Hybrid Origins of Citrus Varieties Inferred from DNA Marker Analysis of Nuclear and Organelle

58 Pages·2016·6.39 MB·English

RESEARCHARTICLE
Hybrid Origins of Citrus Varieties Inferred
from DNA Marker Analysis of Nuclear and
Organelle Genomes
TokurouShimizu1*,AkiraKitajima2,KeisukeNonaka1,TerutakaYoshioka1,
SatoshiOhta1,ShingoGoto1,AtsushiToyoda3,AsaoFujiyama3,TakakoMochizuki4,
HidekiNagasaki4¤,EliKaminuma4,YasukazuNakamura4
1 DivisionofCitrusResearch,InstituteofFruitTreeandTeaScience,NARO,Shimizu,Shizuoka,Japan,
2 ExperimentalFarm,GraduateSchoolofAgriculture,KyotoUniversity,Kizugawa,Kyoto,Japan,3 National
InstituteofGenetics,ComparativeGenomicslaboratory,NationalInstituteofGenetics,Mishima,Shizuoka,
a11111 Japan,4 NationalInstituteofGenetics,CenterforInformationBiology,NationalInstituteofGenetics,
Mishima,Shizuoka,Japan
¤ Currentaddress:DepartmentofFrontierResearch,KazusaDNAResearchInstitute,Kisarazu,Chiba,
Japan
*[email protected]
Abstract
OPENACCESS
Citation:ShimizuT,KitajimaA,NonakaK,
Mostindigenouscitrusvarietiesareassumedtobenaturalhybrids,buttheirparentagehas
YoshiokaT,OhtaS,GotoS,etal.(2016)Hybrid
OriginsofCitrusVarietiesInferredfromDNA sofarbeendeterminedinonlyafewcasesbecauseoftheirwidegeneticdiversityandthe
MarkerAnalysisofNuclearandOrganelle lowtransferabilityofDNAmarkers.Hereweinfertheparentageofindigenouscitrusvarie-
Genomes.PLoSONE11(11):e0166969.
tiesusingsimplesequencerepeatandindelmarkersdevelopedfromvariouscitrusgenome
doi:10.1371/journal.pone.0166969
sequenceresources.Parentagetestswith122knownhybridsusingtheselectedDNAmark-
Editor:DavidDFang,USDA-ARSSouthern
erscertifytheirtransferabilityamongthosehybrids.Identitytestsconfirmthatmostvariant
RegionalResearchCenter,UNITEDSTATES
strainsareselectedmutants,butwefindfourtypesofkunenbo(Citrusnobilis)andthree
Received:August13,2016
typesoftachibana(Citrustachibana)forwhichwesuggestdifferentorigins.Structureanaly-
Accepted:November7,2016 siswithDNAmarkersthatareinHardy–Weinbergequilibriumdeducethreebasictaxacoin-
Published:November30,2016 cidingwiththecurrentunderstandingofcitrusancestors.Genotypinganalysisof101
indigenouscitrusvarietieswith123selectedDNAmarkersinferstheparentagesof22indig-
Copyright:©2016Shimizuetal.Thisisanopen
accessarticledistributedunderthetermsofthe enouscitrusvarietiesincludingSatsuma,Temple,andiyo,andsingleparentsof45indige-
CreativeCommonsAttributionLicense,which nouscitrusvarieties,includingkunenbo,C.ichangensis,andIchanglemonbyallele-sharing
permitsunrestricteduse,distribution,and
andparentagetests.Genotypinganalysisofchloroplastandmitochondrialgenomesusing
reproductioninanymedium,providedtheoriginal
11DNAmarkersclassifiestheircytoplasmicgenotypesinto18categoriesanddeducesthe
authorandsourcearecredited.
combinationofseedandpollenparents.Likelihoodratioanalysisverifiestheinferredparent-
DataAvailabilityStatement:Allrelevantdataare
ageswithsignificantscores.Thereconstructedgenealogyidentifies12typesofvarieties
withinthepaperanditsSupportingInformation
files.Allnucleotidesequencedatathatwere consistingofKishu,kunenbo,yuzu,koji,sourorange,dancy,kobenimikan,sweetorange,
obtainedbytheauthorshadbeendepositedtothe tachibana,Cleopatra,willowleafmandarin,andpummelo,whichhaveplayedpivotalrolesin
publicdatabasewithaccessionnumbersgivenin
theoccurrenceoftheseindigenousvarieties.Theinferredparentageoftheindigenousvari-
thispaper.Accessionnumberscanbefoundinthe
etiesconfirmstheirhybridorigins,asfoundbyrecentstudies.
filetitledS1Table.
Funding:Thisworkwassupportedbyagrantfor
ResearchProject"GenomicsforAgricultural
InnovationNGB1006"fromJapaneseMinistryof
Agriculture,ForestryandFisheriesforTS.Apartof
PLOSONE|DOI:10.1371/journal.pone.0166969 November30,2016 1/58
HybridOriginsofCitrusVarieties
thisworkwasalsosupportedbyKAKEN(No. Introduction
24405025)forAK.
ThegenusCitrusL.(FamilyRutaceae,subfamilyAurantiodeae)coversawiderangeofedible
CompetingInterests:Theauthorshavedeclared
andcommercialvarieties,includingsweetorange,lemon,lime,grapefruit,andmandarins
thatnocompetinginterestsexist.
suchasClementine,Satsuma,King,andponkan[1–4].Theproductionofmajorcitrusvarie-
tiesintropicaltosub-tropicalandtemperatezonesexceeds90milliontons,andthecitrus
industryoccupiesasignificantpositionnotonlyinthefruitindustrybutalsoinglobalagricul-
ture[5,6].Inadditiontotheworldwideproductionofthesemajorcitrusvarieties,numerous
indigenouscitrusvarietieshavealsobeenproducedinspecificregions,andconsumedlocally
[2,7].WidegeneticdiversityobservedinCitrus,however,hasmadeitdifficultfortaxonomists
todrawaclearpictureoftheirclassification.Furthermore,mutantshaveoccasionallybeen
selectedfromlimbsportsornucellarseedlings,andtheseconstitutelargevariantstrains[2,8–
10].Understandinghowthesemoderncitrusvarietiesarosefromtheancestralbasicspecies
wouldbringusimportantinsightsforfuturecitrusbreeding.
Manybotanistsandtaxonomistshaveproposedvariousapproachesfortheclassificationof
awiderangeofcitrusvarieties.Amongthem,twosystemsproposedbySwingle[11]and
Tanaka[7,12]havebeenusedinmanystudies.Thesetwosystemspresumethatmostindige-
nousandcommercialvarietiesarosefromhybridizationofancestralones,butdifferintheway
theytreatindigenousvarietiesandcultivatedvarieties.Swingleprimarilyclassifiedindigenous
varietiesratherthanthecultivatedvarieties,placingtwosubgeneraPapedaandCitrusinthe
genusCitrus[11].ThesubgenusPapedaconsistsofsectionPapedawithfourspecies,andsec-
tionPapedocitruswithtwospecies.HeclassifiedtenspeciesinthesubgenusCitrus,and
regardedmostcultivatedvarietiesasnaturalhybridsoftheseindigenousspecies.Heassigned
mostmandarinvarietiestothescientificnameCitrusreticulata,classifiedtachibanaseparately
asC.tachibana,andalsoclassifiedgrapefruit,whicharosefromachanceseedling[2,9],sepa-
ratelyasC.paradisi.Incontrast,Tanakastressedtheimportanceofbothindigenousvarieties
andcultivatedvarieties,andclassifiedthemequallyasaspecies.Heprimarilyplacedtwosub-
genera(ArchicitrusandMetacitrus)ingenusCitrus.ThesubgenusArchicitrusconsistsoffive
sections(Papeda,Limonellus,Citrophorum,CephalocitrusandAurantium)with111species,
includinggrapefruitasC.paradisi.ThesubgenusMetacitrusconsistsofthreesections(Osmo-
citrus,AcrumenandPseudofortunella)with48species[12].AccordingtoTanaka’ssystem,
individualmandarinvarietiesandtachibanawereclassifiedasaspecieswithindividualscien-
tificnames,andC.reticulatawasassignedtotheponkanmandarin.Tanakaclassified145cit-
russpeciesin22differentcategories[12].Sincethen,hehasaddedseveralindigenousvarieties
tohisclassificationsystem,andhereleasedtheultimatelistconsistingof159speciesin1969
[13].SwingleconsideredC.ichangensisasaspeciesofsubgenusPapeda,anddidnotassigna
scientificnametoyuzubecauseheregardeditasanaturalhybridofC.ichangensis.Incontrast,
TanakaclassifiedC.ichangensisinsubgenusMetacitrussectionOsmocitrus,andclassified
yuzutosubgenusMetacitrussectionEuosmocitrusasC.junos[12].
Bythe1970s,variousstudieshadbeenlaunchedtoclassifycitrusvarietiesusingbiochemi-
calmarkers.In1975,Scorapublishedanovelpaperbasedonhisownchemotaxonomical
studyofcitrustogetherwithasurveyofpastliterature[14].Hepostulatedthreehypothetical
taxa,mandarin(C.reticulata),citron(C.medica)andpummelo(C.maxima,formerlyC.
grandis),astheancestors,andproposedthatmoderncitrusvarietiesarosefromrepeated
hybridizationoftheseancestors.In1976,BarrettandRhodesexaminedcorrelationsamong22
indigenousvarietiesbasedonsimilaritiesfor146traits,thenestimatedtheiraffinitiesaccord-
ingtotheirdeduceddistance[15].Similarchemotaxonomicalstudiesgraduallyrevealedthe
phylogeniesofcitrusvarieties[16–21].WhenDNAmarkertechnologybecameavailable,taxo-
nomicalstudiesattemptedclassificationofcitrususingvariousDNAmarkerssuchasRAPD
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HybridOriginsofCitrusVarieties
[22–26],RFLP[27],AFLP[28,29],ISSR[29–31]andSRAP[8,32].Nicolosiandcolleagues
deducedacitrusphylogenyaccordingtothegenotypesofnuclearandchloroplastmarkers,
anddemonstratedthattheoriginsofcitrusvarietiesproposedbyScora[14]andBarrettand
Rhodes[15]wereacceptable[33,34].Sincethen,theoriginsofsomecitrusvarietieshavegrad-
uallybeenrevealed,andnewclassificationshavebeenproposed[35,36].Nowadays,codomi-
nantprecisionsimplesequencerepeat(SSR)orsinglenucleotidepolymorphism(SNP)
markershavebeendevelopedandusedinmoststudies(seethereviews[34,37–40]).Inaddi-
tion,thechloroplastgenomesequenceofsweetorangehasbeenreleased[41],andgenome
sequencesofmajorcitrusvarietiesarenowpublic[42,43].Thesegenomesequenceresources
enablethedesignofprecisionDNAmarkers,andhaverevealedtheparentageofClementine,
grapefruit,sweetorange,andlimesandlemons[43–48].However,theparentageofmost
indigenousvarietieshasnotyetbeendetermined.
Identifyingthecombinationofseedparentandpollenparentisanotherimportantissueto
besolvedinparentageanalysis.Manystudieshaverevealedthephylogenyofcitrusvarietiesby
evaluatingpolymorphismsinthechloroplastormitochondrialgenome,orboth[33,47,49–57].
However,someofthesestudieshaveonlyevaluatedlocalcitrusvarieties[51,52],orlimited
numbersofvarietiesinthegenusCitrus[50,57,58].Nextgenerationsequencing(NGS)tech-
nologyhasbecomecommonplace,andithasbeenappliedtothegenotypingofcitruschloro-
plastgenomes[56],butitisstillacostlyandtime-consumingapproach.Simplebut
reproducibleandlow-costtechnologiesthatrevealsufficientpolymorphismsareneededfor
theparentageanalysisofawiderangeofcitrusvarieties.
DNAmarkeranalysishasbeenusedinforensicgeneticsforinferringparentageorpater-
nity,andidentifyingmissingpersonsfromtheirremains[59,60].Thesetechniqueshavealso
beenusedtoinfersibshipsofwildpopulations[61–64],andareanticipatedtobeabletoreveal
unknowngenealogyamongindigenouscitrusvarieties.Twobasicapproacheshavebeen
adoptedforparentageestimationwithDNAmarkeranalysis[64].Thefirstusesallele-sharing
teststhatestimatethenumberofallelessharedbetweentwoindividualsatcodominantDNA
markersaccordingtotheMendelianrulesofinheritance.Thesetestsestimatetheprobability
ofparentagefromtheproportionofDNAmarkerswithsharedalleles,andcanalsoeliminate
unrelatedindividuals.Thediscriminatorypowerofthetestisproportionaltothenumberof
locievaluatedandthepolymorphismofeachDNAmarker.However,thesetestsaresuscepti-
bletogenotypingerrors,andmaygivefalsepositiveornegativeresults[64].Anotherapproach
isalikelihoodratioanalysis,whichcomparestheprobabilitiesofalternatehypothesesforthe
parentageoftwoindividuals(e.g.,whethertheyareparentandoffspringorunrelated)then
estimatesanoddsscorebetweenthesetwohypotheses[62–64].Thisisawidelyusedtechnique
forexaminingproposedpaternityorparentageandalsotoidentifyindividuals[59,60,65].The
likelihoodratioanalysisestimatestheprobabilityoftheproposedparentageaccordingtothe
likelihoodofallegedparentsandchild,thencomparesitwithanullrelationbetweenthem
deducedfromtheallelefrequencywithinthepopulation.Thelogarithmoflikelihoodratio
odds(LODscore)isoftenusedtoindicatetheestimatedscore,butthenumberofDNAmark-
ersusedfortheevaluationandtheirallelefrequencyinthepopulationinfluencethescore[64].
Genotypingerrorscanalsoinfluencethescore,anditisthusdifficulttodemonstrateaclear
thresholdfordiscrimination[63].Thesetwomethodseachhaveprosandcons;therefore,an
approachthatfirstexcludesunrelatedindividualsusinganallele-sharingtest,thenexamines
theprobabilityoftheproposedparentageusinglikelihoodratioanalysis,willbeasimplebut
effectivewaytoinferparentageinagivenpopulation.
Becausegenotypingerrorseverelyaffectsthereliabilityofbothmethods,detectingsuch
errorandevaluatingparentagewitherror-freeDNAmarkersisaprerequisiteforreliability.In
thegenotypinganalysisofcitrusvarieties,however,widegeneticdiversityamongnatural
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HybridOriginsofCitrusVarieties
varietiesreducesthetransferabilityofDNAmarkers,resultinginfalsegenotypes[44,46,64,66].
Selectedsomaticmutantscouldalsobeadrawbackbecausesomeofthem,butnotall,have
mutationsintheirgenotypethatmakeitdifficulttoestimatetheiridentity.
Theobjectiveofthepresentstudyistoinferparentageamongvariouscitrusvarietiesusing
DNAmarkeranalysis,andverifytheinferredparentagestatistically.Wehaveattempted1)to
developsufficientDNAmarkersforparentageanalysisandeliminateerroneousDNAmarkers
byexaminingthemwithalargeenoughsetofknownhybridvarieties,2)toestimategenetic
structuresofindigenousvarietiesusingthesecertifiedDNAmarkers,3)todeterminethecyto-
solicgenotypesofindividualvarietiesbyevaluatingchloroplastandmitochondrialgenomes
withDNAmarkeranalysis,4)toinferparentageamongindigenouscitrusvarietiesandverify
itusingalikelihoodratioapproach.
MaterialsandMethods
Plantmaterials
Weselected371citrusaccessionsconsistingof208indigenousvarieties,78hybridvarieties,
and85selectedstrains(Table1andS1Table).Theindigenousvarietiesarefromthecollec-
tionsoftheInstituteofFruitTreeandTeaScience,NARO(NIFTS)thathavebeenmaintained
attheOkitsuCitrusResearchDivisioninShizuokaprefecture,Japan.Thesevarietieswere
selectedfrommajormandarins(C.reticulata,C.tangerina,C.unshiu,C.clementina,C.
Table1. Summaryofcitrussamplesusedinthisstudy.
Category Scientificnames Samples Genotypedsamples Representativesamples
Swingle’ssystem Tanaka’ssystem
Indigenousvarieties 208 269 101
Clementine C.reticulataBlanco C.clementinahort.exTanaka 4 4 1
Dancy C.reticulataBlanco C.tangerinahort.exTanaka 2 2 1
Grapefruit C.paradisiMacf. C.paradisiMacf. 3 3 1
Hyuganatsu C.sinensis(L.)Osbeck C.tamuranahort.exTanaka 5 6 1
Iyo C.sinensis(L.)Osbeck C.iyohort.exTanaka 2 2 1
Kishu C.reticulataBlanco C.kinokunihort.exTanaka 16 21 1
Kunenbo1) C.reticulataBlanco C.nobilisLour.var.kunepTanaka 10 13 4
Natsudaidai C.paradisiMacf. C.natsudaidaiHayata 4 5 1
Ponkan C.reticulataBlanco C.reticulataBlanco 4 5 1
Pummelo C.grandisOsbeck2) C.grandisOsbeck2) 12 14 12
Satsuma C.reticulataBlanco C.unshiuMarcov. 21 33 1
Shiikuwasha C.indica C.depressaHayata 2 3 2
Sourorange C.aurantiumL. C.aurantiumL. 5 6 1
Sweetorange C.sinensis(L.)Osbeck C.sinensis(L.)Osbeck 20 22 1
Tachibana C.tachibanaMakino C.tachibana(Makino)Tanaka 12 13 3
Tankan C.sinensis(L.)Osbeck C.tankanHayata 4 4 1
Willowleafmandarin C.reticulataBlanco C.deliciosaTen. 2 2 2
Others 80 111 66
Hybridvarieties C.spp C.spp 78 83 75
Selectedstrains C.spp C.spp 85 90 85
Total 371 442 261
1)Kunenbo(C.nobilisLour.var.kunepTanaka)includesKingmandarin(C.nobilisLour.)
2)NowclassifiedasC.maximaMerr.
doi:10.1371/journal.pone.0166969.t001
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HybridOriginsofCitrusVarieties
kinokuni,C.tachibana,C.nobilis),pummelos(C.maximaanditshybrids),lemon(C.limon),
sweetorange(C.sinensis),yuzu(C.junos),ichanchii(C.ichangensis)andtheirassumednatural
hybrids.Sixteenvarietiesincludedvariantselectionstoevaluatetheirgeneticidentity:four
Clementines,twovarietiesclassifiedtoC.tangerinehort.exTanaka(DancyandObeni
mikan),threegrapefruits,fivehyuganatsu,twoiyos,16Kishus,10kunenbos,fourponkans,12
pummelos,21Satsumas,twoshiikuwashas,fivesouroranges,20sweetoranges,12tachibanas,
fourtankans,andtwowillowleafmandarins,respectively.Amongthem,kunenboincluded
bothC.nobilisLour.(King)andC.nobilisLour.var.kunepTanaka.Hybridvarietiesusedin
thisstudyarefromthecollectionsofNIFTS.Forty-fiveofthemweredevelopedbyNIFTS,11
byUCRiverside,10bytheUSDA,andtheother12varietiesweredevelopedbysevenother
institutesorbyfarmers.Wealsoused85strainsthatwereselectionsfromvariouscrossesin
NIFTS.
DNAextraction
FullymaturedleaveswerecollectedfromeachsampleinthefieldatOkitsu,Shizuoka,then
providedforDNAextractionusingamodifiedprotocolwithaNucleonPhytopurekit(GE
HealthcareLifeScience,NJ,USA)[67].Forcertainvarieties,severalsampleswerecollected
fromdifferenttrees.Thesewereusedasbiologicalreplicatestoconfirmthereproducibilityof
genotyping(RAinS1Table).DNAconcentrationofthepreparedDNAsampleswasdeter-
minedusingaQubitAssaykit(ThermoFisherScientific,Tokyo,Japan).UVabsorbanceanaly-
siswasusedtoconfirmsamplequality(A /A >1.8,andA /A >2.0),andgel
260 280 260 230
electrophoresisanalysistoverifythesizeandintegrityoftheextractedDNAsamples.
CitrussequenceresourcesforDNAmarkerdesign
NucleotidesequencesofexpressedgenesofcitruswereobtainedfrompubliccDNAsequence
databasesdbEST(http://www.ncbi.nlm.nih.gov/dbEST/),RefSeq(http://www.ncbi.nlm.nih.
gov/refseq/)andHarvEST(http://harvest.ucr.edu/)[68].Citrusgenomesequenceresourcesin
publicdatabases,includingBACendsequencesofClementine[69]andSatsuma[70,71],and
wholegenomeshotgunsequencesofsweetorange‘RidgePineapple’inthetracefilerepository
ofSangerreads(ftp://ftp.ncbi.nlm.nih.gov/pub/TraceDB/citrus_sinensis/),werealsousedfor
DNAmarkerdesign.Preliminaryevaluationofthequalityandlengthofeachofthesedatasets
wascarriedoutusingpregap4[72],thenaconsensussequencesetwasobtainedforeachset
withMiraassembler[73]toreduceredundancy.
NGSanalysisofcitrusvarieties
NGSanalysisofcitrusvarietiesforminingSSRandindelregionswasperformedwithaHiSeq
2000sequencingsystem(Illumina,CA,USA)inpaired-endmode[67].Quality-checkedNGS
readsweremappedtothehaploidClementinereferencesequencev.0.9orv.1.0[43]using
BWA[74].CandidateSSRorindelregionsinthere-sequenceddatawerescoredandidentified
usingSAMtoolsandBCFtools[75],orusingmreps[76].
DNAmarkerdesignforgenotypingnucleargenomes
SSRregionsofeachsequencewereminedusingmreps[76],thencandidateregionswithmotif
lengthbetweentwoandsixnucleotideswereselected.Theidentifiedcandidateregionsfound
inexpressedgenesorgenomicsequenceswereusedforoligonucleotideprimerdesignwith
PerlPrimer[77]orPrimer3[78].PreviouslyreportedSSRmarkersdesignedfromBACend
sequences[46],orfromESTsequences[79,80]werealsousedinthisstudy.
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HybridOriginsofCitrusVarieties
DNAmarkerdesignforgenotypingorganellegenomes
SSRmarkersfordetectingpolymorphismsinthechloroplastgenomeweredesignedfromthe
chloroplastgenomesequenceofsweetorange‘RidgePineapple’(accessionNo.DQ864733)
[41]bysearchingcandidateSSRregionsusingmreps[76]asdescribedintheprevioussection.
Oligonucleotideprimersetsforcitrusmitochondrialgenomes[53],anduniversalprimersets
forthechloroplastgenomesofdicotyledonousangiosperms[81]werealsousedforgenotyping
organellegenomes.
Genotypinganalysis
Allgenotypinganalysisofnuclearororganellegenomesfollowedthemultiplexedandmulti-
coloredpost-labelingmethodinsingletubewithBStagreportedbyShimizuandYano[82].
Post-labelingofthePCRproductwithBStagisasimplebutinexpensivemethodthatdoesnot
requirelargealterationofthePCRprogram,anditreducesthetotalcostofanalysissignifi-
cantly.OneofthesixstandardBStagsequencesoranadditionalBStagsequence(F9TCC:5’-
CTAGTATCAGGACTCC-3’)wasaddedatthe5’endofthedesignedforwardprimer.Ashort
‘pigtail’sequencewasaddedatthe5’endofthereverseprimerinordertosuppressstuttering
ofthedetectedpeak[83].Foreachgenotypinganalysis,fouroligonucleotideprimersetsthat
wereindividuallyattachedtodifferentBStagsequencesweremixedwiththecorresponding
fluorescentlylabeledBStagprimers.AtypicalPCRprogramfortheamplificationandpost-
labelingofthetargetregionofthenucleargenomewas:initialdenaturationat94˚Cfor3min;
32cyclesoftargetamplification(20sat94˚Cfollowedby35sat52–65˚C);thenthreepost-
labelingcycles(20sat94˚Cfollowedby10sat49˚Cand5sat72˚C);andfinalextensionat
72˚Cfor10minthenterminatedat4˚C.EachDNAmarkerwaslabeledseparatelywithoneof
fourdifferentfluorescentdyesinasingletubeatthelabelingstep.Thereactionmixturewas
dilutedtwofoldwithwaterafterthePCR.Then,a0.4-μLaliquotofthedilutedmixturewas
mixedwith0.1μLGeneScan600LIZ1dyeSizeStandard(ThermoFisherScientific,Tokyo,
Japan)andadjustedtobe10μLwithdeionizedformamide,andthenheatdenaturedat95˚C
for4min.ElectrophoresisofthelabeledproductwascarriedoutonanABI3130xlDNA
sequencer(ThermoFisherScientific,Tokyo,Japan)with36cmlengthcapillaryusingthestan-
dardprogram.GenotypesofeachDNAmarker/samplewerecalledusingGeneMapper4.0
software(ThermoFisherScientific,Tokyo,Japan).
Parentagetestandidentitytest
Parentagewasconfirmedforassumedparent–offspringtriadsbyconsideringtheinheritance
ofeachallelefromparentstooffspringaccordingtotheMendelianrule.AnyDNAmarkers
showingdiscrepanciesinknownhybridswereexcludedfromtheanalysis.Theevaluationwas
carriedoutusingafunctionofGUGS(GeneralUtilitiesforGenotypingStudy)software(Shi-
mizu,T.inpreparation).Theidentitytestisasimpleexactmatchtestofeachgenotypetooth-
ersforallcombinations.Ifapairofsamplescoincidedwitheachotherforthegenotypesofall
oftheDNAmarkers,theyweretreatedasidentical.Inthisstudy,wecountedthenumberof
DNAmarkersthatdidnotagreebetweenanygivenpairofsamples.
Statisticalevaluationofthegenotypedata
Observedheterozygosity(H ),expectedheterozygosity(H ,equivalenttotheunbiasedestima-
o e
torofgenediversitygivenbyequation8.4ofNei[84]),numberofuniquealleles,andpolymor-
phicinformationcontent(PIC,representingtheprobabilityofdistinguishingamarkerallele
derivedfromeitheroneoftheparents[85])werecalculatedusingthefrequencyanalysis
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HybridOriginsofCitrusVarieties
functionofCervus[62]andconfirmedwithGUGS.Theprobabilityofmatch(PM),represent-
ingtheprobabilitythatanunrelatedindividualhappenstohavethesamegenotypetoothers
[60]isgivenby:
P
PM ¼ m p2: ð1Þ
k¼1 k
Here,p istheobservedfrequencyofeachuniquegenotypekinthepopulation,andmisthe
k
numberofuniquegenotypesatagivennuclearlocus.Thegenediversity(GD)ofasingleallelic
organellegenotypeatagivenlocuswasevaluatedby
P
GD¼1(cid:0) m x2 ð2Þ
i¼1 i
(equation8.1ofNei[84]).Here,x istheobservedfrequencyoftheithsinglealleleinthepopu-
i
lation,andmisthenumberofallelesatanorganellelocus.Thisparameter(Nei’sGD)isan
equivalentoftheexpectedheterozygosityfordiploidorganisms.Thevaluesoftheunique
genotypes,PMandGD,wereobtainedusingafunctionofGUGS.Wright’sfixationindex(F )
w
wasobtainedbytheequationF =(H −H )/H (equation12.9ofNeiandKumar[86]).
w e o e
Allstatisticalevaluationsofthenormaldistribution(Shapiro–Wilktest)andone-way
ANOVA(Kruskal–Wallistest)wereconductedwiththestatspackageofR(version3.1.3,
https://www.r-project.org/)intheRstudioenvironment(version0.99.893,https://www.
rstudio.com/).Testsforequalvarianceandstochasticequalityoftwosampleswereconducted
accordingtoBrown–ForsythetestandBrunner–Munzeltestusingfunctionslevene.testand
brunner.munzel.testinthelawstatpackage[87].Thep-valueadjustmentformultiplesamples
wascarriedoutbyBenjamini–Hochberg(BH)correctionwiththep.adjustfunctionofR.F-
statisticsforpopulationanalysis(F ,F )[86,88,89]wereestimatedforeachsamplecategory
IT IS
orindividualDNAmarkerusingRpackageshierfstat[90]andpegas[91]incombinationwith
adegenet[92].Additionally,Hedrick’sG'' [88],whichisanequivalentofF extendedto
ST ST
multiallelicDNAmarkers,wasestimatedgloballyorpairwiseusingthemmodpackageofR
[93]incombinationwithadegenet[92].
EvaluationofHardy–Weinbergequilibrium
AnexacttestofHardy–Weinbergproportionsformultiallelicgenotypedatawasestimated
withaMarkovChainMonteCarlo(MCMC)simulationmethoddevelopedbyGuoand
Thompson[94],thatwasimplementedasafunctionofArlequin(version3.5.2.2)[95].The
genotypedatafileusedasinputforArlequinwasformattedwithCONVERTsoftware[96]
withnopriorinferredpopulationstructure.WecontinuedtheMCMCsimulationruns10
timeseachfor1,000,000iterationsinbothinitialburn-inandde-memorizationsteps,and
thentheaverageoftheestimatedp-valueswasprovidedforevaluation.
Factorialanalysisandphylogeneticevaluation
Principalcoordinateanalysis(PCoA)andphylogeneticanalysisoftheobtainedgenotypedata
werecarriedoutwithDARWin(version6.0.13)[97,98].Adissimilaritymatrixwasobtained
fromthegenotypesofeachsamplepairusingasimplematchingmethod(nucleargenotypes)
orfrommodalitiesbyRogersandTanimoto’scoefficient(organellegenotypes).ThePCoA
analysisassumedtwotosixaxes(typicallyfive),anddataforthefirsttwoaxeswereusedto
drawascatterplot.Aconsensusphylogenetictreewasinferredfromthebootstrappeddissimi-
laritymatricesobtainedfrom30,000iterationsforthenucleargenotypedataor5,000iterations
fortheorganellegenotypedatausingtheweightedneighbor-joiningmethod[99],then
obtainedconsensustrees.
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HybridOriginsofCitrusVarieties
Structureanalysis
Structureanalysisfortheinferenceofthebasictaxaandtheirproportionswascarriedout
usingSTRUCTURE[100].Thegenotypedataforthe101representativeindigenousvarieties
obtainedwiththe123selectedDNAmarkerswereformattedusingCONVERTsoftware[96]
withnopriorinferredpopulationstructure.Missingdataweretreatedaslost(assigned‘-9’for
thegenotypedata).Theanalysisassumedtheadmixturemodelforancestryandthatallelefre-
quencieswerecorrelated.Intheestimationofthenumberofbasictaxa(K),wevariedKstep-
wisefromtwototen,thenevaluatedtheprobabilitytentimesforeachKwith100,000
iterationsoftheinitialburn-inand1,000,000MCMCruns.TheinferredproportionsoftheK
populations,andtheestimatedlnPr(X|K),meanlnP(K)anditsvariancewereusedtoobtain
stdevLnP(K),L'(K)and|L''(K)|,thenΔKwasestimatedasthemeanof(|L''(K)|/stdevLnP
(K)),followingEvannoetal[101].WeusedtheStructureHarvesterwebservice[102]athttp://
taylor0.biology.ucla.edu/structureHarvester/forthispurpose.Theinferredproportionsofthe
KbasictaxawerededucedindividuallyfromtheoutputofStructureHarvesterusingthe
GreedyalgorithmofCLUMPP[103].Wecomparedthefullsearchandrandominputorder
runningmodesofCLUMPP,andalsochangedtherunningperiodforthepermutationanaly-
sisfrom1,000to1,000,000,butallresultswereidentical.Wethereforeusedthesimulation
resultsfromCLUMPPruninGreedymodewith100,000permutationruns.Thebarplotof
inferredproportionswasdrawnwithMSExcel.
Allele-sharingtestandstochasticverificationofinferredparentage
Possibleparent-to-offspringrelationshipsbetweenvarietieswereexaminedusinganallele-
sharingtest.ThetestevaluatestheratioofthenumberofDNAmarkersthatshareatleastone
allelebetweentwovarietiestothetotalnumberofDNAmarkers.Anypairofvarietiesin
whichnearlyallDNAmarkerssharedanallelebetweenthetwovarietieswasselectedasa
candidateparent–offspringpair.Whentwovarietieswereassumedtobetheparentsofapar-
ticularoffspringvariety,theparentageoftheassumedtriadwasexaminedusingtheparentage
test.
Theprobabilityoftheinferreddyadortriadbeingtruesingleparent-to-offspringor
parents-to-offspringcombinationswasexaminedbylikelihoodratioanalysisaccordingto
MarshaletalandJonesandArdren[62,63].Inthisanalysis,theprobabilitiesoftwohypotheses
(H andH )arecompared.AssumeP(G|H )istheprobabilityofobservingaparticularpairof
1 2 1
genotypesGunderthehypothesisH ,andP(G|H )istheprobabilityofGunderthehypothesis
1 2
H .TheevaluatedP(G|H )relativetotheevaluatedP(G|H )willgivealikelihoodratioL(H ,
2 1 2 1
H |G)thattheGwillbeobservedunderthetwohypothesesH andH :
2 1 2
PðGjH Þ
LðH ;H jGÞ¼ 1 : ð3Þ
1 2 PðGjH Þ
2
Intheparentagetest,H presumesthataparticularvarietyisanoffspringoftheallegedpar-
1
entorparents,andH presumesthatitisnotanoffspringoftheallegedparentsbutachance
2
seedlingthathasarisenfromagivenpopulation.ThelikelihoodratioLrepresentstheproba-
bilitythattheoffspringwasobtainedfromtheallegedparent(s)ratherthanbeingachance
seedling.
Forthestochasticevaluationoftheparentagetest,letg ,g andg representthegenotypes
S P O
oftheallegedseedparent,allegedpollenparentandoffspring,respectively,ataDNAmarker.
Thelikelihoodratiothattheallegedparentsarethetrueparentsofthegivenoffspringvariety
PLOSONE|DOI:10.1371/journal.pone.0166969 November30,2016 8/58
HybridOriginsofCitrusVarieties
wasestimatedaccordingtoEq(3)fromJonesandArdren[63]:
Tðg jg ;g Þ
LðH ;H jg ;g ;g Þ¼ O S P : ð4Þ
1 2 S P O Pðg Þ
B
Here,thenumeratorT(g |g ,g )isthetransitionprobabilityofg giveng andg .Thisproba-
O S P O S P
bilitywasestimatedfromtheallelefrequenciesandagenotypecombinationaccordingto
Table1ofMarshalletal[62].ThedenominatorP(g )isthefrequencyoftheoffspring’sgeno-
B
typeinaparticularpopulationobtainedaccordingtoTable2ofMarshalletal[62].Thevalue
Listhelikelihoodratiothattheparentageofthistriadiscorrectcomparedtotheoffspring
obtaineditsgenotypefromanunknownhybridcombination.
Inasimilarmanner,anotherlikelihoodratiofortheallegedsingleparenttoanoffspring
wasestimatedaccordingtoEq(2)ofJonesandArdren[63],orEq(5)ofMarshalletal[62]:
Tðg jg Þ
LðH ;H jg ;g Þ¼ O S : ð5Þ
1 2 S O Pðg Þ
B
Here,thenumeratorT(g |g )isthetransitionprobabilityofg giveng ,estimatedfromtheir
O S O S
allelefrequenciesandgenotypecombinationaccordingtoBrenner[104]orTable2ofMar-
shalletal[62].Inmostparentageanalysesofwildplantpopulations,itisunknownwhichvari-
etyistheseedparentorthepollenparent.Thus,aparticularallegedparentsamplewithout
anypriorsupportinginformationwasassignedtoeitherg org arbitrarily.Theprobabilityof
S P
obtainingaparticulargenotypeinapopulationwasestimatedfromtheallelefrequenciesata
givenDNAmarker,asx2forhomozygousgenotype,or2xyforaheterozygousgenotype,
wherexandyaretheallelefrequenciesinapopulation.TheobtainedvalueListheratioofthe
likelihoodthatthisisaparent–offspringdyadtothelikelihoodthattheoffspringisfromsome
unknownhybridcombination.AllDNAmarkersusedintheparentagetestwerepresumedto
beatHardy–Weinbergequilibrium(HWE)inthegivenpopulation.TheLODscore(thenatu-
rallogarithmofthelikelihoodratio,LR)forthesetofgenotypesatmultipleDNAmarkersis
givenbytheproductofLR:
Q
LODscore¼logð k LR Þ; ð6Þ
m¼1 m
whereLR isalikelihoodratioforatriadordyadatthemthDNAmarker.AnyDNAmarkers
m
thatshoweddiscrepanciesintheparentagetestorallele-sharingtestwereexcludedfromLOD
Table2. SummaryofDNAmarkersusedinthisstudy.
Type/source Evaluated Selected (%) Certified (%) Reference
GenomicSSR/INDEL 154 104 67.5% 58 37.7% Thisstudy
EST/cDNASSR 201 110 54.7% 87 43.3% Thisstudy
Ollitrault,Fetal.2010 79 6 7.6% 6 7.6% 1)
Chen,C.etal.2008 106 19 17.9% 12 11.3% 2)
Chen,C.etal.2006 56 7 12.5% 6 10.7% 3)
Total 596 246 41.3% 169 28.4%
1)Ollitrault,Fetal.(2010)Am.J.Bot.e124-e129.
2)Chen,Cetal.(2008)TreeGenet.Genom.4:1–10.
3)Chen,Cetal.(2006)TheorApplGenet.112:1248–1257.
doi:10.1371/journal.pone.0166969.t002
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HybridOriginsofCitrusVarieties
scoreestimation.Therequiredcrosstrialindex(RCI)wasobtainedby:
(cid:18) (cid:19)
1
RCI ¼log Q : ð7Þ
N m f
k¼1 k
Here,Nisthenumberofindividualswithuniquegenotypeintheproposedpopulation,f is
k
theexpectedfrequencyofaparticulargenotypeatthekthDNAmarkerestimatedfromthe
allelefrequenciesofthetwoallelesinthepopulation(equation7.4inNei[84]),andmisthe
totalnumberofDNAmarkersusedfortheevaluation.Singleparent–offspringprobability
(SPP)isnotalikelihoodratiovaluebutacumulativeprobabilitybetweentwoparticularindi-
vidualsassumingthatoneistheallegedparentofaparticularoffspringvarietywithoutprior
informationontheotherparent.TheSPPvaluefortheparticularoffspring(g )andthealleged
O
parent(g )isobtainedfromthetransitionprobabilityT(g |g )ofg giveng inasimilarman-
P O P O P
nertothatdescribedaboveby:
P
SPP¼ m T ðg jg Þ; ð8Þ
k¼1 k O P
wheremisthetotalnumberofDNAmarkersusedfortheevaluation.Thesetests,frequency
analysesandprobabilityestimationswerecarriedoutusingfunctionsofGUGSsoftware.The
inferredgenealogywasdrawnasafamilytreemanually,orusingHelium[105].
Results
DevelopmentandevaluationofDNAmarkersfornucleargenotypingof
citrus
DNAsequencesofcitrusexpressedgenesfromclonedcDNA,EST,andRefSeqinpublic
sequencedatabaserepositoriesortheharvESTcitrusdatabasewereusedforDNAmarker
design.PreliminaryclusteringanalysisofESTsequenceswithasequenceassemblerreduced
duplicationinthesedatasets,andyielded98,869consensussequencesfrom582,270EST
sequences.Anotherclusteringanalysisofwholegenomeshotgunsequencesofsweetorange
‘RidgePineapple’yielded381,909consensussequencesfrom866,700reads,but46,341Clemen-
tineBACendsequenceswerenotusedforassemblybecauseoftheirlowredundancy.SSRmin-
ingofthesedatasetswithmreps[76]identified143,825candidateregionsfromtheconsensus
ESTsequences,314,967fromtheconsensussweetorangewholegenomeshotgunsequences,
and16,159fromtheClementineBACendsequences.SSRminingoftheClementinehaploid
genomesequence[43](https://www.citrusgenomedb.org/)alsoidentified310,413candidate
SSRregionsforbothv0.9(release165)andv1.0(release182)genomes.Thesecandidateregions
wereverifiedwithresequencingdataobtainedfromNGSanalysisof15citrusvarieties(ban-
peiyuA004,ClementineA009,dancyA016,hyuganatsuA036andA038,KingA054,Kishu
A066,ponkanA108,SatsumaA113andA122,sweetorangeA162,willowleaf(Mediterranean)
mandarinA200,‘Encore’B014,‘Harehime’B017,and‘Kiyomi’tangorB031).CandidateSSR
regionsthatweresupportedwithmorethan40×Illuminareadcoveragewereselectedfor
primerdesignbyreferringtheirmotifsize,repeatlength,genomeposition,geneannotation,
specificityandversatilityamongcitrusvarieties.Wealsoidentifiedindelregionsbyreferringto
resequencingdata,andthesewerealsousedforprimerdesign.Consequently,wedesignedSSR
andindelmarkers(S2TablelistsDNAmarkersbytypeandgivestheirsources).
VerifyinggenotypingerrorstoselectcertifiedDNAmarkers
ThegenotypesoftheDNAmarkerswerepreliminarilyevaluatedforpeakheightandpeak
heightratio,productsize,andnumberofallelesinasmallsamplesetconsistingofSatsuma,
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