RESEARCHARTICLE Evaluation of cultivated and wild genotypes of Lens species under alkalinity stress and their molecular collocation using microsatellite markers DharmendraSingh1*,ChandanKumarSingh1,YashPalSingh2,VijayataSingh3, RajendraSingh4*,RamSewakSinghTomar5,SatishKumarSanwal3,SourabhKarwa6, VinayKumarMishra2,SusheelKumarSarkar7,MadanPal6*,ArunKumar8,Rajendra KumarYadav9,ParbodhChanderSharma3 a1111111111 1 DivisionofGenetics,ICAR-IndianAgriculturalResearchInstitute,NewDelhi,India,2 ICAR-Regional a1111111111 StationofCentralSoilSalinityResearchInstitute,Lucknow,India,3 ICAR-CentralSoilSalinityResearch a1111111111 Institute,Karnal,India,4 DivisionsofSoilScienceandAgriculturalChemistry,ICAR-IndianAgricultural a1111111111 ResearchInstitute,NewDelhi,India,5 ICAR-NationalResearchCentreonPlantBiotechnology,Pusa a1111111111 Campus,NewDelhi,India,6 DivisionofPlantPhysiology,ICAR-IndianAgriculturalResearchInstitute,New Delhi,India,7 ICAR-IndianAgriculturalStatisticalResearchInstitute,NewDelhi,India,8 NationalPhytotron Facility,ICAR-IndianAgriculturalResearchInstitute,NewDelhi,India,9 DepartmentofGeneticsandPlant Breeding,ChandraShekharAzadUniversityofAgricultureandTechnology,Kanpur,India *[email protected](DS);[email protected](MP);[email protected] OPENACCESS (RS) Citation:SinghD,SinghCK,SinghYP,SinghV, SinghR,TomarRSS,etal.(2018)Evaluationof Abstract cultivatedandwildgenotypesofLensspecies underalkalinitystressandtheirmolecular collocationusingmicrosatellitemarkers.PLoS Inthisstudy,285lentilgenotypeswerephenotypedunderhydroponicandalkalinefieldcon- ONE13(8):e0199933.https://doi.org/10.1371/ ditions.SignificantgenotypicvariationforalkalinitystresswasobservedamongthesixLens journal.pone.0199933 speciesscreenedhydroponicallyandinthefieldhavingpHupto9.1.Thecrucialparame- Editor:MukeshJain,JawaharlalNehruUniversity, ters,likewholeNa+andK+contentsandtheNa+/K+ratioat40mMNaHCO werefoundsig- 3 INDIA nificantlycorrelatedwithseedlingsurvivabilityunderhydroponics(r=-0.95,r=0.93and Received:February21,2018 -0.97).Genotypes,rankedonthebasesofseedyield,restricteduptakeofNa+withthick Accepted:June15,2018 pitharea,increasedvascularbundles,lessH2O2productionandlowNa+/K+ratio,were foundimportantphysio-anatomicaltraitsforalkalinitystresstolerance.Theproperregula- Published:August13,2018 tionofNa+uptakewasfoundformaintaininghigherK+.Thisrelationshipisprobablythe Copyright:©2018Singhetal.Thisisanopen mainfactorresponsibleforabettermechanismfortolerancetohighpHupto9.1intolerant accessarticledistributedunderthetermsofthe CreativeCommonsAttributionLicense,which breedinglinesPDL-1andPSL-9(cultivars)andILWL-15,ILWL-192andILWL-20(wild permitsunrestricteduse,distribution,and accessions).BasedonUPGMAdendrogram,allthegenotypeswereclusteredintofour reproductioninanymedium,providedtheoriginal diversegroups.DMRTwasimpliedwithinthegrouptodifferentiategenotypesbasedon authorandsourcearecredited. phenotypicresponseunderalkalinitystress.Theseresultscanbeutilizedforselecting DataAvailabilityStatement:Allrelevantdataare diverseparentsfordevelopingalkalinitytolerantgenotypes. withinthepaperanditsSupportingInformation files. Funding:FundedbyIndianAgriculturalResearch Institute,NewDelhi,andProjectno-JAN09/16, (DS),http://www.iari.res.in/. Competinginterests:Theauthorshavedeclared thatnocompetinginterestsexist. PLOSONE|https://doi.org/10.1371/journal.pone.0199933 August13,2018 1/25 PhynotypicandgeneticdiversityevaluationofLensspeciesunderalkalinitystress Introduction Soilsalinityandalkalinityarecommonsevereconstraintstocropproductivity.Intheworld, about830millionhectaresoftheareaareaffectedbysalinityandalkalinity,outofwhich434 millionhectaresareaisalkaline[1].Alkalinesoilisgenerallydominatedbyexcesssodiumon exchangesitesandhasahighconcentrationofcarbonate/bicarbonateanions,whichadversely affectthephysicalandnutritionalpropertiesofthesoil.Thecarbonate/bicarbonateionsare majorcontributorsofsoilalkalinity.PresenceoftheseinexcessNa+,highpHandosmotic stressleadtodrasticreductioninplantgrowthanddevelopment.Oneofthemajorapproaches usedtomanagealkalinesoilsandwater,istodevelopalkalinitytolerantgenotypes.Thiscanbe achievedbyharnessinginter-specificorintra-specificvariability. Lentil(LensculinarisMedikus),isanimportantlegume,providingqualityprotein,carbohy- drates,fibreandmineralsforthehumansandfodderforlivestock.Globally,itiscultivatedon 3.6millionhaareawithaproductionof3.4MT[2].However,itisanalkalinesensitivecrop andyieldisdrasticallyreducedunderhighalkalinity.Highlevelsofalkalinitystressinsoilor irrigationwateradverselyaffectseedgermination,growthandproductivity[3,4].Themost evidentsymptomsofalkalinestressonplantsaretheinductionofleafchlorosisandstunted growthduetohigheruptakeofNa+andloweruptakeofnutrients[5,6,7].Salinity/alkalinity alsodistortsanatomicalstructures.Amajorreductioninthedimensionofvasculartissuewas alsoobservedintherootsofChlorisgayanaKunthundersalinitystress[8].Limitedinforma- tionisavailableontheanatomicaldeformityoflegumeslikepea[9],kidneybean[10]and lotus[11]duetoalkalinitystress. Screeningofgermplasmatseedlingstageunderthehydroponicconditionisareadily acceptabletoolasitisbasedonasimplecriterionofselection[12].Screeningforalkalinitytol- eranceshouldalsobecarriedoutinthefieldatsiteswherealkalinitystressisaproblem.How- ever,screeninginthefieldmaybedifficultbecauseofheterogeneityandisalsoinfluencedbya largenumberofenvironmentalfactors.Thesedifficultiescanbeovercomebyusinghydro- ponicbasedscreeningsystemasitismoreeffectiveasitprovidesconsistentcontroloverpHin cropplants[12,13].Hydroponicscreeningsystemhasbeenfoundmostconvenientandscien- tificbecauseseedlingsrequirelessspaceandtolerantonesmayberecoveredforseedproduc- tionpurpose.Therecanalsobepossibilityofpre-selectionofbreedinglines,progeniesand cultivarsbeforefieldevaluation.Evaluationofcropplantsfortolerancetoalkalinitystresshas beenwelldocumentedinmustard[12]andbeanlines[14]atearlygrowthstageunderhydro- ponicandalkalinefieldconditions,respectively.However,initialscreeninginhydroponicand laterinfieldconditionscanbethebeststrategyforaccuratephenotypingofalkalinitytoler- ance.Therearevariousreportspublishedontheeffectsofsalinitystressonplants,butlimited informationisavailableontheeffectsofalkalinitystress. Molecularanalysisofcharacterizedgenotypescanbedirectlyutilizedbylentilbreedersto selectparentsforcontrastingcharactersassociatedwithalkalinitystresstolerance.Different typesofmolecularmarkershavebeensuccessfullyusedforidentificationofgenotypes,diver- sityandgene/QTLanalysisinlentil[15,16,17,18].Simplesequencerepeat(SSR)markers havebeenextensivelyusedfortheassessmentofgeneticdiversityinlentil[19,20,21]asthey areefficient,easytouseandhavehighreproducibilityandco-dominance.However,nowork hasbeendonesofarontheevaluationofLensspeciesbyusingmorpho-anatomicalandphysi- ologicaltraitsandmolecularmarkers.Therefore,thisstudywasplannedwithfollowingobjec- tives:(1)morho-anatomicalandphysiologicalcharacterizationofgenotypesforalkalinity stresstoleranceunderhydroponics,(2)fieldvalidationofgenotypesforseedyieldandphysio- logicalcharacterizationforalkalinitytoleranceand(3)diversityanalysisofgenotypesusing microsatellitemarkers. PLOSONE|https://doi.org/10.1371/journal.pone.0199933 August13,2018 2/25 PhynotypicandgeneticdiversityevaluationofLensspeciesunderalkalinitystress Materialandmethods Plantmaterials TheexperimentwasconductedatNationalPhytotronFacility,ICAR-IndianAgricultural ResearchInstitute,NewDelhi,India.Twohundredeightyfivegenotypeswereusedforselec- tionofdiversegenotypesunderalkalinitystressconditions.Thedetailssuchasoriginofcoun- try,reactiontothealkalinityofgenotypesarepresentedinTable1.Thewildaccessions includedwereL.orientalis,L.odomensis,L.nigricans,L.ervoidesandL.lamottei,thefirsttwo belongtotheprimarygenepool,whilethelastthreetothesecondary/tertiarygenepool.Air temperatureintheNationalPhytotronFacilitywas22/18˚C(±2˚C),day/night;photoperiod was10/14h(light/dark)andtherelativehumiditywasapproximately45%. Evaluationofgenotypesunderalkalinitystress Seedsofallthegenotypesweresterilizedinsodiumhypochlorite(1%)for2-3minthenwashed threetimeswithdistilledwater.Twentyseedsofeachgenotypewereexposedto20and40mM concentrationofsodiumcarbonate(NaHCO )alongwithacontrolforevaluationforgermi- 3 nation.Thegerminationwasrecordedat12hintervalfor10days.Seedswereconsideredger- minated,whentheradiclehademergedandattained1mmlength.Germinationpercentage wascalculatedwiththeformula:Germinationpercent=(Totalnumberofseedsgerminated/ Totalnumberofseedssown)x100. TheLensspecieswerealsoassessedforalkalinitystresstoleranceatseedlingstage.Seven daysoldseedlingsofcultivatedand14doldseedlingsofwildspeciesweretransferredto hydroponicmediumandbufferedwith20mMand40mMsodiumbicarbonate(NaHCO )to 3 obtaindifferentpHlevels.ThepHofthenutrientsolutionwasobtainedto8.8and9.1and maintainedconstantusing1MKOHonadailybasis.Thenutrientsolutionwithoutbicarbon- atewasusedascontrol(pH7.2)whichwaspreparedaspernutrientcompositionsuggestedby Javidetal.[12].Thesolutionwasrenewedat2dintervaltomaintainnutrientconcentration. Thesolutionwasregularlyaeratedbybubblingairwithanaquariumpumpover15d.After15 dofalkalinitystress(20and40mMNaHCO ),datawererecordedonvisualsaltinjuries,seed- 3 lingsurvivability,seedlinggrowth(rootandshootlength)andbiomassaccumulation(fresh anddryweightofrootsandshoots).Allthegenotypesweregroupedintothreecategories accordingtotheirresponsetoalkalinitystressonplantsurvivalat20and40mMNaHCO 3 concentrationsasperthefollowingcriteria:(1)tolerantgenotypes,showing100%plantsur- vivalat20mMand(cid:20)61%plantsurvivalat40mMNaHCO (2)moderatelytolerantgeno- 3, types,showing(cid:20)87%survivalat20mM,whereasincaseof40mMthesurvivalwasonly (cid:20)50%,(3)sensitivegenotypesshowing(cid:20)67%plantsurvivalat20mMandnoplantsurvivalat 40mMNaHCO .Thesurvivedplantswerekeptinthehydroponicswithoutalkalinitystress 3 foraweekandthentransferredtofieldatnormalconditionstillthematurityforevaluationin termsofseedyield/plant.Forcomparison,thecontrolplants(withoutstress)werealsotrans- ferredtothefieldforrecordingseedyieldperplant.Thereductionpercentinyieldwascalcu- latedbytheformula:(seedyieldofstressedplants-seedyieldofcontrolplants)/(seedyieldof controlplants)x100.Alkalinitystresstolerancewasalsoestimatedonthebasisofvisualsalt injuryofplantsusing1–5scorescale,where:1=healthyplantswithnovisiblesymptomsof alkalinitystress,2=greenplantswithslightwilting,3=leavesturningyellowishgreenwith moderatewilting,4=leavesyellowishbrownwithseverewiltingand5=partialand completelydriedleavesand/orstem.Thescoreswereaveragedandusedfortheassessmentof alkalinitystresstolerancelevelofgenotypes.Eachtreatmentwasreplicatedthreetimes,with sixplantsineachtreatment.Ateachsampling,plantswereseparatedintorootsandshoots.To PLOSONE|https://doi.org/10.1371/journal.pone.0199933 August13,2018 3/25 PhynotypicandgeneticdiversityevaluationofLensspeciesunderalkalinitystress Table1. Genotypeswithdifferentoriginsandsensitivitytoalkalinitystress. S.No. Genotype Origin Type AlkR S.No. Genotype Origin Type AlkR 1 121–12 India GC S 144 ILL-590 Turkey GC S 2 1220–11 India BL MT 145 ILL-6002 ICARDA GC MT 3 210–11 India BL S 146 ILL-7349 Nepal GC S 4 330–12 India GC S 147 ILL-76037 ICARDA GC S 5 BM-4 Bangladesh Cult. S 148 ILL-7978 ICARDA GC S 6 DPL-62 India Cult. S 149 ILL-7979 ICARDA GC S 7 E-153 India GC S 150 ILL-7982 ICARDA GC S 8 FLIP-96-51 ICARDA GC S 151 ILL-8006 Bangladesh GC S 9 IG-109039 ICARDA GC S 152 ILL-8108 Argentina GC S 10 IG-111991 ICARDA LR S 153 ILL-8329 ICARDA GC S 11 IG-111996 ICARDA LR S 154 ILL-91887 ICARDA GC S 12 IG-112078 ICARDA LR S 155 ILL-9841 ICARDA GC S 13 IG-11210 ICARDA LR S 156 ILL-9900 ICARDA GC S 14 IG-112128 ICARDA LR S 157 ILL-9916 ICARDA GC S 15 IG-112131 ICARDA LR S 158 ILL-9941 ICARDA GC S 16 IG-112137 ICARDA LR S 159 ILL-9960 ICARDA GC S 17 IG-116551 ICARDA LR S 160 ILWL-06 Turkey Wild S 18 IG-129185 ICARDA LR S 161 ILWL-09 Syria Wild S 19 IG-129214 ICARDA LR S 162 ILWL-10 - Wild S 20 IG-129287 ICARDA LR S 163 ILWL-100 Turkey Wild S 21 IG-129291 ICARDA LR S 164 ILWL-104 Turkey Wild S 22 IG-129293 ICARDA LR S 165 ILWL-125 Syria Wild S 23 IG-129302 ICARDA LR S 166 ILWL-128 Syria Wild S 24 IG-129309 ICARDA LR S 167 ILWL-13 Italy Wild S 25 IG-129313 ICARDA LR S 168 ILWL-133 Syria Wild S 26 IG-129315 ICARDA LR S 169 ILWL-137 Syria Wild S 27 IG-129317 ICARDA LR S 170 ILWL-142 Syria Wild S 28 IG-129319 ICARDA LR S 171 ILWL-15 France Wild T 29 IG-129372 ICARDA LR S 172 ILWL-165 Syria Wild S 30 IG-129560 ICARDA LR S 173 ILWL-184 Syria Wild S 31 IG-12970 ICARDA LR S 174 ILWL-185 Syria Wild S 32 IG-130033 ICARDA LR S 175 ILWL-192 Syria Wild T 33 IG-130219 ICARDA LR S 176 ILWL-20 Palestine Wild T 34 IG-130272 ICARDA LR S 177 ILWL-203 Turkey Wild S 35 IG-134342 ICARDA LR S 178 ILWL-221 Turkey Wild S 36 IG-134347 ICARDA LR S 179 ILWL-227 Syria Wild S 37 IG-134356 ICARDA LR S 180 ILWL-23 Italy Wild S 38 IG-135424 - Wild S 181 ILWL-237 Syria Wild S 39 IG-135428 - Wild S 182 ILWL-238 Syria Wild S 40 IG-136607 ICARDA LR S 183 ILWL-253 Syria Wild S 41 IG-136608 - Wild S 184 ILWL-269 Turkey Wild S 42 IG-136612 Turkey Wild S 185 ILWL-29 Spain Wild S 43 IG-136614 Italy Wild S 186 ILWL-292 Turkey Wild MT 44 IG-136618 Croatia Wild S 187 ILWL-3 Turkey Wild S 45 IG-136620 Slovenia Wild S 188 ILWL-314 Turkey Wild S 46 IG-136626 Israel Wild S 189 ILWL-320 Turkey Wild MT 47 IG-136637 France Wild S 190 ILWL-321 Turkey Wild MT (Continued) PLOSONE|https://doi.org/10.1371/journal.pone.0199933 August13,2018 4/25 PhynotypicandgeneticdiversityevaluationofLensspeciesunderalkalinitystress Table1. (Continued) S.No. Genotype Origin Type AlkR S.No. Genotype Origin Type AlkR 48 IG-136652 Israel Wild S 191 ILWL-334 Jordan Wild S 49 IG-136653 Israel Wild S 192 ILWL-377 Tajiskistan Wild S 50 IG-136673 Turkey Wild S 193 ILWL-340 Jordan Wild S 51 IG-136788 Syria Wild S 194 ILWL-35 Turkey Wild S 52 IG-140910 Azerbaijan Wild S 195 ILWL-350 Syria Wild S 53 IG-149 ICARDA LR S 196 ILWL-357 Syria Wild S 54 IG-129304 ICARDA LR S 197 ILWL-361 Syria Wild S 55 IG-49 ICARDA LR S 198 ILWL-362 Syria Wild S 56 IG-5320 ICARDA LR S 199 ILWL-366 Syria Wild S 57 IG-69540 ICARDA LR S 200 ILWL-370 Syria Wild S 58 IG-69549 ICARDA LR S 201 ILWL-398(A) Lebanon Wild S 59 IG-70174 ICARDA LR S 202 ILWL-401 Lebanon Wild S 60 IG-70230 ICARDA LR S 203 ILWL-415 Syria Wild MT 61 IG-71352 ICARDA LR S 204 ILWL-418 Syria Wild S 62 IG-71630 ICARDA LR S 205 ILWL-428 Spain Wild S 63 IG-71646 ICARDA LR S 206 ILWL-430 Spain Wild S 64 IG-71685 ICARDA LR S 207 ILWL-436 Turkey Wild MT 65 IG-71710 ICARDA LR S 208 ILWL-437 Turkey Wild S 66 IG-73717 ICARDA LR S 209 ILWL-438 Turkey Wild S 67 IG-73798 ICARDA LR S 210 ILWL-44 Slovenia Wild S 68 IG-73802 ICARDA LR S 211 ILWL-447 Turkey Wild S 69 IG-73816 ICARDA LR S 212 ILWL-462 Turkey Wild S 70 IG-73945 ICARDA LR S 213 ILWL-464 Syria Wild S 71 IG-75920 ICARDA LR S 214 ILWL-472 - Wild S 72 IG-9 ICARDA LR S 215 ILWL-55(2) Israel Wild S 73 IG-936 ICARDA LR S 216 ILWL-58 Turkey Wild S 74 ILL-10030 ICARDA GC S 217 ILWL-60 Turkey Wild S 75 ILL-10031 ICARDA GC S 218 ILWL-69 Uzbekistan Wild S 76 ILL-10032 ICARDA GC S 219 ILWL-83 Turkey Wild S 77 ILL-10034 ICARDA GC S 220 ILWL-95 Turkey Wild S 78 ILL-10040 ICARDA GC S 221 IPL-406 India Cult. S 79 ILL-10041 ICARDA GC S 222 JL-3 India Cult. S 80 ILL-10043 ICARDA GC S 223 L-404 India BL S 81 ILL-10056 ICARDA GC S 224 L-4076 India Cult. S 82 ILL-10061 ICARDA GC S 225 L-4078 India BL S 83 ILL-10062 ICARDA GC S 226 L-4147 India Cult. S 84 ILL-10063 ICARDA GC S 227 L-4578 India BL S 85 ILL-10074 ICARDA GC S 228 L-4590 India Cult. S 86 ILL-10075 ICARDA GC S 229 L-4594 India Cult. S 87 ILL-10082 ICARDA GC S 230 L-4602 India BL S 88 ILL-10133 ICARDA GC S 231 L-4603 India BL S 89 ILL-10234 ICARDA GC S 232 L-4605 India BL S 90 ILL-10266 ICARDA GC S 233 L-4618 India BL MT 91 ILL-10270 ICARDA GC S 234 L-4619 India BL S 92 ILL-1046 ICARDA GC S 235 L-4620 India BL S 93 ILL-10756 ICARDA GC S 236 L-4650 India BL S 94 ILL-10794 ICARDA GC S 237 L-4701 India BL S (Continued) PLOSONE|https://doi.org/10.1371/journal.pone.0199933 August13,2018 5/25 PhynotypicandgeneticdiversityevaluationofLensspeciesunderalkalinitystress Table1. (Continued) S.No. Genotype Origin Type AlkR S.No. Genotype Origin Type AlkR 95 ILL-10795 ICARDA GC S 238 L-5253 India BL S 96 ILL-10804 ICARDA GC S 239 L-7752 India BL S 97 ILL-10805 ICARDA GC S 240 L-7818 India BL S 98 ILL-10806 ICARDA GC S 241 L-7903 India BL MT 99 ILL-10807 ICARDA GC S 242 L-7905 India BL S 100 ILL-10809 ICARDA GC S 243 L-7920 India BL S 101 ILL-10810 ICARDA GC S 244 LC-270-804 India BL S 102 ILL-10811 ICARDA GC S 245 LC-282-1077 India BL S 103 ILL-10812 ICARDA GC S 246 LC-282-1110 India BL MT 104 ILL-10817 ICARDA GC S 247 LC-282-1444 India BL S 105 ILL-10818 ICARDA GC S 248 LC-282-896 India BL S 106 ILL-10819 ICARDA GC S 249 LC-284-116 India BL S 107 ILL-10820 ICARDA GC S 250 LC-284-1209 India BL S 108 ILL-10823 ICARDA GC S 251 LC-285-1344 India BL S 109 ILL-10826 ICARDA GC S 252 LC-289-1444 India BL S 110 ILL-10827 ICARDA GC S 253 LC-289-1447 India BL S 111 ILL-10831 ICARDA GC S 254 LC-292-1485 India BL S 112 ILL-10834 ICARDA GC S 255 LC-292-1544 India BL S 113 ILL-10835 ICARDA GC S 256 LC-292-997 India BL S 114 ILL-10836 ICARDA GC S 257 LC-300-1 India BL S 115 ILL-10837 Turkey GC S 258 LC-300-11 India BL S 116 ILL-10848 Bangladesh GC S 259 LC-300-12 India BL S 117 ILL-10857 ICARDA GC S 260 LC-300-13 India BL S 118 ILL-10893 ICARDA GC S 261 LC-300-15 India BL MT 119 ILL-10894 ICARDA GC S 262 LC-300-16 India BL S 120 ILL-10897 ICARDA GC S 263 LC-300-2 India BL S 121 ILL-10913 ICARDA GC S 264 LC-300-3 India BL S 122 ILL-10915 ICARDA GC S 265 LC-300-4 India BL S 123 ILL-10917 ICARDA GC S 266 LC-300-6 India BL S 124 ILL-10921 ICARDA GC S 267 LC-300-7 India BL MT 125 ILL-10922 ICARDA GC S 268 LC-300-8 India BL S 126 ILL-10951 ICARDA GC S 269 LC-300-9 India BL MT 127 ILL-10953 ICARDA GC S 270 LC-74-1-51 India BL S 128 ILL-10960 ICARDA GC S 271 PAL-3 ICARDA GC MT 129 ILL-10961 ICARDA GC S 272 PDL-1 ICARDA BL T 130 ILL-10963 ICARDA GC S 273 PDL-2 ICARDA BL MT 131 ILL-10964 ICARDA GC S 274 PKVL-1 India Cult. S 132 ILL-10965 ICARDA GC S 275 PL-1 India Cult. S 133 ILL-10967 ICARDA GC S 276 PL-4 India Cult. S 134 ILL-10969 ICARDA GC S 277 PL-406 India Cult. S 135 ILL-10970 ICARDA GC S 278 PL-5 India Cult. S 136 ILL-10972 ICARDA GC S 279 PSL-1 ICARDA GC MT 137 ILL-1970 Ethiopia GC S 280 PSL-7 ICARDA GC MT 138 ILL-358 Mexico GC S 281 PSL-9 India BL T 139 ILL-3829 ICARDA GC S 282 SEHORE-74-3 India Cult. S 140 ILL-4605 Argentina Cult. S 283 SKL-259 India BL S 141 ILL-560 Turkey GC MT 284 VL-507 India Cult. S (Continued) PLOSONE|https://doi.org/10.1371/journal.pone.0199933 August13,2018 6/25 PhynotypicandgeneticdiversityevaluationofLensspeciesunderalkalinitystress Table1. (Continued) S.No. Genotype Origin Type AlkR S.No. Genotype Origin Type AlkR 142 ILL-5722 ICARDA GC MT 285 WBL-77 India Cult. S 143 ILL-5883 Jordan GC S AlkR=AlkanityReaction;GC=Germplasmcollection;BL=BreedingLines;LR=Landraces;S=Sensitive;T=Tolerant;MT=ModeratelyTolerantinAlkR https://doi.org/10.1371/journal.pone.0199933.t001 removetheexternaltreatmentsolutions,rootswerewashedthreetimesindistilledwater.Sur- facewaterwasblottedoffusingpapertowelsandfreshmasswasmeasured.Tissueswereoven driedat65˚Cfor72handdrymasswasdetermined. Analysisofmineral. Formineralanalysis,dryplantmaterialofselectedplantsofcontrol andtreatedgenotypesweregroundinapestleandmortaranddigestedinH SO .Concentra- 2 4 tionofNa+andK+ofthedigestwasdeterminedbyFlamePhotometer(Systronics128,India). Na+andK+werecalculatedonpercentdryweightbasis. DetectionofH O level. Asetof15alkalinetreatedroottipsfromeachtreatmentwere 2 2 collectedandwashedin200mMCaCl solutionfor10min.Theseroottipswereexcisedand 2 placedintoasolutioncontaining200mMCaCl (pH4.4)and10mMFDA(Fluoresceindiace- 2 tate)for15min.TheFDAfluorescencewasthendetectedunderafluorescencemicroscope [22]. Rootandshootanatomy. Forstudyingrootanatomyundercontrolandalkalinitystress, methodfollowedbyKrishnamurthyetal.[23]withslightmodificationwasused.Freehand sectionswerecutandtheselectedsectionswerestainedwith50%toluidineblue.Thesesec- tionswerewashedtoremoveexcessstainandthenmountedindistilledwater.Pictureswere takenusinganopticalmicroscope(Zeiss,AXIOSKOP2)at10×magnification. Evaluationofgenotypesunderalkalinefieldconditions FieldexperimentswereconductedatCentralSoilSalinityResearchInstitute,Regional ResearchStation,Farm,Lucknow,India(80046’32”E26o47’45”Nand120mabovemean sealevelandChandraShekharAzadUniversityofAgricultureandTechnology,Research Farm,Kanpur,India(26.4912˚N,80.3073˚Eand125.9onabovemeansealevel)during 2013–14and2014–15.TheinitialsoilpHduring2013–14atLucknowandKanpursiteswas 9.0and9.1withtheexchangeablesodiumpercentage(ESP)22.0and24.5,respectively.During 2014–2015,thepHvalueattheexperimentalfarmatLucknowwas9.5withthecorresponding ESP40.0.ThepHofnormalsoilwas7.6andESP10.5atKanpur.Torecordseedyieldper plant,atotalof236and224genotypesweretestedatKanpur(pH9.1)duringtheyear2013–14 and2014–15,respectively.Similarly,224genotypeswereevaluatedduring2013–14and2014– 15atLucknowunderpHof9.0and9.5,respectively.Thevariationinanumberofgenotypes wasduetoaninsufficientnumberofseedsproducedbywildaccessionsforfurtherevaluation underalkalinestress.Tenplantsfromeachgenotypewereharvestedat75percentphysiologi- calmaturitytorecordtheseedyield.Thetotalrainfallduringcroppingseasonof2013–14and 2014–15atKanpurandLucknowwas148.8mmand212.0mmand90.2mmand72.6mm, respectively.Themaximumaveragetemperatureduring2013–14and2014–15atKanpurand Lucknowwas23.9˚Cand21.4˚Cand24.7˚Cand24.2˚Candtheminimum8.7˚Cand14.4˚C and11.8˚Cand10.8˚C,respectively.Thecropwasraisedwithrecommendedculturalprac- tices.PlantsweresampledforNa+andK+analysesatseedlingandfloweringstagesandseed yieldwasrecordedatmaturity.Na+andK+wereestimatedfollowingthemethodofJackson’s usingaFlamephotometer[24]. PLOSONE|https://doi.org/10.1371/journal.pone.0199933 August13,2018 7/25 PhynotypicandgeneticdiversityevaluationofLensspeciesunderalkalinitystress Molecularanalysis DNAwasextractedusingmodifiedCTABmethod[25]andwasquantifiedusingaspectropho- tometer.ThirtySSRmarkerswhichwerepolymorphicbetweentwogenotypesthatashowed contrastingresponsetoalkalinitystress,alongwithadditional38arbitrarySSRmarkerswere usedforscreening285genotypes.Thesemarkerswereselectedbasedonearlierlentilreports published[16,26,27]. PCRamplificationswereperformedin10μlreactionvolume,consistingof1XPCRbuffer, 1.5mMMgCl and0.5μMprimerseachofforwardandreverse,1mMdNTP,0.5UTaq DNA 2 polymeraseand50ngtemplateDNA.PCRcyclingconditionswereasfollows:Pre-denatur- ationat94˚Cfor3minfollowedby40cyclesofdenaturationat94˚Cfor30sec,annealingat 55˚Cfor30sec,elongationat72˚Cfor1minwithafinalextensionat72˚Cfor10min.PCR amplifiedproductswereseparatedon3%ultrahighresolutionagarosegelsanddocumented usingVilberLourmatGelDocumentationSystem. Geneticdiversityanalysis. Thegeneticprofileof285genotypeswasscoredonthebasisof differenceinallelesizeusing30SSRmarkerswhichwerepolymorphictoalkalinitytolerant andsensitivegenotypeandthencomparedwiththeresultsobtainedbyadding38moreSSR markerswhichwereselectedarbitrarily.Themajorallelefrequency,PICandgeneticdistance basedclusteringwereperformedwithUnweightedPairGroupMethodforArithmeticaverage (UPGMA)usingPowerMarkerv3.25software[28]andthedendrogramwasconstructedfol- lowingbootstrapanalysiswith1000permutationsforallthegenotypesusingMEGA4.0soft- ware[29].Thepopulationstructurewasdeterminedon285genotypescomprisingbothwild andcultivatedonesusingStructure2.3.4software[30].ThenumberofsubgroupKwasesti- matedby7independentrunsforeachK(1to15)usingtheadmixedmodelwith1,00,000 MonteCarloMarkovChain(MCMC)replicatesafteraburn-inof10,000replicates.Thestruc- tureoutputswerevisualizedusingStructureHarvesterfromwhichEvanoplotswerecon- structed[31]. Statisticalanalysis. Duncan’sMultipleRangeTest(DMRT)(P=0.05)wasfollowedto findoutdifferencesamongclustersforsignificancebyusingSAS9.4software.Dataformor- pho-physiologicaltraitswereanalysedusingtwo-wayANOVAtodetermineifsignificantdif- ferenceswerepresentamongthemeans.Varianceswerecheckedbyplottingresidualvs.fitted valuestoconfirmthehomogeneityofthedata.Differencesamongthemeanvalueswere assessedbyLeastSignificantDifferences(LSD).Relationshipsbetweenindividualvariables wereexaminedusingsimplecorrelationswhichwerealsoperformedusingSAS9.4software. Spearman’srankcorrelationtest(rs)wasusedtoexamineconsistencyintherankingsofgeno- typesforalkalinitytoleranceandseedyieldbetweenthehydroponicandfieldexperiments. Results Phenotypingofalkalinitystresstoleranceunderhydroponics Theadverseeffectsofalkalinitystressongenotypeswerefoundmorepronouncedat40mM NaHCO thanat20mMNaHCO (Figs1and2,S1Table).However,therewasnoadverse 3 3 effectongerminationofwildaccessions(ILWL-15,ILWL-20andILWL-192)andtolerant breedinglines(PDL-1andPSL-9)at40mMNaHCO .Themostsensitivecultivars(L-4076 3 andL-4147)showedamaximumreductioningerminationatasimilarsaltconcentration(Fig 1A).Therewasasignificantreductioninseedlinggrowthandbiomassaccumulationat40 mMNaHCO .However,thereductionwaslowerintolerantwildaccessionsandbreeding 3 linesthanmoderatelytolerantandsensitiveones(Fig1Band1C).Tolerantgenotypescould notexhibitwiltingoranyvisiblesymptomsunderalkalinitystress(20mMNaHCO )upto15 3 PLOSONE|https://doi.org/10.1371/journal.pone.0199933 August13,2018 8/25 PhynotypicandgeneticdiversityevaluationofLensspeciesunderalkalinitystress Fig1. Effectsofalkalinitystress(40mMNaHCO)ongermination(a),seedlinggrowth(b),biomassaccumulation 3 (c),alkalinityscores(d)andsurvivalpercent(e)oflentilgenotypesunderhydroponiccondition. https://doi.org/10.1371/journal.pone.0199933.g001 d,whereasmoderatelytolerantandsensitivegenotypesexhibitedvaryingdegreesofwilting andleafchlorosiswithascoreof1.0to1.3and2.1to2.7,respectively(Fig2).At40mM NaHCO ,tolerantgenotypesexhibitedmoderatesaltinjurycomparedtomoderatelytolerant 3 andsensitivegenotypes(Fig1D)(S1Fig).Alkalinitystressof20and40mMNaHCO concen- 3 trationaffectedtheseedlingsurvivaldifferentlyintolerant,moderatelytolerantandsensitive genotypes.Fivegenotypesamong285,showedhighestseedlingsurvivalattheendofthe experiment(15d)under40mMNaHCO .Tolerantbreedinglines(PDL-1andPSL-9)and 3 wildaccessions(ILWL-15,ILWL-192andILWL-20)exhibitedmaximumseedlingsurvival. Moderatelytolerantgenotypesshowedintermediateseedlingsurvival,whereasthemajorityof thesensitivegenotypesdidnotsurviveatasimilarsaltconcentration(Fig1E).Thesurvived tolerantgenotypesexhibitedlargereductioninseedyieldperplantat40mMNaHCO as 3 comparedto20NaHCO underhydroponicconditions(Fig3). 3 ConsiderabledifferencesinNa+andK+contentsinboththerootsandshootswere observedunderalkalinitystress.ItwasevidentthatNa+accumulatedmoreinrootsandshoots ofsensitivegenotypesthanintolerantonesat20mMand40mMNaHCO (Fig4Aand4B). 3 However,itisworthmentioningherethatmostsensitivecultivars(L-4076andL-4147),accu- mulatedthehighestamountofNa+inrootsandshootsat40mMNaHCO ).TheNa+contents 3 inrootsandshootswereminimumintolerant(PDL-1,PSL-9ILWL-15,ILWL_192and ILWL-20)genotypesat40mMNaHCO .Potassiumconcentrationdeclinedconsiderablyin 3 boththetolerantandsensitivegenotypesatboth20and40mMbicarbonate(NaHCO )con- 3 centration(Fig4Cand4D).TheseresultsevidencedthattheK+concentrationinrootsand shootswasminimuminsensitivecultivars(L-4076andL-4147),whichwasmarkedlyaffected by40mMNaHCO underhydroponics.ThesimilartrendsofaccumulationofNa+andK+in 3 PLOSONE|https://doi.org/10.1371/journal.pone.0199933 August13,2018 9/25 PhynotypicandgeneticdiversityevaluationofLensspeciesunderalkalinitystress Fig2. Effectsofalkalinitystress(20mMNaHCO)ongermination(a),seedlinggrowth(b),biomassaccumulation 3 (c),alkalinityscores(d)andsurvivalpercent(e)oflentilgenotypesunderhydroponiccondition. https://doi.org/10.1371/journal.pone.0199933.g002 rootsandshootswerealsoobservedat20mMNaHCO TheNa+/K+ratiowashigherinsensi- 3. tivecultivarsunderthehydroponiccondition(Fig4Eand4F). Visualizationofhydrogenperoxide(H O )productioninrootsusingFDAproducedgreen 2 2 fluorescentinresponseto40mMNaHCO treatment;theFDAfluorescencewasnegligiblein 3 therootsofcontrolplants,whereasitincreasedmarkedlyunder40mMNaHCO .Thelevelof 3 H O washigherinboththemosttolerantandmostsensitivegenotypesunder40mM 2 2 NaHCO ,whencomparedwiththeirrespectivecontrols.Lowfluorescentsignalswere 3 observedinrootsofthemosttolerantbreedingline(PDL-1),whereasintensegreenfluores- cencewasfoundinrootsofmostsensitivecultigen(L-4076)at40mMNaHCO .Wildtolerant 3 (ILWL-15)accessionexhibitedlessgreenfluorescencewhencomparedtotolerantbreeding line(PDL-1),indicatinglessH O productionatthesimilarlevelofalkalinitystress(Fig5). 2 2 Shootanatomyoftolerantgenotypesunderalkalinitystresscondition(40mMNaCl) showedanenlargedpithareaandincreasedvascularbundlesanddecreasedcorticalarea.In sensitivecultivars,pithareawasshrunkenduetodeformedstemstructure.Instems,deposi- tionwasconfinedwithinthelayersofcortical-sclerenchymaintolerantbreedingline(PDL-1). However,insensitivecultivar(L-4076),depositiondeeplypenetratedmanylayersapartfrom cortical-sclerenchyma,includingthecorticalvascularbundles.Injurywithinthecorticallayers ofsensitivecultivarwasnoticed,whereastolerantbreedinglineandwildaccessionsshowed intactcorticalregionunderthesimilarlevelofalkalinitystress(Fig6).Inroots,depositions wererestrictedtoepidermisandcorticalsclerenchymaonlyinboththetolerantbreedinglines andwildaccessions,whereasdepositionsspreadalloverthecorticalregioninthesensitivecul- tivar.Deformedepidermalandcorticalcellstructureswerenoticedunderalkalinitystresscon- ditionsinsensitivecultivarsincontrasttothetolerantbreedingline,wherethecellswere intactbutslightlyenlargedascomparedtoitscontrol(Fig7). PLOSONE|https://doi.org/10.1371/journal.pone.0199933 August13,2018 10/25