G3: Genes|Genomes|Genetics Early Online, published on March 3, 2017 as doi:10.1534/g3.117.039883
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AutomatedphenotypingindicatespupalsizeinDrosophilaisahighlyheritabletrait
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withanapparentpolygenicbasis
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R.GuyReevesandDiethardTautz
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MaxPlanckInstituteforEvolutionaryBiology,Plön,Germany,
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© The Author(s) 2013. Published by the Genetics Society of America.
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SHORTRUNNINGTITLE
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Pupalsizephenotypingandgenetics
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KEYWORDS
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Automatedphenotyping,Drosophilamelanogaster,pupallength,humanheight,complex
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trait
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Correspondingauthorscontactdetails
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GuyReeves,DepartmentofEvolutionaryGenetics,MaxPlanckInstituteforEvolutionary
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Biology,August-Thienemannstrasse2,24306Plön(Germany)
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[email protected]
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office+494522763376
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lab+494522763296
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ABSTRACT
Theintensefocusonstudyinghumanheighthasdonemorethananyothergeneticanalysis
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toadvanceourunderstandingoftheheritabilityofhighlycomplexphenotypes.Herewe
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describeindetailthepropertiesofapreviouslyunexploredtraitinDrosophila
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melanogasterthatsharesmanysalientpropertieswithhumanheight.Thetotallengthof
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thepupalcasevariesbetween2.8and3.9mmamongnaturalvariantsandwereportthatit
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isamongthemostheritabletraitsreportedinthisspecies.Wehavedevelopedasimple
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semi-automaticphenotypingsystemwithwhichasingleoperatorcanreliablyscore>5000
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individualsinaday.Theprecisionoftheautomatedsystemis0.042mm(±0.030SD).All
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phenotypedindividualsareavailabletobematedinsubsequentgenerationsoruniquely
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archivedforfuturemolecularwork.Wereportbothbroadsenseandnarrowsense
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heritabilityestimatesfortwobiologicallydistinctdatasets.Narrowsenseheritability(h2)
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rangedfrom0.44-0.50andbroadsenseheritability(H2)rangedfrom0.58to0.61.We
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presentresultsformappingthetraitin195recombinantinbredlines,whichsuggeststhat
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therearenolociwith>10%effectsizeinthispanel.Weproposethatpupalsizegeneticsin
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Drosophilacouldrepresentamodelcomplextraitamenabletodeepgeneticdissection
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usingtheautomatedsystemdescribed.
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INTRODUCTION
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Elucidatingthegeneticarchitectureofaphenotypictraitfundamentallyrequiresthatit
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istosomedegreeheritable.Phenotypictraitswithlowheritabilitydonotgenerallyrequire
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additionalexplanationastowhyquantitativetraitlocicannotberobustlyidentified.
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However,whiletraitswithhighheritabilityincreasethepowertoidentifycausativeloci,
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thegeneticarchitectureoftraitsremainsakeyfactorinpredictingsuccessinidentifying
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quantitativetraitloci.Forexample,ahighlyheritabletraitthatisdependentonalarge
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numberofinteractingallelesorlocimaystillrequiresubstantialsamplesizesandcomplex
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analysestoidentifycausativeloci(Visscheretal.2010;Bartonetal.2016).The
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archetypicalexampleofthisishumanheightwhichbyatleasttwodistinctmeasuresof
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heritabilityisamongthemostheritableofquantitativehumantraits,h2≈0.4-0.7using
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regressionoftrios(Galton1886;Hanley2004;Visscheretal.2010)orh2≈0.7usingtwin
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studies(Poldermanetal.2015).Howevertheidentificationofthelociinvolved,andany
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interactionsamongthem,hasprovenfarfromtrivialandsuccessestodatehavebeen
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dependentonutilizingverylargedatasets(upto250,000individuals(Woodetal.2014)).
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Thecurrentunderstandingbeingthatofthe180-4,000lociimplicatedinimpactinghuman
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heightoveritstypicalrangeallhaveadditiveeffectsizesofmuchlessthan1mm(Weedon
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etal.2008;Visscheretal.2010;Woodetal.2014).Althoughstudiesofhumanheight
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representavaluablemodelwithwhichtotesthowcomplexheritablephenotypescanbe
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dissectedtheabilitytoexperimentallycontrolallelefrequenciesandenvironmental
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conditionswouldbeavaluablecapacitythatisnotpossibleinhumanstudies.Whilethisis
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apossibilityinmodelorganismstherearerelativelyfewmorphologicaltraitsthathave
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beenrobustlydemonstratedtobebothhighlyheritableandamenabletoautomatedhigh
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throughputphenotyping(thoughseebelow).Hereinwedescribeindetailanew
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quantitativetrait–lengthofthepupalcase-inDrosophilamelanogasterthatplacesitinthe
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15-20%mostheritablemorphologicaltraitsdescribedinthisspeciesandisamenableto
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reliableandautomatedhigh-throughputphenotyping(RoffandMousseau1987).
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Theutilityofautomatedphenotypingsystemsforawidevarietyoforganismshas
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increasedgreatlyoverthelast10years.Whiletherehavebeenautomatedmorphological
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phenotypingsystemsdevelopedforDrosophila,asyetonlyautomationanalysisofimages
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ofwingshasbeenwidelyemployed(Houleetal.2003).However,acquiringthewing
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imagesrequiresmanualmanipulationtopositionfliesindividuallyandsoisdifficultto
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scaleup.Likewiseasystemtomeasureheartbeatfunctionrequiresthateachflybe
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manipulatedintoposition.Analternativeapproachtophenotypinginaselection
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experimentforgrossbodysizewasachievedusingaseriesofgraduatedsieves(Turneret
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al.2011).Morerecentlyasophisticatedplatformcalledthe‘flycatwalk’wasdescribedthat
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hasthecapacitytoreliablyphenotype700fliesadayforawiderangeofmorphological
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traitssimultaneously(Medicietal.2015).Thisrequiresnousermanipulationofindividual
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fliesandisnon-destructive.Whilethecostofconstructingtheequipmentisnotdetailed
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(https://github.com/IMSB/FlyCatwalk/)itislikelythatitwouldrepresentasignificant
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investmentoftime,expertiseandresourcesformostlabs.
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Oursetupusesaninexpensivecamerainalightproofboxandtheopensourceimage
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analysissoftwareCellprofilerwithwhichasingleusercanphenotype5,000pupaeinaday.
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Pupaearephotographedinsituonflattenedsquaresoftransparentfilmthatlinedthe
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entireverticalsurfaceofthevials.Wedemonstratehowthesystemcanbeusedto(1)
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measurepanelsofrecombinantinbredlines(RILs)and(2)generatelargenumbersof
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parentoffspringtriosthatareparticularlyusefulinexploringcomplextraitsusingartificial
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selectiontechniques.Furthermore,theincreasedthroughputfacilitatesexploringthe
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heritabilityoffamilymeansratherthansingleindividualmeasurementsthatareassociated
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withincreasedmeasurementvariance.Hence,pupalsizecouldbecomeamodelphenotype
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thatwillallowdeepdissectionofitsgeneticarchitecture,sincetheavailabilityofautomated
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phenotypingwillallowtoscreenverylargemappingpanelsortodesignnewcomplex
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mappingstrategies.
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MATERIALSANDMETHODS
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Flystocks
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Theautomatedphenotypingsystemwasappliedtotwoindependentdatasets.
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Thefirstdataset,referredtohereas‘8-way’isacollectionof195recombinantinbred
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lines(RILs)thatarepartoftheDrosophilaSyntheticPopulationResource
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(http://wfitch.bio.uci.edu/~dspr/).TheseRILsarealloriginallyderivedfromacross
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between8globalstocksofDrosophilamelanogaster,theirgenerationisdescribedindetail
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in(Kingetal.2012a;b).Narrowsenseheritabilityh2forthe8-waydatasetwasestimated
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bymeasuringtheprogenyof67singlepairmatingsbetweenindividualsfrom11different
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RILsthatspannedthefullrangeofphenotypemeasurements(IDsofcrossedstocksare
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giveninFileS5,6ofthe67crossesareduplicatesalsoindicatedinFileS5).Broadsense
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heritabilityestimatesH2ofthe8-waydatasetweregeneratedbyrepeatedmeasurementsof
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RILs(IDsgiveninFileS5).
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Theseconddatasettermed‘4-way’wasinitiatedbyacrossbetweentwoJapaneseand
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twoAfricanstocks(SeetableS1).Narrowsenseheritabilityh2wasestimatedbymeasuring
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theprogenyofsinglepairmatingsfromthe2ndto6thgenerationwherephenotyped
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parentswererandomlyselectedfromdifferentvialstoformsubsequentgenerations.The
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followingnumberofsinglepairsweremeasuredineachgeneration[G2,15pairs],[G3,81
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pairs],[G4,78pairs],[G5,88pairs],[G6,154pairs].Noduplicatevialsweregeneratedfrom
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thesamematingpair.Notethatunlikethe8-waydataset,levelsofheterozygosityinthe
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parentsandtheiroffspringarelikelytobesimilar.BroadsenseheritabilityestimatesH2of
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the4-waydatasetweregeneratedbymeasuring83RILsestablishedfromthe4-waycross
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bygeneration37(seeFileS5forstockIDs).Detailsofallprogenitorstocksofthe4-wayand
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8-waydatasetswithestimatesoftheirpupallengtharegiveninTableS1.
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Imageacquisition
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Fliesweremaintainedonstandardfooddispensedinto28.5mmdiameter,95mm
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heightvials(GeneseeScientific).Oncethefoodvialshadfullycooled,10cmx10.5cm
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squaresofoverheadprojectorfilmwereslidintoeachvialliningtheirentireverticalwall
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(nobo,plainpapercopierfilm,33638237).Amoredetaileddescriptionoftheentire
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procedureandequipmentsetupisprovidedasFileS1.Acustomprintedsemi-transparent
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labelincludingauniquebarcodewasaffixedtotheoutsideofeachvial.Vialswere
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incubatedat24°ina12hourlight12hourdarkincubator.Adultswhereremovedafter1-2
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nightsinvials(sometimes3to4nights,iffertilityappearedtobeloworduetoholidays).
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Generallybythe10thdayaftertheparentswereinitiallyintroduced,themajorityof
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offspringinthevialswerepresentaspupaeattachedtothetransparentfilm,fewifany
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larvaeremainedinthefood.Thefilmfromeachvialwasremovedandplacedintoa
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purpose-madeplasticframe(thisframecanbe3DprintedusingafileprovidedasFileS2)
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thatholdsthefilmflatforphotographing.Foodfromthelowerpartofthefilmwasscraped
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awayandanylarvaeoranywhitepupariumstage(P1)removed.Theframewasthen
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photographedusingbottomilluminationinalighttightbox.Batchesoftheresultingimages
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werethenanalyzedusingtheprocedurebelow.
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Automatedimageanalysis
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ACellprofiler(v2.1.0)pipelinewasdevelopedtosimultaneouslyrecognizepupaeandto
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measureavarietyofattributesincludinglength.Theoutputsofallmeasurementsare
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writtento.xlsfilesthatcanthenbeviewedorimportedintoanydatabaseprogram.
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Cellprofilerisfreeopenaccesssoftwareprovidingasuiteofflexibleimageanalysistools
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(Lamprechtetal.2007).InbrieftheCellprofilerpipelinefirstidentifies‘primaryobjects’
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distinctfromthebackgroundwithoutrestrictionontheirsize(module:identifyprimary
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objects).Thenapplyingascalablemodelofpupalshapetoallobjectsthosethatare
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composedofmultipletouchingpupaeareseparatedintodistinctpupae(module:Untangle
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Worms,(Wählbyetal.2012)).Theresultingputativepupaearetheneachshrunkandthen
8
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re-propagatedoutwardstomorepreciselyidentifytheedgesofeachpupabasedon
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boundarychangesinpixelintensity(module:IdentifySecondaryobjects).Finally,pupae
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arecrudelyfilteredonsizeattributesandtheproximityofneighboringobjectstoplace
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theminoneofthethreeconfidenceclassesdescribedintheresults.Thedigitaloutlinesof
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pupaeareoverlaidontoacroppedversionoftheoriginalimagetoallowuserstoeasily
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visuallyassessquality.
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Overlaidimagesandfilesofmeasurementswereimportedinbatchesintoadatabase
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programFileMaker(v14,FileMakerInc.).Auniquebarcodestickeridentifyingthefilmin
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theimagewasautomaticallyreadbythedatabase.Thisenabledtheimagefilestobe
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automaticallyrenamedwiththebarcodeastheirnameforarchiving.Inadditiontopupal
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measurementsa1€centcoin(16.25mmdiameter)presentinallimageswasmeasuredto
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controlforcamerachangesandallowconversionofmeasurementinpixelstomm.Quality
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filteringofpupaeandbasicanalyseswereperformedusingthedatabase.Anannotated
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copyoftheCellprofilerpipelineisprovidedasFileS3.
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Estimatesofheritability
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BroadsenseheritabilityH2wasestimatedusingSPSSversion22withthe‘variance
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components’function,withpupallengthasthedependentvariable,andRILnameasa
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randomfactor.ThiswasdoneusingtheMinimumNormQuadraticUnbiasedmethod
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(thoughANOVAproducedidenticalresultsto2decimalplaces).Themodelforthesingle
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factorwasyij=μ+αj+eij.WhereyijisthejthobservationoftheithRIL,μistheoverallmean,
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αjistherandomfactorandeistheassociatederror.
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Narrowsenseheritabilityh2estimatesweredoneusing‘linearregression’function,with
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pupallengthasthedependentvariableandparent-midpointsasanindependentvariable.
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AllotherstatisticalanalysesandgraphswerealsoperformedusingSPSSorFilemaker.All
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dataforindividualpupaareavailableasFileS4andforvialmeansasFileS5(fieldsusedin
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eachgraphareindicatedintheassociated‘readme’files).
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Humanheightdata
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Estimatesofh2forhumanheightdatawasdoneusingthesamemethodsasforpupaewith
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theoriginaldataofFrancisGaltonfor898individuals,transcribedfromhis1880s
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laboratorynotebooks((Hanley2004),available
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http://www.math.uah.edu/stat/data/Galton.html).The760individualsin123families
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wherefourormoreindividualsweremeasuredwereusedforestimatesoftheh2
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heritabilityofmeanfamilyheight(femaleswerenottransmutedintomales).
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Genomescanforlocioflargeeffectimpactingpupallengthusing8-wayRILs
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ScanswereperformedinR(version3.2.2)forthe195DSPRRILsmentionedabove(Kinget
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al.2012a;b)usingthe‘DSPRscan’commandwithintheDSPRqtl-toolsprocedures
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describedin(Kingetal.2012a;b).Theseutilize1SNPper10kbthroughoutthegenome
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reference(withtheexceptionoftheYchromosomeandthemitochondrialgenome).
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SignificanceLODscorethresholdswereestimatedusingthe‘DSPRperm’commandwith
10
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1,000replicates.ThedatainputfileisavailableasFileS6.Thisscanwasundertakento
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asseswhetheroneorfewlocioflargeeffectcontrolpupallengthi.e.thetraitisnot
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complex.Notethatifthetraitiscontrolledbymanylociofsmallormoderateeffectsize,the
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scanistoounderpoweredtorealisticallyidentifyindividualloci.Butthegoalofthescanis
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toshowthatpupalsizeisnotarelativelysimpletraitcontrolledbyonlyafewmajoreffect
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loci.
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Dataavailability
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AllstartingflystocksareavailablefromthesourcesdetailedinTableS1.RILsareavailable
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fromthewebaddressgivenabove.FilesofrawdataareavailableasFilesS4,S5andS6.
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RESULTS
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Thelargenumbersofindividualsandvialsmeasuredmadepossiblethroughtheuseof
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theautomatedmeasuringsystemprovidesrobustinsightsintohowphenotypicvariationis
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partitionedforpupallength.Whilethemethodautomaticallyidentifiesandmeasures
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pupaeformultipledifferentparameters(seeFileS1forafulllist),thelengthofthepupaeis
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theprinciplefocusofthisstudy.Twodistinctsetsofbiologicalmaterialwereusedtodefine
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thebiologicalpropertiesofpupallength.Thefirstdatasetisallderivedfroman8-waycross
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(Kingetal.2012a;b).Thesecondisfroma4-waycross.Noneofthefoundingstocksare
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sharedbetweenthe8-wayandthe4-waydatasets.BroadsenseheritabilityH2(reflecting
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allpotentiallygeneticcontributionstotraitvariancee.g.,additive,epistatic,dominance,
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maternalandpaternaleffects)wasestimatedthroughtheanalysisofrepeated
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measurementsofrecombinantinbredlines(RILs).Narrowsenseh2heritability(reflecting
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onlytheimpactofadditivegeneticeffects)wasmeasuredbyregressionofmid-parent
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againsttheiroffspringmeasurements.Samplingpropertiesofthe8-wayand4-way
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datasetsaredetailedinTable1andFigure1(seealsoFigureS1forsummaryofrepeat
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measurementsofRILs).
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Dataset Heritability Vial
Vialsmeasured=1184,RILs=195
8-way
H2
Individuals
n=83,402,
meanreplicatesperRIL=6.7(±2.4SD,
meanpupallength=3.2(±0.21SD)
min=4,max=15))
meanvialpupallength=3.2(±0.15SD)
rangeof95%ofpupallengths=2.8-3.5mm
meannumberofmeasuredpupaepervial=70
(±34SD)
crossesmeasured=67
n=3,113,
h2
meanvialpupallength=3.5(±0.11SD)
meanpupallength=3.4(±018SD)
rangeof95%ofpupallengths=3.1-3.5mm
meannumberofmeasuredpupaepervial=47
(±15SD)
Vialsmeasured=436,RILs=81
n=25,356,
4-way
H2
meanreplicatesperRIL=5.54(±0.78SD,
meanpupallength=3.6(±0.24SD)
min=3,max=6))
meanvialpupallength=3.6(±0.15SD)
rangeof95%ofpupallengths=3.4-4.0mm
meannumberofmeasuredpupaepervial=59
(±24SD)
crossesmeasured=363
n=22,487,
h2
meanvialpupallength=3.4(±0.14SD)
meanpupallength=3.4(±0.23SD)
rangeof95%ofpupallengths=3.1-3.7mm
meannumberofmeasuredpupaepervial=62
(±17SD)
Table1Summaryofsamplingof8-wayand4waydatasets.Onlyvialswhere≥15pupaeweremeasuredbythe
automatedsystemareconsidered.ForH2estimatesonlyRILswhere≥3replicatemeasurementswereavailableare
considered.
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A
5,000.0
Frequency
4,000.0
3,000.0
2,000.0
1,000.0
0.0
2.0
2.5
3.5
4.0
4.5
pupal length (mm)
224
225
3.0
B
400.0
Frequency
300.0
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200.0
100.0
0.0
2.0
2.5
3.0
3.5
4.0
4.5
vial average pupal length (mm)
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229
230
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Figure1.Samplingdistributionsofpupallengthforindividualsandvials.A,distributionsforindividualpupal
lengths,4-waydataset(blue)n=47,843,8-waydataset(green)n=86,515.B,Correspondingdistributionsforvialmean
pupallengths,4-waydataset(blue)n=799vials,8-waydataset(green)n=1251vials.Onlyvialswhere≥15pupaewere
measuredbytheautomatedsystemareconsidered.AstockpossessingtheTb1mutationresultinginthewellknown
tubbypupalphenotype(Bloomingtonstock3644)wasmeasuredandfoundtohaveameanpupallengthof2.7mm,which
isatthelowerboundsofthesmallestwildtypeindividualsorvialsshownhere.
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Performanceofautomatedpupalphenotyping.
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Theautomatedpupalrecognitionpipelineattemptstoidentifytheexternaloutlinesof
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threeclassesofobjects.Highconfidencepupae:conformtoasetofexpectedpropertiesand
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thathavenoneighboringpupaewithin20pixels(≈0.7mm,Cellprofilermodule:filter
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objects).Mediumconfidencepupae:conformtothesamesetofpropertiesbutwithcloser
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than20pixelneighboringobjects.Thisoftenoccursaslarvaeselectpupationsitestouching
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eachother,resultinginamorechallengingtargetforimagerecognition.Lowqualityobjects:
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unlikelytobepupae.ExamplesofthethreeclassesareshowninFigure2,(seeFileS1for
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furtherdetails).
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250
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Ofthepupaeretainedforanalysis47%and53%werehighandmediumconfidence
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respectively.Theprobabilityofpupaebeingmanuallyexcludedasaberrantlymeasured
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was0.02forhighqualitypupaeand0.10formediumconfidencepupae(Figure2).Note
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thatnotallimagesweremanuallyexamined,assometimesduetotherobustnessofthe
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automatedestimatesgenerallyonlyvialswithatypicallyhighvarianceofthemeanwere
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examined.
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Theprecisionoftheautomatedsystemwasassessedbyre-measuringasubsetoffilmsafter
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rotatingthemby180°andcomparing516duplicatedmeasurementsofthesamepupae
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(Figure3).Thedifferencebetweenthetwomeasurementswasanaverageof0.043mm
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(±0.030SD).Re-measuringpupaeafterdelaysof15or30hoursalsogeneratedsimilar
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precisionestimates,indicatingthatoncepupaebecomebrownpuparium(P2)thereisno
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detectablechangeinpupallength.Furthermoretheexuviaofeclosedindividualscanalso
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bereliablymeasured(datanotshown).
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Thecountofautomaticallymeasuredpupaeisareasonableproxyfordensitywithinvials.
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Unsurprisinglynotallpupaewerecorrectlycapturedbytheautomatedsystemandbased
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onasampleof148vialsanaverage20%±11.7SDpupaepervialweremissed,calledaslow
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confidenceobjectsormanuallyexcluded(Figures4and2).Whilethereisanincreased
Figure2.Visualoutputofautomatedphenotypingsystem.Blueoutline=highconfidencepupae,redoutline=medium
confidencepupae,yellowoutline=lowqualityobjects.Asmallproportionofpupaeareentirelymissedbytheautomated
system(greenarrow).Othersareaberrantlymeasuredduetocloseproximitytootherpupaeresultingintruncationor
extensionoftheiroutline.Eventhosewithaahighconfidenceassignmentcanalsoappearmalformed(orangearrow).
Theyshouldbemanuallyexcludedfromanalysistoreducenoise(seeFileS1formoreexamplesofaberrantandcanonical
pupae).AllobjectsarenumberedinCellprofileroutputfilestopermitnumericalmeasurementstobeeasilyrelatedto
images.
15
varianceintheproportionofunmeasuredpupaeinhigherdensityvials(e.g.wherethe
271
automatedcountis>80),evenheretheautomatedcountprovidesareasonableproxyfor
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densityofindividualsineachvial.Thisassumesthatfewifanylarvaeremainedonthefood
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surfaceafterfilmtransfer,aswasgenerallythecase(dueinparttotheshortperiodparents
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remainedinthevials).Inadditionthesmallnumberofpupaeremovedfromfilmsthatwere
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obscuredbylarvalfoodrepresentsaconstantproportionacrossallvials(seeFileS1).
pupal length reverse orientation (mm)
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4.5
4.0
3.5
3.0
2.5
2.0
276
277
278
279
280
281
2.5
3.0
3.5
4.0
4.5
pupal length normal orientation (mm)
Figure3.Precisionofautomatedpupallengthestimates.Comparisonofpupallengthmeasurementsoffilmsinthe
normalorientationcomparedtowherethesamefilmwasrotatedby180°Allpupaeisolatedfromothersbyupto15
pixelsinanydirectionwereused(n=516pupae,across10vials).Slope=0.94andR2=0.98.Anx=ylineisshownfor
reference.
Page 1
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estimated vial density (automated
count photographed pupae)
250
200
150
100
50
0
0
50
100
150
200
250
true vial density (manual count of photographed
pupae)
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283
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285
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287
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Impactofvialdensityonpupallength.
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OnecommonmajorenvironmentalcovariateofmanyDrosophilatraitsisdensityof
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individualswithinthevialtheydevelopin.ConsequentlymanyDrosophilaresearchers
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controlforthisinexperimentsbycollectinglargenumbersofzygotesandplacinga
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controllednumberineachvial.Thisisafairlylaboriousprocesstoroutinelyperform,
Figure4.Comparisonofautomatedcountofpupaepervialversusmanualcounting
148vialsfromacrosstheentirerangeoftheautomateddensitiesobservedweremanuallyrecounted.Whileathigher
densitiestheaccuracyoftheautomatedcountdecreasesitremainsareasonableproxyforvialdensityacrosstheentire
observedrange.Slope=0.81andR2=0.92.Anx=ylineisshownforreference.
Page 1
293
whichisconsiderablycomplicatedwheresinglepaircrossesarerequired.
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Heredensitywasonlyindirectlycontrolledthroughlimitingthenumberofparentsused
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pervialandrestrictingthenumberofnightstheyremainedbeforebeingcleared(generally
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2nightsforsinglepaircrosses,andonenightforsmallgroups(n=10-20)ofRIL
17
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individuals,seeFileS1).Throughoutallexperimentstwostockswerecontinuallyre-
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measuredtoactascontrols(stock335and329,seeTableS1).Thelargenumberofrepeat
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measurementsofthesetwostocksacrossarangeofdensitiespermitstheexaminationof
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anyrelationshipbetweendensityandpupallength(Figure5).Furthermore,inthesame
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wayitisalsopossibletoexploretherelationshipbetweendensityandpupallengthusing
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therepeated8-wayand4-wayRILmeasurements(seeFigureS2).AllRILsandcontrol
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stocksexhibitauniformlynegativerelationshipbetweendensityandpupallengththeslope
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variesfrom-0.0006to-0.0041.Thisobservedvariabilitymayreflecteithervariancein
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estimatingslopesoralsothatRILsexhibitdifferentreactionnorms
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.Ifthemodeslopeof-0.002(FigureS2)isusedtocorrectthemeanlengthofvials(or
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individuals)tothatobservedatthemeanobservedvialdensitythiscanbeachievedwith
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thefollowingequation.
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Equation1
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311
312
313
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M=meanvialdensityacrosswholeexperiment
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((D-M)S)+Q=Pupallengthcorrectedforvialdensity
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Applyingthisformulatothe431vialsestablishedassinglepaircrosses>99%ofthem
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wouldrequireacorrectionoflessthan±0.1mmandofthe1,620vialsestablishedfrom
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smallgroupsofRILindividuals95%ofthemrequireacorrectionoflessthan±0.118mm
320
(Figure6).Giventhemodestnumberofvialssubjectedtoalargecorrectionand
S=slopeofregressionofdensityagainstmeanviallength(-0.002)
D=automatedestimateofdensityinvialtobecorrected
Q=individuallengthmeasurementorvialmeantobecorrected
18
uncertainlyaboutwhetherthereisatrulyuniversallinearrelationshipbetweendensity
322
andpupallengthnoneofthemeasurementspresentedinthemanuscripthavebeen
323
analyticallycorrectedfordensity.FurthermorewithrespectstorepeatedRIL
324
measurements,reducinganyconfoundingimpactofdensitycanbeachievedbyeither
325
experimentallyincreasingthenumberofreplicatemeasurementsclosertothemean
326
density,oranalyticallybyexcludingorweightingdownvialswithextremedensityvalues.
Pupa length vial average (mm)
321
4.5
4.0
3.5
3.0
2.5
20
40
60
80
100
120
Automated estimate of pupal density (count of
measured pupae in a vial)
327
328
329
330
331
332
0
Figure5.Relationshipbetweenvialdensityandpupallengthforthetwocontrolstocks.Repeatedmeasurementsof
thesamestocks(oneshortonelong)atdifferentdensitiespermitstheslopeandcorrelationoftherelationshiptobe
estimated.Whilethereisahighdegreeofcorrelationforthelongerstock-329(redcircles)R2=0.48p=>0.001the
correlationfortheshorterstock-335(blacktriangles)isminimalR2=0.07p=0.07
19
Page 1
333
A
B
100%
Cumulative percentage
Cumulative percentage
100%
80%
60%
40%
80%
60%
40%
20%
20%
0%
0%
0.00
0.02
0.04
0.06
0.08
0.10
0.12
Estimate of length correction due to vial density (mm)
0.0
0.1
0.2
0.3
0.4
Estimate of length correction due to vial density
(mm)
334
335
336
337
338
339
340
341
342
343
Varianceintheestimatesofvialmeans.
344
AscriptwaswritteninFilemakertoexploretheextenttowhichestimatesofmeanvial
Figure6.Magnitudeofpotentialcorrectionofpupallengthforvialdensity,usingequation1.
Themoreuniformvialdensitiesfromsinglepairmatings(A)resultedinallbutonevialbeingwithin50pupaeofthe
meanobserveddensityof65,correspondingtoamaximumcorrectionfordensityof0.1mm(n=431vials).Theincreased
varianceofthevialdensitiesresultingfromestablishingvialsfromsmallgroupsofRILindividuals(B)ledto
approximately5%ofvialsbeingcorrectedby>0.1mm(n=1620vials).Acalculatedmeandensity(M)of65wasusedfor
allvials.AslopevalueofS=-0.002wasused,ifasteeperslopeisusedthentherewouldbeacorrespondingincreaseinthe
magnitudeofthecorrections.Allvialwithadensityof<15wereexcluded.
Page 1
Page 1
345
pupallengthbasedonsub-samplesofindividualswithinavialdeviatefromthevialmeans
346
basedonallindividuals.Thisprovidesinsightintoatwhatpointvialswithlowdensities
347
generatemeanestimateswithunacceptablyhighvariance.Figure7indicatesthatwhile
348
selectingsingleindividualstoestimatevialmeansunsurprisinglyresultsinahighdegreeof
349
varianceofupto0.4mm.Whiletheestimatesareunbiasedthisislikelytoprove
350
unacceptablyhighgiventhatthetotalobservedrangeofpupallengthsis1.1mmand0.8-
351
0.9mmwithindatasets.However,forrandomsub-samplesizesequaltoadensityof15or
352
greaterthevarianceisgreatlyreducedtolessthan0.07mmfor95%ofvials(Figure7).On
20
thisbasisonlyvialswithmorethat15measuredpupaewereincludedforanalysisor
354
presentationthroughoutthisstudy.
355
Range of 95% of replicate differences
between the mean vial pupal length
estimate based on all individuals
compared to those based on subsubsamples of individuals (mm)
353
0.4
0.3
0.2
0.1
0.0
-0.1
-0.2
-0.3
-0.4
1
6
11 16 21 26 31 36 41 46 51
Size of sub-sample (count of individuals)
356
357
358
359
360
361
362
363
364
TheimpactofusingsmallernumbersofvialstoestimateRILmeanlength
365
InmeasuringpanelsofRILsitisusefulforpracticalreasonstominimizethenumberof
366
replicatevialsmeasuredforeachRILtoestimateanRILmeanwithanacceptabledegreeof
367
variance.Using8RILsandthetwocontrollinesallofwhichweremeasure12ormore
Page 1
Figure7.Deviationofmeanvialpupallengthofrandomsub-samplescomparedtothatbasedonallindividuals
withinavial.
Eachvialwasresampledselectingonlyasub-sampleofthepupallengthmeasurementswithinavialtocalculateasubsamplemeanlengththatwasthensubtractedfromthemeanlengthbasedonallsampledindividuals.Eachvialwasresampled100timespersub-samplesize.Whiskersrepresentrangeof95%ofsub-sampledeviationsandcirclesthemean
deviationof100replicates.Basedon729vialswithdensitiesrangingfrom51-60measuredpupae.
21
368
timesitispossibletoexploretherelationshipbetweenRILmeansandthenumberof
369
replicatevialmeasurementsused.Figure8indicatesthatsixreplicatedmeasurements
370
generallyresultin95%ofRILestimatesbeinglessthat0.1mmdifferentfromthatbasedon
371
largernumbersofreplicatevialmeasurements.Themeannumberofreplicatevial
372
measurementsperRILinthisstudywas6.4±2.1SD(5.8%theminimum4replicates,
373
18.0%=5replicates,61.2%=6replicatesand15%>6replicates).
374
22
375
376
A
Deviation of sub-sample pupal length
mean from RIL mean (mm)
0.30
0.25
329 (n=64)
335 (n=31)
11021 (n=12)
11210 (n=13)
11229 (n=12)
0.20
0.15
0.10
11236 (n=14)
11237 (n=15)
11257 (n=12)
11259 (n=12)
11265 (n=12)
0.05
0.00
-0.05
-0.10
-0.15
-0.20
-0.25
1
3
5
7
9
11 13 15 17 19 21 23 25 27 29 31 37 43 49 55 61 63
Size of sub-sample (count of individuals)
377
378
B
Deviation of sub-sample pupal length
mean from RIL mean (mm)
0.30
0.25
0.20
0.15
0.10
0.05
0.00
-0.05
-0.10
-0.15
-0.20
-0.25
1
3
5
7
9
11 13 15 17 19 21 23 25 27 29 31 37 43 49 55 61 63
Size of sub-sample (count of individuals)
379
380
Figure8.DeviationofmeanRILpupallengthutilizingrandomsub-samplesofvialscomparedtothatbasedonall
381
measuredvials.ForalleightRILandtwocontrolstockswith12ormorevialreplicatemeasurementstheRILmeanpupal
382
lengthwasrecalculatedusingarandomsub-sampleofvials.EachRILwasresampled100timespernumberofvialsub-
383
samplesizeused.Whiskersrepresentrangeofsub-samplemeans,circlesrepresenttheoverallmeans.(A)providesthe
384
rangeof95%ofsub-samplesand(B)50%ofsub-samples.TotalnumberofreplicatevialmeasurementsperRILwereas
23
Page 1
385
follows,[RILs11229,11257,11259,11265,11021=12replicates],11210=13replicates,11236=14replicates,11237=
386
15replicates,335=31replicates,and329=64replicates.Noteallpupaeinvialswereusedtocalculateeachviallength
387
mean,onlythenumberofvialmeansineachsub-samplewasvariedinestimatinglengthRILmeans.
388
h2(mid-parent
regression)
4-way
8-way
Human
Height
Offspringvialmeans
0.44±0.04SE
R2=0.31
Offspringindividual
0.42±0.08SE
R2=0.10
Offspringvialmeans
0.50±0.09SE
R2=0.33
Offspringindividual
0.54±0.02SE
R2=0.14
Offspringfamily(≥4)means
0.68±0.09SE
R2=0.31
Offspringindividual
0.68±0.06SE
R2=0.11
Vialmeans
0.58
Vialmeans
0.61
Notpossible
Vialmeans
0.61
Vialmeans
0.71
-
H2
ExpectedH2
assuming
additiveonly
model.
(2h2)/(1+h2)a
389
390
391
392
393
Estimatesofnarrowsenseheritabilityh2.
394
Estimatesoftheadditivegeneticimpactonthevarianceofthetraitcanbegainedfromthe
395
slopeofregressingtheparentalmidpoint((lengthoffather+lengthofmother)/2)against
396
thelengthoftheprogeny(Galton1886).Figure9indicatesthatdespitethe8-way(67
397
crosses)and4-waydatasets(363crosses)havingnooverlapinbiologicalmaterialand
398
havingquitedistinctsamplingpropertiestheybothresultinremarkablysimilarestimates
399
ofh2,whenrepresentingalloffspringofacrossasamean.Ifalloffspringarerepresented
400
individuallythentheestimateofheritabilityislargelyunchangedforbothdatasets(Table
401
2).Thisrobustnessintheestimatesissharedwithhumanheight(Table2and(Hanley
402
2004)).Likewise,regressionsthatuseonlythefatherormothermeasurements,ratherthan
a(Mackay2013)
Table2Summaryofheritabilityestimatesforpupallengthandhumanheight.DataforbothHumanandDrosophila
isuncorrectedforanysexualdimorphism.
24
theirmidpoint,confirmthatpaternalandmaternaleffectsareofequalmagnitudeforboth
404
pupallengthandhumanheight(seeFigureS3).
405
Offspring pupa length vial average
(mm)
403
4.5
4.0
3.5
3.0
2.5
2.5
3.0
3.5
4.0
4.5
Parental-midpoint (mm)
406
407
408
409
410
411
412
EstimatesofbroadsenseheritabilityH2
413
Estimatesofallpotentiallygenetic(H2)impactsonthevarianceofthetraitcanbeobtained
414
byestimatingtheproportionofthetotalvarianceinmeanviallengthmeasurements
415
relatedtoRILstock.Table2summarizestheresultswithboththe8-way(195RILs)and4-
416
way(81RILs)datasetsgeneratingverysimilarestimatesofH20.58and0.61respectively.
Figure9Estimatesofmeanviallengthnarrowsenseheritabilityh2for8-wayand4-waydatasets.
Despitenooverlapinthestocksusedtoinitiatebothdatasetsthereisaremarkablesimilarityintheslope(h2)andthe
degreeofcorrelation(R2)amongthem,seeTable2forallparameterestimates.Greenclosedcircles=8-waycrosses,blue
opencircles=4-waycrosses,
Page 1
25
417
ThesignificanceofANOVAtestsreflectsthestronggeneticsignal8-wayp=2.7x10-140(F=
418
10.86,MeanSquare=0.093,df=194,sumofsq=18.3)4-wayp=8.8x10-50((F=9.026,Mean
419
Square=0.087,df=80,sumofsq=6.9)
420
421
Wholegenomescanforlociimpactingpupallength.
422
Using195RILsfromthe8-waydatasetandtheDSPRtoolsagenomescanforgenomic
423
regionsassociatedwithmeanRILpupallengthwasconducted(un-weightedmeanof
424
replicatevialmeans).Thedensityofmarkersusedintheanalysisis1SNPper10kbacross
425
almosttheentirereferencegenome(excludingthemtDNAandYchromosome).NoSNPsof
426
significancewereidentifiedatα=0.05,thiswasalsothecaseifdensitycorrectedRILmeans
427
wereused(Figure10).
428
429
430
431
Figure10GenomescanofRILpupallengthin8-waydataset(195RILsmeansuncorrectedforvialdensity).No
peaksaresignificantatap=0.05thresholdof7.02LOD(estimatedbypermutation).
432
26
433
434
DISCUSSION
435
436
While(Visscheretal.2010)statedthat“Onecouldarguethatheightinhumansisthe
437
equivalentofbristlenumberinDrosophila,intermsofitsroleasamodelphenotype.”we
438
proposethatpupallengthisasgood,ifnotabetteranalogue,basedonitsbiological
439
propertiesdescribedindetailforthefirsttimehere.Thisisinadditiontothecapacityto
440
automatephenotypingusingareliablelowcostsystem.Heritabilityisestimatedusingtwo
441
distinctmeasures,broadsense(H2)andnarrowsense(h2)forbothoftwobiologically
442
independentdatasets.Thisallowstheconsistencyofheritabilityestimatestobeassessed
443
betweentwodatasets,whichlikelyspanmostoftherangeofthetraitwithinDrosophila
444
melanogaster.Furthermorethecontrastbetweenh2(additivegeneticeffectsonly)andH2
445
(additive+non-additiveeffects)permitsthemagnitudeofnon-additiveeffectstobe
446
estimated.Thepropertiesofpupallengthestablishedinthismannerarepotentiallysalient
447
toeffortstodevelopresourceefficientmeanstodissectgeneticallycomplextraitsand
448
similartothosereportedforhumanheight(Table3).Thekeyparameterbeingthehighand
449
consistentheritabilityestimates,whichinpartreflectthereliabilityofautomated
450
measurementsweretheestimatedprecisionofmeasurementsof0.04mmissmallrelative
451
totherangeofthetraitasaspecies(1.1mm)orwithindatasets(0.8-0.9mm).Thereliability
452
ofpupallengthmeasurementsalsolikelycontributestothenormalityofthedistributions
453
observedateveryhierarchicallevelinthedatasets(e.g.Figure1).
27
454
AdultHuman
height
Drosophilamelanogaster
pupallength
(basedonthisstudy)
Amongthemost
Intop80%ofmostheritable
heritablereportedin
traitsinD.melanogaster
Humansh2=0.4-0.7a
h2=0.4-0.5b
Manual
Automated
≈70cm
≈1.1mm
≈1.5cmc
0.042mm(±0.030SD)
≈0.02
≈0.04
Equal
Equal
≈180-4000d
Unknown
Notpossible
Easy
Relativeheritabilityoftrait
Measurementmethod
Typicalrangewithinspecies(ignoring
sexualdimorphism)
Measurementerror
Ratioofmeasurementerror:typical
rangeoftraitinspecies
Paternalandmaternalimpacton
offspringtraitvariance.
Numberoflociestimatedtoimpact
varianceoftrait
Experimentalmanipulationofallele
frequencies
455
456
457
458
Table3Salientpropertiesofpupallengthrelativetohumanheight.
a(Hanley2004;Visscheretal.2010)correctedforsexualdimorphism,bnotadjustedforsexualdimorphism,c(Vossetal.
1990),d(Aulchenkoetal.2009;Vinkhuyzenetal.2013;Woodetal.2014)
28
459
Theeaseofphenotypinglargenumbersofindividualsatadevelopmentalstagewhere
460
theyareobligatorilystationaryfor2-3dayshasenabledrobustinsightintotheheritability
461
ofpupallengthat24°.Theverysimilarestimates(Table2)ofheritabilityfortwo
462
independentdatasets,providesincreasedconfidencethatthistraitrepresentsanexcellent
463
prospectwithwhichtoattempttoidentifythegenesunderlyingthiscomplextrait.
464
Furthermore,thesimilaritybetweentheobservedbroadsenseheritabilityH2estimates
465
andthoseexpectedbasedonapurelyadditivemodelofinheritance(seelastrowinTable
466
2)indicatesthatepistatic,dominanceandpaternalormaternaleffectsarerelatively
467
modest(Mackay2013).Theobservedsimilarityinthetwoh2estimates(andtheassociated
468
correlationR2)betweenthe4-wayand8-waydatasets(Figure9)couldalsobearguedto
469
reflectamodestrolefordominance.Thisisbecausewhiletheparentsandoffspringofthe
470
4-waycrosseshavesimilarlevelsofheterozygosity,the8-wayparentsareinbredRILs
471
whiletheiroffspringareheterozygous.Undersomeformsofdominanceeffectsboththe
472
slopeandthecorrelationoftheregressioncouldbereducedinthelattercase,whichisnot
473
thecaseforpupallength.Withrespectstomaternalandpaternaleffectsthedatapresented
474
here(FigureS3)provideevidencethatanynon-additivepaternalormaternaleffectsare
475
small,asthesizeoftheeffectmothersandfathersexertontheiroffspringissymmetrical
476
(whichisalsoapropertyofhumanheight(Hanley2004)).
477
Ithasbeendetailedabovethattheimpactofdensitywithinvialsonpupallengthis
478
relativelymodest(mostlylessthan0.1mm,Figures5and6)andcanbeconveniently
479
controlledfor.Whileotherenvironmentalvariableswerenotsystematicallyexplored,
480
limiteddatacollectedforthetwocontrollinesindicatesthatbetween18°and24°pupal
29
481
lengthchangesby0.2-0.4mm(FigureS4),withpupallengthbeinggreateratcooler
482
temperatures.Thecorrelationbetweenadultbodyweightandpupallengthwasalsobriefly
483
examinedandfoundtobeR2=0.6forbothmalesandfemales(seeFigureS5).Any
484
correlationbetweenadultbodylengthandpupallengthwasnotexamined,thoughitis
485
potentiallynoteworthythatindividualspossessingtheTb1mutationresultingina
486
strikinglyshorttubbypupaarenotreadilydistinguishedfromwildtypeasadultsbasedon
487
theirbodylength.Giventhelargenumbersofgenesimpactinghumanheight(Table1)itis
488
unsurprisingthatitcanbecorrelatedwithdisparatetraitsofparticularinterest(e.g.cancer
489
risk,cardiovasculardiseaseandlongevity(Nelsonetal.2015;(NCD-RisC)2016)).Future
490
examinationoftraitscorrelatedwithpupallength(includingtheirreactionnorms)may
491
proveinformativewithrespectstotheirgeneticarchitectureandthatofpupallength.
492
Currentlytheautomatedsystemdoesnotattempttodistinguishmalepupaefrom
493
femalepupaebutitislikelythatitcouldbeextendedtogenerateprobabilisticsex
494
assignmentsbasedonthefactthatmalepupaeareonaverage≈8%smallerthanfemales
495
(datanotshown).Whenhumanheightdataiscorrectedforsexualdimorphism(alsoan8%
496
averagedifferencebetweensexes,(Galton1886;Hanley2004)),thereisacorresponding
497
increaseoftheheritabilityestimateby9%(seeTable1in(Hanley2004)).Thisimpliesthat
498
analyzingsexedpupallengthdatawillgenerateheritabilityestimatesthatexceedthe
499
valuesreportedhereforunsexedpupae(Table2).
500
Theobservationthatanaverage20%±11.7SDofpupaeinavialarenotmeasuredby
501
theautomatedsystem(Figure4)isgenerallynotanissueformostexperiments(evenfor
502
selectandre-sequenceapproaches),asitisoftenonlynecessarythatasubstantial
30
503
proportionofindividualsaremeasuredthatissufficienttoprovidearangeofparentsfor
504
thenextgenerationandtoreducethevarianceofmeanvialestimates(Figure7).
505
Furthermore,parametersprovidedinFigures7and8willprovidefutureresearchersthe
506
capacitytoreadilydesignmaximallyresourceefficientexperimentalstrategies.
507
Theobservationthatthegenomescanusing195RILs(8-waydataset)failedtoidentify
508
anysignificantlociimpactingpupallengthshouldbeconsideredinthelightthatwiththis
509
modestnumberofRILsthepowertodetectSNPsof10%or5%effectsizeisonly≈0.37and
510
≈0.08respectively(basedonsimulationsincorporatingtheseexactRILs,seefigure9(King
511
etal.2012b)).Consequently,theresultspresentedhereshouldbeviewedasonly
512
potentiallysufficienttoindicatethatfewifanylocioflargeeffectofsizearelikelytoexist
513
forthistraitamongtheRILsused.Itisofcourseconceivablethatphenotypingmoreofthe
514
>1,600RILscurrentlyavailablewouldidentifysignificantloci(Kingetal.2012a;Huanget
515
al.2014),andthereareseveralinFigure10whichareclosetothe0.05significance
516
threshold.Alternativelyanexpandingvarietyofotherapproachescouldbeappliede.g.
517
selectandre-sequence(Turneretal.2011;NuzhdinandTurner2013;Koflerand
518
Schlötterer2014)orcomparisonsbetweenparallelselectedlines(Chanetal.2012).While
519
thisneedstobefurtherexploredinthefuture,itisclearthattheautomatedphenotyping
520
systemwillbeakeytodesigningmappingstrategiesthatshouldallowtheidentificationof
521
manyloweffectsizegenesforthistrait.
522
Theapparatususedtophotographpupaecanberapidlyassembledforapproximately
523
500€(includingacamera)ormayalreadybepresentinmanylabsasgeldocsystems.
524
Furthermorethenecessarysoftwareisopensourceandfreeandrunsonanystandard
31
525
desktopPCorMAC.Thecapacityoftheautomatedsystemtoprovidealargenumberof
526
phenotypedindividualsprovidesincreasedpowertoexaminethegeneticarchitectureof
527
traitsbymostapproaches.Thisislikelytoprovekeytoidentifyinggenesoralleles
528
influencingmeanpupallengthanditsvariance(Woodetal.2014).Thisisinadditionto
529
potentiallyfacilitatingexploringthepoorlyunderstoodgeneticbasisofsexualdimorphism
530
(Rawliketal.2016))andlociwhichimpactthevarianceoftraits(ratherthantheircentral
531
values,(Ayrolesetal.2015)).Inadditionthecapacitytoselectfromlargenumbersof
532
individualsfromwhichtoestablishfuturegenerationshasthepotentialtoenhanceselect
533
andre-sequenceapproaches(Turneretal.2011;NuzhdinandTurner2013;Koflerand
534
Schlötterer2014).Furthermore,thelargenumbersofpowerfulapproachesbasedonsingle
535
pairmatingsdevelopedbyanimalandplantbreeders(GianolaandRosa2015)mayalso
536
becomeamenabletoexaminecomplextraitsinDrosophilathroughtheuseofthissimple
537
lowcostsystem.Aswithhumanheightoveritstypicalrangethegeneticarchitectureof
538
pupallengthisoflimitedpracticalinterest.However,forthelast130yearstheformer
539
continuestoprovidekeyinsightsintothemethodsthroughwhichcomplextraitscanbe
540
bestunderstood,thisisinpartduetotheeaseandreliabilityofhumanheight
541
measurement.Thehighheritabilityofpupallengthandthecapacitytoeasilyautomate
542
phenotypingcombinedwiththesmallandwell-describedgenomeofDrosophila
543
melanogastercouldmakepupallengthasimilarlyvaluablemodeltrait.
544
545
32
546
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Acknowledgements
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Wewouldliketothankthefollowingindividuals.StuartMacdonaldforhishelpwiththe
687
DRSPstocksandtoolsandformaintainingthisvaluablecommonresource.Masayoshi
688
Watadaforhisinvaluableadviceandaccesstohispersonalflystocks.PredragKalajdzicfor
689
karyotypingthefounderlinesofthe4-waycross.OliverHirschforhelpwiththe
690
developmentofthesemi-transparentbarcodestickers.CarolinaWählbyforhelpwith
691
extendingtheuseofthe‘untangleworms’moduleofCellprofiler.AnitaMoellerforher
692
excellenttechnicalhelpandadvicewithconductingthisexperiment.
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