ExternalScienceReview

BlightResistanceBreedingandResearchProgramsof

TheAmericanChestnutFoundation

NicholasWheeler,Chair

SallyAitken,UniversityofBritishColumbia

JohnDavis,UniversityofFlorida

RichardSniezko,U.S.ForestService

StevenH.Strauss,OregonStateUniversity

SitevisitAugust21-22,2018

ReportsubmittedOctober5,2018

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Summaryoffindings

• Thereviewpanelwasimpressedwiththework,andin-depthself-analysisandplanning,presentedbyTACF.

• ThepanelwaspleasedwithTACFeffortstouseleadingedgescienceandtechnology—specificallytheuseofgenomicselectionandgeneticengineeringtools—toimprovetheefficiencyandrateofimprovementforblightresistance,whilemaintaininglocaladaptationofgermplasminthefaceofclimatechange.

• ThepanelcomplimentedTACFinplanningtobroadentheresistancebaseforbreedingbeyondtheprimaryGravesandClappersourcesandurgedthemtoconsidersubstantiallyexpandingthenumberofsourcesandgenotypesthroughinnovativebreedingmethods(e.g.,useofpollenvs.seeds,earlytestingmethods,anduseofgenomicstoincreaseintrogression/backcrossing(BC)efficiency).

• Thepanelsupportedtheuseofgeneticengineering(GE)toolsandcurrentfocusontheOxOgenebutbelievedthattoomuchrelianceisbeingplacedonasingleinsertioneventanditsderegulation.Asingleeventmayimpairfitnesswhenmadehomozygousduetothehighlikelihoodofexpressionofdeleteriousrarerecessivealleleswhenmadehomozygousinfurtherbreeding,itspossiblesilencing/attenuationoveryearsandgenerations,andthatitmaynotbedurableinthefaceofpathogenevolutioninthelongterm.ThepanelurgedthedevelopmentofalternativeformsofOxOtransgeneswithdistinctpromotersandcodingregionstoavoidgenesilencingwhentheyarecombined,anddevelopmentofalternativegenesthat,amongothers,mightincludethoseforresistancetoPhytophthora.

• ThepanelbelievedthatTACFshouldcompleteitsevaluationandprogenytestsofthebestbackcrossmaterialsinafocusedandrigorousmanner,butgiventhatblightresistancelevelsandtreeformappeartobeshortofhopesforthisstageoftheprogram,considerreducinginvestmentinthesematerialsinthefutureinfavorofinvestmentsintheinfusionandrapidbreedingwithnewsourcesofgeneticdiversity,useofboththecurrentOxOandnovelformsofOxOandothertransgenes,andselectivecombinationofthebestBCandOxOmaterialsinamannerthatmaximizeseffectivepopulationsize(asTACFhasproposed).

• Inadditiontodevelopingresistance,weurgeexpandedstudiesonseedlingculture,chestnutsilviculture,establishmentofhigh-qualityfieldtrials,andreleaseofthebestseedlotsforrestoration.

• ThereviewprocesswouldhavebeenstrengthenedbymoredetailedandintegratedsummariesofprogressandworkillustratingthecomplementaryeffortsbyTACFChaptersandcooperators(breeding,testing,cooperativeactivities,andthesmallgrantsprogram),awardsandimpactsoftheirsmallgrantsprograms,anddatabase/informaticssystems.

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Introduction

AnotablestrengthofTheAmericanChestnutFoundationisitswillingnesstoperiodicallyinviteoutsidescientiststoreviewitsresearchstrategyandprocesses.Previousreviews,in1999and2006,contributedsignificantlytoguidingtheprogram.Withrecentdevelopmentsingenomictechnologiesandresources,andexcitingprogressinbiotechnology,TACFrecognizedtheneedforanewreview.ApaneloffiveseniorscientistswithbackgroundsintreeimprovementandforestgeneticsresearchrelevanttoTACF’sorganizationalgoals,metfortwodaysinAbingdon,Virginia,hostedbyTACFstaff.TheyattendedpresentationsandfieldtripsinthenearbyMeadowviewbreedingandtestingproperties.Subsequently,thepanelconvenedtodiscusstheirobservationsandtodevelopapreliminarylistofrecommendations.TheserecommendationswerethendiscussedindetailwithTACFstaff.

VisitingReviewPanelParticipants

Dr.SallyAitken,UniversityofBritishColumbiaFacultyofForestry,ProfessorandAssociateDean-ResearchandInnovation

Dr.JohnDavis,UniversityofFlorida,AssociateDeanforResearch,AssociateDirectorofFloridaAgriculturalExperimentStation

Dr.RichardSniezko,CenterGeneticist,USDAForestService,DorenaGeneticResourceCenterDr.StevenH.Strauss,DistinguishedProfessor,OregonStateUniversity,CollegeofForestryDr.NicholasWheeler,ReviewCommitteeChairTACFParticipants

Mr.StephenBarilovits(ElectronicsEngineer,SystemsDesign),Chair-ElectofScience&TechnologyCommittee,memberofTACFBoardofDirectors

Ms.LauraBarth,TACFHorticultureandPathologySpecialistMs.SaraFitzsimmons,TACFDirectorofRestorationDr.BrianMcCarthy(OhioUniversity),ChairofScience&TechnologyCommittee,Chair-ElectofTACF

BoardofDirectorsDr.WilliamPowell(SUNY-ESF),memberofScience&TechnologyCommittee,Director,AmericanChestnut

FoundationResearchandRestorationProjectDr.KimSteiner(PennStateUniversity),TACFSeniorScienceAdvisor,Past-ChairofTACFBoardofDirectorsMs.LisaThomson,TACFPresidentandCEODr.JaredWestbrook,TACFDirectorofScience

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Thereviewprocess

ThereviewprocesswaswelldefinedbyTACFandmostoftherelevantreviewmaterialsprovidedwellinadvanceoftheactualreview.Pastreviewsandcurrentsummariesofthestatusofthescienceprogramwerehelpfulinprovidingaworkingframeworkforourownreview.WethankExecutiveCoordinatorMs.CherinMarmon-Saxeforclearandtimelycommunicationsandlogisticalarrangements.

Mostofthereviewpanelcametothismeetingwithonlypassingknowledgeoftheprogramhistoryandscopeofactivities.Thebriefexposure,regardlessofintensity,gaveusrelativelylittletimetofullygrasptheessenceoftheprogram.Futurereviewsmaytakeanextradaytomorefullydevelopthestorytobetold.Aflow-charttypeillustrationofthegeneticresourcescurrentlyusedintheprogramandtheirorigins,andatleastnear-termbreedingplans,wouldhelpthecommittee“seetheforestforthetrees.”Thismightalsobeusefulforeducationandoutreach(e.g.,alargeposterorinteractivemultimediapresentation).MoredetailsontheChapterprograms(strengthsaswellaschallenges),andtheworkandimpactsofthesmallexternalgrantprograms,wouldhelpthecommitteetobetterunderstandhowallTACF,notjustthescienceandMeadowviewcore,operatedandinteracted.Conveyingaclearerideaofwhatendproductstheywishtoputoutwouldbegoodtobothreviewersandtheirpublics:mostresistanceprogramsintrees,andcertainlyforrestorationwillnotwaittohave100%oftheprogenygeneticallyresistant,andwilltrytogettreesonthelandscapesoon(e.g.whitepineblisterrust(multiplespecies),Port-Orford-cedar,Acaciakoa).Gettingtreesintothefield—evenifonlyacertainpercentagewillsurvive—willallowsomelandmanagerstostartusingthespeciesandgivefeedbacktotheprogram.Whatlevelofresistance(survival)hasbeenpromisedtothecooperatorsandisittimetorevisethemessageinapositivemanner?

Evaluation

Thereviewpanelwasimpressedwiththescope,scaleandqualityoftheoverallprogram.Notablefeaturesinclude:

ModelTACF,withitsuniqueorganizationalstructure,haspositioneditselftobeoneoftheworld’smodelprogramsfortherescueandrestorationofaforesttreespeciesfacingfunctionalextinction.Theorganization’ssupportnetwork,fueledbythepassionanddonatedtimeandresourcesofthousandsofcontributors,hascontributedsignificantlytothelong-termsurvivaloftheprogram.Theprogramhasbenefittedgreatlyduetothecontinuityofpurposeandinstitutionalmemoryofitsemployees.Thelackofsuchtraitshascontributedtothedemiseofmanyforesttreeimprovementprogramsaroundtheglobeinthepast(Wheeleretal.2015).

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Weshareyourconcernsregardingthemaintenanceofvolunteersupport(fiscal,in-kind)asmembershipagesandrestorationprogramsaredelayed.StaffTheexistingstaff,fromtoptobottom,appeartobeexcellent.Ourcommendationsbeginwiththeleadershipprovidedbytheprincipals(PresidentandCEO–LisaThomson;ChairofScienceandTechnologyCommittee–BrianMcCarthy;BoardMember–StephanBarilovits;TACFSeniorScienceAdvisor–KimSteiner).Aswithallthoseassociatedwiththisprogram,wecommendtheseindividualsfortheirpassionandexcellenceinleadership.Weareparticularlyhappytoseethelong-termcontinuityofresearchguidanceprovidedbyDr.Steiner.WewereimpressedwiththeapparentlyseamlessadditionofscienceleadDr.JaredWestbrook,supportedoflatebyhorticulturistMs.LauraBarth.Webelievetheirskillssetsarestrongandappropriateandwillbeinstrumentalinmovingtheprogramintothenextphaseofresearchanddevelopment.Dr.BillPowell’scontributionsongeneticengineering(OXOandnewgenes)andrapidbreeding(earlyfloweringandresistancetesting)haveprovidedkeyinnovationsinsupportoftheprogram.SaraFitzsimmons’long-termcontributionsandinstitutionalmemoryareirreplaceable–arealkeytoeventualrestorationsuccess.Thefieldtechniciansareknowledgeableanddeeplyimmersedintheprogram.Theyarecriticallyimportanttoprogramsuccessandcontinuity.KeydocumentsWefindthecurrentStrategicPlanandthe3-BURdocumentstobeexcellentfordefininggoals,strategies,missionsandvisions.Thereshouldbenoconfusionastowherethisprogramseekstogo,andthepaththatwilltakeTACFthere,usingappropriateadaptivemanagementapproachestoscientificmethods,toolsandgeneticmaterials.ProgressivescienceWeapplaudTACF’sadoptionofcutting-edgemethodsandtoolswhichwebelievewill1)yieldincreasedprecisioninthebreeding,testingandselectionofsuperiorindividualsand2)dramaticallyimprovethechancesofdevelopingmultiplesourcesofresistancetobothCryphonectriaandPhytophthora.Notablearetheadoptionofquantitativegeneticmethods,useofgenomictoolsandresourcestoenhanceselectionandtrackpedigrees,andthewell–thought-outacceptanceofbiotechnology(i.e.,geneticengineering)asaresistancebreedingtool.ThekeyinthelattermaycontinuetobediscussionsandcommunicationswithstateChaptersandlandmanagersinterestedinplantingchestnut.

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PhenotypingWeapplaudtheteam’srecognitionthatphenotypingiskeytolong-termprogress.Itshouldreceiveincreasedattentionandresourcestoimprovemethodologiesandincreasetheheritabilityofphenotypicscores.Thiscannotbeemphasizedtoomuch–phenotypingiskeytosuccess.Havinggermplasmdemonstrationsonsilviculturallyappropriatefieldsites(andresistancethatisexpectedtopersist)isstronglyencouraged.Addedbenefitswouldaccruefromgoodfieldtrialsthatrelatetoshort-termtestingtechnologiesthatconfirmtheirutility.TheoldestfieldtrialsarenotTACF’s,butratherwereinstalledbytheUSFS,sostrengtheningthatrelationshipwouldbegood.BasedonconversationsbetweenDr.SniezkoandUSFScolleaguesafterthereview,thereappearstobesomeusefulgeneticresistancefromatleastoneoftheoldest(9-year-old)USFStrials.TACFdoeshaveyoungerfieldtrials,butitwasunclearwhetherthesewereonoptimumsites.ClimatechangeWeagreewiththeteam’srecognitionthatclimatechangewillhaveasignificantimpactonpopulationdevelopmentanddeploymentforrestorationandmustbeplannedforpro-activelyintheirconservation,breedingandrestorationwork.CooperativestudiesWestronglysupportTACF’scontinuedeffortstodevelopcooperativestudies,throughgrantapplicationsorothermeansofleveragingresources.Notablehereareeffortstobetterunderstandexistinggeneticdiversityinsurvivingchestnutpopulationsandtherelationshipofgeneticstoenvironments(ecologicalandprovenancephenotyping,andlandscapegenomics).TheAmericanChestnutCooperators’Foundation(ACCF)isreportedtohaveseedorchardsandisdistributingseed(pureAmericanchestnut)withsomelevelofresistance.CooperationwithACCFshouldbeconsiderediffeasibleandmayhelppublicperceptionsofTACFandACCFjointefforts.Competitivegrantsprogram–Thoughfewdetailswereprovided,thecommitteebelievedthattheTACFcompetitivegrantsprogram,asameansforseedingfutureresearchandleveragingresources,islikelytobeusefulandworthyofcontinuedsupport.Forexample,itcouldaidinilluminatingseveralmodestenquireslikedifferentialreproductivesuccessofpollenpolymixesandcloningmethods.DatabaseTheimportanceoftheprojectdatabase,anditscontinueddevelopmentandmaintenance,can’tbeoveremphasized.Whilethecommitteereceivedonlyabriefoverviewofthesysteminplace,theTACFstaffappearedtoappreciateit'simportance.WerecommendthatTACFensure

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thatthereisqualifiedpersonnel,withbackupexpertise,inplaceforcontinuityandcuration.Itisalsoimportantthatthesystembeconnectedto,andinclude,Chapteractivities.AwrittenoverviewofthefullsystemwouldhelpTACFtocoordinateandmonitorperformance,andwouldhavehelpedthereviewcommitteeinit'sevaluation.TechnicalFeedback

ToprovidereviewcommentsthataredirectedtostrategicquestionsmostimportanttoTACF,belowwespecificallycommentonthestatedprogramobjectives.Wepresenttheobjectivesverbatim(bold),thensummarizethecommittee’sresponsestoeachofthem.

InAppendicesIandIIweconsiderspecificqueriesthatwereprovidedtousshortlybeforeandduringourreview,providingtentativeobservations,asthecommitteedidnotconsiderthemin-depthornecessarilyreachconsensusaboutthem.

Objective1.FinishselectioninClapperandGravesBC3F2seedorchardsatMeadowviewResearchFarms

Objectives1and3constitutetheessenceoftheoriginalbackcrossbreedingplantointroduceandinterbreedresistanceintonativeAmericanchestnut.AssuchtheyhavebeenthefocusofTACFeffortsuntilnow.Theseeffortsareworthyofattention,offeringthebestavailableinformationonfunctionalresistance.Theworkshouldbeconcludedasexpedientlyaspossible,unlessresultsfromotherapproachessuggestsshiftingcoursewillleadtomorerapidprogresssoon.Theseeffortsshouldincludefieldtestingonappropriatechestnutsitestoconfirmefficacyofresistanceandtoinformdevelopmentofsilviculturalprescriptions.

Phenotyping

TACFstaffmaywishtotakeafreshlookatpastmethodsofinoculationandscoring.WeencouragecollaborationwiththePAChaptertoevaluatetherelationshipbetweenshort-terminoculationprotocolsandolderfieldtrialsofnon-inoculatedstock.OnerecentEuropeanchestnutstudysuggestsinoculationstudiesbeforetheageof4maymaskwhatmightotherwisebeusablelevelsofresistance,leadingtounnecessaryearlyculling(Pazitnyetal.2018).Obviously,earlytestingisdesirabletomovetheprogramfasterbutifitissignificantlyinaccurate,itwillbecounter-productive.TheEuropeanresultsshouldbescrutinizedtoevaluatetheimportanceandbenefitvs.riskofthistradeoffandcontactwiththepublishingscientistsmadetoclarifytheirstrengthsandlimitations.

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TACFresponse:Ourmethodsofphenotypingforblighttolerancehavebeenandcontinuetobeasubjectofdebate.Ononesideofthedebate,therearesomethatargueweshouldwaitfifteenyearstoassesslong-termblighttoleranceofindividualtrees.ThisgroupalsoarguesthatblighttoleranceisbestassessedwithnaturalinfectionofstemswithCryphonectriaparasiticaasartificialinoculationcircumventsphysicalbarrierstoblightinfectiononthestem.Attheotherextreme,wehaveartificiallyinoculatedseedlingsintheirfirstyearofgrowthwithahighlypathogenicstrainofC.parasitica.Sofarwehaveperformedthesesmallstemassays(SSAs)primarilyforbackwardselection–meaningweinoculateseedlingprogenytoestimatetherelativegeneticresistanceoftheiropen-pollinatedmothertrees.Wearealsotestingifwecanusesmallstemassaysasaforwardselectionmethodtoeliminatethemostsusceptibletreespriortoplantinginorchards.BeforeweadoptSSAsforforwardselection,wearetestingwhethertheinoculatedseedlingssurviveafterplantinginthefieldandwhetherseedlingcankerseverityandsurvivaliscorrelatedwiththesesametraitsassessedinlaterstagesofgrowth.

Withtheadditionofgenomicselectiontopredictprogenyblighttoleranceonlargenumbersofparenttreesinourseedorchards,wefoundcorrelationbetweenlate-developingblighttolerancephenotypesofparenttreesandthepredictedorobservedaverageshort-termcankerseverityontheirprogeny.Specifically,wefoundthataselectionindexbasedonfivetraitsindicativeoflong-termblighttoleranceofparenttrees(age8to16years)isnegativelycorrelated(r=-0.65)withaveragecankerseverity(predictedorobserved)ontheirtwotothreeyearoldprogenysixmonthsafterinoculationwithC.parasitica(Westbrooketal.2019a).

WehavealsofoundthatblighttoleranceofBC3F2parentsasassessedwithsmallstemassaysonprogenyiscorrelatedwithblighttoleranceofthesameparentsasassessedfromtheaveragecankerseverityondifferentsubsetsofprogenyinoculatedatagethreeinorchards.Specifically,amongAmericanchestnutBC3F3families,withinfamilysurvivalratesoffirstyearseedlingsinoculatedwithahighlypathogenicstrainofC.parasiticaisnegativelygeneticallycorrelated(rgenetic=-0.75±0.30)withfamilyaveragecankerseverityonthreeyearoldseedlingssixmonthsafterinoculationwithweaklyandhighlypathogenicstrainsofC.parasitica(Saielli&Levine2019).

Thesestudiesdemonstratethatblighttoleranceofparentsandprogenyarecorrelatedandfamilyrankingsaresimilarwhetherprogenyareinoculatedintheirfirstyearoratagethree.Inpractice,weselecttreesinourMeadowviewseedorchardsbasedonaselectionindexcomposedofthelong-termblightphenotypesofparenttreesandtheaveragecankerseverityofprogeny,eitherobservedfromprogenytestsorpredictedusinggenomicselection.(Westbrooketal.2019a).

TACFcitedtheirpreferentialuseofahighlyvirulentstrainforcontrolledinoculation;thepanelwasconcernedthatthismighteliminategenotypesandresistancegenesthatareusefulfor

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providingabroaderresistancebase,thusamixedstrategymightbeconsidered(e.g.,twostageormultiplesourceinoculations).

TACFresponse:Weseektobalanceexpedienceandaccuracyinmakingourselections.Ourstandardmethodforselectionwithinseedorchardsistoinoculatetreesatagetwowithaweaklypathogenicstrain(SG2,3)ofC.parasitica.Weremove60%–80%oftreesthatdemonstratesignificantcankerexpansion6monthsto1yearafterinoculation.WehaveperformedadditioncullinginMeadowviewseedorchardsbasedonprogenytesting,genomicselection,andlong-termblightphenotypesasdescribedpreviously.SomechaptersalsoperformasecondinoculationatagefourwithamorepathogenicstrainofC.parasiticatoculladditionalindividualsfromseedorchards.Wehavefoundstrongcorrelationinfamilyrankingsforblighttolerancewhenweinoculatewithaweaklypathogenicv.stronglypathogenicstrainsofC.parasitica(Steineretal.2017;Westbrook&Jarrett2018).Wehavealsofoundcorrelationbetweenparentblightphenotypesandprogenycankerseverity.Withtheadditionofgenomicselectionandthecorroborationbetweenvariousblighttoleranceassessmentmethods,wehavebecomemoreconfidentthatourselectionmethodsareidentifyingthemostblighttoleranttrees.

PhenotypingstudiesattheMeadowviewsiteprovidedthecommitteewithcauseforconcernontwofronts.Thefirstisthesomewhatcomplexnatureofparentagelikelyfoundintheopen-pollinatedprogenyofBC3F2parents,andhowtheresultsofprogenytestsshouldbeinterpreted.TACFstaffreportedapoorcorrelationbetweenBLUP-generatedbreedingvaluesandprogenytestphenotyperesults,andsuggestedtheremaybestrongdeviationsfromrandommatingthatcausederrorsinestimationofbreedingvalues.Werecommendtimeandresourcesbespentoncharacterizingmatingpatternsandlevelsofinbreedingtobetterunderstandhowtheuseofopen-pollinatedprogeniesmayhaveaffectedprogenyphenotypes.Thisproblemappearstobeafunctionofhaphazardplacementofheterogenousresearchmaterials(e.g.,nearbyplantingsofChinesesourcenearsomeparentsandclustersofsiblings).MappingBLUPvaluesspatiallyandconsideringthecompositionofpaternalparentsnearbytomothertrees,mayhelptointerpretandpossiblyadjustBLUPvalues.

TACFresponse:Weareintheprocessofremovingcontaminatingchestnutpollensourcesthatareadjacenttoopen-pollinatedmothertrees.WehaveremovedtheChinesechestnutandAmericanchestnuttreesinthe‘Clapper’BC3F2seedorchardontheDuncanfarm.WearealsointheprocessofremovinganoldBC3F3progenytestthatisadjacenttothe‘Graves’BC3F2seedorchard.Finally,weareremovingChinesechestnutsonthePricefarmthatareadjacenttotheBC3F1parentsoftheBC3F2treesinseedorchards.

Throughgenotyping-by-sequencingofBC3F2treesandreferencepanelsofChinesechestnutandAmericanchestnut,wehaveestimatedtheproportionofBC3F2genomesinheritedfrom

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Chinesechestnutv.Americanchestnut.Throughtheseanalyseswehaveidentifiedanumberof‘pseudo-F1s’inourseedorchards.Thesetreesinherited50%ormoreoftheirgenomefromChinesechestnutandarepresumablytheprogenyofBC3F1treesthatwereopen-pollinatedbyChinesechestnutsonTACF’sPriceFarm.Weplantomonitorthesepseudo-F1treesoverthenextfewyearstodetermineiftheyaremalesterile.Iftheyarenotmalesterile,wewilleventuallyremovethetreesfromtheseedorchardafterwehavecollectedseedfromthesetreestoadvancenewbackcrosslinesfromnovelChinesechestnutsourcesofresistance(seeresponsetoObjective2forfurtherexplanation).

TheBC3F3sinourprogenytestswerealsogeneratedthroughopenpollinationamongtheBC3F2trees.WefoundthataBC3F2motherwithaninferiorblighttolerancephenotype(e.g.,deadmainstem)hadprogenywiththeleastseverecankeringamongallfamiliestestedsofar.ThistreewasplantednearastandofChinesechestnutsontheDuncanfarm,whichwehavesinceremoved.Forrarecaseswherethemothertreewithinferiorblightphenotypehasprogenywithhighblighttolerance,wewillperformcontrolledpollinationswithBC3F2pollenonthemothertreeinquestion.WewillevaluatetheprogenyblighttoleranceagaintoensurethattheapparentlyhighblighttoleranceofthemothertreeisnotanartifactofpollinationbyChinesechestnuttrees.

Weappreciatethereviewerpanel’ssuggestiontogenotypetheBC3F3treesaswelltodeterminelevelsofinbreedingandpercentChinesechestnutancestryinthisgeneration.WeplantogenotypethesetreesafterwehavefinishedcullingsusceptibletreesfromseedorchardsandhaveplantedBC3F3progenyindemonstrationplantingstoassesstheirlevelofblighttolerance.

ThesecondissueposedbytheMeadowviewsiteistherecognitionthatthesiteisnon-optimalforgrowingchestnut.Thesoilsappeartobetoomoistandpoorlydrained,thustreesareunderstressandnotlikelytofullyexpresstheirgeneticpotentialforgrowth,form,andresistingdisease.Thesitemaysufficeforgeneticconservation,plantpropagationandshort-terminoculationpurposes,butseriousconsiderationshouldbegiventomovinglong-termseedorchard,demonstrationplantings1andprogenytrialstomoreoptimalsites.

TACFresponse:WeagreethatMeadowviewisnotanoptimalsiteforgrowingAmericanchestnutsduetothepoordrainageinsomeareasandoccasionallimestoneoutcrops,whichhinderchestnutgrowth,survival,andpossiblyblighttolerance.TACFhasbegunlookingforbetterlandtoplantthenextgenerationofbackcrossandtransgenicseedorchards.

Itwouldbedesirabletoquantitativelydefinetheselectiontarget–oftencalledanideotype.Isthegoaltoproduce(1)treesthatarecanker-freeinthefield,(2)treesthatlivewithcankers,(3)

1Alternatively,demonstrationplantingsatthissitemaybeusedtocontrastresultsatmoreoptimalsites.

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seedlingsthatmighthavesomepartialresistancebuteventuallydie(butmighthavesomevalueinbreedingindifferentcomponentsofresistance),or(4)seedlings(trees)withfewercankers,oralloftheabove?

TACFresponse:Weproposethefollowingselectionideotypestodefinesuccessinthebreedingprogram:

• Mainstemsurvivesindefinitelywithblightinfection.• Blightcankerssuperficialandextensivelycallusedwithminimalexposedwood.• Treesgrowassingle-stemmedtreesatratesandmaximumheightsmoresimilarto

AmericanchestnutthanChinesechestnut.• Leaf,twig,andnutcharacteristicssimilarifnotindistinguishablefromAmerican

chestnut.• Treescompeteandreproduceundercompetitionintheeasternhardwoodforests.

Seedlingrecruitmentisobservednearparenttrees.• Populationsadequatelyrepresentthediversityandadaptivecapacityremaininginthe

post-blightC.dentatapopulation.• AsubsetofthepopulationhastolerancetobothCryphonectriaparasiticaand

Phytophthoracinnamomi.

WehaveobservedanegativecorrelationbetweenpercentofthegenomeinheritedfromAmericanchestnutandblighttoleranceinbackcrosspopulations(Westbrooketal.2019a).Thisresultsuggeststhatallelesforblighttolerancearesegregatingatmoregenomiclocithanpreviouslyassumedandthatphenotypicselectionhasnotbeensufficientlyaccuratetoselectforallresistanceallelesinallbackcrosslinesandgenerations.GeneratinghybridchestnuttreesthatareindistinguishablefromAmericanchestnutyetalsohavehighlevelsblighttoleranceisprovingtobedifficultwithbackcrossbreeding.WeremainoptimisticthatwewillbeabletomeettheseselectioncriteriabyoutcrossingtransgenicAmericanchestnutwiththeOxOgenetopureAmericanchestnutsandbackcrosstrees(seeresponsetoObjective4).WearealsoadvancingnewbackcrosslinesfromadditionalChinesechestnutsourcesofblighttolerancethroughfewerbackcrossgenerations(BC1andBC2)tofindanadequatebalancebetweenblighttoleranceandAmericanchestnutgrowthcharacteristics(seeresponsetoObjective2).

Dependingontheideotype,doesanyparticularphenotypicscreengiveallthenecessaryinformation?Whatinoculumsources(e.g.strains,etc)wouldidentifyparticularideotypesaswellasilluminateunderlyingtypesofresistance?Forexample,inblisterrustresistancework,MGRtyperesistancecanbedetectedininoculationsofveryyoungseedlingsandfairlyquickly(<1yearfrominoculation);whileinlookingforpartialresistanceolder(2year)seedlingsareusedandevaluatedforupto5yearsafterinoculation.ForPort-Orford-cedar,theassessmentperiodwasextended(oninoculated1-yearoldseedlings)from~1year(theprevious‘standard’

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untilthe2005testyear)toupto3yearstopickupthequantitativeresistanceandtoidentifythebestseedlingsineach‘good’family(andtoclearlydelineatewhichfamilieswereMGRvs.quantitativeresistance).

TACFresponse:Wehavenotbeenabletodistinguishmajorgeneresistancefromquantitativeresistanceinourchestnutblighttoleranceevaluations.ThepositivecorrelationbetweenblighttoleranceandtheproportionofbackcrossgenomesinheritedfromChinesechestnutsuggeststhatblighttoleranceisapolygenictraitratherthanprimarilyconferredbymajoreffectalleles.

Cloning

Previousreviewshavecalledfortheuseofcloningtechniquestoenhancetheprogram,whilealsonotingthatthespeciesisratherrecalcitranttosuchefforts,particularlythroughrootedcuttings.Thus,todatecloninghasbeenusedtoaverylimiteddegree.However,asoutlinedbelowwebelievethebenefitsofhavingcloningtoolsaretoogreattoignore.Wesuggestmoreefforttodevelophigh-throughputcloningcapability,centeredaroundmicropropagationand/orgrafting,eitherin-houseorthroughcontracttoalocalhorticulturalcenter.Itwouldbehelpfultoinquireastotheuseofcloninginotherchestnutspeciesprogramsaroundtheworld.

TACFresponse:CloningadiversecollectionofAmericanchestnuthasthusfarprovedtobeelusiveanddifficult.Ittookmorethanadecadetoworkoutmethodsforcloningwithsomaticembryogenesisinchestnut(Merkleetal.1991,Polinetal.2006).TheCanadianChestnutCouncilhasworkedfornearlydecadetoinducerootingfromchestnutscionmaterialthathasbeengraftedontoetiolatedseedlings(Galicetal.2012).Lovat&Donnelly(2019)haverecentlyworkedoutprotocolstocloneadiversecollectionofAmericanchestnutswithaxillaryshootculture;however,theconcentrationofhormonesandculturetemperaturemustbeoptimizedtocloneindividualgenotypes.Ingeneral,whilesomeindividualgenotypeshavebeenclonedwithsomemethods,thesuccessinregeneratingplantshasbeenlowandnotallgenotypescouldbeclonedwithspecificmethods.Toourknowledge,successwithrootingothermembersoftheFagaceaehasbeensimilarlypoororuneven,despiteconsiderablehorticulturalinterestincloningoaks,forexample.

TACFisinterestedsupportingadditionalresearchtofurtherrefinecloningmethodsforAmericanchestnut.Giventhehighriskandpotentiallylowsuccessrates,wewouldpreferthatthesecloningmethodsbeworkedoutbyqualifiedcollaborators,ratherthanbyTACFstaff.TACFcouldpotentiallyprovide“seedfunding”throughitssmallgrantsprogramorotherfundingsourcessupportresearchincloningmethodologies.

GraftingisourcurrentmethodforcloningdiversecollectionsofAmericanchestnutforfutureuseinbreeding.Treesoftengetblightinthegraftunionsand,unlikeseedlings,graftedtreesdonotresprout.Tomitigatetheriskoffailurefromgraftincompatabilitypriortouseinbreeding,

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weplantogrowaportionofgraftedtreesunderhighlightconditionstostimulatecatkin(pollen)production.Wewillthenapplythispollentotransgenicorwild-typeAmericanchestnutstoconservethegeneticsofparenttreeintheseedlingprogeny.Amongfield-growngraftedtrees,wewillalsocollectseedsfromthesetreesassoonastheyflowersowecanplantseedlingprogenyintheeventthatthegraftedparenttreesdie.

Thepotentialusesofcloninginbreeding,andforachievingotherprogramgoals,include:

• Pseudo-cloning–Establishprogeny,fromseed,onoptimaltestsites.Coppicemainstemafteryearone.Save3-4coppiceshootsthatariseinyeartwoandprunetherest.Applydifferenttreatmentstosurvivingshootssuchasdifferentinoculationstrains,controls,andtreatmentstopromoteearlyfloweringcapacity.Thispotentialmethodwillneedtobetestedtoensurethattreatmentsononeshootdoesnotaffectphenotypesonanother.

TACFresponse:WewilltrycoppicingfutureprogenytestsandinoculatingthedifferentstemswithdifferentstrainsofC.parasitica.

• Progenytesting–Clonalpropagationofgenotypesprovidessuperiorestimatesofheritabilityandbreedingvaluesbasedonreplicatedtrials,eitheronasinglesite,oronmultiplesitesacrossarangeofenvironments.Somereplicationsmaybeplacedinforestsettingswithoutinoculation,orinsettingsfreeofblighttoevaluategrowthandform.

TACFresponse:WehaveplantedprogenytestscomposedofBC3F3familiesfromMeadowviewon30+forestandold-fieldsitesacrosstheeasternU.S.Eachsitetypicallyhas20–30families.Sitessharecommonfamilies,althoughnositecontainsallfamiliesplantedamongallofthesites.Wedonotplantoartificiallyinoculateanyofthesetestsandinsteadwillassessblightincidenceandseverityfromnaturalinoculum.Themajorityofthesetestswereplantedin2011through2014andmanyofthetreeshavenotyetbeeninfectedwithchestnutblight.Astreesgetinfectedwithblightinthenextfiveyears,wewillcomparetheaverageblightincidence,severity,andmortalitywithinfamiliestocankerseverityofthesamefamiliesthathavebeenartificiallyinoculatedinMeadowvieworchardprogenytestsandsmallstemassays.

GWAS(genome-wideassociationstudies)orprovenancetrials–Useofreplicationcouldimprovephenotypingcapacityandimprovetheaccuracyandprecisionofgeneticestimates.

• Seedorchards–Considerlocatingorchardsonblight-freesites(e.g.,PacificNorthwest,useofhypovirulence,chemicalcontrol)wherediseasepressureisminimal,andtreescanbegrowntooptimizeseedproduction.Multiplerametsofeachclonecanspeedupbreedingprogressandproductionofmaterialforrestoration.

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SeeresponseinObjective4aboutgrowingAmericanchestnutswestoftheMississippiforgermplasmconservation

Demonstrationplots

ThepanelsupportsfrequentuseofdemonstrationplotsatMeadowviewandChapterlocationsforprogrammarketingpurposes.Rowplantingsofmaterialsofdifferentancestryandresistancewillbetterillustrategeneticimprovementthandatatostakeholdersandthepublic.Suchtrialsshouldbemaintainedaslongastheyprovideanaccuratereflectionofbreedingprogress.Cloningofhighlyresistanttreesofgoodform(andsuitablecomparators),plantedinpairsorrowplots,wouldhelptoestablishconvincingdemonstrations.Ifdemonstrationplotsarereplicatedwithinsites,somereplicatesmaybeinoculatedwhileothersarenot.Ifademonstrationplotdoesnotappropriatelyrepresenttheknownresultsofgenetictesting,theplotshouldberemoved.

TACFresponse:TACFiscurrentlyapplyingforfundingwiththeU.S.ForestServiceLandscapeScaleRestorationprogramtoplantanetworkofrestorationtrialsintheeasternU.S.ThesetrialswouldbecomposedofprogenyfromourmostblighttolerantBC3F2selectionsfromMeadowview,Pennsylvania,andNorthCarolinaseedorchards.WeareplanningonplantingtheserestorationtrialsineightstateswithinthehistoricalrangeofC.dentatarange.Eachplantingwouldconsistof250to500treesfrom10to25BC3F3families.Weareplanningtohavetwoplantingsineachstate.OnlysitesthataresuitableforgrowingAmericanchestnutwillbeselected(i.e.,welldrainedsoilwithapH5-6).Onemajorobjectiveoftheseplantingsistoassessthelong-termblighttoleranceofBC3F3treesafterwecompleteselectioninourBC3F2seedorchards.Asecondobjectiveistoassesstheregionaladaptabilityofbackcrossmaterialwhenweplantitinstatesclosetov.indifferentclimacticzonesfromtheoriginoftheC.dentataparenttrees.

ClarifyingtherelativelevelsofresistanceinBC3F2andBC3F3generations

Thecommitteewasundecidedonwhethertherewasareductionofresistancebetweenthesegenerations.Isthisuncertaintyduetophenotypingdifficulties,oristhereadilutionofresistancefactorsinthemoreadvancedinterbredgenerations?Isresistancesuetomorethanthreemajorgenes?Thus,itisuncleartousifthereisvalueinF4,F5orhighercrosses.Wesuggestconcentratingonhighquality,convincingstudies,supportedatleastinpartbyclonaltrialsofthebestmaterials,beforeincorporationofothersourcesofresistanceintothesematerials(orotherwisegoingtoofardownthisBCpathway).

TACFresponse:WefoundthatthetrueBC3F2selections,onaveragehadblighttolerancethatwassignificantlylessthanpseudo-F1s,butgreaterthanAmericanchestnutinourMeadowviewseedorchards(Westbrooketal.2019a).Anegativecorrelationbetweenproportionof

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backcrosstrees’genomesinheritedfromAmericanchestnutgenomeandblighttolerancesuggeststhatChinesechestnutallelesforblighttolerancesegregateatmorethanthreeloci.Polygenicinheritancecoupledwithlowaccuracyphenotypicselectionforblighttolerancemayexplainwhytherehasbeenadilutionofresistanceallelesacrossbackcrossgenerations.AfterfinishingselectionourClapperandGravesBC3F2seedorchards,weplantocombineBC3F3progenyfromClapperandGravesinanextgenerationseedorchard.WewillusethisseedorchardtodeterminehowmuchblighttolerancecanbeenhancedwithadditionalselectionintheBC3F3generation.Wearenotplanningtopursuerecurrentselectionforallofthechapterbreedingprogramsasthiswouldbeanoneroustaskanditisnotclearatthistimehowmuchrecurrentselectionwillenhanceblighttolerance.

Objective2.Advance10ChinesechestnutsourcesofresistancetoBC2inMeadowviewbreedingprogram

Thereviewcommitteewasunanimousinsupportofthisobjectiveandsuggesteditbegivenhighpriority.However,webelievethestatedobjectiveofadvancing10Chinesesourcesofresistancemaybefartooconservativewhentheprogrameffectivepopulationsize,andthepossibilityofintrogressingnew,distinct,andmorenumerousresistancegenes,isconsidered.Werecommendexpandingthenumberofsourcesofnewresistancebyanorderofmagnitudeormore,viapollenaswellasseed.Furthermore,newsourcesshouldnotberestrictedtoChinesechestnut.ConsiderallAsiansourcesofresistance,aswellasEuropeansweetchestnutsandasmanyLargeSurvivingAmerican(LSA)sourcesthatcanbeobtained.

TACFresponse:Weagreewiththisrecommendation,withacaveatacknowledgingtheoperationaldifficulties.Duringthe2018breedingseasonatMeadowview,TACFstaffperformed25controlledpollinationsandharvested1,350seedsfromthesecrossestoadvanceeightChinesechestnutsourcesofblighttolerancetoBC2F1andBC2F2generations.Also,thestaffharvested525seedsfrompseudo-F1treesinMeadowviewseedorchards.Thepseudo-F1swerepresumablypollinatedbyneighboringBC3F2trees,hencetheirprogenywouldbepseudo-BC1s.TheChinesechestnutgrandparentsofthepseudo-BC1sareunknown,butcouldbetheChinesechestnutprogenyofintercrossesamong19differentChinesechestnutparentsthatarecurrentlyplantedadjacenttotheBC3F1selectionsonTACF’sPriceFarm.WearealsousinglargesurvivingAmericanchestnuts(i.e.,treeswhosemainstemsurvivedtheblightpandemic)asAmericanchestnutparentsinthesecrossesinhopesthattheywillalsocontributetotheblighttoleranceofthebackcrossprogeny.Wearetakingthesenewbackcrosslinesthroughoneortworatherthanthreeorfourbackcrossgenerationstoavoiddilutingoutgenesforblighttoleranceineachbackcrossgeneration.Wewillplanttheprogenyina“mixed-source”breedingorchardandperformstagedinoculationswithincreasinglyvirulentstrainsofC.parasitica.WewillperformfinalselectionstomaximizetheproportionofthegenomeinheritedfromAmericanchestnutsviagenotypingtheselectioncandidates.WewillgeneratelargesegregatingpopulationsofBC1F2andBC2F2treesthroughcontrolledandopen-pollinationsamongthe

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selectionsintheseorchards.TheseBCxF2progenywillbeplantedinaseparateseedorchardwherefurtherselectionforblighttolerancewillbeperformed.SeedsfromtheintercrossesamongBC1F2andBC2F2treeswillbedeployedforrestorationtrials(Westbrook2018).

WeplantoexpandonthisprogrambyadvancingadditionalChinesechestnutandJapanesechestnutsourcesofblighttoleranceatMeadowviewtoBC1orBC2.WearealsoplanningtogenerateBC1F2sandBC2F1sbyintercrossingorbackcrossingBC1F1treesthatdescendedfrom10+sourcesofC.mollissimaresistancethatarecurrentlyreproductiveintheHouseRockorchardinPA.TACF’schaptersthatdonothaveuniquesourcesofblighttolerance(otherthanClapperandGraves)maychoosetoparticipateinthisprogrambypollinatingwild-typetreesintheirregionwithpollenfromselectedbackcrosstreesfromMeadowviewandPA.

Overall,weplantoexpandthismulti-sourcebreedingeffortto20Asiansourcesofblighttoleranceandapproximately100C.dentatabackcrosslines.Wedonotplantoexpandthisprogramto100+Asiansourcesduetothelargeeffortrelativetotheuncertaintyofgain.WhileTACFhasenoughreproductiveF1,BC1,andBC2treesavailablenowtoadvance20additionalsources,wedonothaveenoughuniqueAsianchestnutsourcesofresistancefromthesegenerationstoadvance100additionalsources.Advancingadditionalsources(beyond20)wouldrequiretwoorthreeadditionalgenerationstomakeF1crosseswithAsianchestnutparentsandthenperformoneortwogenerationsofbackcrossingtoC.dentatapriortointercrossingtheselections.Weareplanningtoexpandtheeffectivepopulationsizeoftransgenictreesto>500(Westbrooketal.2019b).ThuswiththeexistingClapperandGravesbackcrossprograms,plustheplannedtransgenicbreedingprogram,ourbreedingprogramstogethershouldadequatelyrepresentdiversityandadaptivecapacitywithinC.dentata.

AcceleratedbreedingmethodssuchasthoserecommendedbyPowell(inducedearlyflowering,juvenileblighttesting)andothers(Meilan1997;Wheeleretal.1982;WheelerandBramlett1990)shouldbeconsideredtoinfusethesenewsourcesasquicklyaspossible.

TACFresponse:Acceleratedbreedingmethodsareusefulforgeneratingpollenfromfirstyearseedlings,butweneedlargetreestogeneratelargenumbersofseeds.Henceacceleratedbreedingmethodshavelimitedapplicabilitytothebackcrossprogram.Wewillneedtogeneratelargenumbersofprogeny(50–200)percrosstoperformselectionwithinbackcrosspopulationssegregatingforblighttolerancealleles.Weneedlargereproductivelymaturetreestogeneratelargenumbersofseed.Furthermore,phenotypicselectionaccuracyforblighttoleranceislimitedgiventhatblightphenotypeshavelowtomoderateheritability(h2<0.5,Westbrooketal.2019a).Thusweplantoprogenytestbackcrossselectioncandidatesthatpassthefirstroundofphenotypicselection.

Westronglyrecommendusingcontrolledcrossesratherthanopenpollinatedmaterialforprogenytesting,totheextentpossible.Thisneednotbebi-parentalcrosses,andinfact,itmaybepreferabletousepolymixestoincreasediversity(Lambethetal.2001;Wheeleretal.2006).

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Concernsaboutdifferentialreproductivesuccessorexclusioncouldbestudiedrelativelyeasilyandquicklywithgeneticmarkers(Apsitetal.1988;NakamuraandWheeler1992a,b)andcouldbeaccomplishedwithoutgreatcostordelaywithlowcostgeneticmarkers(e.g.,byanacademiccollaboration,perhapswithaTACFseedgrant).

TACFresponse:Weplantoprogenytestbackcrossselectioncandidatesthroughcontrolledpollinationswithpolymixes.Wealsoplantogenotypeprogenyofintercrossesamongtheseselectionstoensurethatparticularparentsarenotover-representedamongtheprogeny.

Totheextentpossible,Chapterprogramscouldalsobenefitfromsimilarexpansionsintheirgeneticbaseasopportunitiesarise(thoughweunderstand,andwerepleasedtolearn,thatsomeofthisworkisalreadyunderway).

Objective3.FinishselectioninClapperandGravesseedorchardsinTACF'sChapterbreedingprograms

ThereviewcommitteerecommendssomecontinuedworktodevelopBCpopulationsintheChapters,butsuggeststhattheworkherebelimitedrelativetotheothermajorobjectives,withthefollowingqualifications:

• SelectandguideChapters–EmphasizeasmallnumberofChaptersthathaveuniquecharacteristicsand/orenvironments,suchaszonesofecologicaladaptation,extentofotherdiseases(especiallyPhytophthora),andtheuniquequalityoftheAmericanchestnutparentsemployed.EmphasizeChaptersorregionswheretheextentofprogress,investment,andphenotypequalityarehighest.

TACFresponse:Thesouthernchapters(GA,NC/SC,AL,TN,VA,andKY)areplantingorplantoplantorchardscomposedofbackcrosstreesselectedforresistancetoPhytophthoracinnamomiforeventualbreedingwithblighttoleranttrees(Westbrooketal.2019c).AllchapterswillparticipateingermplasmconservationofAmericanchestnutsintheirregions.TheseAmericanchestnutswillbeusedfortransgenicoutcrossingandtocreatenewbackcrosslinesfromadditionalAsiansourcesofblighttolerance.PreliminarychaptertargetsfornumbersoftreestoconserveareinWestbrook(2018).Wemayrevisethesetargetsafterweobtainresultsfromarange-widestudyofgeneticdiversityandadaptationinAmericanchestnut.

• LeverageMeadowviewresults–UseresultsfromMeadowviewBCstudiesandChapterplantingstoselectprogramstoemphasize.ThisshouldhelpspreadandefficientlyuseresourcesbutshouldbedonecarefullysoasnottodisengageorcreateanimosityamongsttheChapters.Forinstance,hostvolunteersfromtheChapterstohelpwithincreasedbreedingloadsatMeadowvieworotherfutureorchardsites,usingChaptergeneticresources.

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TACFresponse:ConsideringthefindingthattheMeadowviewClapperandGravesBC3F2seedorchardsareexpectedtogenerateprogenywithlowtointermediateblighttolerance(Steineretal.2017;Westbrooketal.2019a),werecommendthatchaptersscalebackonplantingClapperandGravesBC3F2seedorchardsfromnineblocksof20backcrosslinesperchaptertoaminimumofthreeblocks(Westbrook2018).Thesuggestionoforganizingfoundation-widevolunteereventsatMeadowviewisinteresting,andweshallconsiderthepossibilities.

• IntegrateGE–EmphasizeworktointegrateGEtreesintotheBCpopulationsrapidlyinselectedChaptersifthestrength,stability,andderegulationstatusofthetransgenicOxOtreesarefavorableandthereissupportintheChaptersfortransgenicwork.

TACFresponse:TACFstaffhaveadministeredaquestionnairetoassesstowhatextentspecificchapterswouldliketobeinvolvedinatransgenicbreedingprogram.ChapterinvolvementwiththetransgenicbreedingprogrammayvaryfromcollectingscionsfromnaturallyoccurringAmericanchestnutstomaintaininggermplasmconservationorchards,performingpollinationswithtransgenicpollen,andplantingorchardscomposedoftransgenicprogeny.

Objective4.DiversifyGEpopulationsandcombineblightandPRRresistance

Westronglysupportexpandedeffortstoincreasediversityinthetransgenicmaterials.Considerationneedstobegivenbothtothenumberoftransgeniceventsandgenotypesthataretransformed,andtotherangeofgenotypesthatwillbepollinatedwithtransgenictrees.Thebiggestconstrainttoeffectivepopulationsizewillbethenumberoftransgenictreesused.WethinkthisshouldbeincreasedbothbydevelopingOxOtransformantsinseveralmoreAmericanchestnutgenotypes,andpotentiallybydevelopingtransgenicswithresistancefromgenesotherthanOxO.Theobjectivesoutlinedin4b-4gareaggressiveanddesirable.

Additionally:

• Climatechangeconsiderations–Whenexpandingthenumberoftransgenicgenotypesandthenon-transgenictreesthatwillbepollinatedwithtransgenics,selectgoodgenotypesfromacrosstherangeofthespecies,includingbothleadingandlaggingedgesofthecurrentrange.Thiswillcapturetherangeofadaptivediversitywithinnaturalpopulations.

Seeourresponsebelowtothesuggestionforalandscapegenomicsstudy.

• Considerexsituplantingsofcollectionsinwesternrefugia,freeofdiseaseatthistime,toensureavailabilityforcrossinginyearstocome.

TACFresponse:ApermitisrequiredtoimportAmericanchestnutsfromtheeasternUnitedStatesintoOregon,Washington,andCalifornia.CarewouldneedtobetakentodisinfestseedsofblightpropagulespriortoshippingwestoftheMississippiriver.Ifwecanfindreliable

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collaboratorsthatarewillingtomaintainpureAmericanchestnutplantingsonthewestcoast,thentheseblight-freeplantingscouldbeusefulforgermplasmconservationandbreeding.PendingU.S.regulatoryapproval,SUNY-ESFandTACFmaybeabletoshippollengeneratedfromplantsgrowninblight-freegrowthchamberstothePacificNorthwestforpollinatinghealthyAmericanchestnuttreesinthePacificNorthwesttoincreasethediversityinthetransgenicbreedingprogram.

• Linkagedrag–Wesuggestcarefulconsiderationoflinkagedragthatmayresultinexpressionofrecessivedeleteriousalleleswhenindividualtransgeniceventsaremadehomozygousinseedorchards.Diversifyingtransgeniceventswillamelioratethisissue,althoughwerecognizetherearesomepotentialconcernsaroundgenesilencing.Innaturalpopulations,ifgenesilencingoccursinsomegenotypes,naturalselectionwillquicklyremoveorreducethoseinthepopulation.Populationsimulationmodellingcouldbeusedtoevaluatethisrisk.

TACFresponse:Inapaperinreview(Westbrooketal.2019b),wehavesimulatedtransgenicoutcrosspedigreesandmarker-assistedintrogressionoftheOxOtransgene.TheabstractofthepapersummarizesourplanfordiversifyingthetransgenicpopulationofAmericanchestnut:

Summary

• BreedingtransgenicblighttolerantAmericanchestnutswithsusceptiblewild-typetreesispotentiallyanefficientmethodtorescuethegeneticdiversityandadaptivecapacityoftheAmericanchestnutpopulationforlarge-scalerestoration.

• Todevelopabreedingplantodiversifyatransgenicblighttolerantpopulation,wesimulatedpedigreestoestimateinbreedingcoefficientsandeffectivepopulationsizeinscenariosinvolvingoutcrossing1to4transgenicfounderstoamaximumof1500wild-typetreesover1to5generations.Wealsosimulatedmarker-assistedintrogressionscenariostominimizetheextentofthetransgenicfoundergenome,especiallyonthetransgenecarrierchromosome.

• Simulationssuggestthattheeffectivepopulationsizemaybeincreasedto>500and?theaverageinbreedingcoefficientreducedto<0.01byoutcrossingasingletransgenicfounderoverfivegenerationsto2,25,50,150,and450(677total)wild-typeparents.Threegenerationsofmarker-assistedintrogressionwith50to100progenypercrossispredictedtodecreasethelengthoffoundergenometo7%to13%ofthetransgenecarrierchromosomelengthascomparedto42%witheventselectiononly.Transgenicoutcrossprogenymaybeintercrossedtogenerateapopulationoftreesthatishomozygousforblighttolerance.

• AdiversifiedpopulationoftransgenicblighttolerantAmericanchestnutisestimatedtobeavailableforuseinlarge-scaleforestrestoration20to35yearsafterfederalapprovaltodistributethetrees.Incontrast,treesfromearliergenerationswouldbeavailablealmostimmediatelyafterfederalapprovalforpersonalorhorticulturalplantings.Methodstoaccelerateoutcrossingarediscussed.

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• Genediversification–ThecommitteeurgesthatadditionalblightresistantGEeventsareproducedtoprotectagainstunexpectedsilencing,pleiotropy,anddragofdeleteriousalleleswhenhomozygous.ThesecanbealternateformsofOxOoverexpressionbutlackingcontiguousregionsofsequenceidentity.

TACFresponse:WehaveincludedanexcerptfromWestbrooketal.2019bonourplansforcreatingnewtransgenicfounders:

“WecurrentlyplantocreateuptothreeadditionaltransgenicfounderlinesthroughAgrobacterium-mediatedinsertionofOxOintothreeadditionalAmericanchestnuttrees’genomes.Creatingadditionaltransgenicfounderlinesalleviatesthefounderbottleneckoneffectivepopulationsizeoncetransgenicfoundersareoutcrossedtowild-typetrees.HavingadditionalfoundersalsomitigatestheriskthatdeleteriousmutationsinlinkagedisequilibriumwithOxOinasinglefoundertree’sgenomewillhavenegativeeffectsonfitnessamongprogenythatinheritOxOinahomozygousstate.However,outcrossingwithmultipletransgenicfoundersalsocarriestheriskthattheOxOtransgenecouldbesilencedinprogenythatinheritmultiplecopiesofOxOatdifferentlocationsinthegenome.Tomitigatetheriskofgenesilencing,weintendtoexpressOxOwithdifferentpromotersindifferenttransgenicfounders.Inthe‘Darling58’founder,OxOisexpressedwiththeconstitutiveCaMV35spromoter.AdditionaltransgeniclinesofAmericanchestnutwillbedevelopedinwhichOxOisexpressedfromawound-induciblepromoter(win3.12)anddifferentconstitutivepromoter(UBQ11).ExpressingtheOxOtransgenewithdifferentpromotersreducestheriskofsilencingduetomethylationofaspecificpromoterregion,thoughweacknowledgethatpost-transcriptionalgenesilencingwouldnotbeaffectedbyspecificpromoters.Inaforestsetting,blightinfectionwilleventuallykilltreesthathavetheOxOgenesilenced;therefore,naturalselectioncouldmaintaintransgenicblighttoleranceevenifsilencingoccurs.”

”

• ItmaybedesirableinthefuturetoonlycreateT2crossesamongT1individualsfromdifferentgeneticbackgrounds.

TACFresponse:OnceSUNY-ESFhascreatedmultipletransgenicfounderlineswithOxOinsertedatdifferentlocationsindifferentC.dentatageneticbackgrounds,weplantointercrossthesetreesovertwogenerationstocompareOxOexpressionamongprogenythatinherit1,2,3,or4copiesofOxOattwogeneticloci.ThepurposeofthisexperimentwillbetotestifOxOexpressionisreducedorsilencedamongprogenythatinheritmultiplecopiesofOxO.

• Geneinsertioneventstability–WeurgecontinuedstudiesofOXOandnewtransgenes’expressionlevelsandpotentialsilencingastheyaremovedwithadditionalcrosses,astreesageandamongdifferentenvironmentalstresses.Ifthisturnsouttobeinfrequent

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orsporadic,naturalselectionshouldeffectivelycleansethepopulationofmostofthesilenced(blightsusceptible)trees.

TACFresponse:WeacknowledgethatexpressionofOxOmaybeattenuatedorsilencedindifferentC.dentatageneticbackgrounds,differentgenerations,orunderenvironmentalstressconditions.In2019,weappliedforAPHISpermitstooutcrossT1progenyofthe‘Darling58’transgenicfoundertoadditionalwild-typeC.dentataparents.WewillmeasureOxOexpressionamongtheprogenyfromthesecrossestodetermineifexpressionvariesamongprogenyofdifferentC.dentataparents.WeplantoconductfuturestudieswherewewillcomparetheexpressionofOxOamongprogenyofdifferentC.dentataoutcrossgenerationsincludingcomparingOxOexpressioninhemizygousversushomozygoustrees.WealsoplantomeasuretheexpressionofOxOincommonfamiliesplantedindifferentenvironments.

• Landscapegenomics–Thelandscapegenomicstudiesproposedmaybehelpfulforchoosingnativechestnutsourcesfordiversification/outcrossing.Inaddition,becauseoftheuseofcuttingedgemoleculargenotypingtechnologyandinterestfromacademiccollaborators,significantexternalgrantfundingmaybeobtainedtosupportthework.Agenomicapproachmaybeusefulandcost-effectiveinevaluatingtheextenttowhichextantpopulationsarelocallyadaptedtoclimateandmayindicatehowmanyseedzonesareneededonceresistantmaterialisavailableforrestoration.Thisapproachmightsubstituteforprovenancetestingforclimateadaptation(anapproachthatcouldbeexpensiveandpossiblyineffectiveasnon-resistantmaterialfromdifferentlocationsmightsuccumbtoblightbeforereliableadaptivepatternsareexpressedinthosetrialsthatareplantedwithinthecurrentrangeofchestnutandblight).However,theuseofecographicinformation(climateoforigin,e.g.,fromClimateNA)andclimatechangemodelingwillalsobehelpfulforchoosingdiverseandadaptedsourcesforbreedingandfuturerestorationefforts.Theextenttowhichgenomicstudies,orprovenancetrialsonnon-blightlocations,wouldaddtoecographicvariablestopredictadaptivepatternsisunclear,andmightbeinvestigatedinpilotstudies(e.g.,asubsetoflocations,perhapsfundedbyapilotgrant)beforemajorinvestmentsineitherapproachweremade.

TACFresponse:JasonHolliday(VATech)andJaredWestbrook(TACF)receivedagrantfromtheUSDANIFAprogramin2019tosequencethegenomesof500AmericanchestnuttreesfromacrossthehistoricalspeciesrangetostudydiversityandclimateadaptationinremnantAmericanchestnutpopulations.Wehaveincludedanexcerptfromtheproposalthataddresseshowwewillusethegenomicdatatodeveloptargetsforgermplasmconservation:“ResultsfromObjectiveII(delineationpopulationstructure)andObjectiveIII(identificationoflociunderlyinglocaladaptation)willbeusedtodeveloptargetsfornumberofAmericanchestnuttreestopropagatefromeachmanagementunitandadaptiveunit,respectively.Targetsforthenumberofwild-typetreestopropagatewithineachmanagementunitwillbeproportionaltoallelicdiversityamongputativelyadaptivelociandtothepredictedfuture

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spatialextentofsuitableclimateassociatedwitheachadaptiveunit.Ourrationaleforconservinglargernumbersoftreeswithinmoreadaptivelydiversesub-populationsisthatthesesub-populationsareexpectedtorespondmorestronglytonaturalselection.Toincorporatefutureclimateintooursamplingscheme,numericalrangesinclimatevariablesthatdefineadaptiveunitswithinC.dentatawillbeidentifiedbyclippinghistoricalclimatedatabasedonthegeographicextentofeachadaptiveunitinArcGIS.ThetotalgeographicalextentofsuitablehabitatforC.dentatawillbepredictedfortheyear2080withMaximumEntropy(MaxEnt)modeling,whichcombinesgridsofclimatevariableswithhabitatoccupancydatatopredictsuitableconditionsforaspecies.TheinputsforMaxEntpredictionofsuitablehabitatwillbespatialvariationindensityofAmericanchestnutstemsestimatedfromForestInventoryandAnalysisdata;historicalclimatedata(1895–present)forthecurrentrangeofAmericanchestnut(www.prism.oregonstate.edu);soilsurveydata(websoilsurvey.sc.egov.usda.gov),elevationfromtheU.S.GeologicalSurvey3DElevationProgram(nationalmap.gov/3DEP);andIntergovernmentalPanelonClimateChangepredictionsofclimatein2080usingtworepresentationconcentrationpathways(RCP4.5andRCP8.0)forgreenhousegases(http://sedac.ipcc-data.org/ddc/).Thegeographicalareaassociatedwitheachadaptiveunitwillbeestimatedfortheyear2080bypartitioningclimatevariationwithinthepredictedsuitablehabitatforC.dentatain2080rangebyrangesinthehistoricalclimatedataassociatedeachadaptiveunit.”

• GEcaveats--WhileOxOtransgenictreesoffergreathope,theyshouldbeembracedwithcautionastheyhavenotyetbeguntobeusedinfieldtrialsofasizeanddurationthatiscomparabletothoseinforestryoragriculturalbiotechnologybreedingprograms.Theyarealsodependentonasinglegeneinsertioneventthatisthebestofonlyafewdozenthathavebeenproduced.Thus,thecommitteeconsidersUSDAderegulationanditsequivalentatEPA/FDAnotacommitmenttocommercialuse,asiscommoninagriculture,butasalicensetodoessentialbreedingresearch.Iftheyaredeployedatscaleandstarttobeusedinrestoration,caremustbetakentoinsurethefirsteffortsaresuccessful(highqualitysites,management,andoutreach).Earlyfailuresinthiscontentiousareacouldputthefutureoftheprogramatrisk.Thismessageneedstobeconveyedtocooperatorstoavoidoverpromising/underdelivering.

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AppendixI-CommentsonAdditionalQuestionsPosedbyJaredWestbrook

Question1a.DothereviewersseemeritincontinuingplantingandselectioninTACF’sbackcrossprogram(especiallyinthevolunteer-ledChapterprograms)whenintermediateresistancetochestnutblightisexpectedafterselectioniscompleteinBCxF2seedorchards?

WeassumethatthequestionisaskingifTACFshouldmovetotheBC3xF3generationandbeyond(F4,F5).Simulationsthatwerepresentedtothecommitteesuggestedthataveragelevelsofresistanceshouldcontinuetoincrease,butactualresultstodatehavenotreachedfullexpectations2.Therealprospectofinvestingyearsmoreofbreedingandtestingwithlittlereturnexists.AsdiscussedaboveinresponsetotheObjectives,thecommitteebelievesTACFshouldexplorealloptions.ForthemainlineprogramwesuggestintercrossingGravesandClapperassoonaspossible,usingcontrolledcrosses,addingvariablevirulentstrainsforinoculations,andrelyingongenomic/geneticmarker/acceleratedfloweringselectionmethodstoacceleratetheprocess.YoumaywishtofollowtheoriginalplaninselectedChapterprogramsanddisbandingothers.Wealsoproposedinvestinginadditionalsourcesofresistance.

Ifgrantfundscouldbeobtainedbyacollaborator,agenomicapproachcouldbeveryusefulindeterminingthenumberoflociorgenomicregionsinvolvedinresistancefromChinesechestnut,providingapotentialexplanationforwhyresistanceislowerthanexpectedintheBC3F2materials,andinformingthedecisionofwhethertocontinuewithmoregenerationsofbackcrossing.ItcouldalsobeusefulforselectingBC3F2orBC2F2individualsthathavehigherthanaverageAmericanchestnutancestryasancestryshouldbequitevariableaftertwoorthreegenerationsofbackcrossing.Theroleofinbreeding,andtheextentofinbreedingdepression,onblightresistanceandgrowthintheseBCmaterialsalsowarrantsstudy.

Question1b.WhatareprosandconsforcompletingselectionatBCxF2inthebackcrossprogramiftransgenicAmericanchestnuttreeswithpotentiallyhighlevelsofblightresistanceareapprovedforuseinbreedingandrestorationbytheU.S.federalgovernment?

Theobvious“con”isthattransgenicsmaynotbederegulated,orthatthecostforeachaddedeventwillnotbeaffordableorexpeditious.Thatispotentiallyamajorconcern.Wearenotsurewhatlevelofinvestmentisneededfor“completingtheselectionattheBCxF2level”butifitcanbedonein2yearsorless,thenbyallmeansdoitandmoveontotheotherapproachesdiscussedearlierinthisreport,asfundsandtimingpermit.CrossingBCtreeswithtransgenicsshouldhelpbuildgreaterresistancetoblight.

Question2.Dothereviewersseemeritinraisinggrantfundsandphilanthropicinvestmentsforgenomicselection(GS)inChapterbreedingprograms?Alternatively,doyourecommendmostlyphenotypicselectionandlimitedprogenytestinginChapterbreedingprograms?

Thecommitteebelievesthatitwouldbewisetofirstshowproofofconceptinthemainlineprogramsandpenciloutthecost/benefitsbeforemakingdecisionsforChapters.ForChapterprogramsthatreliedonthesamesourcesofresistance,itisalsoessentialtodetermineifonetrainingpopulationispredictiveforotherpopulations—whichweregardasunlikelyunlessthematerialsareidentical.The2SeeStacyClark’sfieldresults.FunctionalresistancemaybegreaterthansuggestedbytrialsatMeadowview.Moreactualfieldresultsongoodchestnutsitesareneededbeforemakingdefinitiveconclusions.

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combinationofgenomicselection,useoflessvirulentstrainsforinoculation(soawidervarietyofresistancegenescanberecognizedandselectedfor),andhopefullycontinuedreductioningenotypingandbioinformaticscosts,maymakethisaviableoptioninthefuture.Nomatterwhethergenomicselectionisusedornot,relianceonprogenytestingwillnotgoawayformanyyears,sinceitisnecessarytoprovidetrainingpopulationsandadvancedgenerationcrosseswithmultiplesourcesofresistance.Considerationshouldbegiventotestingwithandwithoutinoculation.

AsidefromGS,thereisaverystrongroletobeplayedbygenomicsinselectingamongBC2andBC3treesforthehighestproportionofAmericanchestnutancestry,aswellastoreducethesizeofregionsflankingQTLforblightresistanceifsufficientnumbersofindividualscanbegenotypedformappingtheselociorgenomicregions.Genomicapproachescanalsoprovideagoodpictureoftheextentoflocaladaptationtoguidethenumberofbreedingpopulationsneeded.Giventheneedforaccesstolaboratoryfacilitiesandbioinformaticsexpertise,perhapsthebestpathtowardsbringingagenomiccomponentintotheTACFscienceprogramisthroughcollaborationswithacademicpartnerswhocanseekfundingfromfederalgrantprograms.ThepartnershipTACFhasdevelopedwithDr.JasonHollidayisanexcellentexampleofthis.

Question3.DothereviewersseeaddedvalueinpursuingregulatoryapprovalfortransgenictreeswithresistancetoPhytophthoracinnamomi?ConsiderthatbackcrosstreeshavebeendemonstratedtohaveintermediatetohighlevelsofresistancetoP.cinnamomi.

IfnaturalresistancetoPhytophthoraisonlyfoundinraregermplasm,itwillmakebreedingforresistancetoitandchestnutblight,plusselectionforadaptationandgrowth/form,extremelycomplex(asecondaryBCprogram).IfGEapproachestoPhytophthoraresistanceareeffective,theresistancegenescanbecombinedintosingleconstructsorco-introduced,theninsertedandcrossedintodiversebasegermplasm.HavingbothGEandnaturalformswouldalsobehighlydesirablefordurability.

Question4.Towhatextentdothereviewersrecommendbreedingtransgenictreeswithbackcrosstreesandtowhatextentdoyourecommendkeepingthebackcrossandtransgenicprogramsseparate?Inotherwords,whatproportionoftransgenicbreedinglinesdoyourecommendcreatingviaoutcrossingtransgenictreestobackcrosstreesv.crossingtransgenictreestowild-typeAmericanchestnuts?Whatarethesocialimplicationsofkeepingtheseprogramsseparatev.combiningthem?

Assumingtransgenicsarepermitted,thecommitteebelievesthatitwillbenecessarytofollowbothpaths,simplybecausetransgenicswillnotbepermittedeverywhereintherestorationandforestproductmarketlandscape,atleastnotformanyyears.Itmightbewisetomixthelinesinproportiontotheareasinwhichmixingwillbepermitted,asthisshouldprovidethemostdurableresistance.

Question5.Dothereviewersrecommendconsideringlocaladaptationandclimatechangeinourplansforpropagatingwild-typetreesforoutcrossinganddiversifyingtransgenicpopulations?

Thecommitteeunanimouslyagreed,asdiscussedabove.Whenmaterialsarereadyforrestoration,encourage“assistedgeneflow,”climate-basedseedtransfertomatchgenotypeswithnewratherthanlast-centurylocalclimates(AitkenandWhitlock2013;AitkenandBemmels2016).Ifresultsareavailable

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fromecographicorlandscapegenomicsstudiestheycaninformseedtransfer;otherwiseclimatemodelssuchastheUSForestService’sSeedlotSelectionToolcanbeused(https://www.fs.usda.gov/ccrc/tools/seedlot-selection-tool).WhilethedevelopmentofresistantmaterialfaroutweighsclimateadaptationconcernsforAmericanchestnut,ifresistantgenotypesareplantedinenvironmentstheyaremaladaptedtorestorationwillfail,andmostwidespreadtreespeciesshowconsiderablelocaladaptationtoclimate.

Dothereviewersagreewithourapproachofpropagatingtreesfromsubpopulationsinproportiontodiversityatgeneticlocithatpotentiallyunderlielocaladaptationandinproportiontoprojectedrangeareaforeachlocallyadaptedsubpopulation?

Inearlystagesofrestoration,itisnotessentialtomaximizediversityoneachsitebuttomatchgenotypestositestowhichtheyshouldbeabletogrowwell(whichmaybelimitedinearlyplantings).However,somelevelofdiversityishighlydesirable(e.g.,aminimumof5genotypesforspecificlocations).Overtimediversityshouldgrowasnewresistantmaterialsaredevelopedandplanted,andtreesoutcrossinthewild.

Alternatively,wecouldpropagategermplasmtorepresenttherangeofclimateandsoilconditionsoverthehistoricalrangeofAmericanchestnutwithoutconsideringclimatechangeandlocaladaptation.

Asdiscussedabove,inadditiontohistoricrangeandadaptationconsiderations,webelieveitiswisetoconsiderclimatechange,includinglikelychangestocurrentpestsandpathogenranges,inthelightofavailableecologicalandgenotypicinformation.

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AppendixII-CommentsonQueriesfromMs.SaraFitzsimmons

Question1.Howmuchemphasisshouldbeplacedonconservingwildtreesinsituvs.exsitu?

WedonotadvisespendingresourcesoninsituconservationeffortsexceptforlargeAmericansurvivors,butifvolunteersandcooperatorswanttodothis,don’tdiscouragethem.Focussomeeffortonexsituconservationforlaggingedgepopulationsgivenclimatechange(Aitkenetal.2008).Getpollenviahighlightintensityfloralinduction,ortransplanttreesfromnewbasalsproutsfromthefieldandgraftormicropropagate,ifwildtreesdonotlivelongenoughtoprovidepollenoroutbredseeds.

Inaddition,exsituplantingsthatcouldbeusedascommongardensforGWASorprovenancetrialsinblight-freewesternplantations,usingclonallyreplicatedpropagules,couldbehelpful.Plagiotropicgrowthhabitcanbeovercomebycoppicingafteryearoneifrootedcuttingsareemployed.

Question2.Atwhatpointshouldmaterialsbereleasedfor“restoration”?Weurgethattheearlydemonstrationtrialshavehighlevelsofresistance(perhapsselectivelyusingclonesforqualityassurance)beestablishedonhighqualityandpubliclyaccessiblesites.Wesupportusingamixofmaterialsforrestorationonabroaderscale,buttemperexpectationsassomeormanytreeswilldieandotherswon’tlookgreat.PlacesignsneardemonstrationplantingsinawaythatcapturesandamplifiestheeducationalcomponentofTACFanditsmanyprograms.

Question3.Howmuch“diversity”needstoberepresentedinasingle“restorationplanting”.Ifaplantingis300trees,howmanybackgrounds/lines/pedigreesshouldbeestablishedwithinthatplanting?Thecommitteebelievesthathighdiversityisnotessentialinearlyplantings,butnotfewerthanabout5genotypesthatareexpectedtobeadaptedtotheareashouldbeused.Iterativeestablishmentof5ormorenewadaptedgenotypeseveryfewyearsinagivenarea,andasnewmaterialscomeonline,usingboththebestBCandtransgenicsourcesifavailable,isareasonablesilviculturalscheme.Diversityoftrees,andlandscape-leveldiversity,willalsoincreaseovertimewithnaturallyoutcrossingandregeneration.Ofcourse,theminimumnumberofunrelatedgenotypesinthebreedingprogramshouldbemuchhigherthantheminimumnumberinasinglerestorationplanting.

Question4.ShouldTACFcontinuetohavematerialtransferagreementsofsomekindwithpartnerswhoplantmaterials?Iwelcomeanycommentsregardingthestringency/outlineofthesetypesofagreements.Presumingtherearenosignificantpatent,market,orregulatoryconstraints(asthecommitteebelievesthattheTACFdesiresandwillworktoputinplace),werecommendalow-obstacleinstrumentfordistributionandtracking.Thismightbeasimple,one-pagematerialtransferagreementthatrequiresacommitmenttoannualorbiannualreportingofplantingsizeandlocation(atleasttocountylevelasmanywillnotwishtodisclosespecificlocations),andtreehealthandform.TheMTArequirementwouldalsoestablishpaperandemailcontactinformationsoTACForotherscouldqueryaboutstatus,andtoprovidenewinformationtothoseplantingaboutthematerialsasresearchproceeds.

Question5.ShouldTACFplanonoverseeingrestorationactivitiestoensure1)blight-resistantmaterialsarediversifiedtoanappropriatelevelforspeciesrestorationand2)thosematerialsarethenplantedinenoughlocationsandinthemostsuitableareasforthespeciestoresumeecosystemservices.Whatistheminimumnumberoflocationsorareaswhichneedtobeestablished.

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IfTACFisproducingmaterials,thenitcancontrol,toapoint,when,withwhatmaterials,andwhererestorationplantingsareestablished.Thisgoalisunlikelytobefeasibleinallplaces;however,TACFcouldproduceandpublishmaps/locationsthatprioritizehighqualitysitesforAmericanchestnutratherthanmarginalsites,suggestthemmostappropriategeneticmaterials,andupdateusers(seecommentsonquestion4)asnewperformance,genetic,andecologicalinformationbecomesavailable.

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