Cytochrome C Oxidase I (COI)

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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

Plastrum

Testudinis

used

in

traditional

medicine

with

DNA

mini-barcodes

Mingyang

Chen

a

_,

_Xuan

_Han

b

_,

_Jiao

_Ma

c

_,

_Xinyan

_Liu

a

_,

_Meng

_Li

a

_,

_Xiaona

_Lv

a

_,

_Haisheng

_Lu

d

_,

Guangxi

Ren

a

_,

_Chunsheng

_Liu

a,∗

a_School_of_Chinese_Materia_Medica,_Beijing_University_of_Chinese_Medicine,_Beijing,_China

b_Dongzhimen_Hospital,_Beijing,_China

c_School_of_Humanities,_Beijing_University_of_Chinese_Medicine,_Beijing,_China

d_Wangjing_Hospital_of_China_Academy_of_Chinese_Medical_Sciences,_Beijing,_China

a

r

t

i

c

l

e

i

n

f

o

Articlehistory:

Received30December2017 Accepted10April2018 Availableonline14May2018

Keywords:

PlastrumTestudinis

Chinemysreevesii

COI

DNAmini-barcodes

a

b

s

t

r

a

c

t

ThecarapaceofthetortoiseChinemysreevesiiisaningredientof“Guijia”,atraditionalChinesemedicine. However,C.reevesiiisdifficulttoraiseinaquacultureandisrareinthewild.Counterfeittabletsare madefromcarapacesofotherspecies.InadditiontoC.reevesii,otherspeciesincludingMauremyssinensis,

IndotestudoelongateandTrachemysscriptahavebeenusedinPlastrumTestudinisaswell.Afterprocessing,

thesecarapacesaredifficulttoidentifyonthebasisofmorphologicalcharacteristics,whichimpedeslaw enforcement.OurstudyusedDNAbarcodingtechnologytoidentifyC.reevesiianditssubstitutes.We extractedconcentratedgenomicDNAforPCRamplification.Basedontheanalysisof61full-lengthCOI sequences,wedesignedfourpairsofmini-barcodeprimers:Tu-A,Tu-B,Tu-CandTu-D.TheTu-Bprimers sequencedgenomicDNAwithasuccessrateof76.47%,andtheTu-DprimerssequencedgenomicDNA withasuccessrateof88.24%.Theidentificationefficiencyofthesetwomini-barcodeswas70.59%and 64.71%,andtheoverallidentificationefficiencywasapproximately76.47%.Similarly,asetofminibarcode systemswasgenerated,whichmayprovideaneffectiveandlow-costmethodfortheidentificationof authentictortoiseshells.

Introduction

Asa widelyusedChinesemedicine,CarapaxetPlastrum

Tes-tudinisreferstoprocessedmedicinaltabletsthataremadefrom

the carapace of Chinemys reevesii, which are members of the

Testudinidaefamily (ChinesePharmacopoeia, 2015).Carapax et

Plastrum Testudinis is principally derived from C. reevesii bred

by aquiculture in Zhejiang, Guangdong and other provinces in

China, in which carapaces are stripped following capture and

dissection.“Guijia”isa uniqueChinese medicinethat hasbeen

used for thousands of years. According to ancient records and

modernliterature,theshellsin clinicalusearederived fromC.

reevesii(MauremysreevesiiGray,1831).YifeiZhongetal.showed

thatNephrokeli,aChineseherbalformula,whichcontains

“Gui-jia”,mayeffectivelytreatIgAnephropathythroughtheregulation

of the sphingosine-1-phosphate pathway (Zhong et al., 2015).

∗ Correspondingauthor.

E-mail:[email protected](C.Liu).

Nevertheless, to reduce costs and increase profits, counterfeit

tabletsareproducedusingcarapacesofotherspeciesandobtained

from merchants in restaurants, resulting in mixed qualities, of

whichthesafetyand effectivenesscannotbeguaranteed(Yang,

2001;Yi,2010).Additionally,thecarapacesofsomeendangered

species serve in collections and are traded in illegal markets,

which can elude government regulation (Wu, 2012).

Consider-ingtheprotectionforrarespeciesandthestandardregulationof

Chineseherbs,moreresearchisrequiredregardingidentification

approachesforTestudinidaeanimalcarapaces.

Currently,theuniversalDNAbarcodesusedinanimalspecies

identificationareCOIandCytb.COIisamitochondrialDNA

frag-ment650bpinlength(Hebertetal.,2003).Awealthofliterature

hasshownthatCOIbarcodescanpreciselyidentifyvariousanimal

species.However,foroldersamplesorlow-qualityDNA,

amplify-ingtheintactCOIsequenceusingnormalprimersischallenging

andhinderstheidentificationofChineseherbalmedicines(Chen

etal.,2015).Inourstudy,PCRamplificationoftheCytbgenewas

unsuccessfulinmostoftheturtlesamples,whichmaybeduetothe

lowhomologyamongturtlespecies.In2006,theCanadianscholar

https://doi.org/10.1016/j.bjp.2018.04.008

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HebertinventedDNAmini-barcodetechnology(Hajibabaeietal., 2006a,b)toidentifyspeciesinmuseumcollections.Thismethod

allowedforspeciesidentificationbyusing200bpsequencesfrom

COIbarcodes.Inrecentyears,thistechniquehasplayeda

signifi-cantroleinthefoodindustry(Wardetal.,2009;Shokrallaetal.,

2015)andfortheidentificationofTCM(Dongetal.,2014;Chen

etal.,2015).

In2009,Leeetal.appliedmini-barcodingusingthecytbgeneas

aDNAmarkertoidentifyturtleshellsamples(Leeetal.,2009).

However,thesuccessratesof PCRamplificationinoursamples

usingthesame minibarcodes werelow. In recentstudies, COI

sequences have been frequently used because mtDNA is more

conservativethan nuclearDNA, suchthat universalprimersare

easierto design. In this study,we designed miniaturebarcode

primersbasedonCOIsequences,andtheoptimizedsequenceswere

selectedfortheidentificationofshellfragments.

Materialsandmethods

Samplecollection

Tortoise sample collection occurred over two months

(October–November 2015) from the following three provinces

in China: Jiangsu Province, Zhejiang Province and Guangzhou

Province.A total of forty samples werecollected (Table1), all

ofwhich were obtainedfromaquaticproduct marketsin eight

citiesanddeliveredtothelaboratorylocatedinBeijing.To

inves-tigate the intra- and interspecific genetic distances between

these tortoise species, 22 sequences were downloaded from

NCBI(https://www.ncbi.nlm.nih.gov)(Table2).For theanalysis

of mini-barcode primers with commercial products, a total of

seventeenturtleshellproductswereobtainedfromTCMmarkets

(Table3).AllsampleswereidentifiedbyProf.Yang,directorofthe

ChineseMedicineIdentificationInstitution,BeijingUniversityof

ChineseMedicine.Tortoiseswerebredinfishtanks,andbloodwas

collectedandpreparedforDNAextraction.

DNAextraction

DNAfromblood sampleswasextractedusingawholeblood

genomicDNAisolationkit(Biomed,China).Mincedshellsamples

weredecalcifiedwithEDTAsolutionfollowedbyDNAextraction

usingthetissue/cellgenomicDNAextractionkit(Biomed,China).

Eachsamplewasrunintriplicate,andthreeadsorptioncolumns

wereelutedwithmolecularbiologygradewater(50␮l)toincrease

thetemplateDNAconcentrationforPCR.

DNAbarcodelibraryconstruction

PCRwasperformedina25␮lreactionvolumecontaining10×

PCRBuffer(Mg2+_plus)_(2.5

␮l),dNTPs(0.4mMeach),forwardand

reverseprimers(0.2␮Meach),templateDNAandTaqPolymerase

(0.1U). PCR amplifications were performed in a PCR machine

(Bio-Rad Laboratories, TX, USA) utilizing the thermal cycling

parametersdescribedinTable4.ThesequenceofthePCRproducts

Table2

Detailsof22sequencesdownloadedfromNCBI.

No. Speciesandaccession No. Speciesandaccession

1 Mauremysmutica

KP751968

12 PodocnemisunifilisJF802204

2 Mauremysmutica

AF348260

13 PodocnemisunifilisKC751491

3 Geoemydaspengleri

HQ329679

14 PlatysternonmegacephalumGQ867677

4 Geoemydaspengleri

AY562184

15 PlatysternonmegacephalumGQ867679

5 Cyclemysdentata

JX455823

16 IndotestudoelongataDQ080043

6 Cyclemysdentata

JN582334

17 IndotestudoelongataDQ656607

7 Cuoramouhotii

EF011474

18 CuoragalbinifronsAF348266

8 Cuoramouhotii

DQ659152

19 CuoragalbinifronsAY357742

9 Cuoraamboinensis

AY357738

20 ChelydraserpentinaKU985806

10 Cuoraamboinensis

FJ763736

21 ChelydraserpentinaLC145070

11 Pelomedusasubrufa

AF039066

22 DaniorerioNC002333

Table3

Detailsofseventeenturtleshellsamples.

No. Morphological

identificationof species

No. Morphological

identificationofspecies

1 Mauremysreevesii 10 Trachemysscripta

2 Mauremysreevesii 11 Mauremyssinensis

3 Mauremysmutica 12 Mauremyssinensis

4 Mauremysreevesii 13 Mauremyssinensis

5 Mauremysreevesii 14 Cyclemysdentata

6 Mauremysreevesii 15 Mauremysreevesii

7 Indotestudo

elongata

16 Mauremyssinensis

8 Cyclemysdentata 17 Mauremysreevesii

9 Mauremyssinensis

wasdeterminedusinga3730×lDNAAnalyzer(ShanghaiSangon BiologicalTechnologyCo.Ltd.,China).Theobtainedforwardand reverse sequences were aligned using Contig Express software (909ThirdAvenue,#6260,NewYork,NY10150,USA),suchthat the accurate barcode sequence of each sample was acquired. Then, prepared primers were added to the DNAMAN software (Lynnon Biosoft,USA) for comparison. Next, ananalysis of dif-ferencesamongthevariousspecies’COIbarcodesequenceswas made.Finally,theconstructionofageneticNJclusteringtreewas performedbyMEGA5,MolecularEvolutionary GeneticsAnalysis version5.0(Tamuraetal.,2011).

PCRprimerdesignandanalysis

We compared the full-length COI barcodes of all samples

and analyzed the highly conserved sites in turtle species.

In addition, we considered the overall physical structure

Table1

Detailsof40turtlesamplesusedforthefull-lengthCOIbarcodeconstruction.

Species Samplequantity Species Samplequantity

Mauremysreevesii 13 Podocnemisunifilis 1

Mauremysmutica 1 Pelomedusasubrufa 1

Geoemydaspengleri 1 Trachemysscripta 7

Cyclemysdentata 1 Platysternonmegacephalum 2

Cuoramouhotii 2 Indotestudoelongata 2

Mauremyssinensis 5 Cuoragalbinifrons 1

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Table4

PCRamplificationandsequencingprimersusedforDNAmini-barcodingofthePlastrumTestudinissamples.

Primerset Primername Direction Primersequence Barcodelength(bp) Annealingtemp.(◦_C)

COI COI-F_COI-R Forward_Reverse GGTCAACAAATCATAAAGATATTGG_{TAAACTTCAGGGTGACCAAAAAATCA} 691 45

MiniTu-A MA-F_MA-R Forward_Reverse GTGACCAAAAAATCAGAA_{ATTAATATAAAATCCCCAGCC} 215 49

MiniTu-B MB-F Forward ATGTTAATTGCTGTGGTG 215 56

MB-R Reverse ATAAGCTTCTGGCTTCTACC

Mini Tu-C MC-F Forward GGTATGACCATGAAGAAAA 171 47

MC-R Reverse TGGCACCTTATAATTTTG

MiniTu-D MD-F Forward TAGTTAGATCTACAGAGGCG 109 56

MD-R Reverse GGTCATACCTATTATAATCGG

5' 3'

1 691

Cytochrome C Oxidase I (COI)

Tu-C=171bp

Tu-D=109bp

Tu-B=215bp

Tu-A=215bp

Fig.1.Schematicrepresentationofregionsamplifiedbythemini-barcodeprimersdesignedinthisstudy,shownwithinthestandardCOIbarcoderegion.

characteristics and used these data to design four mini-barcode primer sets using online primer design software (http://www.yeastgenome.org/cgi-bin/web-primer – Stanford

University)(Table4).ForfurthertestingwiththePlastrum

Testu-dinissamples,therelativelocationsoftheprimers(comparedto

theCOIbarcodes)areshowninFig.1.Allbarcodespreparedfor

screeningweresubjectedtogradientPCR(annealingtemperature

45–60◦_C)._According_to_the_results_of_agarose_gel_{electrophoresis,}

theoptimum annealingtemperaturewasdetermined (Table4).

Theselectedmini-barcodesamplesmatchedwiththeshellDNA

templates and were amplified. Using sequence accuracy and

identificationefficiencyasparameters,thebestmini-barcodewas

selectedforshellsampleidentification.

Resultsanddiscussion

GenomicDNA of PlastrumTestudinis fromTCMmarketswas

severelydegradedbecauseof processingpriortosale andfrom

preservationoveralongperiodunderconditionsofhigh

tempera-tureandhumidity.Thecellsintortoiseplastronsaresurroundedby

bonetissuesofhighdensity,whichprotectstheDNAfrom

degra-dation.Inthisstudy,althoughDNAextractionwasimproved,only

4of17PlastrumTestudinissamplesfromTCMmarketshad

tem-plateDNAconcentrationsexceeding2.5ng/␮l,andthesuccessrate

forCOIbarcodingwithPCRamplificationwas0%.Inconclusion,

COIbarcoding cannotbe usedforthe identificationofPlastrum

Testudinis.

In total, 61 sequences from fourteen tortoise species were

obtained, including forty from PCR and 21 from GenBank. All

sequencesweretrimmed to691bpand assembled asa dataset

offull-lengthCOIbarcodes.Afteralignment,theresultsshowed

that353locationswereconserved,and338werepolymorphic.The

averageGCcontentwas43.5%.

Intraspecificandinterspecificgeneticdistancesamongthe61

COIsequencesofthefull-lengthbarcodesareshowninTable5.

Morespecifically,intraspecificdistancewasminor,andaveraged

1.12%,whereasinterspecificgeneticdistanceaveraged19.28%.All

fourteenspeciescladesintheNJphylogenetictreeweresupported

with99%bootstrapvalues(Fig.2).Comparedtootherspecies,the

intraspecific geneticdistance inturtles is large, indicating high

variation.Althoughonlyfourteenspeciesfromeightfamiliesand

twelvegenerawerecollectedinthisstudy,weobservedgreater

intraspecificgeneticdistancesthaninbirds,fish,insectsandother

animalsbecauseturtlescomprisealargefamily(Hebertetal.,2004;

Hajibabaeietal.,2006a,b;Wardetal.,2006).Therefore,the

com-pleteCOIbarcodescanbeusedsuccessfullyforturtleidentification

amongthefourteenspeciesinthisstudy.

UsingthemodifiedDNAextractionmethod,DNAextractionwas

conductedwithseventeenshellsamplespurchasedfromthe

mar-ket.Fourpairsofmini-barcodingsequenceswereamplifiedbyPCR,

andthesequencingsuccessratewasrecorded.Tu-Bproductswere

sequencedsuccessfullyatarateof76.47%inthirteensamples,and

Tu-Dhadasuccessrateof88.24%infifteensamples.Tu-AandTu-C

wereeliminatedduetosequencingfailure.

To determine the discrimination ability of the four

mini-barcodingsequences,dataanalysiswasconductedaftertheTu-B

sequencesweretrimmedto215bpandtheTu-Dsequenceswere

trimmedto109bp.Theaverageintra-andinterspecificdistances

oftheTu-Bdatasetwere0.54%and18.80%,respectively,whereas

those of the Tu-D datasetwere 0.51% and 18.60%, respectively

(Tables6and7).Thesedistances wereboth closetothatofthe

691-bpdataset.

TheresultsshowninTable8revealthattheefficiencyof

iden-tification ofTu-Bwas70.59%, Tu-Dwas64.71%,andtheoverall

identificationefficiencywas76.47%.Becauseallthesamplesof

tor-toiseshellswerepurchasedfromtheTCMmarket,sampleswere

selectedthathadbeenstoredforalongtimebutwhosetextural

characteristicswerestillinexistence.Therefore,theresultsofthe

morphologicalidentificationwerereliable.Insamples3,6,and7,

theresultsofthemini-barcodingidentificationwereinconsistent

withthemorphologicalidentification.Itispossiblethaterrorsin

DNAsequencingfromamplifiedintraspecificdifferencesledtothe

deviationoftheidentificationresults.

InChina,theidentificationsystemincludestwobarcodesfor

MauremysreevesiithatqualifyshellspeciesbyPharmacopeia,and

theidentificationsuccessratewas100%.Throughcomprehensive

analysis,turtlespeciessuchasIndotestudoelongataandMauremys

sinensiscanalsobesuccessfullyidentified.Fortheidentificationof

Trachemysscripta,thetwogroupsofbarcodeswereunableto

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Table5

PairwisegeneticdistancesbetweenandwithinfourteenPlastrumTestudinisspeciesbasedonfull-lengthCOIbarcodes.

Species [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14]

[1]Trachemysscripta 0.035

[2]Podocnemisunifilis 0.252 0.003

[3]Platysternonmegacephalum 0.248 0.288 0.005

[4]Pelomedusasubrufa 0.326 0.263 0.349 0.007

[5]Mauremyssinensis 0.163 0.251 0.228 0.297 0.004

[6]Mauremysreevesii 0.160 0.243 0.244 0.278 0.049 0.016

[7]Mauremysmutica 0.177 0.252 0.224 0.280 0.091 0.077 0.001

[8]Indotestudoelongata 0.177 0.234 0.247 0.294 0.158 0.151 0.160 0.006

[9]Geoemydaspengleri 0.177 0.239 0.247 0.310 0.145 0.149 0.146 0.174 0.001

[10]Cyclemysdentata 0.172 0.229 0.218 0.294 0.123 0.116 0.118 0.164 0.151 0.037

[11]Cuoramouhotii 0.187 0.254 0.222 0.292 0.101 0.092 0.102 0.154 0.151 0.133 0.013

[12]Cuoragalbinifrons 0.174 0.273 0.240 0.284 0.098 0.096 0.093 0.164 0.158 0.130 0.079 0.009

[13]Cuoraamboinensis 0.203 0.253 0.231 0.330 0.111 0.119 0.124 0.176 0.182 0.139 0.100 0.106 0.019

[14]Chelydraserpentina 0.183 0.233 0.248 0.263 0.194 0.189 0.187 0.197 0.183 0.182 0.193 0.204 0.201 0.002

P_{odocnemis unifilis KC751491}

Danio rer

io NC002333 P

odocnemis unifilis JF802204

P_{odocnemis unifilis} Pelomedusa subr

uf_{a AF039066}

99

99 99 99

99 99 99 99 99 99 99 Pe_{lomedusa subr}

uf_a Chelydr a ser pentina LC145070 Chelydr a ser pentina Chelydr a ser pentina KU985806 Platyster non megacephalum Platyster

non megacephalum GQ867679

Platysternon megacephalum GQ867677

Geoemyda spengleri HQ329679

Geoemyda spengler i AY562184

Geoemyda spengler i

Indotestudo elongata DQ656607

Indotestudo elongata DQ080043_{Indotestudo elongata}

Indotestudo_elongata Trachem ys scr ipta Trachem ys scr ipta Trachem ys scr ipta Trachem ys scr ipta Trachem ys scr ipta Trachem ys scr ipta Trachem ys scr ipta Cyclem ys dentata JX455823

Cyclem ys dentata JN582334

Cyclem

ys dentata

Cuor

a mouhotti DQ659152 Cuor a mouhotti Cuor a galbinif o_rn s AF348266 Cuor a galbinif or ns A Y357742 Cuor a galbinif orns Cuor

a amboinensis FJ763736 Cuor

a amboinensis A

Y357738 Cuor a amboinensis Cuor a amboinensis Maurem ys m utica KP751968 Maurem ys m utica AF348260 Maurem ys mutica Mauremys sinensis Mauremys sinensis Mauremys sinensis Mauremys sinensis Maurem ys sinensis Maurem ys ree vesii Maurem ys ree vesii Maurem ys ree vesii Maurem ys ree vesii Maurem ys ree vesii Maurem ys ree vesii Maurem ys ree vesii Maurem ys ree vesii Maurem ys ree vesii Maurem ys ree vesii Maurem ys ree vesii Maurem ys ree vesii Maurem ys ree vesii Cuor

a mouhotti EF011474 Cuor

a_mouhotti

Platyster

non megacephalum 2

0.02

Fig.2. Neighbor-joining(NJ)treeoffull-lengthCOIsequencesoffromfourteentortoisespecies.

Table6

PairwisegeneticdistancesbetweenandwithinfourteenPlastrumTestudinisspeciesbasedontheMiniTu-Bbarcode.

Species [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14]

[7]Mauremysmutica 0.210 0.268 0.231 0.240 0.089 0.074 0.000

[11]Cuoramouhotii 0.191 0.264 0.207 0.267 0.096 0.085 0.090 0.112 0.138 0.128 0.005

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Table7

PairwisegeneticdistancesbetweenandwithinfourteenPlastrumTestudinisspeciesbasedontheMiniTu-Dbarcode.

Species [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14]

[7]Mauremysmutica 0.180 0.280 0.228 0.232 0.091 0.077 0.000

[11]Cuoramouhotii 0.192 0.293 0.212 0.235 0.112 0.083 0.107 0.143 0.181 0.143 0.002

[14]Chelydraserpentina 0.171 0.267 0.234 0.233 0.166 0.171 0.165 0.186 0.175 0.146 0.165 0.166 0.161 0.000

Table8

Identificationresultsof17turtleshellsamplesbytheTu-BandTu-Dbarcodes.

a_BLAST_results_report_the_top_bit_score_hit.

Greencellsindicatethattheidentificationresultsbytheminibarcodeswerecorrect,redcellsindicatetheywereincorrect,andyellowcellsindicatePCRfailure.

couldbeaneffectivewaytoidentifydegradedPlastrum Testudi-nissamplesfromwhichthefull-lengthCOIbarcodesareharderto recover.

Conclusions

TheoptimizationofDNAextraction,includingDNAfromshells, wasachieveddespiteseriousdegradation.Additionally, theCOI barcodedatabaseofcommonturtlespeciesin Chinawas estab-lished.TwoCOI-basedDNAmini-barcodeswereselectedforturtle speciesidentification.We developed aminibarcodesystemfor turtle species identification, by which identification success of shellsreached76.47%. Thesampleidentificationsuccessrateof genuineChinesetortoiseshells(conformingtotheChinese Phar-macopeia)peakedat100%.Ourresultsprovideaneffectiveand low-costmethodtoidentifyauthentic tortoiseshells. Addition-ally,thisresearchcontributes tothesafetyand effectivenessof Chineseherbalmedicineand totheprotectionof thisrare tur-tle.

Authorcontributions

Conceivedanddesigned theexperiments:MYCandCSL. Per-formedtheexperiments: MYC,XYL, andJM. Sample collection: MYC,ML, and XNL. Wrote thepaper: MYCand JM. Editedthe manuscript:MYCandXH.

Conflictsofinterest

Theauthorsdeclarenoconflictsofinterest.

Ethicaldisclosures

Protectionofhumanandanimalsubjects. Theauthorsdeclare

thatnoexperimentswereperformedonhumansoranimalsfor thisstudy.

Confidentialityofdata. Theauthorsdeclarethatnopatientdata appearinthisarticle.

Righttoprivacyandinformedconsent.Theauthorsdeclarethat nopatientdataappearinthisarticle.

Acknowledgments

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