QTL analyses for tolerance to abiotic stresses in a common bean

26 Pages·2017·4.45 MB·English

RESEARCHARTICLE
QTL analyses for tolerance to abiotic stresses
in a common bean (Phaseolus vulgaris L.)
population
LucyMilenaDiaz1,JaumerRicaurte1,EduardoTovar2,CesarCajiao1,HenryTera´n3,
MiguelGrajales1,JosePolan´ıa1,IdupulapatiRao1¤b,StephenBeebe1,BodoRaatz1¤a*
1 BiotechnologyUnitandBeanProgram,InternationalCenterforTropicalAgriculture(CIAT),Cali,Valle,
Colombia,2 InstitutodeInvestigacio´ndeRecursosBiolo´gicosAlexandervonHumboldt,Bogota´,Colombia,
3 DuPontPioneer,Salinas,PuertoRico,UnitedStatesofAmerica
a1111111111
a1111111111
¤a Currentaddress:CIAT-InternationalCenterforTropicalAgriculture,Cali,Colombia
a1111111111 ¤b Currentaddress:PlantPolymerResearchUnit,NationalCenterforAgriculturalUtilizationResearch,
a1111111111 AgriculturalResearchService,UnitedStatesDepartmentofAgriculture,Peoria,Illinois,UnitedStatesof
a1111111111 America
*[email protected]
Abstract
OPENACCESS
Commonbeanproductivityisreducedbyseveralabioticstressfactorslikedroughtandlow
Citation:DiazLM,RicaurteJ,TovarE,CajiaoC,
Tera´nH,GrajalesM,etal.(2018)QTLanalysesfor soilfertility,leadingtoyieldlossesparticularlyinlowinputsmallholderfarmingsystemsin
tolerancetoabioticstressesinacommonbean thetropics.
(PhaseolusvulgarisL.)population.PLoSONE13
Tounderstandthegeneticsofstresstolerance,andtoimproveadaptationofcommon
(8):e0202342.https://doi.org/10.1371/journal.
beantoadverseenvironments,theBAT881xG21212populationof95recombinantinbred
pone.0202342
lines(RILs)wasevaluatedunderdifferentabioticstressconditionsin15trialsacrossfour
Editor:RobertoPapa,UniversitàPolitecnicadelle
locationsinColombia,representingtwohigheraltitude(Darie´n,Popaya´n)andtwoloweralti-
Marche,ITALY
tude(Palmira,Quilichao)locations.Stressvsnon-stresstreatmentsshowedthatyields
Received:December14,2017
werereducedindroughttrialsinPalmiraby13and31%,respectively,andobservedyield
Accepted:August1,2018 reductionsinlowphosphorusstresswere39%inQuilichao,16%inPopaya´n,and71%in
Published:August29,2018 Darie´n,respectively.Yieldcomponentsandbiomasstraitswerealsoreduced.Traitslinked
Copyright:©2018Diazetal.Thisisanopen todrymatterredistributionfromstems,leavesandpodstoseed,suchaspodharvestindex
accessarticledistributedunderthetermsofthe andtotalnon-structuralcarbohydrates,werefoundtobeimportantfactorscontributingto
CreativeCommonsAttributionLicense,which
yieldinallconditions.Incontrast,earlymaturitywascorrelatedwithimprovedyieldonlyin
permitsunrestricteduse,distribution,and
loweraltitudelocations,whereasinhigheraltitudesdelayedmaturitypromotedyield.Supe-
reproductioninanymedium,providedtheoriginal
authorandsourcearecredited. riorRILsthatcombinestresstoleranceandhighcross-locationproductivitywereidentified.
Linesthatshowedgoodyieldunderstrongstressconditionsalsoperformedwellundernon-
DataAvailabilityStatement:Allrawdatais
availableondataverse,underthefollowinglink: stressconditions,indicatingthatbreeder’sselectioncanbeappliedforbothconditionsat
https://dataverse.harvard.edu/dataset.xhtml? thesametime.
persistentId=doi:10.7910/DVN/ODYAPF.
Quantitativetraitloci(QTL)analysesrevealedastableyieldQTLonchromosomePv04,
Funding:ThankstotheGenerationChallenge detectedindividuallyinalllocations,severalstresstreatmentsandinbestlinearunbiased
Program(GCP,www.generationcp.org/).And
predictions(BLUPs)acrossalltrials.Furthermore,twoQTLhotspotsformaturitytraitswere
particularlyitscomponentTropicalLegumeI(TL-I)
identifiedonPv01andPv08,whicharethemoststableQTL.TheconstitutiveyieldQTL
project(fundedbytheBill&MelindaGates
Foundation,OPPGD1392)continuedbytheTL3 couldserveasagoodcandidateformarkerdevelopmentandcouldbeusedinmarkerassis-
project(http://tropicallegumes.icrisat.org/, tedselection.
OPP1114827).Theauthorsalsoacknowledgethe
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QTLdetectionforabioticstresstoleranceincommonbean(PhaseolusvulgarisL.)
financialsupportfromtheCGIARresearch Increasedunderstandingofthephysiologyofabioticstresstolerance,combinedwiththe
programonGrainLegumes(http://grainlegumes. availabilityofsuperiorgermplasmandmoleculartools,willaidbreedingeffortsforfurther
cgiar.org/),assumedbyUSAID(https://www.
improvementoftheseplanttraits.
usaid.gov/).Thefundershadnoroleinstudy
design,datacollectionandanalysis,decisionto
publish,orpreparationofthemanuscript.
Competinginterests:Theauthorshavedeclared
thatnocompetinginterestsexist.
Introduction
Commonbean(PhaseolusvulgarisL.)isthemostimportantfoodlegumefordirecthuman
consumptioninthetropicsofLatinAmericaandsouthernandeasternAfrica[1]withatotal
areaharvestedof30.6millionha[2].Itisanimportantsourceofprotein,carbohydratesand
microminerals,particularlyforsmallholderfarmers[2].Itisanidealcropforthesmallholder
farmingsystemsduetoitssymbioticnitrogenfixingabilityandrelativelyshortgrowthcycleof
aboutthreemonths.
Beanproductionisaffectedbyseveralconstraints,mainlyabioticstressesanddiseases.
Hence,nationalaveragesofbushbeanyieldsthroughoutthetropicstypicallyrangefrom500
to700kgha-1,significantlybelowtheyieldpotential[3].Theprincipalabioticstressesthat
limitcommonbeangrainyieldaredroughtandlowsoilfertilitysuchaslowphosphorus(P)
availabilityandaluminum(Al)toxicityinsoil.Theseproblemstendtooccurincombination
[4].
Droughtisestimatedtoaffect60%ofbeanproductioninthetropics,especiallyinregions
suchasCentralAmericaandSouthernAfrica[5,6]andapproximately67%ofgloballyculti-
vatedlandsareaffectedbyPdeficits[5,7].Ithasbeenestimatedthat50%ofbeanproduction
areaworldwidesuffersmoderatetoseverePlimitation[8].LowsoilPavailabilityisexacer-
batedinstronglyacidicoralkalinesoils,mainlyduetoformationofphosphatecomplexeswith
AlandFeinacidicsoilsandCacomplexesinalkalinesoils[4].SoilaciditywithpH(cid:20)5.5affect
morethan50%oftheword’spotentiallyarableland[9],andupto60%oftheacidsoilsinthe
worldoccurindevelopingcountriesinSouthAmerica,CentralAfricaandSoutheastAsia[10].
Conventionalbreedingandtheselectionofsuperiorgenotypesfromsegregatingpopula-
tionshasbeenthetraditionalandpredominantstrategyinbreedingprogramstoimprove
toleranceofcommonbeanfordifferentabioticstressfactors.Pronouncedeffortswereunder-
takentoimprovedroughtresistance(avoidanceandtolerance).Consideringthecomplexityof
thistrait,indirectselectiontraitshavebeenevaluated.Phenotypicdifferencesindroughtresis-
tancewerefoundtobeassociatedwithbiomassproductionatmid-podfilling[11],vigorous
rootsystem[12,13],effectiveuseofwater[14],efficientdrymatterredistributiontograin[14–
16],phenologicalplasticity[17]andearliness[18,19].Earlymaturitywasshowntocorrelate
withyieldunderstressfollowingthestrategyofdroughtavoidanceinseveralstudies(e.g.
[13]),however,earlymaturityalsohasdisadvantagesincludinglimitedyieldpotential.Each
dayofreductioningrowthcyclecouldresultinalossof74kgha-1[20].Thismayvarydepend-
ingongenotypesandconditionsthough,aspositiveaswellasnegativecorrelationsofyield
andmaturityhavebeenreported.Thephenotypicexpressionofthecharacteristicsasdaysto
flowering(DF),daystophysiologicalmaturity(DPM)andpodharvestindex(PHI)couldbe
moreinfluencedbythegeneticconstitutionthanbyenvironmentalstress[16].
AnumberofstudiesaimedatidentifyingbeansadaptedtolowPandacidsoils[5].Several
physiologicalresponsestolowPavailabilityhavebeenreported,suchasmodificationofroot
architecture,associationwithmycorrhizalfungiintherootsystem,andhigheruseefficiency
ofabsorbedPtoproducebiomassandgrainyield[12,21–23].
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QTLdetectionforabioticstresstoleranceincommonbean(PhaseolusvulgarisL.)
Severalyearsoffieldevaluationfordroughtresistanceandacidsoiltoleranceidentifiedthe
landraceG21212tobeadaptedtothesestresses.IncontrastthebreedinglineBAT881issensi-
tivetobothstresses[24–26].BothlinesbelongtoraceMesoamerica.Theauthorsconcluded
thatproductivityunderlowPisheritablebutisbasedonmultiplemechanisms,hence,there
maybeopportunitiestorecombinegenesandmechanismsandobtainhigherlevelsofstress
tolerance.
Inrecentyearsnewgenotypingtechnologieshavebeendeveloped,utilizingseveralthou-
sandsofSingleNucleotidePolymorphisms(SNPs)thathavebecomeavailablethroughnext
generationsequencing(NGS).SNPbasedmarkershavebecomethepreferredchoicefor
geneticstudies,duetotheirabundanceanduniformdistributionthroughoutgenomes[27].
Theyhavebeenappliedtostudydiversity,genomemappingandQTLsdetectionforbioticand
abioticstressesincommonbean[28–30].However,adoptionintobreedingprocesseshasbeen
morelimitedtobioticstressresistance.KompetitiveAlleleSpecificPCR(KASP)genotyping
technologyoffershighprecisionandrobustnessinassaysatlowcost[31].Approximately
~1500KASPmarkershavebeenestablishedforthecommonbeancommunitybytheGenera-
tionChallengeProgram,tosupportgeneticstudiesandbreedingprograms.
Themainobjectivesofthecurrentstudyweretoincreasetheunderstandingofadaptation
ofcommonbeantodifferentstressenvironments,andtoperformmultilocationalevaluations
ofyieldstabilityandstresstolerance.TheMesoamericanRILpopulationBAT881xG21212
(BxG)wasevaluatedforyieldandyieldcomponents,plantvigor,drymatterredistribution
andphysiologicaltraitsunderdifferentconditionsofabioticstress(droughtconditions,low
soilfertilityandmedium-highaluminumlevels).Theobjectiveofthisstudywastoidentify
QTLsassociatedwithagronomictraitsandtoprovidemarkersforuseinmarkerassistedselec-
tion(MAS)employingasaturatedgeneticBxGmap.
Materialsandmethods
Plantmaterials
Atotalof100commonbean(P.vulgaris)genotypeswereusedinthisstudy.Theseincluded95
recombinantinbredlines(RILs)[32],thetwoparents(BAT881andG21212)usedascontrols
andthreeotherlinesusedinthebeanbreedingprogramoftheInternationalCenterforTropi-
calAgriculture(CIAT)asadditionalcontrolsineachtrial.Darie´n,Popaya´n1999andCIAT-
PalmiratrialsusedG3513,G4017(toleranttolowfertility)andDOR364(sensitivetolowfer-
tility)ascontrols;QuilichaotrialsusedDOR364,BAT477andVAX1(toleranttoaluminum
andcommonbacterialblight).RILsweredevelopedbysingleseeddescent(SSD)fromtheF2
totheF5generation,followedbyseedadvancetotheF7generation.BAT881isanadvanced
breedinglineofCIAT,withindeterminatebushgrowthhabit(IIa)andsmalllightbrownseed
thatisresistanttobeancommonmosaicvirus(BCMV)andintermediatelyresistanttothe
leafhopper(Empoascakraemeri)andangularleafspot[32].G21212isasmallblackseeded
Colombianlandrace,growthhabitIIIb,toleranttolowsoilfertility,droughtandwithexcellent
yield,butissusceptibletoBCMV[5,24,32,33].Bothgenotypesshowedintermediatelevelsof
resistancetothemelonthrips[32]andbelongtothesameraceMesoamericaoftheMesoamer-
icangenepool.
Descriptionofexperimentallocations
PhenotypicdatawereobtainedinfourdifferentlocationsinColombiaunderfieldconditions
with10x10balancedlatticedesignwiththreereplicates,exceptthreetrialsatQuilichao
(Q02a_MAl-LP,Q03a_HAL_MP,Q03b_HAL_MP)thathadtworeplicates.Atrialreferstoan
evaluationofall100genotypesinaspecificlocation,season,andmanagement,areplicate
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QTLdetectionforabioticstresstoleranceincommonbean(PhaseolusvulgarisL.)
represents100experimentalplotsharboringallgenotypesandablockreferstoasubsectionof
10plotswithinareplicate.
Descriptionoflocations:Darie´n(Dar,Inceptisol,3˚55’Nlatitude,76˚28’Wlongitude,
1457masl,lowPstress),Popaya´n(Pop,Inceptisol,2˚25’Nlatitude,76˚40’Wlongitude,1730
masl,lowPstress),Palmira(Pal,Mollisol,3˚30’Nlatitude,76˚21’Wlongitude,965masl,
droughtstress)andSantanderdeQuilichao(Qui,Oxisol,3˚06’Nlatitude,76˚31’Wlongitude,
990masl,highAlstress).SpecificsoilconditionsareshowninS1Table.
TwotrialswereestablishedinDarie´n:1997(September-December)withhighPsupply(20
kgha-1supplied,Dar97_HP)andin1998(September-December)withlowandhighPsupply
(9and90kgha-1,Dar98_LPandDar98_HPrespectively).Experimentalunits(4rowsof4m
lengthwithrow-to-rowdistanceof0.6m)weresownmanuallywith10plantspermrow
length.TwotrialsweresowninPopaya´n:1999(April-September)withlowandhighP(0and
40kgha-1,Pop99_LPandPop99_HP,)and2005(JunetoAugust)withhighPsupply(39kg
ha-1,Pop05_HP).Experimentalunits(4rowsof4.9mlengthwithrow-to-rowdistanceof0.6
m)weretractorsownwith14seedsperm.TwotrialswereestablishedinPalmira:2000and
2002inthesamesemester(JunetoSeptember),withtwolevelsofwatersupply(droughtand
irrigated,Pal00_D,Pal00_I,Pal02_D,andPal02_I).Bothenvironmentswereirrigatedto
ensuregoodplantestablishmentatearlygrowthasneededuntilearlypodfilling.Thereafter,
irrigationwasdiscontinuedinthedroughttreatmentwhileirrigatedornon-stress(NS)plots
continuedtoreceivesupplementalirrigationof20mmofwatertwiceaweekforatotalof
additional188and211mmofwaterin2000and2002,respectively(S1Fig).Experimental
units(4rowsof5mlengthwithrow-to-rowdistanceof0.6m)weretractorsownwith14
seedspermrowlength.Finally,fourtrialsweresowninSantanderdeQuilichao:2002(April
toJune)inmoderateAlsaturation(40and64%intop0–10and10–20cm,respectively),low
availablePcontent(3.3and1.9μgg-1)andhighorganicmattercontent(10.7and9.1%),sup-
plementingwithP15kgha-1(Qui02a_MAl_LP).Asecondtrialin2002(OctobertoJanuary
2004),inmoderateAlsaturation(40and45%),moderateavailablePcontent(17.1and6.9μg
g-1)andmoderateorganicmattercontent(5.6and5.2%)soilwithlowPsupply(10kgha-1,
Qui02b_MAl_MP).In2003(MarchtoJune),withhighAlsaturations(61and62%),moderate
availablePcontent(23.5and13.1μgg-1)andmoderateorganicmattercontent(5.9and5.4%)
withlowPsupply(10kgha-1,Qui03_HAl_MP).Lastly,in2003(OctobertoJanuary2004)
twotrialsweresownwithhighAl(61–61%and64–67%),highandmoderateavailablesoilP
content(20.3–17.5μgg-1and13.2–5.5μgg-1),moderateorganicmattercontent(5.6–5.4%
and6.1–5.4%),withlowPsupply(10kgha-1,Qui03_HAl_HPandQui03_HAl_MP,respec-
tively).Experimentalunits(2rowsof3.72mlengthinQui02a_MAl_LP,4rowsof4.9min
Qui02b_MAl_MPand3rowsof4.9mlengthintheotherQuilichaotrials)weretractorsown
with14seedspermrowlengthandrowsseparatedwith0.6m.
InthesupplementalmaterialS1Figtheconditionsofprecipitationandevaporation,mini-
mumandmaximumtemperature,andtotalrainfallwhereavailablearedetailed.Inallloca-
tionsrecommendedagronomicprocedureswerefollowed.
Phenotypictraits
Fivegroupsofplanttraitswereevaluatedinthisstudy:yieldcomponents,plantvigor,drymat-
terredistribution,phenologicaltraits,andmineralnutrients.Notallvariablesweremeasured
ineverytrial.
Yieldcomponents:Yield(Yd,kgha-1)and100seedweight(100SdW,g100seeds-1).Atthe
timeofharvest,0.3m2perplotwereharvestedtomeasureseednumberperarea(SNA,seeds
m-2),podnumberperarea(PNA,podsm-2).Plantvigor:Canopybiomass(CB,kgha-1),leaf
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QTLdetectionforabioticstresstoleranceincommonbean(PhaseolusvulgarisL.)
areaindex(LAI,m2m-2),andleafbiomass(LB),podbiomass(PBMP)andstembiomass
(SBMP,kgha-1)wereevaluatedatmid-podfilling.Plantsfrom0.3m2perplotwereharvested,
leaveswereseparatedfromplantstomeasureleafarea(LI3000;LI-CORmInc.,Lincoln,NE).
Separatedplantpartswereoven-driedat60˚Cfortwodaysuntilestablishingconstantweight,
todeterminedryweight.Drymatterredistribution:Indicessuchasharvestindex(HI),pod
partitioningindex(PPI),podharvestindex(PHI)werecalculated.Seedandshoottotalnon-
structuralcarbohydratescontent(TNCmgg-1)weremeasuredtoexploregenotypicdiffer-
encesindrymatterredistributionandgrainfilling[14,16,18,33,34].Thesedrymatter
redistributiontraitsweredeterminedasHI(%):(seedbiomassasdryweightatharvest)/(total
canopybiomassasdryweightatmid-podfilling)x100;PHI(%):(seedbiomassasdryweight
atharvest)/(podbiomassasdryweightatharvest)x100;PPI(%):(podbiomassasdryweight
atharvest)/(totalcanopybiomassasdryweightatmidpodfilling)x100;seedandshootTNC
weredeterminedbycomparisonwithglucosestandardsaccordingtothemethoddescribedby
[35].Becauseofleaffallincommonbeanduringseedfilling,dryweightoftotalcanopybio-
masswasdeterminedatmid-podfilling[18].Useofdryweightofcanopybiomassandseed
biomassatharvesttimeoverestimatesHIandPPIvaluesincommonbean.Phenological
traits:Daystoflowering(DF,days);daystoharvest(DH,days).DFisdefinedasthenumber
ofdaysafterplantinguntil50%oftheplantshaveatleastoneopenflowerandDHisthenum-
berofdaysafterplantinguntilharvest.Mineralnutrients:Oneplantperplotwasharvestedat
midpodfilling,washedwithdeionizedwater,oven-driedat60˚Cfortwodaysandgroundin
a1mmsieve.Forseedevaluationsseedsfrom0.3m2plotareawereoven-driedat60˚Cfor
twodaystobegroundina1mmsieve.Nutrient(N,P)compositionofplantpartswasdeter-
minedasdescribedby[36].Shootnutrientuptakewascalculatedastheproductofshootdry
matteryieldandnutrientconcentration.Nutrientuseefficiency(NUE_Sh,PUE_Sh)were
estimatedasgramsofshootbiomassproducedpergramofshootnutrientuptake[37]and
nutrientuseefficiencyforseedyield(NUE_Sd,PUE_Sd)wereestimatedasgramsofseedbio-
massproducedatharvestpergramofshootnutrientuptakeatmidpodfilling.Additional
informationondescriptionofphenotypictraitscanbefoundin"TraitDictionariesfor
FieldbookDevelopment"athttp://mbp.generationcp.organdhttp://www.cropontology-
curationtool.org/.
Therawdataisavailableviadataversehttps://doi.org/10.7910/DVN/ODYAPF.
Statisticalanalysis
Varianceandcovarianceanalyseswerecarriedout,andtheresultsofthecovariancearepre-
sentedonlyforthosetraitsthatshowedsignificantco-variationbetweengenotypesandtreat-
ments.Pearson’scorrelationsbetweenalltraitsweredeterminedforalltrialsusingdataof
individualreplications.Datashowninthisworkareadjustedmeansforeachgenotypeand
theenvironment,thatwereobtainedusingSAS(v9.3)PROCMIXEDandPROCCORR
[38],consideringtheeffectsofreplicationandblockswithinreplicationsasrandomand
genotypesasfixed.Alsoleastsignificantdifference(LSD)wascalculatedwiththesamesoft-
waretocomparetraitmeans.Variabilityandfrequencydistributionswererepresentedasvio-
linplotsforeachenvironmentevaluated,generatedbythestatisticssoftwareR[39]withthe
packageggplot2[40].Toestimatetheoveralladjustedmeansandadjustedmeansforeachof
thefourlocations,BestLinearUnbiasedPredictions(BLUP)wereused,accordingto
Resende[41]forthefourmajortraits(yield,100SdW,DF,DH).Weusedthepackagelme4
[42]foranalysisinthestatisticalsoftwareR.Thefollowingmixedlinearmodelwasdefined:
Y=B0+B1trl+B2rep+B3blk+B4+errwhereYisthevectorofobservedvalues,B0is
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QTLdetectionforabioticstresstoleranceincommonbean(PhaseolusvulgarisL.)
intersect,B1istheeffectofeachtrial,B2theeffectofeachrepetition,B3theeffectofeach
blockwithintrialsandreplicates,B4theeffectofeachgenotypeorlineanderrtheresidual
error.Treatmentsandreplicateswereconsideredfixedeffectswhileblocksandgenotypes
wereconsideredrandom.ThenewBLUPvariableswerenamedaftertheirrespectivetraits
(yield,100SdW,DF,DH)acrosseachlocation,e.g.Yd_D,Yd_P,Yd_CandYd_Q,andfor
overalladjustedmeansYd_all.Wealsocalculatedthedifferentialphenotypicresponseunder
stressforyield,expressedaspercentyieldlossunderirrigationvsdrought(%YdL_D)and
highvslowphosphorus(%YdL_LP).Theywerecalculated:%YdL_LP=((BLUP_higher
P–BLUP_lowerP)/BLUPhigherP)(cid:3)100(combiningthetrialsDar98,Pop99,Qui03b);
and%YdL_D=((BLUPirrigated–BLUP_drought)/BLUPirrigated)(cid:3)100(Pal00andPal02
trials).FortheseBLUPsonlydatasetswereusedwherestressvsnon-stressconditionswere
evaluatedinthesameseason.Thesameformulawasappliedforthecalculationofpercent
lossin100SdW,DFandDH.
DNA,markersandQTLanalysis
DNAwasextractedfromRILsandparentsaccordingtothemethoddescribedbefore[43].
DNAqualitywasevaluatedon0.8%agarosegelsfollowedbyquantificationonaHoeferDyNA
fluorometer(DNAQuant™200).ThegeneticmapoftheBxGpopulationwasconstructed
from339markers,fromwhich115markerscorrespondtopreviousanalyses[32]andwe
added53AFLP,2RAPD,42SSRs[44–48].PCRamplificationconditionsweredescribed
before[44,49,50]andPCRswereconductedin96-wellplatesusingaPTC-100thermalcycler
(MJResearch,Watertown,USA).Wealsoadded127SNPsmarkersgenotypedatLGCGeno-
mics(www.lgcgenomics.com,Hoddesdon,UK)withKASPgenotypingtechnology.These
markershadbeenestablishedbytheGenerationChallengeProgram(https://www.
generationcp.org/),basedontheBARKBeanchip[30,51].
ThegeneticmapwasconstructedusingMapDistov.1.7.5(http://mapdisto.free.fr/),using
Kosambimappingfunction,withaminimumLODscoreof3andmaximumrecombination
fractionof0.35;SARFandseriationascriteriafororderingandripple.Allgeneticmapping
wasconfirmedwiththecommandfor‘‘bestorder”inMapDisto.Linkagegroupswere
namedbasedonthechromosomeinformationofPhaseolusvulgarisgenomev2.1https://
phytozome.jgi.doe.gov/pz/portal.html(consultedJuly2018).QTLanalysiswasperformed
throughQTLIciMappingsoftwareV4.0basedontheinclusivecompositeintervalmapping
ofadditive(ICIM-Add)[52].ThemappingparametersofeachstepforICIM-ADDwereset
at1.0cM.LODthresholdvalueof2.96wasappliedforalltraits,determinedthroughcom-
pleting1000permutationswiththeTypeIerrorlevelsetatα=0.05foreachmapping
method.
Results
Phenotypicdatafromfourlocationswithabioticstresstreatments
TheBAT881xG21212RILpopulationwasevaluatedatfourlocations,totaling15trialswith
independentlocation/treatmentcombinations.Twotrialsweregrownunderdroughtstress,
fourinlowPstress,andanotherthreeinhighAlstressenvironments.Atotalof31traitswere
evaluated,onlyyieldandDHwereevaluatedinall15trials(Fig1andTable1),resultingin225
datasets,includingtheaveragesforeachlocalityforthemajortraits(Yd,100SdW,DH,DF)
andtheiroverallaverages.RILspresentedcontinuouspopulationdistributionsoftraitsby
treatment(Fig1andS2Fig).TransgressivesegregationwasalsoevidentinBxGlineswith
lowerorhighervaluescomparedtoparents.
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QTLdetectionforabioticstresstoleranceincommonbean(PhaseolusvulgarisL.)
Fig1.PhenotypicdataoftheBAT881xG21212population,collectedin15trialsoverfourlocationsforthemostfrequentlyevaluatedtraits.On
thexaxisthetrialsappearintheirrespectiveyearandtreatments.Localitiesrepresentedincolors;blueforDarie´n,yellowforPopaya´n,greenfor
PalmiraandorangeforQuilichao.TreatmentsHP,MPandLP:high,moderateandlowphosphorus,I:Irrigation,D:drought,HAlandMAl:Highand
moderatealuminum.Traitvaluesofcontrolsaremarkedwithindicatedsymbols.G3513andG4017wereonlyusedatthelocalitiesofDarie´n,Popaya´n,
Palmira,VAX1andBAT477inQuilichao.LSDisnotedbeloweachplot.
https://doi.org/10.1371/journal.pone.0202342.g001
DroughtandlowPstress
Droughtstressandnon-stresstreatmentswerecomparedintwoseasonsatPalmira(Pal)loca-
tion.Droughtreducedyieldby13%and31%,respectively(Fig1andTable1).Lowestprecipi-
tationamongdroughttrialsduringpodfillingstagewasrecordedin2002(88mmvs147mm,
S1Fig),leadingtostrongeryieldreductionandlowerseedweight(Fig1).Similarly,yieldcom-
ponentsPNoandSdNowerediminishedin2002,by40%byand41%,respectively,whereas
seedsizewasnotaffected(S2Fig).Biomasstraitsweresignificantlyreducedbydroughtinthe
Pal2002trials(CB=42%,LAI=26%LB=28%,SBMP=38%,PBMP=54%).Incontrast,in
Pal2000wherethedroughtwaslessintenseandmorerainfallcoincidedwiththepodfilling
phasenomajorreductionsinbiomasswereobserved.Drymatterredistributiontraitvalues
wereelevatedunderdrought,suggestingbetterallocationofdrymatterresourcestoseed
understress(Table1,S2Fig).ThelackofthisdatainthePalmira2000trialdidnotallowusto
makeacomparisonbetweenthesetraitsbetweenthetwoyears.InmosttrialsshoottraitsLAI
andLBshowedpositiveyieldcorrelations,however,indroughtconditionsnegativeyield
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QTLdetectionforabioticstresstoleranceincommonbean(PhaseolusvulgarisL.)
Table1. PhenotypicevaluationsoftheBAT881xG21212populationin15differenttrialsatfourlocationsinColombia.
Locations Darien Popayan Palmira Quilichao
Year/Environment 97 98 98LP 99HP 99 05HP 00D 00I 02D 02I 02MAl 02MAl 03aHAl 03bHAl 03bHAl
HP HP LP LP MP MP MP HP
Yieldcomponents
Yd,Yield 3077 3737 1069 465 392 2098 1372 1579 671 975 1045 561 527 552 902
100SdW,Seedweight 17.2 14.6 23.3 19.7 19.1 17.9 17.9 28.9 24.4 23.9 20.9 23.2
PNo,Podnumber 274 174 291 149 117 101 146
SdNo,Seednumber 1198 663 1115 570 474 413 654
Plantvigor
CB,Canopybiomass 2792 3656 3305 1912 3305 2188 1561 1105 813 1360
LAI,Leafareaindex 1.809 1.740 2.350 1.955 1.273 0.936 0.950 1.477
LB,Leafbiomass 645 828 689 727 1015 687 448 329 320 470
SBMP,Stembiomass 715 897 673 518 840 634 248 282 267 450
PBMP,Podbiomass 1433 1932 1942 668 1449 866 1313 493 226 440
Drymatterredistribution
HI,Harvestindex 91.3 56.8 49.5 93.6 92.3 81.9 90.4
PHI,Podharvestindex 76.3 75.7 73.8 77.2 77.9 78.6 79.9
PPI,Podpartitioningindex 119.2 75.0 66.5 120.7 118.2 104.1 113.0
TNC_Sh,Totalnon-structural 110 147 342 138 203 103 127
carbohydratesinshoot
TNC_Sd,TNCinseed 276 266 393 376 383 367
Phenologicaltraits
DF,Daystoflowering 40.6 41.0 43.7 45.6 46.8 47.9 38.9 37.5 40.5 39.5 38.9
DH,Daystoharvest 77.9 81.6 82.4 101.3 96.7 104.2 77.6 85.0 80.1 81.4 84.8 78.0 81.5 76.5 76.3
Mineralnutrients
ShN,Shootnitrogen 2.78 2.66 2.45 3.57 2.92 3.54 3.27
SdN,Seednitrogen 4.04 3.60 3.68 3.64 3.47 3.31
NUE_Sh,Nuseefficiencyinshoot 36.8 38.4 42.8 28.5 35.0 28.5 31.0
NUE_Sd,NUEinseed 11.7 10.2 18.3 15.6 16.9 17.8 18.4
ShP,ShootP 0.304 0.345 0.231 0.281 0.298 0.282 0.314
SdP,SeedP 0.416 0.590 0.487 0.645 0.310 0.389 0.393 0.431
PUE_Sh,Puseefficiencyinshoot 338 296 443 361 344 361 324
PUE_Sd,Puseefficiencyinseed 105 78 190 196 167 228 193
Differentialstressresponses
%YdL_D,Yieldlossunderdrought 19.74
%YdL_LP,YieldlossunderlowP 50.87
%100SdWL_D,Seedweightreductionunderdrought 6.67
%100SdWL_LP,SeedweightreductionunderlowP 8.44
%DHL_D,Daystoharvestreductionunderdrought 5.29
%DHL_LP,DaystoharvestreductionunderlowP -0.21
%DFL_LP,DaystofloweringreductionunderlowP -3.97
Phenotypicmeansareshown,moredetailsinS2Fig.HP,MP,LPrepresenthigh,moderateorlowPconditions.D:drought,I:irrigated.MAlandHAl:moderateand
highAl.
https://doi.org/10.1371/journal.pone.0202342.t001
correlationswereobserved(S2Table).SdNwashigherunderdroughtconditions,whereas
SdPwasreduced.
EffectsoflowPstresswereevaluatedcomparingstressandnon-stresstrialsatthreeloca-
tions.YieldreductionsweremoresevereinDarie´n(Dar)wherelowPreducedyieldsby71%
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QTLdetectionforabioticstresstoleranceincommonbean(PhaseolusvulgarisL.)
(Fig1,S2Fig,Table1).InPopaya´n(Pop)yieldwasloweredby16%,inaseasonprobably
affectedbyexcessiverainfall(S1Fig).InPop99_HPexclusivelypositiveyieldcorrelationswith
DFandDHwerefound,incontrast,inPop99_LPallcorrelationswerenegative(S2Table).
Thiscondition-effectwasnotobservedinDarorQuilichao(Qui).IntrialsatQuiyieldsunder
moderatePlevels(MP)werereducedby39%comparedtohighP(HP)conditions.Effectsof
reducedPonyieldcomponents,biomassanddrymatterredistributiontraitswereonlyevalu-
atedinQui.AllwerefoundtobediminishedinmoderatePconditions,includingalldrymat-
terredistributiontraitswhichwasunexpected.Eventhoughshootbiomassproductionwas
reduced,lowervaluesofHI,PHIandPPIindicatethatseedformationandfillingwereeven
morecompromised.SdPwasreducedby9%underlowPconditions.Qui2002trialsunder
moderateAlstressdidnotshowthesametendenciesasthetrialinMPconditionsout-yielded
theHPtreatment,however,theseweresownindifferentseasons,whereclimaticdifferences
likelyhavingalargereffectontraitsthansoilPlevels.Inaddition,goodperformanceof
Qui02_MAl_LPtrialmaybeduetohigherorganicmattercontentinthesoil(S1Table).
Thedifferentialphenotypicexpressionunderstresswasevaluatedforthefourmajortraits
(Fig2).YieldwasreducedmoreunderLPthanunderdroughtacrosstheseexperiments.Most
significantcorrelationsbetweenyieldlossunderstress(%YdL_Dand%YdL_LP)andyield
wereasexpectednegativecorrelationsintherespectivestresstrials(droughtorLP,S2Table).
Furthermore,severalpositivesignificantyieldcorrelationswerefound,solineswithhigh
Fig2.FrequencydistributionsofdifferentialphenotypicresponsesunderdroughtandlowPconditions,expressedas%lossunderstressforfour
majortraits.BLUPswereusedtojoinallavailablestressandnon-stressdatasetswherestressandnon-stressconditionshadbeenevaluatedinthesame
season.%lossunderstressrepresentedincolors;bluefordrought(D)andyellowforlowP(LP).
https://doi.org/10.1371/journal.pone.0202342.g002
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QTLdetectionforabioticstresstoleranceincommonbean(PhaseolusvulgarisL.)
yieldsaremorelikelytoseeyieldsreducedunderstress.Negativecorrelationswereobserved
withpodandseednumberaswellaspodbiomass,suggestingthattheseyieldcomponenttraits
aidstresstolerance.Positivecorrelationswithmaturitytraitsindicatethatlatematuringlines
loosemoreyieldunderstress.Maturitytraitssawmorereductionsindroughttrials,underlow
PDFvalueswereevenextended.Accordingly,manymoresignificanttraitcorrelationswere
foundfor%DHL_Dcomparedto%DHL_LP,generallycontrastingtheDHcorrelations.
Qui2002andQui2003trialswereplantedonsoilsthatwereevaluatedtohavemoderate
andhighAllevels,respectively.NosignificanteffectattributedtodifferencesinsoilAlonyield
andothertraitscouldbedetectedacrossseasons.
SuperiorRILlinesthatperformedwellunderstressandacrosstrialswereidentified.
YieldsundermostseveredroughtandlowPstresstrialswerecomparedtoaverageyields
acrossall15trials(Fig3Aand3B).Highcorrelationsindicatethatmoststresstolerantlines
areamongthemostproductiveinalltrials,displayinghighgeneralyieldstability.Thismeans
thatselectionofbestlinesunderstressconditionsarealsogenerallywellperformingunder
optimalconditionsandviceversa.RILsR9,47,154,arethebestlinestocombinedrought
andcross-locationyieldstability,andanothersevenlinesareexcellentforboth.ForlowPtol-
erancethecorrelationwasalsopositive,butlessclearasthetop5%oflinesforeachcondition
donotoverlap.Thebestlinesthatcombinecross-locationyieldstabilityandlowPtolerance
areR30,45,80,andtheparentallineG21212,whichisexcellentinlowP.Takentogether,
droughtandlowPstresstreatmentshadsignificanteffectsonproductivity,yieldcomponents
andbiomass,however,variabilitybetweentrialsduetoclimaticandotherconditionswas
alsolarge.RILs1,80,and154canbeusefulasparentstodevelopmultiplestressresistant
genotypes.
Amongtheparentallines,G21212parentshowedhigheryieldandearlinessthanBAT881
underLPstressinPopandQui,droughtandcombinationsofhighAlandmoderateP(Fig1).
BAT881ontheotherhand,wassuperiorunderfavorableconditionsunderliningthecontrast-
ingbehavioroftheparentallines.TheadditionalcontrolsG3513andG4017usedintheDar
andPoplocalitiesweresimilarinyield,theirperformancebeinggenerallyintermediate
betweenthetwoparentsunderlowP.G3513performspoorlyinPal.Theadditionalchecks
DOR364,BAT477andVAX1selectedforcontrastingaluminumstressresponsearemostly
inferiortoG21212.
Effectoflocationsontraits
Comparisonsofphenotypicdatasetsshowedstrongcorrelationsamongtrials.DHshowedsig-
nificantpositivecorrelationsbetweennearlyalldatasets(Fig4A),demonstratingthatthedata
setisusefulforcomparativeanalysisandvariableresponseishighlystable.Yieldcorrelations
betweenlocationswereexpectedlylower,allsignificantcorrelationswereexpectedlypositive
(Fig4A).BestcorrelationswerefoundbetweentrialsatDarandPop,whichareathigheralti-
tudes(1457–1730masl,respectively)comparedtoloweraltitudesitesPalandQui(965–990
masl).FewersignificantyieldcorrelationswereobservedbetweenPalandQuilocations,prob-
ablyduetotheircontrastingsoilfertilityconditions.
Phenotypicdataformajortraitsvariedwidelyamongtrials(Fig1).Highestyieldswere
observedinDarlocationundernon-stressenvironmentswithaverageyieldsof3.7t/ha
(Table1).Popwasthemostvariable.LoweryieldswereobservedinPalandQui.Highaltitude
locationPoprecordedmostdelayedfloweringandmaturitywithhighestDFandDHvalues.
HighestseedsizeswereobservedatQuilocation,atraitnotevaluatedatDar.
Siteregressionanalysisbasedonyielddata(Fig4B)resultedinPop05_HPbeingthemost
distincttrial,Dar98_HPandDar97_HPappearclusteredtoeachother,differentfromallother
PLOSONE|https://doi.org/10.1371/journal.pone.0202342 August29,2018 10/26