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THE COMPLETE CHLOROPLAST DNA SEQUENCE OF ELEVEN GRAPE CULTIVARS. SIMULTANEOUS RESEQUENCING METHODOLOGY

VazhaTABIDZE 1,2 ,GrigolBARAMIDZE 1,2 ,IaPIPIA 1,2 ,MariGOGNIASHVILI 1,2 , LevanUJMAJURIDZE 2,TengizBERIDZE 1,2* ,AlvaroG.HERNANDEZ 3 andBarbaraSCHAAL 4 1:FreeUniversityofTbilisi,D. AgmashenebeliAl.13thkm,0159,Tbilisi,Georgia 2:InstituteofMolecularGenetics,AgriculturalUniversityofGeorgia,D. AgmashenebeliAl.13thkm, 0159,Tbilisi,Georgia 3:RoyJ.CarverBiotechnologyCenter,UniversityofIllinoisatUrbana-Champaign,IL61801,USA 4:WashingtonUniversityinStLouis,MO63130-4899,USA

Abstract Résumé Aims :ThechloroplastDNAsequenceofeightGeorgian Objectifs :Laséquenced’ADNchloroplastiquedehuit grapecultivars(Rkatsiteli,Saperavi,MeskhuriMtsvane, cultivarsGéorgiens(Rkatsiteli,Saperavi,Meskhuri Chkhaveri,Aladasturi,Krakhuna,Tsitska,Tsolikouri)and Mtsvane,Chkhaveri,Aladasturi,Krakhuna,Tsitska, threeFrenchcultivars(Chardonnay,GouaisBlanc, Tsolikouri)ettroiscultivarsFrançais(Chardonnay,Gouais Chasselas),belongingtofourdifferenthaplogroups(AAA, Blanc,Chasselas),appartenantàquatrehaplogroupes ATT,ATA,GTA),wasdeterminedbyIllumina différents(AAA,ATT,ATA,GTA),aétéobtenuepar resequencingofgenomicDNA.ThechloroplastDNA reséquençagedel’ADNgénomiqueselonlatechnologie sequenceoftheMaxxacultivarwasusedasreference. Illumina.Laséquencedel’ADNchloroplastiqueducultivar Maxxaaétéutiliséecommeréférence. Methods and results :Thecomparisonofsequenced chloroplastDNAgave100 %identitytoChardonnayand Méthodes et résultats :LacomparaisondesADN GouaisBlanc,differingfromMeskhuriMtsvanebytwo chloroplastiquesséquencésarévéléuneidentitéde100 % insertions/deletions(indels)(allATAhaplogroup).The entreChardonnayetGouaisBlanc,différentedecellede differencebetweenChasselasandSaperaviwasasingle MeskhuriMtsvanededeuxinsertions/délétions(indels) insertion(bothATThaplogroup),whileMaxxa,Chkhaveri, (appartenanttousàl’haplogroupeATA).Chasselaset Aladasturi,Krakhuna,TsitskaandTsolikouriwereall Saperavi(del’haplogroupeATT)sontdifférenciésparune identical(allmembersoftheGTAhaplogroup).Forty-seven seuleinsertion,tandisqueMaxxa,Chkhaveri,Aladasturi, identicalsinglenucleotidepolymorphisms(SNPs)were Krakhuna,TsitskaetTsolikouri(tousmembresde detectedintheAAA,ATAandATThaplogroupsin l’haplogroupeGTA)sonttousidentiques.Quarante-sept comparisontothereferenceDNA.Additionally,18SNPs polymorphismesmononucléotidiques(SNPs)identiquesont weredetectedfortheATThaplogroup,4forAAA,6for étédétectéschezleshaplogroupesAAA,ATAetATTen ATAand11forbothAAAandATA.Thephylogenetic comparaisondel’ADNderéférence.Enoutre,18SNPsont resultsshowthattheATT,AAAandATAhaplogroupsare étéidentifiéspourl’haplogroupeATT,4pourAAA,6pour morecloselyrelatedtoeachotherthantotheGTA ATA,et11communspourAAAetATA.Lesanalyses haplogroup. phylogénétiquesmontrentqueleshaplogroupesATT,AAA etATAsontplusétroitementliéslesunsauxautresqu’avec Conclusion :InthesequencingdataofgrapegenomicDNA l’haplogroupeGTA. atthecoverage(readdepth)ofchromosomalDNA30-40, thecoverageofchloroplastDNAreachesseveralthousand Conclusion :Leséquençagedel’ADNgénomiquederaisin readsperbpduetothehighnumberofchloroplastDNA àpartirdefragmentsd’ADNchromosomiquesde30-à40 copiesingenomicDNA,muchhigherthannecessaryfor delongpermetunrecouvrementdel’ADNchloroplastique resequencing.Basedonthesedata,anewmethodologyof deplusieursmilliersdelecturesparbpenraisondunombre simultaneousresequencingoflargenumberofchloroplast decopiesélevéd’ADNchloroplastique,nettementsupérieur DNAwasdevelopedwithoutpreliminarychloroplast àcequiestnécessairepourlereséquençage.Unenouvelle isolationorchloroplastenrichment. méthodologiedeséquençaged’ADNchloroplastiqueaété élaboréesansisolementouenrichissementdechloroplastes. Significance and impact of the study :Thismethodhas greatpotentialforexpandingbothphylogeneticand Signification et impact de l’étude :Cetteméthodeaunfort populationgeneticinformationontheevolutionof potentielpouraccroîtrenosconnaissancesengénétiqueeten domesticatedcrops. phylogéniesurl’évolutiondesculturesdomestiquées. Key words :grapeDNA,Illumina,resequencing,indels,SNP Mots clés :ADNderaisin,Illumina,reséquençage,indels, SNP

manuscript received 16th January 2014 - revised manuscript received 21st April 2014

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INTRODUCTION coast.SixotherGeorgiancultivarsexhibitedthe “Saperavi”(ATT)haplotype.Amongthesewasthe Georgiaishometoover500grapecultivars well-knownSaperavicultivar,whichisnowmainly (Ketskhoveli et al., 1960).ThegreaterCaucasus distributedinEasternGeorgiabutisbelievedtohave regioninwhichGeorgialiesiswidelybelievedtobe originatedinsouth-westCaucasus.OnlytwoGeorgian theareawheregrapedomesticationbegan,andthe cultivarsexhibitedthe“Meskhuri”grouphaplotype studyofgeneticdiversityinthisregionisviewedasa (ATA),asthisgroupcomprisesmainlyWestEuropean keytounderstandinggrapedomesticationingeneral cultivars(Beridze et al., 2011).BoththeBandC (Negrul1946). chlorotypesofArroyo-Garcia et al. (Arroyo-Garcia et al., 2006)arecombinedinthehaplotypeATA Theplastidgenomeisaneffectivetoolfor accordingtothesedata(Beridze et al., 2011). interspecificphylogeneticandintraspecific phylogeographicstudiesofangiosperms(Aoki et al., ThegenomicDNAofGeorgiangrapecultivarswas 2006 ;Gutiérrez-Rodríguez et al., 2011).Fewpapers alsostudiedbynuclearmicrosatelliteanalysisbased aredevotedtograpechloroplastDNA.Arroyo-Garcia on20nuclearmicrosatellitesandnocloserelationship et al. (2006)analyzedchloroplastDNAvariationat betweenGeorgianandWesternEuropeancultivars ninepolymorphicmicrosatellitelociof1201 wasfound(Imazio et al., 2013). V. vinifera genotypesbelongingtoboth sativa and sylvestris subspecies.Genotypicanalysesforthese Inthepresentinvestigation,thewholechloroplast microsatellitelociidentifiedeightdifferent DNAoffourGeorgiangrapecultivars,onefromeach chlorotypes(AtoH)(Arroyo-Garcia et al., 2006). haplotypegroup(asdefinedby(Beridze et al., 2011)), Amongthem,onlyfour(A,B,CandD)hadglobal wassequenced.Thesecultivarswere :Rkatsiteli frequenciesgreaterthan5 %.Theintermediate (haplotypeAAA),Saperavi(haplotypeATT), relationshipofchlorotypeBtoallotherchlorotypes MeskhuriMtsvane(haplotypeATA)andChkhaveri suggeststhatitcouldbeanancestral V. vinifera (haplotypeGTA). chlorotype. InthesequencingdataofthefourGeorgiangrape PlastidDNAofCaucasian(Georgian)grapevarieties cultivars(coverageofchromosomalDNA30-40),the wasstudiedbysequencingofsomenoncodingregions coverageofchloroplastDNAreachesseveralthousand ofgrapechloroplastDNA(Beridze et al., 2011). becauseofthehighnumberofchloroplastDNAcopies Duringtheinvestigationof113samplesofaworld- ingenomicDNA,muchhigherthannecessaryfor widesetofgrapecultivarsincludingGeorgian resequencing.Theideaofthenewmethodologyof cultivars,fourplastidDNAhaplotypeswereevident simultaneoussequencingoflargenumberof andweredesignatedbytheircharacter-statesateach chloroplastDNAwasdevelopedbymixingand ofthethreepolymorphicpositions(Beridze et al., sequencingthegenomicDNAfrommanycultivarsin 2011)(Table1). oneIlluminalaneuponindividuallybarcodingeach librarywithoutpreliminarychloroplastisolationor TheAAAplastidhaplotypewasfoundonlyinthe chloroplastenrichment.ThechloroplastDNAoften cultivarsfromGeorgia.Morespecifically,twenty- differentgrapecultivarsandspecieswassequenced three(57.5 %)ofthe40includedGeorgiancultivars simultaneouslyinoneIlluminarun.Asaresult,the exhibitedthishaplotype,ofwhichthe“Rkatsiteli” numberofsequencedchloroplastDNAofgrape cultivarsoriginatingfromEasternGeorgiaprevailed. cultivarsincreasedtoeleven :tothefourGeorgian Thiscontrastswiththeninecultivars(22.5 %)ofthe grapecultivarsmentionedabove,sevenothers,three “Chkhaveri”group(GTA),whicharemostly French-Chardonnay,GouaisBlanc(bothATA cultivatedinWesternGeorgianeartheBlackSea haplogroup)andChasselas(ATThaplogroup)-and

Table 1. Haplogroup definition for investigated cultivars. Nucleotide position Haplogroup Investigated cultivars 205 86715 86721 AAA (1 haplotype) A A A Rkatsiteli ATA (2 haplotypes) A T A Chardonnay, Gouais blanc, Meskhuri Mtsvane ATT (2 haplotypes) A T T Chasselas, Saperavi GTA (1 haplotype) G T A Pinot noir, Maxxa, Chkhaveri, Aladasturi, Krakhuna, Tsitska, Tsolikouri

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fourGeorgiancultivars-Aladasturi,Krakhuna, Biosystems3100or3700geneticanalyzer TsitskaandTsolikouri(allGTAhaplogroup)-were (LaboratoryServicesDivisionoftheUniversityof added. Guelph,ON,Canada).Sequencesweremanually alignedinSe-Al(Rambaut2002)andhaplotype MATERIALS AND METHODS networksweregeneratedusingTCS1.18(Clement et 1. Plant material and DNA isolation al., 2000). Thegrapecuttingsoffourgrapecultivars(Rkatsiteli, 3. Construction of shotgun genomic DNA libraries Saperavi,MeskhuriMtsvaneandChkhaveri)were ShotgungenomicDNAlibrarieswereconstructed collectedattheSaguramoNationalCentrefor usingtheTruSeqDNASampleprepkit(Illumina,San GrapevineandFruitTreePlantingMaterial Diego,CA).Briefly,1 µgofgenomicDNAwas Propagation(Mtskheta,Georgia)andgrowninwater sonicatedonaBioruptor(Diagenode,NJ)for atroomtemperature.TotalgenomicDNAwas 20 cyclesof30 secondesONand90secOFF.After extractedfromyounggrapeleaves.Theleaveswere sonication,DNAwasblunt-ended,3’-endA-tailed groundinliquidnitrogenandDNAwasisolatedusing andligatedtoindexedadaptors.Theadaptor-ligated theCTAB-basedmethod(Lodhi et al., 1994).Allfour DNAwassizeselectedwithAMPure-beadsusingthe DNApreparationswerepreliminarilycheckedby gel-freeprotocoldescribedintheTruSeqDNA sequencingofthreenoncodingplastidDNAregions SamplePrepmanual.Size-selectedDNAwas (the trnH-psbA intergenicspacer,the accD -psa I amplifiedbyPCRtoselectivelyenrichforthose intergenicspacerandthe rpl 16intron). fragmentsthathaveadaptersonbothends. TheDNAofothergrapecultivars(MuscatBlancà Amplificationwascarriedoutfor6cycleswiththe PetitsGrains,Chardonnay,CabernetSauvignon, KapaHiFipolymerase(KapaBiosystems,Woburn, CabernetFranc,VeltlinerRot,Mourvedre,Alvarelhao, MA)toreducethelikelinessofmultipleidentical TraminerRot,GouaisBlanc,Chasselas,Aladasturi, readsduetopreferentialamplification.Thefinal Krakhuna,Tsitska,Tsolikouri)wasalsoanalyzed.The librarieswerequantitatedbyquantitativeqPCRonan originofthesecultivars(totalof59cuttingsreceived ABI7900(LifeTechnologies,GrandIsland,NY). fromtheInstitutNationaldelaRecherche FinalamplifiedlibrarieswerealsorunonAgilent Agronomique(INRA),Montpellier,France)andDNA bioanalyzerDNA7500LabChips(Agilent,Santa isolationprocedurehavebeendescribedinour Clara,CA)todeterminetheaveragefragmentsizeand previouspublication(Beridze et al., 2011). toconfirmthepresenceofDNAoftheexpectedsize range. 2. PCR conditions and Sanger sequencing 4. Sequencing on an Illumina HiSeq 2000 ThePCRconditionsincludedinitialdenaturingat 94 °Cfor1min,30cyclesof94 °Cdenaturing Thelibrarieswerepooledinequimolarconcentration (1 min),55 °Cannealing(2 min)and72 °Cextension andloadedontoonelaneofan8-laneflowcellfor (2 min),followedbyafinalextensionstepat72 °C clusterformationandsequencedonanIlluminaHiSeq (5 min).Sigma-Aldrichchemicalswereusedforthe 2000.Thelibrariesweresequencedfrombothendsof PCRreactions.PCRproductswerepurifiedwith themoleculestoatotalreadlengthof100ntfrom GenElutePCRClean-UpKit(Sigma-Aldrich),dye- eachend.Therawbclfileswereconvertedinto labeledusingaBigDyeTerminatorKit(Applied demultiplexedcompressedfastqfilesusingCasava Biosystems)andanalyzedoneitheranApplied 1.8.2(Illumina).

Table 2 . R egions s equenced by capillary electrophoresis Primers Forward Reverse Primer1: 54 bp deletion (position 30,133 – 30,186) (haplotypes - AAA, ATA, ATT) CTACTGGCCCGGTCTTGAAT TGGAATCGATGGTGCAGAGT Primer 2: 54 bp deletion (position 30,133 – 30,186) (haplotypes - AAA, ATA, ATT) GGGTGTCGCCTGATCAACAA TTAAAGCAGCCCAAGCGAGA Primer 3: 33 bp duplication (position 6,658 - 6,691) (haplotypes AAA, ATA, ATT) TCAAGTCGCACGTTGCTTTC GATGTAATGATGTCGAAGTAGTCAA Primer 4: 18 bp insertion (position 10,001 - 10,019) (haplotype ATT) TTATTCCCACGGCCCGGATA TTGTGCAAGAATCCATAGTTCCC Primer 5: 18 bp duplication (position 19,527-19,544) (haplotype AAA) GGCTGTTCCTAAAGGACCCAA TCGAAAAGACCCATGCTTCC

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ThesequencingofSaperavi,MeskhuriMtsvaneand nucleotidepolymorphisms(SNPs)andinsertions/ ChkhaveriDNAwascarriedoutatthefacilitiesof deletions(indels). RoyJ.CarverBiotechnologyCenter,Universityof IllinoisinUrbana-Champaign(USA) ;RkatsiteliDNA AllofalignedreadsallowedbyBowtie2were wassequencedattheLaboratoryServicesDivisionof consideredandweightedequally.Thisinformation GuelphUniversity(Canada).Fortheassemblyof wasextractedintompileupfilesusingSAMtools(Li Georgiangrapecultivargenome,theMaxxaplastid et al., 2009a).Theveryhighcoverage(rangingfrom DNAsequencewasused(http://www.ncbi.nlm.nih. 8,000to20,000readsperbp)gaveustheflexibilityof gov/nuccore/91983971)(Jansen et al. 2006).Some usingevenlowerqualityreads,relyingonthelawof disputedregionsofchloroplastDNAswere largenumberstomakevariantcallsforhigh-level additionallysequencedbycapillarysequencing mutationswhilealsodetectingpotentiallower-level (Table 2). mutations.Toverifythisapproachcomputationally, wealsoappliedamoretraditionalcomputational 5. Computational analysis pipelinetovariantcalling(filteringonreadqualities andapplyingvariousvariantcallingprogramswith RawFASTAQdatafileswereusedtoalignpaired-end consequentrealignments)toobtainnear-identical readstotheMaxxachloroplastreferencegenome results.Inaddition,lookingattotalreadcounts usingBowtie2(version2.1.0)(Langmead et al., allowedustoeasilyidentifyalarge54-bpdeletionin 2012)usingdefaultalignmentcriteria,inparticular : threeoutoffourcultivars(seeFigure 1).Thefiltered chloroplastreadswereassembledinparallelby Readswithmorethan15ambiguouscharacters(Ns SOAPdenovocomputerprogramandcoinciding and.s)werefilteredout. resultswerereceived(Li et al., 2009b). Readswithgapswithin4positionsofreadendswere ThechloroplastDNAsequencesoftheGeorgian disallowed. grapecultivarshavebeendepositedintheDNAData Traditionally,variantcallingprogramsaregeared BankofJapan.Accessionnumbers :AB856289 towardsnuclearDNAofdiploidorganismsand Rkatsiteli ;AB856290Saperavi ;andAB856291 relativelylowreadcoverage.Incaseofchloroplasts, MeskhuriMtsvane. however,thereisamultitudeofDNAmoleculesper RESULTS chloroplastaswellasamultitudeofchloroplastsper cell.Thisresults,ononehand,inaveryhighcoverage Weidentified86SNPsinthreeGeorgiangrape ofchloroplastDNA(takenasone)duringHiSeq cultivarsincomparisontothereferenceMaxxa sequencingand,ontheotherhand,inthepossibility chloroplastDNA(Table3).Forty-sevenidenticalSNPs ofheteroplasmy(presenceofmultiplechloroplast werecharacteristicforallthreecultivars(Rkatsiteli+ genomeswithinacell).Becauseofthis,weuseda Saperavi+MeskhuriMtsvane),theadditionalSNPs somewhatdifferentapproachinidentifyingsingle were :18forSaperavi,4forRkatsiteli,6forMeskhuri

Figure 1. Read counts (depth coverage) for the alignment of Meskhuri Mtsvane cultivar chloroplast DNA. The reads of Meskhuri Mtsvane were aligned against the reference genome of Maxxa chloroplast. The graph shows the depth coverage for each position of the reference sequences after initial alignment performed by Bowtie 2. The dip at around 30,000 bp corresponds to a 54-bp deletion in Meskhuri Mtsvane chloroplast genome.

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Mtsvaneand11forbothRkatsiteliandMeskhuri 6-aa-longpeptideduplicationoccurred(TLLNRN ; Mtsvane.Thenumberofnoncodingsubstitutionswas position934-939intherpoC2protein). 67andcodingsubstitutions19.In8cases, nonsynonymoussubstitutionswereobserved,which Toillustratetheevolutionaryrelationshipbetweenthe alteredtheaminoacidsequence(Table3).In13cases, studiedcultivars,aphylogenetictreebasedonthe synonymoussubstitutionsweredetected.Ingeneycf1, multiplealignmentswasconstructedusingNCBI 5SNPs(3nonsynonymousand2synonymous)were BLAST’sTreeViewDisplayoptionthatconstructsa observed. phylogeneticdistancetreebasedonfastminimum evolutionapproach(Desper et al., 2002).Figure 3 Shortindelswerealsodetected :inSaperavi-11 representstheresultingphylogenetictree. insertionsand15deletions ;inRkatsiteli-15insertions and9deletions ;andinMeskhuriMtsvane- Chloroplastisolationorchloroplastenrichmentfrom 13 insertionsand9deletions.InChkhaveri,no leavesofgrapecultivarsor Vitis speciesisagreat insertionsordeletionsweredetectedincomparisonto problemduetothelargeamountofphenolic referenceDNA.Asaresult,thelengthofsequenced compoundspresentinleaves,whichnegatively chloroplastDNAvaried,withRkatsiteliat160,927, influenceDNAisolation.Therefore,amethodfor Saperaviat160,928andMeskhuriMtsvaneat160,906 determiningchloroplastDNAsequencedirectlyfrom bpinlength. genomicDNAwasdeveloped. Intheregionsurroundingposition30,100-30,200bp, Insequencingdata,thenumberofreadsforchloroplast adrasticfallofreadnumberswasobservedinall DNAcan,insomecases,reach20,000perbasedueto cultivarsexceptChkhaveri(Figure 1).Thisregionwas ahighnumberofchloroplastDNAcopiesingenomic subsequentlysequencedbycapillaryelectrophoresis. DNA,whereasforresequencingofDNA,40-50reads A54-bpdeletionatposition30,133-30,186(trnC- aresufficient.Onthebasisofthesefacts,thenew GCA–petNintergenicspacer)wasdetectedin methodologyofchloroplastDNAresequencingwas Saperavi,RkatsiteliandMeskhuriMtsvane,butnotin developed.TheideawasmixingthegenomicDNA Chkhaveri.Theexistenceofthisdeletionwaschecked frommanycultivarsandsequencingtheminone inothergrapecultivarsincludingWestEuropeans.It Illuminalane,barcodingeachlibrarysothatreads wasestablishedthatthis54-bpdeletionistypicalfor comingfromeachcultivarcanbeidentified.The allcheckedAAA,ATAandATThaplogroupsofthe numberofchloroplastDNAreadsisquitehigh, world-widesetofgrapecultivars,butnotforGTA sufficienttocomputechloroplastDNAsequence,but haplogroup(Figure 2). readsfromchromosomalarelowandareignored. Additionally,threeotherlongindelsweredetectedby Duringmanuscriptpreparation,otherpapersappeared capillarysequencinginchloroplastDNA(Table4). wherenext-generationsequencingtechniquesalso Twowereinintergenicspacers(Rps16–trnQ-UUG haveallowedforsimultaneousgenomicstudiesof andtrnS-GCU–trnG-GCC) ;theotherwasan18-bp mitochondrialandchloroplastDNA(Hancock-Hanser duplicationintherpoC2geneofRkatsiteli(AAA et al., 2013 ;Middleton et al., 2014).Here,ten haplotype ;position19,527-19,544).Asaresult,a chloroplastDNAsofdifferentgrapecultivars

Figure 2. 1% agarose gel electrophoresis of PCR-amplified grape chloroplast DNA fragment 29,945 - 30,506. Lanes: 2. Muscat Blanc à Petits Grains (ATT); 3. Chardonnay (ATA); 4. Cabernet Sauvignon (ATT); 5. Cabernet Franc (ATT); 6. Veltliner Rot (ATT); 7. Mourvedre (GTA); 8. Alvarelhao (GTA); 9. Traminer Rot (ATT); and 10. Chkhaveri (GTA). Lanes 1 and 11, 100-bp DNA marker.

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Table 3. The intravarietal SNPs and amino acid substitutions in Georgian grape chloroplast DNA.

Nucleotide Amino Acid Locus Saperavi Rkatsiteli Meskhuri Mtsvane Position Substitutions 205 Intergenic trnH-psbA G-A G-A G-A 1552 Intergenic psbA - trnK-UUU G-A 4527 Intergenic trnK-UUU - rps16 T-G 4547 Intergenic trnK-UUU - rps16 A-T 5591 Intron rps16 A-C 5978 Intron rps16 T-G T-G 8114 Intergenic rps16 - trnQ-UUG A-G A-G A-G 8175 Intergenic rps16 - trnQ-UUG T-C T-C T-C 9996 Intergenic trnS-GCU - trnG-GCC A-G A-G A-G 10266 Intergenic trnS-GCU - trnG-GCC A-T 11120 Intron trnG-GCC A-G A-G 11121 Intron trnG-GCC A-T 11625 Intergenic trnR-UCU - atpA T-A T-A T-A 14416 Intron gene atpF A-G A-G A-G 14794 Intergenic atpF - atpH T-A T-A T-A 20840 Gene rpoC2 C-T C-T C-T S - N 21046 Gene rpoC2 G-T G-T G-T F - L 22321 Gene rpoC2 A-G A-G A-G syn 24437 Intron rpoC1 C-A C-A C-A 28707 Intergenic rpoB - trnC-GCA T-G 29232 Intergenic rpoB - trnC-GCA T-C 29507 Intergenic rpoB - trnC-GCA C-T C-T C-T 29571 Intergenic rpoB - trnC-GCA G-T G-T G-T 30783 Intergenic petN - psbM T-C T-C T-C 32670 Intergenic psbM - trnD-GUC C-G C-G C-G 34037 Intergenic trnE-UUC - trnT-GGU C-A C-A C-A 36031 Intergenic trnT-GGU - gene psbD C-A 36397 Gene psbD A-C A-C A-C syn 39357 Gene psbZ T-A T-A T-A syn 39584 Intergenic psbZ - trnG-GCC T-G T-G T-G 39585 Intergenic psbZ - trnG-GCC C-G C-G C-G 39586 Intergenic psbZ - trnG-GCC C-A C-A C-A 39883 Intergenic psbZ - trnG-GCC T-A T-A T-A 39885 Intergenic psbZ - trnG-GCC C-T C-T C-T 39961 Intergenic psbZ - trnG-GCC T-G T-G T-G 42474 Gene psaB C-T C-T C-T G - S 43685 Gene psaA T-G syn 43910 Gene psaA C-G syn 50339 Intergenic trnT-UGU - trnL-UAA T-G 50627 Intergenic trnT-UGU - trnLUAA T-A T-A T-A 51700 Gene trnL-UAA A-T 54999 Intergenic ndh3 - trnV-UAC T-G T-G 55094 Intergenic ndh3 - trnV-UAC T-C T-C T-C 59143 Intergenic atpB - rbcL A-T

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Table 3. The intravarietal SNPs and amino acid substitutions in Georgian grape chloroplast DNA.

Nucleotide Amino Acid Locus Saperavi Rkatsiteli Meskhuri Mtsvane Position Substitutions 61186 Intergenic rbcL - accD T-G T-G T-G 63478 Intergenic accD - psaI T-A T-A T-A 63819 Gene psaI A-G A-G A-G syn 67651 Intergenic petA - psbJ T-C T-C T-C 70248 Intergenic psbE - petL A-C A-C 70595 Intergenic psbE - petL C-T C-T C-T 70921 Intergenic petL - petG G-A G-A 71588 Intergenic trnP-UGG - psaJ C-T C-T C-T 73579 Gene rpl20 C-T syn 73765 Gene rpl20 G-A syn 75398 Intron clpP C-T 77099 Intergenic clpP - psbB C-T C-T C-T 80022 Intron petB A-C A-C A-C 80194 Intron petB C-T 80276 Intron petB C-T C-T C-T 86715 Intron rpl16 T-A 86721 Intron rpl16 A-T 89112 Gene rps19 C-T syn 99217 Intergenic ycf2 - trnL-CAA A-T A-T 99218 Intergenic ycf2 - trnL-CAA T-G T-G 99219 Intergenic ycf2 - trnL-CAA C-A C-A 99220 Intergenic ycf2 - trnL-CAA A-T A-T 115531 Intergenic ycf1 - ndhF A-T 117310 Gene ndhF T-G T-G T-G syn 117791 Intergenic ndhF - rpl32 T-A T-A T-A 119481 Intergenic rpl32 - trnL-UAG G-T G-T G-T 119571 Intergenic rpl32 - trnL-UAG T-G T-G T-G 121732 Intergenic ycf5 - ndhD A-C A-C A-C 123321 Intergenic ndh4 - psaC A-T A-T A-T 123367 Intergenic ndhD - psaC T-A 123664 Intergenic psaC - ndhE T-G T-G 123690 Intergenic psaC - ndhE C-A 124258 Intergenic ndhE - ndhG G-A G-A G-A 125681 Gene ndhI G-A G-A G-A syn 126020 Gene ndhA G-T L - M 128420 Gene ndhH C-T E - K 131820 Gene ycf1 T-C T-C T-C Q - R 133211 Gene ycf1 T-G T-G syn 133359 Gene ycf1 A-C L - W 133934 Gene ycf1 G-T G-T G-T syn 133982 Gene ycf1 A-C I - M

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(Chardonnay,GouaisBlanc,Chasselas,Aladasturi, Krakhuna,Tsolikouri,TsitskaandRkatsiteli(asa control))andspecies( V. champinii and V. rupestris (notpresentedinthisarticle))weresequenced simultaneouslyinoneIlluminalane.Preliminary resultsshowthat,togetherwithchloroplastDNA, grapemitochondrialDNAsequencesof10samples canbecomputed.Inaverage,thenumberofreadsper baseforeachchloroplastDNAsamplevariedbetween 1000-2000.Therefore,itispossibletoincreasethe Figure 3. Complete chloroplast genome phylogeny amountofsequencedchloroplastDNAby2-3times of grape cultivars. The GenBank accessions used for the analyses are NC_007957.1 (Maxxa), basedonthefactthatfordeterminationofchloroplast Chkhaveri, Rkatsiteli, Saperavi DNAsequence,50-100readsaresufficient. and Meskhuri Mtsvane. ThecomparisonofsequencedchloroplastDNAgave withoutasinglemismatch)tothereferenceMaxxaand 100 %identitytoChardonnayandGouaisBlanc, consequentlytoPinotNoirchloroplastDNA.Saperavi differingfromMeskhuriMtsvaneby1-bpinsertion andChasselaschloroplastDNAdifferbyoneinsertion. (position5415)and1-bpdeletion(position121,616) (allATA) ;thedifferencebetweenChasselasand MeskhuriMtsvanechloroplastDNAdiffersbytwo Saperaviwasa1-bpinsertion(position39554)(both indelsfromChardonnayandGouaisBlancchloroplast ATT) ;Maxxa,Chkhaveri,Aladasturi,Krakhuna, DNA.Ingeneral,itcanbeproposedthatallGTA,ATA TsitskaandTsolikouri(allGTA)gave100 %identity. andATThaplotypescontainpracticallyidentical chloroplastDNA.ThesinglehaplotypeAAA,to DISCUSSION whichmorethanhalfoftheanalyzedGeorgian cultivarsbelong,hadnoanalogsintheworld-wideset Todate,asequenceofchloroplastDNAisavailablefor ofgrapecultivarsuntilnow(Beridze et al., 2011). onlyonegrapecultivar, V. vinifera L.cvMaxxa(Jansen et al., 2006).Tothatend,abacterialartificial Oneofthedifferencesbetweenthefourhaplotypesof chromosome(BAC)libraryofMaxxagenomicDNA grapecultivars,besidesSNPs,isa54-bpdeletionin wasconstructed,BACclonescontainingthechloroplast thetrnC-GCA ‒ petNintergenicspacer(position genomeinsertswereisolated,andthenucleotide 30,133-30,186),whichisobservedinAAA,ATAand sequencesoftheBACclonesweredeterminedbythe ATThaplotypesandabsentinGTAhaplotype. shotgunmethod(Jansen et al., 2006). Additionally,AAA,ATAandATThaplotypesshowa Duringthedeterminationofahighqualitydraft 33-bpduplicationintheRps16–trnQ-UUGintergenic genomesequenceofacultivatedcloneof V. vinifera L. spacer(position6,658-6,691).InATThaplotype,an cvPinotnoir,Velasco et al. showedthatthechloroplast 18-bpduplicationinthetrnS-GCU–trnG-GCC DNAsequenceofPinotnoirwasidentical(withouta intergenicspacer(position10,001-10,019)was singlemismatch)tothatofMaxxa(Velasco et al., detected. 2007). Thesecondsubstantialdifferencebetweenthestudied Themosti nterestingresultofthepresentstudyisthat grapecultivarsisthepresenceofan18-bpduplication thechloroplastDNAsequenceofGeorgiangrape (TATTTCTATTTAATAACG ;position19,527- cultivarsispracticallyidenticaltothatofWest 19,544 ;rpoC2gene)inRkatsiteli(AAAhaplotype) Europeancultivars :thechloroplastDNAofChkhaveri chloroplastDNA.Asaresult,a6-aa-longpeptide andfourotherGeorgiancultivars(Aladasturi, (TLLNRN ;position934-939intherpoC2protein) Krakhuna,Tsitska,Tsolikouri)isidentical(also duplicationisobserved.

Table 4. Long indels in grape chloroplast DNA.

Nucleotide position Locus Haplogroup Indellength Indeltype 6,658-6,691 Rps16 – trnQ-UUG intergenic spacer AAA, ATA, ATT 33 bp Duplication 10,001-10,019 trnS-GCU – trnG-GCC intergenic spacer ATT 18 bp Duplication 30,133 - 30,186 trnC-GCA –petN intergenic spacer AAA, ATA, ATT 54 bp Deletion 19,527-19,544 rpoC2 gene AAA 18 bp (6 aminoacid) Duplication

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Thephylogeneticschemebasedonindelpriorityof UnfortunatelythecultivarsfromSouthCaucasuswere grapechloroplastDNAhaplotypesispresented notanalyzedinthisinvestigation,thoughtheroleof (Figure 4).Comparativeanalysisshowsthatthe SouthCaucasusingrapedomesticationwas separationofChkhaveri(GTAhaplotype)andthree underlined.LaterDNAsequencediversitywas othercultivars(AAA,ATA,ATThaplotypes)occurred investigatedinagroupof40wildgrape( Vitis vinifera bya33-bpduplicationanda54-bpdeletioninAAA, subsp. sylvestris )samplesfromtheSouthCaucasusat ATA,ATThaplotypes(noother Vitis speciesshowthis fourplastidregions( accD-psa Iintergenicspacerwas 54-bpdeletion).Furtherseparationoccurredbetween added ;63,186-T,C).Thisgroupincluded22 ATTandthetwohaplotypesATAandAAAbyan18- samplesfromGeorgia,9samplesfromAzerbaijan,2 bpduplicationandbetweenATAandAAAagainbyan samplesfromArmeniaand7samplesfromTurkey 18-bpduplication. (thebaseintheparenthesiscorrespondsto accD-psa I intergenicspacer)(Pipia et al., 2012). 8of11grapechloroplastDNAsweresequencedby thenewmethodologyofsimultaneousresequencing. Asinthecaseoftheworld-widesetofgrapecultivars, Thismethodologycanbeusedalsofordetermination fourplastidhaplotypeswereevidentinthe40wild ofmitochondrialDNAsequences.Ithasgreatpotential samples :AAA(T)–22samples,ATT(T)–6samples, forexpandingbothphylogeneticandpopulation GTA(C)–1sample,andATA(T)–11samples.The geneticinformationontheevolutionofdomesticated AAA(T)haplotypewasrestrictedtoGeorgiaand crops. Azerbaijan,theATA(T)haplotypewasdistributed acrosstheentirestudyarea,theATT(T)haplotype Myles et al. findsupportforaNearEastoriginof wasdistributedinthesouthernpartofthestudyarea vinifera andpresentevidenceofintrogressionfrom fromtheBlackSeatotheCaspianSea,andthe local sylvestris asgrapesmovedintoEurope(Myles et GTA(C)haplotypewasonlyfoundinsouthwestern al., 2011).ButexactDNAdataofgrapedomestication Georgia.TheAAA(T)haplotypeisrestrictedtoboth isstillabsent.Thesequencingofwildsamplescanshed wild( V. vinifera subsp. sylvestris )andcultivated( V. lightongrapedomestication.Arroyo-Garcia et al. vinifera subsp. vinifera )grapesamplesfromthe suggestedtheexistenceofatleasttwooriginsforgrape Caucasus.TheobservationthathaplotypeAAA(T)is cultivars,oneintheNearEastcharacterizedby foundonlyamongGeorgiangrapecultivarsandis chlorotypesC(ATAhaplotypeinthepresent restrictedtowildsamplesofGeorgiaandAzerbaijan publication)andD(ATThaplotype)andawesternone suggeststhatthishaplotypewasdomesticatedin relatedtoIberianPeninsulacultivarsandcharacterized SouthCaucasus.Itwasproposedthattheinitialgrape bychlorotypeA(GTAhaplotype)(Arroyo-Garcia et dispersaltoGreeceandEgyptmayhaveoccurrednot al., 2006).Thegeographicdistributionobservedfor onlybylandbutalsobysea,becausetheAAA(T) somechlorotypesin sylvestris groupsisstillobserved haplotypewasformedintheAlazanihearth(farfrom incultivatedgroups.CultivarswithchlorotypeAare theBlackSea)andthishaplotypeisabsentinthe highlyabundantinWesternEuropewhiletheyarenot world-widesetofcultivars(Pipia et al., 2012). observedintheNearandMiddleEastsamples. Similarly,chlorotypesCandD,whicharevery AsmentionedbyArroyo-Garcia et al. manyofthe commonamongtheNearEastandMiddleEast currentgrapevarietiescanbetracedbackhundredand cultivars,arelessfrequentamongIberianPeninsula eventhousandyearsbasedonhistoricalrecords ;they cultivars. areprobablyseparatedfromtheirwildrelativesbya

Figure 4. The phylogenetic scheme based on single nucleotide polymorphisms (SNP) and insertions/deletions (indel) priority of grape chloroplast DNA haplogroups.

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lownumberofsexualgenerations(Arroyo-Garcia L. BulletinoftheGeorgianNationalAcademyof et al., 2006).Regardinggrapedomestication, Sciences 5 (N1) :98-103. currentlyonlyoneevidenceseemscredible :AAA ClementM.,PosadaD.andCrandallK.A.,2000.TCS :a haplotypeisrestrictedtobothwildandcultivated computerprogramtoestimategenegenealogies. grapesamplesinSouthCaucasus(Pipia et al., 2012). Molecular Ecology 9 (10) :1657-1659. Thishaplotypeseemstohavebeendomesticatedin DesperR.andGascuelO.,2002.Fastandaccurate SouthCaucasus. phylogenyreconstructionalgorithmsbasedonthe minimum-evolutionprinciple. J. Computational CONCLUSIONS Biology 9 (5) :687-705. Todate,acompletesequenceofchloroplastDNAhas Gutiérrez-RodríguezC.,OrnelasJ.F.andRodríguez- GómezF.,2011.ChloroplastDNAphylogeography beenpublishedforthegrapecultivarMaxxa . Inthe ofadistylousshrub( Palicourea padifolia ,Rubiaceae) presentinvestigation,thechloroplastDNAsequence revealspastfragmentationanddemographic ofeightGeorgiangrapecultivars(Rkatsiteli,Saperavi, expansioninMexicancloudforests. Molecular MeskhuriMtsvane,Chkhaveri,Aladasturi,Krakhuna, Phylogenetics and Evolution 61 (3) :603-615. Tsitska,Tsolikouri)andthreeFrenchcultivars Hancock-HanserB.L.,FreyA.,LeslieM.S.,DuttonP.H., (Chardonnay,GouaisBlanc,Chasselas),belongingto ArcherF.I.andMorinP.A.,2013.Targetedmultiplex fourdifferenthaplogroups(AAA,ATT,ATA,GTA), next-generationsequencing :advancesintechniques wasdeterminedbyIlluminaresequencingofgenomic ofmitochondrialandnuclearDNAsequencingfor DNA.Anewmethodologyofsimultaneous populationgenomics. Molecular Ecology Resources resequencingoflargenumberofchloroplastDNAwas 13 (2) :254-268. developedwithoutpreliminarychloroplastisolationor ImazioS.,MaghradzeD.,deLorenzisG.,BacilieriR., chloroplastenrichment.Thismethodhasgreat LaucouV.,ThisP.,ScienzaA.andFaillaO.,2013. potentialforexpandingbothphylogeneticand Fromthecradleofgrapevinedomestication : molecularoverviewanddescriptionofGeorgian populationgeneticinformationontheevolutionof grapevine( Vitis vinifera L.)germplasm. Tree domesticatedcrops. Genetics & Genomes 9 (3) :641-658. Acknowledgements :Theauthorswouldliketothank JansenR.K.,KaittanisC.,SaskiC.,LeeS.-B.,TomkinsJ., M. K.Bendukidzeforpermanentinterestandsupport.This AlversonA.andDaniellH.,2006.Phylogenetic researchwasfundedbyKnowledgeFund.KnowledgeFund analysesof Vitis (Vitaceae)basedoncomplete isthefundingorganizationoftheFreeUniversityofTbilisi chloroplastgenomesequences :effectsoftaxon andAgriculturalUniversityofGeorgia. samplingandphylogeneticmethodsonresolving relationshipsamongrosids.BMCEvolutionary REFERENCES Biology 6 (1) :32. KetskhoveliN.,RamishviliM.andTabidzeD.,1960. AokiK.,MatsumuraT.,HattoriT.andMurakamiN.,2006. Ampelography of Georgia .MetsnierebaPublishing ChloroplastDNAphylogeographyof Photinia glabra House,Tbilisi,Georgia. (Rosaceae)inJapan. Am. J. Botany 93 (12):1852- 1858. LangmeadB.andSalzbergS.L.,2012.Fastgapped-read alignmentwithBowtie2. Nature Methods 9 (4) :357- Arroyo-GarciaR.,Ruiz-GarciaL.,BollingL.,OceteR., 359. LopezM.A.,ArnoldC.,ErgulA.,SöylemezoğluG., UzunH.I.,CabelloF.,IbanezJ.,AradhyaM.K., LiH.,HandsakerB.,WysokerA.,FennellT.,RuanJ., AtanassovA.,AtanassovI.,BalintS.,CenisJ.L., HomerN.,MarthG.,AbecasisG.,DurbinR.and CostantiniL.,GorislavetsS.,GrandoM.S.,Klein G.P.D.P.Subgroup,2009a.TheSequence B.Y.,McGovernP.E.,MerdinogluD.,PejicI.,Pelsy Alignment/MapformatandSAMtools. Bioinformatics 25 (16):2078-2079. F.,PrimikiriosN.,RisovannayaV.,Roubelakis- AngelakisK.A.,SnoussiH.,SotiriP.,TamhankarS., LiR.,YuC.,LiY.,LamT.-W.,YiuS.-M.,KristiansenK. ThisP.,TroshinL.,MalpicaJ.M.,LefortF.and andWangJ.,2009b.SOAP2 :animprovedultrafast Martinez-ZapaterJ.M.,2006.Multipleoriginsof toolforshortreadalignment. Bioinformatics 25 (15) : cultivatedgrapevine( Vitis vinifera L.ssp. sativa ) 1966-1967. basedonchloroplastDNApolymorphisms. Molecular LodhiM.A.,YeG.-N.,WeedenN.F.andReischB.I.,1994. Ecology 15 (12) :3707-3714. AsimpleandefficientmethodforDNAextraction BeridzeT.,PipiaI.,BeckJ.,HsuS.-C.,GamkrelidzeM., fromgrapevinecultivarsand Vitis species. Plant GogniashviliM.,TabidzeV.,ThisP.,BacilieriR., Molecular Biology Reporter 12 (1) :6-13. GotsiridzeV.,GlontiM.andSchaalB.,2011.Plastid MiddletonC.P.,SenerchiaN.,SteinN.,AkhunovE.D., DNAsequencediversityinaworldwidesetof KellerB.,WickerT.andKilianB.,2014.Sequencing grapevinecultivars( Vitis vinifera L.subsp. vinifera ). ofchloroplastgenomesfromwheat,barley,ryeand

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