population structure of the brachypodium species complex ...€¦ · 11/1/2018 · 71 these traits...

1

PopulationstructureoftheBrachypodiumspeciescomplexandgenome1

wideassociationofagronomictraitsinresponsetoclimate.2

3

Authors:4

PipWilson1*,JaredStreich1*,KevinMurray1*,SteveEichten1,RiyanCheng1,Niccy5

Aitkin1,2,KurtSpokas3,NormanWarthmann1,AccessionContributors4,Justin6

Borevitz17

*co-firstauthors8

Affiliations:1TheCentreforPlantEnergyBiology,ResearchSchoolofBiology,9

AustralianNationalUniversity,Canberra,Australia;2Ecogenomicsand10

BioinformaticsLab,ResearchSchoolofBiology,AustralianNationalUniversity,11

Canberra,Australia;3SoilandWaterManagement,AgriculturalResearchService,12

USDA,Minnesota,USA;4ListofAccessionContributorsatendofmanuscript13

14

Abstract15

Thedevelopmentofmodelsystemsrequiresadetailedassessmentofstanding16

geneticvariationacrossnaturalpopulations.TheBrachypodiumspeciescomplex17

hasbeenpromotedasaplantmodelforgrassgenomicswithtranslationalto18

smallgrainandbiomasscrops.Tocapturethegeneticdiversitywithinthis19

speciescomplex,thousandsofBrachypodiumaccessionsfromaroundtheglobe20

werecollectedandsequencedusinggenotypingbysequencing(GBS).Overall,21

1,897sampleswereclassifiedintotwodiploidorallopolyploidspeciesandthen22

furthergroupedintodistinctinbredgenotypes.AcoresetofdiverseB.23

distachyondiploidlineswereselectedforwholegenomesequencingandhigh24

resolutionphenotyping.Genome-wideassociationstudiesacrosssimulated25

seasonalenvironmentswasusedtoidentifycandidategenesandpathwaystied26

tokeylifehistoryandagronomictraitsundercurrentandfutureclimatic27

conditions.Atotalof8,22and47QTLswereidentifiedforfloweringtime,early28

vigourandenergytraits,respectively.Overall,theresultshighlightthegenomic29

structureoftheBrachypodiumspeciescomplexandallowpowerfulcomplextrait30

dissectionwithinthisnewgrassmodelspecies.31

32

33

.CC-BY 4.0 International licenseavailable under awas not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made

The copyright holder for this preprint (whichthis version posted January 11, 2018. ; https://doi.org/10.1101/246074doi: bioRxiv preprint

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Introduction34

Climatechangeisimpactingtheproductionoffoodworldwide(1)while35

increasingglobaldemandwillsoonoutstriptherateofimprovementincrop36

yieldbytraditionalbreedingmethods(2).Toaddressfoodandclimatesecurity,37

thereisaneedforagriculturalinnovationacrossarangeofscientificdisciplines,38

fromgenomicstophenomicsinnewspeciesacrossthelandscape(3).Keyto39

breedingformorevariablefutureclimates,aswellasforbroadadaptability,is40

understandingtheplasticityofthegeneticarchitectureofagronomictraits41

acrossenvironments.Theuseofcontrolledgrowthcabinets,thatcanmimic42

regionaldiurnalandseasonalclimatetypes(e.g.4),allowsustoexaminethe43

geneticarchitectureunderlyingcomplexadaptivetraitsacrossfieldlike44

environments.45

46

Twocomplextraitsthathavealargeimpactonyieldareearemergenceand47

earlyvigour.Thetimingofearemergenceiscruciallyimportanttoyieldinmany48

graingrowingregions,includingAustraliawhereearlyfloweringmayleadto49

cold-inducedsterilitywhilelatefloweringmayresultinheatstressorlackof50

waterlimitinggrainfilling.Earlyvigour,definedasanincreaseintheabove51

groundbiomasspriortostemelongation,isabeneficialtraitinmany52

environmenttypes,especiallywhencombinedwithincreasedtranspiration53

efficiency(5).Sincevapourpressureislowinwinter,increasedbiomassduring54

earlygrowthimprovesplantwateruseefficiency.Earlyvigouralsoincreases55

competitionagainstweeds,reducessoilevaporationandmayimproveyieldsby56

increasingtotalseasonalbiomass(6).Energyuseefficiencyisarelatively57

understudiedcomponentofplantgrowththatrepresentstheefficienttransferof58

energy,acquiredthroughphotosynthesis,tothegrain,andmaysignificantly59

affectyield.Earlystudiesindicatethatenergyefficiency,vialowerrespiration60

rates,arecorrelatedwithanincreaseinbiomassinmonocotspecies(7,8).61

Identificationofthegeneticarchitectureofenergyuseefficiency,timingof62

headingandearlyvigourtraits,aswellasthegeneticsensitivitytofuture63

temperatureprofiles,couldacceleratebreedingincropspeciesviaselectionfor64

improvedpredictedyieldsinthefuture.65

66



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GenomeWideAssociationStudies(GWAS)combinedensegeneticmarkers67

identifiedvianextgenerationsequencingandhighthroughputphenotypingto68

identifythecausativeallelesandtopredictcomplexquantitativetraits(9).The69

improvementofcropyieldinvolvesmanycomplextraitsandtheexpressionof70

thesetraitscanbehighlydependentonthegrowthenvironment.GWASisan71

excellentmethodformappingandpredictingyield-relatedtraitsandtheir72

interactionwiththeenvironment.GWAShasbeenundertakeninanumberof73

cropspecies;fordozensofagronomictraitsindiploidspeciessuchasrice,barley74

andcorn(forreviewsee10),andhasevenbeusedreasonablysuccessfullyin75

wheatdespitetheaddedcomplexityofahexaploidgenome(e.g.11).76

77

BrachypodiumdistachyonisamodelspeciesfortemperateC3grasscropssuchas78

wheat,barley,ryeandoatsasitisalsolocatedinthePooideaefamilyandhasa79

numberofadvantageouscharacteristicsasamodelspecies(12–15).B.80

distachyonalsohasanumberofadvantagesovertherelateddomesticPooideae81

foraGWASapproachasitisawildspecieswithawideclimaticdistribution,82

resultingindiversephenotypes,aswellaswidegenomicdiversity,fortraits83

involvedinlifestrategyandabioticstresstolerance.B.distachyonhasafully84

sequencedsmallgenomeof270Mb(16)comparedtothe16Gbofwheat(17)or85

5.1Gbofbarley(18).Italsocontainsalowpercentageofrepetitivenon-coding86

DNAat21.4%ofnucleotidescomparedtomorethan80%inwheat(19)and87

84%inbarley(18).ThismeansthatsequencereadsfromB.distachyonaremuch88

easiertoidentifyandaligncomparedtowheat,withalargerproportionofthe89

sequencingprovidingusefulreads.Finally,andperhapsmostimportantly,isthe90

shortstatureofB.distachyonwhichallowslargenumbersofplantstobetaken91

throughfulllifecyclesincontrolledgrowthconditions.92

93

Brachypodiumiswidespreadthroughouttemperateregions,includingitsnative94

MediterraneanrangeandintroducedrangeinAustralia,SouthAfricaand95

westernUSA(20,21).Alargenumberofaccessionshavebeencollected96

throughouttheworldbytheBrachypodiumcommunitybuttheuseofthese97

collectionsingenomicassociationstudieshasbeendelayedbythecrypticnature98

oftheBrachypodiumspeciescomplex.Thethreespeciesinthiscomplexare99



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difficulttodistinguishinthefieldandincludethediploidB.distachyon;the100

diploidB.stacei;andtheallotetraploidB.hybridumwhichcontainsoneB.101

distachyon-likegenomeandoneB.stacei-likegenome(22–24).Toaddtothe102

complexitythereisevidenceofdistinctsubgroupsorsubspeciesofB.distachyon,103

(21,25).WhilethegenomeoftheBd21genotypeofB.distachyonwaspublished104

in2010,thegenomeofB.staceiandotherSNPcorrectedgenomeswerereleased105

onlinein2016(DOE-JGI,http://phytozome.jgi.doe.gov/).Recently,aB.106

distachyonpangenomewaspublishedidentifyinggeographicdiversityandmany107

newgenesnotidentifiedintheinitialreference(26).Priortoourstudy,species108

identificationhascommonlybeenundertakenbymorphoanatomical109

classification,asmallnumberofmarkersorcytology(e.g.17,23).Thereisaneed110

forarapididentificationofspecies,subspeciesandgenotypelineages,withinthe111

BrachypodiumspeciescomplextoaidtheselectionofHapMapsetsandtoenable112

landscapegenomicstudiesofmigrationandadaptation.113

114

Inthisstudy,weaimedto1)characterisethespecies,genotypeandpopulation115

structureofaBrachypodiumglobaldiversitysettoselectacorehaplotype116

mappingsetforGWASinB.distachyonand2)identifythegeneticarchitecture117

andplasticityoftheagriculturallyrelevanttraitsofheadingdate,earlyvigour118

andenergyuseefficiencyinresponsetoclimate.119

120

Results121

CrypticBrachypodiumSpecies,diversegenotypesandpopulationstructure122

identifiedusingGenotypingbySequencing123

Toestablishadiversesetofgermplasm,thousandsofBrachypodiumaccessions124

werecollectedontripstosouth-westEurope,south-easternAustralia,western125

USAandthroughcollaborationswiththeinternationalBrachypodiumcommunity126

(https://github.com/borevitzlab/brachy-127

genotyping/blob/master/metadata/brachy-metadata.csv).Outofthese,1968128

accessionsweregrowntoproducesingleseeddescentlinesinthegreenhouses129

atANUforsubsequentgenomicanalysis.Areducedrepresentationapproach,130

PstIdigest,genotyping-by-sequencing(GBS;26–28)wasusedtogenetically-131

profiletheaccessions.132



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133

Althoughoncedescribedasasinglespecies,B.distachyonhasmorerecently134

beenshowntoexistasaspeciescomplexconsistingofa5ChromosomeB.135

distachyon,10ChromosomeB.stacei,anda15chromosomeallopolyploidB.136

hybridum(22).Tocategoriseeachaccessionintospecieswithinthe137

Brachypodiumcomplex,GBStagsweremappedtoamergedreferencegenome138

consistingofB.distachyon(Bd21-3)andB.stacei(ABR114)[v1.1DOE-JG,139

https://phytozome.jgi.doe.gov].Mostaccessionswerereadilydistinguishedas140

havingreadsthatalignedtoeitherorbothreferencegenomes(seemethods;141

Supp.S01).Themajorityofaccessions,56%(1100/1968)wereidentifiedasB.142

hybridum.Incontrast,only3%(60/1968)wereclassifiedasB.stacei,while35%143

(698/1968)wereB.distachyon.Theremaining6%(110/1968)couldnotbe144

definitivelyassigned.Mappingoftheaccessions’geographiclocationsshowed145

thatB.hybridumhasexpandedacrossthegloberepresentingessentiallyallthe146

collectionsoutsidethenativerange(Fig1A).Conversely,B.distachyonislargely147

limitedtothenativeMediterraneanandWesternAsianregions,withB.staceiin148

thesameareabutwaslesscommon.149

150 Figure1.DistributionandGenomicDiversityoftheB.distachyoncomplex151 A)Geographicdistributionof1573Brachypodiumcomplexaccessionsclassifiedbyspecies:pink152 =B.distacyhon,blue=B.staceiandpurple=B.hybridumB)StructureplotoftheB.distachyon153 species,K=3;andC)GeographicstructureofB.distachyonacrossIberianPeninsulaandTurkish154 region.ProportionsofpiesrepresentthenumberofeachB.distachyonsubgroup(fromB)ateach155 site.ThearrowfromCtoAshowstheAustraliaB.distachyon(WLE2-2)andthenearidentical156 accessionfromTurkey(BdTR9f).157

A. B. 20 40 60 80 100 120

C.

Cas2Bd1-1

WLE2-2Bdis23-2Bdis23-1Bdis23-5

Bdis22-10Bdis22-5Bdis22-1Bdis22-6

ABR5Sig2

Mur3Mig3

Bar1020Bdis31-1Bdis32-2

Cas1Pal1

Bdis05-1Bdis05-7

ABR4Bdis03-6Pal2032

Yas3Gal1Rei7

Bd30-1ABR2

USDA4006Bd21

Bd21-3Bd3-1Koz-3

BdTR13bBdTR3aKoz-2

Bd18-1Gaz-2

BdTR1cGaz-3Adi-15

BdTR11aAdi-16Adi-9

Adi-18BdTR10c

Adi-6BdTR9aAdi-1

BdTR12cBdTR5gAdi-8Kah-6Kah-1Kah-5

Bdis25-4Bdis25-8Bdis25-1Bdis25-5

Bdis25-10Bdis28-7Bdis31-2



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158

DuetothehighlyselfingnatureofallBrachypodiumspecies,wenextsoughtto159

categoriseaccessionsintouniquewholegenomegenotypesrepresentingasingle160

inbredlineage.WeusedtheSNPRelatepackage(31)tocluster72genotypes161

from490highqualityB.distachyonaccessionsgenotypedat81,400SNPs(see162

methods;https://github.com/borevitzlab/brachy-genotyping-notes;Supp.S02).163

Recombinantinbredlines,includedaspositivecontrols,wereoftencalledunique164

genotypesasexpected,butwereexcludedfromsubsequentanalysisofnatural165

populationstructure.166

167

Wholegenomevariation168

Wholegenomesequencingwasperformedonasetof107B.distachyon169

accessionstodeterminehighdensityvariationatmultiplelevels,patternsof170

linkagedisequilibrium,andtoenablegenomewideassociationstudies(GWAS).171

Thesamplesrangedacrossthesubspecies,populationandfamilylevels172

revealing2.65Mpolymorphicsites(1%diversity,θ,on260Mb).Mostsamples173

weretheAsubspecieswith3clearoutliers(Bd1-1,Cas2,WLE2-2)belongingto174

theBsubspecies.Thesubspeciesshowedfixeddivergenceat6.5%ofsites(169k175

with<100expectedbychanceamong3accessionoutliers).WithintheA176

subspeciestherewere2clearpopulationswith1.5%fixeddivergenceamong177

groups.Accessionswithinthesamegenotypiclineagedivergedatbetween0.1-178

to0.4%ofSNPshighlightingtherelativeamountofrarevariationwithina179

uniquegenotype.Abalancedsetofrepresentativeaccessionsacrossthe180

genotypelineageswithinjusttheAsubspecieswasselectedforfurthergenomic181

andphenomicanalysis(Supp.S03).182

183

PreviousgeneticanalysisonsmallerdatasetshadshownB.distachyontohave184

substantiallevelsofpopulationstructure(20,25,26,32,33).Wesoughttorefine185

theancestralpopulationstructureofB.distachyonbyreducing107accessionsto186

63highlydiversegenotypesusing2,648,921SNPs.Toreducedatacomplexity187

SNPsweresubsampledtoevery100thsitetocreateafinalSNPmatrixof26,490188

variantsthatwerefedintoSTRUCTUREv.2.3.4(34;Supp.S04).STRUCTURE189

analysisidentifiedthreemainsubgroupsamongB.distachyongenotypesand190



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sevenadmixedlines(Fig1B).Theyellowlineagewasthemostdivergedand191

representssubspeciesB,withthebrownandredstructuregroupsrepresenting192

thepredominantlyEastandWestpopulationsoftheAsubspecies.Tovisualise193

thegeographicdistribution,theancestralgroupcompositionwassummed194

acrossaccessionsforeachgeographicsite(Fig1C).ThesingleB.distachyon195

accessionfromAustraliaWLE2-2wasnearlyidenticaltoBdTR9f(GBSdata,196

Supp.S02)fromsouthernwesternTurkey,whereitmayhaveoriginatedfrom.It197

isshowninitsancestrallocation(Fig1Carrow).198

199

Linkagedisequilibrium(LD)wascalculatedforconsecutivewindowsof2000200

SNPsacrossthegenome.TherewaslargevariationinLDacrossthegenome201

(Supp.S05)withthemedianLD113kb(50–235kbinterquartilerange)andthe202

maximumwasgreaterthan2.4Mb.203

204

DeterminingthebesttraitsandclimaticconditionsforGWASinB.205

distachyon206

ForourGWASstudywewantedtoidentifyhighthroughoutnon-destructive207

phenotypicmeasureswithhighheritability.Wealsowantedtodeterminethe208

bestenvironmentalconditionstocharacteriseourtraitofinterest.Hencetwo209

preliminaryexperimentswereundertaken,oneforfloweringtimeandonefor210

earlyvigour.211

212

FloweringtimewaschosenasanidealtraitforGWASasithashighheritabilityin213

manyspeciesincludingArabidopsis(35)andbarley(36).Inpreviousstudiesof214

B.distachyonithasbeenfoundthatthedependenceoffloweringtimeon215

vernalisationandphotoperiodvariesbetweenaccessions(37–40).Thisstudy216

aimedtoidentifyQTLforearlinessperseinflowering,i.e.thoseresponsivetothe217

accumulationofthermaltime.Henceapreliminaryexperimentwasundertaken218

todetermineifourconditionscouldmeetthevernalisationrequirementsofallB.219

distachyonaccessionsandtodeterminewhichaccessionshadstrong220

vernalisationrequirementsinourconditions.Todothis266diverseAandB-221

subspeciesaccessions,with5accessionsreplicated5-6timesweregrownin222

bothasimulatedwintersowing,starting01June,andaspringsowing,starting223



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01September,inWaggaWagga,NSW,Australia(Supp.S06).Earemergencewas224

monitored,asasurrogatemeasureforfloweringtimeasfloweringoccurslargely225

withintheearinB.distachyonsoishardtoaccuratelyrecord(Supp.S07).Outof226

the266accessionstherewere17accessionsthatdidnotflowerintheSpring227

condition,indicatingastrongvernalisationrequirement(Supp.S08A).Alllines228

floweredintheWintercondition,indicatingthatnighttemperaturesof4oCwere229

sufficienttomeetvernalisationrequirement.Asexpected,daystoearemergence230

showedastrongheritabilityinthewintercondition,ascalculatedfromthe231

replicatedlines(h2=0.96).Thethermaltimetofloweringwascalculatedto232

determinethedependenceoffloweringontheaccumulationofthermaltime.The233

fastcyclingaccessions,thatwerenotvernalisationrequiring,stillrequireda234

largerthermaltimeaccumulationthanthevernalisationrequiringaccessions235

(Supp.S08B).Thisindicatesthattheseeitherhavesomelow-levelrequirement236

forvernalisationthatisnotbeingfullymetintheSpringconditionorthatthe237

photoperiodisalsoafactorinthisrelationship.Asthisstudyaimedtoidentify238

QTLforearlinessperseinflowering,i.e.thoseresponsivetotheaccumulationof239

thermaltime,weattemptedtoexcludevernalisationandphotoperiodeffectsby240

focusingonthewinterconditionfortheGWASexperiment.241

242

Intemperategrasscropssuchaswheatandbarley,earlyvigourcanresultinan243

increasedyieldinshortseasonsorinseasonswherethereishighrainfall244

(reviewedin6).Oftenthedimensionsofseedlingleavesaremeasuredasanon-245

destructivesurrogatemeasureforearlyvigour(41,42).Toconfirmthatthiswas246

alsoanappropriatesurrogatemeasureforearlyvigourinB.distachyon,ahighly247

replicated(n=10)validationexperimentwasperformedonsixdiverseB.248

distachyonlines(Supp.S09A)inasimulatedWaggaWagga,seasonalclimate249

startingon01September(Spring).Aftersevenweeks,whenplantshadbetween250

fourandfivemainstemleaves,thedimensionsofleaf#3,seedlingheight,total251

leafarea,andabovegrounddryweightweremeasuredandphenotypic252

correlationswerecalculated(Supp.S09B).Narrowsenseheritabilitywasalso253

calculatedtodeterminewhichearlyvigourtraitwouldprovidethemostpower254

formappingQTLswithGWAS(Supp.S09C).Leaf#3widthandlengthcorrelated255

wellwithabovegroundbiomass(r2=0.46,p<0.01andr2=0.48,p<0.01,256



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respectively)andhadquitehighheritabilitiesofh2=0.60andh2=0.64,257

respectively,ascomparedtoabovegrounddrymass,h2=0.51.Interestingly,258

seedlingheightalsohadastrongcorrelationwithabovegroundbiomass259

(r2=0.74,p<0.01)withaheritabilityofh2=0.74.However,thistraitwasalsomore260

highlycorrelatedwithdevelopment,asindicatedbythenumberofleaves261

(r2=0.21,p<0.01),thanthedimensionsofleaf#3.Togetthemostdirectmeasure262

ofearlyvigourthedimensionsofleaf#3werechosenasthefocusfortheGWAS.263

264

SelectionofglobalHapMapset265

High-levelpopulationstructureconfoundsGWASwhentherearefew266

segregatingSNPsincommonbetweenancestralgroupsrelativetovariation267

withineachsubgroups(43).HerewefocusedonsubspeciesAwhichcontainsa268

majorityofuniquegenotypes,resultinginaHapMapsetof74genotypes.Within269

theAsubspeciesthereisstillclearpopulationstructure,butfurthersubset270

selectionwouldlimitboththesamplesizeandthephenotypicandgenotypic271

diversity,reducingtherateoftruepositiveresults.Thisresidualrelatedness272

betweenlineswasaccountedforbyincludingakinshipmatrixintheGWAS273

model.274

275

Earlyvigourandearemergenceshowsgenotypicvariationinresponseto276

differentsimulatedenvironments277

Todeterminethegeneticarchitectureforearemergencedate,earlyvigour,anda278

rangeofotheragronomictraits,therefinedandbalancedHapMapsetof74B.279

distachyonaccessions(Supp.S10),withfourbiologicalreplicates,weregrownin280

twosimulatedconditionsinspeciallymodifiedgrowthchambers(4).To281

determinetheeffectofanincreaseintemperatureinlinewithclimatechange282

predictionsonthetraitsofinterest,theconditionsmodelledapresent(2015,Fig283

2A)andafuture(2050,Fig2B)temperatureprofileatWaggaWagga,NSW,284

Australia.Theappropriateincreaseinaveragemaximumandminimum285

temperatureforeachmonthwasdeterminedusinganaverageof12global286

climatechangemodelsdeterminedtobehighconfidenceforsoutheastAustralia287

usingtheClimateFuturesTool(Fig2C,42;Supp.S11).288

289



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290 Figure2.Climatechamberswereusedtocomparetheresponseofagronomictraitsto291 smallchangeintheclimateforaWintersowingintheWaggaWaggaregion,south-eastern292 Australia.TheGWASHapMapsetweregrowninA)2015temperatureclimateandB)a2050293 temperatureclimate.Photosshowrepresentativeplantsafter16weeksofgrowth.Climate294 chamberswereprogrammedtohaveC)diurnalandseasonalchangesintemperatureresultingin295 differentratesofaccumulationofthermaltimeD)inthe2015and2050climates.Timingofear296 emergencewascomparedbetweenchambersforbothE)daystoearemergenceandF)the297 accumulationofthermaltimetoearemergence.298



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Asexpected,theaccessionsdevelopedquickerandgrewlargerinthe2050299

temperatureprofile(Fig2A,B)asisconsistentwithaquickeraccumulationof300

thermaltime(Fig2D).Earlyvigourparametersandenergyuseefficiencytraits301

weremeasuredwhenthemajorityofplantswereatafour-leafstage.Growth302

stages,tillernumberandearemergencedateweremonitoredtwiceaweek303

(Supp.S12,S13,S14).Theexperimentwasceasedafter200daysofgrowth,at304

whichtimetherewere5and7linesthatdidnotflowerinthe2015condition305

and2050conditions,respectively.Theremaininglinesreachedearemergenceat306

asimilarnumberofdaysinboththepresentandfutureconditions(Fig2E).307

However,whenconvertedtothermaltimethoselinesinthe2015temperature308

conditionrequiredlessthermaltimethanthoseinthe2050temperature309

condition(Fig2D,F).Thisindicatesthatthereisgenerallymoredependenceon310

photoperiodinthispopulationthanonthermaltimetotriggerthetransitionto311

flowering.Therewasvariationbetweengenotypesintheplasticityintheir312

responsetothetwoconditions(Fig2E&F),indicatingthatitwouldbe313

worthwhilemappingthegenotypebyenvironmentinteraction.314

315

Determiningthegeneticarchitectureofearlygrowth,earemergenceand316

energyuseefficiencytraitsinresponsetoenvironment317

GWASwasperformedonrawandderivedtraitsasdescribedinthemethods(Fig318

3;Supp.S15&S16).Forearemergence,eightsignificantQTLswereidentified.319

EarEmerg_QTL3.1explains62%ofthephenotypicvariationinthermaltimeto320

earemergenceinthe2015temperatureconditionwhiletwoQTLs,321

EarEmerg_QTL3.1andEarEmerg_QTL5.3,explain56%and10%ofthe322

phenotypicvarianceinthermaltimetoearemergenceinthe2050temperature323

condition,respectively.NoQTLswerefoundtobesignificantinbothconditions324

butEarEmerg_QTL5.3wassignificantinthe2050temperatureconditionand325

wasjustunderthesignificantthresholdinthe2015temperaturecondition(Fig326

4A,Supp.S17).Withinthe100kbregionofthisSNPthereare15genesofwhich327

severalcouldberelevanttotheregulationoffloweringincludingaYABBY328

transcriptionfactor(Bradi5g16910),anoapicalmeristem(NAM)protein329

(Bradi5g16917)andanexpressedgenecontainingaRNArecognitionmotif330

(Bradi5g16930).Interestingly,thereweretwoQTLsthatweresignificantfor331



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thermaltimetoearemergence,EarEmerg_QTL3.1andEarEmerg_QTL4.2,but332

notfordaystoearemergence.ThereweresixQTLsidentifiedforthegenotype333

byenvironmentinteraction,explaininginpart,thevariationamonglinesin334

responsetofutureclimate.335

336 Figure3.QTLwereidentifiedforarangeofagronomictraitsphenotypedinthe2015337 temperatureand2050temperatureclimatesandtheGxEinteraction.338 Atotalof73significantQTLwereidentifiedbyGWAS.TherewaslittleoverlapbetweenQTLfor339 differenttraitsbut2robustQTLwereidentifiedinbothenvironmentswhile16QTLwere340 identifiedforagenotypebyenvironment(GxE)interaction.GxE-genotypebyenvironment341 interaction;EarEmerg-earemergence;TT-thermaltime;PTU-photothermalunits;L3Width-342 leaf3width;L3Length-leaf3length;GR-growthrate;GR-growthrate;EV-earlyvigour;phyll-343 phyllacroninterval;AvgQY-averagequantumyield;DM-drymass;EUE-energyuseefficiency344 345

Forearlyvigour,22significantQTLswereidentifiedfor5traitsacrossthetwo346

climateconditions(Supp.S15).TwoQTLswereidentifiedinbothconditions,347

EarlyVigour_QTL1.1andEarlyVigour_QTL3.1,andbothofthesewereforleaf#3348

length.The100kbregionsurroundingtheseQTLscontained19and13genes,349

respectively(Fig4BandC).TherewasahighlysignificantQTLonchromosome3350

GxE_EUE42050_EUE42015_EUE4GxE_EUE32050_EUE32015_EUE3GxE_EUE22050_EUE22015_EUE2GxE_EUE12050_EUE12015_EUE1

GxE_Respiration_DM2050_Respiration_DM2015_Respiration_DMGxE_Respiration_area2050_Respiration_area2015_Respiration_area

GxE_AvgQY2050_AvgQY2015_AvgQY

GxE_Phyll_PTU2050_Phyll_PTU2015_Phyll_PTUGxE_Phyll_TT2050_Phyll_TT2015_Phyll_TTGxE_Huan_GR22050_Huan_GR22015_Huan_GR2GxE_Huan_GR12050_Huan_GR12015_Huan_GR12050_EVHeight2015_EVHeightGxE_L3Length2050_L3Length2015_L3LengthGxE_L3Width2050_L3Width2015_L3Width

GxE_EarEmerg_PTU2050_EarEmerg_PTU2015_EarEmerg_PTUGxE_EarEmerg_TT2050_EarEmerg_TT2015_EarEmerg_TTGxE_EarEmerg_days2050_EarEmerg_days2015_EarEmerg_days

QTL

1.1

QTL

1.1

QTL

1.2

QTL

1.3

QTL

1.4

QTL

1.2

QTL

1.5

QTL

1.6

QTL

1.7

QTL

1.8

QTL

1.9

QTL

1.10

QTL

1.11

QTL

1.3

QTL

1.12

QTL

1.13

QTL

1.14

QTL

2.1

QTL

2.1

QTL

2.2

QTL

2.3

QTL

2.4

QTL

2.1

QTL

2.2

QTL

2.5

QTL

2.3

QTL

2.4

QTL

2.6

QTL

3.1

QTL

3.1

QTL

3.2

QTL

3.3

QTL

3.4

QTL

3.1

QTL

3.5

QTL

3.2

QTL

3.6

QTL

3.3

QTL

3.7

QTL

3.8

QTL

3.9

QTL

3.10

QTL

3.12

QTL

3.11

QTL

3.13

QTL

3.14

QTL

3.15

QTL

3.16

QTL

3.6

QTL

3.17

QTL

3.8

QTL

3.7

QTL

3.9

QTL

4.1

QTL

4.1

QTL

4.1

QTL

4.2

QTL

4.3

QTL

4.4

QTL

4.5

QTL

4.6

QTL

4.3

QTL

4.7

QTL

4.3

QTL

4.4

QTL

5.1

QTL

5.2

QTL

5.1

QTL

5.1

QTL

5.2

QTL

5.3

QTL

5.3

QTL

5.2

6420

Maximum LOD Score Ear Emergence QTLEarly Vigour QTLEnergy Use Efficency QTL



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13

forgrowthrate1,EarlyVigour_QTL3.3,ameasureoftherateofdevelopmentof351

theseedlingatthetwoleafstage,butonlyinthe2015temperaturecondition.352

The100kbregionsurroundingthisQTLcontained13genes(Supp.S18).Atotal353

of6QTLswereidentifiedforthegenotypexenvironmentinteractionacrossthe354

twoconditionsforearlyvigourtraits.355

356 Figure4.PutativecandidategeneswereidentifiedunderQTLofkeyinterest357 A)TheearemergenceQTL,EarEmerg_QTL5.3,wassignificantfordaystoearemergenceinthe358 2050temperatureconditionandonlyjustunderthesignificancethresholdforthe2015359 condition.LikelycandidategenesincludeaYABBYtranscriptionfactorBradi5g16910.B)The360 earlyvigourQTL,EarlyVigour_QTL1.1forleaf#3lengthwasfoundtobesignificantinboth361 conditions.Thisregioncontainsanethylenesensitivetranscriptionfactor,Bradi1g00666.C)The362 earlyvigourQTL,EarlyVigour_QTL3.1wasalsoidentifiedforleaf#3lengthinboth363 environments.D)AstrongQTLwasidentifiedforphotosyntheticefficiency,Energy_QTL3.2,364 whichwassignificantonlyinthe2015temperaturecondition.Likelycandidategenesincludea365 heatshockprotein,Bradi3g01477,andaLowPhotosystemsIIAccumulation3(LPA3)protein,366 Bradi3g01550.367 368

Fortheenergyuseefficiencytraits,atotalof47QTLswereidentifiedacrossthe369

twoconditionsforthethreemeasuredtraitsandfourderivedtraits(Supp.S15).370

OftheseQTL,nonewerefoundinbothenvironments.However,astrongQTL,371

Energy_QTL3.3,wasidentifiedforaveragequantumyield,ameasureof372

photosyntheticefficiency,inthe2015temperatureenvironment.The100kb373

0

1

2

3

4

5

20250 20300 20350 20400 20450

Location on Chromosome 5 (Kb)

LOD

2015 Temperature2050 Temperature

0

1

2

3

4

5

480 510 540


LOD

0

1

2

3

4

5

3150 3175 3200 3225 3250


LOD

0

1

2

3

4

900 925 950 975


LOD

A

C D

B

Bradi5g16900 Bradi5g16910 Bradi5g16930 Bradi5g16950 Bradi5g16970 Bradi5g16990 Bradi5g17010

Bradi5g16960 Bradi5g16980 Bradi5g17005

EarEmerg_QTL5.3 - Days to Ear Emergence

EarlyVigour_QTL3.1 - Leaf #3 Length

EarlyVigour_QTL1.1 - Leaf #3 Length

Energy_QTL3.2 - Average Quantum Yield

Bradi1g00580 Bradi1g00600 Bradi1g00620 Bradi1g00666 Bradi1g00690 Bradi1g00710 Bradi1g00740

Bradi1g00630 Bradi1g00730

Bradi3g04617 Bradi3g04630 Bradi3g04650 Bradi3g04655 Bradi3g04681 Bradi3g04690

Bradi3g04660

Bradi3g01427 Bradi3g01450 Bradi3g01477 Bradi3g01483 Bradi3g01515 Bradi3g01550

Bradi3g01419 Bradi3g01460 Bradi3g01510


2015 Temperature





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regionaroundthisQTLcontained24genesincludingalowPSIIaccumulation3374

chloroplasticprotein(Bradi3g01550),aHeatShockProtein(Bradi3g01477)and375

severaltranscriptionfactors(Fig4D,Supp.S19).376

377

Discussion378

379

ThankstotheinternationalBrachypodiumcommunity,inadditiontoourown380

collections,herewewereabletoprovidethemostcomprehensivesurveyof381

Brachypodiumspeciescomplexdiversitytodate.With1897accessionsacross382

theglobethisisagreaterthan10-foldincreasefrompreviousstudies(25,32).383

384

Sincebeingdescribedasthreeseparatespeciesin2012(22),species385

identificationintheBrachypodiumspeciescomplexhasbeenachievedby386

morphology,PCRofaselectsetofmarkersorDNAbarcoding(e.g.41,42).Here387

wepresentauniquesystematicmethodofdeterminingthespeciesofan388

accessionusinglowcoveragegenotypingbysequencingandbioinformatics,389

providingahighthroughputandlowcostalternativeforspeciesidentification.390

WefoundthatthemajorityofouraccessionswereinfactB.hybridum(56%)391

includingthevastmajorityofaccessionsinAustraliaandNorthAmerica(Fig392

1A).Thewidedispersionofthisspeciesmaybeduetothebenefitofthemultiple393

genomesresultingfrompolyploidisation(47).TherewererelativelyfewB.stacei394

(3%),whichwerelimitedtotheMediterraneanregion(Fig1A).395

396

WithinB.distachyonitselfwefoundsignificantpopulationstructureincluding397

highlevelsubspeciessplits,with6.5%divergencebetweensubspecies,whichis398

greaterthanthatfoundbetweenindicaandjaponicariceat1.4%divergence399

(48).Whilemanypreviousstudieshavefocusedonindividualregions(32,33),400

thecollectionof490highqualityB.distachyonaccessionscombinedwith81,400401

highqualitySNPspresentedherehasallowedustofurtherdistinguish402

subgroupswiththeB.distachyonsubspecies,withandeasternandwestern403

Europeangroupineachsubspecies.Anumberofgeographicallydiversehighly404

relatedgenotypiclineageswerealsoidentifiedwhichshowedwithinlineage405

divergenceofbetween0.1-to0.4%ofSNPs.Thegeographicspreadofthese406



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lineageshighlightstheinbreedingnatureandhighdispersalabilityofB.407

distachyon.408

409

ThehierarchicallevelsofgeneticvariationwithintheBrachypodiumspecies410

complexcanbeattributedtoallopolyploidisationandsubspeciation,possibly411

duringthemostrecenticeage;east/westisolationbydistanceinEurope;and412

thehighlevelsofself-fertilisationinthespecies(21).Theselevelsofpopulation413

structurehavebeenseeninArabidopsis(9),andotherhighlyselfingplant414

speciessuchasbarley(49),butaremoreextremeinBrachypodium.Inrice,415

eithertheindicasub-species(50)orjaponicasub-species(51),wereseparately416

usedforGWAS.Similarly,todealwiththepopulationstructureinthisstudy,the417

HapMapsetwaslimitedtotheAsubspeciesofB.distachyonwithremaining418

relatednessincludedintheGWASanalysisusingmixedmodels(QTLrel;47).419

420

ThelackofrecombinantgeneticdiversitywithsubspeciesandpopulationsofB.421

distachyonalsolimitsthepowerofGWASanalysis.TheHapMapsetcontainsa422

largeamountofgenomicdiversity(>1%ofbasesarevariable)butthesample423

sizeislowandtheextentoflinkagedisequilibriumishigh,limitingmapping424

resolution.However,thepatternsaresimilartoricewhereGWASisvery425

effectiveassamplesizeincreases(10).TheconstructionofaNestedAssociation426

Mapping(NAM)populationforB.distachyonwouldbeadvantageoustobreak-up427

thepopulationandfamiliallineagesandtoincreasethefrequencyofminor428

alleles.Thishasbeenasuccessfulapproachinotherspeciessuchasmaizeand429

wheat(53,54).430

431

Infieldconditions,determiningtherelationshipbetweenvariousphysiological432

traitsandtheirimpactonyieldisdifficultduetoin-seasonenvironmental433

variabilityandthepresenceofarangeofabioticandbioticstresses.However,434

experimentsingrowthchambersoftenhavelittlerelevancetofieldconditions435

duetotheunrealisticandstaticnatureoftheconditions.Theuseofclimate436

chambersprovidesarealisticrangeoftemperatures,whichresultsinmore437

translatableresultstothefield(4,55),whileremovingthevariabilitycausedby438

weatherandstresses.Theuseofclimatechambersalsoallowstheimpactof439



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16

smallchangesinclimatetobeobservedandthedissectionofwhichcomponents440

oftheclimatehavethelargestinfluenceonatraitofinterest.Inthisstudy,we441

examinedtheeffectofanincreaseintemperatureinlinewithclimatechange442

modelpredictionsfor2050insoutheasternAustralia.Unexpectedly,therewas443

generallyashortdelayoffloweringtimeinthe2050temperatureconditionwith444

variationintheextentofdelayindifferentgenotypeswhiletherewaslittle445

dependenceoffloweringontheaccumulationofthermaltime.Thisindicatesthat446

theremaybesomevernalisationrequirementsinB.distachyonthatarenotbeing447

metinthe2050temperaturecondition.Thelackofvernalisationisalsoevident448

inthefactthatsevenlineshadnotfloweredbytheendofthe2050temperature449

conditionwhile5linesdidnotflowerinthe2015temperaturecondition.While450

thisGWASanalysisdidnotidentifyknownfloweringtimelocithatregulate451

vernalisation-inducedfloweringsuchasVRN1,VRN2andFT(37,56),theQTL452

mayrepresentmoresubtlevernalisationprocessesthatwouldbeimportantfor453

facultativevarieties.Perhapslargelytothedifferenceingrowthconditions,the454

QTLinthisstudydidnotoverlapwiththosefoundinapreviousGWASof455

floweringtime(25).Candidategenesidentifiedforfloweringtimehereincluded456

severaltranscriptionfactors,includingaYABBYtranscriptionfactorwhose457

closestorthologinrice,Os04g45330,ismosthighlyexpressedintheshootapical458

meristemanddevelopinginflorescence(RiceGeneExpressionAtlas)andwhole459

closestorthologinArabidopsis,At2g45190,isinvolvedinregulationofthefloral460

morphology(57)underEarEmerg_QTL5.3.ThisQTLwassignificantinthe2050461

temperatureconditionsandwasonlyjustbelowthesignificancethresholdinthe462

2015temperaturecondition(Fig4A).463

464

EarlyvigourisanimportanttraitinmanypartsofAustralia,andtheworld,465

wherethereiscompetitionfromweedsandashorterseason.Despitethehighest466

correlatingnon-destructivemeasureofearlyabovegroundbiomassbeing467

seedlingheight,themostrobustQTLsacrossenvironmentswereactually468

identifiedbyleaf#3length.TwoQTLsidentifiedforleaf#3lengthwere469

identifiedinbothenvironments,indicatestheycouldpotentiallybeusefulfor470

breedingforearlyvigourinmultipleenvironmenttypes.Oneofthese,471

EarlyVigour_QTL1.1islocatedinanareaofsyntenytootherareaswhereearly472



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17

vigourQTLshavebeenidentifiedattheendofchromosome3inrice(16,58,59)473

andChromosome4inwheat(60).WithinEarlyVigour_QTL1.1thereisa474

candidategene,Bradi1g00666,thatisdescribedasanethylene-responsive475

transcriptionfactor.ThemaincandidategeneintheQTLonChromosome3in476

ricewasalsoanethyleneresponsivegene(58).TheEarlyVigour_QTL3.1forleaf477

#3lengthwasalsofoundtobesignificantacrossbothenvironments.Therewere478

noobviouscandidategenesforthisQTLbutanumberofsignallingproteinsthat479

couldbeinvolvedinmolecularcontrolofleafsize(Fig4C,Supp.S18).480

481

Thebalanceofenergyproductionanduseinplantsishighlylinkedtothe482

conditionsthattheplantisgrownunder,howevergeneticvariationcontrolling483

theenergyefficiencyofplantscouldbeusedtoincreaseyieldpotentials.The484

quantumyieldisanindicatorofphotosyntheticefficiency,theproportionof485

energyharvestedthroughthelightharvestingcomplexesthatgoestowards486

producingphotosynthates(61).NoQTLwereidentifiedincommonacrossboth487

environments,buttherewere11QTLthatwereidentifiedfortheGxE488

interaction.Thismaybeduetothesensitivityoftheseenergyprocessestothe489

subtledifferenceinenvironmentsoraresultofbeingmeasuredondifferentdays490

toallowcomparisonofplantsatthesamedevelopmentalstage.AstrongQTL491

wasidentifiedforquantumyield,ameasureoftheefficiencyofphotosystemII,492

inthe2015climatebutinterestinglynotinthe2050climate.Candidategenes493

underthisQTLincludedagenewith66%homologytotheLowPSII494

Accumulation3(LPA3)geneinArabidopsis,whichhasbeenshowntobe495

importantinPhotosystemsIIassembly(62).Furtherstudiesintotheimportance496

ofthisQTLindifferentconditions,aswellastheotherphotosynthesisand497

respirationQTL,wouldbeworthwhile.498

499

Inconclusion,theBrachypodiumspeciescomplexisheavilystructuredatthe500

ploidy,subspecies,population,andfamilylevels.Thislimitstheabilitytoidentify501

thegeneticbasisofadaptationasrelativelyfewrecombinantgenotypeswere502

obtained.Despitetheselimitations,thisstudyindicatesthepotentialtouse503

Brachypodiumdistachyon,amodelforPooideaegrasscrops,toidentifygenetic504

variationinkeypathwaysunderlyingagriculturaltraitsthroughGenomeWide505



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18

AssociationStudies.Furtherwildcollectionsand/orthedevelopmentofaNested506

AssociationMapping(NAM)populationscouldaddressthelimitationof507

recombinantgenotypesandresultinveryhighpowermappingpopulation508

typicalof1000genomeprojects.Asitnowstands,Brachypodiumisagoodmodel509

forbothpolyploidisation,withlikelymultipleeventsamongsmalldivergent510

genomes,andforinvasionbiologywithmultiplewidespreadgenotypes511

identifiedacrosscontinents,regionsandsites.512

513

MaterialsandMethods514

515

GenotypingbySequencingandSpeciesIdentification516

517

Genotypingbysequencing(GBS)wasundertakenasdescribedbyElshireand518

colleagues(28)usingPstIenzymeandalibraryofhomemadebarcodedadaptors519

(seehttps://github.com/borevitzlab/brachy-genotyping;27,28).Approximately520

384samplesweremultiplexedtorunonasinglelaneinanIlluminaHiSeq2000521

withamediannumberof564000100bpreadpairspersample522

(https://github.com/borevitzlab/brachy-genotyping).Sequencingrunswere523

undertakenbytheBiomolecularResourceFacility(JCSMR,ANU).524

525

Axe(63)wasusedtodemultiplexsequencinglanesintolibraries,allowingno526

mismatches.AdapterRemoval(64)wasusedtoremovecontaminantsfrom527

reads,andmergeoverlappingreadpairs.ReadswerealignedusingBWAMEM528

(65,66)totheBd21-3(B.distachyon)andABR114(B.stacei)referencegenomes529

(Phytozomev.12.1),andtoaB.hybridumpseudo-referencegenomecreatedby530

concatenatingtheB.staceiandB.distachyonreferencegenomes(Supp.S01).531

Variantswerecalledusingthemultiallelicmodelofsamtoolsmpileup(67)and532

bcftoolscall(68).Variantswerefilteredwithbcftoolsfilter,keepingonlySNPsof533

reasonablemappingandvariantqualities(>=10)andsequencingdepthacross534

samples(>=5readsacrossallsamples).535

536

Todeterminethespeciesofeachoftheaccessions,wecomputedtheproportion537

ofeachChromosomeintheB.hybridumpseudo-referencecoveredwithatleast3538



https://doi.org/10.1101/246074


19

reads,excludingreadswhichmappedtomultiplelocationsinthepseudo-539

reference,usingmosdepth(69).TheproportionsoftheB.distachyon/B.stacei540

genomescoveredwerenormalisedtobein[0,1],andthenusedtoassign541

samplesintothresholdgroups:B.stacei(<0.03),intermediateB.stacei/B.542

hybridum(<0.28),B.hybridium(<0.34),intermediateB.hybridum/B.distachyon543

(0.94)andB.distachyon(>0.94);anadditionalgroupconsistedoflowcoverage544

samples(<100000readsintotal).Samplesfromintermediateandlowcoverage545

groupswereexcluded,andonlyvariantsintherespectivegenomeswereusedto546

allocatethethreespeciesgroups.547

548

PopulationStructureofB.distachyon549

TodeterminethepopulationstructureofB.distachyonapairwiseIdentityBy550

Stategeneticdistancewascalculatedtoidentifyamong490highqualitysamples551

acorediversitysetof72distinctgenotypesusing82,800SNPsderivedfromGBS552

dataandtheSNPRelatepackageusingaz-scoreof3.5.Occasionally,when553

genotypesarecloselyrelated,noisebetweentechnicalreplicatesofanaccession554

willresultinthembeingsplitacrosstherelatedgenotypes.Therefore,wekeep555

replicate(s)fromthegenotypewiththemajorityofreplicatesforthataccession,556

breakingtiesbykeepingthereplicatewiththelowestmissingdata.Inaddition,557

29accessionswhosegeographicoriginwassuspectwerealsoexcluded.558

559

Toavoidbiasfromincludingupto30inbredaccessionsofthesamegenotype,a560

reducedsetwasinputintoSTRUCTUREV.2.3.4(34).Atotalofsixreplicateswere561

runofpopulation(K)1-13withaburninsettingof10,000sets,and100,000562

permutationsperrun(Fig1B,Supp.S04).TheoptimalKwasdeterminedasK=3563

byEvanno’sDeltaK,processedviaStructureHarvesterandCLUMPP(70–72).564

Barplotsandpiechartsweregeneratedviain-housedevelopedRscripts565

availablethroughgithub(https://github.com/borevitzlab/brachy-genotyping-566

notes).567

568

ForB.distachyonthepairwisedistancebetweengenotypeswasalsocalculatedin569

Randplottedasadendrogram(Supp.S02).Fromthisasetof107accessions570



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20

wereselectedtorepresentthegenotypicdiversityofthespeciesforwhole571

genomesequencingtomaximiseSNPcoverageacrossthegenome.572

573

WholeGenomeSequencing574

575

ForWholeGenomeSequencing(WGS),sequencinglibrariesforindividual576

sampleswerepreparedfrom6nggenomicDNAwiththeNexteraDNALibrary577

Prepkit(Illumina,SanDiego,CA,USA).Librarieswereenrichedandbarcoded578

withcustomi5-,andi7-compatibleoligosandQ5®High-FidelityDNA579

Polymerase(NEB,Ipswich,MA,USA).Librarieswerepooledandsequencedin580

onelaneonaNextSeq500sequencer(Illumina,SanDiego,CA,USA).581

582

Trimit(73)wasusedtocleanWGSreadsofadaptors,andmergeoverlapping583

readpairs.BWAMEMwasthenusedtoalignthesereadsagainsttheBd21-1584

referencegenome(version314_v3.1;16).Variantswerecalledusingfreebayes585

(74)withdefaultparameters.Variantswerefilteredsuchthatonlyvariants586

meetingthefollowingcriteriawerekept:variantquality>20,minorallele587

frequency>=2%.Heterozygousvariantcallswerechangedtomissing;duetothe588

inbrednatureoftheseaccessions,heterozygouscallswerealmostcertainly589

erroneous.https://github.com/borevitzlab/brachy-genotyping590

591

LinkageDisequilibrium(LD)wascalculatedacrosstheB.distachyongenome592

usingconsecutivewindowsof2000SNPsfromthewholegenomedataofthe593

HapMap74set(http://github.com/borevitzlab/brachy-genotyping-notes).594

595

PlantGrowth596

597

Individualgrainofeachgenotypewereplanted2.5cmdeepinsquareplasticpots598

(5cmwidth,8cmdeep)inamixof50:50soil:washedriversandwhichhadbeen599

steampasteurised.Potswerethenplacedat4oCinthedarkforthreedaysto600

stratifytheseedbeforebeingmovedtospeciallymodifiedclimatechambers(see601

4).Inbrief,thesechambershavebeenfittedwith7LEDlightpanelsandare602

controlledtochangethelightintensity,lightspectrum,airtemperatureand603



https://doi.org/10.1101/246074


21

humidityevery5minutes.ClimaticconditionsweremodelledusingSolarCalc604

software(75).TheWaggaWaggaregionwascentredaround-35S,147Ewithan605

elevationof147m.PlantswerefertilisedwithThrive(N:P:K25:5:8.8+trace606

elements,Yates)andwateredwithtapwaterasneeded.Growthstageswere607

recordedbasedontheHuandevelopmentalstage(76)upuntilstemelongation608

andthereaftertheZadoksscalewasused.Totalleafareawasmeasuredwitha609

Li-1300AreaMeter(Li-COR).Fordryweight,leaftissuewasdriedinapaper610

envelopeat60oCforfivedaysbeforeweighing.611

612

ConversionsofPhenotypicData613

614

Thermaltimewascalculatedfromtheloggedconditionwithineachchamber615

withthefollowingformula:616

617

𝐼𝑓𝑇𝑒𝑚𝑝.) > 2℃, 𝑡ℎ𝑒𝑛𝑇𝑇1 = 𝑇𝑇) +(𝑇𝑒𝑚𝑝.1− 2)×𝛥𝑇𝑖𝑚𝑒1:)618

619

whereTTiisaccumulatedthermaltimeataparticulartimepointiandTemp.iis620

theairtemperatureataparticulartimepointi.621

622

Photothermalunits(PTU)werecalculatedusingtheloggeddatafromaPAR623

sensorinthemiddleofthechamberandthefollowingformula:624

625

𝑃𝑇𝑈) = 𝑇𝑇)×𝑃𝐴𝑅)626

627

whereTTistheaccumulatedthermaltimeattimepointiandPARisthe628

measuredphotosyntheticallyactiveradiationattimepointi.629

630

Growthrates(GR)werecalculatedas:631

632

𝐺𝑅 = ∆𝐺𝑆BC:BD∆𝑇𝑖𝑚𝑒BC:BD

633

634



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22

whereGSistheHuanGrowthStageandT1wasapproximatelyoneleafforthe635

initiallineargrowthstage(GR1)T1wasapproximatelyoneleafandthreeleaves636

andthefastergrowthstage(GR2)betweenthreeleavesandfiveleaves.The637

Phyllachroninterval,thetimetakentogrowoneleafwascalculatedas:638

639

𝑃ℎ𝑦𝑙𝑙𝑎𝑐ℎ𝑟𝑜𝑛𝐼𝑛𝑡𝑒𝑟𝑣𝑎𝑙 = 𝑇1 − 𝑇L𝐺𝑆1

640

641

whereT2istheunitoftimeatapproximatelythreeleafstageandTeistheunitof642

timeatseedlingemergenceforthatparticularplant.GS2istheHuanGrowth643

StageatT2.644

645

Finalgrowthefficiencywascalculatedwhenplantsreachedearemergence.The646

finalgrowthefficiency1wascalculatedas:647

648

𝐹𝑖𝑛𝑎𝑙𝐺𝑟𝑜𝑤𝑡ℎ𝐸𝑓𝑓𝑖𝑐𝑖𝑒𝑛𝑐𝑦1 =𝐵𝑖𝑜𝑚𝑎𝑠𝑠𝑎𝑡𝑒𝑎𝑟𝑒𝑚𝑒𝑟𝑔𝑒𝑛𝑐𝑒(𝑔)

𝛥𝑇ℎ𝑒𝑟𝑚𝑎𝑙𝑡𝑖𝑚𝑒649

650

whereaccumulatedthermaltimeiscalculatedfromseedlingemergencetoear651

emergence.Thefinalgrowthefficiency2wascalculatedas:652

653

𝐹𝑖𝑛𝑎𝑙𝐺𝑟𝑜𝑤𝑡ℎ𝐸𝑓𝑓𝑖𝑐𝑖𝑒𝑛𝑐𝑦2 =𝐵𝑖𝑜𝑚𝑎𝑠𝑠𝑎𝑡𝑒𝑎𝑟𝑒𝑚𝑒𝑟𝑔𝑒𝑛𝑐𝑒(𝑔)

𝛥𝑃ℎ𝑜𝑡𝑜𝑡ℎ𝑒𝑟𝑚𝑎𝑙𝑢𝑛𝑖𝑡𝑠654

655

whereaccumulatedphotothermalunitsiscalculatedfromseedlingemergenceto656

earemergence.657

658

EnergyUseEfficiencyTraits659

660

Energyuseefficiencytraitsweremeasuredonplantsfromthe2015-2050661

Temperatureexperimentata4-5leafstage.Photosyntheticparameterswere662

measuredusingaTrayscansystem(PSI)incorporatingPulseAmplitude663

Modification(PAM)ChlorophyllFluorescencemeasuresofQuantumEfficiency664



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23

(61).Theparametersmeasuredincludedphotosyntheticefficiency,non-665

photochemicalquenchingandphoto-inhibition.SeeSupp.S20forProtocol.666

667

DarkrespirationratewasmeasuredusingtheQ2system(AstecGlobal)asin668

Scafaroetal(77).Inbrief,thissystemusesanoxygensensitivefluorescentdye669

embeddedinacaptomonitortheoxygendepletionwithatubecontainingthe670

sample.A3cmfragmentsinthecentreofthelastfullyexpandedleafofeach671

plantwasusedtomeasuredarkrespirationperunitareaandperunitdrymass.672

673

Severalenergyuseefficiencyformulaswerecalculated.Theseincludedaratioof674

darkrespirationtophotosynthesisandmeasuresofgrowthperunitdark675

respiration.Thesewereasfollows:676

677

𝐸𝑛𝑒𝑟𝑔𝑦𝑈𝑠𝑒𝐸𝑓𝑓𝑖𝑐𝑖𝑒𝑛𝑐𝑦1 = 1 −�𝑒𝑠𝑝𝑖𝑟𝑎𝑡𝑖𝑜𝑛𝑝𝑒𝑟𝑢𝑛𝑖𝑡𝑎𝑟𝑒𝑎

𝐴𝑣𝑒𝑟𝑎𝑔𝑒𝑃ℎ𝑜𝑡𝑜𝑠𝑦𝑛𝑡ℎ𝑒𝑡𝑖𝑐𝐸𝑓𝑓𝑖𝑐𝑖𝑒𝑛𝑐𝑦678

679

𝐸𝑛𝑒𝑟𝑔𝑦𝑈𝑠𝑒𝐸𝑓𝑓𝑖𝑐𝑖𝑒𝑛𝑐𝑦2 = 𝑆𝑒𝑒𝑑𝑙𝑖𝑛𝑔𝐻𝑒𝑖𝑔ℎ𝑡

𝑅𝑒𝑠𝑝𝑖𝑟𝑎𝑡𝑖𝑜𝑛𝑝𝑒𝑟𝑔𝑑𝑟𝑦𝑤𝑒𝑖𝑔ℎ𝑡680

681

𝐸𝑛𝑒𝑟𝑔𝑦�𝑠𝑒𝐸𝑓𝑓𝑖𝑐𝑖𝑒𝑛𝑐𝑦3 = 𝐿𝑒𝑎𝑓#3𝐿𝑒𝑛𝑔𝑡ℎ

𝑅𝑒𝑠𝑝𝑖𝑟𝑎𝑡𝑖𝑜𝑛𝑝𝑒𝑟𝑔𝑑𝑟𝑦𝑤𝑒𝑖𝑔ℎ𝑡682

683

𝐸𝑛𝑒𝑟𝑔𝑦𝑈𝑠𝑒𝐸𝑓𝑓𝑖𝑐𝑖𝑒𝑛𝑐𝑦4 = 𝑆𝑒𝑒𝑑𝑙𝑖𝑛𝑔𝐻𝑒𝑖𝑔ℎ𝑡

𝑅𝑒𝑠𝑝𝑖𝑟𝑎𝑡𝑖𝑜𝑛𝑝𝑒𝑟𝑢𝑛𝑖𝑡𝑎𝑟𝑒𝑎684

685

686

Heritability687

688

Narrow-senseheritabilitywascalculatedfromthephenotypedatausingthe689

nlmepackageinR.690

691

GWASanalysis692

693



https://doi.org/10.1101/246074


24

InpreparationforGWAS,thegenotypedatawasfilteredtoremovenon-variant694

SNPsandredundantSNPs(i.e.SNPswhosegenotypesarenotdifferentfrom695

adjacentSNPsbuthavemoremissingdatapoints).ThenSNPswithaminorallele696

frequencyof<3%werefilteredout.Astherewas18.5%missingdatainthe697

originaldataset,imputationwasundertaken.First,iftheobservedgenotypesof698

twoadjacentSNPswerenotdifferent,thenthemissinggenotypeofoneSNPwas699

replacedbytheobservedgenotypeoftheotherSNP.Secondly,nearest700

neighborhood(NN)methodwasimplementedtoimputetheremainingmissing701

genotypesbasedonHuangetal(78)withsomemodifications.Thenearest50702

SNPsfromeachsideoftheSNPunderimputationwereselectedtoestimate703

similaritybetweeneachpairofaccessions,andthenthemissinggenotypeofan704

accessionwasreplacedbytheobservedmajoritygenotypeoftheclosest5705

accessions.Theseparametersweredeterminedbysimulationstoachievean706

optimalimputationsuccessrate,whichwas97.95%forourdata.Finally,SNPs707

withaminorallelefrequency<5%werefiltered.708

709

Linearmixed-effectmodelswereemployedtoidentifygeneticvariants710

underlyingphenotypesofinterest711

712

𝒚 = 𝒙𝜷 + 𝒛𝜸 + 𝒖 + 𝝐 713

714

wherey=(y1,y2,…….,yn)’denotesphenotypicvalues,x=(xij)nx(k+1)represents715

interceptandkcovariates(ifany)witheffectsβ,zisavectorofthecoded716

genotypesatascanninglocuswitheffectγ,u=(u1,u2,……,un)’represents717

polygenicvariation,andϵ=(ϵ1,ϵ2,……,ϵn)theresidualeffect.Itwasassumedthat718

u~N(0,K𝜎c1),ϵ~N(0,Is1)anduwasindependentofϵ.Thegenetic719

relationshipmatrixKwasestimatedbyidentify-by-state(IBS)fromgenotypic720

datawithmarkersontheChromosomeunderscanbeingexcludedtoavoid721

proximalcontamination(79,80).EstimationofKandgenomescanwere722

performedinRpackageQTLRel(52).723

724

Todetermineasignificancethreshold,thepermutationtestwasimplementedon725

1000permutationsofthephenotypedatatoestimatethegenome-wide726



https://doi.org/10.1101/246074


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significancethresholdat0.05forthetraitofdaystoearemergence.The727

significancethresholdwasdeterminedtobeaLOD(LogarithmofODds)of728

4.43583.729

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SupplementaryMaterials1009 1010 S01.CoverageofB.staceiandB.distachyonGenomesforSpeciesID1011 S02.StructureofB.distachyoncollectionassequencedbyGBS1012 S03.DendrogramofreducedB.distachyonsetusedforSTRUCTURE1013 S04.B.distachyonreducedsetSTRUCTUREplotsfork=21014 S05.LinkagedisequilibriumacrosstheB.distachyongenome1015 S06.Graphicalrepresentationofclimaticconditionsfromthefloweringtime1016 experiment1017 S07.Phenotypedatafromfloweringtimeexperiment1018 S08.Floweringtimeexperimentacross266B.distachyonaccessions1019 S09.Phenotypiccorrelationoftraitsfromtheearlyvigourvalidationexperiment1020 S10.ListofHapMap74genotypes1021 S11.AssessmentofclimatechangemodelsforSouthEastAustralia1022 S12.Phenotypedatafrom2015temperatureand2050temperatureclimate1023 experiment1024 S13.Heritabilityoftraitsofinterestfrom2015temperatureand20501025 temperatureclimateexperiment1026 S14.Histogramsofalltraitsin2015temperatureand2050temperatureclimate1027 experiment1028 S15.ListofQTLidentifiedfortraitsofinterestin2015temperatureand20501029 temperatureclimateexperiment1030 S16.ManhattenPlotsforalltraitsin2015temperatureand2050temperature1031 climateexperiment1032 S17.ListofcandidategenesforstrongearemergenceQTL1033 S18.ListofcandidategenesforstrongearlyvigourQTL1034 S19.ListofcandidategenesforstrongenergyQTL1035 S20.ProtocolformeasuringphenotypicparametersusingaPAM1036 1037 1038 1039



https://doi.org/10.1101/246074


population structure of the brachypodium species complex ...€¦ · 11/1/2018 · 71 these traits...

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