47thTomatoBreedersRoundtable

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AcknowledgementsMysincerethankstoeachandeveryoneofyouforattendingthe47thTomatoBreeders

Roundtable.IamhappytohostyouinOhio,andpleasedtofeatureTheOhioStateUniversity’sfacilities,facultyandstudentsinourprogram.Wewilltrytostrikeabalancebetweenformalpresentations,hands-onactivities,anddiscussion.Aportionofthemeeting,focusedontomatorootstocks,willbelive-streamedaswebinarandwillfeatureworksupportedthroughtheUSDA’sSpecialtyCropsResearchInitiative.

TheOhioStateUniversity,theOhioAgriculturalExperimentStationandtheOhioAgriculturalResearchandDevelopmentCenterinWooster,OHhasalonghistoryoftomatoresearchencompassinginnovationinpathologyandbreeding.NotablemilestonesincludediscoveryanddeploymentoftheTm2alocusbyLeonardAlexander,ProfessorofPlantPathology;discoveryofresistanceandmanagementofFusariumCrownRotbyteamsfromPlantPathologyandHorticulture;andthedevelopmentofhumid-environmentadaptedprocessinggermplasmbyDr.StanBerry,ProfessorofHorticulture.TheTm2alocusisthemostwidelydeployedresistanceintomatoandhasbeeneffectivefornearly50years.Dr.Berry’svariety,OH8245,hasbeenusedextensivelyinBrazilasAG45,andasaparentincommercialhybridsinCA,Italy,theMidwesternUS,andBrazil.Dr.Berry’searlyseasonOH7983survivedasanOP/inbredvarietyintothelate1990’sandmayhavebeenthelastinbredprocessingvarietyusedbythecanningindustry.Thesebreedingcontributionshavebeensupportedbyaresearchnetworkthatencompassed“fieldtofactory”contributionsfromPlantPathologyandFoodScienceandTechnology.Inthelastdecade,throughtheleadershipofDr.StevenSchwartz(FoodScienceandTechnology)andDr.SteveClinton(JamesCancerCenter)theresearchmodelhasexpandedto“fieldtoclinic”,encompassingproduction,breeding,management,humannutrition,health,andmedicalresearch.ThefieldtoclinicresearchparadigmlivesincollaborationsthroughtheFoodInnovationCenter(FIC),theCenterforAdvancedFunctionalFoodsResearchandEntrepreneurship(CAFFRE),theWilburA.GouldFoodIndustriesCenter,andourUniversityDiscoveryThemes.

DavidFrancis,Host.

CoverArt.EdMarthey,Wooster,OH.pencilsketchprint,allrightsreserved.RegistrationandWebPage.ChristineECooley,UniversityofFlorida,AssistanttoCenterDirector,MediaCoordinator,EventCoordinator.FisherConferenceCenter.DebbieShaffer,ConferenceCoordinator,OARDC,FisherAuditoriumCatering.LindaPatin,Linda’sLocalThymeHiltionGardenInn.AimeeWelsh,DirectorofSales,HiltonGardenInn,WoosterLogisticalSupport.Thetomatoteam:Jihuen(Jin)Cho,TroyAldrich,EduardoBernal,SeanFenstemaker,MichaelDzakovich.FiscalAdministrativeSupport.KimberlyNollettiandLauraWilliams,HorticultureandCropScience,OSU.

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Schedule47thTomatoBreedersRoundtableApril4thOpeningreception5:30-8:00HiltonGardenInnLightHorsd’OeuvresandBarApril5th47thTomatoBreedersRoundtableFisherAuditoriumSouthExhibitArea8:00Registration8:30-9:30Roundtablecheckin/Areareports9:30-10:00Break10:00-12:00TomatoRootstocksMattKleinhenz,ModeratorChieriKubota,OSU"graftingtechnologiesandtheirtrends"(KeynoteSpeaker)CaryRivard,KSU“assessingscioncompatibilityintomatoasafunctionofyieldimprovement”SeanFenstemaker,OSU“vigor,resistance,androotstocks”JonathanKressin,NCSU“colonizationdynamicsofthetomato-bacterialwiltpathosystemanditsimplicationsforresistanceselection”RafaelLacaz-Ruiz,Monsanto“rootstockforthecomputercontrolledenvironment,anindustryperspective”BryanZingel,Sakata“industryperspective”12:00-1:30Lunch2:00-5:00BreedingTechnologyArronCarter“phenotypingandgenomicpredictioninwheatbreeding”(KeynoteSpeaker)ErinSteerandLeahBenedict,LGC“KASPservicetechnologyforSNPgenotyping”

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ScottWeigel,AgriPlexGenomics“PlexSeq:atargetedampliconbasedsequencingmethodforSNPgenotyping”PaulChometandDavidNeuman,NRGene“Pan-Genome:Movingbeyondasinglereferencegenome:GenoMagicTM,anovelsolutiontodescribeandmanagegenomicvariation”3:15-3:30BreakBarbaraE.Liedl,WVSU,“understandingsegregationdistortionandreproductivebarrierstoimprovetransferoftraitsfromS.pennelliitocultivatedtomato”EduardoBernal,OSU,“backgroundgenomeselectionforrapidintrogressionandevaluationofquantitativetraitloci(QTL)forresistanceintomatotomultiplexanthomonasspp.”AndikaGunadi,“rapidprogressofCRISPRfortargetedcropmodification”DavidFrancis,OSU“experiencewithgenomicselectioninprocessingtomato”Eveningactivities:Dinneronown5:00-6:30DiscussionofPanGenomeProject(HiltonGardenInn,BuckeyeBoardRoom)7:00-9:00TomatoCGC(123WilliamsHall,ZOOMlinkforcomputervideoortelephoneconnection)

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April6th47thTomatoBreedersRoundtableFisherAuditoriumSouthExhibitArea8:00-8:50PestResistanceJohnSnyder,UKY,“progressreport:introgressionofiingibereneandtypeIVtrichomedensityfromSolanum.HabrochaitesLA2329intoS.lycopersicum”(KeynoteSpeaker)MohammadHDawood,UKY“anewseqquiterpenealcoholfromwildtomatorepelledthetwo-spottedspidermite,Tetranychusuritcae”AmmarAL-Bayati,UKY“theroleoftrichomesecretionsanddensitiesinspidermitebehaviorforaninterspecificpopulationoftomato”MarthaMutschler,Cornell,“prebreedingtomatoforoptimizedacylsugar-mediatedresistancetoinsects”8:50-9:00Break9:00-11:00DiseaseResistance

SamHutton,UFL“virusresistance”(KeynoteSpeaker)JasmineLopez,UFL.,“identificationofamajorgraywallresistancelocusonchromosome9oftomato”JessicaL.Chitwood,UFL.,“discoveryandimprovementofnovelandknownresistancetofusariumwiltracethreeoftomato.”RobynRoberts,BoyceThompsonInstitute,“ascreenofgeneticallydiversetomatoaccessionsrevealsarangeofunusualresponsestoPseudomonassyringaepv.Tomato’ RebeccaL.Wente,UFL,“finemappingandcandidategenecharacterizationofthepepperbacterialspotresistancegenebs6”PragyaAdhikari,NCSU,“bacterialspotoftomato:pathogenstoryandoureffortsoftomatoimprovementinNC”TaylorAnderson,CornellUniv.,“Pyramidingresistancestobacterialspotandbacterialspeckinelitefreshmarkettomato”

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11:00-12:00QualityinWhole-PeelandDicedTomatoProducts11:00-11:10ValenteAlvarez,OSU“TheWilburA.GouldFoodIndustriesCenter”11:10-11:25KenMartin,Furmano’s“qualityevaluationforwholepeelanddiced(discussion)”12:00-1:00Lunch1:00-2:45TomatoQuality

JessicaCooperstone,OSU“tomatoes,health,andthemetabolome”(KeynoteSpeaker)StevenLoewen,UofGuelph“breedingforprocessingtomatoquality”DilipR.Panthee,NCSU,“tomatoimprovementforfruitqualityanddiseaseresistanceatNCStateUniversity”EdgarSierraOrozco,UFL.,“finemappingofalocusinchromosome12controllingflatandglobefruitshapeinfresh-markettomato”RezaShekasteband,UFL.,“fruitweightvariationcausedbytheknownFW2.2,FW3.2,FAS,andFW11.3alleleinfreshmarkettomato”2:30-3:30DiscussionNextPlace,NextTime3:30-5:00FacilitiesToursSeenextpage5:00-7:00NetworkingEvent,MillerPavilion,SecrestArboretum(33onCampusMap)Dinneronown

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3:30-5:00FacilitiesTours47thTomatoBreedersRoundtableClosed-ToeShoesarerecommendedWewilldivideintogroups,eachwithatourleader.Pleasestaywithyourgroup.Station1MolecularandCellularImagingCenter(Building4,CampusMap,SelbyHallBasement)SequencingandGenotypingEquipment(Dr.TeaMeulia)MCICComputationalBiologyLaboratory(MCBL)(MichaelDzakovich(expressionanalysis),SarangaWijeratne(SNPpipeline))Station2WilliamsGreenhouse(Building7,CampusMap,South/EastWing)TomatoBacterialSpotScreen(EduardoBernal)Assessmentofbeneficialbacteriainpetuniadiseasescreen(KayleeSouth)Station3WilliamsGH(NorthWestWing,HeadHouse,andGourleyGreenhouse)WheatBreeding(NellyArguello-Blanco,WilliamsGreenhouse,N.Westwing)DigitalPhenotyping(NathanNordstedt,WilliamsHeadHouse)BarleyBreedingandGenetics (Dr.EricStockinger,GourleyGreenhouse) 5:00-7:00NetworkingEvent,MillerPavilion,SecrestArboretum(33onCampusMap)DinneronownApril7,OSUPlantScienceSymposium

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ABSTRACTSRootstockBreeding

AssessingScionCompatibilityinTomatoasaFunctionofYieldImprovement

DavidE.LoewenandCaryL.RivardDepartmentofHorticultureandNaturalResources

KansasStateUniversity AlthoughresearchwithgraftedvegetablesisfairlycommonaroundtheUS,mostofthesetrialsaredesignedtocomparerootstocksorevaluatetheeffectivenessofspecificgrowingpracticesorenvironments.Littleresearchhasbeenconductedtodetermineifgraftingwithaparticularrootstockareconsistentacrossdifferentscioncultivars.Therefore,ourobjectiveswereto1)examinetheeffectsofasinglerootstockonmultiple(10)scionvarieties,2)determinewhichscionvarietiesshowedahighdegreeofcompatibilitywiththerootstock,and3)identifygraftedornongraftedvarietiesthatshowcommercialpotentialforhightunnelproduction.Ourstudyemployed‘Maxifort’astherootstockandevaluatedten,determinate,redslicertomatocultivarsasscion.Thestudywasconductedduring2016and2017,andtookplaceinonebayofathree-season,multi-bayhightunnel.Nongraftedandgraftedtreatmentswereincludedforeachvariety.ThestudyutilizedaRCBDwithfourreplicationsandfiveplantsweregrownineachplot.Yearhadaverystrongeffectonyield.Alltenvarietiesprovidedsomenumericalbenefitfromgraftingwith‘Maxifort’rootstockandaveragemarketableyieldimprovementsrangedfrom8.2%to34.6%.‘RedDeuce’,‘Skyway’,and‘TastiLee’maybegoodcandidatesforgraftingwith‘Maxifort’inadditionto‘BHN589’.Thesevarietiesdisplayedthehighestdegreeofgraftingcompatibilityasitrelatestofruityieldandrangedfrom28.6%to34.6%.Interestingly,‘RedDeuce’graftedonto‘Maxifort’rootstockdisplayedthehighestmarketableyieldsofanycombinationtested,butthebenefitofgraftingwas28.6%whencomparedtonongraftedplants.Similarly,‘PrimoRed’showedinsignificantimprovements(8.2%)inyieldasaresultofgraftingwith‘Maxifort’,butwasthehighestproducingnongraftedvarietyinbothyears.Elucidatingtheinteractionsthatoccurbetweenscionandrootstockandtheirimpactonfruityieldmaybeonequantitativewayofassessingrootstock/scioncompatibilityintomato.

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Vigor,Resistance,andRootstocks

SeanFenstemaker,NancyHuarachiMorejon,MohammedMasud,MattKleinhenz,

StephanieWedrykandDavidFrancis,TheOhioStateUniversity.FrankLouws,NCStateUniversity

Graftingtorootstocksisbecomingpopularinannualvegetableproductiontocontrolsoilbornediseases,replacefumigation,andimpartvigor.Weinitiatedarootstock(RS)breedingprogramin2007toaddressthefeasibilityofusinggraftedplantsinsoil-basedproduction,includingcertifiedorganicsystems.Atotalof110rootstocksweredevelopedthroughpollinationof10cultivated(Solanumlycopersicum)parentallinesasfemaleplantsand11accessionsofwildspeciesasmaleparents.UnderopenfieldconditionsusingtheF1scion‘Celebrity’wefoundlittleevidenceforyieldincreaseswiththeexceptionoftrialswheresoil-bornediseasereducedyields.IncontrastRSperformancetrialsconductedwithheirloomscionsorunderseasonextensionexhibitedyieldincreases.WedeterminedthatsomeRSwereabletoimpartvigor,measuredasincreasedgrowthandcanopydensity.TherewasapositivecorrelationwiththegeneticdistancebetweenparentsusedtocreatehybridRSandvigor.Therewerenegativecorrelationsassociatedwithgeneticdistanceandtraitsassociatedwithseedquality.Environmentalandgeneticeffectsonseedqualitywereevaluatedduringtwoseedproductionseasonsandbyharvestingfruitatdifferentmaturationstages.Seedqualitywasassessedbasedonseedsizeandgerminationrates.Seedsizewasinfluencedmorebyparentalgeneticsthanenvironment.Seedgerminationwasinfluencedbygeneticsandenvironmentalfactors.UsingseedweightasselectioncriterioninbreedingmightallowearlyselectionforhighqualityRS.Keyselectionpointsinthebreedingofnewtomatorootstockcultivarswereidentified.

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4-DIMENSIONALCOLONIZATIONDYNAMICSOFTHEGRAFTEDTOMATO-BACTERIALWILT

PATHOSYSTEMANDTHEIRIMPLICATIONSFORRESISTANCESELECTION

*JonathanKressinz,NorthCarolinaStateUniversityDept.ofHorticulturalScienceandDept.ofEntomologyandPlantPathology;DilipR.Panthee,NorthCarolinaStateUniversityDept.of

HorticulturalScience;FrankJ.Louws,NorthCarolinaStateUniversityDept.ofEntomologyandPlantPathology;MarcPlanasz,NuriaSanchez-Coll,andMarcValls,CentreforResearchin

AgriculturalGenomics.zFirstco-authors

Thebreedinghistoryoftomato(SolanumlycopersicumL.)resistancetobacterialwilt(Ralstoniasolanacearumspeciescomplex)hasbeenriddledwithenigmas,conflictingreports,anddecadesoflowgeneticgain.Themostefficaciousresistancehasbeenavailableformorethan40years,yetithasonlybeeneffectivelydeployedinrootstockvarietiesbecauseoftheextremedifficultyofintrogressinghighlevelsofresistanceintolarge-fruited,pleasanttastinggeneticbackgrounds.These resistant rootstocks have been a primary driver of the adoption of grafted tomatoproductioninNorthCarolinaandelsewhere,yetthequantitativeresistanceintomatodoesnotprevent thebacteria from invading rootsandhypocotyl tissuewhere thegraftunion resides;ratheritpredominantlyrestrictstheabilityofthepathogentocolonizeadjacentxylemcells.Weexplored this enigmatic aspect of bacterial invasion patterns over time and space in graftedtomato combinations (susceptible to highly resistant) with luciferase- and green fluorescentprotein-labeledGMI1000strainstobetterunderstandwhyvegetablegraftingenduresasaviablemanagementstrategy.Throughdestructivesamplingandnovelimagingtechniques,ourresultssuggest that tomato resistance induces a series of tug-of-war arenas that are sequentiallyovercome for thebacteria toeffectively colonize theplant—root invasion, rootproliferation,radial vascular colonization, vertical xylary translocation (unclear), and pith/cortical tissueinvasion.Theworkalsoclearlyhighlightskeyaspectsofthisquantitativetug-of-wardynamicthatresultsinamostlybinarydiseaseresponse—wilt.Wiltdevelopmentismostrelatedtothetug-of-wardynamicinthebasalhypocotyl,whereeffectiveresistancesuppressesbundleinvasion,notjustinvasionwithinbundles.Wehavethenexploredwaystomeasurekeyaspectsofthatinteractioninamorehigh-throughputmannerthatwouldberequiredforbreedingprograms,specificallythevascularbrowningsymptomology.Althoughdestructive,itcouldprovidearapidvisualmetricforassessingcolonizationresistanceatthevasculartissue-systemlevel.Themeritsanddrawbacksofthismethodwillbediscussedbriefly.Ourworkprovidesbreederswithnewtoolsandaframeworkformeasuringandselectingforresistanceateachimportantanatomicallocation.

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Rootstockforthecomputercontrolledenvironment,anindustryperspective

RafaelLacaz-Ruiz.Ag.Eng.UniversityofSãoPaulo.TomatoTechnicalDevelopmentRepresentativeforDeRuiterinNorthAmerica.

ThedemandforconstantsupplyoftopqualityfreshtomatoesfromhightechgreenhousesinNorthAmericahasbeengrowingconstantlyinthepastdecade.Consumersareleadingthisdemand,lookingformoreandbetterproduce.Consequently,therehasbeenmorecompetitionamongstmajorsupermarketersthatseeinthismarketanopportunityforincreasedsales.Inresponse,theindustryhasbeenaggressivelyexpandingitsareainMexico,CanadaandUSA,causinganaturalpricecompetition.Underthisscenarioofpressureandopportunitychase,Growershavebeeninnovatingaggressivelybyincreasingtheirefficiencyandincreasingthesizeofgreenhousestomaintainprofitabilityandremaincompetitive.Theuseofrootstocksiscurrentlywidelyadoptedandofupmostimportanceinthegreenhouseindustrybecauseitprovidesadditionalvigorforlongcropsanddiseaseresistancesthatarecommonlypresentingreenhouses.Understandingwhatarethemajorchallengesandneedsrelatedtouseofrootstocksthegreenhouseindustrytocontinuedeliveringtherighttoolsforgrowersiskeytoansweringthisdynamicgrowth.

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BreedingTechnologyUNDERSTANDINGSEGREGATIONDISTORTIONANDREPRODUCTIVEBARRIERSTOIMPROVE

TRANSFEROFTRAITSFROMSOLANUMPENNELLIITOCULTIVATEDTOMATO

BarbaraE.Liedl,DepartmentofBiology,WestVirginiaStateUniversity,Institute,WV,25112

Thegermplasmofmanycrops including tomato isnarrowdue toconstraints imposedduringdomesticationandspread,thusincreasingtheimportanceofwildrelativesasasourceofgenetic variation. However, segregation distortion, linkage drag and reproductive barriersimpede the transfer of desirable traits from thewild to cultivated species. This projectwasundertaken to understand segregation distortion and reproductive barriers in the F2 andbackcross(BC)populationsfromtheinterspecificcrossbetweentomato,S.lycopersicum,andthewildspecies,S.pennellii.

The three populations (F2, BCLyc and BCPen) derived from crosses between S.lycopersicumM82andS.pennelliiLA716andtheirinterspecificF1wereevaluatedfor%pollenstainability,and%seedgerminationaswellasiftheplantshadproducedflower,fruitorseed.Percentstainablepollenvariedgreatlyfortheparents:99.22%M82,16.31%LA716and85.7%oftheinterspecifichybrid,F1.Bothbackcrosspopulationshadsimilarmeans,57.9%BCLycand48.9%forBCPen,whiletheF2meanwas31.7%butvariedfromzeroto99.2%.TheF2,asexpectedfrompreviousresearchhadonly27.75%ofthepopulationproducingfruit,ofwhich,only9.43%producedseed.Asexpected,alloftheBCLycfloweredand98%producedfruitwithgerminableseed. Conversely, themajority of the BCPen produced flowers, but only 8.7% of the plantsproducedself-fruitandonly3.26%producedseed.Onlytwopopulations,F2andBCLycexhibitedanyabnormalseedgermination(18.8%and1.1%respectively),withallofthegerminatedseedfromtheF2beingabnormal.

Ofthe1000SNPmarkers,nodatawasobtainedfor181markerswiththehighestfailurerateonchromosome6(28.7%).Anadditional184markerswerenotusedbasedonnovariationor incorrect markers found, leaving at most 635 markers to be used for analysis. The F2population, as expected,had43%of themarkersdeviate from theexpected1:2:1 ratio. TheBCLycpopulationhadover63%oftheirmarkersdeviatingfromtheexpected1:1ratiowithexcesstomato alleles and all of the markers on two chromosomes (1 and 8) skewed. The BCPenpopulation,only21.9%ofthemarkersdisplayeddistortedsegregationandonechromosome(1)didnotdisplayanyskewingfromtheexpectedratio. Inaddition,mostoftheskewingBCPenmarkersfavoredhomozygousLA716combination,butafewskewedtowardstheheterozygouscombinationonchromosomes5and9. Eachpopulationwasexamined for thepercentofS.pennelliigenomepresent.FortheF2,theexpectationwas50%andweobserved54.4%(rangingfrom18.6to75.48).Thetwobackcrosspopulationswereprojectedtohave25%(BCLyc)and75%(BCPen),ouranalysisfoundanaverageof17.8%forBCLyc(range0-35.9)and77.6%forBCPen(range63.7-90.7).Mapsofeachpopulationweremade,butproblemswiththelargeamountofskewedmarkersintheBCLyclimitedthenumberofmarkersthatcouldbeusedtocreateamap.Analysisofthedataisongoing.

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Time-saving,cost-effectiveSNPgenotypingstrategiesfromLGCGenomicsLeahBenedict,ClientExecutive,GenomicsLGC,3600MinnesotaSt.Alexandria,MN56308

Singlenucleotidepolymorphisms(SNPs)havebecomethemolecularmarkerofchoice

formanycropimprovementprograms.Historically,alackofsufficientSNPmarkershasbeenasignificantobstaclefortomatobreeders.Commendableeffortsmadeinthelast20yearshaveunlockedthispowerfulmoleculartool,allowingforfocustoshifttoidentificationoftime-saving,cost-effectiveSNPgenotypingstrategies.

Inthistechnologyshowcase,wewilldescribeanautomatedSNPgenotypingworkflowdevelopedtominimizehands-onoperationtime,reducereagentcosts,andboostproductivity.TheoKtopure™,aDNAextractionrobotoptimizedforusewithoursbeadex™chemistrywiththecapacitytoextracteight96-wellplatesinasinglerun,willbehighlighted.Additionally,wewillprovideanoverviewoftheIntelliQube®,ourfully-automatedPCRplatformthatintegratesliquidhandling,sealing,thermalcycling,signaldetection,anddataanalysisonasingleinstrument.High-throughputSNPgenotypingbecomesstreamlinedwhenpairingtheIntelliQubewithourKompetitiveAlelleSpecificPCR(KASP™)genotypingchemistry.TheKASPtomatopanel,developedbytheSolanaceaeCoordinatedAgriculturalProject,furthersimplifiestheadoptionofKASPfortomatobreeders.Thesetechnologiesproviderobustandeconomicalsolutionstoovercomethroughputandcostchallengesthatcanhindertomatobreedingprograms.

PlexSeq:atargetedampliconbasedsequencingmethodforSNPgenotypingScottWeigel,SalesDirectorAgriPlexGenomics

AgriPlexGenomicsisafullservicegenomicslaboratorylocatedinCleveland,OH.OurPlexSeqsystemcanscreen10to3000SNP'sacross100to100,000samplessimultaneously,makingPlexSeqanextremelyrapid,low-cost,versatilesystemforplantbreeders.

Movingbeyondasinglereferencegenome:GenoMagicTM,anovelsolutiontodescribeandmanagegenomicvariation

PaulChometandDavidNeuman,NRGeneNextGenerationsequencingtechnologieshaveopenedthedoortomultiplegenomeanalysesandanincreasedunderstandingofthevariationspresentinpopulations.Todate,mostofthegermplasmanalyseshavereliedonthecomparisonofsequencereadstoonereferencegenomeassembly,limitingourunderstandingofgenomicvariation.NRGenehasdevelopednovelanalyticsandapproachestoefficientlydenovo-assemblegenomesandtodescribetherelevantvariationacrossgermplasmusingapan-genomeapproach.Wearepromotingapangenomeconsortiumthatwillenablefullgenomecomparativeanalysesfortomatogermplasm.Thelonger-termgoalisforthepan-genometoserveasareferencetofullycatalogthediversityinSolanumlycopersicumthroughsequence-basedhaplotypes.Thistalkwillfocusontheneedandadvantageofapangenomeandsequence-basedhaplotypestoforbreedingandgenediscoveryapplications.

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BACKGROUNDGENOMESELECTIONFORRAPIDINTROGRESSIONANDEVALUATIONOFQUANTITATIVETRAITLOCI(QTL)FORRESISTANCEINTOMATOTOMULTIPLE

XANTHOMONASSPP.*EduardoBernal,DeboraLiabeuf,andDavidFrancis,TheOhioStateUniversityDepartmentof

HorticultureandCropSciences.BacterialSpotoftomatoisafoliardiseasecausedbyfourspeciesofXanthomonas.Identificationofgeneticresistance inwildtomatoesandbreedinghasbeenafocusofourcontrolstrategy.Three independent sources of resistance, Hawaii 7998, PI 114490 and LA2533 have beendiscovered, and genetic studies have identified amajorQTLmapping to the same region onchromosome11.Genomeresequencingandanalysissuggeststhattheselociarenotidentical,thoughcurrentresolutiondoesnotallowustodistinguishallelesfromlinkedgenes.ToassesswhetheroneQTLprovidesbetterresistancetomultiplespecies,wedevelopednearisogeniclines(NILs)usingmarker-assistedselectionandbackgroundgenomeselection.Theresistantsourceswereindependentlyintroducedintoasusceptibleparent,OH88119.Insertion/DeletionmarkerswereusedtoselectfortheQTLregionandapanelofSNPmarkersassayedontheKASPplatformwereusedforbackgroundgenomeselection.ThisapproachallowedustorapidlydevelopNILsthatare95%-99%geneticallyidentical,exceptfortheQTLonchromosome11.In2016and2017weassessedmultiple linesdeveloped fromeachsource in independent field trials inoculatedwiththreespeciescausingbacterialspot(X.perforans,X.euvesicatoria,X.gardneri).TheNILswereevaluatedusingtheHorsfall-BarratScale(1-12),whichestimatesthepercentageofdiseaseonthefoliage.LinearModelswereusedtomakecomparisonsbetweenQTLsourcesandalleliceffectswithinsource.Theresultsshowthattherearesignificantdifferencesinbothcases,withHawaii 7998 providing the highest level of resistance to all three species. NILs resistant tomultiplespecieswillbereleasedforusebyprivateandpublicbreedingresearchprograms.

TherapidprogressofCRISPRfortargetedcropmodificationAndikaGunadiandJohnJ.Finer,PlantTransformationandGeneExpressionLaboratory,DepartmentofHorticultureandCropScience,TheOhioStateUniversity,Wooster,Ohio

RecentlyavailablegenomeeditingtoolssuchasCRISPRhaveshowngreatpromisefortheintroductionofvaluabletraitsintotargetedgeneticlocationswithinplants.Theabilitytodelete,insert,andsubstituteDNAofvarioussizesintospecificlocationswithinaplantgenomehavefar-reachingapplicationforcropimprovements,beyondthecapabilitiesofconventionaltransgeneintroductionandbreedingmethods.Withmorethan3,000totalpeer-reviewedpublicationsonCRISPRin2017,therapidpaceintheapplication,development,andramificationsofthistechnologyinvariousfieldsoflifesciencescanbebothexcitingandalarming.ThispresentationwillprovideabriefoverviewontheprocessofplantgenomeeditingusingCRISPR.Recentadvancementsinthetechnologywillbehighlighted,andseveralcurrentapplicationsincropimprovementwillbediscussed.

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ExperiencewithGenomicSelectioninProcessingTomatoDavidFrancis,TheOhioStateUniversity,OARDC,Wooster,OH

TheOhioStateUniversityprocessingtomatobreedingprogramispredominatelyfocusedontrait-basedselectionforyield,qualityanddiseaseresistanceunderhumidgrowingconditions.Atthesametime,weareinterestedinselectionstrategiesthatleveragesequenceresourcesandtheabilitytoefficientlyquerysequencevariationinthegenome.RobustSNPresourcesdevelopedtodetectpolymorphismwithincultivatedbreedingpopulationshavemadeseveralapproachesaccessible.Theseincluderoutineuseofmarker-assistedbackgroundgenomeselectiontoincreasetheefficiencyoftraitintrogression.UsingBCpopulationsof192plants,selectingforatraitofinterestandapplying2-4backgroundmarkersperchromosomearmon96selectedplantshasledtorecoveryofprogenythatareafullgenerationaheadduringeachcycle.WehavealsoappliedGenomicSelection(GS)strategiestoourbreedingeffortsforbacterialspotandyield.Wefindthatmarkercoverageintherangeof20-300markersissufficientforgainsthatexceedphenotypicselection.Usingpriorknowledgeoflinkagereducesthenecessarynumberofmarkers.IncorporatingknowledgeofgeneactionintoGSmodelsincreasesprediction.CrossvalidationofGSmodelswithininbredpopulationsshowstrongprediction(0.4-0.6foryieldtraits).Post-hocanalysisofhybridsdevelopedfromthesameinbredsusedtocreatetrainingmodelsshowpredictionintherangeof0.2.RecentlywehaveappliedGSstrategiesforparentchoiceindesigningnewhybridsusinginbredlinesthatwerenotintheoriginaltrainingpopulation.Predictionofperformancewasstatisticallysignificant(0.03)thoughpredictionwaslow(0.1).GSstrategiesleadtoyieldincreasesof1.2T/Arelativetophenotypicselection.CombiningGSandphenotypicselectionleadtoyieldincreasesof4.3T/Arelativetophenotypicselectionalone.OurexperiencereinforcestheroleofmarkersandGSstrategiesasatooltosupplementbreederknowledgeandimprovedecisionmakingduringtheselectionprocess.

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PestResistance

IntrogressionofZingibereneandTypeIVTrichomeDensityfromSolanum.HabrochaitesLA2329intoS.lycopersicum–ProgressReport

JohnSnyder,AmmarAl-BayatiandMohammadDawood,Dept.ofHorticulture,Universityof

KentuckyInsummer2012,wegrewoutapproximately1500BC2F1individuals.DonorwasaBC1F1havinghighzingibereneconcentration,asesquiterpenehydrocarbonpresentintrichomesecretionsonleaves,hightypeIVtrichomedensityandhighresistancetospidermites.AllBC2F1plantswereevaluatedfortypeIVdensity,fruitsetinthefieldandextentofspidermiteinfestationinthefield.Approximately10%oftheplantshadtypeIVtrichomedensitysimilartothatofthewildaccession,andapproximately10%hadfruitsetinthefield.Cuttingsof19individualsofinterestwererootedandthengrowninthegreenhouseinfallof2012andwinter2013.Trichomes,trichomesecretioncompositionandabundanceandselffruitsetwereassessedonthesegenotypes.Ofthe19genotypes,only10producedsufficientseed(BC2F2)forfieldproductionin2013.TheseplantswereevaluatedfortypeIVtrichomedensity,fruitsetandtrichomesecretioncompositionandabundance.Cuttingswereobtainedfromafewoutstandingindividuals.Afteradditionalevaluationinthegreenhouse,weidentifiedoneindividualhavingaconcentrationofzingibereneandtypeIVtrichomedensitysimilartothoseofthewildprogenitor,butnofruitset.ThisdonorplantwasusedasfemaletogeneratetheBC3generation.AtpresentthepopulationisatBC5.Progresstodate,includingmethodsusedforassessingzingibereneconcentrationandtypeIVtrichomedensitywillbepresented.TheoverallgoalistocombinereliablefruitandseedsetwithhighzingibereneconcentrationandhightypeIVtrichomedensity.

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ANEWSESQUITERPENEALCOHOLFROMWILDTOMATOREPELLEDTHETWO-SPOTTEDSPIDERMITE,TETRANYCHUSURTICAE

MohammadHDawoodandJohnC.Snyder,

UniversityofKentucky,DepartmentofHorticulture,Lexington,KY40546,USATomato(SolanumlycopersicomL.)isoneofthemosteconomicallyimportantworld-widegrownvegetable. However, tomato is a host for numerous pests that reduce productivity. Tomatobreedershavefocusedmoreonincreasingfruitquantityandqualityandlessonenhancingcropresistance toherbivores.Manyaccessionsof thewild tomato relative,Solanumhabrochaitesstrongly resist arthropod attacks. The monocyclic sesquiterpene hydrocarbon known aszingibereneisfoundinSolanumhabrochaitestypeIVglandulartrichomes.Toinvestigateifothercomponents of type IV trichome exudates are responsible for S. habrochaites resistance toarthropods, seedsofS.habrochaites LA2329weregerminatedandgrownundergreenhouseconditions.Thequestionpursuedwas“Doothercompoundspresentintrichomesecretionshaverepulsiveactivitiesagainst thetwospotted-spidermiteTetranychusurticae, similar to thatofzingiberene?Accordingly,LA2329trichomeleafexudateswereextractedandaportionoftheirextracts was injected into a gas chromatograph equipped with a mass selective detector(GC/MSD) to separate and identify trichome exudates of LA 2329. The results revealed thepresenceoftwopredominatechromatographicpeaks.Oneofthesewas7-epizingibereneandthe other was an unidentified compound, but, based on its mass spectrum is likely asesquiterpene-alcohol,relatedtozingiberene.Toinvestigatetherelativeactivitiesofthesetwomajorchemicalconstituents,crudeextractsoftrichomesecretionswereseparatedbysilicagelcolumnchromatographyandtheresultingfractionswereevaluatedforrepellencytospidermiteusing bridge bioassays. Results revealed that the activity of sesquiterpene alcohol wassignificantlyhigherthanthatforzingiberene.Theseresultssupporttheideathatthedegreeofrepellency may differ among components of trichome exudates and also highlighted thepotentialvalueofintegratingthesesquiterpenealcoholintocultivatedaccessionsoftomatotoimprovearthropodresistanceincommercialtomatocultivars.Keywords:Trichome,Zingiberene,Sesquiterpene-alcohol,Tomato,Spidermites,Repellency

21

THEROLEOFTRICHOMESECRETIONSANDDENSITIESINSPIDERMITEBEHAVIORFORAN

INTERSPECIFICPOPULATIONOFTOMATO

AmmarAL-Bayati,JohnSnyder,Dept.ofHorticulture,UniversityofKentucky,Lexington,KYThereissignificantnecessityforassayplatformstoassessandidentifiedlevelsofresistanceandinsectperformance,asdemonstratedbyrecentpublicationsreportingthissubject.Thewholeleafbioassayrequiresverylimitedphysicalspaceandlabortoassessandidentifylevelsofplantresistanceandinsectbehavior.AninterspecificpopulationbetweenSolanumlycopersicumandawildrelative,SolanumhabrochaitesLA2329,providedtheplantmaterialusedinthisresearchandwasmaintainedinthegreenhouseandfieldinsummer2017attheUniversityofKentucky,Lexington,Ky.Thepopulationhasbeenproducedtoimprovearthropodresistanceoftomatobyintrogressionofhighzingiberene(ZGB)concentrationandhightypeIVtrichomedensityfromthewildtocultivatedtomato.Theobjectiveofthisresearchwasto(1)observethedifferencesofmitebehavior amonga rangeof hybrids compared to thepositive andnegative controls, (2)identifytheinteractionsbetweengenotypesandleafsurfaceswithspidermiteswithrespecttopresence and absence of type IV trichomes and chemical profiles and, (3) determine theinteraction of zingiberene and type IV trichomes on mite performance. Thirteen tomatogenotypeswiththreereplications(BC3F3&BC3F4)wereselectedbasedonpresenceorabsenceoftrichometypeIV,andZGB.Awholetomatoleaf,consistingoffiveleafletswastransferredtothelaboratory,insertedintoa250-mlflaskfilledwithtapwaterandsuppliedwithfluorescentlight.,.Thenabeanleafinfestedbyaboutthirtytwo-spottedspidermites,TetranychusurticaeKoch,wasplacedatthebaseofthetomatoleaf.Leafletposition(1-5)andsurfaceinfestedbymites,mitewebbing score (0-3), feedingdamagescore (0-3), leafletpositionofwebbingandfeedingdamage(1-5),eggdensitypercm2aswellwererecordedandanalyzedbySASusingGLM&CORRprocedures.ThreeleafletsfromtheleafadjacenttothebioassayedleafwereanalyzedforZGBandmonoterpene(MTP)byGC-FIDplusanotherleafletwastakenforcountingnumberof type IV trichomes on abaxial and adaxial surfaces using a microscopic grid. There weresignificantnegativecorrelationsbetweentotaltypeIVtrichomedensitiesandnumberofleafletsandsurfaces infestedbymites,mitewebbing,numberof leafletswithmitewebbing, feedingdamage, number of leafletswith feeding damage, egg counts and density per cm2. ZGBwasnegativelycorrelatedwitheggcountsanddensities.Basedonreducedmitesuccessonsomeofthegenotypes,weconcluded that resistancehasbeen successfully transferred from thewildaccessionstotheinterspecificpopulation.Thisbioassaydemonstratedbehavioraldifferencesofmitesassociatedwiththepresenceorabsenceofleafcompoundsandtrichomedensities,aswellassupportedtheideathatintrogressionoftypeIVtrichomeandZGBwillleadtogreaterspidermiteresistance.Newplantversionsthatcanproducetoxicorrepellentchemicalsontheirowncandefendthemselvesagainstcertaintypesofarthropodsandinsectswhichinturnreduceoreliminateextensivesyntheticpesticideutilizationandcost.

22

PrebreedingTomatoforOptimizedAcylsugar-MediatedResistancetoInsects

MarthaMutschler1,DarleneDeJong1,JohnSmeda2,TaylorAnderson1,DianeUllman3

1PlantBreedingandGeneticsSection,SIPS,CornellUniversity2FormerlyPlantBreedingandGeneticsSection,SIPS,CornellUniversity;currentlyFSU3Dept.OfEntomology,UC-Davis

AcylsugarsproducedbythewildtomatoSolanumpennelliicontrolmajorpestsoftomato.Priorbreedingcreatedthefirsttomatolinesproducingacylsugarlevelsimpactingmajorpests.Tomatolinesproducingdifferentlevelsofthesameacylsugarsshowedtheimportanceofacylsugarlevel.UsingthebenchmarklineCU071026,tomatolineswerebredthatpossessadditionalS.pennelliiintrogressionscontainingQTLthatimpactfattyacidcomponentsofacylsugars;theselinesvaryforthefattyacidprofilesorsugarsoftheiracylsugars.Testingthesetomatolinesperformedbycooperatorshaslinesthatproducedifferentacylsugarchemotypesalsovaryinefficacyofinsectcontrol. Furthermore, the optimal lines for control of vector insect species also reduce thelikelihoodofviruspresenceafterplant/planttissueisexposedtoviliferousinsects.

Use of acylsugar mediated resistance in commercial hybrids requires that any negativehorticultural traitsmaintained inacylsugar lines through linkagedragare removed.Themostsevere of these negative traits was lack of normal fruit set; this trait has been resolved byalterationsonchromosome3andchromosome8,resultingincreationofthenewbenchmarklineCU17NBL-1.DeconstructionofCU070126createdaseriesofmonoanddiintrogressionlines,and recovered plantswith recombinations in each of the 4 targeted introgressions, allowingmappingofQTLresponsiblefortheothernegativehorticulturaltraits:smallfruit,shortbranchyplanttype,andoffflavortofruit,facilitatingtheirremovalofeachofthesetraits.Thisworkisprogressingveryrapidly. SmallfruitQTLwerelocatedonchromosomes2,3and10;a line inwhichthechromosome10smallfruitQTLhasbeenremovedhasalreadybeencreated.Theoffflavor trait is governedby aQTLon chromosome3; several lines lacking thisQTLhavebeenidentified.Aseachcaseoflinkagedragisrectified,thealteredintrogressionisbackcrossedintoa common line, which eventually will possess all of the altered introgressions needed foracylsugarproductioninalinewithgoodhorticulturaltype.

23

DiseaseResistanceDiscoveryandimprovementofnovelandknownresistancetoFusariumwiltracethreeof

tomato.

JessicaL.ChitwoodandSamHutton,GulfCoastResearchandEducationCenter,UniversityofFlorida,Balm,FL,USA.

Tomato(Solanumlycopersicum)isoneofthemostimportantvegetablesintheU.S.,butbothfresh-marketandprocessingtomatoproductionisincreasinglythreatenedbyFusariumwiltrace3(Fol3)causedbythesoil-bornefungus,Fusariumoxysporumf.sp.lycopersici.Fumigationhastraditionallybeenusedtohelpmanagemanysoil-bornediseasesincludingfusariumwilt,butthephase-outofmethylbromidelefttheindustrywithoutaneffectivereplacementandculturalcontrolmethodsareinadequate.HostresistancetoFol3throughtheI-3geneisthemosteffectivemanagementstrategy.However,producersoftenchoosetogrowsusceptiblecultivarsduetotheassociationofI-3withdetrimentaltraits,includingbacterialspotsensitivityandsmallfruitsize.WeareusingadualstrategytoproducemoredurableFol3resistancethatisfreefromtheseproblems.Inordertoaddresspotentiallinkage-drageffects,wereducedtheI-3introgressionfrom5Mbtoapproximately120Kbthroughsuccessiverecombinantscreeningandcrossingefforts.ThereducedintrogressionwascrossedintoeliteFloridabreedinglinesandevaluatedforitseffectsonbacterialspotsensitivityandfruitsize.Currentdatashowsthereducedintrogressionresultsinsignificantlylessbacterialspotandlargerfruitsizethantheoriginal4Mbintrogression,andithasnoeffectcomparedwithFol3susceptibility.Takentogether,theseindicatethatlinkagewiththesenegativetraitshasbeenbroken.Topromotegreaterdurabilityofresistance,wealsosoughttoidentifynovelFol3resistance.ThewildtomatorelativeS.pennelliihasbeenshowntobehighlyresistanttoallracesofFusariumwilt,andwesubjected42accessionstodiseasescreenstoidentifyresistance.MolecularmarkerswereusedtoscreenawayfromtheI-3andI-7resistancelociasFol3resistancewasintrogressedfromtheseaccessionsintoelite,susceptibletomatobackgroundsviaseedlingdiseaseassaysandbackcrossing.Afterfourgenerationsofbackcrossing,resistantplantswereevaluatedinthefieldforhorticulturaltraits.F2progenyofsingle-plantselectionsrepresenting36oftheaccessionswerescreenedforresistancetoFol3,andratiosofhealthytoinfectedplantswereusedtoidentifyaccessionsmostlikelytohavecontributedsingle,dominantresistancegenes.Phylogeneticrelationshipswerealsoconsidered,andfourbackcrosspopulationswithresistancederivedfromaccessionsinthreephylogeneticclusterswerechosenformappingnovelresistancealleles.Thenovelalleleswillbecharacterizedforefficacyagainstraces1and2aswellasacollectionofdiverseFol3isolates;andwillalsobepyramidedwithcurrentlyavailablealleles.TheseeffortswillresultinthedevelopmentofimprovedFol3resistantcultivarsandmoredurableresistanceagainstthispathogen.

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ASCREENOFGENETICALLYDIVERSETOMATOACCESSIONSREVEALSARANGEOFUNUSUALRESPONSESTOPseudomonassyringaepv.Tomato

*RobynRoberts,SamanthaMainiero,AdrianF.Powell,SarahR.Hind,SusanR.Strickler,Boyce

ThompsonInstitute;andGregoryB.Martin,BoyceThompsonInstituteandtheSchoolofIntegrativePlantScience-PlantPathologyandPlant-MicrobeBiology,CornellUniversity

Pseudomonas syringae pv. tomato (Pst) causes bacterial speck disease of tomato. The onlygeneticresistancetoPstisconferredbytheRgenePto,whichrecognizesthePsteffectorsAvrPtoandAvrPtoB.However,Pststrainshaverecentlyemergedwhichlacktheseeffectors,renderingPto-mediatedresistanceineffective.OurstudyaimstoidentifynewsourcesofgeneticresistanceagainstPstthatcanbeincorporatedintotomatovarietyimprovementprograms.Usingavailablewhole-genome resequencing data, we leveraged natural variation among tomato heirlooms,breedinglines,andawildspeciestoscreen216accessionsfornewsourcesofresistance.Weinoculated the accessions by spraying Pst wildtype and mutant strains which lack specificeffectorsand/orflagellintohelpelucidatewhetherobservedhostresponses involveeffector-triggered(ETI)orpattern-triggered(PTI)immunepathways.Interestingly,ourscreenuncoverednewPstdiseasephenotypesbeyondthetypicalspecksymptoms,andwehavefoundthatsomeofthesephenotypesaresimplyinherited.UsinganassaythatmeasurestheproductionofPTI-related reactive oxygen species, we discovered new accessions that have increased, orconversely,noresponsetotheflagellinpeptidesflg22orflgII-28,whicharerecognizedbythepattern recognition receptors FLAGELLIN SENSING 2 (FLS2) or FLAGELLIN SENSING 3 (FLS3),respectively.Theseresultsprovideagatewayforthestudyofthemolecularmechanismsactingdownstreamofthesetwopatternrecognitionreceptors.Wearecurrentlyusingavailablewhole-genome resequencingdataandgeneticapproaches to identifygenecandidates thatunderlienovelresistanceorsusceptibilitytoPst.

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FINEMAPPINGANDCANDIDATEGENECHARACTERIZATIONOFTHEPEPPERBACTERIALSPOTRESISTANCEGENEbs6

*RebeccaL.Wente,JianLi,SamuelF.Hutton,GulfCoastResearchandEducationCenter,

UniversityofFlorida;JeffreyB.Jones,GeraldV.Minsavage,RobertE.Stall,DepartmentofPlantPathology,UniversityofFlorida

Bacterial spot, causedbymultipleXanthomonas spp., is amajordiseaseof tomato (SolanumlycopersicumL.)andpepper(CapsicumannuumL.) inwarm,humidenvironmentalconditions.The disease is characterized by necrotic lesions on the leaves and fruit, and can result insubstantial crop losses. Host resistance has been an objective in many tomato and pepperbreedingprograms;anddominantresistancegenes thatelicitahypersensitiveresponsehavebeenidentifiedinbothcrops.However,thesegeneshavelackeddurabilityinthefield,resultingin continued efforts to pyramid known resistance genes and to identify novel sources ofresistance. Conversely, the recessive pepper resistance gene, bs5, has demonstrated moredurable resistance toX. euvesicatoria races 0-10. A second recessive gene,bs6, is known toprovide a higher level of resistancewhen combinedwithbs5, andmay further promote thedurabilityof resistance to thispathogen.Whereas thebs5 locuswas recentlydiscovered, thelocation of bs6 remains unknown. To map and fine map bs6, an F2 population and F2:3recombinant inbred lines (RILs)werecreated fromthecrossbetweenthesusceptiblecultivar'EarlyCalwonder'(ECW)andanECWnear-isogeniclinethatcontainsbs6(60R).GenotypingbysequencingofECW,60R,and93F2plantsmappedbs6toa27Mbregiononchromosome6.RILswere phenotyped by successive disease screens and genotypedwithmarkers saturating thisinterval,delimitingbs6toa666Kbinterval.Candidategenesweresequencedandsubjectedtogene expression experiments for characterization. These results will improve breeding andselectioneffortsinpepper,andfurtherworktoclonethebs6genecouldaccelerateeffortstoengineerbs6-basedresistanceintomato.

26

TomatoImprovementforFruitQualityandDiseaseResistanceatNCStateUniversity

DilipR.PantheeDepartmentofHorticulturalScience,NorthCarolinaStateUniv.,455ResearchDrive,Mills

River,NC28759

Asacontinuousefforttoimprovetomatoesfordiseaseresistanceandfruitquality,wearemakingsomeprogressinlarge-fruited,plumandgrapehybriddevelopmentatNCStateUniversity.Asaresult,wehavedevelopedNC13506whichisresistanttoVerticilliumwilt(VW;Vegene),Fusariumwilt(FW;races1,2and3)(IandI-2andI-3genes),Tomatospottedwiltvirus(TSWV;Sw-5gene),Tomatomosaicvirus(ToMV;Tm2gene),androot-knotnematode(RKN;Migene).BothparentsofNC13506carrytherecessiveogcgeneforcrimsonfruitcolorresultinginexpressionofimprovedredcolorandincreasedlycopenecontentintheF1hybrid.ThecombinationofahighlevelofresistancetotheTSWValongwithresistancestoVWandraces1,2and3ofFW,thehighmarketableyieldoflargefruitdesiredforvine-ripeproductionandimprovedredcolormakeNC13506auniquehybrid.Moreimportantly,thishybridmaturesaboutaweekearlierthanstandardcontrol,whichmaybebeneficialfortomatogrowers.

AnewplumpotentialhybridtomatoNC11265hasbeendeveloped,whichisresistanttoVW,FW(IandI-2genes),lateblight(LB;Ph-2andPh-3genes),andTSWV.BothparentsofNC11265carrytherecessiveogcgeneforcrimsonfruitcolorresultinginexpressionofimprovedredcolorintheF1hybrid.ThecombinationofahighlevelofresistancetoTSWV,VWandraces1and2ofFW,earlymaturity,andimprovedredcolormakesNC11265uniquehybrid.ResultsfrommultiplereplicatedtrialsindicatedthattheyieldperformanceofNC11265washighlystableinNorthCarolina.

IntheimprovedgrapetomatohybridNC10259,wehavecombineddiseaseresistanceincludingFWrace3(I-3gene),TSWVandLB(Ph-2andPh-3genes)resistance.Yieldperformanceofthishybridwasconsistentlyhigherthancontrol,andtotalsolublesolids(TSS)contentwascomparabletootherhybrids.Thesehybridsmaybeanewadditiontooureffortstoimprovetomatoforfruitqualityanddiseaseresistanceinourprogram.

27

BACTERIALSPOTOFTOMATO:PATHOGENSTORYANDOUREFFORTSOFTOMATO

IMPROVEMENTINNC

*PragyaAdhikari,DilipR.Panthee,HamidAshrafi,ChristopherC.Gunter,NorthCarolinaStateUniversityDepartmentofHorticultureScience;FrankJ.Louws,NorthCarolinaStateUniversity

DepartmentofEntomologyandPlantPathology;ConsueloArellano,NorthCarolinaStateUniversityDepartmentofStatistics

BacterialspotoftomatoisaseriousdiseaseinNCandmanyotherStates,causedbymultiplebacterialspeciesandphysiologicalraceswithinthegenusXanthomonas.Thediseasepotentiallycausesupto66%yieldloss.However,practicalmanagementofthisdiseaseischallengedbylackof effective chemical compounds and commercial resistant cultivar. Field surveys wereconductedinmajortomatoproductionareasofwesternNorthCarolinaduring2015/2016fieldseasonstoassessthepathogendiversitycausingbacterialspotdisease(Bsp)oftomato.Atotalof284Bspstrainswerecollectedandassessedforsensitivitytocopperandstreptomycin(100and200ppm)andgeneticdiversityusingBOX-PCRassay.Forty-six representativeBsp strainsweresubjectedtoreal-timePCRandassessedforahypersensitiveresponse(HR)todifferentiatespeciesandraces.Overall,nearly95.0%oftheXanthomonasstrainswereresistanttocopperand25%-44%resistanttostreptomycin.AllXanthomonasisolatesanalyzedinbothyearshadsimilargeneticprofiles,suggestingthatthesebacterialisolatesfromNCaregeneticallysimilar,despitefrom different seed and transplant sources. Real-time PCR results demonstrated that thebacterialspotpathogensinNCbelongtoasinglespeciesX.perforans.HRassayidentified93.5%ofXanthomonasstrainsasraceT4and6.5%asraceT3.Resultssuggestthatemergenceofcopperand streptomycin insensitive bacterial phenotypes in NC and a need for the investigation ofalternativestrategiesforcropmanagement.GeneticresistancetoraceT4hasbeenidentifiedinthe S. pimpinellifolium LA3707 derived line NC22L-1(2008) through greenhouse and fieldscreeninginpreviousstudies.WecarriedoutQTLanalysisusing110recombinantinbredlines(RIL) froma cross betweenNC-NC30P x 22L-1 (2008.A linkagemapwith 886 SNP lociwasconstructed covering 739.5 cM of 12 chromosomes of tomato, with an average of 0.83 cMbetweenmarkers.TwomajorQTLswereidentifiedonchromosome6atpositions11.83cM-14.33cMand27.49cMwithLODvaluesof4.0and8.0,respectivelyexplaining16%-26%ofthetotalphenotypicvariance.TheseQTLswereconsistentinatleasttwoenvironments.TwoQTLsweredetectedonchromosome1atpositions49.58cMand95.21cMwithLODvalueof3.0,explaining23%and16%ofphenotypicvariance,respectively.AnotherQTLisdetectedonchromosome12explaining9%-14%ofphenotypicvariance.ThemarkerslinkedtotheseQTLsmaybevaluableinamarker-assistedtomatobreedingprogramagainstbacterialspotdisease.

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PYRAMIDINGRESISTANCESTOBACTERIALSPOTANDBACTERIALSPECKINELITEFRESHMARKETTOMATO.

*TaylorAnderson,PlantBreedingandGeneticsSection,SIPS,CornellUniversity;MarthaA.Mutschler,PlantBreedingandGeneticsSection,SIPS,CornellUniversity;DavidFrancis,The

OhioStateUniversity

Significant losses are incurred in tomato due to the bacterial diseases bacterial speck andbacterialspot.Theneedforresistantbreedingstocksisincreasinglyimportantgivenwidespreadresistancetocopper-basedcontrol.TheCornellUniversity,incooperationwiththeOSUbreedingprogram,iscurrentlypyramidingseveraldiseaseresistancelocitotheunderlyingpathogensinfreshmarkettomato.Characterizedresistancelocitobacterialspeckandspot,onchromosomes5 and 11, are being pyramided in an elite, disease-resistant freshmarket breeding line thatalreadycontainsresistancestotheoomycetediseaselateblight,andthefungaldiseasesearlyblight,andSeptoria leafspot.Themarker-assistedbackcrossbreedingapproachpairsforwardandreverseselectiononallchromosomestoacceleratetherecoveryoftheelite,recurrentlines.Severalofthesegenesareinrelativelycloseproximitytoeachother,reducingthenumberoflinkagegroupswhichmustbetrackedtomovetheresistancestonewbreedingmaterials.Thefirstofthelines,possessingabacterialspotresistancegeneandresistance-associatedQTLonchromosome11,werecompletedinthewinterof2017,andwillbetrialedinthesummerof2018toevaluatetheimpactoftheselocionbacterialspotdiseasecontrolandonfreshmarkettomatomarket qualities. The lines possessing the chromosome 5 bacterial spot and bacterial speckresistancesarenearingcompletionandwouldbetestedstarting2019.

29

Quality

PURÉETOPEAKSIN15MINUTES:ARAPIDCAROTENOIDEXTRACTIONANDUHPLC-PDAANALYSISWORKFLOWFORTOMATOBREEDING(POSTER)

*MichaelP.Dzakovich,TheOhioStateUniversityDepartmentofHorticultureandCropScience;KenM.Riedl,TheOhioStateUniversityDepartmentofFoodScienceandTechnology;StevenJ.Schwartz,TheOhioStateUniversityDepartmentofFoodScienceandTechnology;JessicaL.

Cooperstone,TheOhioStateUniversityDepartmentofHorticultureandCropScienceandTheOhioStateUniversityDepartmentofFoodScienceandTechnology;DavidM.Francis,TheOhio

StateUniversityDepartmentofHorticultureandCropScience

Tomatoes(Solanumlycopersicum)areaneconomicallyandnutritionallyimportantcropthatowetheircolortocarotenoidpigmentssuchaslycopeneandβ-carotene.Publicandprivatebreedingprograms have focused on manipulating fruit carotenoid content to improve consumeracceptability and enhance potential health benefits associated with consuming tomatoes.However, accurate phenotyping of carotenoids requires lengthy extraction protocols andchromatographic separationmethods creating analytical bottlenecks that can reduce geneticgainperyear.Wedevelopedbotharapidtomatocarotenoidextractionprotocolandanultra-high performance liquid chromatography photodiode array detector (UHPLC-PDA) analysismethod. To validate our extraction and quantitation methods, backcross populations ofprocessingandcherrytomatoeswerecreatedwithvaryingfruitconcentrationsoflycopeneandβ-carotene by exploiting natural variation in the fruit-specific lycopene beta cyclase (Cyc-B).Thesepopulationswerephenotypedusingestablishedextractionandhighperformanceliquidchromatographyphotodiodearraydetector(HPLC-PDA)methodsinadditiontoourrapidtomatocarotenoid workflow. We estimated variance components for genetic and environmentalvariables forbothmethodsanddeterminedthatournewextractionandUHPLC-PDAanalysismethod performed comparably to established protocols while taking substantially less time.Notably,ourmethodswereapproximately2x(~5minutes/sample)and5x(4.2minutes/sample)fasterthancurrentextractionandHPLC-PDAmethods,respectively.OurUHPLC-PDAmethodwasable to resolvemost cis-carotenoid isomers from all-trans carotenoids.Our rapid carotenoidextractionandanalysisworkflowcouldgreatlyenhancetomatobreedingprogramsbydrasticallyincreasingthroughputwithoutsacrificinganalyticalperformanceorheritability.

30

BREEDINGFORPROCESSINGTOMATOQUALITY

SteveLoewen,UniversityofGuelphRidgetownCampus

Thepre-breedingeffortattheUniversityofGuelph’sRidgetownCampushastheoverarchinggoalofincreasinggeneticdiversityforprocessingtomatoes.TheworkbuildsontheaccomplishmentsofpreviousintrogressionofwildtomatospeciesintocultivatedtomatobyVainoPoysa.Througha strategy of backcrossing and phenotypic selection, the semi-wild lines are developed intobreeding lines that retain genetic diversity from thewild specieswhile resembling regionallyadapted,processingtomatoes.Historicallytheworkhasbeendevelopingbreedinglinestargetedtoward whole-peel end-use. The fruit quality attributes required for this end-use will bediscussed.Earlyresultsofourworkusinghp-1toimprovecolourandnutritionalvalueoffruitwillbepresented.Wehavemadesomeprogressimprovingfruitmaturity.Theprogressinourworkwithhigh-anthocyaninfruit inaprocessingbackground,developedindependentlyofthefresh-marketworkbyothers,willbereviewed.Studieshavebeenconductedthatbegintolinkanthocyaninsinfreshtomatoes,andpossiblyprocessedtomatoproducts,withpotentialbenefitsforhumanhealth.Thediscussionwillconcludewithsomereflectionsonbreedingforflavourintomatoes.Despitetomatobreeders’goodintentions,publicdiscourseabouttomatoflavourisfraughtwithchallenges.Weproposethatformanyyearstomatoflavourhasbeenasymbolforeverythingthatpeopledislikeaboutmodernagriculture.

31

IDENTIFICATIONOFAMAJORGRAYWALLRESISTANCELOCUSONCHROMOSOME9OFTOMATO

*JasmineLopez,RezaShekasteband,JayW.Scott,andSamuelF.Hutton,GulfCoastResearchandEducationCenter,UniversityofFlorida,Balm,FL,USA.

Graywall (GW) isadiseaseofunknownetiology thataffects ripeningof freshmarket tomato(SolanumlycopersicumL),causingunevenripeningandvascularbrowningonthefruits.Thereare no biotic or abiotic factors known to cause GW, but incidence is associated withenvironmental conditions such as low light, cool weather during fruit ripening, and nutrientmanagementpracticessuchashighnitrogen,orlowpotassium.Symptomsusuallyappearaftermaturegreenfruithavebeenharvested,storedanddeliveredtotheretaillevel.GWcancausesubstantial losses to marketable yield, and the only effective control strategy is the use ofresistant cultivars. Resistance breeding efforts rely on phenotypic selection in seasonswhendiseasesymptomsareexpressed.However,seasonalvariationindiseaseincidenceandseveritycontributetoreducedselectionaccuracy,resultinginalengthierandmoreexpensiveselectionprogram.AnunderstandingofthegeneticsofGWresistance,togetherwiththeavailabilityofmolecularmarkerslinkedtoresistancegeneswouldgreatlyimproveresistancebreedingefforts.Field observations made in fall 2015 suggested that GW resistance/susceptibility may beassociatedwith the Frl locus on chromosome 9. To test this hypothesis, 20 resistant and 15susceptiblerecombinantinbredlines(RILs)fromthecrossbetweenFla.8000(GWsusceptible)and Fla. 8111B (GW resistant)were genotypedwith 9markers spanning chromosome9 andcorresponding to SNPs developed through the USDA SolCAP project. Single marker analysisconfirmedlinkageofamajorGWresistancelocuswiththreemarkersontheshortarmofthechromosome (P ≤ 0.0005), near the Frl locus. Trials conducted inwinter 2016-2017 likewisedemonstratedcouplingphaseorrepulsionphaselinkageofGWresistancewithFrl inthe(Fla.8828xFla.8590)and (Fla.7946xFla.8442)F2populations, respectively.Usingamap-basedapproachforfinemapping,35recombinantscorrespondingtoa6Mbpintervalwereidentifiedfroman(Fla.8000xFla.8111B)F3population.ProgenyofrecombinantsweregenotypedtoselectplantsthatwerehomozygousfortherecombinationeventsforthedevelopmentofRILs.ResultsfromevaluationofRILsinwinter2017-2018delimitedtheresistancelocusinFla.8111Btoa1.5Mbpinterval.ResistanceinFla.8111Bisdominant,basedontestingofthisparentalongwithFla.8000andtheF1inwinter2016-2017and2017-2018seasons.Thechromosome9locuswasalsotestedinaseparatepopulationdevelopedfromthecrossbetweenFla.8570(GWresistant)andFla. 8059 (GW susceptible). 25 resistant and 22 susceptible RILs from this population weregenotypedwith the initial9markerscorresponding tochromosome9.Singlemarkeranalysisindicatednosignificantassociationofchromosome9markerswithGWresistance,suggestingthat resistance in Fla. 8570 may be controlled by a separate locus (loci). Mapping of GWresistanceinthispopulationiscurrentlyunderway.

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FINEMAPPINGOFALOCUSINCHROMOSOME12CONTROLLINGFLATANDGLOBEFRUIT

SHAPEINFRESH-MARKETTOMATO*EdgarSierra-Orozco,RezaShekasteband,SamuelHutton;UniversityofFlorida,GulfCoastResearchandEducationCenter;EudaldIlla-Berenguer,EsthervanderKnaap;UniversityofGeorgia,DepartmentofHorticultureRelative to wild tomato, fruit size of cultivated tomato (Solanum lycopersicum L.) has beentremendouslyenlarged,anditsshapehasgreatlydiversifiedduringthedomesticationprocess.About30QTLshavebeenfoundtoaccount formostof thisvariation.Withinspecific tomatomarkets,however,fruitmorphologyislessexplored,anditisbelievedthatratherthanknownmajor QTLs, other loci are responsible for variation in shape and size. Within large-fruitedgermplasm,breedershave long recognizeddifferences in flat (oblate)andglobe (round) fruitshape,especiallyasthisisthoughttoinfluencesizeandmarketabilitycharacteristics.Ingeneral,globefruitstendtobelarger,whereasflatfruitsretainbettermarketability.Breedersoftenmakecrossesbetweenflatandglobeparents,andintheresultinghybrids,flatshapeisobservedtobedominant,while size is intermediate. Large fruit size is an important characteristic formanymarkets,andinsomeofthose,growersreceivehigherpricesforlargerfruit.Knowledgeofthelocationofthegene(s)controllingthistraitmayimprovecultivardevelopmenteffortsforfresh-markettomato.Theobjectivesof thisstudywere(1) to identifyandfinemapthe locus (loci)controllingflatandglobefruitshapeinlarge-fruitedgermplasm,and(2)tocharacterizetheeffectofthistraitonfruitshape,size,andmarketability,aswellasonothermorphologicaltraits.Elevenflatand12globe-fruitedF6recombinantinbredlines(RILs)fromtheFla.8000(flat)xFla.8111B(globe) populationwere genotyped using the 7,720 SNP Illumina Infinium SolCAP SNP array.Compositeintervalmapping(CIM)detectedasinglelocusontheupperarmofchromosome12controllingfruitshape,andfurtherCIMusing240F2plantsfromthesamecrossconfirmedthisregion. Using a map-based approach, recombinant plants for this interval were identified,evaluatedforfruitshape,andgenotypedusingmarkerssaturatingtheregion.Resultsdelimitedthelocustoanapproximately400KBintervalcontainingasinglecandidategene.Inasurveyof29 flat and 21 globe UF/IFAS breeding lines, a marker corresponding to this gene alwayssegregatedwith shape. For threedifferentbackgrounds,homozygous flat, heterozygous, andhomozygousglobenear-isogeniclineswereusedtocharacterizetheeffectsofthislocus.FruitshapeattributesweremeasuredusingtheTomatoAnalyzer2.2software.Significantdifferenceswereobservedamonggenotypesforfruitweightandformultiplefruitshapetraitssuchasfruitheight,fruitshapeindex,andshouldercharacteristics.Heterozygousgenotypesweregenerallyintermediatetothehomozygousgenotypesforfruitweightandothertraits,butthesignificanceofthosedifferencesdependedonthetraitandonthebackground.Fortwoofthethreenear-isogenicbackgrounds,plantbiomasswaslowerforglobegenotypescomparedtoflatgenotypes.Inallbackgrounds,pedicelswerelongerandthinnerforglobegenotypesthanforflatgenotypesand pedicels of heterozygous genotypes were intermediate but very similar to those of flatgenotypes.Basedononeseasonoftrialing,thislocushadnoconsistenteffectonfruitdefectssuchascrackingandchecking.

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FRUIT WEIGHT VARIATION CAUSED BY THE KNOWN FW2.2, FW3.2, FAS, AND FW11.3 ALLELES IN FRESH MARKET TOMATO

Reza Shekasteband, University of Florida Department of Horticulture; Eudald Illa Berenguer, Esther van der Knaap, University of Georgia, Department of Horticulture; and Samuel F Hutton, University of Florida Department of Horticulture Previous studies have shown that the mutant alleles of FW2.2, FW3.2, and FW11.3 account for the majority of fruit weight increase in cultivated tomato; FW2.2 is practically fixed, but the last two genes are not, even though they are common in cultivated tomato germplasm. The mutant allele of FAS also regulates locule number and fruit weight, resulting in significantly larger fruit size. However, this allele is relatively rare within fresh market germplasm due to excessive fasciation and rough blossom end scarring, resulting in fruit that are too distorted and unmarketable. We sought to study potential interactions among these fruit weight genes in order to determine if introduction of wild type (WT) alleles at FW2.2, FW3.2 and/or FW11.3 might allow the utilization of FAS for increased fruit size without compromising fruit marketability. We developed two BC3 populations in the large-fruited Fla. 8059 and Fla. 8111B backgrounds for studying these effects: ‘LYC1894’ was used as the donor of the mutant FAS allele (linked with WT FW11.3) and the WT FW2.2 allele for the Fla. 8059 background; and ‘Rosato Tondo’ was used as the donor of the mutant FAS allele and the WT FW3.2 allele for the Fla. 8111B background. For each background, BC3F2 plants were genotyped at seedling stage to select the nine possible genotypic combinations of mutant and WT alleles. Recurrent parents and twenty-four plants per genotype were transplanted to the field at GCREC in a randomized complete-block design with three blocks. The trial was harvested twice at maturity, and fruit were graded for size and marketability. In the Fla. 8111B background, homozygosity for the WT FW3.2 allele reduced fruit size by 59 g relative to homozygosity for the mutant allele. Although homozygosity for the mutant FAS allele gave an increase in average fruit weight, misshapen fruit and rough blossom scars resulted in approximately 50 percent culls. Heterozygosity for the mutant FAS allele had no effect on fruit size when plants were homozygous for the WT FW3.2 allele, but resulted in an average increase of 39 g when plants were either heterozygous or homozygous for the mutant allele at FW3.2. In the Fla. 8059 background, FW2.2 had no significant effect on fruit size. Homozygosity for the mutant FAS allele again resulted in increased fruit weight, but for the Fla. 8059 background, 100 percent of the fruit were unmarketable. Heterozygosity for the mutant FAS allele had no effect on fruit size for plants that were homozygous for the WT FW2.2 allele, but an average 18 g increase in fruit weight was observed for plants that were either heterozygous or homozygous for the mutant allele at FW2.2. Heterozygosity for the FAS mutant allele had no significant effect on total marketable yield in either background. Results suggest that utilization of the mutant FAS allele in heterozygous state may help to increase fruit size without compromising marketability, but the substitution of WT alleles at FW2.2 and FW3.2 provides little or no advantage in this.