RNA-Seq Analysis of Abdominal Fat in Genetically Fat and Lean Chickens Highlights a Divergence

41 Pages·2015·5.96 MB·English

RESEARCHARTICLE
RNA-Seq Analysis of Abdominal Fat in
Genetically Fat and Lean Chickens Highlights
a Divergence in Expression of Genes
Controlling Adiposity, Hemostasis, and Lipid
Metabolism
ChristopherW.Resnyk1,ChumingChen2,HongzhanHuang2,CathyH.Wu2,JeanSimon3,
ElisabethLeBihan-Duval3,MichelJ.Duclos3,LarryA.Cogburn1*
1 DepartmentofAnimalandFoodSciences,UniversityofDelaware,Newark,Delaware,UnitedStatesof
America,2 CenterforBioinformaticsandComputationalBiology,UniversityofDelaware,Newark,Delaware,
UnitedStatesofAmerica,3 INRAUR83RecherchesAvicoles,37380,Nouzilly,France
*[email protected]
OPENACCESS
Citation:ResnykCW,ChenC,HuangH,WuCH, Abstract
SimonJ,LeBihan-DuvalE,etal.(2015)RNA-Seq
AnalysisofAbdominalFatinGeneticallyFatand Geneticselectionforenhancedgrowthrateinmeat-typechickens(Gallusdomesticus)is
LeanChickensHighlightsaDivergenceinExpression
usuallyaccompaniedbyexcessiveadiposity,whichhasnegativeimpactsonbothfeedeffi-
ofGenesControllingAdiposity,Hemostasis,andLipid
Metabolism.PLoSONE10(10):e0139549. ciencyandcarcassquality.Enhancedvisceralfatnessandseveraluniquefeaturesofavian
doi:10.1371/journal.pone.0139549 metabolism(i.e.,fastinghyperglycemiaandinsulininsensitivity)mimicovertsymptomsof
Editor:JaeYongHan,SeoulNationalUniversity, obesityandrelatedmetabolicdisordersinhumans.Elucidationofthegeneticandendocrine
REPUBLICOFKOREA factorsthatcontributetoexcessivevisceralfatnessinchickenscouldalsoadvanceour
Received:March10,2015 understandingofhumanmetabolicdiseases.Here,RNAsequencingwasusedtoexamine
differentialgeneexpressioninabdominalfatofgeneticallyfatandleanchickens,which
Accepted:September14,2015
exhibita2.8-folddivergenceinvisceralfatnessat7wk.IngenuityPathwayAnalysis
Published:October7,2015
revealedthatmanyof1687differentiallyexpressedgenesareassociatedwithhemostasis,
Copyright:©2015Resnyketal.Thisisanopen endocrinefunctionandmetabolicsyndromeinmammals.Amongthehighestexpressed
accessarticledistributedunderthetermsofthe
genesinabdominalfat,acrossbothgenotypes,were25differentiallyexpressedgenes
CreativeCommonsAttributionLicense,whichpermits
unrestricteduse,distribution,andreproductioninany associatedwithdenovosynthesisandmetabolismoflipids.Over-expressionofnumerous
medium,providedtheoriginalauthorandsourceare adipogenicandlipogenicgenesintheFLchickenssuggeststhatinsitulipogenesisin
credited.
chickenscouldmakeamoresubstantialcontributiontoexpansionofvisceralfatmassthan
DataAvailabilityStatement:TheRNA-Seqreadsin previouslyrecognized.Distinguishingfeaturesoftheabdominalfattranscriptomeinlean
SequenceReadArchive(SRA)formatwere
chickenswerehighabundanceofmultiplehemostaticandvasoactivefactors,transporters,
depositedintotheNationalCenterforBioinformatic
andectopicexpressionofseveralhormones/receptors,whichcouldcontrollocalvasomotor
InformationGeneExpressionOmnibus(NCBIGEO)
databaseundertheaccession#GSE42980.Further toneandproteolyticprocessingofadipokines,hemostaticfactorsandnovelendocrinefac-
datasetssupportingthepresentresultsareincluded tors.Over-expressionofseveralthrombogenicgenesinabdominalfatofleanchickensis
withinthearticleandinsupportinginformationfiles
quiteoppositetothepro-thromboticstatefoundinobesehumans.Clearly,divergentgenetic
(S2andS4–S6Tables).
selectionforanextreme(2.5–2.8-fold)differenceinvisceralfatnessprovokesanumberof
Funding:Thisworkwassupportedbyagrantfrom
novelregulatoryresponsesthatgoverngrowthandmetabolismofvisceralfatinthisunique
theUnitedStatesDepartmentofAgriculture,Initiative
avianmodelofjuvenile-onsetobesityandglucose-insulinimbalance.
forFutureAgriculturalandFoodSystems(http://www.
PLOSONE|DOI:10.1371/journal.pone.0139549 October7,2015 1/41
AdiposeTranscriptomeDivergenceinFatandLeanChickens
csrees.usda.gov/about/offices/compprogs_ifafs.html)
Introduction
AnimalGenomeProgram(Award#00-52100-9614)
toLACandJS.Additionalfundingwasobtainedfrom
Thedomesticchicken(Gallusdomesticus)servesadualpurposeasaworld-widesourceof
theAvianBiosciencesCenter(http://ag.udel.edu/abc/
high-qualitydietaryproteinandasanimportantmodelorganismfordevelopmentalbiology
index.html)andaCoreFeeWavier(toLAC)froman
InstitutionalDevelopmentAward,Networkof andgenomicsresearch[1–4].Inparticular,thechickenhasbeenusedextensivelyasabiomedi-
BiomedicalResearchExcellenceattheUniversityof calmodeltounderstandbasicmechanismscontrollingembryonicdevelopment,immunesys-
Delaware(http://bioinformatics.udel.edu/node/107).A temfunction,nutrientutilization,hormonesensitivity,andadiposity.Chickenshaveseveral
portionofthisworkwassupportedbytheNational
uniquemetabolicattributeswhichmakethemanattractivemodelforobesityrelatedstudies.
ScienceFoundation,ExperimentalProgramto
Unlikemammals,chickensnaturallyexhibithyperglycemia(>200mg/dLduringfasting)and
StimulateCompetitiveResearch/Institutional
survivelargedosesofexogenousinsulin,indicatinganinnateinsensitivitytoinsulin,particu-
DevelopmentAward(http://www.nsf.gov/od/iia/
programs/epscor/nsf_oiia_epscor_index.jsp)toCHW. larlyinadiposetissuewhereinsulinexertsonlymarginaleffectsontheuptakeofglucoseby
Thefundershadnoroleinstudydesign,data isolatedadipocytes[5,6].Anotheruniquefeatureofmetabolicregulationinthechickenisthe
collectionandanalysis,decisiontopublish,or disruptionofsyntenicgenomiclocioffivemajormammalianadipokines[leptin(LEP)[7],
preparationofthemanuscript. plasminogenactivatorinhibitor–1(PAI–1),tissuenecrosisfactoralpha(TNFA),resistinand
CompetingInterests:Theauthorshavedeclared omentin[8]].Theabsenceofthesekeyadipokinesinthechicken,particularlyLEP[9],indi-
thatnocompetinginterestsexist. catesthatalternativemechanismsmustexisttoregulatetheirfeedintakeandthebalance
Abbreviations:FL,Fatline;LL,Leanline;DE, betweenenergyexpenditureandstorage.Despiteamajorclassdifferenceinabsence/presence
differentiallyexpressed;HE,highestexpressed;wk, ofadipokines,chickensdoshareseveralkeymetaboliccharacteristicswithhumans,including
weekofage;IPA,IngenuityPathwayAnalysis;qRT- thefactthattheliveristheprimarysiteofdenovosynthesisoflipids[10–13],whicharethen
PCR,quantitativereal-timepolymerasechain
transportedastriglyceridestoadiposetissueforstorageandrelease.Inchickens,abdominal
reaction;h,hour(s).
fatnessisahighly-heritablepolygenictraitregulatedbymultiplebehavioral,environmental
andhormonalfactors[14–21].
Recenthigh-densitymicroarraystudieshaveshownthatlipogenicgenesarereadilytran-
scribedinchickenadiposetissue[22,23]anddevelopmentallyregulatedingeneticallyfat(FL)
andlean(LL)chickens[24].Usingacombinedmetabolomicsandtranscriptomicapproach,Ji
etal.[23]comparedgeneexpressionandmetaboliteprofilesinabdominalfatofrelatively
leaner-chickenbreeds(LeghornandFayoumi)againstfatterandheaviercommercialbroiler
chickensatthesameage(7wk).Theirmainconclusionswerethatabdominalleannessinthe
LeghornandFayoumibreedswasachievedbyenhancedlipidcatabolismandreducedlipid
synthesisinabdominalfat,whereasenhancedadipogenesisandgreaterfatnessinbroilerchick-
ensreflectareductioninbothfatty-acidoxidationandliberationofnon-esterifiedfattyacids
byvisceralfat.Incontrast,theFLandLLchickensusedinthepresentstudyweredivergently
selectedoversevengenerationsforeitherhigh(FL)orlow(LL)abdominalfatnessatsimilar
bodyweightsandfeedintake[25].TheseuniqueFLandLLchickensserveasvalidgenetic
models[26–28]ofleannessandjuvenile-onsetobesitywitha2.5-folddifferenceinabdominal
fatnessbetween3to11weeksofage(wk)[25,29].Ourrecenttime-coursemicroarrayanalysis
ofabdominalfatinFLandLLchickens[24]hasrevealednumerousdifferentiallyexpressed
(DE)genesinvolvedinseveralprocesses,whichultimatelyproduceeitheralipolytic(LL)or
lipogenic(FL)state.AmongtheDEgenes,wefoundextensiveoverexpressionofendocrine,
hemostatic,lipolytic,andlipidexportgenesinthediminishedabdominalfatofLLcockerels,
especiallyat7wk.Ontheotherhand,visceralfatoftheFLchickensshowhighexpressionof
multipletranscriptionfactors,enzymesandtransportersinvolvedinadipogenesisandlipogen-
esis[24].Inanothertranscriptionalstudyofadiposetissueinjuvenilechickensofacommercial
broilercross,short-termfasting(5h)resultedinalteredexpressionof1780genes,whileacute
insulinimmunoneutralizationaffectedonly92adiposegenes[22].Thisrelativelyshortperiod
offastingwassufficienttodownregulateadiposegenesassociatedwithsynthesis,elongation
anddesaturationoffattyacids.However,agapremainsinourunderstandingofadipogenesis
inchickens,especiallytheimportanceofdenovosynthesisoflipidsinvisceraladiposetissue
andthefunctionofnumerousendocrinefactors/receptorsexpressedbyabdominalfat.
PLOSONE|DOI:10.1371/journal.pone.0139549 October7,2015 2/41
AdiposeTranscriptomeDivergenceinFatandLeanChickens
Formorethanthreedecades,scoresofpapershavedescribedvariousaspectsofgrowthand
nutrientmetabolisminthedivergently-selectedFLandLLchickensoriginallydevelopedby
Leclerqetal[25].Ingeneral,theFLandLLcockerelshavesimilargrowthrateswitha2.5-fold
differenceinabdominalfatnessandhigherbreastmuscleweightsintheLL.TheFLchickens
alwaysexhibitalowerplasmaglucoselevelwithoutoverthyperinsulinemiafoundinmammals,
apeculiarconditionwhichSimonetal.[30,31]describedasa“glucose-insulinimbalance”.
HypertriglyceridemiaoftheFLchickensindicateshigherrateofhepaticlipogenesisfromcar-
bohydratemetabolism,mostlikelytheconsequenceofasmallincreaseininsulin-sensitivityin
theFLchickens[26,31].TheFLandLLchickensareabletomaintaintheirrespectivefator
leanphenotypeindependentlyofalteredenergysources[32],emphasizinggeneticregulation
ofphenotypicexpression.Thesemetabolicpeculiaritiesinourpolygenicmodelofjuvenile-
onsetobesityhavebeenextensivelyexaminedbynutritionalandmetabolicperturbations
[26,33–36].Furthermore,transcriptionalprofilingofmultipletissues[2,18,24,37–41]and
high-throughputsurveysofvariationsingenomesequenceandstructure,includingextensive
quantitativetraitloci(QTL)andexpression(eQTL)analyses[17,19,20,42–44]intheFLand
LLchickenshavebeguntoidentifycausalgenesandtounravelthegeneticandmolecularbasis
fortheirdivergenceinlipidmetabolismandvisceralfatness.
Thepresentdescriptivetranscriptomicsstudy,usingRNAsequencing(RNA-Seq)analysis,
wasdesignedtoexpandourcatalogofexpressedadiposegenesat7wkwithadualgoal(1)to
determinethemosttranscriptionally-activebiologicalprocessesinabdominalfatand(2)to
establishmajorfunctionaldifferencesbetweentheabdominalfattranscriptomesofFLandLL
chickens.First,afunctionalcharacterizationofthe900highestexpressed(HE)adiposegenes
independentofgenotypewasprovidedbyabioinformaticsanalysis.Second,weidentified1687
differentially-expressed(DE)genesfromthecomparisonoftranscriptsinabdominalfatofFL
andLLchickens.IngenuityPathwayAnalysishasrevealedtheover-expressionofnumerous
genesinvolvedinhemostasis,lipidcatabolism,andendocrinesignalingintheLL.Incontrast,
theup-regulationofseveralkeyadipogenicandlipogenicgenesinabdominalfatoftheFL
chickenssuggeststhatinsitulipogenesiscouldmakeamoresubstantialcontributiontothe
expansionofadiposemassinthechickenthanpreviouslyrecognized.
MaterialsandMethods
Animalsandtissuepreparation
TheFLandLLchickenswerebredandraisedatINRAUE1295Pôled'ExpérimentationAvicole
deTours,F–37380Nouzilly,France,asdescribedpreviously[24].Briefly,8birdsfromeach
genotype(FLandLL)wererandomlyselectedfortissuesamplingatsixages(1,3,5,7,9,and
11wk),weighed,bledintoheparinizedsyringes,andkilledbycervicaldislocation.Abdominal
fatwasquicklydissected,weighed,asamplewasimmediatelysnapfrozeninliquidnitrogen,
andstoredat−75°Cforfurtherprocessing.Allanimalprocedureswereperformedunderthe
strictsupervisionofaFrenchgovernmentveterinarianandinaccordancewithprotocols
approvedbytheFrenchAgriculturalAgency,theScientificResearchAgency,andtheInstitu-
tionalAnimalCareandUseCommitteeatINRA,Nouzilly,France.Theseprocedureswerealso
incompliancewiththeUnitedStatesDepartmentofAgricultureguidelinesontheuseofagri-
culturalanimalsinresearchandapprovedbytheUniversityofDelawareAgriculturalAnimal
CareandUseCommittee.
RNAextraction,librarypreparationandRNAsequencing
Abdominalfatsamplesfromeightindividual7-wk-oldchickens(4FLand4LL)werehomoge-
nizedandcellularRNAextractedusingguanidinethiocyanateandCsClgradientpurification
PLOSONE|DOI:10.1371/journal.pone.0139549 October7,2015 3/41
AdiposeTranscriptomeDivergenceinFatandLeanChickens
[45]followedbyDNaseItreatment.SamplequalitywasanalyzedwithanRNA6000Nano
AssaykitandtheModel2100Bioanalyzer(AgilentTechnologies;PaloAlto,CA).TherRNA
ratio(28S/18S)wasdeterminedandallsampleshadanRNAintegritynumber(RIN)greater
than9.0.Sequencinglibrariesweremadefrom1μgoftotaladiposeRNAwiththeIllumina
RNASamplePrepKitv2followingstandardIlluminaprotocols.IndividualRNAsampleswere
indexed(bar-coded)toenablemultiplexingoflibrarieswithinsequencinglanes.Librarieswere
pooledandsequencedusinganIlluminaHiSeq2000SequencingSystemattheDelawareBio-
technologyInstitute,UniversityofDelaware.Threeseparateschemeswereusedforpaired-end
(101bp)sequencingof8libraries(4FLand4LL)acrosstwosequencinglanesperrun.In
SchemeA,twosequencinglaneswereusedformultiplexingoftwoFLandtwoLLsamplesper
lane(n=4/lane).Twolibraries(1FLand1LL)insequencinglane2ofSchemeAhadlowqual-
itycontrol(QC)scoresandwereeliminatedfromfurtheranalyses.Consequently,thetwolow
QClibrarieswerere-sequencedinindividuallanesinSchemeB(n=1/lane).Finally,alleight
(4FLand4LL)librariesweremultiplexedandsequencedintworeplicatelanesinSchemeC
(n=8/lane).AllsamplesinSchemes1–3weremergedintoonefileof12samplesfromtheFL
and12samplesfromtheLLcockerelsforfurtheranalysis.
RNAsequence(RNA-seq)analysis
Allreadsgeneratedfromthethreesequencingschemes(12FLand12LL)describedabove
wereincludedintheRNA-SeqanalysisusingCLCGenomicsWorkbench5.1software(CLC
bio,Cambridge,MA).Thedataanalysisincludedsequencedatafiltering,readmapping,
transcriptandgeneidentification,analysisofdifferentialgeneexpression,andfunctional
annotation.
Sequencedatafiltering. Twenty-fourshort-read(101basepairs)sequencingsamples
(12FLand12LL)fromthe3sequencingschemeswerede-multiplexedandimportedintoCLC
GenomicsWorkbench,separately.SeveralQCtrimmingmethodswereusedwithintheCLC
GenomicsWorkbenchsoftware,includingqualitytrimming,ambiguitytrimmingandadapter
trimmingwithdefaultsettingsappliedbeforemappingtothereferencechickengenome.
Readmappingandtranscript/geneidentification. Thereferencegenomeforthechicken
(Gallusgallus,build2.1)inFASTAformatandthecorrespondingannotationfileinGTFfor-
matwereobtainedfromEnsembl(ftp.ensembl.org/pub/release–64),whichrepresents17,934
genesand22,298transcripts.Twohundrednucleotidesofflankingregionupstreamanddown-
streamofknowngeneswerealsoincludedintheanalysis.Theshortpaired-endreadsequences
(101bpx2)weremappedtothereferencechickengenomesequence,withmappingparame-
tersthatenforced:(1)amaximumoftwomismatchesand(2)readsmustmapwith(cid:1)90%of
thebasesalignedtothereferencesequencewith(cid:1)80%similarity.Non-specificmatches(reads
mappedtomultipleplacesinthereferencegenome)wereexcludedfromtheanalysis.
Differentialexpressionanalysis. Theuniqueexonreadscount(includingtheexon-exon
andexon-intronjunctions)forthereadsmappedtoageneanditsflankingregionswereused
astherawexpressionvalueforthatgene.Thisrawexpressionvaluewasnormalizedtothe
medianofthetotalmappedreadsacrossthe24samplestoaccountforvariationinoriginal
libraryconcentrationandmultiplexingnumber.The24sequencingsamplesweredividedinto
twogenotypes(FLandLL),resultingin12replicatesforeachgenotype.Normalizedexpression
valueswereanalyzedasabeta-binomialmodel[46]todetectdifferentialexpression.Thetwo-
sidedP-valuewascorrectedusingthefalsediscoveryrate(FDR)adjustmenttoaccountfor
multiplehypothesistestingprocedures[47].GeneswithFDR-adjustedP-value((cid:3)0.05)were
consideredtobestatisticallysignificant.Toensurethebiologicalrelevance,aconditionoffold
change(cid:1)1.2(or(cid:3)-1.2)wasaddedontopofFDR-adjustedP-value((cid:3)0.05);Geneswith
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AdiposeTranscriptomeDivergenceinFatandLeanChickens
FDR-adjustedP-value((cid:3)0.05)andfoldchange(cid:1)1.2(or(cid:3)-1.2)wereconsideredtobediffer-
entiallyexpressed(DE)inthisstudy.Thefold-changethreshold(±1.2-fold)forDEgenesis
basedonourextensiveexperienceinfunctionalgenomicsandtranscriptionalprofilingofmul-
tipletissuesfromvariouschickenmodels.ArecentRNA-Seqstudyofbreastmuscleinchick-
ensafflictedwith“WoodenBreast”disease[48]usedasimilarsignificancelevel[FDR-adjusted
P-value((cid:3)0.05)and±1.3fold-change]toidentifyDEgenesusingdefaultsettingsfortheCuff-
diffprocedureintheopen-sourcesoftware,Cufflinks(http://cole-trapnell-lab.github.io/
cufflinks/).
Availabilityofsupportingdata
TheRNA-SeqreadsinSequenceReadArchive(SRA)formatweredepositedintotheNational
CenterforBiotechnologyInformationGeneExpressionOmnibus(NCBIGEO)database
undertheaccession#GSE42980.Furtherdatasetssupportingthepresentresultsareincluded
withinthearticleandinsupportinginformationfiles.
QuantitativeRT-PCRanalysis
Forverificationofexpression,quantitativereal-timePCR(qRT-PCR)analysiswasperformed
onasubsetof47DEgenesidentifiedbyRNA-Seqanalysis.First-strandcDNAsynthesiswas
performedbyincubationofa13μlreaction(containing1μgoftotalDNase-treatedRNA,1μl
of100μMoligodT ,1μlof10mMdNTPmix,andwaterto13μltotalvolume)for5minat
20
70°Candplacedonicefor2min.Amastermixcontaining5μlof5xfirst-strandsynthesis
buffer,1μlof0.1MDTT,1μlofRNaseOUT,and200UofSuperScriptIIIreversetranscriptase
(Invitrogen,Carlsbad,CA)wasadded(finalreactionvolumeof20μl).Primersweredesigned
forqRT-PCRusingPrimerExpressv2.0software(AppliedBiosystems,FosterCity,CA).
Detailedinformationforeachprimerpair,includinggenename,genesymbol,forwardand
reverseprimersequences,GenBankaccessionnumberandampliconsize,areprovidedin
S1Table.
AnABIPrismSequenceDetectionSystem7900HTwasusedtoperformtheqRT-PCR
assays,using10ngoftotalRNA,PowerSYBRgreenPCRmastermix(AppliedBiosystems,
FosterCity,CA),and400nMofeachprimerpair(Sigma-Aldrich,St.Louis,MO)induplicate
wells.Adisassociationstepwasusedtovalidatespecificamplificationandverifyabsenceof
primerdimers.PCRproductswereanalyzedusingagarosegelelectrophoresistocompare
productsizetotheexpectedampliconsize.Thecycletime(Ct)foreachsamplewasnormalized
tothecorrespondingsamplegeometricmeanofhousekeepinggenes[49].Weselectedtwo
housekeepinggenes[pantothenatekinase1(PANK1)andribosomalproteinL14(RPL14)]
basedontheirinvariabilityinqRT-PCRanalysisandidentifiedasthemoststably-expressed
genesusingRefFindersoftware(http://www.leonxie.com/referencegene.php).The2-(ΔΔCt)for-
mulawasusedtocalculaterelativeabundanceoftranscripts[50].Thestatisticalanalysisof
normalizedqRT-PCRdataacrossthreeages(3,7and11wk)wasperformedusingageneral
linearmodel(GLM)procedureinStatisticalAnalysisSystem(SASv9.3;Cary,NC).Thesedata
wereanalyzedusingatwo-factoranalysisofvariancetodeterminemaineffects(P(cid:3)0.05)of
genotype(G),age(A),andtheinteractionofagewithgenotype(A×G).Wheregeneswere
onlyusedforqRT-PCRanalysisatoneage(7wk),astudent’sT-testwasusedtoidentifysignif-
icant(P(cid:3)0.05)differencesbetweentheFLandLLgenotypes.
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AdiposeTranscriptomeDivergenceinFatandLeanChickens
Table1. SummaryofRNA-SeqanalysisofabdominalfatindivergentFLandLLchickensat7wk.
Scheme(samples/lane) Totalinput Paired-endreads Totalreads Totalreads Expressed Expressed
reads aftertrimming mapped unmapped Genes Transcripts
A(n=3)*:FL 59.13M 58.71M 32.54M 26.59M 12,959 14,358
A(n=3)*:LL 67.11M 27.54M 36.02M 31.09M 13,187 14,599
B(n=1):FL 123.68M 122.2M 54.64M 69.05M 13,890 15,550
B(n=1):LL 187.85M 182.7M 71.05M 116.8M 14,134 15,853
C(n=8):FL 40.26M 34.22M 19.12M 21.14M 12,810 14,081
C(n=8):LL 35.71M 35.34M 17.67M 18.04M 12,612 13,902
AverageAcrossSchemes:FL 74.36M 71.71M 35.43M 38.92M 13,220 14,663
AverageAcrossSchemes:LL 96.89M 81.86M 41.58M 55.31M 13,311 14,785
AverageAcrossGenotypes 85.62M 76.78M 38.51M 47.12M 13,265 14,724
andschemes(A,B,C)
Readtrimming,readmappingandexpressiondataareprovidedforthreedifferentsequencingschemesutilizedforpaired-endsequencingof8libraries(4
FLand4LLcockerels).Valuesareaveragedacross3–4individualbirdspergenotype.InSchemeA,twosequencinglaneswereusedforassignmentof
twoFLandtwoLLlibrariesperlane(n=4/lane).
*Twolibraries(1FLand1LL)insequencinglane2(SchemeA)hadalowQCscore;therefore,theirlow-qualitydatawereeliminatedfromfurther
RNA-Seqanalysisunderthisscheme.Consequently,thesetwolibrarieswereeachre-sequencedinindividuallanesinSchemeB(n=1/lane).Finally,all
eightlibraries(4FLand4LL)weresequencedintworeplicatelanesinSchemeC(n=8/lane).SchemeCprovidesthemostrobustRNA-Seqdesign,the
balancedblockdesign;where,alleightlibraries(4FLand4LL)aresequencedintworeplicatesequencinglanes.Thisdesignallowsthetargeted
biologicalvariationtobepartitionedfromtechnicalerrorasdescribedindetailbyAuerandDoerge[51].AdditionalinformationonRNA-Seqanalysisis
providedinS2Table.
Abbreviations:fatline(FL),leanline(LL),andmillion(M).
doi:10.1371/journal.pone.0139549.t001
Results
Mappedreadsanddetectionofgenesandtranscripts
Sequencedatafromthe24samplesweremappedtothereferencegenome(Gallusgallus,build
2.1).Table1presentsasummaryoftheRNA-Seqanalysisincludingthenumberofmapped
readsanddetectionofcorrespondingchickengenesandtranscripts(seeS2Tableformore
details).Theoriginalsequencingrun(SchemeA)wascompletedbymultiplexing2FLand
2LLsamples(N=4)intwoseparatesequencinglanes.Twosamples(1FLand1LL)inLane
2hadlowqualityscores;therefore,thelow-qualitydatawaseliminatedfromfurtherRNA-Seq
analysis.Nonetheless,SchemeAprovidedanaverageof32.5MmappedreadsfortheFL
(N=3)and36MmappedreadsfortheLL(N=3),whichalloweddetectionof73%genesand
65%oftranscriptsacrosstheFLandLLgenotypes.Subsequently,thetwolowqualityscore
librarieswerere-sequencedinseparatelanesinanothersequencingrun(SchemeB),which
gavethehighestaveragedetectionlevelsof78%forgenesand70%fortranscriptsacrossboth
genotypes.InSchemeC,the8libraries(4FLand4LLsamples)weremultiplexedand
sequencedinduplicatelaneswithinthesamesequencingrun.SchemeCrepresentsthebal-
ancedblockdesignwithtwotechnicalreplicatesasdescribedbyAuerandDoerge[51]for
properstatisticalanalysisofRNA-Seqexperiments.Comparingsequencingdepths(averaged
acrosstheFLandLLchickens),SchemeCalloweddetectionof71%ofgenesand63%oftran-
scriptsbymultiplexing8librariesperlanewhichweresequencedinduplicatelanes.Theaver-
agenumberofreadsmappedacrossSchemeA,BandCwasgreaterfortheLL(41.6M)than
theFL(35.4M),whichisreflectedintheslightlyhighernumberofexpressedgenesfoundin
abdominalfatoftheLLcockerels.Theoverallaverageacrossgenotype(FLandLL)and
sequencingschemes(A,BandC)showsthat45%(38.5M)ofthetotalreadsweremapped,
PLOSONE|DOI:10.1371/journal.pone.0139549 October7,2015 6/41
AdiposeTranscriptomeDivergenceinFatandLeanChickens
whichequatestoidentificationof74%ofgenes(13,265/17,934)and66%oftranscripts
(14,724/22,298)basedonthereferencechickengenome.Genesfromthereferencechicken
genomeweremappedtoUniProtKBaccessionnumbersbytheProteinInformationResource
(PIR)IDmappingservice[52].Theassignedfold-changevalues(i.e.,FL/LLexpressionratios)
werebasedonthenumberofnormalizedreadsfromtheRNA-Seqanalysis.
ApoweranalysiswasperformedonthisRNA-Seqdatasetusingtheweb-basedsoftware
program“Scotty”(http://euler.bc.edu/marthlab/scotty/scotty.php)[53].Thisanalysisdemon-
stratesthatoursamplesizeoffourbirds/genotype(n=4),sequencedacrossthreedepths[38.5
millionmappedreadspersampleaveragedacrossdepths(Table1)],hadpowertodetect80%
ofgenesexpressedineachgenotype(P(cid:3)0.01)at(cid:1)1.5folddifferenceandgreaterthan90%at
afold-changeof(cid:1)2(S1Fig).Further,the“Scotty”programperformedhierarchicalclustering
usingSpearmancorrelationasthedistancemetric.Thiscorrelationanalysisgroupedthetwo
genotypesdistinctly,wheretheindividualswithineachgenotype(FLandLL)wereclosely
linked.
AbdominalfattranscriptomeoffedFLandLLchickens
First,the900highest-expressed(HE)genes,definedasgeneswithanaverage(acrossboth
genotypes)of>4289reads/gene,wereidentifiedfromRNA-Seqanalysisofabdominalfatin
FLandLLchickensat7wk(S3Table).OftheHEgenes,164wereexpressedhigherintheFL
(>1.2-folddifference),while155HEgeneswereup-regulated(<-1.2-folddifference)invis-
ceralfatoftheLL.Second,weidentified1687DEgeneswithaFDR-correctedP-value
(P(cid:3)0.05)andfoldchange(cid:1)1.2(or(cid:3)-1.2);andofthese,1182DEgeneswereexpressedhigher
inabdominalfatofLLchickens,whereasonly505DEgeneswereexpressedhigherinFL
chickens(S4Table).Aworkinglistof607functionalgenes,associatedwithlipidmetabolism
(lipogenesis,lipolysis,lipidtransport,etc.),wascompiledfromtheRNA-Seqdatasetsusing
IngenuityPathwayAnalysissoftware(S5Table).TheVenndiagram(Fig1)showstheintersec-
tionamongHEgenes,DEgenes,andlipidmetabolismgenes,with25genesincommonacross
allthreegenesets.Atotalof164DEgenesweresharedbetweenHEandDEgenelists.And,
108DEgenesaresharedbetweenthe1687DEgenesandthe607lipidmetabolismgenes.Fur-
ther,87genesaresharedbetweenthe900HEgenesandthe607lipidmetabolismgenes.
IngenuityPathwayAnalysis(IPA)ofgeneexpression
AnalysisofhighestexpressedgenesinabdominalfatofFLandLLchickens. The900
highest-expressed(HE)genesinabdominalfat(S3Table)weresubmittedtotheIngenuity
KnowledgeBase(http://www.ingenuity.com/)forfunctionalannotation,mappingofgenesto
canonicalpathways,andidentifyinggeneinteractionnetworks.Therewere828DEgenesiden-
tifiedbyIPAas“Analysisready”(i.e.,annotatedintheIngenuityKnowledgeBase).Asummary
oftheover-representedIPAfunctionalcategoriesfoundfor828“Analysisready”HEgenesis
presentedinTable2.ThemajorIPAcategoriesrepresentedbytheHEgenesincludethe“Top
CanonicalPathways,DiseasesandDisorders,MolecularandCellularFunctions,Physiological
SystemDevelopmentandFunction,TopGeneInteractionNetworks,andTopTox[Toxicol-
ogy]Lists”.SeveralsubcategoriesundertheIPAcategory“DiseasesandBioFunctions”reveal
themostprevalentbiologicalprocessesfoundinabdominalfat.Forexample,severalsubcate-
goriesarerelatedtotheregulationofadiposity[“MetabolicDisease”(193genes),“Endocrine
SystemDisorders”(50genes),“mTORSignaling”(31genes),“InsulinReceptorSignaling”
(12genes),and[“Type2DiabetesSignaling”(10genes)].Additionalsubcategoriesofinterest
arethoseinvolvedinmetabolism[“LipidMetabolism”(164genes),“CarbohydrateMetabo-
lism”(65genes),“GlycolysisI”(8genes)]andhemostasis[“ThrombinSignaling”(23genes)].
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Fig1.Venndiagramillustratingoverlapamongfunctionalgenelists.TheVenndiagramshowsthe
intersectionsofhighlyexpressed(HE)genesinabdominalfat(S3Table),differentiallyexpressed(DE)genes
(S4Table),andIngenuityannotatedgenesknowntobeinvolvedinlipidmetabolism(S5Table).The
numbersinoverlappingarcsindicatethenumberofgenessharedbetweenandamongthesethree
categories.
doi:10.1371/journal.pone.0139549.g001
SeveralcategoriesundertheIPATopToxLists(Table2)arealsoofparticularinterest.Of
the21HEgenesassociatedwith“HepaticFibrosis”,17HEgenesareexpressedhigherin
abdominalfatoftheLLchickens,including5collagengenes(COL3A-COL6A),fibronectin1
(FN1),spondin1and2(SPON1,SPON2),fibrillin1(FBN1),transforminggrowthfactor,beta
receptorII(TGFBR2)andthrombospondin1(THBS1).The“PPAR/RXRActivation”category
includes28genesthatarehighlyexpressedintheFLandinvolvedinlipidsynthesis[thyroid
hormoneresponsivespot14alpha(THRSPA),stearoyl-CoAdesaturase(SCD1),fattyacid
synthase(FASN),sterolregulatoryelementbindingfactor2(SREBP2),lipoproteinlipase(LPL)
andglycerol-3-phosphatedehydrogenase1(GDP1)].Likewise,the“MechanismofGeneRegu-
lationbyPPARs”categoryincludes18HEgenes.
ManyHEgenesfoundinabdominalfatoftheFLandLLchickensarekeytranscriptional
regulatorsoflipogenesisandadipogenesis(Fig2).Forexample,severaltranscriptionfactors
[SREBF2,THRSP,nuclearreceptorsubfamily1,groupH,member3(NR1H3)orliver-acti-
vatedreceptoralpha(LXRA),andperoxisomeproliferator-activatedreceptorgamma
(PPARG)]interactwitheachotherandultimatelyeffectthetranscriptionofseveraldown-
streamtargetgenes(Fig2A).SomeHEtargetsofSREBF2includefattyaciddesaturase2
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Table2. IngenuityPathwayAnalysisofhighest-expressed(HE)genesinabdominalfatofFLandLL.
TopCanonicalPathways p-value Overlap Ratio
EIF2Signaling 1.60E-27 27.00% 50/185
IntegrinSignaling 1.00E-18 20.90% 42/201
EpithelialAdherensJunctionSignaling 8.09E-18 24.00% 35/146
ILKSignaling 3.91E-15 19.40% 36/186
Caveolar-mediatedEndocytosisSignaling 3.58E-13 29.60% 21/171
DiseasesandDisorders p-value #Genes
Cancer 1.21E-08–1.20E-28 747
OrganismalInjuryandAbnormalities 1.21E-08–1.20E-28 754
InfectiousDisease 1.08E-08–1.20E-27 196
DevelopmentalDisorder 4.85E-09–1.86E-13 160
HereditaryDisorder 9.81E-09–373.E-26 107
MolecularandCellularFunctions p-value #Genes
CellularGrowthandProliferation 1.22E-08–2.9.E-51 420
CellularMovement 1.21E-08–4.98E-48 302
CellDeathandSurvival 1.28E-08–5.35E-37 372
CellularDevelopment 1.15E-05–1.67E-31 385
CellularAssemblyandOrganization 5.40E-09–2.95E-30 271
PhysiologicalSystemDevelopmentandFunction p-value #Genes
CardiovascularSystem 1.25E-08–7.38E-36 208
OrganismalDevelopment 7.47E-09–9.24E-34 342
OrganismalSurvival 3.50E-10–5.38E-32 282
TissueDevelopment 8.09E-09–5.35E-27 313
ImmuneCellTrafficking 6.36E-09–5.58E-20 120
TopGeneInteractionNetworks Score
Cancer,OrganismalInjuryandAbnormalities,RespiratoryDisease 45
MetabolicDisease,NeurologicalDisease,PsychologicalDisorders 42
CellMorphology,RNAPost-TranscriptionalModification,ConnectiveTissue 42
NucleicAcidMetabolism,SmallMoleculeBiochemistry 42
EmbryonicDevelopment,OrganismalDevelopment,TissueDevelopment 40
TopToxicologyList p-value Overlap Ratio
CardiacHypertrophy 1.11E-11 12.20% 48/395
HepaticFibrosis 3.61E-10 12.20% 21/99
RenalNecrosis/CellDeath 9.46E-10 10.30% 1/494
PPAR/RXRActivation 1.38E-09 15.30% 28/183
MechanismofGeneRegulationbyPPARs 4.22E-08 18.90% 18/95
Atotalofthe900highest-expressed(HE)genesfromRNA-SeqanalysisweresubmittedtoIPA,which
provided828“AnalysisReady”HEgenesforfunctionalannotationandmappingtocanonicalpathwaysand
geneinteractionnetworks.P-valuesweredeterminedbyIPAsoftwareusingFisher’sExactTestas
describedbyIngenuity.Thepercentoverlapandratiowerecalculatedfromthenumberofobservedgenes
comparedtothenumberofknowngenesforthatcategoryintheIngenuityKnowledgeBase.
doi:10.1371/journal.pone.0139549.t002
(FADS2),acetyl-CoAcarboxylasealpha(ACACA),SCD,FASN,ATPcitratelyase(ACLY),peri-
lipin2(PLIN2),isocitratedehydrogenase1(IDH1)andTHRSP.Thetranscriptionfactor
THRSP,whichitselftargetsACACA,FASNandSCD,isalsoregulatedbyPPARG.Further,
PPARGhasnumerousHEtargetgenesthatregulatelipidmetabolism[acyl-CoenzymeAoxi-
dase1,palmitoyl(ACOX1),fattyacidbindingprotein3–5(FABP3,FABP4andFABP5),
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