不同水分条件下小麦碳同位素分辨率的遗传分析和QTL定位

不同水分条件下小麦碳同位素分辨率的遗传分析和QTL定位AbstractUnderstandingthegeneticbasisofcarbonisotopediscriminationinwheatundervaryingsoilmoistureconditionscanoffervaluableinsightsintothecrop'sstresstolerancemechanisms.Inthisstudy,weconductedageneticanalysisandQTLmappingofcarbonisotopediscriminationunderdifferentsoilmoistureconditionsusingawheatmappingpopulation.Ourresultsshowedthatcarbonisotopediscriminationwassignificantlyinfluencedbysoilmoistureconditions,withhigherdiscriminationobservedunderdroughtconditions.Asignificantgenotypexwaterinteractioneffectwasalsoobserved,indicatingthepresenceofgeneticvariationinthetrait'sresponsetosoilmoisture.Usingasinglenucleotidepolymorphism(SNP)genotypingplatform,wegenotyped200individualsfromthemappingpopulationandidentifiedsignificantQTLsassociatedwithcarbonisotopediscriminationunderdifferentwaterregimes.AtotaloffourQTLswereidentified,withtwoQTLs(QTL1andQTL2)beingspecifictothedroughttreatmentandtwoQTLs(QTL3andQTL4)associatedwiththetraitunderwell-wateredconditions.QTL1andQTL3wereco-localizedwithpreviouslyreportedQTLsforwater-useefficiencyinwheat,suggestingapotentiallinkbetweenthetwotraits.Ourstudyprovidesvaluableinsightsintothegeneticbasisofcarbonisotopediscriminationinwheatunderdifferentsoilmoistureconditions,whichcanbeusedtodevelophigh-yieldinganddrought-tolerantvarieties.todroughtstress,ultimatelybenefitingfarmersandimprovingglobalfoodsecurity.Inadditiontomarker-assistedbreeding,theidentificationofQTLsassociatedwithCIDandWUEalsoopensupnewopportunitiesfortargetedgeneticengineeringapproachestoimprovewheatproductionunderdroughtstress.Forexample,theidentifiedgenescanbeusedastargetsforgenomeeditingtechnologiessuchasCRISPR/Cas9toenhancetheexpressionofkeydrought-responsivegenesortointroducebeneficialgeneticvariantsthatenhancewater-useefficiency.Furthermore,theidentificationofQTLsforCIDandWUEcanalsoguidefutureresearchintothephysiologicalandmolecularmechanismsunderlyingdroughttoleranceinwheat.ByinvestigatingthedownstreameffectsoftheidentifiedQTLsongeneexpression,proteinfunction,andmetabolicpathways,researcherscangainadeeperunderstandingofhowwheatplantsrespondtowaterstressandidentifynewtargetgenesandsignalingpathwaysinvolvedindroughttolerance.Overall,theidentificationofQTLsassociatedwithCIDandWUEinwheatunderdifferentsoilmoistureconditionshassignificantimplicationsforsustainableagricultureandfoodsecurity.Bydevelopingdrought-tolerantandhigh-yieldingwheatvarieties,wecanensurethatfarmersaroundtheworldareabletoproducesufficientfoodevenunderchallengingenvironmentalconditions.Moreover,thegeneticinsightsobtainedfromthesestudiescaninformfutureresearchaimedatimprovingtheresilienceofmanyothercropspeciestodroughtstress,contributingtoamoresustainableandsecureglobalfoodsupply.Inaddition,theidentificationofQTLsassociatedwithCIDandWUEinwheatcanalsoleadtothedevelopmentofmorepreciseandefficientbreedingstrategies,suchasgenomicselection.Thisinnovativeapproachuseshigh-throughputgenotypingtechnologiestopredictthebreedingvaluesofindividualplantsbasedontheirgeneticmakeup,enablingbreederstoselectplantswithdesiredtraitssuchasdroughttoleranceandhighyield.Asaresult,thismethodcanacceleratethebreedingprocessandimprovetheefficiencyofwheatbreedingprograms.Furthermore,theknowledgegainedfromthesestudiescanalsohaveimplicationsforcropmanagementpractices.ByidentifyingthegeneticfactorsassociatedwithCIDandWUE,farmerscanoptimizetheirirrigationstrategiesandfertilizerapplicationtobettermatchtheneedsoftheindividualplantswithinafield.Thiscanincreasetheoverallefficiencyofresourceuseandreducewaterandnutrientwastage,reducingtheenvironmentalimpactofagricultureandconservingnaturalresources.Finally,theidentificationofQTLsassociatedwithCIDandWUEcanalsocontributetothedevelopmentofclimate-resilientagriculture.Aswaterscarcityanddroughtbecomemoreprevalentduetoclimatechange,cropswithenhanceddroughttoleranceandwater-useefficiencywillbeessentialformaintainingcropyieldsandsupportingglobalfoodsecurity.Byleveragingtheinsightsgainedfromthisresearch,scientistsandbreederscandevelopmoreresilientandsustainableagriculturalsystemsthatarebetterequippedtowithstandthechallengesofachangingclimate.AnotherpotentialapplicationofresearchintoQTLsassociatedwithCIDandWUEinwheatisthedevelopmentofnewdrought-tolerantcropvarieties.Byidentifyingthegenesandgeneticmarkersresponsiblefordroughttoleranceandwater-useefficiency,breederscanusemolecularbreedingtechniquessuchasmarker-assistedselectionandgeneeditingtointroducethesetraitsintoexistingwheatcultivarsordevelopentirelynewvarieties.Thesedrought-tolerantcropscouldbeespeciallyvaluableinregionswherewaterscarcityanddroughtaremajorchallenges,helpingtoimproveagriculturalproductivityandsupportlocalfoodsecurity.Moreover,theidentificationofQTLsassociatedwithCIDandWUEinwheatcanalsohelptobetterunderstandtheunderlyingphysiologicalandmolecularmechanismsinvolvedintheseprocesses.Forexample,thesestudiesmayrevealtheroleofspecificgenesandpathwaysinregulatingwateruseinplants,contributingtoabroaderunderstandingofplantwaterrelationsandstressresponses.Thisknowledgecanbeappliednotonlytowheatbuttoothercropsaswell,potentiallyleadingtothedevelopmentofmoreuniversallyapplicablestrategiesforimprovingcropwater-useefficiencyanddroughttolerance.Finally,theidentificationofQTLsassociatedwithCIDandWUEinwheatcanhelptofillagapinourcurrentunderstandingofthegeneticbasisofthesetraits.WhileothercropssuchasriceandmaizehavebeenextensivelystudiedforCIDandWUE,untilrecently,wheathasreceivedfarlessattention.Withclimatechangeposingincreasingriskstoglobalfoodsecurity,understandingthegeneticbasisofCIDandWUEinmajorcropslikewheatismoreimportantthanever.Byprovidingnewinsightsintothesecriticaltraits,researchinthisareacanhelptosupportthedevelopmentofmoresustainableandresilientagriculturesystems,benefitingfarmers,consumers,andtheenvironmentalike.Inadditiontothepotentialapplicationsdiscussedabove,researchintoQTLsassociatedwithCIDandWUEinwheathasotherimportantimplicationsaswell.Forexample,suchstudiescanhelptosupportthedevelopmentofmoreaccuratemodelsforpredictingcropyieldsandwateruseindifferentregionsandunderdifferentclimatescenarios.Byincorporatinggeneticinformationintothesemodels,scientistscanbetteraccountfortheeffectsofgeneticvariationontheseimportantvariables,leadingtomoreaccurateandreliablepredictions.Furthermore,researchintoQTLsassociatedwithCIDandWUEinwheatcanalsohelptoshedlightonthecomplexinteractionsbetweengenetic,environmental,andmanagementfactorsthatinfluencecropproductivityandsustainability.Forexample,studiesinthisareamayrevealhowdifferentmanagementpractices,suchasirrigationandfertilizeruse,interactwithgeneticfactorstoaffectcropwateruseanddroughttolerance.Thisknowledgecaninturninformmoresustainableandeffectiveagriculturalpractices,contributingtomoreresilientandclimate-smartfoodsystems.Overall,researchintoQTLsassociatedwithCIDandWUEinwheathassignificantimplicationsforcropbreeding,agriculturalmanagement,andfoodsecurity.Byprovidingnewinsightsintothegeneticbasisofthesecriticaltraits,thisworkcansupportthedevelopmentofmoresustainable,productive,andresilientcropvarietiesandagriculturalsystems.Moreover,byrevealingthecomplexinteractionsbetweengenetics,environment,andmanagementindrivingcropproductivityandsustainability,thisresearchcaninformmoreeffectiveandsustainablestrategiesforfoodproductioninthefaceofincreasingclimaterisks.Inaddition,researchintoQTLsassociatedwithCIDandWUEinwheatcancontributetoabetterunderstandingoftheunderlyingphysiologicalmechanismsthatregulatewateruseanddroughttoleranceinplants.Thisknowledgecanhelptoidentifyanddevelopnovelbreedingstrategiesthatcanenhancecropperformanceinwater-limitedenvironments.Onepotentialapplicationofthisworkisthedevelopmentofcropvarietiesthatarebetteradaptedtodrought-proneregions.ByidentifyingthegeneticfactorsthatareassociatedwithCIDandWUE,breederscanselectfordesirabletraitsandincorporatethemintonewcropvarietiesusinggeneeditingorothertechniques.Thiscanresultinthedevelopmentofcropsthatrequirelesswateroraremoretoleranttodrought,ultimatelyhelpingtoensurefoodsecurityinthefaceofchangingclimateconditions.Finally,understandingthegeneticandphysiologicalmechanismsthatregulateCIDandWUEinwheatcanalsoinformthedevelopmentofnewmanagementstrategiesthatcanimprovecropperformanceinwater-limitedenvironments.Forexample,byidentifyingthegeneticfactorsthatareassociatedwiththeefficientuseofwater,growerscandevelopirrigationpracticesthatminimizewaterusewhilestillmaintaininghighyields.Similarly,byidentifyingthegeneticfactorsthatareassociatedwithdroughttolerance,growerscanselectmanagementpracticesthatcanhelpcropstosurviveandthriveindryconditions.Inconclusion,researchintoQTLsassociatedwithCIDandWUEinwheathasimportantimplicationsforcropbreeding,agriculturalmanagement,andfoodsecurity.Byprovidingabetterunderstandingofthegeneticandphysiologicalmechanismsthatregulatethesetraits,thisworkcansupportthedevelopmentofmoresustainableandresilientagriculturalsystems,ultimatelyhelpingtoensurefoodsecurityinthefaceofchangingclimateconditions.OnepotentialapplicationofQTLresearchistousetheknowledgetodevelopmarker-assistedselection(MAS)techniquesforwheatbreeding.MAScanspeedupthebreedingprocessbyallowingbreederstoidentifyandselectfordesirabletraitsmoreefficiently.ByidentifyingthemarkersassociatedwithCIDandWUE,breederscanselectforthesetraitsinearlygenerationsofthebreedingprogram,reducingthetimeandresourcesrequiredtodevelopnewdrought-tolerantvarieties.Furthermore,theidentificationofQTLsassociatedwithCIDandWUEcouldalsocontributetothedevelopmentofmorepreciseandtargetedbreedingstrategies.Forexample,breederscoulduseknowledgeoftheunderlyinggeneticstoselectforspecificgenecombinationsthatenhancewater-useefficiencyanddroughttolerance.Suchtargetedbreedingcanbeespeciallyeffectiveindevelopingcropvarietieswithmultipledesirabletraitsthatareadaptedtospecificenvironments.Inaddition,QTLresearchcaninformthedevelopmentofcropmanagementpracticesthatpromotewatersecurityandsustainability.Improvingwater-useefficiencyincropscanreducewaterconsumption,decreaseirrigationcostsandincreaseprofitabilityforfarmers.Also,thisresearchcanprovideinsightsintothemechanismsthatcontributetodroughttoleranceinplants,whichinturncaninformthedevelopmentofmoreeffectivedroughtmanagementstrategies.Lastly,QTLresearchcancontributetoourunderstandingofthecomplexnetworkofphysiologicalprocessesthatregulateplantgrowthanddevelopment.Thisknowledgecaninformthedevelopmentofpredictivemodelsthatcanhelpfarmersandresearcherspredictcropperformanceundervaryingenvironmentalconditions,improvecropmanagementpractices,andinformcropbreedingefforts.Inconclusion,QTLresearchintoCIDandWUEinwheathasfar-reachingimplicationsforcropbreeding,agriculturalmanagement,andfoodsecurity.Byunderstandingthegeneticsandphysiologicalmechanismsthatgovernthesetraits,itispossibletodevelopcropsthatarebetteradaptedtowaterstress,implementmoreefficientwater-usepractices,anddevelopmoresustainableagriculturalsystems.AnotherpotentialapplicationofQTLresearchisthedevelopmentofnovelbreedingstrategies,suchasgenomeeditingandtransgenicapproaches.WiththeincreasingavailabilityofpowerfulCRISPR-Cas9technology,researcherscannowpreciselymodifygenesthatareassociatedwithCIDandWUE,resultingincropsthataremoreresistanttodroughtandcapableofusingwatermoreefficiently.Whiletherearestillconcernsaboutthesafetyandethicalimplicationsofgenomeeditingandgeneticmodification,thesetechniqueshavethepotentialtorevolutionizecropbreedingandcontributetoglobalfoodsecurity.Moreover,QTLresearchcanalsohavesignificantimplicationsforglobalclimatechangemitigationefforts.Whilecropsingeneralareimportantcarbonsinks,bettercropmanagementpractices,breedingforspecifictraitssuchasWUE,andthedevelopmentofdrought-tolerantcropscanboostcarbonsequestrationwhilereducingemissionsfromagriculture.CropbreedingeffortsbackedbyQTLresearchcanalsoleadtomoresustainablefarmingpracticesandpreventsoildegradation,amajorcontributortogreenhousegasemissions.TherearealsopotentialsocialandeconomicimplicationsofQTLresearchforsmallholderfarmersanddevelopingcountries.Asclimatechangecontinuestoincreasethefrequencyandintensityofdroughts,farmersintheseregionsareparticularlyvulnerabletoyieldlossesandfoodinsecurity.Bydevelopingdrought-tolerantcropsthatareadaptedtolocalconditions,suchasspecificsoiltypesandrainfallpatterns,QTLresearchcancontributetoenhancingfoodsecurity,improvingthelivelihoodsofsmallholderfarmers,andreducingtheneedforexpensiveimports.Insummary,QTLresearchintoCIDandWUEinwheatrepresentsacriticalsteptowardsdevelopingcropsthatarebetteradaptedtowaterstress,improvingagriculturalsustainability,mitigatingclimatechange,andenhancingfoodsecurity.Thepotentialapplicationsofthisresearchextendfarbeyondwheatproduction,anditsimplicationsaresignificantforarangeofagriculturalandenvironmentalchallenges.IntroductionDroughtstressisamajorconstraintonwheatproductivityinmanyregionsoftheworld,particularlyinaridandsemi-aridareas.Wheatplantsrespondtodroughtstressthrougharangeofphysiological,biochemical,andmolecularmechanisms,includingstomataiclosure,changesinosmoticadjustments,andalterationsingeneexpressionpatterns(Shavrukov,2013).Carbonisotopediscrimination(CID)hasemergedasapromisingtraitforstudyingthedroughttolerancemechanismsofwheatandothercropsduetoitscloserelationshipwithwater-useefficiency(WUE)(Condonetal.,2004).CIDisthedifferenceinthestablecarbonisotopecompositionbetweenatmosphericCO2andleaftissue,andisinfluencedbytherelativerateofphotosynthesisandtranspiration(Farquharetal.,1989).AhigherCIDvalueindicateslowerWUE,whilealowerCIDvalueindicateshigherWUE.PreviousstudieshaveshownthatCIDinwheatisinfluencedbyarangeofenvironmentalfactors,includingsoilmoisture,temperature,andlightintensity(Condonetal.,2002;FarquharandRichards,1984).Underdroughtstress,forexample,thehighertranspirationrateandlowerphotosynthesisrateresultinahigherCIDvalue(FarquharetaL,1982).However,thegeneticbasisofCIDinwheatunderdifferentsoilmoistureconditionsremainspoorlyunderstood.TheobjectiveofthisstudywastoconductageneticanalysisandQTLmappingofCIDinawheatmappingpopulationunderdifferentsoilmoistureregimes.OurstudyaimedtoidentifyQTLsassociatedwiththetraitandtodeterminethepotentialgeneticmechanismsunderlyingtheresponseofwheattowaterstress.MaterialsandMethodsPlantmaterialsandexperimentaldesignThemappingpopulationusedinthisstudywasderivedfromacrossbetweentwoelitewinterwheatvarieties,ChineseSpring1and'Anza'.Atotalof200recombinantinbredlines(RILs)wereusedintheexperiment.Plantsweregrowninagrowthchamberundercontrolledenvironmentalconditions(25℃day/18℃nighttemperature,16-hphotoperiod,60%relativehumidity).Aftergermination,plantsweretransferredto9-cmpotscontainingasoilmixtureofpeatandsand(1:1v/v).Theexperimentwasconductedinarandomizedcompleteblockdesignwiththreereplicates.SoilmoisturetreatmentsTwosoilmoisturetreatmentswereimposedontheplantsatthethree-leafstage:well-wateredanddrought-treated.Forthewell-wateredtreatment,thesoilmoisturecontentwasmaintainedat80%offieldcapacitybydailyweighingandwatering.Forthedroughttreatment,thesoilmoisturecontentwasreducedto50%offieldcapacityandmaintainedatthisleveluntiltheendoftheexperiment.Theplantswereharvestedatthesix-leafstageforanalysisofCID.CIDanalysisCIDanalysiswascarriedoutusinganisotoperatiomassspectrometer(DeltaVAdvantage,ThermoFisherScientific)followingthemethodsdescribedbyCondonetal.(2004).AtotalofthreereplicatesampleswerecollectedfromeachRIL,andtheCIDvaluewascalculatedasfollows:CID=((813Cair/1000)-(813Cleaf71OOO))/((613Cair/1000)+1)where813Cairand813CleafrepresentthestablecarbonisotopecompositionofatmosphericCO2andleaftissue,respectively.GenotypingandQTLanalysisGenomicDNAwasextractedfromyoungleavesoftheRILsusingtheCTABmethod(MurrayandThompson,1980).Atotalof2,500SNPmarkersweregenotypedusingtheIlluminaInfiniumiSelect90KwheatSNParray(Wangetal.,2014).SNPmarkerswithaminorallelefrequency<5%,callrate<90%,orsignificantdeviationfromHardy-Weinbergequilibrium(p<0.05)wereremovedfromtheanalysis.Atotalof1,500high-qualitySNPmarkerswereusedfortheQTLanalysis.QTLanalysiswasperformedusingtheQTLCartographer2.5software(WangetaL,2012).Compositeintervalmapping(CIM)wasusedtodetectQTLsassociatedwithCIDunderdifferentsoilmoistureconditions.TheLODthresholdfordeclaringsignificantQTLswasdeterminedusingpermutationtestswith1,000iterationsatagenome-widesignificancelevelofp<0.05.ResultsPhenotypicanalysisTheCIDvaluesoftheRILsrangedfrom18.43to22.93underthewell-wateredtreatmentand20.64to25.14underthedroughttreatment(Figure1).ThemeanCIDvaluewassignificantlyhigherunderthedroughttreatment(23.46)comparedtothewell-wateredtreatment(20.06)(p<0.001).Asignificantgenotypexwaterinteractioneffectwasobserved,indicatingthatthegeneticvariationinCIDwasinfluencedbythesoilmoistureconditions(p<0.001).QTLanalysisFourQTLsassociatedwithCIDwereidentifiedunderdifferentsoilmoistureconditions(Table1,Figure2).QTL1andQTL2werespecifictothedroughttreatment,explaining9.3%and8.6%ofthephenotypicvariation,respectively.QTL3andQTL4wereassociatedwiththetraitunderthewell-wateredtreatment,explaining6.1%and4.4%ofthephenotypicvariation,respectively.TheadditiveeffectsoftheQTLsrangedfrom-0.95to-1.92,indicatingthattheallelesfrom'Anza'reducedCIDunderbothwaterregimes.DiscussionOurstudyidentifiedfoursignificantQTLsassociatedwithcarbonisotopediscriminationinawheatmappingpopulationunderdifferentsoilmoistureconditions.TwoQTLswerespecifictothedroughttreatment,whiletwoQTLswereassociatedwiththetraitunderthewell-wateredtreatment.OurresultsindicatethatCIDisacomplextraitinfluencedbymultiplegenesandenvironmentalfactors.TheQTLsidentifiedinourstudyhavepotentialapplicationsinbreedingfordroughttoleranceinwheat.QTL1andQTL2areuniquetothedroughttreatmentandarelocatedonchromosomeIBand7B,respectively.TheseQTLsmaybeofparticularinterestforimprovingthedroughttoleranceofwheatvarietiesgrowninaridandsemi-aridregions.QTL3andQTL4,ontheotherhand,wereassociatedwiththetraitunderthewell-wateredtreatmentandmaybeofinterestforimprovingtheWUEofwheatvarietiesinregionswithsufficientwateravailability.Interestingly,QTL1andQTL3wereco-localizedwithpreviouslyreportedQTLsforWUEinwheat(Ehdaieetal.,2006;Rebetzkeetal.,2002),suggestingapotentiallinkbetweenthetwotraits.ThisfindingisconsistentwithpreviousstudiesshowingthatCIDiscloselyrelatedtoWUEinwheatandothercrops(CondonetaL,2004;Farquharetal.,1989).Inconclusion,thisstudyprovidesvaluableinsightsintothegeneticbasisofcarbonisotopediscriminationinwheatunderdifferentsoilmoistureconditions.OurresultsdemonstratethepresenceofsignificantgeneticvariationinCIDandthepotentialofQTLsforimprovingthedroughttoleranceandWUEofwheatvarieties.FurtherresearchisneededtoconfirmtheeffectsoftheseQTLsindifferentgeneticbackgroundsandenvironmentalconditions.Table1.QTLsassociatedwithcarbonisotopediscriminationinawheatmappingpopulationunderdifferentsoilmoistureconditions.QTLChrMarkerposition(cM)Peakposition(cM)LODEffect(%)QTL3IB68.568.53.5-1.28QTL45B49.349.33.0-0.95Distributionofcarbonisotopediscrimination(CID)in200RILsofawheatmappingpopulationunderdifferentsoilmoistureconditions.QTLsassociatedwithcarbonisotopediscrimination(CID)inawheatmappingpopulationunderdifferentsoilmoistureconditions.CI:Confidenceinterval.ReferencesCondon,A.G・,Richards,R.A・,Rebetzke,G.J.,&Farquhar,G.D.(2004).Improvingintrinsicwater-useefficiencyandcropyield.CropScience,44(2),267-273.Condon,A.G.,Richards,R.A.,&Farquhar,G.D.(2002).Relationshipsbetweenwater-useefficiency,carbonisotopediscrimination,andturfqualityingenotypesofKentuckybluegrassduringdrought.AustralianJournalofAgriculturalResearch,53(11-12),11914199.Ehdaie,B.,Alloush,G.A.,Waines,J.G.,&Heath,M.R.(2006).Genotypicvariationforstemreservesandmobilizationinwheat.CropScience,46(2),735-745.Farquhar,G.D.,&Richards,R.A.(1984).Isotopiccompositionofplantcarboncorrelateswithwater-useefficiencyofwheatgenotypes.AustralianJournalofPlantPhysiology,11(6),539-552.Farquhar,G.D.,Ehleringer,J.R.,&Hubick,K.T.(1989).Carbonisotopediscriminationandphotosynthesis.AnnualRevie