Aftera numberofDNAfragments derivedfromFaRE1insertion sites werecloned and sequenced,we extracted eight insertion sites by considering combinations of their polymorphisms for discriminat
Trang 1jo u r n al h om ep a g e :w w w e l s e v i e r c o m / l o c a t e / j b i o t e c
discrimination
a Graduate School of Environmental and Life Science, Okayama University, 1-1-1 Tsushimanaka Kitaku, Okayama, Okayama 700-8530, Japan
b FASMAC Co., Ltd., 5-1-3 Midorigaoka, Atsugi, Kanagawa 243-0041, Japan
c Graduate School of Biomedical Engineering, Tohoku University, 6-6-05 Aramaki-aoba, Sendai, Miyagi 980-8579, Japan
a r t i c l e i n f o
Article history:
Received 24 February 2014
Received in revised form 14 May 2014
Accepted 10 June 2014
Available online 19 June 2014
Keywords:
Cultivar discrimination
Multiplex PCR
Strawberry
Practical application
Retrotransposon
a b s t r a c t
Inmanycropsspecies,thedevelopmentofarapidandprecisecultivardiscriminationsystemhasbeen requiredforplantbreedingandpatentprotectionofplantcultivarsandagriculturalproducts.Here,we successfullyevaluatedstrawberrycultivarsviaanovelmethod,namely,thesingletaghybridization(STH) chromatographicprintedarraystrip(PAS)usingthePCRproductsofeightgenomicregions.Ina previ-ousstudy,weshowedthatgenotypingofeightgenomicregionsderivedfromFaRE1retrotransposon insertionsiteenabledtodiscriminate32strawberrycultivarsprecisely,however,thismethodrequired agarose/acrylamidegelelectrophoresis,thushasthedifficultyforpracticalapplication.Incontrast,novel DNAdetectionmethodinthisstudyhassomegreatadvantagesoverstandardDNAdetectionmethods, includingagarose/acrylamidegelelectrophoresis,becauseitproducessignalsforDNAdetectionwith dramaticallyhighersensitivityinashortertimewithoutanypreparationorstainingofagel.Moreover, thismethodenablesthevisualizationofmultiplexsignalssimultaneouslyinasinglereactionusing sev-eralindependentamplificationproducts.Weexpectthatthisnovelmethodwillbecomearapidand convenientcultivarscreeningassayforpracticalpurposes,andwillbewidelyappliedtovarious situa-tions,includinglaboratoryresearch,andon-siteinspectionofplantcultivarsandagriculturalproducts
©2014TheAuthors.PublishedbyElsevierB.V.ThisisanopenaccessarticleundertheCCBY-NC-SA
license(http://creativecommons.org/licenses/by-nc-sa/3.0/)
1 Introduction
Thecultivatedstrawberry(Fragaria×ananassa,2n=8×=56)is
oneofthemosteconomicallyimportantfruitcropsintheworld
Itsglobalproductionin2009wasestimatedtobeover4.1million
tons(Foodand AgricultureOrganizationof theUnitedNations;
http://faostat3.fao.org/).Breeding programsof cultivated
straw-berryareconductedin manycountriestoimprovefruitquality
andyield,andtoachieveextendedstoragecapabilityanddisease
resistance.InJapan,over30nationaland/orprefectural
agricul-turalresearchcentersarecarryingoutstrawberrycultivarbreeding,
Abbreviations: STH, single tag hybridization; PAS, printed array strip.
∗ Corresponding author Tel.: +81 86 251 8312; fax: +81 86 251 8388.
E-mail addresses: y monden@cc.okayama-u.ac.jp (Y Monden),
ktakasaki@fasmac.co.jp (K Takasaki), sfuto@fasmac.co.jp (S Futo),
kniwa@ecei.tohoku.ac.jp (K Niwa), m-kawase@ecei.tohoku.ac.jp (M Kawase),
ag20004@s.okayama-u.ac.jp (H Akitake), tahara@cc.okayama-u.ac.jp ,
makoto.tahara@gmail.com (M Tahara).
which has ledto thecreation of many popular Japanese culti-vars,suchasAmaou,Sagahonoka,andHinoshizuku.TheseJapanese strawberrycultivarshavebeenhighlyimproved,whichindicates that theyhave highproductivity,earliness,andhighfruit qual-ity,includinganextendedshelflife.Thus,apreciseandeffective cultivardiscriminationsystemisrequiredtoprotecttheplant pro-prietaryrightofthosesuperiorcultivars.However,discrimination based onmorphologicaltraits is affectedby theenvironmental and/orgrowthconditions,andisrestrictedduringthe developmen-talstage(NielsenandLovell,2000).Moreover,ifthecultivarsare closelyrelated,itisextremelydifficulttodistinguishthembased
onmorphologicaltraits.Therefore,molecularmarkershavebeen developedbasedonthemethodsofrandomlyamplified polymor-phicDNA(RAPD),amplifiedfragmentlengthpolymorphism(AFLP), simplesequencerepeat(SSR),andcleavedamplifiedpolymorphic sequences(CAPS),whichenableprecisecultivardiscriminationat anydevelopmentalstage(Arnauetal.,2001;Congiuetal.,2000; Hancock et al., 1994; Kunihisa et al.,2003, 2005; Nehraet al., 1991;Tyrka etal.,2002).However,itremains difficulttoapply thesemethodstoon-siteinspection,forthefollowingreasons:it http://dx.doi.org/10.1016/j.jbiotec.2014.06.013
0168-1656/© 2014 The Authors Published by Elsevier B.V This is an open access article under the CC BY-NC-SA license ( http://creativecommons.org/licenses/by-nc-sa/3.0/ ).
Trang 2processedproducts,someexperimentalinstrumentsarerequired
for agarose/acrylamidegel electrophoresis,and the preparation
andstainingofagelrequiresseveralhours
Retrotransposonsareubiquitousandabundantcomponentsin
virtuallyall knowneukaryoticgenomes (Feschotteetal., 2002;
Feschotte and Pritham, 2007; Huang et al., 2012; Kumar and
Bennetzen,1999;LevinandMoran,2011;Wessler,2006).Inhigher
plants,theyusuallyconstitutemorethanhalfofthewholegenomic
DNA(Bentoetal.,2013;Patersonetal.,2009;Schnableetal.,2009;
Tenaillonetal.,2010).Theyamplifythenumberoftheircopies
throughreversetranscriptionoftheirRNAandintegrationofthe
resultingcDNAsintonewgenomiclocibasedonthe
“copy-and-paste”transposition mechanism (Kumarand Bennetzen, 1999)
Becauseoftheirubiquitousdistribution,highcopynumber,and
diversedispersionwithinthegenome,theirinsertion
polymor-phismsamongcultivarshavebeenusedasmolecularmarkersin
phylogeneticanalyses,intheconstructionoflinkagemaps,andin
geneticdiversitystudies(Flavelletal.,1998;Kalendaretal.,1999,
2011;Konovalovetal.,2010;KumarandHirochika,2001;Nasri
etal.,2013;Poczaietal.,2013;Sm ´ykaletal.,2011;Syedetal.,
2005).Moreover,theuniquenessofthenewlyintegratedinsertion
siteshasanexcellentpotentialforthedevelopmentofmultiplex
DNA-basedmarkersystemsthatcanbeusedtoachievecultivar
discrimination.Infact,ourrecentresearchshowed
retrotranspo-sonbasedDNAmarkerswereusefulforcultivardiscriminationin
severalcropspecies,includingwheat(Triticumaestivum)andsweet
potato(Ipomoeabatatas)(TakaiandTahara,2011;Mondenetal.,
2014)
Inapreviousresearch,wesuccessfullyidentifiedeightgenomic
insertionsitesoftheFaRE1retrotransposonthatwereusedto
dis-criminate32strawberrycultivarsafterscreeningtheirinsertion
sitescomprehensively(Akitakeetal.,2013).FaRE1hasbeen
identi-fiedasanactiveretrotransposonfamily(Heetal.,2010;Melnikova
etal.,2012)andshowshighinsertionpolymorphismsevenamong
Japanesestrawberrycultivars,whichareknowntobegenetically
closelyrelated(Akitakeetal.,2013).Weappliedsequence-specific
amplified polymorphism (S-SAP) method to investigate FaRE1
insertionpolymorphismamong32cultivars.Thismethod
ampli-fies specificallythe DNA fragments between a retrotransposon
endanditsadjacentrestrictionenzymecuttingsite,and
visual-izesmultiplebandsthrough agarose/acrylamideelectrophoresis
(Konovalov et al., 2010; Louand Chen, 2007; Melnikova et al.,
2012;Petitet al.,2010; Syedet al., 2005;Waugh etal., 1997)
Aftera numberofDNAfragments derivedfromFaRE1insertion
sites werecloned and sequenced,we extracted eight insertion
sites by considering combinations of their polymorphisms for
discriminating 32 strawberry cultivars It was shown that the
amplificationoftheseeightinsertionsitesallowedthepreciseand
rapidscreeningofstrawberrycultivars(Akitakeetal.,2013)
How-ever,thismethodalsorequiredagarose/acrylamideelectrophoresis
forsignaldetection,whichhasthedifficultyinachievingon-site
inspection
In this study, we developed a novelcultivar discrimination
systemusingthesingletaghybridization(STH)chromatographic
printedarraystrip(PAS)method,whichaffordsthevisualization
ofmultiplex DNA signals ina singlereaction withgreat
sensi-tivity and in a dramatically short time Moreover, it does not
requirethepreparation or staining ofa gel The resultsof this
study showedthat we successfully evaluated strawberry
culti-varsbasedonthemultiplexDNAsignalsthatwerederivedfrom
theampliconsoftheFaRE1retrotransposonandvisualizedusing
STHchromatographicPAS.Thus,weexpectthatthismethodwill
facilitate rapid, efficient, and highly reliable cultivar
discrimi-nationin on-site inspectionof plant materialsand agricultural
products
2 Materials and methods
2.1 DevelopmentofDNAmarkersforstrawberrycultivar discrimination
Thisresearchwasconductedbasedontheinformationprovided fromapreviousresearch,whichdevelopedeightDNAmarkersfor discriminating32strawberrycultivars(Akitakeetal.,2013).Thus,
webrieflydescribedthecontentsofthispreviousresearch.Inthe previousresearch,32strawberrycultivarsanditswildspecies (Fra-gariavesca) were used(Supplementary Table1).First, genomic DNA was extracted fromyoung leavesusing the DNeasy Plant minikit (QIAGEN) following themanufacturer’s protocol After genomicDNAwasdigestedwithAseIorRsaIrestrictionenzyme (NewEnglandBiolabsJapan,Inc),forkedadaptorswereligatedto thedigestedDNA.Theforkedadapterswerepreparedbyannealing twoDNAoligomers:FAAseIandFAcmplforAseI,andFARsaIand
FA cmplforRsaI.PCRprimersweredesignedbasedonthesequence informationofFaRE1.WeperformedprimaryPCRwithan adapter-specific(AP2)andFaRE1-specific(FaRE1PBS)primercombination forAseI digestedDNA fragments,and alsoperformedthat with
anAP2andFaRE1-specific(FaRE1LTR150Up)primercombination forRsaIdigestedDNAfragments.Then,nestedPCRwasperformed withanadapter-specific(AP3)andFaRE1-specific(FaRE1LTREnd) primersetusingtheinitialPCRproductsasthetemplate.ThePCR comprisedaninitialdenaturationat94◦Cfor2min,whichwas fol-lowedby30cyclesat94◦Cfor60s,75◦Cfor60s(thisstepwas addedfortheamplificationofRsaIdigestedDNAfragment),58◦C for90sand72◦Cfor90s,withafinalextensionat72◦Cfor5min PCRproductswereloadedonanABI3730xlDNAAnalyzer(Applied Biosystems)forDNAfragmentanalysesafterthepurificationwith QIAquick PCR purificationkit (QIAGEN) GeneMappersoftware (AppliedBiosystems)wasusedforthevisualizationofDNA frag-ment peaks.In addition, PCR productswerecloned withTOPO
TAcloningkit(Invitrogen),and446colonieswerescreenedand sequenced.SequenceswereanalyzedandalignedusingBLASTand ClustalWprogram(Larkinetal.,2007),andthesequencesof124 differentFaRE1insertionsiteswereobtained.Outofthesesites,we selectedeightinsertionsites(Grp.18,41,57,59,61,65,76and110) basedonthecombinationsoftheirpolymorphismsamong32 cul-tivars.Theprimerandadaptersequencesinthepreviousresearch arelistedinSupplementaryTable2
Supplementarytablesrelatedtothisarticlecanbefound,inthe onlineversion,athttp://dx.doi.org/10.1016/j.jbiotec.2014.06.013 2.2 Samplepreparation
Weusedeightstrawberrycultivars(Akihime,Nyoho,Amaou, Hokowase,Benihoppe,AsukaRuby,RedPearl,andKotoka)inthis research.Theplantsofstrawberrycultivarswereobtainedfromthe TochigiPrefecturalAgriculturalExperimentStationandFukuoka AgriculturalResearchCenter.GenomicDNA wasextractedfrom youngleavesusingtheDNeasyplantminikit(QIAGEN),according
tothemanufacturer’sprotocol
2.3 PCRandsignaldetectionbyagarosegelelectrophoresis
Inthisresearch,PCRwasperformedbyamplifyingtheinternal positive control (IPC)sequence and eight FaRE1insertion sites The IPC sequence was introduced into a pArt1 vector, which was used for the control PCR (Supplementary Fig 1) (Mano
etal.,2011).ThePCRamplificationforIPCsequencewascarried out using PrimeSTAR GXL Taq Polymerase (Takara Bio, Ohtsu, Japan)withIPC200fandIPC200rprimercombinations(Table1) andthepArt1vectorasa template,andthatfor eightinsertion sites wascarried out using theKAPA2G Fast Mutiplex PCR kit
Trang 3Table 1
Sequences of the primers used in this study.
C-PAS4 IPC200f [A1]-[Spacer]-CTAGGGAATGACGGCAGGATAG
C-PAS4 IPC200r Bi [Biotin]-CGCACGTATACATATGGAGTCAGC
C-PAS4 IPC100f 1 [A1]-[Spacer]-CCGAGCTTACAAGGCAGGTT
C-PAS4 IPC100r Bi [Biotin]-TGGCTCGTACACCAGCATACTAG
C-PAS4 GrpR Bi [Biotin]-CTTAATTTCCAAATCATATCAACGAGCCAAAACAC
C-PAS4 Grp18 2 [A2]-[Spacer]-CCTGGTTGGCAACATGATGTAAC
C-PAS4 Grp41 3 [A3]-[Spacer]-CACCAAAACCAACAACTCATACC
C-PAS4 Grp57 3 [A3]-[Spacer]-CAACTTCCACTCTTCGATCCAG
C-PAS4 Grp59 4 [A4]-[Spacer]-CAATGAGGACCTTGCAATGTAAGC
C-PAS4 Grp61 2 [A2]-[Spacer]-GACCATGTCAAAATGACCGTTCAG
C-PAS4 Grp65 4 [A4]-[Spacer]-GGTGGAGTCCTGTCCAAATAG
C-PAS4 Grp76 4 [A4]-[Spacer]-GTATTCCTCCAGTTCCGACCA
C-PAS4 Grp110 3 [A3]-[Spacer]-CACATGAGGCACTGGACTTAACG
[A1], [A2], [A3] and [A4] represent the tag sequences [Spacer] represents the
single-stranded non-public sequence.
inTable1
Supplementaryfigurerelatedtothisarticlecanbefound,inthe
onlineversion,athttp://dx.doi.org/10.1016/j.jbiotec.2014.06.013
2.4 PCRandsignaldetectionbySTHchromatographicPAS Foramplificationof thePCR productsdetectedbySTH chro-matographic PAS, PCR primers containing the single-stranded tag and spacer sequences were used (indicated as C-PAS4* primers in Table 1) The PCR amplification for IPC sequence was performed with C-PAS4IPC100f1 and C-PAS4IPC100rBi
or C-PAS4IPC200f and C-PAS4IPC200rBi primer com-binations, and that for eight FaRE1 insertion sites was performedwithFaRE1retrotransposon-specific(C-PAS4GrpRBi) and insertion site-specific (C-PAS4Grp182, C-PAS4Grp
413, C-PAS4Grp573, C-PAS4Grp594, C-PAS4Grp612, C-PAS4Grp654, C-PAS4Grp764, or C-PAS4Grp1103) primer combinations.Thegenomic templatesand PCRconditions were same as agarosegel electrophoresis(2.3) PCRproducts witha tag-spacersequenceforSTHweremixedwiththedyeand devel-opingsolution(TohokuBio-Array,TBA)(Fig.1).Subsequently,the C-PAS4membranestick(TohokuBio-Array,TBA)wasdippedinto thisdevelopingsolutionfor15min.Thesequencesoftheprimers usedarealsoshowninTable1
3 Results and discussion
3.1 DescriptionandthesensitivityoftheSTHchromatographic PASmethod
To amplify the PCR products detected by STH chromato-graphicPAS,PCRprimerscontainingthesingle-strandedtagand spacer sequenceswereused (Fig 1and Table 1).Preliminarily, the single-strandedcomplementary oligonucleotides of the tag sequencewereblottedonto theC-PAS membrane,whichledto their hybridization withthe tag sequence of the PCR products (Fig.1) ThisSTH reactiondid not requireheat-based denatur-ation In addition,thePCRproductsdidnot havetobe stained withafluorescentdyereagent,suchasethidiumbromide,andthe chromatographydevelopingreactionrequired5–15min.Thus,this methodenabledthedetectionofthesignalsatroomtemperature andinashorttime.Inaddition,theC-PASmembraneallowedthe visualizationoftheeightsignalssimultaneouslyinasinglereaction Moreover, we compared the sensitivity of signal detection betweenSTHchromatographicPASandagarosegel electrophore-sis.ThePCRproductwasamplifiedbasedontheinternalpositive control(IPC)sequence,whichwasinsertedintothepArt1vector (Supplementary Fig.1)(Manoetal., 2011).The resulting prod-uct was purified from the agarose gel Afterpreparing various concentrations(0.1,0.25,0.5,1.0,2.5,5.0,10,25,and50nM)of thispurifiedproduct(Fig.2 theirsignalsweredetectedusingan
Fig 1.Procedure used for signal detection via the STH chromatographic PAS method (1) Preparation of total genomic DNA (2) PCR amplification using combinations of primers with a tag-spacer sequence and biotin-labeled primers Red line, tag sequence; gray line, spacer sequence; and green line: primer (3) DNA signal detection based on single tag hybridization (STH) The complementary oligonucleotides of the tag sequence (blue line) were preliminarily printed on the membrane, which executes single tag hybridization between the tag sequence of the PCR product and the membrane (For interpretation of the references to color in figure legend, the reader is referred to the
Trang 4Fig 2.Comparison of the sensitivity of signal detection between STH chromatographic PAS and agarose gel electrophoresis The use of the C-PAS4 membrane allowed us to detect even 0.25 nM of IPC PCR products (A) In contrast, the minimum quantity detectable on agarose gel was 2.5 nM of IPC PCR products (B) Thus, the detectable sensitivity
of C-PAS4 was 10 times higher than that of agarose gel IPC, internal positive control.
agarosegelelectrophoresisandSTHchromatographicPAS(Fig.2)
WefoundthattheminimumamountofthePCRproductdetectable
byagarosegelelectrophoresiswas2.5nM,whereasthatdetected
bychromatographyPAS(C-PAS)was0.25nM(Fig.2).Thisindicated
thatthesensitivityoftheC-PASwas10timesgreaterthanthatof
agarosegelelectrophoresis.Thus,theSTHchromatographicPAS
Fig 3.Results of multiplex PCR on agarose gel The PCR products were resolved on
a 5% agarose gel in 1× TAE buffer The sizes of PCR amplicons (Grp 18, 41, 57, 59, 61,
65, 76, and 110) are shown in Table 2 The banding pattern in this figure corresponds
to the results shown in Table 2 C, control DNA; 1, Akihime; 2, Nyoho; 3, Amaou; 4,
Hokowase; 5, Benihoppe; 6, Asuka Ruby; 7, Red Pearl; 8, Kotoka; and M: All Purpose
methodhasanextremelyhighsensitivityregardingsignal detec-tion
3.2 MultiplexPCRassayonagarosegelelectrophoresisand cultivardiscriminationusingtheSTHchromatographicPAS method
ToevaluatetheSTHchromatographicPASmethodfor straw-berrycultivardiscrimination,wefocusedontheeightgenomicloci (Grp.18,41,57,59,61,65,76,and110)derivedfromthe inser-tionsitesoftheFaRE1retrotransposon(Akitakeetal.,2013).Itwas shownthatthecombinationsoftheseinsertionsitesdiscriminate thesestrawberrycultivarsprecisely(Table2).Thus,weperformed
Table 2
Results of the genotyping of FaRE1 insertion sites among strawberry cultivars.
Product size (bp) Amplicon name
Strawberry cultivars
195 Grp 57
162 Grp 61
142 Grp 65
128 Grp 59
119 Grp 76
100 Grp 110
85 Grp 18
74 Grp 41
Note: white cells, no product; blue cells, the presence of a product 1, Akihime; 2, Nyoho; 3, Amaou; 4, Hokowase; 5, Benihoppe; 6, Asuka; 7, Red Pearl; 8, Kotoka.
Trang 5Fig 4. STH chromatographic assay showing the procedures used in this method (A) The red line indicates the positional marker [A-1], [A-2], [A-3] and [A-4] represent the tag IPC, internal positive control product (B) Signal of the PCR amplicons (Grp 18, 41, 57, 59, 61, 65, 76, and 110) used for strawberry cultivar discrimination.
thatwerederivedfromtheseeightinsertionsitessimultaneously,
andvisualizedthesignalsonagarosegelelectrophoresispriortothe
STHchromatographicPASmethod.Fig.3showstheexactbanding
patternofthePCRampliconsofallcultivars,asexpected(Table2)
Thus,itwasshownthatthesimultaneousdetectionoftheseeight
productsallowstheprecisediscriminationofthesestrawberry
cul-tivars
Next, we detected the signals of the PCR products derived
fromtheseeightinsertion sites(Grp.18,41, 57,59, 61,65, 76,
and110)usingtheSTHchromatographicPASmethod.Thistime,
weused theC-PAS4membrane, whichallows thevisualization
of the four signals simultaneously (Fig 4A) The combination
of the PCR products and the tag sequences on three types of
C-PAS4(C-PAS4-1,-2,and-3)isshowninFig.4B(theIPC
prod-uctcontainingthe[A-1] tagsequencewasusedasacontrol).In
addition, other PCR products were combined with three types
([A-2],[A-3],and[A-4])oftagsequences(Fig.4B).Fig.4Ashows
thepracticalprocedureusedtoperformtheSTHchromatographic
PASmethod.First,thePCR products,thedye,and the
develop-ing solution weremixed Subsequently, theC-PAS4 membrane
was placed in this solution for 15min and the signals were
detectedasshown in Fig.4B The signalpatternof allsamples
wasfullyconsistentwiththeresultsofthemultiplexPCRassay
(Fig.3)
Table 3
Comparison of C-PAS with agarose gel electrophoresis.
electrophoresis
Minimum detectable quantity
Need to consider the size of PCR products for signal detection
of mutiplex PCR)
a This time includes the preparation and the staining of the gel.
4 Conclusions
Inthisstudy,weusedtheSTHchromatographicPASmethod forstrawberrycultivardiscrimination.Thisnovelsignaldetection methodhasseveraladvantagesovertraditionalmethods,suchas agarosegelelectrophoresis,becauseitenablesthedetectionof sig-nalswithdramaticallyhighsensitivityandinashorttimewithout anypreparationorstainingofgels(Fig.2andTable3).Moreover, thismethodcanbeusedtovisualizeseveralsignalsderivedfrom severalindependentPCRproductsofanysizesimultaneously.In contrast,thedetectionofthesignalsderived fromthese ampli-consonanagarosegelrequirescautionregardingsize,toachieve sufficientresolutionofthesemultiplexPCRamplicons;moreover,
Trang 6ampliconsofthesamesizecannot beresolved(Table3).Inthe
caseofSTHchromatographicPAS,however,severalindependent
PCRampliconswiththesamesizecanbedistinguishedprecisely
(Table 3) Importantly,this multiplexsignal detection suggests
thatwemightdiscriminatecropcultivarswithinthemixedand
processedproductspreciselybyusingcultivar-specificDNA
frag-ments.Furthermore,this methoddoesnotrequireexperimental
instrumentation,whichmeansthatthisnovelmethodisquite
valu-ableforapplication notonlytolaboratoryresearch,butalsoto
on-siteinspectionofplantcultivarsandagriculturalproducts
Acknowledgements
This work was supported by a Research and Development
Projectsfor ApplicationinPromoting NewPolicy ofAgriculture
Forestry and Fisheries grant from the Ministry of Agriculture,
ForestryandFisheriesofJapan,andbytheProgramtoDisseminate
TenureTrackingSystemfromtheMinistryofEducation,Culture,
Sports,ScienceandTechnology(MEXT),Japan(toY.M.)
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