Rev. bras. farmacogn. vol.26 número5

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w ww.e l s e v i e r . c o m / l o c a t e / b j p

Original

Article

Identification

of

reference

genes

for

gene

expression

normalization

in

safflower

(

Carthamus

tinctorius

)

Fei

Liu

a

_,

_Dan

_Guo

a

_,

_Yan

_Hua

_Tu

a

_,

_Ying

_Ru

_Xue

a

_,

_Yue

_Gao

b,∗

_,

_Mei

_Li

_Guo

a,∗

a_Department_of_{Pharmacognosy,}_College_of_Pharmacy,_Second_Military_Medical_University,_Shanghai,_China

b_Teaching_Experimental_Center,_College_of_Pharmacy,_Second_Military_Medical_University,_Shanghai,_China

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received5June2015

Accepted24May2016

Availableonline16June2016

Keywords:

Safflower

Referencegene

RT-qPCR

a

b

s

t

r

a

c

t

Safflower(CarthamustinctoriusL.,Asteraceae)isanimportantoilcropandmedicinalplant.Gene expres-sionanalysisisgainingimportanceintheresearchofsafflower.QuantitativePCRhasbecomeapowerful methodforgenestudy.ReferencegenesareoneofthemajorqualificationrequirementsofqPCRbecause theycanreducethevariability.Toidentifythereferencegenesinsafflower,ninecandidategenesof thehousekeepinggeneswereselectedfromtheESTlibraryofsafflowerconstructedbyourlab:CtACT (actin),CtGAPDH(glyceraldehyde3-phosphatedehydrogenase),CtE1F4A(elongationfactor1alpha), CtTUA(alpha-tubulin),CtTUB(beta-tubulin),CtPP2A (serine/threonine-proteinphosphatase),CtE1F4A (eukaryoticinitiationfactor4A),CtUBI(Ubiquitin),andCt60S(60Sacidicribosomalprotein). Expres-sionstabilitywasexaminedbyqPCRacross54samples,representingtissuesatdifferentfloweringstages andtwochemotypeofsafflowerlines.Weassessedtheexpressionstabilityofthesecandidategenesby employingfourdifferentalgorithms(geNorm,NormFinder,Ctapproach,andBestKeeper)andfound thatCtUBIandCt60Swerethehighlyrankedcandidategenes.CtUBIandCt60Swereusedasreference genestoevaluatetheexpressionofCtFAD2-10andCtKASII.OurdatasuggestCtUBIandCt60Scouldbe usedasinternalcontrolstonormalizegeneexpressioninsafflower.

Introduction

Safflower(CarthamustinctoriusL.,Asteraceae)isathistle-like, self-compatible, annual, diploid (2n=24) herbaceous crop that thrives in hot, dry climates and survives on minimal surface moisture.Thesafflowercultivarsaredistributedfromthe Mediter-raneantothePacificOceanatlatitudesbetween20◦_S_and₄₀◦_N,

whereverahot,dryclimatesuitsthecrop.Insomecountries, saf-flowerhasbecomeanimportantcropduetotherichcontentof edibleoil,whichhasthehighestpolyunsaturated/saturatedratios ofanyoilavailable(Gecgeletal.,2007;Yeilaghietal.,2012). Saf-flowerisalsoavaluablemedicinalplant.Theflowersofsafflower canbeusedforthetreatmentofcardiovascularand cerebrovascu-lardiseases(Tianetal.,2010;AsgarpanahandKazemivash,2013) andtheextractsfromsafflowercanbeusedasanti-inflammatory agentsaswell(Junetal.,2011).Thereareseveralstudiesabout thegeneticvariationofsafflowercultivarsusingvariousmolecular DNAmarkers,suchasSNP(ChapmanandBurke,2007),SRAP(Peng etal.,2008),ISSR(Chapmanetal.,2009;Golkaretal.,2011),and

∗ Correspondingauthors.

E-mails:[email protected](Y.Gao),[email protected](M.L.Guo).

AFLP(Zhangetal.,2009;Fengetal.,2010;Lietal.,2010). Mean-while,inthepreviousstudyofourlab,wehavefoundthatobvious differentiationhasoccurredinsafflowerpopulationsfromexterior appearancetoinnerchemicalconstituent,duetothelong natu-ralandartificialselection(Pengetal.,2008;Zhangetal.,2009; Fengetal.,2010;Lietal.,2010).Fromourconclusion, hydroxysaf-floryellowA(HYSA),astheactivecompound,isthemainfactorto determinethediversityofsafflower(Yangetal.,2011).

Assecondarymetabolitesinsafflower,theflavonoidsaremajor componentsoftheextractsfromtheflowerswithmedicinal func-tion(AndersenandMarkham,2010;AsgarpanahandKazemivash, 2013).HSYA,oneofthemostimportantflavonoidswithaunique presenceintheflowerpetalsofsafflower,playsamajorrolein thepharmacologicaleffectsofflavonoids(Fengetal.,2013;Sun et al., 2013; Wang et al., 2013). The flowering process of saf-flowerisacomplexdevelopmentassociatedwiththebiosynthesis offlavonoids,particularlythecolorchange(Tanakaetal.,2010). Insafflowerseeds,theidentificationandinitialcharacterizationof theFAD2genefamilywithelevenmembersprovidesaninsight intotheprincipaldeterminantsofsynthesisoflinoleicacidin saf-flowerseedoil(Caoetal.,2013;Liuetal.,2013).AndtheCtFAD3

enzymeactivityisimportantforfattyaciddesaturationinsafflower flower(Guanetal.,2014).Theunderstandingoftheexpressionof

http://dx.doi.org/10.1016/j.bjp.2016.05.006

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thekeygeneswillhelpinvestigatethemechanisminvolvedinthe biosynthesisofflavonoidsandfattyacids.

Quantitativereal-timePCR(qPCR)isaroutinetoolwithhigh sensitivityand specificityfor thequantificationofgene expres-sion(Gachonetal.,2004).Whenwestudythegeneexpression, thereferencegenescanbeusedinthenormalizationof experi-mentalvariations,suchastheamountofmaterial,extractionof RNA,efficiencyofreversetranscription, andsoon(Silver etal., 2006;Gutierrezetal.,2008;Bustinetal.,2009;Guéninetal.,2009). Itisimportanttonormalizetheexpressionofthetargetgenein ordertoobtainreliableand accurateresultsbyusingthe refer-encegenes. Housekeeping genes areoften considered asbeing constantlyexpressedandarealwaysusedasinternalstandards. Butmanyresearchesshowedthattheexpressionofhousekeeping genesvariesindifferentmaterialsandeveninthesame materi-alsduringdifferenttreatments(Thellinetal.,1999;Schmidtand Delaney,2010).Sotheevaluationofreferencegenesindifferent planttissuesduringbioticorabioticstressisnecessaryfor expres-sionstudies.

Housekeepinggenes,suchasactin(Basetal.,2004),elongation factor1alpha(SchmidtandDelaney,2010;Lietal.,2012), alpha-tubulin(JarosovaandKundu,2010;Wanetal.,2011),beta-tubulin (JarosovaandKundu,2010),serine/threonine-proteinphosphatase (Liu et al., 2012), eukaryotic initiationfactor 4A (Silveiraet al., 2009),ubiquitin(Infanteetal.,2008), and60Sacidic ribosomal protein(Leetal.,2012),arecommonly usedasreferencegenes forgene expressionstudies.Therearestudieswhich haveused referencegenesforthenormalizationofgeneexpressionin saf-flower(Lietal.,2012;Caoetal.,2013).Butthesereferencegenes were not evaluated for qPCR data in safflower tissues. In this study,nine housekeepinggenesnamelyCtACT(actin), CtGAPDH

(glyceraldehyde 3-phosphate dehydrogenase), CtE1F4A (elonga-tionfactor1alpha),CtTUA(alpha-tubulin),CtTUB(beta-tubulin),

CtPP2A(serine/threonine-proteinphosphatase),CtE1F4A (eukary-oticinitiationfactor4A),CtUBI(Ubiquitin),andCt60S(60Sacidic ribosomal protein) were selected as candidate reference genes fromourconstructededtranscriptome dataof safflower.Atthe sametime,tworepresentativechemotypeofsafflowervarieties, with-HSYA(yellowflower)andwithout-HSYA(whiteflower)were selectedtoidentifyreferencegenes.Theexpressionofthesegenes in the bract, ovary, stem, leaf, calyx,petal (I–IV, fourdifferent stages ofdevelopmentduring flowering)of twosafflower lines wereanalyzed using four differentalgorithms, that is, geNorm (Vandesompeleetal.,2002),NormFinder(Andersenetal.,2004),

Ctapproach (Silveret al.,2006), andBestKeeper (Pfaffl etal., 2004).Ourstudywillhelptoachievemoreaccurateandreliable resultsinawidevarietyofsafflowersamples.

Materialsandmethods

Plantmaterial

Theseedsoftwolinesofsafflower,CarthamustinctoriusL., Aster-aceae(ZHH0082withyellowflowerandXinHonghuaNO.7with whiteflower)fromtheChinesepopulationswerecultivatedinthe fieldatthemedicinalbotanicalgardenofSecondMilitary Medi-calUniversity.Thesamples(bract,ovary,stem,leaf,calyx,petal (I–IV))werecollectedfromthreedifferentfloweringplants (bio-logicaltriplicates)andimmediatelyfrozeninliquidnitrogen.All 54sampleswerethenstoredat−70◦CforRNAextraction.

TotalRNAextraction

Total RNA was isolated using the RNA Extraction Plant Mini Kit (LifeFeng, Shanghai, China) according to the protocol

providedbythemanufacturer.RNAconcentrationandqualitywere measuredwiththeNanoDropspectrophotometer(NanoDrop Tech-nologies)andagarosegelelectrophoresis.OnlytheRNAsamples withA260/A280 ratiosbetween1.9 and 2.1 and A260/A230 ratios

greaterthan2.0wereusedforcDNAsynthesis.

FirststrandcDNAssynthesis

FirststrandcDNAsweresynthesizedaccordingtothe manufac-turer’sinstructionsofTransScript®_One-Step_gDNA_Removal_and

cDNASynthesisSuperMix(TransGenBiotech,Beijing,China).Total RNA(1␮g),suitablevolumesofH2O,and1␮lanchoredOligo(dT)

18primer(0.5␮g/␮l)weremixedandincubatedat65◦_C_for₅_min

followedbycoolingonice.Thereversetranscriptasereactionswere startedafteradding10␮l2×TSreactionmix,1␮lEnzymeMix,and

1␮lgDNARemoverat42◦_C_for₄₅_min._And_then_the_mixture_was

heatedfor5minat85◦_C_for_inactivating_the_enzymes._All_cDNA

sampleswerediluted1:10withRNase-freewaterbeforebeingused astemplatesintheqPCRanalysis.

Q-PCR

Ninesequenceswereselectedascandidatereferencegenesfrom thepetalESTlibrariesofC.tinctorius(Table1).TheqPCRprimers weredesignedusingtheBeaconDesignerv8.0software.Amplicon lengthsvariedfrom75to200bp,withmeltingtemperatures(Tm) varyingbetween52◦_C_and₆₀◦_C_and_primer_lengths_between₁₈

and22bp.PrimerpairsweretestedforspecificitybyqPCR,followed byadissociationcurveandagarosegelelectrophoresis(Fig.S1). PCRreactionswereperformedin96-wellplateswiththeCFX96 TouchTM_Real-Time_PCR_Detection_System_(Bio-Rad_{Laboratories,}

CA,USA) andABI7500Real-Time PCR System(Applied Biosys-tems,FosterCity,USA).TheCFX96wasusedtoobtaintheqPCR dataofninecandidategenesandABI7500wasusedtoobtainthe qPCRdataofCtFAD2-10(KC257456,F-CTTTACCGTATGGCTTTAG, R-GTGTGTTGAAGGTATGTG), CtKASII (KC257458, F-GACAGGTTTAT-GCTCTAC, R-CAATCAGAACTCCACATC), and two stable reference genes.Eachreactioncomprising20␮lofthefollowingwas pre-paredasfollows:0.3␮mforwardprimer,0.3␮mreverseprimer, 10␮l 2× TranStartTM Top Green qPCR SuperMix (TransGen

Biotech,Beijing, China),2␮lcDNA,0.4␮lpassivereference dye II(onlyused inABIsystem), and suitablevolumesof H2O.The

thermocyclingconditionsweresetat95◦_C _for₃₀_s,_followed_by

40cyclesof10sat95◦_C_for_template_{denaturation,}₁₅_s_at_Tm_for

annealing,and20s(30sforABIsystem)at72◦_C_for_extension_and

fluorescencemeasurement.Afterwards,thedissociationcurvewas obtainedbyheatingtheampliconfrom60◦_C_to₉₅◦_C _and

read-ingateach0.5◦_C_increase_(0.2◦_C_for_ABI_system)._Three_technical

replicateswereperformedforeachPCRreaction.

Dataanalysis

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Table1

NinecandidatereferencegenesandtheirprimersequencesforqPCR.

Geneidentification/genedescription EvalueID(%) Primersequence Ampliconsize Amplification efficiency

GeneBank Accession Number

Tm

CtACT 0.0 F-ACTGGTGTTATGGTAGGA 89 1.85 KJ634809 60

actin 100 R-GGATACTTCAAGGTAAGGATA

CtGAPDH 0.0 F-GTTGTGGACTTAACCGTAA 78 1.83 KJ634805 53

glyceraldehyde3-phosphatedehydrogenase 93 R-TTCTGATTCCTCCTTAATAGC

CtEF1 0.0 F-CCAAGAGACCATCAGACAA 76 1.88 KJ634806 58

elongationfactor1alpha 98 R-GGCACAGTTCCAATACCA

CtTUA 0.0 F-CTACACCAACCTCAATCG 91 1.89 KJ634803 58

␣-tubulin 99 R-AGTCACATCCACATTCAAG

CtTUB 0.0 F-GGAAGAGGAGTATGATGA 93 1.88 KJ634802 54

␤-tubulin 99 R-AATGGCAGTTGAGATTAC

CtPP2A 0.0 F-GAGAACCTGATGTAACGAGAC 80 1.89 KJ634804 58

serine/threonine-proteinphosphatase 95 R-ACCACCAAGCAAGCAATC

CtE1F4A 0.0 F-CGCTGATTACATTAAGATG 90 1.85 KJ634807 54

eukaryoticinitiationfactor4A 98 R-AATTGGAAGATATCGTAGAT

CtUBI 3e−87 _{F-TCACTTATGTTTACCAGAA} ₁₅₀ _1.85 _KJ634808 ₅₄

Ubiquitin 98 R-GCTTTCAATTTCAACTCA

Ct60S 9e−165 _{F-CATCCATTATCCAACAATC} ₉₂ _1.86 _KJ634810 ₅₄

60Sacidicribosomalprotein 88 R-AAGAGTAATCAGTCTCCA

Results

SelectionofputativereferencegenesforqPCRexperiments

Inordertofindthebestreferencegenesinsafflower,nine

house-keeping genes, CtACT, CtGAPDH, CtE1F4A, CtTUA, CtTUB, CtPP2A,

CtE1F4A,CtUBI,andCt60S,wereselectedasputativereferencegenes (Table 1).These genes were usedto BLAST search against a C. tinctoriusEST(expressedsequencetag)libraryconstructedbyour laboratoryfromthepetalduringflowering.Thehousekeepinggene commonlyusedasreferencegeneisthe18SrRNA.Inthisstudy, however,theCtvaluesof18Sinthesafflowertissueswereatleast 10cycleshigherthantheCtvaluesoftheothercandidategenes (datanot shown).Such highCtvalues make 18Sunsuitable for useasareferencegene(Lietal.,2012).Thedissociationcuresof ampliconsoftheseninecandidategenesexhibitedasinglepeak showedthattheprimerpairswerespecificity,whichwerealso ver-ifiedbyagarosegelelectrophoresisresults(Fig.S1).Theexpression ofthesegenesinbract,ovary,stem,leaf,calyx,petal(I–IV,four dif-ferentstagesofdevelopmentduringflowering,Fig.2,TableS1)was analyzed.Sincefloweringbeginsintheoutercircleoffloretsand progressescentripetallytowardthecenterofthecapitulum,the

fourstagesofpetalscanbefoundinonecapitulum.Thereasonwe differentiatethefourstagesofpetalsisthatthecompoundshave significantvariationduringthedevelopmentof theC. tinctorius

flowers(Salemetal.,2011)andthegeneexpressionsaredifferent aswell(Mallonaetal.,2010).Inourstudy,atotalof54samples(two linesofsafflower(ZHH0082andXinHonghuaNO.7),nineorgans, threebiologicalreplicates)wereusedtoevaluatethestabilityof putativereferencegenes.Thesignificantdifferencebetweenthe ZHH0082andXinHonghuaNO.7wasthecolorofflowers.The flow-ersofZHH0082wereyellowandtheflowersofXinHonghuaNO.7 werewhite.

Expressionstabilityofputativereferencegenesviadifferential statisticalanalyses

Thecyclethreshold(Ct)valuesprofilingofcandidategenesin differentorgansandsafflowerlinesareshowninFig.3.Themean ofeachoftheninecandidategenesin54samplesareshownin

TableS2.Inthisstudy,geNorm,NormFinder,Ctapproachand BestKeeperwereusedtoanalyzethestabilityofgeneexpression.

GeNormcalculatedthegeneexpressionstabilityvalueM(the average pairwise variation of a particular gene with all other

Table2

RankingofthecandidatereferencegenesaccordingtotheirvaluescalculatedbygeNorm,NormFinder,CtapproachandBestKeeperinAS,YandW.

Rank Allsamples(AS) Yellow(Y) White(W)

geNorm (M) NormFinder (SV)

Ct

approach

BestKeeper geNorm (M) NormFinder (SV)

Ct

approach

BestKeeper geNorm (M) NormFinder (SV)

Ct

approach

BestKeeper

1 CtUBI Ct60S Ct60S CtPP2A Ct60S CtUBI Ct60s CtPP2A CtACT CtPP2A CtUBI CtPP2A

0.776 0.061 0.671 0.774 0.063 0.670 0.756 0.062 0.643

2 Ct60S CtACT CtACT CtUBI CtUBI Ct60s CtACT CtUBI CtTUB CtEF1 CtTUB CtUBI

0.777 0.066 0.673 0.781 0.070 0.672 0.759 0.072 0.649

3 CtACT CtUBI CtUBI Ct60S CtTUB CtACT CtTUB CtE1F4A CtGAPDH CtACT Ct60S Ct60S

0.788 0.068 0.681 0.788 0.081 0.683 0.767 0.092 0.651

4 CtTUB CtGAPDH CtTUB CtTUB CtACT CtE1F4A CtUBI Ct60S CtUBI Ct60S CtACT CtTUB

0.837 0.076 0.724 0.804 0.081 0.689 0.770 0.113 0.660

5 CtE1F4A CtEF1 CtE1F4A CtE1F4A CtEF1 CtTUB CtGAPDH CtTUB Ct60S CtGAPDH CtE1F4A CtE1F4A

0.839 0.078 0.736 0.814 0.085 0.748 0.773 0.123 0.699

6 CtGAPDH CtE1F4A CtGAPDH CtACT CtE1F4A CtGAPDH CtEF1 CtACT CtE1F4A CtTUA CtGAPDH CtGAPDH

0.845 0.085 0.748 0.815 0.088 0.772 0.780 0.137 0.700

7 CtEF1 CtTUA CtEF1 CtGAPDH CtGAPDH CtEF1 CtPP2A CtEF1 CtPP2A CtUBI CtPP2A CtACT

0.851 0.127 0.785 0.878 0.103 0.863 0.856 0.144 0.766

8 CtPP2A CtTUB CtPP2A CtTUA CtPP2A CtPP2A CtE1F4A CtGAPDH CtEF1 CtE1F4A CtEF1 CtTUA

0.975 0.127 0.818 1.021 0.148 0.869 0.880 0.146 0.781

9 CtTUA CtPP2A CtTUA CtEF1 CtTUA CtTUA CtTUA CtTUA CtTUA CtTUB CtTUA CtEF1

1.104 0.138 0.957 1.130 0.168 0.974 1.077 0.154 0.926

RankAggrega _Ct_60S/_Ct_UBI/_Ct_A_CT _Ct_UBI/_Ct_60S/_Ct_TUB _Ct_UBI/_Ct_60S/_Ct_TUB

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7

A

B

30

25

20

15

10

5

0 6

5

4

CtUBI+Ct60S CtUBI Ct60S 3

2

1

0

Relativ

e quantification of

CtF

AD2-10

Relativ

e quantification of

CtKASII

C

B G J L T I II III IV B C G J L T I II III IV

Fig.1. RelativequantificationofCtFAD2-10andCtKASIIexpressionusingdifferentinternalcontrolsanalyzedbythe2−Ct_method_in_all_samples._Relative_{quantification}_of

CtFAD2-10andCtKASIIexpressionweredetectedusingCtUBIandCt60Sbothindividuallyandincombination.TherelativeexpressionofpetalIwassetto1.(A)CtUBI,Ct60S

andthegeometricaverageofCtUBIandCt60SwereusedasinternalcontrolsforCtFAD2-10expression.(B)CtUBI,Ct60SandthegeometricaverageofCtUBIandCt60Swere

usedasinternalcontrolsforCtKASIIexpression.B,bract;C,calyx;L,leaf;O,ovary;S,stem;I–IV,petalI–IV.

A

B

C

3 cm

2 cm

1 cm

3 cm

2 cm

1 cm

I II III IV

F

D

E

Fig.2. Twolinesofsafflower.(1)A,B,CaretheZHH0082(Yellow);(2)D,E,FaretheXinHonghuaNO.7(White);(A,D)capitulumofsafflower;(B,E)longitudinalsectionof

capitulum;(C,F)fourdifferentstagesofthepetalduringflowering.Blackarrowspointtothesamplescollectedfromsafflower.B,bract;O,ovary;T,stem;L,leaf;C,calyx;P, petal.

controlgenes)for areferencegene. ThelowertheM value,the morestablyexpressedthegeneis.NormFinderusedamodel-based approachfor identifyingtheoptimalnormalizationgene(s).The intra-andintergroupvariationswerecalculatedandincludedin thegeneexpressionstabilityvalues. Geneswiththelowest val-ueshadthemoststableexpression.GeNormandNormFinderused the linear scale expression quantities, which can be calculated fromtheCtvaluesbyusingastandardcurveorcomparativeCT method(Schmittgenand Livak, 2008).Ctapproach compared theCtvariationofpairsofhousekeepinggeneswithinindividual

samplestoidentifythereferencegenes.ThegeneswithlowerCt

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16

CtA CT

CtEF1 CtTU A

CtT UB

CtPP2A CtE1F4A

CtUBICt60S CtGAP

DH

18 20

Cycle threshold v

alues (Ct)

Cycle threshold v

alues (Ct)

Cycle threshold v

alues (Ct)

22 24 26 28 30 32 34

16

CtA CT

CtEF1 CtTU A

CtT UB

CtPP2A CtE1F4A

CtUBICt60S CtGAPDH

18 20 22 24 26 28 30 32 34

A

B

C

16

CtA CT

CtEF1 CtTU A

CtT UB

CtPP2A CtE1F4A

CtUBICt60S CtGAPDH

18 20 22 24 26 28 30 32 34

Fig.3. Ctvaluesofcandidatereferencegenes.ExpressiondatashowedtheCtvalues(blackspots)ofeachgene.Theverticalboxesrepresentthe25thand75thpercentiles

witherrorbars(whiskers).Thelinesacrosstheboxesaredepictedasthemedian.(A)Allsamplesofsafflower,(B)samplesofZHH0082(Yellow),and(C)samplesofXin

HonghuaNO.7(White).

InAS(all54samplesofthetwolinesofsafflower),CtUBI,Ct60S, andCtACTwerethethreemoststablyexpressedcandidategenes calculatedbygeNorm,NormFinder andCt.In thelistof Best-Keeper,thetopthreewereCtPP2A,CtUBI,andCt60S.Themoststably expressedgeneidentifiedbyBestKeeperwasCtPP2A,whichhad thehighestvariationintheresultsfromothermethods(geNorm ranked8,NormFinderranked9,andCtranked8).Thetopthree aggregateordercalculatedbyRankAggregwasCt60S/CtUBI/CtACT

inAS(Table2).

TheresultsofASwereanalyzedbysegregatingintotwo sub-groups, Y and W. The Y group included 27 samples collected fromZHH0082(Yellow)and Wcontained27samplesfromXin HonghuaNO.7(White).Thefouralgorithmsproducedsimilar rank-inglistsinASand Y.Butthere weremoredifferencesbetween two lines, Yand W.In Y,Ct60S was thefirst, second, and the first best-ranked gene in the geNorm, NormFinder, and Ct

approach,respectively.However,Ct60Swasrankedatfifth,fourth, and third in W. In the analysisof NormFinder, theposition of

CtPP2AhadremarkablevariationbetweenYandW,whichwasthe eighthinYbutthefirstinW.Althoughtherewerediscrepancies amongtheordersproducedbythesealgorithmsinthetwolines, theaggregateorders werethesame, namelyCtUBI/Ct60S/CtTUB

(Table2).

Theresultsanalyzedbyalgorithmsweredifferentinall Flow-ers(F),Yellow-Flower(YF,flowersofZHH0082),andWhite-Flower (WF,flowersof XinHonghua NO.7).Accordingtotheresultsof NormFinder,theCtE1F4Awasthesecondmoststablyexpressed geneinF,seventhinYF,andfirstinWF.Inparticular,CtE1F4Awas themoststablyexpressedgeneinYFcalculatedbythegeNorm, NormFinder,andCtapproach.TheupgradedpositionsofCtE1F4A

resultedinchangingoftheaggregateordersinSA,F,andYF.But theorderinWFwasCt60S/CtUBI/CtACT,whichwasthesameasthat inAS(Table3).

Ithasbeensuggested thattheuseoftwo ormorereference genesforRT-qPCR studiesmightgenerate morereliableresults (Vandesompeleet al.,2002).To determinetheoptimalnumber ofgenesrequiredforaccuratenormalization,pairwisevariations

Vn/Vn+1werecalculatedbasedonthenormalizationfactor(NFnand

NFn+1)valuesaccordingtothegeNormalgorithm.IftheVn/Vn+1of ngeneswerebelowthecut-offvalue,theadditionalhousekeeping gene(n+1)wasnotnecessaryforreliablenormalization.The cut-offvalueofpairwisevariationsVn/Vn+1wassetat0.15.Intheresults

ofthesixgroupsanalyzed,atleastfourreferencegenescanbeused foraccuratenormalizationinAS(V2/V3=0.173,V3/V4=0.176),Y

(V2/V3=0.193,V3/V4=0.175)and F(V2/V3=0.193,V3/V4=0.183).

InW(V2/V3=0.22)andYF(V2/V3=0.203), thebestnumberwas

three.Twogenesweresufficientforexpressionnormalizationin WF(V2/V3<1.5)(Fig.4).

QuantificationofCtFAD2-10andCtKASIIexpressionwithstable referencegenes

TheexpressionofCtFAD2-10,oneoftheFAD2genefamilyin safflower,wasnormalizedusingKASIIinanearlystudy(Caoetal., 2013).Ketoacyl-acylcarrierproteinsynthaseII(KASII)alsoplays arolein thefattyacidbiosynthesisinplants.Theexpressionof

KASIIwasnotevaluatedforusingasaninternalcontrolfor nor-malization.Whencomparedwithorgansobtainedfromdifferent individuals,singlehousekeepinggeneRNAlevelswerenot appro-priatetobeusedfornormalizationofRNAlevels(Tricaricoetal., 2002).Tofurtherverifythesuitabilityofreferencegenesselected inthepresentstudy,CtFAD2-10andCtKASIIexpressionlevelswere detectedinsafflower(Fig.1).Therelativeexpressiondatawere cal-culatedusing2−Ct_method._The_internal_control_genes_were_the CtUBIandCt60SandthegeometricaverageofCtUBIandCt60S.

Itspattern of expressionwasassessed in allsamples(bract, ovary,stem,leaf,calyx,petal(I–IV)).Similarexpressionpatterns weregeneratedwheneitheroneortwoofthemoststablegenes wereusedfor normalization(Fig.1A and B).Whennormalized thegeometricaverageofCtUBIandCt60S,transcriptabundance ofCtKASIIgraduallyincreasedindifferentdevelopmentalstages offlower,peaking attheStageIV(Fig.1B).It suggeststhatthe

0.25

V2/3 V3/4 V4/5 V5/6 V6/7 V7/8 V8/9

0.20

0.15

0.10

0.05

0.00

AS Y W F YF WF

P

a

irwise v

a

riations

Fig.4. OptimalnumberofreferencegenesfornormalizationaccordingtogeNorm

results.Pairwisevariation(Vn/Vn+1)wasmeasuredbetweenthenormalization

fac-torsNFnandNFn+1.Theinclusionofanadditionalreferencegenewasnotrequired

belowthecut-offvalueof0.15(thechartreuseline).AS,allsamples;Y,ZHH0082

(Yellow);W,XinHonghuaNO.7(White).F,flower;YF,yellowflower;WF,white

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Table3

RankingofthecandidatereferencegenesaccordingtotheirvaluescalculatedbygeNorm,NormFinder,CtapproachandBestKeeperinF,YFandWF.

Rank All flowers (F) Yellow-Flower (YF) White-Flower (WF)

geNorm(M) NormFinder (SV)

Ct

approach

BestKeeper geNorm(M) NormFinder (SV)

Ct

approach

BestKeeper geNorm(M) NormFinder (SV)

Ct

approach

BestKeeper

1 Ct60S CtACT Ct60S CtPP2A CtE1F4A CtE1F4A CtE1F4A CtPP2A Ct60S CtEF1 Ct60S CtPP2A

0.705 0.073 0.625 0.727 0.061 0.635 0.604 0.039 0.550

2 CtE1F4A CtEF1 CtACT CtUBI Ct60S CtACT Ct60S CtUBI CtE1F4A Ct60S CtACT CtUBI

0.742 0.086 0.650 0.755 0.071 0.665 0.657 0.050 0.583

3 CtUBI CtUBI CtE1F4A Ct60S CtTUB CtGAPDH CtTUB Ct60S CtACT CtPP2A CtTUB Ct60S

0.749 0.097 0.661 0.764 0.079 0.665 0.671 0.060 0.591

4 CtACT CtE1F4A CtUBI CtTUB CtUBI Ct60S CtACT CtE1F4A CtTUB CtUBI CtE1F4A CtTUB

0.777 0.097 0.665 0.768 0.098 0.687 0.678 0.060 0.593

5 CtTUA Ct60S CtGAPDH CtE1F4A CtGAPDH CtTUB CtUBI CtTUB CtTUA CtACT CtUBI CtE1F4A

0.806 0.100 0.707 0.819 0.109 0.692 0.683 0.091 0.594

6 CtGAPDH CtGAPDH CtTUB CtACT CtACT CtUBI CtGAPDH CtACT CtUBI CtTUA CtTUA CtGAPDH

0.808 0.112 0.709 0.829 0.110 0.697 0.687 0.098 0.637

7 CtTUB CtTUB CtTUA CtGAPDH CtEF1 CtEF1 CtEF1 CtEF1 CtPP2A CtE1F4A CtPP2A CtACT

0.809 0.123 0.754 0.836 0.130 0.767 0.745 0.126 0.666

8 CtEF1 CtPP2A CtPP2A CtTUA CtTUA CtTUA CtPP2A CtGAPDH CtGAPDH CtGAPDH CtGAPDH CtTUA

0.875 0.127 0.755 0.888 0.146 0.817 0.755 0.158 0.677

9 CtPP2A CtTUA CtEF1 CtEF1 CtPP2A CtPP2A CtTUA CtTUA CtEF1 CtTUB CtEF1 CtEF1

0.885 0.128 0.760 0.981 0.183 0.826 0.900 0.164 0.737

RankAggrega _Ct_UBI/_Ct_E1F4A/_Ct_60S _Ct_E1F4A/_Ct_60S/_Ct_TUB _Ct_60S/_Ct_UBI/_Ct_A_CT

a_Top_three_ranked_genes_were_shown.

CtKASIIwasnotastablegeneforgeneexpressionnormalization. TheexpressionlevelsofCtFAD2-10werehighinpetals,especiallyin thefloweringstageIV(Fig.1A).Whenonlyonereferencegenewas employed,expressionprofilesofCtKASIIweresimilar(Fig.1B),but differenceswereevidentinestimatedtranscriptabundance,which washigherwhennormalizedagainstCtUBIthanagainstCt60S.

Discussion

Extensivestudiesarebeingcarriedoutonthesafflower,because itisbothanoilcropandamedicinalplant.Furthermore,research onthebiosynthesisoftheseflavonoidsandfattyacidmetabolism werealsoreportedrecently(Caoetal.,2013;Lietal.,2012).Because itisasensitive,specific,reproducible,andconventionalmethod, qPCR has become an essential tool for gene expression analy-sis,especially thegene expressionofthesecondary metabolites pathway(Al-Ghazietal.,2009).

Althoughsomereferencegeneshavebeenusedforthe normal-izationofgeneexpressioninsafflower,butthesereferencegenes werenotevaluated forqPCR datainsafflower tissues(Lietal., 2012;Caoetal.,2013).Recently,thesafflowerreferencegeneswere reportedinourprocessofrevising,buttheirtranscriptomedata wereonlyfromtheseedandalgorithmsforthereferencegenes evaluationwereincomplete(Lietal.,2015).Inthisstudy,two repre-sentativechemotypeofsafflowerlines,with-HSYA(yellowflower) andwithout-HSYA(whiteflower),waschosentoidentifythe saf-flowerreferencegenes.Theninehousekeepinggeneswereselected asthecandidategenesfromtranscriptomedataofdifferent devel-opmentalfloweringstageofsafflowerconstructedbyourlab.The profilingofthesegeneswascarriedoutusingfouralgorithmsand wasrankedbyRankAggreg.All54sampleswereseparatedintosix groupsinordertoevaluatethevariationofthesecandidategenesin differentlines,organs,andflowers.Althoughthereweredifferent listsofthemoststablyexpressedgenesfromtheresultsofgeNorm, NormFinder,CtapproachandBestKeeper,theCtUBIandCt60S

wereinthetopthreerankedgenesaccordingtotheRankAggreg analysisinthefivegroups(excepttheYF).Ourfindingswerein accordancewiththeresultthatUBQ(ubiquitin)showedhighly sta-bleexpressioninArabidopsis(Czechowskietal.,2005).Similarly,

UBQwastherecommendedhousekeepinggenefornormalization inpoplar(Brunneretal.,2004)andrice(Jainetal.,2006).However,

UBQwasnotsuggestedusingasinternalcontrolstonormalizegene expressioninsoybean(Jianetal.,2008).Asfor60S,itwasfoundto

bethebestreferencegeneindifferenttissuesandundervarious stressconditionsinsoybean(Leetal.,2012).Therefore,weused

CtUBIandCt60Sasreferencegenesandevaluatedtheexpression ofCtFAD2-10andCtKASIIinsafflower.CtKASIIhadbeenusedasthe referencegenetoprofiletheCtFAD2geneexpression.But accord-ingtoourstudy,theCtKASIIwasnotsuitableforgeneexpression normalizationbecauseitsexpressionswerehighlyvariable.

Ourresultsindicatedthatthestabilityofreferencegene expres-sionmustbevalidatedforeachlineandthedevelopmentstages offloweringinsafflower.Basedontheseresults,westrongly sug-gestthatCtUBIandCt60Sshouldbeusedasreferencegenesfor geneexpressioninsafflower.Theidentificationofthesetwostable referencegeneswillenableaccurateandreliablegeneexpression studiesrelated tofunctionalgenomicsandmetabolomics about safflower.

Ethicaldisclosures

Allauthorsdeclaretoourmanuscriptdidnotinvolveanyethical issues.

Authors’contribution

XYRandGDDwereresponsibleforthecollectionofplant sam-ple.LFandTYHdevelopedtheanalyticalmethodology.LFandGY wereresponsiblefordataanalysis.LFandGMLdesignedthestudy andwrotethemanuscript.Alltheauthorsreadthefinalmanuscript andapprovedthesubmission.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

(7)

AppendixA. Supplementarydata

Supplementarydataassociatedwiththisarticlecanbefound,in theonlineversion,atdoi:10.1016/j.bjp.2016.05.006.

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