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Frdric CHOULETThe IWGSC: Strategies & Activities to Sequence the Bread Wheat GenomeINRA GDEC Clermont-Ferrand, France

Streptomyces8 Mb

1234567ABD17 GbHexaploid Bread Wheat Genome

Launched in 2005on the initiative of wheat growerswww.wheatgenome.org

Executive directorCo-chairsJ. DvorakY. OgiharaR. AppelsC. FeuilletB. GillB. KellerK. EversoleCoordinating committee65 membersGeneral members>800 members22 sponsorsArcadia BiosciencesACPFGBayer CropScienceBiogemmaCrales ValleCIMMYTCSIRODow AgroSciencesDuPontEvogeneFlorimond DesprezGRDCGraminorHPIINRAKansas WheatKWSLimagrainMonsantoPunjab Ag UnivRAGTSyngenta The IWGSCJ. RogersHigh quality annotated genome sequenceOrdered sequence linked to markers & phenotypes Why? Vision The IWGSCLay a foundation to accelerate wheat improvementYield potential and stability

Adaptation to climate changeDurable resistance to pathogensQuality of grains Wheat Improvement Is ComplexImproved wheat varietiesGenomics ResourcesPhysical mapsGenome/TranscriptomeHT genotyping platformsBioinformaticsGenetics ResourcesMapping pop.Association panelsGenebanksMutant pop.Phenotyping datain controlled conditionsin the fieldsimaging technologiesMethodologiesModelingGM approachesAgronomyagricultural practices, policiesBreeding methods An integrated toolboxLink to genetic maps and phenotypesOf what grows in the fieldsProduce a useful sequence:Reduce the genome complexity1234567ABDADBTriticum aestivumTriticum urartuAegilops speltoidesAegilops tauschiiFlow sorting chromosomesPhysical map (BACs)Sequence BACs Goal and Strategy

Flow sorting

J. Dolezel1234567ABD600-1000 Mb300 Mb 2014 all BAC libraries available @ CNRGVaneuploid linefluorescence intensity Current status Physical maps

www.wheatgenome.org10 IWGSC strategy1234567ABDRef Sequencesorted chromosomesBACs11

% repeated kmersk

Wheat genome (17 Gb)Rice genome (400 Mb)E. coli genome (4 Mb)Wheat 3B (1 Gb)Wheat BAC contig (4 Mb)

GenesTEsWhole genome shotgun assemblyBACs1234567ABDRef Sequencesorted chromosomesBACs14 WGSADBTriticum urartuAegilops speltoidesAegilops tauschii1234567ABDWGSBrenchley et al. Nature 2012Roche/454 depth 5xWGSIllumina (PE+MP lib.)Ling et al. Nature 2013WGSJia et al. Nature 2013Scaffold N50: 50 kb15Chr. Survey Sequences

Illumina 80x (PE 2x100bp) Survey Sequences- TGAC, Norwich- IEB, Olomouc- MIPS IBIS, Munich1234567ABDRef Sequencesorted chromosomesBACs3B16 Survey Sequences1 assembly per chr. arm10 Gb10 million contigscontig N50: 2.4 kbAnnotation:124,000 genes (HC)AA: 40,000BB: 45,000DD: 39,000"GenomeZipper" (Mayer et al, MIPS)WheatBrachy.RiceSorghum66,000 genes "zipped"+9,000 genes anchored with POPSeq approach (Stein et al, IPK) Survey SequencesHigh level of "small scale duplications"3.5 M markers mapped13 M SNPs from POPSeqNo subgenome dominanceResultsPhylogeny A-B-DCore and pan genome of wheatLimited gene loss after polyploidizationHomoeolog-specific gene expression1234567ABDRef Sequencesorted chromosomesBACs 3BSEQ- INRA GDEC (C. Feuillet)- CEA-IG-Genoscope (P. Wincker)- INRA URGI (H. Quesneville)- INRA CNRGV (H. Berges)

3B193B #BACs8452 #BAC pools922 #Roche 8 kb MP lib.922 #GS-FLX runs150 bp coverage (Roche/454)36x

BAC-ends (Sanger)42,551 3B Survey Seq (Illumina)82x 3B MTP-Sequencing3BBAC-poolsScaffolds 1358 scaff 774 Mbpseudomoleculeunlocalized7% 2808 scaff 833 Mb 0.9 Mb (N50)Finishing93%NNNNNNNNOrdering Building a pseudomolecule

protein coding genestransposable elements(pseudogenes:pseudomolecule7,26427%)234,606(85%) Annotation

10RecombinationLD

5centromeric retrotransposons %0774 Mb68 Mb59 Mbpericentromere122 Mb Structure, Recombination

9070(%)155(# transcripts)

Gene density(/10 Mb)0100200

(# conditions)158Expression774 MbAlt. splicingTE densitySynteny

rice01774 MbWheat3BBrachy.chr2Ricechr1Sorghumchr3Wheat3BBrachy.chr2Ricechr1Sorghumchr316%18%15%37%intra-chr. duplicates Lots of small-scale duplications# syntenic genes3400-36003899# syntenic genes3400-36003899# nonsyntenic genes150-2102065Wheat3BBrachy.chr2Ricechr1Sorghumchr335%Wheat3BBrachy.chr2Ricechr1Sorghumchr36%5%4% Lots of small-scale duplicationsinter-chr. duplicatesintra-chr. duplicates

B. distachyon chr2O. sativa chr1S. bicolor chr33B Nonsyntenic gene distribution3B Structural and functional PartitioningDisease resistance genes (Sr, Lr, Yr, Stb)Solid stem (saw fly)YieldDrought toleranceBoron transporterFlowering timeNitrogen Use EfficiencyChromosome pairing 13 gene cloning projects Map-based cloning on 3B Pseudomolecules, current status

Current Status1A2A3A4A5A6A7A1B1D2B2D3B3D4B4D5B5D6B6D7B7D31

1234567ABDRef Sequencesorted chromosomesBACs IWGSC resource accesshttp://wheat-urgi.versailles.inra.fr/Seq-Repository/http://plants.ensembl.org/Triticum_aestivumSurvey Seq3B pseudomoleculeGbrowse @ URGI32

Acknowledgments 3B team

Sbastien TheilNatasha GloverJosquin DaronLise PingaultPierre SourdilleEtienne PauxPhilippe LeroyJacques Le GouisNicolas GuilhotNelly CubizollesCatherine FeuilletFranois BalfourierM. AlauxL. CoudercV. JamillouxH. QuenesvilleURGIH. BergesA. BellecCNRGVBIAA. AlbertiV. BarbeJ. PoulainS. DurandS. MangenotJM. AuryA. CoulouxP. WinckerGenoscopeJ. DolezelJ. SafarIEBK. VandepoeleK. Mayer et al.P. SchnableS. RounsleyD. WareC. GaspinSABVIBMIPSTGACJ. Rogers, M. Caccamo et al.J. RogersK. EversoleElodie Rey

TACACCCAAGTTGGTGAGCTGAAAGTCCACCTCCTCCTTCTCTTCTCCTCGGACATGTGTATGATGATATTTGGACATGTGGCCTTTATTTGCAGGGAACACCTCCAAGAGTGTATGATGATATTTTATGGACATGTAATTAGTAGACAGCTAGGTGTTTGTGCCAACCACGTGTAACTCATGACTATTATATAAGGTCACCACGGAGTATCTAAATAGACTAGAAAAAAAGCTAGTAGGATGATGTCATAATTCAACATAAAACGTAGGGAGGTCCGCTATGTGATTGGGTGCACAAAACACATAGGATTGTTCGAGCTCTGCTTAAAAGAAGTAGGAAAAGTAATCGGGTTGATAACAATCGACAGTTTAGCTCCCTGCAAAATACGAGCATTCA__IWGSC__TATTATTTCACAAAGCTGATGACAGATGTTTAATTTACCATATACAAGATTGTCTATAAACCTTTAAATTTCATGCTTATGCAGTTGCCTTGCACGTAAACATTCAAGGCAAGAAAATGACTACGAGATTAATAGTATACTTCTAATTATCAAGACAACTACACGGTCAGATGTTTCAGTTATCTCAGTTGAACATGTAATGCATGAGCTCGGTTACAAGTGGCACACAATAATCTGCCCTGCAACCTGAGGTGGTACCTGATCAATATTAAACTCACATCCAAATAGTAGATAACTACTCTCTCCGTTCCAAATTACTCGTCGCAGAAATGGTTGTATCTAGAATTAAAATACATCTAGATACATCTATAGATGCAACAAGTAATTCGGAACGGAGGGAGTAACCGAGAAGACTATGATCCCTCCTGGGTTTGCCATGATCTAATTAGTACCATATGAATAGTGGGTGATCAAC__KLAUS_MAYER__GTTCCGAGTCCATTCACATTACCAGTTTTAGTCAGCCCCCATCATTCAAG__KELLYE_EVERSOLE__ACAAGGTGAAAATGTCGCGCGAATTTGGAACAAGGAGGACGCCGATCACT__JANE_ROGERS__TGCTGATCGATCAGAGGTGTTGAAACCAGGGAGGTGTAGGAATATCAGGT__JAROSLAV_DOLEEL__GTGTGCGAGCCGATTGCATTCGGGAGAACCCCTCTCTGGGCGGATTCCGG__CURTIS_POZNIAK__GGGACCGAATCTCGCCAAACACATCCCGGACCATGACACGTCAAGATCAA__CATHERINE_FEUILLET__AGAGAATACTATTGCTGGTGGCCGCTCGTAGGGGTATGGAACCGTACTAC__EDUARD_AKHUNOV__TATATATTTCCGCGGCGTATACCTTGGTAAAACTTCACAAAAATTCGGTT__BIKRAM_GILL__TACTCAGGGTGGAGTACCTTTTCATCTACCAAAGTCTCGCATGCGATTGC__TAKASHI_ENDO__AAGGTATCTGTCGGATAGCGGGGTTGTTGCTGCCCGGCCGCAAAGGCTAA__MARIO_CACCAMO__CTACCTGAGCATATAGGCTTTCCTTGGTTCACTGTATAGTTTCGGTCGCC__ETIENNE_PAUX__ACTTACTATTCCCTCCCGATGGTCCAATTATGTCAGATAGATGCGGAGGT__PATRICK_WINCKER__TCTGTGGTGCATCACCGCGTGTATAAAATCAGATAAATCGCCCGATAGCT__HIROKAZU_HANDA__ACACCTTATTAAATCAGGGCTGTAGCGTACGAGAGGTAGGGGCTCCGCAC__ANDREW_SHARPE__CAGGTCAAGATCTGGTCAGGAAAGTCACTAAAAGTCTGGAACCGCTAGCG__ODDARNE_OLSEN__TCCGTAGATTCATTTCAAACCATAAACAAGTTGTTGGCTAACCCTGTGGT__SIMEN_SANDVE__ACACGCTGTGCTTCAGCATGCCCTACATCGCCCCCCATTGTGTTAACTAC__BURKHARD_STEUERNAGEL__TACTAAACCGGGAATTCAGCCCCCCCAACATAATCATGCGGCAGATGGAA__NILS_STEIN__CATCGAAGAGCATAATGAACGCTTGCCTGCAATAACAACATGACCGAAGA__MICHAEL_ALAUX__CCACATGCGGGTTTTATGCCCAGCGATAGAGCACCTTATAGGCGTGAACT__HADI_QUESNEVILLE__GGCTTTACGATTAGGGTTGGTAAGAACGCGAGAACGCCGGCTTAAACTGA__LUIGI_CATTIVELLI__GGAGTCAAGCCGAAGCTGGTACGTTCTGAAGCCTCTTAAGGCTATAAAAC__RUDI_APPELS__GATGACTCCATCACAAGTGACATGAGTTCATATGAGAGGTTACCGTGCCG__KAI_BADER__ACACGGAAGACTGCTTAAAAAATAGCAGCATCAGTAGGGTCAAGGGCGAC__HEIDRUN_GUNDLACH__CTGATAATTTGGGCTAGGACTGCAAATTATCCGGTAGCGGAAGCCGCAAT__MIHAELA_MARTIS__CTTACTTATCTCCATTGGTCTCGACGCGGTGGAGGCAACACATGTCTACT__THOMAS_NUSSBAUMER__TTGTCCTCGCGGGCGGTATGACCGGGGTCGATATCACGGTGCCCGATGTG__MATTHIAS_PFEIFER__AGGTCGTGGTTTAGTGACCAGGGTCAGGGCAGCTCATACGCTAGCTCGGT__SAPNA_SHARMA__TGCGTTACGTTAAGTCTCATGATATCCTTCCCCCGCAGAGGAGGCCGGAA__MANUEL_SPANNAGL__ACAAAAAAATCAGACTCTGCGTAAGGCTCCCTATCATTTAGAAGTATTTC__JARMILA_HALKOV__GATACTAAGCCTGGGACTAGCCCCTGTTGTCTGAAGGCTTAAGCGCGGGA__ZDEKA_DUBSK__TTGCCATGAAAGACGACGCATACGAGAGCCTGGTACTCAGGAGTTATGTA__MARIE_KUBALKOV__CAGTAGACTGTGCCCCTTGTTAGTTCGACGTGGGGCTGGGGATGGTGCTG__HANA_IMKOV__TAGCTGCCGCCTGAAAGTCTAAGATGAGAAGCGGGATGTAACGCTTCATG__ROMANA_PERKOV__GAGTTAACGCGCCTCCGGCCGTTTCAGGGCGCTCACGTGGGGGCGTAAAA__JAN_VRNA__CATTAGCACTGAGGAGTCGAAGGAGACTTCGTTGGAAAGTGAAGCGAGCA__RON_MACLACHLAN__ACAGTCGGTGCCAATAGAAGGCGGCAGTGAACGTGTACCCCAGCCCGCGC__SHICHEN_WANG__ATCCAAGACCCCACATTACTCTTCAAACGTTAAGGGCGGACCGACGGGAA__ADAM_LUKASZEWSKI__GCGCCAAAGGCCTTAAAAATGGTCTTATTCATATGGCATCCACTTCGAGA__MELANIE_FEBRER__GGTTTACATACTAAGAGGAAATTGCTCTTTGGGTAACCGGGCTAGGAAGA__SARAH_AYLING__AATCGACTCACGTCTGGGTGTTGAGCGCAAGATGTTCCGACAGTTGGAGT__BERNARDO_CLAVIJO__GCTCGACAAGGAAGATCCGGCGAAGCCTAGGCGGTGCTAATTTACAGTGC__LEAH_CLISSOLD__TTCAGCTAGATTGAACATAGCCCCGCGTAAGTGAGACCGCGATCTCGTTC__KIRSTEN_MCLAY__AAGCCCCAGTGAAATCCGGCTGTCAACGCTACGGGTAGGATTAATATTCC__RICARDO_RAMIREZGONZALEZ__TTTTACGCTCTGTTTCAGCACCCGCGGCTGGGTAACCATCCTAACGGTCC__JONATHAN_WRIGHT__CGTTGTCGTCGCAGCTAAGGTTCACGGGGAGCTCCTTTTTCATCTCCTTT__PARVEEN_CHHUNEJA__GAGACTGAAAAGTGTCCGGGGTGAGACTTTATGGAGCCGGTCTAGGTAGG__KULDEEP_SINGH__TATCCCCACCACTTCTACTCCCTCCGGCAACGGTTGTCCTGGAAGCCAGC__NAGENDRA_SINGH__CGTCTCAGCGATAGGTGGGTGGTACGCGTATCGAATAGGGGATGTTGGTC__JITENDRA_KHURANA__TGGAGGTCTTTGTCTCGTCAACGTGGCTGCAATTTGACATCTGATTTCTT__FRDERIC_CHOULET__ATCAGAAGAAGAGCATAGTGTCTTGTCTGCCTCGATAGGATACCCCCGTA__NATASHA_GLOVER__CTACAAGCCAGTCTTAGGAGAGCCCCCTCGGTCAAGGAACCAGCCTAAAG__NICOLAS_GUILHOT__CGGGAGTTCCGTGTTGATGAACCATGCGCATCACGAGCTGAACTACACAA__LISE_PINGAULT__TGTGTATGGGGTGTACGACAATTCATGCCCTTACCCTGAAATTTCAATGG__HLNE_RIMBAULT__CCCGGTCGGTATGCCGTTAAAAGGTTGGTTTTCGCGGCACCTTGTAGGTC__ADRIANA_ALBERTI__CCTTAGTCTATTGACCGAAGTTGAGATGTGCCGTCTTGCAGATCGTGGTC__VALRIE_BARBE__AATTGTCTACTAGAATCGCGTGTGTATACGCTCCGAAGAATAAGCTCCTT__TAKESHI_ITOH__CAAGCTTCCCGTACTCACCAATAACCGGGGTAAAACCATTGTAGCATTAA__HIROYUKI_KANAMORI__CTTTAACACTCTCCAATAAAAGTAAGATTCGTAAATCTAATTCACTTGTT__FUMINORI_KOBAYASHI__GAGGTTTAACGGGGACTGAAGACGGAGTCTTGAAGGTACCCCGGCATGTA__TAKASHI_MATSUMOTO__TTGCGTACGGTTCGGGGATGACGGCCCTTCATCTTCATGCGATTACCTCC__HIROAKI_SAKAI__AAGGTCCTGTGCTAAGGCCATTGTCGGAGGGATGTACCAGTCGGAACAGT__TSUYOSHI_TANAKA__TACAGGGCGGATCACTGCTGGCAGCTTAAGTCAATCAACACAGGGGCGCC__JIANZHONG_WU__TGAGTAAGGAGCTAAGTCCCCGTGAAATTCCAGGAGTCAGGTTAATGTGT__YASUNARI_OGIHARA__GTCTGCTGCCGTTTGATAACGAGCACTGCGAGGGACTGATGCGTCGAACT__DARRIN_KLASSEN__TTTGTAGAAATTATGAATGTACATGTGTTACAGGCTGAATGTCATATTTG__JACQUELINE_BATLEY__CATGGCACTTAGTCACACCGGACGGTCCAGCTAGGTCTCTAGTGCCGCAG__DAVID_EDWARDS__ACACTTGCCAGGAAGTAGGAGACAGATCATAAAGTCGAGATACCTAATTT__SIGBJRN_LIEN__GCCATCGTTTCCTGGCATAGCTGGTAAAGACCCCGATCGAGAAGGGCTGT__BRANDE_WULFF__GGATGAACAAATTCGCGTTCACGATGGCACTAAATGAACAATCAGAGATA__MARTIN_MASCHER__AGCTGGGCTGTGGACGCGTTCAGACCATTCCCTTCCGCGGAGCGACTCCC__UWE_SCHOLZ__GGTGGCGCGATTGGATTTTACGAATAAGTCTTAAAAGCCCCAATTTGCGT__KERRIE_BARRY__TAAGGGGTCATGCGCTTTCCGAGCGCTTAGGTCCAAAGTGCCTGAGCCGC__JARROD_CHAPMAN__TACGTTCTGACCATACCGTCCGTCCTCACATGCAAAGTCCTGACTTGCGG__DANIEL_ROKHSAR__TTATATGTTTCCGCACATCGGTACCCCTCAAATCGGCACATGACTTTGCA__JESSE_POLAND__CACACTATGCCGAGGATAGTCGCACGAAGACATTCTGGCCCAAGGAGACG__ROBBIE_WAUGH__TCCTGTGCGTCGATTGGTCCACTTTAAAACGCCGTGTTTGTTTATGAATT__GARY_MUEHLBAUER__ACTGAACTTAAACGGGTCCTAGTAGTACGGGGCCCTCGGATCATTCGAGC__FRANOISE_ALFAMA__CCTAGAGGACCAAGGCTCATATTATCCCCGCTAATCGAATCCCTCATGGG__LOC_COUDERC__AAATCCGAAACATGCGAGAGGACGCGCCCAAGAAACCTGCCCAGGTGATC__VRONIQUE_JAMILLOUX__AGAGGTAGCACAGCAACAACGGCTCTAGATAATGCTTCCACCTATATCTA__MIKAL_LOAEC__CCCGGGTCGGATAAGCGTCCTTGGCGGTGCCCCATTACGTCGATGTAAAT__CLAIRE_VISEUX__TGCCCAACCTTGCGGGTCCTCAGAATGATCATCGCGGAGGGGTTGTCAGT__MATTHIAS_ZYTNICKI__GCAGGACTGGCATACAAGTCTTGCGGGGCCAAAGGGGTTCGGCGAATCAT__BEAT_KELLER__TGACTGACACTCACTAGTAGCAATTCGGCGGTTTGCAACCACATAGTAGC__THOMAS_WICKER__TAGTCACATCAACAACGTGTGTCAGTGACGCGGGAGTAATCAGCACGTTT__MORENO_COLAIACOVO__CTGCTCTGCGGTGAACTTAACATGAGAGGTTGCTTCTCTCCCTACGAGCG__PRIMETTA_FACCIOLI__TGCACTGATCTGCCCTTACAAGCTGGCGGCCATGTGGTAGTCCCAAAACG__ANTONIO_STANCA__GTGTTGTTGGAATCGCTGCCACCTTATGGCATAGTCTACAGGGTTAGCGT__HIKMET_BUDAK__GTTCTCTAGTGCTGAGCTGTCCTGTTCTAGGGTCGCTTTGCCCAGCCCCC__MATTHEW_BELLGARD__AGGACCGGAATTACATGAGACAAGACAGCCGAGTTCTGGGGTTAATTACC__BRETT_CHAPMAN__ACCAACCTTGAAAAGCTTTATTTTTTAAGAGAATTGTGCTATGACAGTAG__RON_KNOX__TCTTATTTCCATCTCGATCTGCTTCGGATACCACACTGAGTGGATGAGAA__ANDRZEJ_KILIAN__ACGAAAATCAGGGATCCGCGTCACTGCGGAGTCTGTGGGCCCGCATAAGC__SEBASTIEN_PRAUD__AGTCAGTACTCCTGGCGGCAGCTCTTCATGGATGGTGACTGGTGTTATCTGTACGTACCAAGCTGCTATGAGTATACCTCATTGGCGAGAGGCCATGGAGCAGGAGTATCATGCTCTTCTTCGTAACAAGACATGGACTCTTGTTCCTCCACCACCCCGTGTTAATGTCATTGATTCTAAATGGATTTTCAAAGTGAAGAAGCATTCTGATGGTTCTATTGAGAACTACAAGGCGCGGCTGGTTGATAGAGGTTTTCGGCAGCGTTATGGTCTTGATTATGAAGACACCTTCAGTCCAGTGGTTAAACCTACTACTATCAGGCTTCTTCTCTCTCTTGCAGTTACTCGGGGATGGTCACTTCGTCAGCTTGATGTGCAGAATGCTTTTCTCCATGGTGTTCTGGAAGATCTCACTAAAGCACTCTATGGTCTGAAGCAAGCTCCCCGTGCTTGGCATGCTCGTCTTGCGACATCCCTTCGTGCTCATGGTTTTGCATCATCAGCTGCTGACTCTTTGTTATTTCTTCTACAAAGGCCCGAAGTTACCATGTATCTGCTGGTCTATGTGGATGATATTATACTCGTCAGTTCTTCTCAGTCGGCTGCTGCTGCTCTTGTTCGGTCTCTTGGTGCTGACTTTGCGGTCAAAGATCTTGGGCAGCTTCACTACTTCCTTGGTGTGGAAGTTGCTTCTGTTTCCAATGGTATTGTTATGACGCAAAGAAG__IWGSC__TACTCTTTGGATTTGTTGTAGCGGGATGGGATGCTTAAGTGCAAACCGACTACTACACCTATGTCTGCTACTGACAGGATCACTGCTGTCGATGGTGATCTCCTGCCTTCTGCTGATGCGACACAGTACAGGAGCATTGTTGGTGGGTTTCAGTACTTGACGATCACATGTCCAGATATATCCTTTGTTGTCAACAGAGTATGTCAGTATCTCCAAGCACCTCGTGACACTCATTGGTCTGCGGTGAAGTGCATCTTGCGCTACATTCGCTTCAAGTTGTCCTTGGTTTGCACATTCGACCGAACCCCTCTAGGGTCATTTCGGCGTATTCTGATGTGGATTGGGTTGGCAGTCCGGATGACAGGCGATCCACGGGAGGCTCTGCTGTATTTTTTGGTTCTACCTTGATCGCCTGGAGTGCTCGGAAACAGGCTACTGTTTCGCGTAGCAGTACTGAAGCTGAGTATAAGGCTGTGGCTAATGCAACTGCAGAAATTATTTGGATACAGTCTTTGCTTCAGGAGTTGGGAATATCTAATCACAGTCTCCTGTTCTTTGGTGTGATAACATCGGTGCTACATACCTATCTGCTAATCCGGTATTCCATGCCCGAATGAAACACAACGAAGTGTACTATCATTTTGTACAGGAACGGGTATCTCAGAAGCAACTACAGATCAAGTTCATTTCTTCCAAGGATCAACTTGGGTAGTATATACGTTTGGAATGCACTAAAGCATTCATTTGAGTAGTAGGTTGCATGCATTATACACGTAGCAAAATATTTTAATTGAACATAACATGAATTCAAAGTTTTGAATGACATTTGTAGTGCGCATTGATTTGGTACAGTACACGTTAGGCTTGTCCGGAAATTTCAACCCGCTCCTTGTTAGCCCCAAATATTTAAGATATATCTTTGTCTCGTTGTGCTCCGACAACTCCCTCCATCTCAACCCGCGCCTTGCTATTCCGAAATTACAACGCGCGCGAAAACTCCCTCCTCCTGTGAAATCCCGACACGCGAAATGCTCGTGGTACCCCTGAACCGAAAGAACCGCCTCAAATCGGTGGGGGGTACTTTCGTAACTTACCCCACATTTCGGACAAGCGCGTCTCTAAGCCATGGTTCCCCACAGCCATCCCATCCGCCCACCCATTCGTACACCGAGGCCGCGAAAACCCGCGACGAAACTCCACACCCTGCTCCGTCCGCCACCCAGCCGGAGCTTCTTCCCCGACGACATCGTCCACAGCAACACCTCGATGTCCCTCATCCACCGTACCGGATGAGGATCCGTCATCGATCTCATCGTCCCGCCGGTTCAGCCACCCCGTCCTCCACCTCCAAGGAGCTGCCCCGACGTTCCCCTCGTCTTTCGCGCCACCTCCATTCCCACACCGCCGTCTTCACCTGCACCACCGGAAGAGCATCATCATCACCGTTTCCTCGGATGAAGCTGCGGCCTAATCGGCGCCACCAAAGAGGTTGTACACTAATCGCCGCATTTTCTTCTTGATTCGATCTCACGGTGCTGCCGACGTTCGGTCCCAAGCCGACACGGCGCGGCTCCATCCACGGCAGCGTCGCCGGCCACTTCCTCCACGACGTCGCTCCCTCGGCGGCGACGTCCACATCAGTAGGAGCTGCTGCTGCTTTAGCTCTCGCTCACGCTGCTGCTCTGCTCTTGCTCTCGGTCCCCTGCCTGGTCTGCTCTTGCTATTGCTCTTGCTCGCGCTGCTTCTCTTGCTCTTGCTCTTGCTTGCGATGCTGCTCCAATTTGGCTACACTTCAGTCGACTGAATCGACTTTTGGGTCAGTCGATTTCCAAGGGGTGGGCTCGCCGGGGTGAAGGAAGAACCAGCAGCAGCAGGGAGGGGGGCTCGCTGGAGAGGTACCCCACTATCTATCTTAGGGTTCAGGATGGGGGCGGTGGTCGCCGACGGTAGCGGGGAGTTGGCGGGGAGGTGGCGTGGGGATCGCCGGAGAAAAAGCTCGGCACGGGGGGGCCTAGAGGGATGGCCGGGGCGGCAGTGGACCGGCGGTGGGGAGTGTTTTTGGGCCGGGTAGGGAGGTGCACCGCCGGCCGCGGGCGGCGTGGGGCTGCTGTTTGGCCGGTGGTGGCTGGCGGCTCAGGGGGGGTGGAGGTTCAAGATGAACCGTAGGCTCTTGATTTCGTATCCAACGGCTGCAAAATCGACTGACCAGAGATGAAAAAGTCAGTCGACCGATGTGTAGCCTCACCTTGCTAGTGTGTTCAGTTTCGATAGCAAAATTCAGTTTCGACAGTTAGGTTCAGTTTCGACAATTAAATTCAGTTTCGACAGTTAAGTTCAGAGATGAGCGGCAGATGTATTTTACATCTAGCACTTTGTGGTTATGTATTTTACGTCATGTATTGGAGATGCTATTAGAATTCCACTCAGGTTAACGTTGTTCATTGTCTTTCTTGTCCTGCTTCAGCCTCGGCGTCGTACAACTCACTGTAGCTGCTCCAACGACGAAACCCTCCATCACAGCGGAGCAGTACCTTGGGATCCATCTCCAGACGCCTAAGATCTTCTTCCCCTCCTCCGACAGCAAAGTCAGCCGGAGCATCCTCACTGGCCAACCCAATCATGCTGAGATCGACATGGCTTCGCCAAAGAGGTTGTACACTTGTTGCATCTCATTTTTACTCTACATGTTAGTATGTCAGTATCTCCAAGCACCTCGTGACACTCATTGGTCTGCGGTGAAGTGCATCTTGCGCTACATTCGCTTCAAGTTGTCCTATGGTTTGCACATTCGACCGAACCCCTCTAGGGTCATTTCGGCGTATTCTGATGTGGATTGGGTTGGCAGTCCGGATGACAGGCGATCCACGGGAGGCTCTGCTGTATTTTTTGGTTCTACCTTGATCGCCTGGAGTGCTCGGAAACAGGCTACTGTTTCGCGTAGCAGTACTGAAGCTGAGTATAAGGCTGTGGCTAATGCAACTGCAGAAATTATTTGGATACAGTCTTTGCTTCAGGAGTTGGGAATATCTAATCACAGTCTCCTGTTCTTTGGTGTGATAACATCGGTGCTACATACCTATCTGCTAATCCGGTATTCCATGCCCGAATGAAACACAACGAAGTGTACTATCATTTTGTACAGGAACGGGTATCTCAGAAGCAACTACAGATCAAGTTCATTTCTTCCAAGGATCAACTTGGGT34