What is in your cup of tea? DNA Verity Test to characterize black and green commercial teas

17 Pages·2017·1.47 MB·English

RESEARCHARTICLE
What is in your cup of tea? DNA Verity Test to
characterize black and green commercial teas
OlgaDeCastro1*,MariaComparone1,AntoniettaDiMaio1,EmanueleDelGuacchio2,
BrunoMenale1,JacopoTroisi3,FrancescoAliberti4,MarcoTrifuoggi5☯,MarcoGuida4☯
1 DepartmentofBiology,BotanicalGarden,UniversityofNaplesFedericoII,NaplesItaly,2 Botanical
Garden,UniversityofNaplesFedericoII,NaplesItaly,3 DepartmentofMedicineandSurgeryandDentistry,
UniversityofSalerno,Salerno,Italy,4 DepartmentofBiology,UniversityofNaplesFedericoII,NaplesItaly,
5 DepartmentofChemicalSciences,UniversityofNaplesFedericoII,Naples,Italy
☯Theseauthorscontributedequallytothiswork.
*[email protected]
a1111111111 Abstract
a1111111111
a1111111111 Inthisstudy,weusedseveralmoleculartechniquestodevelopafastandreliableprotocol
a1111111111
(DNAVerityTest,DVT)forthecharacterizationandconfirmationofthespeciesortaxapres-
a1111111111
entinherbalinfusions.Asamodelplantforthisprotocol,Camelliasinensis,atraditional
teaplant,wasselectedduetothefollowingreasons:itshistoricalpopularityasa(healthy)
beverage,itshighsellingvalue,theimportationofbarelyrecognizablerawproduct(i.e.,
crushed),andthescarcityofstudiesconcerningadulterantsorcontamination.TheDNA
OPENACCESS VerityTestincludesboththesequencingofDNAbarcodingmarkersandgenotypingof
Citation:DeCastroO,ComparoneM,DiMaioA, labeled-PCRDNAbarcodingfragmentsforeachsampleanalyzed.Thisprotocol(DVT)was
DelGuacchioE,MenaleB,TroisiJ,etal.(2017)
successivelyappliedtoverifytheauthenticityof32commercialteas(simpleoradmixture),
Whatisinyourcupoftea?DNAVerityTestto
andthemainresultscanbesummarizedasfollows:(1)theDVTprotocolissuitableto
characterizeblackandgreencommercialteas.
PLoSONE12(5):e0178262.https://doi.org/ detectadulterationinteamatrices(contaminationsorabsenceofcertifiedingredients),and
10.1371/journal.pone.0178262 themethodcanbeexportedforthestudyofothersimilarsystems;(2)basedontheBLAST
Editor:MassimoLabra,UniversitadegliStudidi analysisofthesequencesofrbcL+matK±rps7-trnV(GAC)chloroplastmarkers,C.sinensis
Milano-Bicocca,ITALY canbetaxonomicallycharacterized;(3)rps7-trnV(GAC)canbeemployedtodiscriminate
Received:February13,2017 C.sinensisfromC.pubicosta;(4)ITS2isnotanidealDNAbarcodeforteasamples,reflect-
ingpotentialincompletelineagesortingandhybridization/introgressionphenomenain
Accepted:May4,2017
C.sinensistaxa;(5)thegenotypingapproachisaneasy,inexpensiveandrapidpre-screen-
Published:May23,2017
ingmethodtodetectanomaliesintheteatemplatesusingthetrnH(GUG)-psbAbarcoding
Copyright:©2017DeCastroetal.Thisisanopen
marker;(6)twoherbalcompaniesprovidednoauthenticproductswithacontaminantor
accessarticledistributedunderthetermsofthe
withoutsomeofthelistedingredients;and(7)theleafmatricespresentinsometeabags
CreativeCommonsAttributionLicense,which
permitsunrestricteduse,distribution,and couldbeconstitutedusinganadmixtureofdifferentC.sinensishaplotypesand/oralliedspe-
reproductioninanymedium,providedtheoriginal cies(C.pubicosta).
authorandsourcearecredited.
DataAvailabilityStatement:Allrelevantdataare
withinthepaper.
Funding:Theauthorsreceivednospecificfunding
Introduction
forthiswork.
Competinginterests:Theauthorshavedeclared AnancientEnglishproverbstates“Thepathtoheavenpassesthroughateapot”,andthisprov-
thatnocompetinginterestsexist. erbissadlytrueinrecentyears.Infact,consideringthelatestdramaticnewsconcerningfood
PLOSONE|https://doi.org/10.1371/journal.pone.0178262 May23,2017 1/17
CommercialblackandgreenteasanalyzedusingtheDNAVerityTest
fraudandsoilcontamination(e.g.,soilcontaminationinIndianteaestatessoil;[1,2]),itis
likelythat“thepathtoheaven”canbeacceleratedbywhatyoudrinkoreat(e.g.,thetragiccase
ofthe“soiloffire”insouthernItaly;[3,4]).Currently,theadulterationoffoodisfrequent,and
thescientificcommunityhasusedbeenadvancedtechnologiestoprotecthumansfromincor-
rectpracticesinfoodordrinkproduction(e.g.,[5,6,7,8,9,10]).Indeed,someproductscan
representrealanddangerous“mirrorforlarks”intermsofhealth,asthepresumednaturalor
biologicalalimentsorsupplementspurchasedbyconsumerstoprotecttheirhealthlackvalid
qualitycontrols[11].Moreover,thesecontrolsareoccasionallydifficulttoperform,reflecting
theintrinsicnatureoftheproduct(e.g.,foodsupplementsorherbalmedicine[12,13,14]).
Theinternationaltradeofherbalproductsisincontinuousdevelopmentforbothalimentary
andpharmaceuticalpurposes[15];infact,manyplantsareuseddailyinthepreparationof
foodsandherbalteas(e.g.,[16,17]).Specifically,teainfusionsarewidelyusedasbothpleasant
drinksandfortheirmanybeneficialproperties[18],andanaccuratedefinitionofthecom-
poundspresentintheseteasisimportantnotonlyfortheconsumerbutalsofortheproducers
andcontrolauthorities[19].Becauseofthefragmentationorpulverizingofthevegetalmate-
rial,itisoftendifficulttoidentifythespeciesamongtheingredientsusingtraditionalanalyses
(i.e.,macroscopicandmicroscopicmorphology)[20,21].Thus,aDNAbarcodingtechnique
hasrecentlybeenusedtoaddressthisproblem,resultinginausefulsystemforthedetectionof
theplantsemployedintheseteasandthecharacterizationofthepossibleadulterationand/or
contaminationinawiderangeofplant-basedfoods(e.g.,[22,23]),becomingauniversal
adoptedapproachinthelastfewyears[24,25].
Inthisstudy,weusedamulti-facetedDNAbarcodingapproachtodevelopafastandreliable
protocol(DNAVerityTest,DVT)forthetaxonomiccharacterizationandconfirmationofherbal
infusions.Asamodelplantforthisprotocol,Camelliasinensis(L.)Kuntze,thetraditional
teaplant,wasselectedfor(1)itshistoricalpopularityasa(healthy)beverage[26,27,28],(2)
itshighsellingvalue(cfr.[27]),(3)theimportationofbarelyrecognizablerawproduct(i.e.,
crushed)[29],and(4)thescarcityofstudiesconcerningadulterantsorcontaminations[30,31].
TheDNAVerityTest wassuccessivelyappliedtoverifytheauthenticityof32commercial
teapackages(simpleoradmixture).Briefly,theDNAVerityTest verifiedboththesequencing
ofDNAbarcodingmarkersandthegenotypingoflabeled-PCRDNAbarcodingfragmentsfor
eachsampleanalyzed.
ThemethodologyofDNAVerityTest presentedhereisverydetailedandisapromisingtool
forcheckingtheauthenticityofteasamplesanditcouldbealsosuitableforapplicationindif-
ferentstudysystems.
Inaddition,thedevelopmentofDNAVerityTest ispartofawiderprojectcomprisinga
multi-facetedpilotstudywiththepurposeofanalyzing32brandsofEuropeanandItalian
commerciallyavailableteas(16blackand16greenteas)usingdifferentanalyticalapproaches
evaluatethepresenceofmycotoxins,microbialcontaminants,heavymetalsandphthalates(O.
DeCastro,unpublisheddata).
Materialsandmethods
Teasamplingdesign
Currently,teaproductsareavailableatavarietyofmainstreamoutlets,suchassupermarkets,
healthfoodstores,drugstoresandonlinefromherbalsupplycompanies.Inthepresentstudy,
atotalof32teapackages(C.sinensis)werepurchasedfrommarketsinNaples(southernItaly)
andfromonline-shops(Table1)andsubsequentlytestedusingablindexperimentforthe
analysesreportedbelow.Allproductswerealsoavailabletoconsumersthroughonline-shops,
representing17Italianorinternationallyfamousbrands(sevenandten,respectively).Within
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CommercialblackandgreenteasanalyzedusingtheDNAVerityTest
Table1. ListofItaliancommercializedblackandgreenteapackagesanalyzedinthepresentstudy(NandVsamples,respectively). Information
foreachaccessionaboutthemarketingquality(high,mediumandlow),salesnetwork(D=discountsupermarket;H=herbalistshop;S=supermarket;
P=drugstore),price((cid:15)){(A),<1(cid:15);(B),1<(cid:15)<2;(C),2<(cid:15)<4;(D),4<(cid:15)<6;(E),>6(cid:15)}andmolecularresultsforrbcLandrps7-trnV(GAC)sequences(presence
ofaSNPinthe68bpcodingregionofrbcL,A=adenine,C=cytosine;rps7-trnV(GAC),239bp=Camelliasinensis,226bp=C.pubicosta;insmallerfont,the
nucleotide/fragmentlessrepresented).
Code Marketing Sales Price Labellinginformation rbcLhaplotype(68 rps7-trnV(GAC)
quality* network bpSNP) haplotype(bp)
Blackteas
N1 Medium H D OrganicblacktealeavesofCameliasinensis(L.)Kuntze A/c 239
(100%)
N2 Medium-good P,H D Tealeaves[Camelliasinensis(L.)Kuntze] C 239
N3 Good S B Blacktea,aromas C 239
N4 Low-medium S C Notreported C 239
N5 Good P,H C Blackcertifiedtea(100%) C/a 239
N6 Good S B Tea C 239/
226
N7 Good S C Decaffeinatedtea C 239
N8 Good S B Notreported C 239/
226
N9 Good S C Blacktea(94%),lemonaroma(6%) C/a 239/226
N10 Low S B Blacktea(100%) C 239/
226
N11† Good S D Decaffeinatedblacktea,caffeine<0.1% C/a 239/
226
N12 Low S B Blacktea C 239/
226
N13 Low S B Blacktea(95%),naturallemonaroma(5%) C/a 239/
226
N14 Low S B Blacktealeaves C 239/
226
N15 Medium H C Decaffeinatedorganicblacktea,caffeine<0.1% C 239
N16 Good S C Sugar,acidifier(citricacid),decaffeinatedteaextract, N.d. N.d.
aromas,lemonjuicepowdered(0.5%);glutenfree
Greenteas
V1 Good P,H E Pureleavesofgreentea(CameliasinensisKuntze) A 239
V2 Medium P,H E Greentealeaves(Cameliasinensis)(99%),bergamot A 239
essentialoil(1%)
V3 Medium H D Organicgreentealeaves[Camelliasinensis(L.)Kuntze] A 239
(100%)
V4 Medium P,H D Greentealeave A 239
V5 Medium-good P,H D Unfermentedorganictealeaves(CamelliasinensisL.) A/c 239
V6 Medium H C GreenteacertifiedbyFairtrade C 239/
226
V7 Medium H D Decaffeinated;ingredientnotlisted A 239
V8 Good S C Greentea(100%) C 239/
226
V9 Good S C Greentea,aromas,andcitruspeels(2.1%:grapefruit,lemon, C 239/226
lime,andorange)
V10† Good S C Greentea,aromas,andspices(2%)(anise,cinnamon,and C 239/226
licorice)
V11 Good S C Greentea,aromas,andmint(7.9%) C 239/226
V12 Good S C Greentea A/c 239
V13 Low S B Greentea A 239
V14 Low S B Organicgreentea A/C 239
V15 Low D A Greentea A/C 239
V16 Medium S A Sugar,acidifier(citricacid),greenteasolubleextract, N.d. N.d.
aromas,andginsengsolubleextract
*,valuedeterminedbyaquestionnaireconductedon25people(13femalesand12males)
†,adulteratedproduct;N.d.,nodatum.
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CommercialblackandgreenteasanalyzedusingtheDNAVerityTest
these17brands,16sampleswerefermentedtea(onlyblacktea)and16sampleswererawtea
(onlygreentea).Decaffeinated,soluble,admixture(i.e.,thepresenceofotherplantmaterial)
andflavoredsampleswerealsoconsidered(Table1).Theteasampleswereselectedconsider-
ingthetradenetwork(supermarket,drugstoreandherbalistshop),theprice(cheapand
expensive),themarketingquality(packaging,publicityandbrand)andthepresenceoffilters
inthepackages(exceptforonetypeofsolublegreentea).Thisinformation(exceptthebrand
name)isreportedinTable1.Theteasampleswerestoredatroomtemperaturepriortothe
molecularanalyses,andduplicatesforeachteapackage-lotwereanalyzed.Asapreliminary
molecularanalysis,specimensofC.sinensis(CS-DNAs)wereobtainedfromtheBotanicalGar-
denofNaples(BGN).Thetissuesofotherspeciesindicatedintheadmixtureteasampleswere
obtainedfromBGNasreferencesamples{i.e.,MenthaL.sp.(peppermintandcornmint),Cit-
rusL.sp.(lemon,orange,grapefruit,andlime),PimpinellaanisumL.(anise),Cinnamomum
verumJ.PreslorC.cassia(L.)J.Presl(cinnamon),andGlycyrrhizaglabraL.(licorice)}.
DNAVerityTest(DVT)procedure
Barcodemarkers. CandidateDNAbarcodingmarkerswereselectedbasedonprevious
tests,whichhadbeenconductedusinguniversalprimers[25,31,32,33,34],GenBankdataand
referenceC.sinensisDNAfromBGN.Thefollowingfourcriteriaforselectinganidealnucleo-
tidesequencesbarcodewereconsidered:(1)highlyefficientamplification,(2)highquality
sequences(e.g.nounambiguoussequencescausedbydoublepeaksorstutteringeffect),(3)an
exhaustivesequencedatabasepubliclyavailable,and(4)highspeciesdiscriminationcapability.
ThefollowingtwomoleculartechniqueswereusedfortheDNAVerityTest (DVT):
sequencingviaSangerchemistryandgenotypingoffluorescedamplifiedfragmentviacapil-
laryelectrophoresis.Afterinsilicoandlaboratorytests,thecandidatemarkersforthesequenc-
ingapproachincludedthefollowing:genesandplastidintergenicspacers{rbcL,matKand
trnH(GUG)-psbA,rps7-trnV(GAC),respectively}andanuclearintergenicspacer(ITS2).Forthe
genotypinganalyses,theconsideredmarkerswere:anintron{P6loopofthetrnL(UAA)},plastid
intergenicspacers{trnH(GUG)-psbAandrps7-trnV(GAC)},andanuclearintergenicspacer
(ITS2).Theuniversalbarcodeprimersandthespecificbarcodingprimersdesignedinthe
presentstudyarereportedinTable2.
TeaDNAextraction. Atotalof30mgofshreddedmaterialpresentinteabagsfromeach
packagewasusedforDNAextraction.AimingatselectingthebestDNAisolationprocedure
toobtainhighyieldandqualityofextractedDNA[42],apreliminaryanalysisonthreesamples
(green,blackandadmixturetea)wasperformedusingfourcommercialkits{PowerPlantPro
DNAIsolationKit(MoBio),PlantGenomicDNAExtractMiniKit(FisherMolecularBiology),
ZRPlant/SeedDNAMicroPrep(ZymoResearch),GeneAllExgenePlantSVkit(GeneAllBio-
technology)}andtwodetergentprotocols[43,44].TheisolatedDNAwasanalyzedusingbotha
spectrophotometerNanodrop2000(ThermoFisherScientific)toquantifyitspuritygrade(260/
280and260/230)andaQubit3FluorometertodeterminethepreciseDNAconcentration(Life
Technologies,ThermoFisherScientific).Inaddition,avisualestimatewasobtainedusing1%
agaroseelectrophoresiswithGelRedstrained(Biotium)bandintensitiesandGeneRuler1kb
PlusDNALadder(ThermoFisherScientific).Afterpreliminaryanalysis,allextractedgenomic
DNAwereestimatedusingbothfluorometricandelectrophoresisanalyses.
ForfreshplantreferencesfromBGN,100mgofleaftissuewasusedfortheDNAextraction
usingGeneAllExgenePlantSVkit(GeneAllBiotechnology)accordingtothemanufacturer’s
instructions.
PCRamplification. Molecularmarkerswereamplifiedusingahigh-fidelityDNApoly-
merase,andtheprimersarelistedinTable2.ThePCRswereperformedusing10ngof
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CommercialblackandgreenteasanalyzedusingtheDNAVerityTest
Table2. PrimersusedinthepresentstudyforDNAbarcodinganalyses.
Locus Primername Sequence(5’-3’) Ta Primernote Reference
rbcL rbcLa_F ATGTCACCACAAACA 55 Universal [35]
GAGACTAAAGC
rbcLajf634R GAAACGGTCTCTCCA Universal [36]
ACGCAT
CS_rbcL-300rev TAAAGGATACGCTAC 55 SpecificforCamelliasinensis;usedtochecktheSNP-M(68bp)in Inthepresent
ATAAGC sequencingPCR study
matK 1R_KIM-f ACCCAGTCCATCTGG 55 Universal Ki-JoongKim,
AAATCTTGGTTC pers.comm.
3F_KIM-r CGTACAGTACTTTTG
TGTTTACGAG
trnH(GUG)- psbA3’f GTTATGCATGAACGT 55 Universal [37]
psbA AATGCTC
trnHf* GTTATGCATGAACGT [38]
AATGCTC
Cinnamon_2psb- ACGAGTCGTTGAAGG 56 SpecificforCinnamomumverumandC.cassia(cinnamon) Inthisstudy
for ATCAAT
Cinnamon_2trnH- TGCAGGTTGGTACAG
rev AAGAA
Citrus_psbA-in GTATTGATCCGTTAT 57 SpecificforCitrussp.(lemon,orange,grapefruit,andlime) Inthisstudy
TTAGTCG
Citrus_trnH-in ATCTTATCTTACTTA
TGAAGAACC
Glycyrrhiza_psbA- GTTTTAAAGAAGGAT 55 SpecificforGlycyrrhizaglabra(licorice) Inthisstudy
in ACGAGG
Glycyrrhiza_trnH- TACATTCGCCCTTCT
inA TATAC
Mentha_2psb_for TTCCAGGCAAGTCCA 56 SpecificforMenthasp.(M.aquatica,M.arvensis,M.canadensis,M. Inthisstudy
ATACT longifolia,P.pulegium,M.suaveolens,M.pulegium,andM.x
Mentha_2trnH_rev TGTGTAGGAGTTTTT piperita,M.spicata)
GAAAATAGAC
Piminella_psbA-in ACCTAGCTGCTGTTG 60 SpecificforPimpinellaanisum(anise) Inthisstudy
AAGCTC
Pimpinella_trnH-in GGAGCAATATCGCTT
TCTTGATAGA
rps7- CS_rps7_1 GTGCTATGGCTCGAA 67 SpecificforCamelliasp.;usedtodiscriminateC.sinensisfromC. Inthisstudy
trnV(GAC) TCCGT pubicosta
CS_trnV(GAC)_1* ACCACGTCAAGGTGA
CACTC
P6loop g GGGCAATCCTGAGCC 55 Universal [39]
trnL(UAA) AA
h* CCATTGAGTCTCTGC
ACCTATC
ITS2 S2F ATGCGATACTTGGTG 55 Universal [40]
TGAAT
S3R* GACGCTTCTCCAGAC
TACAAT
BELL-1 GGDGCGGAKAHTGGC 55 Universal [41]
CYCCCGTGC
BELL-3 GACGCTTCTCCAGAC
TACAAT
*,labeled
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CommercialblackandgreenteasanalyzedusingtheDNAVerityTest
genomicDNA,5XPhusionHFbufferand0.5uMofeachprimerwithPhusionHotStartII
DNAPolymerase(ThermoFisherScientific)accordingtothemanufacturer’sinstructions.
TheamplificationoftherecalcitrantDNAtemplateswasperformedusingAmpONEFastPfu
DNApolymerase(GeneAllBiotechnology).
Sequencingapproach. Accordingtothesequencingapproach,theamplifiedfragments
longerthan350bp(e.g.,rbcL/matK)werepurifiedusingPEG8000precipitation(PEG20%,2.5
MNaCl).Incontrast,polymorphicPCRfragmentsshorterthan350bp{i.e.,trnH(GUG)-psbA/
rps7-trnV(GAC)/ITS2}werepurifiedusingMonarchPCRandtheDNACleanupkit(New
EnglandBioLabs).Approximately10ng/100bpofthepurifiedtemplatesweresequenced
accordingtoDiMaioandDeCastro[45]usingafluorescentdye(BrightDyeTerminatorCycle
SequencingKit,ICloning).ThereactionswerepurifiedusingBigDyeXTerminatorPurification
Kit(AppliedBiosystems,ThermoFisherScientific)andreadusinganautomatedsequencer
(3130GeneticAnalyzer,LifeTechnologies,ThermoFisherScientific).Thesequenceswereana-
lyzedusingABDNASequencingAnalysisversion5.2software(AppliedBiosystems,Thermo
FisherScientificInc.),editedinChromaslitever.2.1.1.software(http://technelysium.com.au/?
page_id=13),andassembledandalignedinBioEditver.7.2.5software[46].PCRfragmentswith
multiplepeakswithinthesequencewereclonedusingtheCloneJETPCRCloningKit(Thermo
FisherScientific)accordingtothemanufacturer’sinstructions.Transformationwasperformed
usingStrataCloneSoloPackCompetentCells(AgilentTechnologies).Thebacteriawerecultured
inLBmediumat37˚Cfor30minandsubsequentlytransferredtoLBagarplatescontaining100
ug/mlampicillin.Ninety-sixrandomlyselectedclonesfromeachtransformationwereamplified
usingthecorrespondingDNAbarcodingprimers.ForeachPCRfragmentpolymorphicfrom
theC.sinensisreference(e.g.,CS ref=510bp),fivePCRsamplesweresequenced.
trnH-PsbA
Theidentificationofunknownsequencebarcodesfromtheteasampleswasconducted
usingtheBasicLocalAlignmentSearchTool(BLAST;[47])implementedintwobarcode
libraries{GenBankandTheBarcodeOfLifeDatasystem(BOLD);[48,49]}.Tooptimizecor-
rectidentifications,theclosestmatchforeachmolecularmarkerwasdefinedasthetargetwith
thehighestpercentageidentityusinganarbitrarycut-offof95%andanE-value<1e-4or
greaterintermsofoverlapwiththequerysequence.
Genotypingapproach. Regardingthegenotypingapproach,theamplificationprocedure
fortheP6loop,trnH(GUG)-psbA,rps7-trnV(GAC)andITS2markerswasthesameasthat
reportedabove,exceptfortheuseof0.5uMoffluorescentlylabeledreverseprimer(Table2).
Fivemicroliters{ca.20ng(P6loop),40ng(rps7-trnV(GAC)}and100ng{trnH(GUG)-psbAand
ITS2}ofamplifiedlabeledfragmentswerepurifiedusing2uLofCleanSweepPCRPurification
Reagent(AppliedBiosystems,ThermoFisherScientific);0.5uL{ca.3and7.5ng,respectively;
RelativeFluorescenceUnits(RFU)>6000}and1uLof1:5dilution(ca.1and3ng,respec-
tively;RFU(cid:21)3000)foreachpurificationwereloadedontoa3130GeneticAnalyzerwith0.4
uLoffluorescentlylabeledinternalsizestandard(GeneScan1000ROXor600LIZdyeSize
Standards,AppliedBiosystems,LifeTechnologies).CamelliasinensisPCR-labeledstandards
werealsogeneratedforeachmarkerused(CS-ref)andfortheotherplantspeciespresentin
theadmixtureteainfusions(i.e.,mint,lemon,orange,grapefruit,lime,anise,cinnamonand
licorice,Table1).RawdatawerescoredwithaninternalsizestandardusingPeakScannerver-
sion1.0software(AppliedBiosystems,LifeTechnologies).
Resultsanddiscussion
DNAVerityTest(DVT)protocol
DNAextraction. First,weobtainedastandardandrapidmethodtoachievegoodquality
genomicDNAfreefrominhibitors(e.g.,polysaccharides,polyphenolandphenoliccompounds)
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CommercialblackandgreenteasanalyzedusingtheDNAVerityTest
thatcouldinterferewiththeactivityofDNApolymeraseinthePCRamplification,aspreviously
reported[50].Indeed,accordingtoGraham[51],somecompoundsarepresentathighconcen-
trationsintheleavesofC.sinensis(e.g.,polysaccharides>12%,secondarymetabolites=40%
ondryleafweight)andcanbeco-precipitatedwithDNAdeterminingfailureinPCRamplifica-
tionreactions.ThegenomicDNAfromtheteasampleswasisolatedusingtheExgenePlantSV
Kit,whichproducedabetterquantity(70.3ng/uL±4.3SE)andqualityofDNAamplification
(A260/A280=1.82±0.005SE;A260/A230=1.47±0.02SE)comparedwithotherkits.The
DNAwaselutedin100uLofnuclease-freemolecularbiologygradewater(Ambion,Thermo
FisherScientific).
PCRamplification. InC.sinensisreferenceDNAs(CS-DNAs),rbcLoligosproducea
fragmentof654bp;matKoligosproduceafragmentof889bp;trnH(GUG)-psbAoligosproduce
afragmentof510bp;rps7-trnV(GAC)produceafragmentof239bp;P6loopoligosproducea
fragmentof90bpandITS2oligosproducefragmentsof475bp(ITS2-Chenoligos)and345
bp(ITS2-Chiouoligos).HighefficiencyDNAamplificationwasobtainedforallthemarkers,
exceptforthematKgeneregion(i.e.,low-medium),asalsoreportedintheliterature(e.g.,[31,
33,34]).Polymerasestutteringeffectswereeliminatedusinghigh-fidelityDNAPolymerase
(PhusionHotStartII),particularlyintrnH(GUG)-psbA,whereafixedpolA waspresentatthe
13
endofsequence{5’-trnH(GUG)-psbA-3’}[52].
PCRsequencing. ThePCRproductsfromCS-DNAscorrespondingtotheaboveDNA
markersweresequencedandhigh-qualitysequenceswereobtainedforallmarkers(i.e.,no
doublepeakscausedbymoresequences),exceptforthenuclearmarkersITS2-Chenand
ITS2-Chiou[40,41].Comparingtherawdataofelectropherogramsobtainedusingthediffer-
entITS2oligos(Chenvs.Chiou),theITS2-Chioussequencespresentlessnoise,butdouble
peakswereconsistentlypresent.Itishighlylikelythatthedoublepeaksreflectthedifferent
designoftheforwardprimer(BELL1)intoaconservedpartofITS2,whichamplifiedapartial
ITS2,determininglessnoisecomparedwiththetotalITS2ofChenetal.[40],wherethefor-
wardoligoisdesignedbeforetheITS1(i.e.,5.8SrRNA).ThepresenceofdifferentITS2
sequenceswasalsodemonstratedthroughthecloningofthePCRfragmentfromthereference
DNAoftwodifferentaccessionsofC.sinensis;tenpositivecoloniesforeachsamplewere
sequencedandblastedusingtheGenBankdatabase,confirmingtheabsenceofDNAasacon-
taminant(GenBankaccessions:fromKY928288toKY928307).Thepresenceofamulti-family
ofITSsequencesintheCS-DNAswasalsoobservedinanadditionalassessmentofthetea
samples,whichdidnotshowcontaminationphenomenaoradulterationdetectedwiththe
othermolecularmarkersemployed.Thesedata(i.e.,intra-variabilityofITS)wereconsistent
withtheliterature[42,53,54],whereaprobableincompletelineagesortingtogethertoahigh
anthropicmanipulationofthespecies(e.g.,domesticationandcultivation)wouldamplifythe
geneticvariabilityofthisnuclearmolecularmarker.Thus,ITS2hasbeendiscardedfromthe
analysesoftheteasamplesinthepresentstudy,reflectingpotentialdoublepeaksandnoisein
therawdata(i.e.,ambiguoussequences)andthusreflectingitsintrinsicnatureandnotthe
presenceofactualcontaminationsoradulterants.Indeed,forthesimplecharacterizationof
thespecies,theconsensussequencecouldbeusedasreportedinLeeetal.[55],evenifcareful
manualeditingmustbeperformedtoobtainthesequences.
AnothermolecularmarkerdiscardedasanidealsequencingbarcodewastrnH(GUG)-psbA.
Evenifthismarkeriseasilyamplified,itssequencewasnotdiscriminantamongcongeneric
Camelliaspecies,incontrasttorbcL+matK.Indeed,BLASTtrnH(GUG)-psbAsequencecom-
parison(excludingpolyA )showed100%identityinadditiontoC.sinensis,withmorethan
n
tenCamelliataxa[e.g.,C.cuspidata(Kochs)H.J.Veitch,C.danzaiensisH.T.Chang&K.M.
Lan,C.elongata(Rehder&E.H.Wilson)Rehder,C.euryoidesLindley,C.fraternaHance,C.
forrestii(Diels)Cohen-Stuart,C.grandibracteata HungT.Chang&F.L.Yu,C.leptophyllaS.
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CommercialblackandgreenteasanalyzedusingtheDNAVerityTest
YeLiangexHungT.Chang,C.oleiferaAbel,C.pitardiiCohen-Stuart,C.pubicostaMerr.,C.
reticulataLindleyandC.yunnanensis(PitardexDiels)].Someofthesetaxaresultedinincom-
pletesequences,resultinginincorrectcomparisonoutputs(e.g.,C.cuspidata,C.elongata,C.
euryoidesC.fraterna,C.forrestii,C.oleifera,C.reticulate,andC.yunnanensis).Notably,a
polyA sequencewasdetectedintrnH(GUG)-psbA,representingapotentiallydiagnosticmarker
n
todiscriminatespeciessimilartoC.sinensis(e.g.,C.sinensisA vs.C.pubicostaA )butshow-
13 14
ingtheintrinsicproblematicnatureofapolyN(e.g.,stuttering,homoplasy).Thus,thismarker
wasnotusedfortaxonomicaldiscriminationinthepresentstudy.
Incontrast,therbcLmarkerwasused,reflectingbothgooddiscriminationpoweranduni-
versalprimeramplification[31]thatcanalsodetectthemacro-contaminationofotherspecies
[56].Thismarkerresultedintwohaplotypesforasinglenucleotidepolymorphism{SNP-A
andSNP-C(M)}inthe68bpcodingregionofrbcL(5’-AAATTGA-M-TTATTA-3’,GenBank
reference:KJ806281,57209bp;C.sinensisvar.sinensis,completechloroplastgenome),consis-
tentwiththereportofStoeckleetal.[31].Indeed,accordingtotheseauthors,whengeography,
teatypeandtaxonomicalcharacterizationswereavailableforC.sinensisreferencesamples,
thisnucleotidevariation(SNP-Cvs.SNP-A)wasstronglyassociatedwithproductsfromIndia
incomparisonwithChina,withblackvs.greenteaandvar.assamicavs.var.sinensis,respec-
tively.Notably,basedontheincreaseofnewaccessionsinGenBank(e.g.,newCamelliachlo-
roplastgenomes[57,58,59],currentlytheSNP-Cshowsalowertaxonomicaldiscrimination
levelcomparedwithSNP-A.InsubsequentBLASTsearches,haplotypeSNP-Adiscriminates
forC.leptophylla,C.oleiferaandC.sinensis;incontrast,haplotypeSNP-Cisalsoassociated
withC.sinensis,C.crapnellianaTutcher,C.cuspidata,C.grandibracteata, C.granthamiana
Sealy(=C.albogigasHu),C.impressinervisH.T.Chang&S.Y.Liang,C.japonicaL.,C.kissii
Wall,C.oleifera,C.petelotii(Merr.)Sealy,C.pubicosta,C.sasanquaThunb.,C.taliensis(W.
W.Sm.)Melch.,C.tonkinensis(Pit.)Cohen-StuartandTutcheria hirta(Hand.-Mazz.)H.L.Li
(=Pyrenariahirta(Hand.-Mazz.)H.Keng).
AccordingtothematKmarker,evenifitsprimersdidnotshowgooduniversality[31,33,
34],thismarkerwasusedtoconfirmthepresenceofC.sinensiswhenrbcLresultsinSNP-Aor
tonarrowthetaxonomicalfieldintheteabagswhentheSNP-CrbcLhaplotypewaspresentin
thesample.BLASTanalysessuggestthatthisbarcodingmolecularmarkerwasabletodiscrimi-
natethefourCamelliaspecies(C.grandibracteata, C.leptophylla,C.pubicostaandC.sinensis)
with100%identity.
Basedontheseresults,anadditionalandexclusivemarkerhasbeendemonstratedtodis-
criminateC.pubicostafromC.sinensis,whenthetemplatesrepresentSNP-CinrbcL,assug-
gestedabove.Throughthealignmentofthechloroplastgenomesofthesetwospecies,the
intergenicregionbetweenrps7andtrnV(GAC)wasselectedforthepresenceoftwoexclusive
deletionsinC.pubicosta(7and6bp;102651and102718bp,respectively;GenBankreference:
KJ806277,C.pubicosta,completechloroplastgenome).Theoligosweredesignedbetween
thesetwodeletions,amplifyinga226bpfragmentinC.pubicostaanda239bpfragmentinC.
sinensisforbothsequencingandgenotypinganalyses(Table2).
PCRcapillarygenotyping. Accordingtocapillarygenotypingelectropherograms,ITS2
wasexcludedduetothepresenceofmultiplepeaks,asconfirmedbyprevioussequencing
results.EvenifthetrnH(GUG)-psbAintergenicspacershowedanucleotidesequenceshared
withotheralliedCamelliaspecies,thismolecularmarkerwasdemonstratedasabettermolecu-
larmarkerthantheP6loop.Infact,trnH(GUG)-psbA,whencomparedwiththeP6loop,
showedabetterdiscriminantpowerintermsoflength(e.g.,ineudicotyledons,thetrnH(GUG)-
psbArangeisfrom152bpto851bp[60];andtheP6looprangevariedfrom22bpto122bp,
[61]),andnucleotidevariability[39,60,61].Surely,theP6loopcanbeusefulwithahigher
PLOSONE|https://doi.org/10.1371/journal.pone.0178262 May23,2017 8/17
CommercialblackandgreenteasanalyzedusingtheDNAVerityTest
concentrationofdegradedDNA(i.e.,ancientDNA),whereitisdifficulttoobtainfragments
longerthan150bp[39,62].
Inconclusion,trnH(GUG)-psbAwasemployedasanimportantpre-screeningmarkerto
detectpotentialadulterantsviagenotyping,asthismarkercanindividuatethepolymorphisms
ofPCRasfragmentsofdifferentlengths(observableasanomalouspeaks)comparedtotheref-
erencestandardofC.sinensisand/oralliedBLASTspecies,whichresultinthesamelengthin
bp(CS -ref=510bp).Inparallel,rps7-trnV(GAC)wasalsousedtodiscriminateC.pubicosta
TP
(226bp)fromC.sinensis(239bp)forthedifferenceinlengthoftheamplifiedfragment(see
aboveinparagraph“PCRsequencing”).
IntheteatemplateswithonlyC.sinensiscertifiedonthelabel(Table1),theanomalous
peakswithanRFU<5%,(comparedtothepeakofC.sinensis)wereinterpretedascasual
micro-contaminationsandthereforeexcluded.Anydeviationfromthestandard(i.e.,reference
profileofthelistedingredients)hasbeenfurtherinvestigatedthroughcloningandsequencing
theanomalousfragments.
DNAVerityTest(DVT)appliedtocommercialteabags
DNAextraction(ExgenePlantSVkit). AllDNAextractedfromteasamplesshoweda
degradedpatternonelectrophoreticanalysis;lowerconcentrationswereobservedinblacktea
andindecaffeinatedteasamples(N11,N15andV7).Inthesetemplates,electrophoreticanaly-
sisshowedamoredegradedDNAgenomicpatternthantheothers.NoDNAwasdetectedin
solubleteasamples(N16andV16).
PCRamplification{trnH(GUG)-psbAandrps7-trnV(GAC)fluorescentlylabeled;rbcLand
matK}. Noamplificationofanytestedmarkerwasobtainedfromthetwosolubleteasamples
(N16andV16).CasesofrecalcitrantPCRamplificationswereobservedforalldecaffeinated
samples(V7,N7,N11andN15)andsomeblackteasamples(N1,N4,N7,N11,N15).These
recalcitrantDNAsampleswereamplifiedusingAmpONEFastPfuDNAPolymerase,which
wasmorerobustthanPhusionDNAPolymerase.
PCRgenotyping{trnH(GUG)-psbAandrps7-trnV(GAC)fluorescentlylabeled}. Usingthe
genotypingoftrnH(GUG)-psbAforpre-screening,allthesamplesshowedapeakat510bp,cor-
respondingtotheC.sinensisreference(CS -ref).However,severalsampleswerecharacter-
TP
izedbasedonanomalouspeaks.Forexample,amongtheteabagsthatlistedonlyC.sinensis,a
blackdecaffeinatedsample(N11)resultedinthepresenceofanadditional,highlyvisiblepeak
at365bp{RFU(365bp:510bp)=ca.1:2};incontrast,intheteasampleswithadditionalmaterial
fromotherplants(admixtureteas,V9-V11),thecapillarygenotypingdatadidnotcorrespond
withthenumberandintensityofpeaksdeducedbyingredientscertifiedonthelabel,except
fortheconfirmedpresenceofC.sinensis(peakat510bp).Forexample,V10presentsonlyan
additionalpeak,whichcouldbeduetoanise{ca.317bp;RFU(317bp:510bp)=ca.1:3},while
thepeaksofothercertifiedingredients,namelycinnamon(523bp)andlicorice(433bp),were
notdetected.Lowpeaks(0.5%<RFU<1.2%),assumetobefromCitrusspecies(lemon,
orange,grapefruit,andlime,ca.552–577bp)wereindividuatedintheV9greenteasample.A
similarpatternwasobservedintheV11sample(greentea),whereapeakcorrespondingto
mint(Menthasp.)(ca.436bp)showedanRFUofapproximately1.7%,asreferencedtotheC.
sinensispeak{i.e.,RFU(436bp:510bp)=ca.1:57}.
Asaconsequenceoftheseunexpectedresults,twofurtherprocedureswereused:(1)the
trnH(GUG)-psbAofsampleswithanomalouspeaksnumberswassequencedinadditionand
successivelycloned;and(2)theadmixturesamples(V9,V10andV11)withalowernumberor
lessmarkedpeakscomparedwiththereferenceprofilesofthelistedingredientswereamplified
usingtheprimersforthetrnH(GUG)-psbAspecificallydesignedinthepresentstudy(Table2)
PLOSONE|https://doi.org/10.1371/journal.pone.0178262 May23,2017 9/17
CommercialblackandgreenteasanalyzedusingtheDNAVerityTest
(seeparagraph,“Anomalousteatemplate”).ThetrnH(GUG)-psbAmarkerwasalsosequenced
inthesampleswithnormalcapillarygenotypingdata(onepeakat510bp)togeneraterefer-
enceSangerchromatogramsforcomparisonandnovariabilitywasobservedamongtheacces-
sions(GenBankaccession:KY989996).
Inaddition,thegenotypingofrps7-trnV(GAC)canbeasuitableadditionalpre-screening
shortmarkerforeventualcontaminants,evenifthismolecularregionisnotyetwellrepre-
sentedinGenBank;incontrast,ithasbeenusefultodiscriminatebetweenC.sinensisandC.
pubicosta.Thecapillarygenotypingpatternsconfirmedtheresultsobtainedbythesequencing
approach(seeresultsinthenextparagraph).Surely,thegenotypingapproachwasfasterand
moreefficientcomparedwithSangersequencing.
PCRsequencing{rbcL+matK+rps7-trnV(GAC)}. Accordingtothechromatogramsof
therbcLsequences,theteasamplesshowedthepresenceofthetwohaplotypes(SNP-Aand
SNP-C;GenBankaccession:KY989997andKY989998,respectively)(Table1),asalsoreported
byStoeckleetal.[31].Inthetemplatesanalyzedinthepresentstudy,amongthegreenteas,six
samplesshowedthepresenceofSNP-A,onesampleindicatedthepresenceofSNP-Candfour
showedheterozygouspeaks(SNP-M),withadeninemorerepresentedthancytosine(V5and
V12,SNP-A/c)orsimilarlyrepresented(V14andV15,SNP-A/C).Alltheblackteasamples
showedthepresenceofSNP-C,exceptforfiveaccessions,characterizedbyaheterozygous
peak(SNP-M)(Table1),withcytosinemorerepresentedthanadenine(N5,N9,N11andN13,
SNP-C/a)andviceversa(N1,SNP-A/c).Anadditionaloligo(CS_rbcL-300rev)wasdesigned
asanadditionalassessmentofthisSNP(Table2).
IntherbcLsequencesofthesampleswithadditionalpeaksinthetrnH(GUG)-psbAgenotyp-
ingprofile,thecorrespondingSangerrbcLsequencesresultedinmultiplepeaks(N11,V10)or
decreasedbackgroundnoise(V9,V11).
RegardingthematKsequences,novariabilitywasobservedamongtheteaaccessions(Gen-
Bankaccession:KY989999),andnomultiplepeakswereevidentfortheanomalousand/or
admixturetemplatesasinthecorrespondingrbcLsequences,confirmingthelowuniversality
oftheseprimerstodetectpotentialDNAcontaminationsordifferentDNAintheadmixture
teatemplates(e.g.,[34]).
Rps7-trnV(GAC)wasamplifiedandsequencedtodiscriminateC.sinensis(haplo-CS239bp;
GenBankaccession:KY990000)fromC.pubicosta(haplo-CP226bp;GenBankaccession:
KY990001)inteatemplateswheretheSNP-ChaplotypesofrbcLwerepresent.Thismarker
wasalsosequencedinsampleswithSNP-AhaplotypesofrbcLtogeneratereferenceSanger
chromatogramsforcomparison.
Sevenblacktemplates(N1-N5,N7andN15)revealedthepresenceofrps7-trnV(GAC)
belongingtoC.sinensis;incontrast,severalteatemplatesshowedasequencewithmultiple
peaks,initiatedbythefirstindelpresentinC.sinensisvs.C.pubicostaalignment.Insometem-
plates,thesequenceswereunreadableonaccountofthesameheightofthepeaks(V9,V10,
V11andN9),butintheotherones,thesequencemostrepresentedcanbededuced(V6,V8,
N6,N8,N10-N14)(Table1).Inthelattercase,themostrepresentedsequencewaseasilyana-
lyzedandcorrespondedtothatofC.sinensis.Forthetemplateswithpeaksofthesameinten-
sity,a2%agarosegelelectrophoresisshowedclearseparationofthetwobands;incontrast,in
theothertemplates,thePCRwasclonedandsequenced.TheanalysesofSangerelectrophero-
gramsappliedtothesefragmentsconfirmedthepresenceofbothC.pubicostaandC.sinensis
inthesameamplifiedtemplates(Table1).Asanticipatedinthepreviousparagraph,thesedata
wereconfirmedusingagenotypingapproach.
Anomalousteatemplate. Accordingtothepreviouscapillarygenotypingdata,thefol-
lowingtwotypologiesofanomalousteatemplatesoccurinoursampling:(I)(simple)sample
withadditionalpeak(s),evenifC.sinensiswascertifiedastheonlyingredient(N11)and(II)
PLOSONE|https://doi.org/10.1371/journal.pone.0178262 May23,2017 10/17