,wheretheheterozygoteNILfellintothesamephenotypicclassasthe
397
resistantNILcarryingtwocopiesoftheTx303allele.Preliminarydatafromone
398
environmentshowedthatthepan1‐emsmutationwasalsodominantforresistance.
399
Furtherdataisneededtoconfirmthisobservation.Tofurthertestwhetherpan1underlies
400
qNLB1.06,wecomparedpan1geneexpressionlevelsinmatureleavesofthesetwolinesvia
401
RT‐PCR.Consistentwithreducedpan1functioninqNLB1.06Tx303,wefoundpan1tocontrol
402
actinsignalratiosweredecreasedfrom0.915+/‐0.168inqNLB1.06B73to0.553+/‐0.035
403
inqNLB1.06Tx303(+/‐standarderrors,p<0.05usingStudent’st‐test).However,further
404
workwillbeneededtodeterminewhetherthereductioninpan1expressionlevelin
405
qNLB1.06Tx303iscausallyrelatedtotheincreaseinNLBresistanceseeninthisline.
406
407
DISCUSSION
408
Locithatunderlieanumberoftraitspresentanopportunitytoinvestigatethe
409
complexrelationshipbetweenvariationfortraits,genomestructure,recombination,and
410
causativegenes.Maizebin1.06isonesuchlocusthatisassociatedwitheffectsondiverse
19
411
traits.Thischromosomalregionofinteresthasbeendescribedasayield‐stabilizinglocus
412
associatedwitheffectsonresistancetoseveraldiseases,rootarchitecture,plantheight,
413
floweringtimeandyieldacrossdifferentsoilmoisturelevelsandgeneticbackgrounds
414
(Tuberosaetal.2002;Landietal.2002;Wisseretal.2006;Landietal.2010).Thislocusis
415
arelativelyQTL‐densesegmentofthemaizegenome,withmorethandoubletheaverage
416
numberofQTL(35QTLv.anaverageof15QTL/bin),butofaveragegeneticsizebasedon
417
theNAMgeneticmapandbasedonphysicalsize(McMullenetal.2009;Andorfetal.2010).
418
TheelevatednumberofQTLinthisbin,coupledwithanaveragegenecontentandgenetic
419
size,indicatethatthisbinisimportantformaizebreedingacrossabroadsetoftraits.
420
Hence,itisofinterestnotonlytoidentifythegenesunderlyingthosetraits,including
421
multiplediseaseresistance,butalsotoinvestigatethegenomedynamicsshapingthe
422
region.
423
ConsistentwiththeobservationofhighQTLdensity,variationsinthetargeted
424
intervalwereshowntobeunderselectionduringdomesticationandsubsequentvarietal
425
improvement.Candidategenesfordomesticationsyndromeinthisregion,identifiedas
426
geneslyinginextendedregionswithallelefrequencydifferentiationbetweenlandraces
427
andZeamaysssp.parviglumis,includegenessuchasaputativelipoxygenase,putative
428
frataxin,andaputativezincfingeramongothers,while“improvementcandidates”(those
429
thatcontrastforimprovedlinesversuslandraces)includeputativeproteinkinases,
430
putativeEF‐handproteins,aputativealcoholdehydrogenase,andaputativeantifreeze
431
protein,amongothers(Huffordetal.2012).Theobservedpatternsofselectionmaybe
432
relatedtodiseaseresistance,asthetranscriptionalrewiringofthemaizetranscriptome
433
duringdomesticationsuggeststhatgenesrelatedtobioticstressareoverrepresented
20
434
amongthegroupofgenesup‐regulatedduringdomestication(Swanson‐Wagneretal.
435
2012).Indeed,someofthesecandidategenes,suchasthelipoxygenase,serine/threonine
436
proteinkinase,andtheantifreezeprotein,couldbeinvolvedinoneofthemanydisease
437
resistancesconditionedbythislocus.
438
ThisregionharboringnumerousQTLfordiversetraitsshowssignsofhighgenome
439
complexityandplasticity.Alowrecombinationrateinthefine‐mappingintervalwas
440
observedintheNILs,withalimitednumberofrecombinantsidentifiedbetween184.6Mb
441
and187.6Mb,butanaveragegenedensityascomparedtothemaizegenomeasawhole
442
(Schnableetal.2009).Thefine‐mappinginterval,includingtheregionoflow
443
recombination,co‐localizeswiththeyield‐stabilizingQTLreportedbyLandietal.(2010).A
444
reducedrecombinationrateinthisintervalwasobservedinmanyoftheNAM
445
subpopulations,providingsupportforthehypothesisthatB73hasalackofsyntenywith
446
othermaizelinesatthisregion,withtheexceptionofHp301,whichrecombineswithB73
447
inthisregion.Thelowrecombinationcouldbeduetosmallinversions,indels,transposon
448
insertion,orprescence/absencevariation(McMullenetal.2009).Suchdifferencesthat
449
suppressrecombinationmaybeselectedupontoconservetheyield‐stabilizinghaplotype
450
locatedatthisregion.
451
Increasingly,copynumbervariation(CNV)hasbeenfoundtounderlietrait
452
variation,includingbioticandabioticstresstolerance(Cooketal2012a;Maronetal.
453
2013).ACNVpolymorphismwassignificantlyassociatedwithNLBinthe1.06interval
454
basedontheNAMGWAS.Togetherwiththelackofrecombinationinpartofthefine‐
455
mappingpopulation,thissuggeststhatgenomecontentvariationacrossdiversemaize
456
germplasmmayunderliethedifferencesindiseaseresponse.Totestthishypothesis,
21
457
structuraldifferenceswereexploredbyexaminingreaddepthvariationacrosstheregion.
458
PronouncedvariationinthenumberofTx303readsthatmappedtothereferencewas
459
found,whichcanbeinterpretedasevidenceforduplicationsorgenomeexpansionatthis
460
regioninTx303.Thisisnotuncommon,asthemaizegenomeishighlyplastic,withread
461
depthvariationin90%ofthegenome(Chiaetal.2012)andpresence/absencevariation
462
thoughttobeamajordriverofphenotypicvariationinmaize(Wallaceetal.2014).
463
Giventhecomplexgeneticbasisofquantitativetraitsandthisregion,afine‐
464
mappingapproachwastakentorefinethegenomicregionassociatedwithresistanceto
465
NLBandStewart’swilt,complementedbyassociationmappingtoidentifycandidategenes.
466
Thefine‐mappingapproachallowedforthedissectionofthemulti‐traitnatureofthisQTL.
467
ResistancetoStewart’swiltwaslocalizedtoa95.9‐kbregionwithinthelarger3.60Mb
468
NLBfine‐mappinginterval.WhilethemajorityofmajorQTLhavebeenshownnottobe
469
pleiotropicinnature(Wallaceetal.2014),apleiotropicbasisofdiseaseresistancecannot
470
beexcludedatthislocus.Thebreakpointanalysisforbothdiseaseswassimilar,although
471
theconfidenceintervalofNLBwascalculatedtobelargerthanthatforStewart’swilt.
472
ThecandidateregionforStewart’swiltcontainsthreegenesintheB73genome
473
sequence:agenewithaputativezincfingerandtwouncharacterizedgenes.Both
474
uncharacterizedgeneslackhomologsandonelacksexpressionevidence(Dongetal.2004;
475
Senetal.2010).However,thegeniccontentoftheregionmaydifferinTx303.Aphysical
476
mapassemblyforTx303acrossthefine‐mappingregionwouldclarifythis.Anumberof
477
mappingstudieshaveimplicatedtheregiononChr.1between180and190Mbacross
478
diversepopulationsforresistancetoNLB.TheqNLB1.06Tx303regionhasbeensuccessfully
479
narrowedto3.6Mb.While117candidategenesfromtheB73genomesequencewere
22
480
implicatedthroughfine‐mapping,itisagainlikelythatTx303differsinthegeniccontentof
481
thisregion.AsubsetoftheNLBcandidategeneswasparticularlycrediblebasedon
482
previousknowledgeofplantdefense,includingfourputativeleucine‐richrepeatprotein
483
kinases.Othercandidatesincludeaputativefrataxin,aputativeABCtransporter,anda
484
putativelipoxygenase.
485
Associationmappingprovidesacomplementaryapproachforidentifyingcandidate
486
genes.SignificantassociationswithintheqNLB1.06Tx303fine‐mappingintervalwere
487
detectedusingboththeNAMandGoodmandiversitypanels(Flint‐Garciaetal.2005;
488
Polandetal.2011;Wisseretal.2011;Chiaetal.2012).Themostsignificantassociation
489
withinthisregionintheNAMwasanintergeniccopynumbervariation,whichhadaBPP
490
valueof44(p‐value=0.0000737),oneofthemosthighlysignificantassociationsfromthe
491
analysis(Polandetal.2011;Chiaetal.2012).AmongthegenesimplicatedbyNAM,the
492
putativeA20/AN1zincfingerwasthestrongestcandidate;agenedomainthatisassociated
493
withstresstoleranceinplantsandtheimmunesysteminanimals(VijandTyagi2008).
494
ThispolymorphismisclosetotheStewart’swiltfine‐mappinginterval,lendingsupportto
495
thehypothesisthataregionpresentinTx303butnotB73maycontaingene(s)for
496
resistancetobothdiseases.AssociationanalysisusingtheGoodmandiversitypanel
497
revealedasignificantintergenicSNPwithinthefine‐mappinginterval,approximately800
498
KbfromtheNAMCNV(Flint‐Garciaetal.2005;Wisseretal.2011).TheNLB‐associated
499
CNVandSNPpolymorphismsmaybeinlinkagedisequilibriumwithoneormoregenesin
500
thevicinity.Candidategenesfromassociationmappingcanbefurtherinvestigatedthrough
501
expressionanalyses,re‐sequencing,andtestingacrossdifferentgermplasmsets.
23
502
Withinthefine‐mappingintervalshowninFigure1,thesignificantGoodmanpanel
503
SNPwas38kbfrompan1.WetestedthepanmutantsforbothNLBandStewart’swilt
504
becausethemappingresultsforthetwodiseasesweresimilar(Figure1).Thesmaller
505
confidenceintervalforStewart’swiltmayreflectthestrongerphenotype,whilethesimilar
506
profileindicatesthattheremaybemultiplegenesunderlyingtheQTLforbothdiseases.
507
TwoindependentnullmutantallelesofthisgeneconferredresistancephenotypesforNLB
508
andStewart’swilt(Figure5),demonstratingthatmutationsinpan1itself(notalinked
509
genepresentinoneorothermutantbackground)increaseresistanceforthetwodiseases.
510
Thisfindingsuggeststhatalossoffunctionalleleofpan1derivedfromTx303may
511
contributetothediseaseresistancephenotype(s)conferredbyqNLB1.06Tx303and
512
qSW1.06Tx303.PreliminarydatashowedthattheQTLandpan1mutantsshowedsimilar
513
inheritancepatternswithdominantresistanceinbothcases.Thisisunexpectedforaloss
514
offunctionmutation.Furtherworkisneededtoconfirmthegeneactionofthepan1
515
mutantsandtodissecttherelationshipbetweentheMDRQTLandpan1.
516
PAN1hasbeenshowntoplayaroleinpromotingfeaturesofactinorganizationthat
517
supportasymmetriccelldivision(Cartwrightetal.2009).Interestingly,linescarrying
518
mutationsforpan2,whichalsoshowasymmetriccelldivision,werenotsignificantly
519
differentfromB73forNLB,indicatingaberrantstomatafoundinbothmutantlinesarenot
520
theunderlyingmechanismofresistanceinthepan1mutants.Susceptibilityconditionedby
521
wild‐typepan1couldbeduetoapassivemechanism,suchasalteredanatomicalstructures,
522
oranactiveprocess,suchasactinre‐organizationduringpathogenattack.
523
524
Wehavesuccessfullyrefinedq1.06Tx303,identifiedcandidategenes,and
demonstratedaroleforpan1inmultiplediseaseresistance.NLBandStewart’swilt
24
525
resistanceregionshavebeennarrowedsufficientlythatthemarkerswithintheNLBand
526
Stewart’swiltintervalscanbeusedformarker‐assistedselection.Thesedatastrongly
527
suggestthatstructuralvariationunderliesthislocusandpan1,ageneinwhichlowered
528
expressioniscorrelatedwithhigherresistancelevels,mayunderlietheNLBandStewart’s
529
wiltQTL.Lossofsusceptiblepan1allelescouldbeusedtodecreasemaizesusceptibilityto
530
diversepathogens.Throughfine‐mapping,examiningrecombinationratesandre‐
531
sequencingdata,andevaluatingmutantlines,wewereabletodissectacomplexlocusand
532
identifiedaroleforpan1inplantdefense.Thisapproachshedlightonalocusknownforits
533
complexityandquantitativeeffect.
534
ACKNOWLEDGEMENTS
535
WewouldliketoacknowledgeDr.SantiagoMiderosforbioinformaticssupport,and
536
537
Drs.RandallWisser,BodeOlukolu,andChia‐LinChungforhelpfuldiscussionsand
538
technicalassistance.WewouldliketoacknowledgeLauraMorales,AlyssaCowles,William
539
Miller,ChrisMancuso,KatharineConstas,ArielFialko,andXingyuLuofortheirassistance
540
withfieldwork,andYeriParkforhelpwiththepan1RT‐PCRexperiments.Thisworkwas
541
fundedbyNationalScienceFoundationaward1127076,theMcKnightFoundation,USDA
542
AgriculturalExperimentalStation(HATCH)AwardNYC‐153418,andCornellUniversity.
543
25
LITERATURECITED
544
545
Andorf,C.M.,Lawrence,C.J.,Harper,L.C.,Schaeffer,M.L.,Campbell,D.A.,etal.,2010The
546
locuslookuptoolatMaizeGDB:identificationofgenomicregionsinmaizeby
547
integratingsequenceinformationwithphysicalandgeneticmaps.Bioinformatics
548
26:434‐436.
549
Belcher,A.R.,Zwonitzer,J.C.,SantaCruz,J.,Krakowsky,M.D.,Chung,C.L.,etal.,2012
550
Analysisofquantitativediseaseresistancetosouthernleafblightandofmultiple
551
diseaseresistanceinmaize,usingnear‐isogeniclines.TheorApplGenet124:433‐
552
445.
553
554
555
Bent,A.F.,1996Plantdiseaseresistancegenes:Functionmeetsstructure.PlantCell8:
1757‐1771.
Bradbury,P.J.,Zhang,Z.,Kroon,D.E.,Casstevens,T.M.,Ramdoss,Y.,etal.,2007TASSEL:
556
softwareforassociationmappingofcomplextraitsindiversesamples.
557
Bioinformatics23:2633‐2635.
558
559
560
561
562
563
Broman,K.W.,Wu,H.,Sen,S.,Churchill,G.A.,2003R/qtl:QTLmappinginexperimental
crosses.Bioinformatics19:889‐890.
Buckler,E.S.,Holland,J.B.,Bradbury,P.J.,Acharya,C.B.,Brown,P.J.,etal.,2009The
geneticarchitectureofmaizefloweringtime.Science325:714‐718.
Canaran,P.,Buckler,E.S.,Glaubitz,J.C.,Stein,L.,Sun,Q.,etal.,2008Panzea:anupdateon
newcontentandfeatures.NucleicAcidsRes36:D1041‐1043.
564
Cartwright,H.N.,Humphries,J.A.,Smith,L.G.,2009PAN1:areceptor‐likeproteinthat
565
promotespolarizationofanasymmetriccelldivisioninmaize.Science323:649‐
566
651.
26
567
Chia,J.M.,Song,C.,Bradbury,P.J.,Costich,D.,deLeon,N.,etal.,2012MaizeHapMap2
568
identifiesextantvariationfromagenomeinflux.NatGenet44:803‐807.CookJP,
569
McMullenMD,Holland,J.B.,TianF,BradburyP,etal.,2012Geneticarchitectureof
570
maizekernelcompositioninthenestedassociationmappingandinbredassociation
571
panels.PlantPhysiol158:824‐834.
572
Chung,C.L.,Jamann,T.,Longfellow,J.,Nelson,R.,2010aCharacterizationandfine‐mapping
573
ofaresistancelocusfornorthernleafblightinmaizebin8.06.TheorApplGenet
574
121:205‐227.
575
Chung,C.L.,Longfellow,J.M.,Walsh,E.K.,Kerdieh,Z.,VanEsbroeck,G.,etal.,2010b
576
Resistancelociaffectingdistinctstagesoffungalpathogenesis:useofintrogression
577
linesforQTLmappingandcharacterizationinthemaize‐Setosphaeriaturcica
578
pathosystem.BMCPlantBiol10:103.
579
Chung,C.L.,Poland,J.,Kump,K.,Benson,J.,Longfellow,J.,etal.,2011Targeteddiscoveryof
580
quantitativetraitlociforresistancetonorthernleafblightandotherdiseasesof
581
maize.TheorApplGenet123:307‐326.
582
Cook,D.E.,Lee,T.G.,Guo,X.,Melito,S.,Wang,K.,etal.,2012aCopynumbervariationof
583
multiplegenesatRhg1mediatesnematoderesistanceinsoybean.Science338:
584
1206‐1209.
585
Cook,J.P.,McMullen,M.D.,Holland,J.B.,Tian,F.,Bradbury,P.,etal.2012bGenetic
586
architectureofmaizekernelcompositioninthenestedassociationmappingand
587
inbredassociationpanels.PlantPhysiol158:824‐834.
588
589
Dong,Q.,Schlueter,S.D.,Brendel,V.,2004PlantGDB,plantgenomedatabaseandanalysis
tools.NucleicAcidsRes32:D354‐359.
27
590
591
592
Doyle.J.J.,Dickson,E.E.,(1987)PreservationofplantsamplesforDNArestriction
endonucleaseanalysis.Taxon:715‐722.
Flint‐Garcia,S.A.,Thuillet,A.C.,Yu,J,,Pressoir,G.,Romero,S.M.,etal.,2005Maize
593
associationpopulation:ahigh‐resolutionplatformforquantitativetraitlocus
594
dissection.PlantJ44:1054‐1064.
595
Freymark,P.J.,Lee,M.,Woodman,W.L.,Martinson,C.A.,1993Quantitativeandqualitative
596
traitlociaffectinghost‐plantresponsetoExserohilumturcicuminmaize(Zeamays
597
L.).TheorApplGenet87:537‐544.
598
Gallagher,K.,Smith,L.G.,2000Rolesforpolarityandnucleardeterminantsinspecifying
599
daughtercellfatesafteranasymmetriccelldivisioninthemaizeleaf.CurrBiol10:
600
1229‐1232.
601
Goff,S.A.,Vaughn,M.,McKay,S.,Lyons,E.,Stapleton,A.E.,etal.,2011TheiPlant
602
Collaborative:Cyberinfrastructureforplantbiology.FrontPlantSci2:34.
603
604
605
606
607
Gore,M.A.,Chia,J.M.,Elshire,R.J.,Sun,Q.,Ersoz,E.S.,etal.,2009Afirst‐generation
haplotypemapofmaize.Science326:1115‐1117.
Gurung,S.,Bonman,J.M.,Ali,S.,Patel,J.,Myrfield,M.,etal.,2009Newanddiversesources
ofmultiplediseaseresistanceinwheat.Cropscience49:1655‐1666.
Hufford,M.B.,Xu,X.,vanHeerwaarden,J.,Pyhajarvi,T.,Chia,J.M.,etal.,2012Comparative
608
populationgenomicsofmaizedomesticationandimprovement.NatGenet44:808‐
609
811.
610
611
Jennings,P.,Ullstrup,A.,1957AhistologicalstudyofthreeHelminthosporiumleafblights
ofcorn.Phytopathology47:707‐714.
28
612
Johnson,E.B.,Haggard,J.E.,StClair,D.A.,2012Fractionation,stability,andisolate‐
613
specificityofQTLforresistancetoPhytophthorainfestansincultivatedtomato
614
(Solanumlycopersicum).G3(Bethesda)2:1145‐1159.
615
Jones,J.D.,Dangl,J.L.,2006Theplantimmunesystem.Nature444:323‐329.
616
Krattinger,S.G.,Lagudah,E.S.,Spielmeyer,W.,Singh,R.P.,Huerta‐Espino,J.,etal.,2009A
617
putativeABCtransporterconfersdurableresistancetomultiplefungalpathogensin
618
wheat.Science323:1360‐1363.
619
Kump,K.L,,Bradbury,P.J.,Wisser,R.J.,Buckler,E.S.,Belcher,A.R.,etal.,2011Genome‐
620
wideassociationstudyofquantitativeresistancetosouthernleafblightinthemaize
621
nestedassociationmappingpopulation.NatGenet43:163‐168.
622
623
624
Kosambi,D.D.,(1943)TheestimationofmapDistancesfromrecombinationvalues.Annals
ofEugenics12:172‐175.
Kump,K.,Holland,J.,Jung,M.,Wolters,P.,Balint‐Kurti,P.,2010Jointanalysisofnear‐
625
isogenicandrecombinantinbredlinepopulationsyieldsprecisepositional
626
estimatesforquantitativetraitloci.ThePlantGenome3:142‐153.
627
628
Lai,J.,Li,R.,Xu,X.,Jin,W.,Xu,M.,etal.,2010Genome‐widepatternsofgeneticvariation
amongelitemaizeinbredlines.NatGenet42:1027‐1030.
629
Landi,P.,Giuliani,S.,Salvi,S.,Ferri,M.,Tuberosa,R.,etal.,2010Characterizationofroot‐
630
yield‐1.06,amajorconstitutiveQTLforrootandagronomictraitsinmaizeacross
631
waterregimes.JExpBot61:3553‐3562.
632
633
Landi,P.,Sanguineti,M.,Darrah,L.,Giuhani,M.,Salvi,S.,etal.,2002DetectionofQTLsfor
verticalrootpullingresistanceinmaizeandoverlapwithQTLsforroottraitsin
29
634
hydroponicsandforgrainyieldunderdifferentwaterregimes.Maydica47:233‐
635
243.
636
Li,H.,Handsaker,B.,Wysoker,A.,Fennell,T.,Ruan,J.,etal.,2009TheSequence
637
Alignment/MapformatandSAMtools.Bioinformatics25:2078‐2079.
638
Lopez,C.E.,Acosta,I.F.,Jara,C.,Pedraza,F.,Gaitan‐Solis,E.,etal.,2003Identifying
639
resistancegeneanalogsassociatedwithresistancestodifferentpathogensin
640
commonbean.Phytopathology93:88‐95.
641
Manosalva,P.M.,Davidson,R.M.,Liu,B.,Zhu,X.,Hulbert,S.H.,etal.,2009Agermin‐like
642
proteingenefamilyfunctionsasacomplexquantitativetraitlocusconferringbroad‐
643
spectrumdiseaseresistanceinrice.PlantPhysiol149:286‐296.
644
Maron,L.G.,Guimaraes,C.T.,Kirst,M.,Albert,P.S.,Birchler,J.A.,etal.,2013Aluminum
645
toleranceinmaizeisassociatedwithhigherMATE1genecopynumber.ProcNatl
646
AcadSciUSA110:5241‐5246.
647
McHale,L.K.,Haun,W.J.,Xu,W.W.,Bhaskar,P.B.,Anderson,J.E.,etal.,2012Structural
648
variantsinthesoybeangenomelocalizetoclustersofbioticstress‐responsegenes.
649
PlantPhysiol159:1295‐1308.
650
McMullen,M.D.,Kresovich,S.,Villeda,H.S.,Bradbury,P.,Li,H.,etal.,2009Genetic
651
propertiesofthemaizenestedassociationmappingpopulation.Science325:737‐
652
740.
653
654
McMullen,M.D.,Simcox,K.D.,1995Genomicorganizationofdiseaseandinsectresistance
genesinmaize.MolPlantMicrobeInteract8:811‐815.
30
655
Ming,R.,Brewbaker,J.,Moon,H.,Musket,T.,Holley,R.,etal.,1999IdentificationofRFLP
656
markerslinkedtoamajorgene,sw1,conferringresistancetoStewart'swiltin
657
maize.Maydica44:319‐323.
658
Olukolu,B.A.,Negeri,A.,Dhawan,R.,Venkata,B.P.,Sharma,P.,etal.,2013Aconnectedset
659
ofgenesassociatedwithprogrammedcelldeathimplicatedincontrollingthe
660
hypersensitiveresponseinmaize.Genetics193:609‐620.
661
Paterson,A.,DeVerna,J.,Lanini,B.,Tanksley,S.,1990Finemappingofquantitativetraitloci
662
usingselectedoverlappingrecombinantchromosomes,inaninterspeciescrossof
663
tomato.Genetics124:735‐742.
664
Poland,J.A.,Bradbury,P.J.,Buckler,E.S.,NelsonR.J.,2011Genome‐widenested
665
associationmappingofquantitativeresistancetonorthernleafblightinmaize.Proc
666
NatlAcadSciUSA108:6893‐6898.
667
668
669
RDevelopmentCoreTeam,2013R:Alanguageandenvironmentforstatisticalcomputing.
Vienna,Austria:RFoundationforStatisticalComputing.
Ramalingam,J.,VeraCruz,C.M.,Kukreja,K.,Chittoor,J.M.,Wu,J.L.,etal.,2003Candidate
670
defensegenesfromrice,barley,andmaizeandtheirassociationwithqualitativeand
671
quantitativeresistanceinrice.MolPlantMicrobeInteract16:14‐24.
672
Romay,M.C.,Millard,M.J.,Glaubitz,J.C.,Peiffer,J.A.,Swarts,K.L.,etal.,2013
673
ComprehensivegenotypingoftheUSAnationalmaizeinbredseedbank.Genome
674
Biol14:R55.
675
676
Roper,M.C.,2011Pantoeastewartiisubsp.stewartii:lessonslearnedfromaxylem‐
dwellingpathogenofsweetcorn.MolPlantPathol12:628‐637.
31
677
Rossi,C.,Cuesta‐Marcos,A.,Vales,I.,Gomez‐Pando,L.,Orjeda,G.,etal.,2006Mapping
678
multiplediseaseresistancegenesusingabarleymappingpopulationevaluatedin
679
Peru,Mexico,andtheUSA.MolecularBreeding18:355‐366.
680
681
Schnable,P.S.,Ware,D.,Fulton,R.S.,Stein,J.C.,Wei,F.,etal.,2009TheB73maizegenome:
complexity,diversity,anddynamics.Science326:1112‐1115.
682
Sen,T.Z.,Harper,L.C.,Schaeffer,M.L.,Andorf,C.M.,Seigfried,T.E.,etal.,2010Choosinga
683
genomebrowserforaModelOrganismDatabase:surveyingthemaizecommunity.
684
Database(Oxford)2010:baq007.
685
Springer,N.M.,Ying,K.,Fu,Y.,Ji,T.,Yeh,C.T.,etal.,2009Maizeinbredsexhibithighlevels
686
ofcopynumbervariation(CNV)andpresence/absencevariation(PAV)ingenome
687
content.PLoSGenet5:e1000734.
688
Steinmetz,L.M.,Sinha,H.,Richards,D.R.,Spiegelman,J.I.,Oefner,P.J.,etal.,2002
689
Dissectingthearchitectureofaquantitativetraitlocusinyeast.Nature416:326‐
690
330.
691
692
693
694
Storey,J.D.,2002Adirectapproachtofalsediscoveryrates.JournaloftheRoyalStatistical
Society:SeriesB(StatisticalMethodology)64:479‐498.
Studer,A.J.,Doebley,J.F.,2011DolargeeffectQTLfractionate?Acasestudyatthemaize
domesticationQTLteosintebranched1.Genetics188:673‐681.
695
Swanson‐Wagner,R.,Briskine,R.,Schaefer,R.,Hufford,M.B.,Ross‐Ibarra,J.,etal.,2012
696
Reshapingofthemaizetranscriptomebydomestication.ProcNatlAcadSciUSA
697
109:11878‐11883.
698
699
Szalma,S.J.,Hostert,B.M.,Ledeaux,J.R.,Stuber,C.W.,Holland,J.B.,2007QTLmapping
withnear‐isogeniclinesinmaize.TheorApplGenet114:1211‐1228.
32
700
Tuberosa,R.,Sanguineti,M.C.,Landi,P.,MichelaGiuliani,M.,Salvi,S.,etal.,2002
701
IdentificationofQTLsforrootcharacteristicsinmaizegrowninhydroponicsand
702
analysisoftheiroverlapwithQTLsforgrainyieldinthefieldattwowaterregimes.
703
PlantMolecularBiology48:697‐712.
704
VanEsbroeck,G.,Smith,M.E.,Balint‐Kurti,P.J.,Jung,J.,Yang,J.,2010Useofamaize
705
advancedintercrosslineformappingofQTLfornorthernleafblightresistanceand
706
multiplediseaseresistance.CropScience50:458‐466.
707
Vij,S.,Tyagi,A.K.,2008A20/AN1zinc‐fingerdomain‐containingproteinsinplantsand
708
animalsrepresentcommonelementsinstressresponse.FunctIntegrGenomics8:
709
301‐307.
710
711
712
Wallace,J.G.,Larsson,S.J.,Buckler,E.S.,2014Enteringthesecondcenturyofmaize
quantitativegenetics.Heredity(Edinb)112:30‐38.
Welz,H.G.,Xia,X.C.,Bassetti,P.,Melchinger,A.E.,Lubberstedt,T.,1999QTLsforresistance
713
toSetosphaeriaturcicainanearlymaturingDentxFlintmaizepopulation.Theor
714
ApplGenet99:649‐655.
715
716
717
718
719
Williams,K.J.,2003Themoleculargeneticsofdiseaseresistanceinbarley.Australian
JournalofAgriculturalResearch54:1065.
Wisser,R.J.,Balint‐Kurti,P.J.,NelsonR.J.,2006Thegeneticarchitectureofdisease
resistanceinmaize:asynthesisofpublishedstudies.Phytopathology96:120‐129.
Wisser,R.J.,Kolkman,J.M.,Patzoldt,M.E.,Holland,J.B.,Yu,J.,etal.,2011Multivariate
720
analysisofmaizediseaseresistancessuggestsapleiotropicgeneticbasisand
721
implicatesaGSTgene.ProcNatlAcadSciUSA108:7339‐7344.
33
722
Wisser,R.J.,Sun,Q.,Hulbert,S.H.,Kresovich,S.,Nelson,R.J.,2005Identificationand
723
characterizationofregionsofthericegenomeassociatedwithbroad‐spectrum,
724
quantitativediseaseresistance.Genetics169:2277‐2293.
725
Xu,X.,Liu,X.,Ge,S.,Jensen,J.D.,Hu,F.,etal.,2012Resequencing50accessionsofcultivated
726
andwildriceyieldsmarkersforidentifyingagronomicallyimportantgenes.Nat
727
Biotechnol30:105‐111.
728
Zhang,X.,Facette,M.,Humphries,J.A.,Shen,Z.,Park,Y.,etal.,2012IdentificationofPAN2
729
byquantitativeproteomicsasaleucine‐richrepeat‐receptor‐likekinaseacting
730
upstreamofPAN1topolarizecelldivisioninmaize.PlantCell24:4577‐4589.
731
Zipfel,C.,2008Pattern‐recognitionreceptorsinplantinnateimmunity.CurrOpinImmunol
20:10‐16.
732
733
Zipfel,C.,Rathjen,J.P.,2008Plantimmunity:AvrPtotargetsthefrontline.CurrBiol18:
R218‐220.
734
735
Zwonitzer,J.C.,Coles,N.D.,Krakowsky,M.D.,Arellano,C.,Holland,J.B.,etal.,2010
736
Mappingresistancequantitativetraitlociforthreefoliardiseasesinamaize
737
recombinantinbredlinepopulation‐evidenceformultiplediseaseresistance?
738
Phytopathology100:72‐79.
739
740
741
742
743
34
744
745
FigureLegends:
746
Figure1MappingofqNLB1.06andbreakpointanalysesforqNLB1.06.
747
(A)LocationofintrogressionTBBC3_19Eisshownwithmarkernames.(B)Breakpoint
748
analysisforNLBandStewart’swilt.(C)Selectedrepresentativerecombinantsandtheir
749
associatedphenotypes.Forgenotypes,darkshadingindicatestheTx303allele,whilewhite
750
indicatestheB73allele.Lightgreyshadingindicatestheregionofarecombinationevent.
751
Forphenotypes,blueshadingindicatesamoreresistantline,whileredindicatesamore
752
susceptibleline.
753
Figure2Recombinationratesacrossfine‐mappinginterval.
754
RecombinationrateswerecalculatedbetweenninemarkersfortheNAMsub‐populations.
755
NAMfoundersareshownontheleftandthegenecountsfortheeightsub‐intervalsacross
756
thetop.Redindicatesregionsofhighrecombinationandblueindicatesregionsoflow
757
recombination.
758
Figure3ReaddepthvariationacrossqNLB1.06fine‐mappingregion.
759
BluebarsindicatethenumberofB73Illuminareadsthataligntothegivenbin,whilered
760
barsindicatethenumberofTx303Illuminareadsthataligntothegivenbin.Binsare327
761
KbandthestartpositionofthebininnotedontheX‐axis.
762
Figure4Diversitypanelassociation.
35
763
AssociationanalysisforNLBinthefine‐mappingregionusingtheGoodmandiversitypanel
764
(Flint‐Garciaetal.2005;Wisseretal.2011).ThesignificantSNPat188,018,070bp(p‐
765
value=1.72x10‐4,q‐value=0.136)ishighlightedinblue.
766
Figure5Mutantphenotypesofpanmutants.
767
Plantshomozygousforbothpan1‐emsandpan1‐Muallelesaresignificantlydifferentfrom
768
B73,thebackgroundforthemutants,whilepan2mutantsarenot.Lettersdenote
769
significancewithStudent’st‐test(p‐value<0.0001).pan2mutantsareshowninblue,while
770
pan1mutantsareshowningreen.B73isshowninred.
771
FileS1Genotypicandphenotypicdata
772
Dataforfine‐mappingstudyisincludedinthisfile.
36