DuplicationDetector, a light weight tool for duplication detection using NGS data

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DuplicationDetector, a light weight tool for duplication

detection using NGS data

G. Djedatin, C. Monat, S. Engelen, François Sabot

To cite this version:

G. Djedatin, C. Monat, S. Engelen, François Sabot. DuplicationDetector, a light weight tool for

duplication detection using NGS data. Current Plant Biology, Elsevier, 2017, Plant Development,

9-10, pp.23-28. �10.1016/j.cpb.2017.07.001�. �hal-03070294�

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ContentslistsavailableatScienceDirect

Current

Plant

Biology

j ou rn a l h o m ep a g e :w w w . e l s e v i e r . c o m / l o c a t e / c p b

DuplicationDetector,

a

light

weight

tool

for

duplication

detection

using

NGS

data

夽

Gustave

Djedatin

a,b,∗∗

_,

_Cécile

_Monat

b,c,d,1

_,

_Stefan

_Engelen

e

_,

_Francois

_Sabot

b,c,d,∗

a_BIOGENOM_Laboratory,_FAST/DASSA,_BP₁₄_{Dassa-Zoumé,}_Benin

b_DIADE_UMR_{IRD/UM–Centre}_IRD_de_Montpellier,₉₁₁_av_Agropolis_BP_604501,_F-34₃₉₄_Montpellier_Cedex_5,_France c_South_Green_{Bioinformatics}_Platform,_Agropolis_Campus,_Montpellier,_France

d_Université_de_Montpellier,_Place_Eugène_Bataillon,_34000,_Montpellier,_France

e_Commissariat_à_l’Energie_Atomique_(CEA),_Institut_de_Génomique_(IG),_Genoscope,_BP5706,_F-91057_Evry,_France

a

r

t

i

c

l

e

i

n

f

o

Articlehistory: Received19May2017

Receivedinrevisedform18July2017 Accepted19July2017 Keywords: Duplication NGS Rice

a

b

s

t

r

a

c

t

Duplicationsareoneonthemainevolutionaryforcesinangiosperm,especiallyinPoaceae.Alargenumber ofgenesinvolvedinvariousmetabolismsandpathwaysoriginatefromsuchduplications(wholegenome, segmentalorsinglegene).However,todetectsuchduplicationmaybecomplicated,costlyandgenerally requiresheavyhumanandmaterialinvestments.Here,weproposeanalternativeapproachfordetecting putativerecentsegmentalduplicationsinhaploidordiploidhomozygousorganismsbasedonNGSdata. Werelyonabusivemappingsofparalogoussequencesthatincreaseapparentheterozygouspointsata givenlocustoidentifysuchduplicatedgenomicregions.Wetestourtoolonsimulateddata,thenontrue ricegenomicsequencesandwereabletoidentifyabout200candidateduplicatedgenesinAfricanrice (Oryzaglaberrima)lineagecomparedtoAsianone(O.sativa).

1. Introduction

Duplicationisanimportantfeatureoftheplantgenome archi-tecture,andcaninvolveasinglegene,achromosomesegment,an entirechromosomeoreventhewholegenome[1].Itwasshownfor instancethatangiospermsundergonelargescaleduplicationsand multiplewholegenomeduplicationsallalongtheirevolution[2]. Wholegenomeduplication,i.e.doublingtheamountofthe com-pletegeneticmaterialofanindividualwithoutcrossing,appears asasourceofevolutionandbiologicalcomplexity[1,3,4].Inthe sameway,segmentalduplicationscreatelocalvariationsoffering newopportunitiesfornaturalselectiontooccur[5].Therefore,gene andgenomicduplicationsplayanimportantroleintheevolutionof plantphenotypes[6],andduplicatedgenescouldundergodifferent behaviors:(i)neofunctionalization–retentionofbothdivergent

夽 Thisarticleispartofaspecialissueentitled“PlantDevelopment”.

∗ Correspondingauthorat:DIADEUMRIRD/UM–CentreIRDdeMontpellier,911 avAgropolisBP604501,F-34394MontpellierCedex5,France.

∗∗ Correspondingauthorat:BIOGENOMLaboratory,FAST/DASSA,BP14 Dassa-Zoumé,Benin.

E-mailaddresses:djedatingustave@yahoo.fr(G.Djedatin),francois.sabot@ird.fr

(F.Sabot).

1 _Current_address:_{Domestication}_Genomics_Group,_IPK_Gatersleben,_OT

Gater-sleben,Corrensstrasse3,D-06466Seeland,Germany.

copiesbutwithanewfunctionforoneofthem–,(ii) subfunction-alization–retentionofbothcopieswithconservedfunctionbutin anothertissue/organ/timeframeforone–,or(iii) nonfunctionaliza-tion/pseudogenization–largenumberofmutationsaccumulation in oneof thecopies [1]. Thetwo ﬁrst case (neo-and subfunc-tionalization)mayleadtonewexpressionpattern,oreven new regulatorypathway[7].

In cultivated Asian rice (Oryza sativa), for instance,genome duplicationprovidedimprovedrootresistance[8],seed germina-tionandseedlinggrowthtosaltstress[8,9].Inaddition,tandem duplications were evidenced, amplifying adaptively important resistancegenesencodingmembraneproteinsandfunctionrelated toabioticandbioticstress[10].Hence,segmentalduplicationand tandemduplicationleadtoHAP(HeterotrimericHemeActivator Protein)geneduplicationregulatingriceheadingdate[11].

However,detectinggenomeorsegmentalduplicationsisa com-plextask.Differentapproachesandtechniquesareused,suchas molecularonesthatgather(time-consuming)techniquese.g. com-parativegenomehybridization(CGH)[12],FISH,and arrayCGH

[13].Recently,duetotheavailabilityofNextGeneration Sequenc-ingtechnologiesandoftheirlowcost[14–16],morecomputational sequencing-based approaches weredeveloped (e.g. [17]).These methods rely mainlyonDepth of Coveragevariations(DoC) to identifyduplicationsrelative toa referencegenome,asa dupli-cated regions are expected to be twice more sequenced than

https://doi.org/10.1016/j.cpb.2017.07.001

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24 G.Djedatinetal./CurrentPlantBiology9–10(2017)23–28

non-duplicatedregions[18].However,molecularapproachessuch asDoCmethods requirehighly preciseexperiments,repetitions andheavycomputationaltimes,aredesignedtocompareone tar-getindividualtoagivenreferenceone,andthuscannotbeapplied onalargenumberofindividuals.

Inthisstudy,weproposeanewmethodologybasedontheuseof apparentexcessofheterozygousloci(AEH)ongenomicintervalsin autogamousdiploidspecies.Thismethodologywasimplemented inatoolcalledDuplicationDetector,andwastestedonasetof simu-lateddataandshowntobefastandrobust.Inaddition,weappliedit oncultivatedandwildAfricanrices,respectivelyOryzaglaberrima andO.barthii,todetectduplicatedgenescomparedtotheAsian riceOryzasativa.

2. Materialsandmethods

2.1. Material

2.1.1. Simulatedsequencedataforvalidation

Afragmentofchromosome7(1Mb)ofOryzasativasspjaponica cvNipponBareIRGSP1.0wasextractedfromposition1,000,000to 1,999,999usingtheextractseqtoolfromEMBOSS[19].Three dupli-cations(namelyduplications1–3)wereartiﬁciallycreatedwithin thissequenceusinghome-madePerlscript(availableondemand), respectivelyatpositions300–1500;350,000–390,000(containing another initialduplication) and 800,000 to 810,000. A total of ninevirtual‘clones’wereconstructed.Clone1,withoutduplicated sequences,wasconsideredasidenticaltothereference.Clone2has thethreeduplicatedsequenceswithoutmutation.Clones4–6have theduplicatedsequencesandmutationswith3%of supplemen-tarydivergence,whileclones7–9havetheduplicatedsequences withthesamemutationsand6%ofdivergence.Inadditionweadd inclones3–9additionalcommonmutationsinthenonduplicated sequence.Allmutationswereinducedusingthemutatednatool fromtheSMS2suite[20].Thesequencesfromeachvirtualclone werethenusedtosimulateFASTQdatausingaRTsimulationtool

[21],specifyingasoptionsHiSeq2500machine,100pair-end frag-ment,depthof35,insertsizeof200,10%ofinsertsizedivergence. aRTincludesanempiricerrormodelthatallowsaverygood simu-lationofsequencingdata[21].

2.1.2. SequencedataforOryzaglaberrimaandO.barthiiand initialqualitycontrol

Eight accessions of African cultivated rice Oryza glaberrima (TOG5307, TOG5307f, TOG5321, TOG5666, TOG5887, TOG7291, UB06, UG26), and six wild relatives Oryza barthii (B88, IG05, IRGC106302,MB323,TB41,TG57)wereusedinthisstudy(see[22]

formore informationsaboutthoseaccessions). Asian cultivated riceO. sativaIR64(sspindica)and Azucena(ssp japonica)were alsoincludedascontrol.AllsamplesweresequencedatGenoscope (France),intheframeoftheIRIGINproject(http://irigin.org),as follows:

Sequencing:Librarieswere preparedusing theNEBNext DNA ModulesProducts(NewEnglandBiolabs,MA,USA)witha‘onbeads’ protocoldevelopedattheGenoscope,thusreducingthecostsand increasingtheyields.Briefly,aftergDNAfragmentationwiththe E210Covarisinstrument(Covaris,Inc.,USA),endrepair,A-tailing andligationwithadaptedconcentrationsofNextflexDNAbarcodes (BiooScientific,Austin,TX,)wereperformedonthesameAMPure XPbeadsthatwasusedforthefirstpurificationafterendrepair. Aftertwoconsecutive1xAMPureXPcleanup,theligatedproduct wasamplifiedby12cyclesPCRusingKapaHifiHotstartNGSlibrary Amplificationkit(KapaBiosystems,Wilmington,MA),followedby 0.6xAMPureXPpurification.Librariestraceswerevalidatedon Agi-lent2100Bioanalyzer(AgilentTechnologies,USA)andquantified

byqPCR usingtheKAPALibraryQuantiﬁcationKit (KapaBiosys-tems)onaMxProinstrument(AgilentTechnologies,USA).Libraries weresequencedonanIlluminaHiSeq2000orHiSeq4000instrument (Illumina,USA),at2×101bpor2×151bp.respectively.About50 billionusefulpaired-endreadswereobtainedperrun.

QCand initial treatments: Low quality clusters werefiltered duringthesequencingrunbyRealTimeAnalysis(RTA)software. FilteringstepswereperformedonwholepairedFASTQfiles: Illu-minaadaptersandprimerswereremoved,nucleotideswithquality valuelowerthan20weretrimmedfrombothendsandsequences between the second unknown nucleotide (N) and the end of thereadweretrimmed.Readsshorter than30 nucleotidesafter trimmingwerediscarded. These trimmingstepswereachieved usingfastxtend (http://www.genoscope.cns.fr/fastxtend/), a soft-ware based on the FASTX library [23]. The filtered reads and theirmatesthatmappedontorunqualitycontrolsequences(PhiX genome)wereremovedusingSOAPaligner[24].

2.1.3. Referencesequence&annotation

The reference sequenced genome IRSGP-1.0/MSU7.0 and its annotationfromMSUv7[25]wereusedforanalysisasdescribed above.TheinitialVCF(VariantCallFormat)ﬁlesareavailableat

http://bioinfo-storage.ird.fr/2017/CPB/Djedatin/.

2.2. Methods

2.2.1. Mappingapproach&initialSNPcalling

For VCF creation, cleaned paired FASTQ data were mapped uponthereferenceinitialsequenceusingBWA0.7.12(aln/sampe legacyalgoritm)[26].SAM(SequenceAlignment/Map)fileswere cleanedandfilteredforlowqualitymappingandabnormal map-ping,mergedandrealignedusingcombinationofSAMtools[27]and PicardTools[28].Afterrealignment,SNPwerecalledusingtheGATK HaplotypeCaller[29]understandardconditions.Callingwas per-formedperindividualchromosometooptimizecalculationtime. AllstepswereperformedusingtheTOGGLEpipeline[30]toensure repeatabilityand traceability.The standard defaultvalues were chosenrespectingtwocriteria:I)THEIRIC/3Kgenomesstandards formapping/callingandII)numeroushomemadetestsand evalua-tionsofconditionsusingcontrolsamplesindifferentanalyses(such asinMonatetal.[30],GBE)thatprovidethebestresults.Detailed optionsareshowninsuppData,aswellasTOGGLEconfiguration file.

2.2.2. HeterozygousSNPrecovery

VCFwereﬁlteredoutforrecoveryoflinescontaining heterozy-gousSNPsbasedonstandarddefaultﬁlters(depthforeachsample, maximumnumberofmissingdata,minimalcallingqualityvalue, maximumMQ0value,homozygouscontrols).

2.2.3. Genomicsintervalsrecomposition

Extracted VCF lines were recompiled in genomics intervals respectinga specifiedmaximal distance between2 SNPs tobe consideredasrelated, aminimalblocksize,andaminimal het-erozygousSNPdensity.Resultingfilesare3columnsBED-likefiles.

2.2.4. DuplicatedgenesidentiﬁcationandSNPpotentialeffect identiﬁcation

GenomicsintervalﬁleswerecrossedwithGFFﬁlecontaining annotationusingintersectBEDfromtheBEDtoolssuite[31].Selected heterozygousSNPswerethenannotatedfortheirpotentialeffect usingsnpEffsoftware[32].Aschematicviewofthewholepipeline isdetailedinSuppdata.

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Fig.1. ApparentExcessofHeterozygouswillappearifreadscomingfromaduplicatedregionareabusivelymappedonareferencegenomewithouttheduplication.

2.3. Availability

All codes, installation instructions and manual for Dupli-cationDetector are available, under the GPLv3/CeCiLL-B double licenses, on the GitHub of the project: https://github.com/

SouthGreenPlatform/duplicationDetector.

3. Results

3.1. Descriptionoftheapproach

We rely on AEH genomic intervals to detect duplicated sequences(Fig.1).Basically,wedetectabusivemappingofreads comingfromduplicatedregionsinasequencedindividualwhen theyaremappedonareferencegenomewithouttheduplication (i.e.harboringonlyasinglecopy).Suchabusivemappingwilllead theSNPcallingtoproduceanApparent ExcessofHeterozygous locus,i.e.toomanyheterozygouslociinashortregion.Ifmany indi-vidualsaresequencedandmappedinthesameexperiment,such AEHlociwillappearfor(almost)eachindividualinthesame loca-tion,indicatingthusthattheregionisduplicatedinthesequenced genomes compared to thereference one. Userscan manage in DuplicationDetectorthelevelofstringencyforselectionofAEHloci usingdifferentcriteria:

• Minimaldepthperindividuals(defaultat30)

• Minimalnumberofindividualstobeheterozygousforapointto bechosen(defaultat10)

• MaximalnumberofMQ0reads(thatcanbemappedattwo posi-tionswithidenticalscore;defaultat0)

• Controlindividuals(somesamplesthatmaynotbeheterozygous) Thegenomicintervalcreationcanalsobesetupusingfollowing criteria:

• Maximumsizebetweentwoheterozygousloci(defaultat1kb) • Minimumsizeofthegenomicinterval(defaultat100bp) • Minimumdensityofheterozygouslociinthegenomicinterval

(defaultat25basesbetweeneachSNP).

IfuserprovideaGFFfileforgeneannotation,duplicatedgenes will beidentified throughoverlapping withidentified genomic intervals.

Intermsofspeed,acompletescanfromarawVCFof16African riceindividuals (12chromosomes,∼380Mb)athighsequencing depth(∼35x,seeMaterialsandmethods),withtwoAsianrice indi-vidualsascontrol,willbeperformedonasinglerecent64-bitscore inlessthan2h.

3.2. Resultsonsimulateddata

Onsimulateddata,wewereabletopartially(∼40%)identifythe duplication1directlyandalmostentirely(∼95%)theduplication

3(Table1)asfragmentedblocks.Wewereabletolimitthosetwo

duplicationswithaquitegoodresolution,i.e.capacityofcorrectly identifytheborders(max500bpoferrorinlimitating;seeTable1). Thedifferenceofrecoverylevelbetweenthetwoduplicationsis mainlyduetotheirsize,asforduplication1(1.2kb),thenon recov-eredfragmentisof193bpin5and522bpin3on1200,whilefor duplication3thenon-recoveredsizeisof142/355bp.The fragmen-tationeffectmaybeduetothefactthatduplication3isalargeblock butwithalowvariationdensity,andAEHlocidensityparameteris quiteconservative.

Duplication2wasnot detected,asit containsanother older (non-simulated) nestedduplication,and AEH loci inthis region wereremovedbasedonthemaximumMQ0parameter.Indeed, readscomingfromaregionalready duplicatedin thereference genomecouldbemappedonanyofthetwocopiesonthe refer-encewiththesameprobability.ThiswillincreasetheMQ0level,

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Table1

Statisticsaboutsimulateddata.Positionsaregiveninbasepair.

Duplication Start Stop Start(recovered) Stop(recovered) Resolutioninbp/Nbblocks Recovery

1 300 1500 493 978 193<->522/1block 40.42%

2 3,50,000 3,90,000 NA NA NA NA

3 8,00,000 8,10,000 8,00,142 8,09,675 142<->355/8blocks 95.33%

Table2

StatisticsaboutduplicatedregionsinAfricanricescomparedtoAsian.Sizesaregiveninbasepair.

BlockSize AEHloci AEHlocifreq

Min Mean Max Min Mean Max Min Mean Max

Chr1 142 795 2770 9 37 111 9.33 10.99 13.03 Chr2 106 623 2031 5 31 106 8.58 10.75 12.75 Chr3 110 293 825 5 15 40 8.78 10.67 13.3 Chr4 103 794 2079 6 40 132 8.33 10.31 13 Chr5 119 719 2668 6 37 113 9.62 11.07 13.25 Chr6 107 1057 3560 7 50 169 8.44 11.02 13.15 Chr7 147 903 3198 7 49 154 8.94 11.19 13.17 Chr8 111 925 3336 8 44 158 8.1 11.29 13.36 Chr9 127 634 1057 7 35 64 10.07 11.37 13 Chr10 127 937 2817 7 47 170 9.14 11.21 13.29 Chr11 124 716 2055 6 35 92 8.68 10.91 12.89 Chr12 101 744 1804 5 37 85 8.79 10.62 12.05

andthusthoseregionswillbeﬁlteredoutwiththecurrentversion ofDuplicationDetector.

Nofalsepositivesregions wereobtainedfromthesimulated data.

3.3. Resultsonexperimentaldata

Wethentest ourtool onanexperimentalsetof Africanrice individualssequencedatrelativelyhigh-depth(seeMaterialsand methods).AfricancultivatedriceOryzaglaberrimaanditswild rela-tiveO.barthiidivergedfromAsiancultivatedriceO.sativaancestor almost 1 million year ago [33,34], and may harbor duplicated regionscomparedtoAsianrice.Wethustested8cultivatedand 6wildsamplestoevaluateourtoolonrealdata.Toavoid individ-ualeffectduetothereferencegenome(i.e.identiﬁcationofAEH locionlybecauseofalossofduplicationinNipponbare),weaddas controltwootherO. sativasample,IR64(indicasubspecies)and Azucena (japonicasubspecies), sequencedas described in 2.2.1. Thosetwoindividualsmustbehomozygousandsimilartothe ref-erenceforanAEHlocusontheAfricansamplestobevalidatedas such.

ThewholeanalysisontherawVCFrepresentingvariationson allthe12chromosomesofNipponbarereferencespendlessthan 2husingasinglecore,withaveryshortmemoryfootprint(max

2Mbytesperﬁle),andprovided786putativelyduplicatedregions inAfricanricelineagerelativetoAsianone(seeTable2).Fromthose putativelyduplicatedregions,wecanidentify200annotatedgene feature(i.e.taggedas‘gene’intheMSU7GFFﬁle;seeSuppData).The blocksrangedfrom102to3560bases,with5–170SNP(11SNP/kb onaverage)withAEHineachblock(Table2).

Theduplicatedregionsarewidelyspreadallalong the chro-mosomes, without any main locations (Fig. 2 and SuppData). Theputativelyduplicatedgenesthemselvesseemstoberelated partiallytostressresponse,butnogeneraltrendwereobserve con-cerningGene Ontology (datanotshown).We observeda mean valueof38AEHlociperregion,showingthattheseduplications arequite recent,but dating from beforetheradiation between O.glaberrimaandO.barthii.Indeed,ifapplyingameanvalueof 1.3×10−8mutationpermillionyears(ascalculatedin[35]),the meanageoftheputativeduplicatedregionsisof∼800,000years, spanningfrom∼605,000to972,000years,asexpectedfromthe twogroupseparation[33,34].

DetailedanalysisofmutationinducedbytheAEHlociin dupli-catedgenesshowedthatmostofthemutationsoccurredoutside ofthegenecodingregions(7–20%onlyinexonicsequences; Sup-pData). For exonicmutation, a mean Ka/Ks ratio of 1.53±0.45 (1.01–2.75)wasobserved,indicatingalowlevelofglobal diver-gent selection. The mean observed Transition vs Transversion

Fig.2. ChromosomallocationofduplicationsinAfricanricescomparedtoAsianonChromosome1.Inredaresymbolizedduplicationinvolvingannotatedgenes,ingreen regionwithoutannotatedgenes.

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ratio is around 2 (1.7–2.3), as expected for genomic mutation

[36,37].

Whenfocusingonindividualgenes,wewereabletoidentify asputativelyduplicatedtheLOCOs07g09900gene(Chromosome 7,from5,263,409to5,267,310),DiseaseresistanceproteinRPM1, locatedunderamajorQTLofresistancetoAfricanstrainof Xan-thomonas(qABB-7,from[38]).Asithasbeenshowninotherplant species,duplicationofresistancegenesmayincreasedisease resis-tance.

3.4. Limitsoftheapproach

In a recent paper, Hutin et al. [39] shown a recent partial duplicationof3.2kboftheLOCOs11g31190geneOsSWEET14[39]

(Chromosome11,from18,171,678to18,174,478),involvedinan increasedresistancetoXanthomonasoryzaepvoryzae.Asetof12 highlevelvariantpositions,includingthe18bpdeletionbetween theoriginalcopyandtheduplicatedone[39],wereidentiﬁedin thevariantselectionstep.However,post-ﬁltrations(especiallythe minimalSNPdensity,hereof177bpbetweentwoSNPinsteadof 25)didnotallowtorecoverthisduplicatedblockinourcurrent test.

4. Discussion

Detectionofduplicatedregionsbetweenindividualsisa chal-lengingtaskusingmoleculartools,andacostingcomputingtask withsequencedata.Uptonow,useofthelatterapproach(mainly using NGS) was based on raw mapping, DoC divergence com-putationand CopyNumberVariation(CNV)analyses.Numerous toolsexperimentedsuchapproaches,withmoreorlessefﬁciency

[14–16],butallofthemrequiresalongcomputationtimeand

can-notcomparemassivelydifferentindividuals.

In thepresent study,we rely onabusivemapping and sub-sequentAEH loci to identifythe duplicatedregions. Moreover, ourtooldoesnotrequireintensere-calculationormapping,asit reliesdirectlyontherawVCFdata(alreadygeneratedin numer-ousgeneticanalyses)toidentifythoseAEHloci.Thisapproachis fastandallowstoworkonlargesamples;inaddition,itoffersthe possibilitytoincludenegativecontrolswhichallowusersto iden-tifyduplicationsexistinginonlyasubsampleoftheirsequenced individuals.Ourapproachishoweverquiteconservative,asshown onsimulateddata,andwillnotdetectforinstancenewcopiesof analreadyexistingrepeatedsequenceinthereferencegenome. Inthesameway,itcannotidentifytoorecentduplications,due tothelownumberofmutationsbetweenthetwocopies(asfor OsSWEET14).However,appliedtothedivergencebetweenAfrican andAsianrices,wewereabletoidentifymorethan200putatively duplicatedgenesandalmost780totalregions.Thedetectedgenes arewidelyspreadallalongthechromosomes,generallyrelatedto stressresponse,atleastmarginally,andunderaquitelowdivergent selection.

5. Conclusion

DuplicationDetectoristhusaveryefﬁcienttooltodetect dupli-cationinhaploidandhighlyhomozygousdiploidorganisms,such asrice(testedhere),butalsobacteria,yeasts,autogamousplants, haploidfungi,andsoon.Thefuturedevelopmentofourtoolwill includetheimplementationofdetectioninheterozygousor poly-ploidorganismsorboth,aswellasadditionalcriteriaforﬁltering (suchashard-clippinglevel).

Author’scontribution

GD and FS manage the whole study and wrote the whole pipeline.SE&Genoscopeperformedthesequencingandinitialdata

treatmentsandQC.FSperformedthesimulation,GDandCM per-formedthebasicdataanalyses,andGDanalyzedtheresults.GDand FSwrotethemanuscript,andallauthorscorrectedandapprovethe currentversion.

Acknowledgments

GD was supported by an IRD grant (2013–2017 BEST FellowShip). CM was supported by ANR (AfriCrop project #ANR-13-BSV7-0017)andNUMEVlabex(LandPanToggle #2015-1-030-LARMANDE).AuthorswanttothanksGenoscopemembers forthesequencingofallricedata.Thisworkwassupportedby FranceGénomiqueFrenchNationalinfrastructure,fundedaspart of“Investissementd’avenir”programmanagedbyANR (#ANR-10-INBS-09),intheframeoftheIRIGINproject(http://irigin.org).

Conﬂictofintereststatement

Theauthorshavenoconﬂictofinterest.

AppendixA. Supplementarydata

Supplementarydataassociatedwiththisarticlecanbefound,in theonlineversion,athttp://dx.doi.org/10.1016/j.cpb.2017.07.001.

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