acetal.netcms

TheorApplGenet(2016)129:181–199DOI10.
1007/s00122-015-2621-yORIGINALARTICLEGeneticvariationandinheritanceofphytosterolandoilcontentinadoubledhaploidpopulationderivedfromthewinteroilseedrapeSansibar*OasecrossLishiaTeh1·ChristianMllers1Received:20June2015/Accepted:9October2015/Publishedonline:30October2015TheAuthor(s)2015.
ThisarticleispublishedwithopenaccessatSpringerlink.
comtraits,betweentwoandsixQTLforfourfattyacids,fiveQTLforoilcontent,fourQTLforproteincontentofdefat-tedmeal,andthreeQTLforseedweight.
ColocalizationsofQTLfordifferenttraitsweremorefrequentlyobservedthanindividualisolatedQTL.
MajorQTL(R2≥25%)werealllocatedintheAgenome,andthepossiblecandi-dategeneswereinvestigatedbyphysicallocalizationoftheQTLtothereferencegenomesequenceofBrassicarapa.
KeywordsBrassicanapus·Phytosterols·Oilcontent·Fattyacids·QTLmappingIntroductionOilseedrape(BrassicanapusL.
;genomeAACC,2n=38)istheworld'sthird-leadingsourceofvegetableoilforhumannutritionandindustrialproducts.
Almostalloftheoilseedrapecultivationis"doublelow"or"canola"qual-ity"withlowcontentoferucicacidintheoilandglucosi-nolatesintheseeds(FriedtandSnowdon2010).
Whileoil-seedrapebreedinghasachievedremarkablesuccessoverthepastfewdecades,thereisstillmuchtolearnaboutthegenesregulatingseedoilcontentandqualitytraits.
Increas-ingconcernsaboutrapidpopulationgrowth,demandsforimprovednutritionaloil,andexpansionofbiofuelproduc-tionhavealsoledtothecallforfurtherenhancementinquantityandqualityofseedoil.
Inthecaseofacomplextraitlikeseedoilcontent,thenumberofQTLasreportedbynumerousstudiesvariedbetween3and27QTLandwerefounddistributedamong17ofthe19chromosomesinB.
napus(Rahmanetal.
2013).
Inaddition,theseQTLindividuallyexplainedbetween2and10%ofthepheno-typicvariancewhiletheadditiveeffectrangedfrom0.
2%tomorethan1.
0%(Rahmanetal.
2013).
Therefore,AbstractKeymessageIdentificationofQTLforphytosterolcontent,oilcontent,fattyacidscontent,proteincontentofdefattedmeal,andseedweightbymultipleintervalmappinginaBrassicanapusDHpopulation.
AbstractPhytosterolsareminorseedconstituentsinoil-seedrapewhichhaverecentlydrawnwide-interestfromthefoodandnutritionindustryduetotheirhealthbenefitinloweringLDLcholesterolinhumans.
Tounderstandthegeneticbasisofphytosterolcontentanditsrelationshipwithotherseedqualitytraitsinoilseedrape,QTLmappingwasperformedinasegregatingDHpopulationderivedfromthecrossoftwowinteroilseedrapevarieties,SansibarandOase,termedSODHpopulation.
Bothparentallinesareofcanolaqualitywhichdifferinphytosterolandoilcontentinseed.
AgeneticmapwasconstructedforSODHpopulationbasedonatotalof1638markersorganizedin23linkagegroupsandcoveringamaplengthof2350cMwithameanmarkerintervalof2.
0cM.
TheSODHpopula-tionandtheparentallineswerecultivatedatsixenviron-mentsinEuropeandwerephenotypedforphytosterolcon-tent,oilcontent,fattyacidscontent,proteincontentofthedefattedmeal,andseedweight.
MultipleintervalmappingidentifiedbetweenoneandsixQTLforninephytosterolCommunicatedbyL.
Jiang.
ElectronicsupplementarymaterialTheonlineversionofthisarticle(doi:10.
1007/s00122-015-2621-y)containssupplementarymaterial,whichisavailabletoauthorizedusers.
*LishiaTehlteh@gwdg.
de1DepartmentofCropSciences,Georg-August-UniversittGttingen,VonSieboldStr.
8,37075Gttingen,Germany182TheorApplGenet(2016)129:181–199increasingoilcontentthroughbreedingwouldhavetorelyonprogressivestackingofpositivealleles.
Besidesconsid-eringtheenvironmentalinfluenceontheQTL,identify-ingtheunderlyingcandidategenesaswellasrecognizingthepleiotropiceffectorcorrelationbetweentraitswouldgreatlyincreasetheefficiencyofbreeding.
Severalstud-ieshaveshownthatoilcontentisinfluencedbythefattyacidcompositionorviceversa(Eckeetal.
1995;MllersandSchierholt2002;Hobbsetal.
2004;Zhengetal.
2008).
Sincetriacylglycerolsconstituteabout90%oftheoil,thefattyacidcompositionwhichrepresentstheoverallcom-positionofthetriacylglycerolsisanimportantqualityparameterdeterminingthevalueandsuitabilityoftheoilfornutritionalorindustrialapplications.
Althoughthecan-olaqualityoilseedrapepossessesanearlyidealfattyacidprofile,thereisstillroomforimprovementonthethermalstabilityofoilbyfurtherincreasingoleicacidandreducingthepolyunsaturatedfattyacidscontent.
Recently,someminorsalutaryoilconstituentssuchascarotenoids(Shewmakeretal.
1999;Yuetal.
2008;Weietal.
2010),phytosterols(Amaretal.
2008b),andtocophe-rols(Marwedeetal.
2005;Fritscheetal.
2012;Wangetal.
2012b)havealsodrawntheattentionamongplantbreed-ersandresearcherstostudyandimprovethecontentandcompositionduetotheirconferredhealth-benefitingprop-erties.
Phytosterolsarewidelyknownfortheircholesterolloweringpropertiessince1950s(Peterson1951;Pollak1953).
Aneffectivedoseof1–3gday1leadstoreduc-tionbetween8and15%inLDLcholesterol(Quilezetal.
2003).
Otherpromisingeffectsincludeanti-cancer(Woy-engoetal.
2009),anti-atherosclerosis(Moghadasianetal.
1997),anti-inflammation(Bouic2001),andanti-oxidation(VanRensburgetal.
2000).
Thesehealth-promotingprop-ertieshaveledtothedevelopmentoffunctionalfoodsenrichedwithphytosterolsasbioactiveingredients.
Avari-etyoffoodsfortifiedwithphytosterols,includingmarga-rines,mayonnaises,vegetableoils,saladdressings,milk,dairyproducts,beverages,andsnackbars,arenowwidelyavailableinthemarket(Bergeretal.
2004).
Themostcom-monsourcesofphytosteroladdedtofoodsaretalloil—abyproductofthepulpingindustrythatisrichinsitosterolandsitostanol(Jonesetal.
1998)—anddistillatefractionfromvegetableoilrefining.
Whilemostcrudevegetableoilscontainabout1–5gkg1ofphytosterol,cornoilcon-tainsabout8–16gkg1andoilseedrapeoilcontainsabout5–10gkg1(Piironenetal.
2000).
Thehighamountofphytosterolinoilseedrapemeansthatitmayserveasvalu-ablebasestockforthehealthandnutritionindustry.
Phytosterolsincludeawidevarietyofmoleculesthatarestructurallysimilartocholesterol.
Thestructuralvari-ationsofphytosterolsarisefromdifferentnumberofcar-bonatomsonC-24inthesidechainaswellasthenum-berandpositionofdoublebondsinthetetracyclicskeleton(Fig.
1).
Inoilseedrape,thephytosterolprofileconsistsmainlyofsitosterol,campesterol,brassicasterol,andave-nasterol,whilecholesterolandstigmasteroloccuronlyintraceamounts(Appelqvistetal.
1981).
BrassicasterolisacharacteristicsterolofBrassicaceaespeciesandinoilseedrape,itamountstoabout13%oftotalphytosterolcontent.
Amongtheadaptedwinteroilseedrapepopulations,mod-erncultivarswithcanolaqualitycontainhigheramountoftotalphytosterolsthanthegeneticallydiverseorresynthe-sizedlinesthatareofnon-canolaquality.
Thisobserva-tionisduetotheclosenegativecorrelationbetweentotalphytosterolcontentanderucicacidcontent(Amaretal.
2008b).
InawinteroilseedrapeDHpopulationsegregat-ingforerucicacid,QTLmappingshowedthattwoofthethreeQTLidentifiedfortotalphytosterolcontentcolo-calizedwithtwoerucicacidgenes(Amaretal.
2008a).
Basedonthefactthatcytoplasmicacetyl-CoAisrequiredinthesynthesisofbotherucicacidandphytosterols,colo-calizationsofQTLaremostlikelyattributedtopleiotropiceffectexertedbytheerucicacidgenes.
Tofurtherinvesti-gatetheinheritanceofphytosterolsandtheirrelationstootherimportantseedqualitytraits,aDHpopulationcon-structedfromthetwocanolaqualitywinteroilseedrapecultivars,SansibarandOase,wasusedinthisstudy.
Theparentallineswereshowntodifferwithrespecttophytos-terolandoilcontentbasedonapreviousscreening(Amaretal.
2009).
ItwasanticipatedthatthisDHpopulation,whichdoesnotsegregateforerucicacid,mayhavegreaterpowerfordetectionofQTLwithnovelallelesforphytos-terolcontentthanpreviousstudies.
Themainobjectivesofthisstudywere(1)toidentifyQTLforphytosterolcontent,fattyacids,oilcontent,proteincontentofdefattedmeal,andseedweight;(2)toinvestigatethecorrelationbetweentheanalyzedtraits;and(3)toinspectforpossiblecandidategenesunderlyingthemajorQTL.
MaterialsandmethodsPlantmaterialTheexperimentalpopulationconsistedof226F1micro-spore-derivedDHlinesderivedfromtheSansibar*Oasecross.
Thetwoparentallineswereamongthe27canolaqualitywinteroilseedrapecultivarsanalyzedbyAmaretal.
(2009)andwerechosenduetotheircontrastingtotalphy-tosterolcontentandoilcontentinseed;Sansibarhadthehighesttotalphytosterolcontent(~480mg100g1seed)andlowestoilcontent(43%),whileOasehadthelowesttotalphytosterolcontent(~360mg100g1seed)andhigh-estoilcontent(46%).
TheDHpopulationwasdevelopedintheDivisionofPlantBreedingatGeorg-August-Univer-sittGttingenandwasnamedasSODHpopulation.
183TheorApplGenet(2016)129:181–199FieldexperimentsTheSODHpopulationandtheparentallineswerecul-tivatedinsixenvironments:twoenvironmentsatGt-tingen,Germanyduringgrowingseasons2009/2011and2010/2011;oneenvironmentatEinbeck,Germanyduringgrowingseason2010/2011byKWSSaatAG;oneenvi-ronmentatAsendorf,Germanyduringgrowingseason2011/2012byDeutscheSaatveredelung(DSV)AG;andtwoenvironmentsatSvalv,Swedenduringgrowingsea-sons2010/2011and2011/2012byLantmnnenSWSeed.
Thefieldtrialswerecarriedoutinsmallplotsinacompleterandomizeddesignwithoutreplication.
Seedsoftenopenpollinatedplantsfromeachlinewereharvestedandbulkedforanalyses.
MolecularmarkersGenomicDNAoftheSODHpopulationandtheirparentallineswereisolatedfromyoungleavesof4–5week-oldgreenhouse-grownseedlingsusingNucleonPhytoPureplantextractionkits(GEHealth-care,Illustra)accordingtomanufacturer'sinstruc-tions.
DNAwasquantifiedusingBio-RadFluorescentDNAQuantificationKit(Bio-RadLaboratoriesCA,USA).
Simplesequencerepeats(SSR)andamplifiedfragmentlengthpolymorphism(AFLP)markersSSRanalysiswascarriedoutfollowingtheM13-tailingPCRtechnique(Schuelke2000).
PCRreactionswereper-formedin96-wellPCRplateswithavolumeof20μlperreaction,containing25ngofgenomicDNA,0.
05μMofforwardprimerwithaM13tailof19bpatthe5′end,0.
05μMofreverseprimer,0.
05μMofM-13primer,2.
5mMMgCl2,0.
2mMofeachdNTP,1*PCRbuffer,and1UofTaqDNApolymerase.
Atwo-steptouchdownPCRprogramwasperformedinaBiometraT1Thermocycler(BiometraGmbH,Gttingen,Germany):95°Cfor2min;5cyclesof95°Cfor45s;68°C(2°C/cycle)for5min,72°Cfor1min;5cyclesof95°Cfor45s,58°C(2°C/cycle)for1min,72°Cfor1min;27cyclesof95°Cfor45s,47°Cfor30sand72°Cfor1min;and72°Cfor10min.
Atotalof350primerpairsobtainedfromvarioussourceswerescreenedforpolymorphismsbetweenthepar-ents.
TheSSRprimerpairsprefixedwith"BRA"and"CB"weredevelopedbyCeleraAgGenconsortium,andprefixedFig.
1Simplifiedphytosterolbiosyntheticpathwayinplants.
Solidanddashedarrowsindicatesingleandmultiplebiosyntheticsteps,respectively.
AdaptedfromBenveniste(2002),Schaller(2003).
HMGSHMG-CoAsynthase,HMGRHMG-CoAreductase,SMT1C-24sterolmethyltransferase1,SMT2C-24sterolmethyltrans-ferase2Acetoacetyl-CoAHMG-CoA24-methylenelophenol24-ethyldienelophenolCampesterolBrassicasterolBrassinosteroidpathwaySgmasterol2Acetyl-CoAHMGSSqualene24-methylenecycloartenolCycloartenolAvenasterol24-methylenecholesterolSitosterolHMGRMalonyl-CoASMT2SMT124-epi-campesterolC22-desaturaseMevalonateIPPC18:1-CoAC20:1-CoAC22:1-CoAElongaonoffayacidC22-desaturase184TheorApplGenet(2016)129:181–199with"MR"and"MD"weredevelopedbyDivisionofPlantBreedingatGeorg-August-UniversittGttingen.
AFLPanalysiswasperformedbyadaptingthemethoddescribedbyVosetal.
(1995).
Atotalof16primercombinationsmadeupfrom8EcoRIfluorescence-labeledprimersand4MseIprimerswereused:E32M48,E37M50,E36M51,E36M59,E39M48,E38M50,E37M51,E37M59,E44M48,E40M50,E38M51,E38M59,E45M48,E44M50,E44M51,andE44M59.
ThePCRproductsofAFLPandSSRwereseparatedontheABIPRISM3100geneticanalyzer(AppliedBiosystems)withGeneScan-500ROXsizestand-ard(AppliedBiosystems)using36cmcapillaryarrays.
TheresultswereanalyzedwithGeneScansoftwareversion3.
7(AppliedBiosystems)andscoredusingGenotypersoftwareversion3.
7NT(AppliedBiosystems).
Singlenucleotidepolymorphism(SNP)markersAtotalof125polymorphicSNP,designatedwithprefix"ra"weregenotypedbythebreedingcompanyKWSSaatAGandwerekindlyprovidedtousformapconstruction.
Diversityarraystechnology(DArT)andSilicoDArTmarkersTheSODHpopulationwasgenotypedwiththeB.
napusv1.
0DArTmicroarraycomprising3072markers,desig-natedwiththeprefix"brPb.
"Asubsetof183linesfromtheSODHpopulationwasgenotypedwith4787Silico-DArTmarkers(www.
diversityarrays.
com/dart-application-dart-seq-data-types),designatedwiththesuffix"|F|0.
"Geno-typingwithDArTandSilico-DArTmarkerswasperformedbyDiversityArrayTechnologyPtyLtd,Yarralumla,Aus-tralia.
ThesequencesforDArTmarkerswereretrievedfromhttp://www.
diversityarrays.
com/dart-map-sequenceswhilethesequencesforSilico-DArTcloneswerepro-videdbyDiversityArrayTechnologyPtyLtd,Yarralumla,Australia.
KompetitiveallelespecificPCR(KASP)markersFromtheIlluminaInfiniumBrassica60KSNParray,asubsetof32markersthatwerepolymorphicbetweentheparentallines,SansibarandOase,wereselectedforKASPgenotyping(TraitGeneticsGmbH).
Ofthe32markers,13werephysicallycloselylinkedtopromisingcandidategenesforphytosterolbiosynthesisand19wereassociatedwithoilcontentinSGDH14*Express617DHpopulation(NinaBehnke,personalcommunication).
ThesequencesforSNPmarkerswereprovidedbyIsobelParkin(AAFC,Saskatoon,Canada).
ThephysicalpositionswerebasedonreferencegenomeofB.
rapav1.
5genomedatabase(BRAD;http://www.
brassicadb.
org/brad/)(Wangetal.
2011)andB.
oleraceav1.
0genomedatabase(Bolbase;http://www.
ocri-genomics.
org/bolbase/).
KASPmarkerswereprefixedwith"Bn-.
"CandidategenebasedmarkersFivecandidategenesinvolvedintheregulationofphytos-terolsynthesisandonecandidategeneinvolvedinthetria-cylglycerolsynthesiswereselectedtodevelopcandidategene-basedmarkers.
ThedetailsofthecandidategenesaredescribedinSupplementaryTable1withtheaidofthephytosterolbiosyntheticpathwaydepictedinFig.
1.
Theapproachinvolvedfirstdesigningalocus-specificmarkertodifferentiatebetweenhomologsbasedonlocus-specificSNP,followedbysequencingoftheampliconstoscreenforallelicSNPbetweentheparentallines.
IfanallelicSNPwasfound,anallele-specificmarkerwasdevelopedforthepertaininghomolog.
Duetothelimitednumberoflocus-specificSNPandalackofpolymorphismsbetweentheparentallines,onlyfourcandidategene-basedmarkersweredeveloped:HMG1A07-O1for3-hydroxy-3-methylglutaryl-CoAreductase1(HMG1)andHMG2A10-2forhydroxy-3-methylglutaryl-CoAreductase2(HMG2),andD120E-3andDx-3fordiacylglycerolacyltransferase1(DGAT1).
TheDGAT1primerpairswerekindlyprovidedbyDr.
RenateSchmidtfromIPKGatersleben.
Primersequenceofcandidategene-basedmarkersarelistedinSupplementaryTable2.
LinkagemapofSODHpopulationLinkagemapwasconstructedusingMAPMAKER/EXP3.
0(Lincolnetal.
1992)withtheaidofapurpose-builtPerlscript(unpublished;WolfgangEcke,personalcommunica-tion)thatautomatesthemappingprocess.
Segregationofeachmarkerwastestedbyχ2analysis(P=0.
05)toassessthegoodness-of-fitfortheexpectedsegregationratio(1:1).
Markerswhichweresignificantlydeviatingfrom1:1seg-regationratiowereregardedasskewedsegregatedmarkerswhilemarkerswhichwerenotsignificantlydifferentfrom3:1orbeyondweredefinedasstronglyskewedsegregatedmarkers.
Markerswithstronglyskewedsegregationwereinitiallyexcludedformapconstructionandwereattemptedformappingaftertheinitialmapwasbuilt.
Markerswereassignedtolinkagegroupstoconstructacoremapbythe"group"commandwiththeminimumLODscoreparametersetto4andthemaximumdis-tanceparametersetto35cM.
Themostprobablemarkerorderwithineachgroupwasdeterminedbythecommand"order"andtheresultinghigh-fidelitymapwasbuiltuponbyaddingmarkersusingthecommand"try.
"Markersthatshowedmorethanthepredeterminednumbersofcrosso-verswereexcludedinthehigh-fidelitymap.
Markersthat185TheorApplGenet(2016)129:181–199werenotsupportedbyaLODscoreof3inthehigh-fidelitymapwereplacedattheirmostlikelypositioninthelink-agegroup.
Followingthis,the"ripple"commandwasusedtofindtheoptimalmarkerorderinthelinkagegroups.
GeneticdistancesbetweenlociwerecalculatedusingtheKosambimappingfunction(Kosambi1944).
Theresultingmapconsistedofhigh-fidelitymarkerswhicharesupportedbyaLODscoreofatleastthreeandplacedmarkerswhicharesupportedwithLODscoreoflessthanthree.
Themapwasfurtheroptimizedbyconstructingeachlinkagegroup200timeswitharandomsubsetoffivehighlyinformativemarkersaccordingtoMAPMAKER/EXP3.
0commandordertoobtainthepossiblevariantofahigh-fidelitymap.
Theoptimalvariantwasselectedtohaveasmanymarkersaspossible,asfewdoublecrossoveraspossible,andthatthemarkerswereasevenlydistributedaspossible.
ThemapwasalignedwithcommonmarkerlocionestablishedgeneticmapsbasedonSSR(Piquemaletal.
2005;Radoevetal.
2008;SharpeandLydiate,unpublisheddata),DArT(Ramanetal.
2013),andSNP(KWSSaatAG,unpublisheddata).
LinkagegroupswerenamedaccordingtothenomenclatureofParkinetal.
(1995)asA01–A10andC01–C09.
ForQTLmappingpurpose,asubsetofmarkerswereselectedfromthehigh-fidelitymarkersonthebasisthatthedistancebetweenadjacentmarkerswasabout5–10cM.
ThetermframeworkmapwasusedtorefertothemapusedforQTLmapping.
PhenotypicanalysisPhytosterolsPhytosterolcontentwasanalyzedbyadaptingtheprotocolofAmaretal.
(2008b)andFernández-Cuestaetal.
(2012),followingadirectalkalinehydrolysismethodwhichinvolvesthreemajorsteps:alkalinehydrolysis(saponifica-tion),extractionofthenon-saponifiablematter,andderivat-izationofthesterolstotrimethylsilyl(TMS)-etherderiva-tives.
Themainadvantageofusingthismethodisthatitbypassesthelipidextractionstep,facilitatinglargenumberofseedsamplestobeanalyzedmoreeconomically.
Thedownsideofthismethodisthatalkalinehydrolysiscouldonlyquantifyfreesterolsandsterylesters,butnotsterylglycosides.
Thehydrolysisofacetalbondbetweenphytos-terolandthecarbohydratemoietyrequiresacidicconditionwhichmaybedestructivetothecompoundandlaboriousforroutineanalysis.
Hence,itispossiblethatthepresentanalysiswouldunderestimatethetotalphytosterolconcen-trationintheseedsample.
Foreachsampleanalysis,200mgofseedwasweighedandplacedinapolypropylenetube.
Twomilliliterof2%potassiumhydroxide(CarlRoth,Germany)inethanol(w/v)wasaddedforalkalinehydrolysis,followedby200μlof2%cholesterol(99%purity,Sigma-Aldrich,Germany)inhexane-ethanol(3:2)solution,usedasaninternalstandardtoquantifyphytosterolcontent.
Byplacingonestainlesssteelrod(1.
1cmlength;0.
4cmdiameter)ineachtube,seedswerecrushedandhomogenizedusingacustom-builtverticalhomogenizer(InstituteofAppliedPlantNutrition,Georg-August-UniversittGttingen)for3minataspeeddeemedsufficienttohomogenizetheseeds.
Thetubesweresubsequentlyincubatedfor15minat80°Cinawaterbathandcooledatroomtemperaturefor30min.
Toextractthephytosterols,1.
0mlofhexaneand1.
5mlofdistilledwaterwereadded,brieflyvortexed,andcentrifugedfor10minat4000rpm.
Theupperhexanelayerwastransferredtoanewtubeandleftovernightonahotplateat37.
5°Ctoevapo-rate.
Theresidueobtainedafterevaporationwasdissolvedwith80μlhexaneandderivatizedwith20μlofsilylatingagent,composedofhexamethyldisilazane(Flukaanalytical):trimethylchlorosilane(Sigma-Aldrichpurum>98%;GCgrade)3:1.
ThesolutionwaspipettedintoaGCvial,capped,andincubatedatroomtemperaturefor20min.
Tosettletheprecipitate,thederivatizedsampleswerecentri-fugedfor10minat3000rpmpriortoGCanalysis.
Analysisofderivatizedsterolswasperformedusingcap-illarygas–liquidchromatograph(ChrompackCP-9003),equippedwithautosampler,splitinjector(320°C;injec-tionvolumeof3μlwithasplitratioof100:1),andflameionizationdetector320°C,withfusedsilicacapillarycol-umnofmediumpolarity(SE-54,50mlong,0.
1μmfilmthickness,0.
25mmi.
d.
coatedwith5%-phenyl-1%-vinyl-methylpolysiloxane)(IVAAnalysentechnik,Meerbusch,Germany).
Hydrogen(carriergas)pressurewassetat150kPa.
Initialoventemperaturewassetat240°Cwithanincrementof5°Cpermintofinaloventemperatureat275°Candheldfor20min.
Totalanalyticaltimewas25min.
Phytosterolcontentwasexpressedasmg100g1seedThephytosteroltraitsevaluatedinthisstudyincludecon-tentsofbrassicasterol,campesterol,sitosterol,avenasterol,totalphytosterol,24-methylsterol,24-ethylsterolandcampesteroltositosterolratio,and24-methylto24-ethylsterolratio.
Totalphytosterolcontentwascalculatedasthesumofbrassicasterol,campesterol,sitosterol,andavenas-terolcontents.
24-Methylsterolwascalculatedasthesumofbrassicasterolandcampesterolcontents.
24-Ethylsterolwascalculatedasthesumofsitosterolandavenasterolcontents.
FattyacidsFattyacidcompositionwasanalyzedbygaschroma-tographyusingamethodadaptedfromThies(1971).
186TheorApplGenet(2016)129:181–199Approximately200mgofseed,1mlofNa-methylate-methanol(0.
5moll1),andonestainlesssteelrod(1.
1cmlength;0.
4cmdiameter)wereaddedinapropylenetube.
Theseedswerethenhomogenizedusingacustom-builtverticalhomogenizer(InstituteofAppliedPlantNutrition,Georg-August-UniversittGttingen)for3min.
Followingincubationfor20minatroomtemperature,300μlisooc-taneand100μl5%NaHSO4inwaterwereadded,brieflyvortexed,andcentrifugedfor3minat4000rpm.
About200μloftheupperphasewaspipettedintoaGCvialand3μlwasinjectedintoagaschromatograph(ThermoTraceGCUltra),equippedwithautosampler,splitinjector(splitratio70:1),flameionizationdetector(320°C),andcapil-laryFFAP-phase(0.
25mm*25m;Macherey&Nagel).
Hydrogen(carriergas)pressurewassetat100kPa.
Oventemperaturewassetat210°C.
Totalanalyticaltimewas6min.
Thefattyacidcontentreportedinthisstudyincludepalmiticacid(C16:0),oleicacid(C18:1),linoleicacid(C18:2),andlinolenicacid(C18:3),expressedaspercent-ageoftotalfattyacids(includingotherminorfattyacids)inmatureseeds.
OilandproteincontentofdefattedmealOilandproteincontentinseedswereestimatedbyNIRSusingcalibrationraps2012.
eqaprovidedbyVDLUFAQualittssicherungNIRSGmbH(Teichstr.
35,D-34130Kassel,http://h1976726.
stratoserver.
net/cms,accessedSep-tember24,2015).
Oilcontentandproteincontentofdefat-tedmealwereexpressedasapercentageofseeddrymattercontentat9%moisture.
ProteincontentofdefattedmealwascalculatedbyusingtheestimatedseedoilcontentandseedproteincontentobtainedfromtheNIRSpredictionasfollows:SeedweightThousandseedweightwasobtainedfromweightconver-sionof500seeds.
Theseedswerecountedusingaseedcounter(Model:Contador,PfeufferGmbH,D-97318Kitz-ingen,http://www.
pfeuffer.
com).
StatisticalanalysisVariancecomponents,heritability,andmeanswereesti-matedusingPLABSTATsoftwareversion3A(Utz2011).
ThemodelimplementedinANOVAanalysiswasasfollows:%Proteinofdefattedmeal=%Seedprotein100%Seedoil*100%.
Yij=+gi+ej+εij,whereYijisthetraitvalueoftheithgenotypeinthejthenvironment,isthegeneralmean,giistheeffectofithgenotype,ejistheeffectofjthenvironment,andεijistherandomerrormeanoftheithgenotypeinthejthenviron-mentconfoundedwithresidualerrorandgenotype*envi-ronmentinteraction.
Thegenotypewastreatedasfixedeffect,whereasenvironmentwastreatedasrandomeffect.
Broad-senseheritabilityh2wasestimatedasfollows:whereσ2Gandσ2Earevariancecomponentsforgenotypeandrandomerror;nereferstonumberofenvironment.
MeanvaluesacrossallenvironmentswereusedtocalculateSpearman'srankcorrelationcoefficientsbetweentraits.
QTLmappingQTLdetectionwasperformedwithWinQTLCartographersoftwarever.
2.
5(Wangetal.
2012a)usingmeansofphe-notypicdataobtainedfrom6environmentsandaframe-workmapconsistingof273markers.
QTLwereinitiallydetectedwithcompositeintervalmapping(CIM)usingthedefaultmodel(model6)thatselectscertainmarkersascontrolmarkersbyusingadditionalparameters.
Foreachtrait,theLODsignificancethreshold(α=0.
05)wereestimatedby1000permutationtests.
Fivemarkersselectedbyaforwardandbackwardregressionmethodwereusedascofactors.
CIMtestswereperformedat1-cMstepswitha10-cMwindowsize.
PeaksweretreatedasseparateQTLwhenthedistanceismorethan5cMandtheminimumLODvalueexceedsonebetweenanytwoadjacentpeaks.
Subsequently,multipleintervalmapping(MIM)wasperformedtorefinetheQTLpositions,tosearchformoreQTL,andtoinvestigateepistaticeffectsamongthedetectedQTL(Kaoetal.
1999).
TheMIMmodelwasbuiltuponapriorimodelfromCIManalysisandprogressivelyrefinedusingtheBIC-M2=2ln(n)criterion.
QTLpositionsthatdidnotremainsignificantwhenfittedwithotherswerethendroppedfromthemodel.
QTLeffectsandtheirpercentageofphenotypicvarianceexplainedbyindividualandalltheQTLwereestimatedwiththefinalmodelfittedinMIM.
Aone-LODdropfromthepeakpositionwasusedasaconfi-denceintervalforeachQTL.
InsilicomappingofsequenceinformativemarkersontheB.
napusgenomeSequencesofDArT,silico-DArT,SSR,andKASPmarkerswereusedtosearchintheB.
napusDarmor-bzhgenomesequenceassembly(Chalhoubetal.
2014)usingthenucle-otideMEGABLASTalgorithm.
Thewordsizewassetath2=σ2Gσ2G+σ2εne,187TheorApplGenet(2016)129:181–19928andthecutoffe-valuewassetat1e-10.
Whenmultiplehitswereobtained,thephysicalmarkerpositionwaspre-dictedbasedonthealignmentwiththegeneticmap.
IdentificationofpossiblecandidategenesformajorQTLBasedonknownkeyregulatorygenesfromtheliterature,anattemptwasmadetoinvestigatewhetherthepredictedgeneswereinfactunderlyingthemajorQTL.
SinceallthemajorQTLwerelocatedintheAgenome,sequencesofpredictedArabidopsisgenesweresearchedinbothB.
rapaChiifu(Wangetal.
2011)andB.
napusDarmor-bzh(Chal-houbetal.
2014)genomesequenceassemblies.
Likewise,markerswithinthemajorQTLregionweresearchedagainstbothB.
rapaandB.
napusgenomesequenceassem-bliesusingthenucleotideMEGABLASTalgorithmwithwordsizeof28andcutoffe-valueat1e-10.
ResultsPolymorphismofmolecularmarkersandlinkagemapdevelopmentofSODHpopulationDifferenttypesofmolecularmarkerswereusedinthecon-structionofthegeneticmapfortheSODHpopulation:AFLP,SSR,DArT,Silicor-DArT,SNP,KASP,andcandi-dategene-basedmarkers.
With16AFLPprimercombina-tions,atotalof75polymorphicmarkerscouldbescoredintheSODHpopulation.
Ofthe350SSRprimerpairsscreened,23(0.
07%)werefoundpolymorphicbetweentheparentsandexhibitedclearandunambiguousamplifica-tion.
Sevenofthe23SSRprimerpairsamplifiedmorethanonepolymorphiclocus,resultingin32SSRloci.
Approxi-mately13%(407/3072)ofDArTand42%(2005/4787)ofsilico-DArTmarkerswerepolymorphicbetweentheparents.
Afterremovalofmarkerswithaminorallelefrequencyoflessthan10%,atotalof2555markerlociwereavail-ableformapconstruction.
TheresultinglinkagemapforSODHpopulationhas1638markersmappedonto23linkagegroupsandcovered2350.
2cMwithameaninter-valdistanceof2.
0cMbetweenmarkers.
Theunmappedmarkerswereeitherambiguouslylinkedtovariouslink-agegroups,unlinked,orformedsmalllinkagegroupsthatwereexcludedforestimationofthelinkagemaplength.
About50%(457/913)oftheunmappedmarkersshowedskewedsegregationofwhich47%(217/457)showedstronglyskewedsegregation.
Thenumberofmarkers,mapsize,markerdensity,andmeandistancebetweenmarkersaresummarizedinTable1andthegeneticmapisshowninSupplementaryFig.
1andSupplementaryTable3.
AlllinkagegroupscouldbeassignedwithchromosomenamesaccordingtothenomenclatureofParkinetal.
(1995)asA01–A10andC01–C09.
The23linkagegroupsrepre-sented19chromosomesinB.
napus,additionalfourlink-agegroups(A08-II,C02-II,C03-II,andC04-II)wereformedduetolooseornolinkagetotheirmainlinkagegroups.
Themaphasanaveragedensityof0.
70markerpercMwithdistributionofmarkersvaryingfrom0.
20to1.
37cMacrossthelinkagegroups(Table1).
TheAgenomecom-prisedmoremarkers(987)ascomparedtotheCgenome(655),withameanintervaldistancebetweenmarkersof1.
6cMintheAgenomeand2.
4cMintheCgenome.
Thenumberofmarkersmappedinanindividuallinkagegrouprangedfrom7(A08-II)to164(A07).
About44%ofthemappedmarkers(718)showedsig-nificant(P=0.
05)segregationdistortionwiththemajor-ity(76%)ofthemarkersfavoringtheSansibarallele.
LociwithskewedsegregationfavoringtheSansibaralleleweremostlyfoundonlinkagegroupsA07,A10,C03,andC05;whilelociwithskewedsegregationfavoringtheOaseallelewereclusteredmainlyonlinkagegroupsA05,C01,C03-II,andC04-II.
Threecandidategene-basedmark-ers(HMG1A07-O1,HMG2A10-2S,andD120E-3)weremappedonA07andanother(Dx-3)wasmappedonC09(SupplementaryTable3).
Atotalof910sequence-informativemarkerswerephysicallymappedtotheB.
napusgenomesequence.
ThealignmentoftheSODHgeneticmapandthephysicalmapofB.
napusgenomesequencewasgenerallyinagree-mentalthoughsomeregionsshoweddisruptionofcolin-earitywhichmaysuggestchromosomalrearrangements,erroringenomesequenceassembly,orinaccuraciesofthemap(SupplementaryFig.
2).
Thegenomiclocationsofsequence-informativemarkersandtheirhomologyarepro-videdinSupplementaryTable4.
PhenotypicanalysisHighlysignificanteffectsforthegenotypeandtheenvi-ronmentwerefoundforalltraitsintheSODHpopulation(Table2).
Broad-senseheritability(h2)estimateswerehigh,rangingfrom0.
80to0.
90,indicatingthatmuchofthephenotypicvarianceweregeneticallydetermined.
Thetotalphytosterolcontentrangedfrom311.
2to486.
9mg100g1seed,withameanof401.
9mg100g1seed(Table3).
Amongthefourquantifiedend-productsofthesterolpathway,sitosterolwasthemostprominentsterol,followedbycampesterol,brassicasterol,andavenasterol.
The24-ethylsterolcontent,whichincludessitosterolandavenasterol,washigherthanthe24-methylsterolcontent,whichcomprisescampesterolandbrassicasterol.
Betweentheparents,Sansibarconsistentlyshowedahigher188TheorApplGenet(2016)129:181–199phytosterolcontentthanOasewhileOasehadahigher24-methylto24-ethylsterolratiothanSansibarandonlyasmalldifferencewasobservedforthecampesteroltositosterolratio.
Theoilcontentwashighinthispopula-tion,rangingfrom41.
2to48.
6%,withameanof46.
3%.
Betweentheparents,OasehadahigheroilcontentthanSansibar.
Highlysignificantcorrelations(P=0.
01)wereobservedbetweentotalphytosterolandthefourindividualsterols(Table4).
Allninephytosteroltraitswerepositivelycor-relatedtoC16:0whilebrassicasterolinparticularwascorrelatedtoallthemajorfattyacids.
Oilwaspositivelycorrelatedwithtotalphytosterolandoleicacidandnega-tivelycorrelatedwithlinoleicandlinolenicacids.
Exceptforbrassicasterol,nosignificantcorrelationwasobservedbetweenphytosterolsandproteincontentofthedefattedmeal.
QTLmappingMultipleintervalmappingidentifiedbetweenoneandsixQTLforninephytosteroltraits,betweentwoandsixQTLforfourfattyacids,fiveQTLforoilcontent,fourQTLforproteincontentofdefattedmealandthreeQTLforseedweight(Table5).
TheseQTLweredistributedon13link-agegroupsasshowninFig.
2.
ColocalizationsofQTLfordifferenttraitsweremorefrequentlyobservedthanindivid-ualisolatedQTL.
PhytosterolsQTLforphytosterolscontentweredistributedonninelinkagegroups:A01,A02,A03,A04,A06,A07,C03-II,C05,andC08(Table5;Fig.
1).
MajorQTL(R2≥25%)wereidentifiedforbrassicasterolonlinkagegroupTable1Markerdistribution,size,density,andmeandistancebetweenmarkersofeachlinkagegroupinthelinkagemapofSODHpopulationaCG:candidategene-basedmarkersbCo-segregatingmarkersarerepresentedasasinglemarkerinthecalculationofmeandistancebetweenmarkersLinkagegroupNo.
ofmarkersperlinkagegroupSize(cM)Markerdensity(cM1)Averagedistancebetweenmarkers(cM)bAFLPCGaDArTKASPSilico-DArTSNPSSRTotalA0126158437498.
00.
761.
50A02352633740.
70.
911.
30A037193107151152224.
10.
681.
70A042148451106194.
20.
552.
10A05272635281163.
80.
492.
20A064182955124128.
00.
971.
20A07139213415164133.
71.
230.
90A082913124537.
81.
191.
10A08-II1679.
70.
721.
90A09382492165130.
20.
502.
50A10811107312994.
11.
370.
90C01424413377.
50.
432.
70C02111111448.
20.
294.
40C02-II1116112097.
90.
205.
40C0325272384111.
30.
751.
40C03-II351456161134.
80.
452.
40C04119222479.
50.
303.
60C04-II53475693101.
00.
921.
20C052114545392.
10.
581.
90C062514976495.
20.
671.
60C078829175121142.
40.
851.
40C0842723352.
50.
631.
80C0911344415463.
50.
851.
20A3431071376059119841254.
30.
791.
57C281371251847126541095.
90.
602.
42Whole6241442512781062316382350.
20.
702.
01189TheorApplGenet(2016)129:181–199Table2VariancecomponentsandheritabilityoftheSODHpopulation(n=226)**DenotessignificanceatP=0.
01aOriginalvalues(ratio)*100TraitVariancecomponents(σ2)HeritabilityGenotype(G)Environment(E)εh2Phytosterolmg100g1seedBrassicasterol14.
28**5.
06**16.
120.
84Campesterol315.
99**160.
43**150.
540.
93Sitosterol267.
43**36.
23**310.
050.
84Avenasterol48.
38**94.
09**52.
440.
85Totalsterol1139.
02**706.
69**934.
690.
8824-methylsterol330.
13**188.
89**192.
910.
9124-ethylsterol412.
95**206**368.
780.
87Campesterol:sitosterola89.
77**24.
56**41.
650.
9324-methyl:24-ethylsterola62.
9**8.
29**33.
990.
92OthertraitsC16:0(%)0.
1**0.
04**0.
070.
90C18:1(%)2.
57**0.
52**1.
480.
91C18:2(%)1.
24**0.
1**2.
200.
93C18:3(%)0.
43**0.
15**0.
310.
89Oil(%)1.
65**3.
52**1.
940.
84Proteinofdefattedmeal(%)1.
59**8.
01**2.
310.
81Thousandkernelweight(g)0.
20**0.
23**0.
260.
85Table3DescriptivestatisticoftheparentsandtheSODHpopulation(n=226)LSD5%leastsignificantdifferenceatthelevelof5%**DenotessignificanceatP=0.
01aOriginalvalues(ratio)*100TraitParentsDoublehaploidpopulationSansibarOase(n=226)MeanMinMaxMeanFvalueLSDPhytosterolmg100g1seedBrassicasterol50.
446.
432.
759.
548.
86.
3**4.
6Campesterol157.
2114.
987.
8192.
7136.
513.
6**13.
9Sitosterol226.
7167.
5154.
9251.
6193.
46.
2**20.
0Avenasterol23.
819.
79.
852.
325.
46.
5**8.
2Totalsterol461.
7352.
4311.
2486.
9401.
98.
3**34.
624-methylsterol207.
7161.
3130.
2214.
0185.
311.
3**15.
724-ethylsterol250.
4187.
2170.
1252.
7218.
87.
7**21.
8Campesterol:sitosterola69.
468.
747.
399.
771.
013.
9**7.
324-methyl:24-ethylsterola82.
986.
262.
3108.
285.
312.
1**6.
6OthertraitsC16:0(%)5.
04.
63.
85.
64.
89.
6**0.
3C18:1(%)58.
863.
157.
365.
461.
611.
5**1.
4C18:2(%)21.
018.
717.
124.
119.
914.
1**0.
9C18:3(%)9.
89.
07.
511.
89.
69.
3**0.
6Oil(%)43.
746.
341.
248.
645.
46.
1**1.
6Proteinofdefattedmeal(%)29.
332.
927.
335.
230.
55.
1**1.
7Thousandkernelweight(g)5.
55.
64.
47.
85.
85.
8**0.
6190TheorApplGenet(2016)129:181–199Table4Spearman'srankcorrelationoftraitsintheSODHpopulation(n=226)*and**DenotessignificanceatP<0.
05and0.
01Brassi-casterolCampes-terolSitosterolAvenas-terolTotalphytos-terol24-Methylsterol24-EthylsterolCampesterol:sitosterol24-Methyl:24-ethylsterolC16:0C18:1C18:2C18:3OilProteinofdefattedmealCampesterol0.
03Sitosterol0.
15*0.
32**Avenasterol0.
040.
78**0.
36**Totalsterol0.
20**0.
84**0.
74**0.
80**24-methylsterol0.
24**0.
98**0.
34**0.
77**0.
86**24-ethylsterol0.
14*0.
53**0.
95**0.
65**0.
88**0.
54**Campesterol:sitosterol0.
080.
77**0.
35**0.
53**0.
34**0.
73**0.
1124-methyl:24-ethylsterol0.
120.
49**0.
61**0.
15*0.
010.
50**0.
45**0.
89**C16:00.
29**0.
31**0.
18**0.
26**0.
33**0.
36**0.
24**0.
18**0.
15*C18:10.
43**0.
060.
090.
040.
110.
15*0.
060.
010.
120.
53**C18:20.
27**0.
020.
020.
090.
030.
080.
010.
020.
110.
31**0.
83**C18:30.
37**0.
100.
010.
090.
120.
18**0.
040.
080.
13*0.
29**0.
68**0.
34**Oil0.
100.
20**0.
16*0.
30**0.
24**0.
17**0.
23**0.
09**0.
060.
020.
48**0.
51**0.
23**Proteinofdefattedmeal0.
27**0.
060.
010.
050.
010.
000.
020.
060.
030.
18**0.
090.
060.
16*0.
43**Seedweight0.
120.
080.
19**0.
130.
15*0.
050.
20**0.
05**0.
15*0.
070.
110.
14*0.
060.
38**0.
16*191TheorApplGenet(2016)129:181–199Table5QTLdetectedforphytosterolcontentsmg100g1seed,fattyacidcomposition(%),oilcontent(%),proteincontentofdefattedmeal(%),andseedweight(g)inSODHpopulationTraitQTLnameLGPeak(cM)CIa(cM)LODAdditiveeffectbR2TotalR2BrassicasterolDE-Bra.
1A017974–855.
30.
924.
562.
6DE-Bra.
2A03172167–1786.
81.
035.
1DE-Bra.
3A049591–9731.
72.
6138.
3DE-Bra.
4A074741–535.
40.
944.
7DE-Bra.
5A07116100–1313.
30.
772.
6DE-Bra.
6C03-II8279–888.
11.
137.
5CampesterolDE-Camp.
1A049587–996.
55.
8311.
737.
8DE-Camp.
2A066659–708.
66.
5913.
8DE-Camp.
3A074638–523.
94.
565.
1DE-Camp.
4C08120–204.
74.
747.
3SitosterolDE-Sito.
1C058778–896.
06.
1511.
323.
2DE-Sito.
2A069492–996.
56.
0612.
0AvenasterolDE-Ave.
1C08141–205.
92.
5911.
811.
8TotalphytosterolDE-TPC.
1A072713–383.
510.
498.
214.
2DE-TPC.
2C08140–353.
18.
796.
124-methylsterolDE-Methyl.
1A066459–699.
97.
7315.
931.
1DE-Methyl.
2A074638–524.
75.
406.
9DE-Methyl.
3C08131–205.
25.
448.
324-ethylsterolDE-Ethyl.
1A069491–1003.
65.
927.
27.
2Campesterol:sitosterolDE-CSratio.
1A016865–863.
20.
012.
271.
5DE-CSratio.
2A049385–9812.
80.
0314.
7DE-CSratio.
3A066462–7730.
40.
0533.
3DE-CSratio.
4C058377–8616.
50.
0416.
4DE-CSratio.
5C08172–206.
10.
024.
924-methyl:24-ethylsterolDE-MEratio.
1A018379–925.
20.
023.
670.
3DE-MEratio.
2A02250–54.
90.
024.
5DE-MEratio.
3A049283–1024.
20.
025.
6DE-MEratio.
4A066361–6632.
30.
0538.
7DE-MEratio.
5C058479–8916.
50.
0317.
8C16:0DE-16:0.
1A017371–776.
20.
084.
859.
0DE-16:0.
2A0910099–10321.
70.
3028.
8DE-16:0.
3C058773–893.
90.
074.
0DE-16:0.
4C0882–1712.
20.
1210.
4DE-16:0.
5C092624–308.
10.
1010.
9C18:1DE-18:1.
1A018482–8916.
50.
8726.
343.
6DE-18:1.
2A074741–543.
70.
395.
6DE-18:1.
3C0840–158.
10.
6011.
7C18:2DE-18:2.
1A017472–7711.
20.
5318.
830.
6DE-18:2.
2A094234–525.
00.
4311.
8C18:3DE-18:3.
1A018681–8920.
00.
3627.
357.
0DE-18:3.
2A0310488–1233.
90.
154.
8DE-18:3.
3A043521–493.
20.
167.
4DE-18:3.
4C058680–894.
70.
173.
5DE-18:3.
5C078281–925.
00.
165.
3DE-18:3.
6C081411–198.
20.
218.
7192TheorApplGenet(2016)129:181–199A04(DE-Bra.
3),forcampesteroltositosterolratioand24-methylto24-ethylsterolratioonlinkagegroupA06(DE-CSratio.
3andDE-MEratio.
4).
ThemajorQTLDE-Bra.
3onA04wasatthesamepositionastheQTLforcampesterol,andoverlappedwithQTLforthecampesteroltositosterolratioand24-methylto24-ethylsterolratio;theadditiveeffectofDE-Bra.
3wasnegativeasopposedtotheotherthreeQTL.
Onthecontrary,minorQTLforbrassicas-terolonA01(DE-Bra.
1)andA07(DE-Bra.
4)overlappedwithQTLforphytosterolrelatedtraitswiththesamedirec-tionofadditiveeffect.
ThemajorQTLDE-CSratio.
3andDE-MEratioonA06overlappedwithQTLforcampesteroland24-methylsterol;thesefourQTLshowednegativeeffects,indicatingthattheallelesincreasingthetraitvalueswerederivedfromOase.
Fortotalphytosterolcontent,twoQTLwithpositiveadditiveeffectsweredetectedonA07andC08;theQTLDE-TPC.
1onA07waslocatedatthetopofthelinkagegroup,closetoagenomicregionwithmanyQTL(Table5;Fig.
2).
TheQTLDE-TPC.
2onC08over-lappedwithQTLfordifferentphytosterolsaswellasforfattyacidsandoilcontent.
Fattyacids,oilcontent,andproteincontentofthedefattedmealBetweentwoandsixQTLwereidentifiedforfattyacids,fiveQTLforoilcontent,andfourQTLforproteincontentofthedefattedmeal(Table5;Fig.
2).
MajorQTLweredetectedforC16:0onA09(DE-16:0.
2)andforoleicacidandlinolenicacidonA01(DE-18:1.
1andDE-18:3.
1).
OnlyminorQTLweredetectedforoilcontentandforproteincontentofthedefattedmealwhichindividuallyexplainedbetween4.
3and6.
7%andbetween8.
0and12.
9%ofthephenotypicvariance,respectively.
ThereweretwoQTLhotspotregionsworthmentioning.
ThefirstoneconsistsofQTLDE-16:0.
1,DE-18:1.
1,DE-18:2.
1,DE-Oil.
1,andDE-Pro.
1onA01.
Thedirectionoftheaddi-tiveeffectssuggeststhattheOasealleleledtoadecreaseofC16:0,C18:2,C18:3,andproteincontentofthedefattedmeal,andtoanincreaseinC18:1andoilcontent.
Thesec-ondQTLhotspotregionconsistsofQTLDE-16:0.
4,DE-18:1.
3,DE18:3.
6,andDE-Oil.
5onC08;thedirectionsoftheadditiveeffectswereidenticaltotheQTLatthefirstQTLhotspotregion.
AllofthefiveQTLforoilcontenthadnegativeadditiveeffects,indicatingthattheallelesincreas-ingtheoilcontentwerederivedfromOase.
Interestingly,thelargestQTLDE-Oil.
3onA07waslocatedwithintheconfidenceintervalofQTLDE-Bra.
5forbrassicasterolwiththesamedirectionoftheadditiveeffect.
SeedweightThethreeQTLdetectedforseedweightwerefoundonlinkagegroupsA02,A07,andC03-II(Table5;Fig.
2).
IndividualQTLexplainedbetween6.
1and10.
7%ofthephenotypicvariance,whichcollectivelyaccountedfor27.
1%ofthetotalphenotypicvariance.
AdditiveeffectswerepositiveforQTLlocatedonA02andC03-IIandnegativeforQTLlocatedonA07.
TheQTLDE-SW.
1onLGlinkagegroupaCI1-LODconfidenceintervalbAdditiveeffectisthesubstitutioneffectofoneOaseallelebyoneSansibaralleleTable5continuedTraitQTLnameLGPeak(cM)CIa(cM)LODAdditiveeffectbR2TotalR2OilcontentDE-Oil.
1A017368–7930.
314.
327.
5DE-Oil.
2A022116.
5–2650.
396.
3DE-Oil.
3A07124120–12750.
446.
7DE-Oil.
4C03-II5034–6630.
374.
7DE-Oil.
5C08170–3430.
305.
5ProteinofdefattedmealDE-Pro.
1A017668–885.
20.
408.
738.
1DE-Pro.
2A074444–486.
70.
448.
5DE-Pro.
3C03-II3327–374.
60.
368.
0DE-Pro.
4C03-II9289–976.
90.
4612.
9SeedweightDE-SW.
1A022321–295.
50.
1510.
327.
1DE-SW.
2A074744–545.
50.
1610.
7DE-SW.
3C03-II8578–933.
00.
126.
1Fig.
2QTLassociatedwithphytosteroltraits,fattyacids,oilcontent,proteinofdefattedmeal,andseedweightinSODHpopulation.
Asteriskonmarkernameindicatescandidategene-basedmarker.
Italicfontofmarkernameindicatesplacedmarker.
PlusandminusindicatethatthetraitvalueisincreasedbythealleleSansibarandOase,respec-tively193TheorApplGenet(2016)129:181–1993091853|F|00.
03083750|F|06.
8CB10097b10.
03091606|F|016.
23094873|F|020.
1CB1009932.
4E39M48-19434.
73115206|F|045.
3100000853|F|053.
8E38M59-28557.
53094588|F|064.
83133092|F|068.
0brPb-66153673.
33195628|F|078.
93114358|F|088.
5brPb-83988692.
2CB1057296.
7DE-Bra.
1(+)DE-CSratio.
1(+)DE-MEratio.
1(+)DE-16:0.
1(+)DE-18:1.
1(-)DE-18:2.
1(+)DE-18:3.
1(+)DE-Oil.
1(-)DE-Pro.
1(+)A01ra00578s010.
0brPb-8099178.
7100005036|F|011.
7E38M51-15721.
4brPb-67077724.
93081349|F|027.
9brPb-83904840.
7DE-MEratio.
2(+)DE-Oil.
2(-)DE-SW.
1(+)A023131550|F|00.
0E32M48-35420.
1brPb-84217924.
0ra02449s0130.
7ra02025s0134.
4ra00558s0142.
0brPb-67121763.
5brPb-83888577.
1ra02551s0182.
0ra02122s01108.
2ra03222s01117.
3ra00720s01143.
6ra00109s01156.
2E37M50-87159.
9E44M51-62172.
23093734|F|0196.
3brPb-840964203.
5E36M51-388220.
6DE-18:3.
2(+)DE-Bra.
2(+)A033115752|F|00.
03097895|F|04.
1100002422|F|012.
53115220|F|027.
93100326|F|050.
83096579|F|067.
83076080|F|074.
4100000708|F|082.
0ra00529s0188.
0brPb-66199998.
9brPb-841775104.
23102738|F|0110.
4brPb-660337117.
2brPb-658275124.
5ra00443s01132.
1ra02159s01137.
1100001117|F|0143.
2E44M50-368167.
4DE-18:3.
3(-)DE-MEratio.
3(+)DE-CSratio.
2(+)DE-Camp.
1(+)DE-Bra.
3(-)A04ra00669s010.
0brPb-8417366.
73085327|F|011.
23097489|F|022.
2brPb-80884430.
33077601|F|037.
83080133|F|045.
03156714|F|050.
13111072|F|058.
2brPb-66235465.
83114544|F|070.
6brPb-65911975.
6100003367|F|080.
93091100|F|084.
13129180|F|090.
6brPb-83896994.
4brPb-658875101.
63075341|F|0105.
53112388|F|0114.
13096863|F|0120.
13216051|F|0128.
0DE-MEratio.
4(-)DE-Sito.
2(+)DE-Methyl.
1(-)DE-Ethyl.
1(+)DE-CSratio.
3(-)DE-Camp.
2(-)A06HMG2A10-2*(HMG2)0.
0D120E-3*(DGAT1)11.
53110517|F|018.
03126880|F|032.
83201696|F|037.
7brPb-67108344.
3ra00106s0149.
3ra00123s0155.
4ra00476s0161.
8ra00366s0165.
5ra00069s0168.
3ra04226s0187.
4ra00688s01101.
0E40M50-263109.
53122992|F|0117.
5HMG1A07-O1*(HMG1)120.
03124997|F|0123.
63174697|F|0127.
23100149|F|0133.
7DE-TPC.
1(+)DE-Pro.
2(-)DE-Bra.
5(-)DE-Methyl.
2(+)DE-Oil.
3(-)DE-Camp.
3(+)DE-SW.
2(-)DE-Bra.
4(+)DE-18:1.
2(-)A07100001587|F|00.
03128791|F|024.
63123759|F|030.
7100000807|F|036.
4100001207|F|053.
03129027|F|059.
8100010105|F|063.
8100001522|F|076.
33086664|F|078.
9brPb-84010299.
3brPb-661741107.
83078836|F|0115.
63106147|F|0120.
0DE-18:2.
2(+)DE-16:0.
2(-)A09E40M50-3550.
0ra02841s0127.
4brPb-65940032.
93087789|F|038.
43151023|F|041.
83132883|F|066.
33101542|F|074.
2brPb-67077982.
23115435|F|088.
93205118|F|094.
3ra00281s0199.
0ra00115s01102.
33083936|F|0107.
63208561|F|0134.
8DE-Pro.
2(-)DE-Oil.
4(-)DE-Bra.
6(+)DE-SW.
3(+)DE-Pro.
3(-)C03-II100001020|F|00.
0100001059|F|04.
13187836|F|013.
7ra00447s0123.
2ra00237s0138.
83084373|F|055.
33091164|F|063.
5ra00678s0169.
4100001393|F|086.
2100002303|F|092.
1DE-CSratio.
4(-)DE-MEratio.
5(-)DE-18:3.
4(-)DE-Sito.
1(+)DE-16:0.
3(-)C05E36M51-2140.
0E36M51-21722.
7brPb-65788633.
9E37M59-13440.
13083814|F|049.
0100001027|F|055.
0100001167|F|066.
9brPb-80867574.
9CB1001482.
1brPb-67055593.
03113313|F|0100.
0E38M50-142S113.
13122303|F|0119.
93119656|F|0127.
2CB10425a132.
2ra03178s01142.
4DE-18:3.
5(-)C07Dx-3O*(DGAT1)0.
03074850|F|016.
23080644|F|024.
23092949|F|030.
93104273|F|035.
03163621|F|040.
5100001132|F|044.
5ra02032s0163.
5DE-16:0.
5(+)C09O3133904|F|00.
0ra03760s0110.
8E38M51-6518.
63159566|F|021.
3E32M48-25341.
6DE-18:1.
3(-)DE-16:0.
4(+)DE-Camp.
4(+)DE-Methyl.
3(+)DE-TPC.
2(+)DE-Ave.
1(+)DE-18:3.
6(+)DE-Oil.
5(-)DE-CSratio.
5(+)C08194TheorApplGenet(2016)129:181–199A02overlappedwithQTLDE.
Oil.
2withoppositeeffects,indicatingthattheOaseallelewasincreasingoilcontentanddecreasingseedweight.
TheQTLDE-SW.
2onA07overlappedwithQTLDE-Bra.
4,DE-18:1.
2,DE-Methyl.
2,DE-Camp.
3,andDE-Pro.
2;thedirectionoftheadditiveeffectsindicatethattheOasealleleledtoanincreaseinseedweight,oleicacid,andproteincontentofthedefattedmealandtoadecreaseinphytosterols.
TheQTLDE-SW.
3onC03-IIoverlappedwithQTLDE-Pro.
4andDE-Bra.
6;thedirectionsoftheadditiveeffectsindicatethattheOaseallelereducedseedweightandBrassicasterolcontentbutincreasedproteincontentofthedefattedmeal.
IdentificationofpossiblecandidategenesformajorQTLSequencesofmarkersassociatedwiththemajorQTLandthepredictedgenesweresearchedingenomesequencesofbothB.
rapaChiifu(Wangetal.
2011)andB.
napusDar-mor-bzh(Chalhoubetal.
2014).
ThecongruencyofmarkerorderswasbetterwithB.
rapaChiifugenomesequenceassemblywhilesomemarkersandmostofthepredictedgeneswerefoundmatchingonrandom,non-anchoredscaf-foldsinB.
napusDarmor-bzhgenomeassembly.
Therefore,theB.
rapaChiifugenomesequenceassemblywasusedasareferencefortheinvestigationoftheunderlyingcandidategenes.
ThealignmentsofthemajorQTLonA01,A04,A06,andA09withB.
rapaareprovidedinSupplementaryFigs.
3–6.
Withinthegenomicregionof64.
8–92.
2cMonA01,majorQTLforC18:1andC18:3werefoundcolocal-izedwithFAD2(notannotatedinB.
rapa)whiletheminorQTLforC16:0,C18:2,andoilcontentwerecolocalizedwithLPAAT(Bra037553).
ThemajorQTLforbrassicast-erolonA04colocalizedwithtwoorthologsofCYP710A2whichareannotatedasCYP710A1inB.
rapa(Bra021916andBra021917).
OnA06,majorQTLforcampesteroltositoterolratioand24-methylto24-ethylsterolratiocolo-calizedwithSMT2.
OnA09,themajorQTLforC16:0colocalizedwiththehomologofFATB(Bra031631).
DiscussionMolecularmarkersandlinkagemapThetwoparentallines,SansibarandOase,revealedanar-rowgeneticbackgroundbasedontheirlowlevelofpoly-morphismsformostmarkertypes.
Thenumbersofpoly-morphicmarkersweregreatlyincreasedwitharray-basedhigh-throughputDArTandsilico-DArTmarkerswhichatthesametimewerealsosequenceinformative.
Asa10-cMintervalbetweenmarkerlociiscommonlyusedforQTLanalysis,theSODHmapcanbeconsideredsuitableforper-formingQTLanalysis.
HighlevelofsegregationdistortionsobservedinthisstudyhavealsobeenreportedinotherB.
napusmaps(Kauretal.
2009;Zhangetal.
2011;Delourmeetal.
2013;Ramanetal.
2013).
Suchphenomenonappearstobecommoninmapsofmicrospore-derivedDHpopula-tions,whichmaybeduetothedifferentialresponsivenessbetweenthetwoparentallinestomicrosporeculturedur-inginvitroandrogenesisandplantletregeneration(forareviewseeFerrieandMllers2011).
TheSODHmaphasahighernumberofmarkersmappedontheAgenomethanontheCgenome,similartoafewreportedstudies(Ban-croftetal.
2011;Delourmeetal.
2013;Ramanetal.
2013).
PhenotypicanalysisResultsfromthephenotypicanalysisrevealedarela-tivelylargeandsignificantphenotypicvariationforallthetraits.
Totalphytosterolcontentwhichrangedfrom311.
2to486.
9mg100g1seedwascomparabletotherangefrom356.
6to480.
0mg100g1seedreportedin27mod-ernrapeseedcultivars(Amaretal.
2009)andhigherthantherangefrom257to410mg100g1seedreportedinaDHpopulationsegregatingforerucicacidcontent(Amaretal.
2008b).
Bytakingoilcontentintoconsideration,thetheoreticalphytosterolcontentinoilrangedfrom718to1123mg100g1oilintheSODHpopulation,whichwaslowerthantherangefrom766to1402mg100g1oilin12differentspringcanolavarieties(Abidietal.
1999)buthigherthantherangefrom464to807mg100g1oilinninecanolalines(VlahakisandHazebroek2000)andtherangefrom448to928mg100g1oilinthreedifferentDHpopulationsofwinteroilseedrape(Amaretal.
2008b).
ThehightotalphytosterolcontentfoundintheSODHpopulationmaybeattributedtothelowerucicacidcontentoftheseedoilasanegativecorrelationbetweenthetwotraitshasbeenreportedbyAmaretal.
(2008b).
Amongtheindividualphytosterols,sitosterolwasthemostprominentsterol,followedbycampesterol,brassicasterol,andavenas-terol,whichisinaccordwiththerelativecontentsreportedfromliteratures(VlahakisandHazebroek2000;Verleyenetal.
2002;Amaretal.
2008a,b,2009).
ThetransgressivesegregationforindividualandtotalphytosterolcontentoftheDHpopulationcanbeexplainedbythefactthatbothparentscontributedpositivealleles.
Therangeofseedoilcontentfrom41.
2to48.
6%waswithintherangeofcom-mercialcultivarswhichusuallycontainabout40–50%ofoil.
SignificantgenotypicvariationandhighheritabilityobservedinalltraitssuggestthatSODHpopulationissuit-ableforQTLanalysis.
Fromanutritionalpointofview,negativecorrelationsbetweenoilcontentandpolyunsatu-ratedfattyacidssuchaslinoleicandlinolenicacidsandpositivecorrelationsbetweenoilcontentandbotholeic195TheorApplGenet(2016)129:181–199acidandtotalphytosterolcontentaredesirableasreducedlevelsofpolyunsaturatedfattyacidsandincreasedlevelofoleicacidwillincreaseoxidativestabilityofoilwhilephy-tosterolscanlowerLDLcholesterols.
QTLmappingResultsfrommultipleintervalmapping(MIM)indicatethatadditiveeffectswerethemainfactorscontributingtovariationinalltraitsasnosignificantepistaticinteractionwasdetectedinanycase.
Fortotalphytosterolcontent,thepresentstudyidentifiedonlytwominorQTLlocatedonA07andC08whileAmaretal.
(2008a)detectedtwomajorQTLonA08andC03andaminorQTLonC08.
ThedisappearanceoftwomajorQTLinthepresentstudycor-roboratethefindingsofAmaretal.
(2008a)whofoundthatthetwomajorQTLfortotalphytosterolcontentweremostlikelyduetopleiotropiceffectsexertedbytheerucicacidgenes.
Asamatteroffact,thepresentstudydidnotdetectanyQTLonA08andC03foralltheninephytosteroltraitsexceptfortheoneminorQTLforbrassicasterolidentifiedonC03-II(DE-Bra.
6).
BydisregardingtheQTLonA08andC03fromthestudyofAmaretal.
(2008a,b),thenum-berofQTLwasalmostthesameasdetectedinthepresentstudyexceptforavenasterolinwhichfouradditionalQTLweredetectedinthestudyofAmaretal.
(2008a).
Simi-larly,thepresentstudyshowsthatmoreQTLweredetectedforindividualphytosterolcontentthanfortotalphytosterolcontent.
OftheeightlinkagegroupsthatharboredQTLforphytosterolsinthisstudy,onlytwolinkagegroups(A02andA07)werenotfoundtohaveQTLinthestudyofAmaretal.
(2008a).
MajorQTLandpossibleunderlyingcandidategenesOfthe16traitsanalyzed,QTLwithmajoreffectswerefoundforC18:1andC18:3onA01,brassicasterolonA04,campesteroltositosterolratioand24-methylto24-ethylsterolratioonA06,andC16:0onA09.
AgoodcolinearityobservedbetweenthegeneticandtheB.
rapaphysicalmappositionsenabledtheinvestigationofthepossibleunderly-ingcandidategenes.
OnA01,majorQTLforC18:1andC18:3colocalizedwithFAD2whichencodestheenzymeendoplasmicΔ-12oleatedesaturasethatdesaturatesC18:1toC18:2.
InB.
napus,fourlocilocatedonA01,A05,C01,andC05havepreviouslybeenreportedforFAD2(Schierholtetal.
2000).
However,inthereferencegenomeofB.
napus,homologsofFAD2wereonlyfoundonA05,C05,andscaffoldchro-mosome.
Becausethereweremanycaseslikethis,wherethepositionofthecandidategenecouldnotbedeter-mined,thereferencegenomeofB.
rapawasusedfortheinvestigationofcandidategenes.
About2–3cMabovethemajorQTLforC18:1andC18:3,therewereminorQTLforC16:0,C18:2,andoilcontentwhichwerefoundcolo-calizedwiththeLPAATgene,whichencodesthesecondenzymeoftheKennedypathwaythatacylatesthesn-2hydroxylgroupoflysophosphatidicacidtoformphospha-tidicacid.
InArabidopsis,expressionoftheoilseedrapemicrosomalLPAATisozymehasshownenhancementofseedoilcontentandseedmass(Maisonneuveetal.
2010).
Moreover,studieshaveshownthatLPAATisasubstrate-specificacyltransferaseandappearstodiscriminateagainstsaturatedacylCoAinmostoilseeds(NortonandHarris1983;Sunetal.
1988;Lassneretal.
1995),whichsomehowexplainstheoppositedirectionofadditiveeffectsobservedbetweenQTLforoilcontent(DE-Oil.
1)andQTLforpal-miticacidandoleicacid(DE-16:0.
1andDE-18:2.
1).
Inaddition,thenegativecorrelationbetweenpalmiticacidandoleicacid(rs=0.
53**)inSODHpopulationmaysuggestanenhancedfluxindenovofattyacidsynthesispathway.
AsreportedbyMllersandSchierholt(2002),anenhancedC16/C18-fattyacidratiooftheseedoilmayindicateanimprovedseedoilsynthesisbyatop-downcon-trolmechanism.
Inmaize,simultaneousenhancementofoilandoleicacidcontentsinseedarelinkedtoageneencod-ingdiacylglycerolacyltransferase(DGAT)thatcatalyzesthefinalcommittedstepintheKennedypathwayleadingtotriacylglycerolproduction(Zhengetal.
2008).
Similarly,expressionofDGATinwild-typeArabidopsisthalianaandthehighlinoleicacidfad3fae1mutanthavebothshownastrikingincreaseinseedoleicacidcontent(Zhangetal.
2013).
OneexplanationfortheinfluenceofDGATonfattyacidcomposition,ormorespecificallytheincreasedoleicacidcontent,isthattheenhancedtriacylglycerolsynthesismediatedbyDGATlimitsthefluxthroughthephosphati-dylcholine-baseddesaturationreactions(Aznar-Morenoetal.
2015).
Inourstudy,weshowthatthepositivecorrela-tionbetweenoilandoleicacidcontentcouldalsobeduetocloselinkagebetweenFAD2andLPAAT(SupplementaryFig.
3).
Becausethenatureofmetaboliccontrolissuchthatasinglegene(orenzyme)rarelyleadstoahugeeffectforacomplextraitlikeoilcontent,recognizingthepleiotropiceffectandcloselinkageoftheinvolvedgenesmayfacili-tategenestackingforimprovingseedoilcontentaswellasthequalitytraits.
Therefore,itwouldalsobeofinter-esttoidentifythefunctionalpolymorphismsofLPAATandFAD2betweenSansibarandOaseaswellastoinvestigatetheallelicdiversityusingabroadergermplasmtodevelopfunctionalmarkerformarker-assistedselection.
ThemajorQTLDE-Bra.
3onA04collocatedwithtwoorthologsofCYP710A2,annotatedasCYP710A1inB.
rapa.
TheCYP710AgeneshavebeenknowntoencodecytochromeP450enzymethatcatalyzestheC-22desatu-rationreaction,convertingboth24-epi-campesterolandsitosteroltobrassicasterolandstigmasterol,respectively196TheorApplGenet(2016)129:181–199(Morikawaetal.
2006).
InArabidopsis,threeC-22steroldesaturasesencodedbyCYP710A1,CYP710A2,andCYP710A4(Morikawaetal.
2006;Arnqvistetal.
2008)areabletocatalyzethesynthesisofstigmasterolwhileonlyoneC22-desaturaseencodedbyCYP710A2isabletoproducebrassicasterol(Morikawaetal.
2006).
ThefactthatQTLDE-Bra.
3overlappedwiththreeotherQTL(DE-Camp.
1,DE-CSratio.
2andDE-MEratio.
3)withoppositeeffectsalsosuggeststhatCYP710A1geneexertsapleio-tropiceffectonthecompositionofphytosterols.
Giventhatbrassicasterolissynthesizedviatwoenzymaticstepsfrom24-methylenecholesterol,andcampesterolissynthesizeddirectlyfrom24-methylenecholesterol,atrade-offbetweencampesterolandbrassicasterolisusualinthecaseofpar-allelbiosyntheticpathways(Fig.
1).
OverlappingQTLbetweenbrassicasterolandcampesterolonA04aswellastheoppositeadditiveeffectsweresimilarlyobservedinthestudyofAmaretal.
(2008a),indicatingthattheymaybethesamelociinbothpopulations.
OnA06,twomajorQTLforcampesteroltositosterolratioand24-methylto24-ethylsterolratiocolocalizedwithtwootherminorQTLforcampesteroland24-methylsterol(Table5;Fig.
2).
About30cMbelowthisgenomicregion,therewasacolocationoftwominorQTLforsitosteroland24-ethylsterolwithpositiveadditiveeffectsasopposedtotheuppergenomicregion.
AlignmentbetweenthegenomicregionandphysicalmapofB.
raparevealedthattheSMT2genewaswithinthegenomicregionofmajorQTL(Supple-mentaryFig.
5).
TheSMT2geneencodestheenzymesterolmethyltransferase2whichregulatestheratiobetweencampesterolandsitosterolorbetween24-methylsteroland24-ethylsterol.
Campesteroltositosterolratioisofinter-estbecauseitisimportantinplantgrowthanddevelopment(Schaefferetal.
2001)andinhumans,itdeterminestheefficacyofcholesterolloweringability(Miettinen2001).
Theabilityofplantstosynthesizesterolswithbranchedethylgroups(asinsitosterolandstigmasterol)hasalsobeenproposedtobepartofanevolutionaryadaptationpro-cesstocopewithwidertemperaturefluctuations,andtomaintaintheessentialmembraneassociatedmetabolicpro-cesses,ascomparedtoanimals(Dufourc2008).
Consider-ingthefactthatnounfavorablepleiotropiceffectorcloselinkagewithotherqualitytraitswereobserved,independ-enteffectofthismajorQTLorSMT2onA06couldbeofinterestformodifyingphytosterolcomposition.
OnA09,themajorQTLDE-16:0.
2forpalmiticacidwasfoundcolocalizedwiththeFATBgene(Table5;Sup-plementaryFig.
6),whichencodestheenzymethathydro-lyzesthethioesterbondofC16:0-ACPandreleasesC16:0fromacyl-ACP(Bonaventureetal.
2003).
Acyl-ACPthi-oesterasesareknowntoberesponsibleforregulatingthechainterminationduringdenovofattyacidsynthesisandinchannelingcarbonfluxbetweentheplastidandcytosolinplants.
TheFATBgenebelongstooneofthetwoisoformsofacyl-ACPthioesterasewhichprimarilyhydrolyzesC8–C16-saturatedacyl-ACPs(Jonesetal.
1995).
GiventhatnootherQTLwerefoundoverlappingwithDE-16:0.
2,thisfurthersupportsthehypothesisthatFATBistheunderlyingcandidategene.
SimilarassumptioncanalsobemadeforQTLDE-16:0.
5onC09butthehomologsofFATBinB.
napusCsubgenomewerelocatedonC05andC08.
MinorQTLIncontrasttothelargeeffectsofQTLidentifiedforphy-tosterolsandfattyacids,fiveminorQTLdistributedonfivelinkagegroupswereidentifiedforoilcontent.
TheallelesincreasingoilcontentwereallderivedfromOase,thepar-entwithahighoilcontent,whichexplainswhyonlyslighttransgressivesegregationwasobservedintheSODHpopu-lation.
TheSODHpopulationissimilartotheRNSLpopu-lationusedinthestudyofDelourmeetal.
(2006)astheparentswerealsochosenfromtheelitewinteroilseedrapegermplasmwhichdiffersinoilcontent.
Thestudyreportedatotalof10genomicregionsassociatedwithoilcontentwhichweredistributedon10linkagegroups.
AcomparisonbetweenthetwopopulationsshowedthatQTLweresimi-larlydetectedonfivelinkagegroups(A01,A07,A08,C03,andC08)butitcouldnotbeconfirmediftheywerethesamelociinbothpopulationsasthegeneticmapsdonotshareanycommonmarkers.
Inthisstudy,theQTLwiththelargesteffectwaslocatedonA07(DE-Oil.
3)andwasfoundcolocalizedwithQTLforbrassicasterol(DE-Bra.
5)andthecandidategene-basedmarkerofHMG1(HMG1A07O)at120cM.
GiventhatHMG1geneisresponsibleforregulat-ingthecarbonfluxintotheisoprenoidpathwayandbothdenovofattyacidsynthesisandphytosterolsynthesissharethesameprecursor(acetyl-CoA)(Fig.
1),thecollocationofbothQTLwithHMG1maybecausedbyadownstreameffectofHMG1geneoralternatively,itmightbeduetocloselinkagebetweenthecausativegenesandHMG1.
BesidestheHMG1,twoothercandidategenes,HMG2andDGAT1,werealsomappedonA07butwerenotfoundcolocatedwithanyQTL.
AbovetheoverlappingQTLforoilandbrassicasterolonA07liesagenomicregion(38–54cM)whichharboredsixQTLassociatedwithdifferenttraits(brassicasterol,campesterol,24-methylsterol,oleicacid,proteinofdefattedmeal,andseedweight).
AllofthesixQTLshowedminoreffects;however,QTLforproteincontentofdefattedmealandseedweightweretheindi-vidualQTLwhichhavethelargesteffectintheirrespectivetrait.
Particularlyforseedweight,numerousstudieshaveconsistentlydetectedQTLonA07indifferentpopulationswithdiversegeneticbackgrounds(Quijadaetal.
2006;Udalletal.
2006;Shietal.
2009;Basunandaetal.
2010;Caietal.
2012)whileinthelateststudy,12candidate197TheorApplGenet(2016)129:181–199genesunderlying8QTLforseedweightwereidentifiedthroughcomparativemappingamongArabidopsisandBrassicaspeciesbutnocandidategenescouldbeinferredforthetwomajorQTLdetectedonA07(Caietal.
2012).
Inconclusion,theresultsshowthatphytosterolcompo-sitionandcontentcanbeimprovedwithouthamperinggeneticprogressinimprovingseedoilcontent.
MajorQTLwerefoundexclusivelyontheAgenomeandtheidentifiedcandidategeneswouldneedtobeconfirmedinfuturestud-iesforimplementingmarker-assistedselection.
Notably,thecolocationofQTLforoilcontentandfattyacidswithLPAATandFAD2onA01couldeitherbeduetoindepend-entorcombinedeffectsofthegenes.
AcknowledgmentsThisstudywasfundedbytheDeutscheForschungsgemeinschaft(DFG)(GrantNumber:MO604/8-1).
SpecialthankstoKWSSAATAG,DeutscheSaatveredelungAG,andLantmnnenSWSeedforcarryingoutfieldtrialsandtoGundaAsselmeyer,CarmenMensch,RosiClemens,andUweAmmermanfortheirexcellenttechnicalsupport.
AuthorcontributionstatementCMdesignedtheexperimentanddevelopedthemicrospore-derivedDHmappingpopulation.
LTper-formedtheexperiment.
CMsupervisedtheoverallstudyandalongwithLTwrotethemanuscript.
Bothauthorsreadandapprovedthefinalmanuscript.
CompliancewithethicalstandardsConflictofinterestTheauthorsdeclarethattheyhavenocompetinginterests.
OpenAccessThisarticleisdistributedunderthetermsoftheCreativeCommonsAttribution4.
0InternationalLicense(http://crea-tivecommons.
org/licenses/by/4.
0/),whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedyougiveappropriatecredittotheoriginalauthor(s)andthesource,providealinktotheCreativeCommonslicense,andindicateifchangesweremade.
ReferencesAbidiSL,ListGR,RennickKA(1999)Effectofgeneticmodificationonthedistributionofminorconstituentsincanolaoil.
JAmOilChemSoc76:463–467AmarS,EckeW,BeckerHC,MllersC(2008a)QTLforphytosterolandsinapateestercontentinBrassicanapusL.
collocatewiththetwoerucicacidgenes.
TheorApplGenet116:1051–1061AmarS,BeckerHC,MllersC(2008b)Geneticvariationandgeno-type*environmentinteractionsofphytosterolcontentinthreedou-bledhaploidpopulationsofwinterrapeseed.
CropSci48:1000–1006AmarS,BeckerHC,MllersC(2009)Geneticvariationinphytos-terolcontentofwinterrapeseed(BrassicanapusL.
)anddevel-opmentofNIRScalibrationequations.
PlantBreed128:78–83AppelqvistL-D,KornfeldAK,WennerholmJE(1981)SterolsandsterylestersinsomeBrassicaandSinapisseeds.
Phytochemistry20:207–210ArnqvistL,PerssonM,JonssonLetal(2008)OverexpressionofCYP710A1andCYP710A4intransgenicArabidopsisplantsincreasesthelevelofstigmasterolattheexpenseofsitosterol.
Planta227:309–317Aznar-MorenoJ,DenolfP,VanAudenhoveKetal(2015)Type1diacylglycerolacyltransferasesofBrassicanapuspreferen-tiallyincorporateoleicacidintotriacylglycerol.
JExpBot.
doi:10.
1093/jxb/erv363BancroftI,MorganC,FraserFetal(2011)Dissectingthegenomeofthepolyploidcropoilseedrapebytranscriptomesequencing.
NatBiotechnol29:762–766BasunandaP,RadoevM,EckeWetal(2010)Comparativemap-pingofquantitativetraitlociinvolvedinheterosisforseedlingandyieldtraitsinoilseedrape(BrassicanapusL.
).
TheorApplGenet120:271–281BergerA,JonesPJH,AbumweisSS(2004)Plantsterols:factorsaffectingtheirefficacyandsafetyasfunctionalfoodingredients.
LipidsHealDis19:1–19BonaventureG,SalasJJ,PollardMR,OhlroggeJB(2003)DisruptionoftheFATBgeneinArabidopsisdemonstratesanessentialroleofsaturatedfattyacidsinplantgrowth.
PlantCell15:1020–1033BouicPJD(2001)Theroleofphytosterolsandphytosterolinsinimmunemodulation:areviewofthepast10years.
CurrOpinClinNutrMetabCare4:471–475Bouvier-NaveP,HusselsteinT,BenvenisteP(1998)Twofamiliesofsterolmethyltransferasesareinvolvedinthefirstandthesecondmethylationstepsofplantsterolbiosynthesis.
EurJBiochem256:88–96CaiG,YangQ,YangQetal(2012)IdentificationofcandidategenesofQTLsforseedweightinBrassicanapusthroughcomparativemappingamongArabidopsisandBrassicaspecies.
BMCGenet13:105ChalhoubB,DenoeudF,LiuSetal(2014)Earlyallopolyploidevolu-tioninthepost-NeolithicBrassicanapusoilseedgenome.
Sci-ence345:950–953ChyeML,TanCT,ChuaNH(1992)Threegenesencode3-hydroxy-3-methylglutarylcoenzymeAreductaseinHeveabrasiliensis:hmg1andhmg2aredifferentiallyexpressed.
PlantMolBiol19:473–484DelourmeR,FalentinC,HuteauVetal(2006)Geneticcontrolofoilcontentinoilseedrape(BrassicanapusL.
).
TheorApplGenet113:1331–1345DelourmeR,FalentinC,FomejuBFetal(2013)High-densitySNP-basedgeneticmapdevelopmentandlinkagedisequilibriumassessmentinBrassicanapusL.
BMCGenom14:120DemontyI,HaddemanE,vanderPutNetal(2007)Spreadsfortifiedwithabrassicasterol-richphytosterolmixturefromrapeseedoillowerserumtotalandLDL-cholesterolconcentrationsinmildlyhypercholesterolemicsubjects.
FASEBJ21(847):12DufourcEJ(2008)Sterolsandmembranedynamics.
JChemBiol1:63–77EckeW,UzunovaM,WeisslederK(1995)Mappingthegenomeofrapeseed(BrassicanapusL.
).
II.
Localizationofgenescontrol-lingerucicacidsynthesisandseedoilcontent.
TheorApplGenet91:972–977EnjutoM,BalcellsL,CamposNetal(1994)Arabidopsisthalianacontainstwodifferentiallyexpressed3-hydroxy-3-methylglu-taryl-CoAreductasegenes,whichencodemicrosomalformsoftheenzyme.
ProcNatlAcadSci91:927–931Fernández-Cuesta,Aguirre-GonzálezMR,Ruiz-MéndezMV,VelascoL(2012)Validationofamethodfortheanalysisofphytosterolsinsunflowerseeds.
EurJLipidSciTechnol114:325–331FerrieAMR,MllersC(2011)HaploidsanddoubledhaploidsinBrassicaspp.
forgeneticandgenomicresearch.
PlantCellTissOrganCult.
104:375–386198TheorApplGenet(2016)129:181–199FriedtW,SnowdonRJ(2010)Oilseedrape.
In:VollmannJ,IstvanR(eds)Handbookofplantbreeding,vol4:oilcropsbreeding.
SpringerVerlag,Dordrecht,pp91–126FritscheS,WangX,LiJetal(2012)Acandidategene-basedasso-ciationstudyoftocopherolcontentandcompositioninrapeseed(Brassicanapus).
FrontPlantSci3:129HarkerM,HolmbergN,ClaytonJCetal(2003)EnhancementofseedphytosterollevelsbyexpressionofanN-terminaltruncatedHeveabrasiliensis(rubbertree)3-hydroxy-3-methylglutaryl-CoAreductase.
PlantBiotechnolJ1:113–121HobbsDH,FlinthamJE,HillsMJ(2004)GeneticcontrolofstorageoilsynthesisinseedsofArabidopsis.
PlantPhysiol136:3341–3349.
doi:10.
1104/pp.
104.
049486JonesA,DaviesHM,VoelkerTA(1995)Palmitoyl-acylcarrierpro-tein(ACP)thioesteraseandtheevolutionaryoriginofplantacyl-ACPthioesterases.
PlantCell7:359–371JonesPJH,HowellT,MacDougallDEetal(1998)Short-termadmin-istrationoftalloilphytosterolsimprovesplasmalipidpro-filesinsubjectswithdifferentcholesterollevels.
Metabolism47:751–756KaoC-H,ZengZ-B,TeasdaleRD(1999)Multipleintervalmappingforquantitativetraitloci.
Genetics152:1203–1216KaurS,CoganNOI,YeGetal(2009)GeneticmapconstructionandQTLmappingofresistancetoblackleg(Leptosphaeriamaculans)diseaseinAustraliancanola(BrassicanapusL.
)cultivars.
TheorApplGenet120:71–83KosambiDD(1944)Theestimationofmapdistancesfromrecombi-nationvalues.
AnnEugen12:172–175LassnerMW,LeveringCK,DaviesHM,KnutzonDS(1995)Lysophosphatidicacidacyltransferasefrommeadowfoammedi-atesinsertionoferucicacidatthesn-2positionoftriacylglyc-erolintransgenicrapeseedoil.
PlantPhysiol109:1389–1394.
doi:10.
1104/pp.
109.
4.
1389LiR,YuK,HildebrandD(2010)DGAT1,DGAT2andPDATexpres-sioninseedsandothertissuesofepoxyandhydroxyfattyacidaccumulatingplants.
Lipids45:145–157.
doi:10.
1007/s11745-010-3385-4LiaoP,WangH,HemmerlinAetal(2014)Pastachievements,currentstatusandfutureperspectivesofstudieson3-hydroxy-3-methyl-glutaryl-CoAsynthase(HMGS)inthemevalonate(MVA)path-way.
PlantCellRep33:1005–1022LincolnS,DalyM,LanderE(1992)MappinggeneticmappingwithMAPMAKER/EXP3.
0.
CambWhiteheadInstTechRepLiuQ,SilotoRM,LehnerRetal(2012)Acyl-CoA:diacylglycerolacyltransferase:molecularbiology,biochemistryandbiotechnol-ogy.
ProgLipidRes51:350–377MaisonneuveS,BessouleJ-J,LessireRetal(2010)ExpressionofrapeseedmicrosomallysophosphatidicacidacyltransferaseisozymesenhancesseedoilcontentinArabidopsis.
PlantPhysiol152:670–684MarwedeV,GulMK,BeckerHC,EckeW(2005)MappingofQTLcontrollingtocopherolcontentinwinteroilseedrape.
PlantBreed124:20–26MiettinenTA(2001)Phytosterols—whatplantbreedersshouldfocuson.
JSciFoodAgri81:895–903MoghadasianMH,McManusBM,PritchardPH,FrohlichJJ(1997)"Talloil"–derivedphytosterolsreduceAtherosclerosisinApoE-deficientmice.
ArterThrombVascBiol17:119–126MllersC,SchierholtA(2002)Geneticvariationofpalmitateandoilcontentinawinteroilseedrapedoubledhaploidpopulationseg-regatingforoleatecontent.
CropSci42:379–384.
doi:10.
2135/cropsci2002.
0379MorikawaT,MizutaniM,AokiNetal(2006)CytochromeP450CYP710AencodesthesterolC-22desaturaseinArabidopsisandtomato.
PlantCell18:1008–1022NortonG,HarrisJF(1983)Triacylglycerolsinoilseedrapeduringseeddevelopment.
Phytochemistry22:2703–2707.
doi:10.
1016/S0031-9422(00)97676-3ParkinIAP,SharpeAG,KeithDJ,LydiateDJ(1995)IdentificationoftheAandCgenomesofamphidiploidBrassicanapus(oilseedrape).
Genome38:1122–1131PetersonDW(1951)Effectofsoybeansterolsinthediesonplasmaandlivercholesterolinchicks.
ProcSocExpBiolMed78:143–147PiironenV,LindsayDG,MiettinenTAetal(2000)Plantsterols:bio-synthesis,biologicalfunctionandtheirimportancetohumannutrition.
JSciFoodAgri80:939–966PiquemalJ,CinquinE,CoutonFetal(2005)Constructionofanoil-seedrape(BrassicanapusL.
)geneticmapwithSSRmarkers.
TheorApplGenet111:1514–1523PollakOJ(1953)Successfulpreventionofexperimentalhyper-cholesterolemiaandatherosclerosisintherabbit.
Circulation7:696–701QuijadaPA,UdallJA,LambertB,OsbornTC(2006)Quantitativetraitanalysisofseedyieldandothercomplextraitsinhybridspringrapeseed(BrassicanapusL.
):1.
Identificationofgenomicregionsfromwintergermplasm.
TheorApplGenet113:549–561QuilezJ,Garcia-LordaP,Salas-SalvadoJ(2003)Potentialusesandbenefitsofphytosterolsindiet:presentsituationandfuturedirections.
ClinNutr22:343–351RadoevM,BeckerHC,EckeW(2008)Geneticanalysisofheterosisforyieldandyieldcomponentsinrapeseed(BrassicanapusL.
)byquantitativetraitlocusmapping.
Genetics179:1547–1558RahmanH,HarwoodJ,WeselakeR(2013)Increasingseedoilcon-tentinBrassicaspeciesthroughbreedingandbiotechnology.
LipidTechnol25:182–185.
doi:10.
1002/lite.
201300291RamanH,RamanR,EckermannPetal(2013)Geneticandphysicalmappingoffloweringtimelociinoilseedrape(BrassicanapusL.
).
TheorApplGenet126:119–132SchaefferA,BronnerR,BenvenisteP,SchallerH(2001)TheratioofcampesteroltositosterolthatmodulatesgrowthinArabidopsisiscontrolledbySTEROLMETHYLTRANSFERASE2;1.
PlantJ25:605–615SchierholtA,BeckerHC,EckeW(2000)Mappingahigholeicacidmutationinwinteroilseedrape(BrassicanapusL.
).
TheorApplGenet101:897–901SchuelkeM(2000)AneconomicmethodforthefluorescentlabelingofPCRfragments.
NatBiotechnol18:233–234ShewmakerCK,SheehyJA,DaleyMetal(1999)Seed-specificover-expressionofphytoenesynthase:increaseincarotenoidsandothermetaboliceffects.
PlantJ20:401–412ShiJQ,LiRY,QiuDetal(2009)UnravelingthecomplextraitofcropyieldwithquantitativetraitlocimappinginBrassicanapus.
Genetics182:851–861SunC,CaoYZ,HuangAH(1988)Acylcoenzymeapreferenceoftheglycerolphosphatepathwayinthemicrosomesfromthematur-ingseedsofpalm,maize,andrapeseed.
PlantPhysiol88:56–60.
doi:10.
1104/pp.
88.
1.
56ThiesW(1971)DerEinfluderChloroplastenaufdieBildungvonungesttigtenFettsureninreifendenRapssamen.
Fette,Seifen,Anstrichm73:710–715UdallJA,QuijadaPA,LambertB,OsbornTC(2006)Quantitativetraitanalysisofseedyieldandothercomplextraitsinhybridspringrapeseed(BrassicanapusL.
):2.
Identificationofallelesfromunadaptedgermplasm.
TheorApplGenet113:597–609UtzHF(2011)PLABSTATAcomputerprogramforstatisticalanaly-sisofplantbreedingexperimentsVersion3AVanRensburgSJ,DanielsWMU,VanZylJM,TaljaardJJF(2000)AComparativestudyoftheeffectsofcholesterol,beta-sitosterol,beta-sitosterolglucoside,dehydroepiandrosteronesulphate199TheorApplGenet(2016)129:181–199andmelatoninoninvitrolipidperoxidation.
MetabBrainDis15:257–265VerleyenT,ForcadesM,VerheRetal(2002)Analysisoffreeandesterifiedsterolsinvegetableoils.
JAmOilChemSoc79:117–122VlahakisC,HazebroekJ(2000)Phytosterolaccumulationincanola,sunflower,andsoybeanoils:effectsofgenetics,plantingloca-tion,andtemperature.
JAmOilChemSoc77:49–53VosP,HogersR,BleekerMetal(1995)AFLP:anewtechniqueforDNAfingerprinting.
NuclAcidsRes23:4407–4414WangXX,WangHHH,WangJJJetal(2011)Thegenomeofthemes-opolyploidcropspeciesBrassicarapa.
NatGenet43:1035–1039WangH,NagegowdaDA,RawatR,Bouvier-NavePetal(2012a)OverexpressionofBrassicajunceawild-typeandmutantHMG-CoAsynthase1inArabidopsisup-regulatesgenesinsterolbio-synthesisandenhancessterolproduction.
PlantBiotechnolJ10:31–42WangS,BastenCJ,ZengZB(2012a)WindowsQTLcartographer2.
5WangX,ZhangC,LiLetal(2012c)UnravelingthegeneticbasisofseedtocopherolcontentandcompositioninrapeseedBrassicanapusL.
).
PLoSONE7:e50038WeiS,YuB,GruberMYetal(2010)Enhancedseedcarotenoidlev-elsandbranchingintransgenicBrassicanapusexpressingtheArabidopsismiR156bgene.
JAgriFoodChem58:9572–9578WoyengoTA,RamprasathVR,JonesPJH(2009)Anticancereffectsofphytosterols.
EurJClinNutr63:813–820YuB,LydiateDJ,YoungLWetal(2008)EnhancingthecarotenoidcontentofBrassicanapusseedsbydownregulatinglycopeneepsiloncyclase.
TransgenicRes17:573–585ZhangL,YangG,LiuPetal(2011)Geneticandcorrelationanalysisofsilique-traitsinBrassicanapusL.
byquantitativetraitlocusmapping.
TheorApplGenet122:21–31ZhangC,IskandarovU,KlotzETetal(2013)Athraustochytriddia-cylglycerolacyltransferase2withbroadsubstratespecificitystronglyincreasesoleicacidcontentinengineeredArabidopsisthalianaseeds.
JExpBot64:3189–3200.
doi:10.
1093/jxb/ert156ZhengP,AllenWB,RoeslerKetal(2008)AphenylalanineinDGATisakeydeterminantofoilcontentandcompositioninmaize.
NatGenet40:367–372