ContentslistsavailableatScienceDirect
Industrial
Crops
and
Products
j ou rn a l h o m epa g e :w w w . e l s e v i e r . c o m / l o c a t e / i n d c r o p
Bioenergetic
potential
and
genetic
diversity
of
elephantgrass
via
morpho-agronomic
and
biomass
quality
traits
João
Romero
do
Amaral
Santos
de
Carvalho
Rocha
a,
Juarez
Campolina
Machado
b,∗,
Pedro
Crescêncio
Souza
Carneiro
a,
Jailton
da
Costa
Carneiro
b,
Marcos
Deon
Vilela
Resende
c,
Francisco
José
da
Silva
Lédo
b,
José
Eustáquio
de
Souza
Carneiro
aaUniversidadeFederaldeVic¸osa,CampusUniversitário,Vic¸osa36570-000,Brazil bEmbrapaGadodeLeite,RuaEugêniodoNascimento610,JuizdeFora36038-330,Brazil cEmbrapaFlorestas,EstradadaRibeira,Km111,Colombo83411-000,Brazil
a
r
t
i
c
l
e
i
n
f
o
Articlehistory: Received23March2016 Receivedinrevisedform 21September2016 Accepted2October2016 Availableonline9November2016
Keywords: Pennisetumpurpureum Bioenergy Ethanol Combustion Mixedmodels
a
b
s
t
r
a
c
t
Elephantgrasshasbeenanotableoptionasbioenergyplant.However,foritsbioenergeticuse,the quan-tificationofgeneticdiversitybasedonbiomassqualitytraitshasnotbeencommonlyreportedinthe literature.Theobjectiveofthisstudywastoquantifythegeneticdiversityamong100accessionsofthe ActiveElephantgrassGermplasmBank(BAGCE),bymeansofmorphological(flowering,height,vigorand stalkdiameter),agronomic(totaldrybiomass)andbiomassqualitytraits(drymatterconcentration, cel-lulose,lignin,hemicellulose,invitrodigestibility,nitrogen,ash,andcalorificvalue),andtheultimategoal wastousetheelephantgrassasabioenergyfeedstock.Byusingmixedmodelmethodologyandgenetic diversityanalyses,itwasfoundgeneticvariabilitybetweenelephantgrassaccessions,whichisthebasic premisetostartanybreedingprogram.TheBAGCEpresentedgreatergeneticvariabilityforthebiomass qualitytraits,whencomparedwithmorpho-agronomictraits.Theaccessionsweredividedinto6clusters ofgeneticsimilarity,withpotentialforuseinsecondgenerationethanolproductionanddirectbiomass combustion,besidesforageuses.Furthermore,topotentiateelephantgrassasbioenergeticplant,crosses amongdivergentindividualsfromdistinctclusterswererecommended.Thus,thegeneticvariabilityof BAGCEcanbeexploitedtoproducesuperiorcombinationsthatcanmaximizesecondgenerationethanol conversionandbiomassdirectcombustion.Inaddition,theseactionscanincreasethecontributionof elephantgrassforasustainableenergeticmatrixdiversification.
©2016TheAuthors.PublishedbyElsevierB.V.ThisisanopenaccessarticleundertheCCBY-NC-ND license(http://creativecommons.org/licenses/by-nc-nd/4.0/).
1. Introduction
Elephantgrass (Pennisetum purpureum Schum.) has been a notableoptionforbioenergyproduction.Thespecies,whichis tra-ditionallyusedasforageplant,hasattractedconsiderableattention asoneofthepromisingcropsforuseasbioenergetic feedstock, especially for its photosynthetic efficiency (C4 carbon fixation mechanism),highbiomass production, longevity, rapid growth, broadadaptation,inadditiontoitschemicalproperties(Anderson
∗ Correspondingauthor.
E-mailaddresses:[email protected]
(J.R.d.A.S.d.C.Rocha),[email protected](J.C.Machado),[email protected]
(P.C.S.Carneiro),[email protected](J.d.C.Carneiro),
[email protected](M.D.V.Resende),[email protected]
(F.J.d.S.Lédo),[email protected](J.E.d.S.Carneiro).
etal.,2008;Moraisetal.,2009;Zeng-HuiandHong-Bo,2010;Ra etal.,2012;Fontouraetal.,2015),andforitsbiologicalnitrogen fixationability,sinceitscontributionsrangesfrom18to70%ofthe nitrogenusedbytheplant(Moraisetal.,2009,2012).
Manyofthecultivarswhicharecurrentlyinusewereselected foranimalfeed,placinganemphasisonhighpercentageofleaves, highnitrogenconcentration,andlowfiberlevels.Biomass produc-tionwasoftenasecondaryfactorinregardstoobtainingincreased nutritionalquality(Rengsirikuletal.,2013).Ontheotherhand, forbioenergyproduction,theobjectiveistoobtainthemaximum biomassyield,withadequatequalityfordirectcombustionorfor biofuelconversion(Strezovetal.,2008;Prochnowetal.,2009;Naik etal.,2010;Naetal.,2016a).
Thebiomassusedasasourceofthermalenergyinthe com-bustionprocessshouldpresenthighconcentrationsofligninand cellulose (Gani and Naruse, 2007), high carbon/nitrogen ratio,
http://dx.doi.org/10.1016/j.indcrop.2016.10.060
0926-6690/©2016TheAuthors.PublishedbyElsevierB.V.ThisisanopenaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4. 0/).
highcalorificvalue,and low levelsofmoisture,ash, and nitro-gen(McKendry,2002; Longetal.,2006; Jaradat,2010).Forthe production ofcellulosic ethanol, highcellulose/ligninratio, and highhemicellulosecontentaredesirabletoprovidehighethanol productionpertonofbiomass.Moreover,inthefermentative pro-cesses,itisdesirablethatthebiomasspresentshighconcentration of carbohydrateswith low molecularweight in unpolymerized state(Porteretal.,2007).
EmbrapaDairyCattleResearchCentermaintainsanActive Ele-phantgrass Germplasm Bank (BAGCE) with 160 accessions. Of these,101accessionsareofthePennisetumpurpureum,19 acces-sions are of the species of the tertiary gene poolof the genus (Pennisetumspp.),and40 accessionsareofaworkcollectionof P.glaucum.Thepre-breedingexpansioneffortsofelephantgrass, suchas theactivities of characterization and evaluation of the germplasminregardstobiomassquality,favoritsuseasa bioen-ergysource.
Inthepre-breedingstage,geneticdiversityanalysesare note-worthy,andinrelationtoelephantgrass,itshouldbementioned studiesofgeneticdiversitybasedonmorphologicalandagronomic traits(VandeWouwetal.,1999;Shimoyaetal.,2001,2002), cyto-genetictraits(Techioetal.,2002),andmoleculartraits(Struwig etal.,2009;Harrisetal.,2009;Azevedoetal.,2012;Wanjalaetal., 2013;Lópezetal.,2014).However,forbioenergeticuse,the quan-tificationofgeneticdiversityinelephantgrassbasedonbiomass qualitytraitshasnotbeencommonlyreportedintheliterature.
Theobjectiveofthisstudywastoquantifythegeneticdiversity amongelephantgrassaccessionsoftheBAGCE,aimingatusingit asbioenergeticfeedstock,bymeansofthedescriptionof morpho-agronomicandbiomassqualitytraits.
2. Materialandmethods 2.1. Experimentconduction
Theexperimentwascarried outattheexperimentalfield of EmbrapaDairyCattleResearchCenter,locatedinthemunicipality ofCoronelPacheco,MG,Brazil(21◦3318s,43◦1551W,at417m asl).Theplantingwascarriedout inDecember,2011,in0.20m deepfurrows,and80kgha−1P2O5fertilizerwasappliedatplanting.
Aftertheestablishmentstage,30daysafterplanting,elephantgrass werecutto0.30mstubbleheight(uniformityharvest).Thefirst oftwo250daygrowthperiodsstartedatthistime.Maintenance fertilizationwascarriedoutwith300kgha−1 oftheN-P2O5-K2O
formulation(20:05:20blendedgranularfertilizer),afterthe uni-formityharvest,andafterthefirstevaluationcutting.Fertilization wascarriedoutaccordingtothesoilanalysis.
Twoevaluationcuttingswerecarriedoutforthisstudy.Aiming atusingthemasbioenergeticfeedstock,weadoptedcuttingsevery 250days.
2.2. Geneticmaterialandexperimentaldesign
OnehundredaccessionsoftheActiveElephantgrassGermplasm Bank(BAGCE)wereevaluated.Plots(1.5m×4m)consistedofa sin-gle4mrow.Therowswereplantedsidebyside,spaced1.5mapart. Plotswereallocatedinasimplexlatticedesign,withtwo replica-tions.Simplelatticedesignisapartiallybalancedlattice,a type ofresolvableincompleteblockdesignthatwasdevelopedforthe comparisonoflargenumberoftreatments(clones)inagricultural experiments.
2.3. Morpho-agronomictraits
Thefollowingmorpho-agronomictraitsweremeasured: Flow-ering(days)–wasdeterminedbythenumberof daysfromthe
standardizedharvestuntilthefloweringof50%ofthe experimen-talplot;meanheight(m)–wasobtainedfromthearithmeticmean oftheheightofthreerandomlyselectedplants,ineachplot, mea-suredfromthegroundleveltothecurveofthelastcompletely expandedleaf;phenotypicvigor(1–5)–wasobtainedusinga grad-ingscale,whichrangedfrom1to5(5=highvigor;1=lowvigor); stalkdiameter(mm)–wasobtainedfromthearithmeticmeanof fiveplantsintheusefulplot,takenatrandom,measuredat10cm fromthegroundlevelwithadigitalcaliperrule;totaldrybiomass (Mgha−1)–wasobtainedfromacutat7.5cmstubbleheightin a3msectionfromthemiddleofrows,usingagasolinepowered trimmer,andafterthat,itwasharvestedbyhand.The3msection wasimmediatelyweighedinthefieldtoprovideestimatesoffresh biomass.Totaldrybiomasswasquantifiedbymultiplyingthefresh biomassandthedrymatterconcentrationgivenaspercentage. 2.4. Biomassqualitytraits
Beforecuttingtheexperimentalplots,randomsamplesofthree completeplantsfromeachplotwerecollected.Then,thesesamples weredriedinaforcedaircirculationovenat56◦Cfor72h.After drying,sampleswereground(1mm)inaWileytypegrinderand senttothebiomassanalysislaboratoryforthechemicalanalysis describedbelow:
Cellulose(gkg−1),lignin(gkg−1)andhemicellulose(gkg−1)– weredeterminedfollowingthemethodologyproposedbyGoering and Van Soest (1967). In vitro digestibility of the dry biomass (gkg−1)– was determinedfollowing the methodology usedby TilleyandTerry(1963).Nitrogen(gkg−1)–wasdetermined fol-lowing the methodologyproposed bythe Association of Official AnalyticalChemical(AOAC,1975).Ash(gkg−1)–wasdetermined according to the methodology proposed by Silva and Queiroz (2002).Calorificvalue(MJkg−1)–wasdeterminedusingaIKA C-5000calorimeter.Drymatterconcentration(gkg−1)–wasobtained bythesamplingofthreecompleteplantsfromeachplot,which weredriedinakilnafterweighing(freshweight)untilweight sta-bilization.Sampleswereweighed(dryweight)again,andthenthe drymatterconcentrationwasdeterminedbytheratiobetweendry weightandfreshweight.Thistraitwasusedasacommon denom-inatorfortheestimationofcellulose,lignin,hemicellulose,invitro drymatterdigestibility,nitrogen,ash,andcalorificvalue.
2.5. Statisticalanalyses
Duetothestructuralcomplexityofthedata(repeatedmeasures throughouttimeorlongitudinaldata),itwasadoptedthemixed modelstatisticalanalysesviaREML/BLUP(restrictedresidual max-imumlikelihoodandbestlinearunbiasedprediction),accordingto PattersonandThompson(1971)andHenderson(1975).
For the deviance analysis, it was used the statistical model denotedby:y=Xm+Zg+Wb+Ti+Op+,inwhich
y=datavector
m=vectoroftheeffectsofthemeasurement-replication combi-nation(assumedtobefixed)addedtotheoverallmean;
g=vectorofthegenotypiceffects(assumedtoberandom); b=vectoroftheeffectsofblocks(assumedtoberandom); i=vectoroftheeffectsofthegenotype×measurements; p=vectorofthepermanentenvironment(random); ε=vectoroferrorsorresiduals(random)
X,Z,W,T,and Qrepresenttheincidencematrices forthese effects.
Fortherandomeffectsofthemodel,thesignificanceforthe Like-lihoodratiotest(LRT)wastestedusingthechi-squaretestwithone degreeoffreedom.BLUP(BestLinearUnbiasedPrediction)means wereestimatedforeachofthe100accessionsbasedonthe13traits evaluated.
Table1
Jointdevianceanalysisintwocuttingsforthe13traitsevaluatedin100 elephant-grassaccessions.
Traits Effects
Clonesa Clones×Cuttingsb
Flowering(days) 70.74** 120.09**
Averageheight(m) 15.79** 22.76**
Phenotypicvigor(1–5) 22.02** 0.89NS
Stalkdiameter(mm) 23.58** 11.42**
Totaldrybiomass(Mgha−1) 30.31** 11.80**
Drymatterconcentration(gkg−1) 12.06** 34.08**
Cellulose(gkg−1) 5.67* 4.60*
Lignin(gkg−1) 8.61** 1.86NS
Hemicellulose(gkg−1) 1.77NS 3.28NS
Invitrodigestibilityofthedrybiomass(gkg−1) 10.59** 1.87NS
Nitrogen(gkg−1) 12.02** 0.44NS
Ash(gkg−1) 10.03** 1.79NS
Calorificvalue(MJkg−1) 12.56** 2.61NS
Chi-squaredtabulated:3.84and6.63forlevelsofsignificanceof5%and1%, respec-tively.
**,*significantto1and5%respectively,bythechi-squaredtest. NSnotsignificantto5%,bythechi-squaredtest.
aLikelihoodratiotest, usingthechi-squared testwithonedegreeoffreedom,
reducedmodelwithoutcloneseffects.
bLikelihoodratiotest,usingthechi-squared testwithonedegreeoffreedom,
reducedmodelwithoutclones×cuttingseffects.
The Tocher clustering method(Rao, 1952) was used in the
quantificationof thegeneticdiversity among 100accessionsof theBAGCE,basedonthegeneticdissimilaritymatrix,obtainedby themeanstandardizedEuclideandistanceoftheBLUPmeans.For devianceanalysis,estimates oftheBLUPmeansand dissimilar-itymatrixwereobtainedusingthesoftwareSelegen-REML/BLUP (Resende,2007).
The relative importance of the traits in quantifying genetic divergencewasestimatedbasedonthemethodproposedbySingh (1981),usingtheGENESsoftware(Cruz,2013).
To show the genetic diversity of BAGCE, graphical analysis dendrograms(graphicalanalysis)wereconstructedbasedonthe hierarchical methodology of single linkage (neighbor joining), usingtheRprogram(RDevelopmentCoreTeam,2015).
3. Resultsanddiscussion 3.1. Geneticvariability
Significantcloneeffects (p<0.05)weredetectedbythejoint deviance analysis in the two cuts evaluated for the morpho-agronomic traits and for the biomass quality trait, with the exceptionof hemicellulose(Table1).These resultsindicate ele-vated genetic variability among 100accessions (clones) of the BAGCE.
Regardingtheclones x cuttings interaction,it wasobserved significanteffect(p<0.01)forfour(flowering,meanheight,stalk diameter,andtotal drybiomass)ofthefivemorpho-agronomic traits. For the traits of biomass quality, there was significant effect (p<0.01) for two (dry matter concentration and cellu-loselevel)oftheeightevaluatedtraits(Table1).Non-significant clones×cuttingsinteractionindicatescoincidentperformanceof clones in relationtothedifferentcuttings. Therefore, it canbe inferredthatselectioncanbecarriedoutinanyofthecuttings, especiallywhenthetraitsofbiomassqualityaretakenintoaccount. Sincethecloneseffect,basedonthemeansofthecuts,isgiven bythemeansofthecloneseffectsineachcuttingsubtractedby theinteractioneffect(clones×cuttings),itmaybestatedthat sig-nificanteffectofclonesinthepresenceofsignificantinteraction confirmsthehighvariabilityofthe100accessionsoftheBAGCE.
3.2. Morpho-agronomicclustering
Consideringthevariabilityofthemorpho-agronomictraits eval-uatedinthe100accessions,theformationofthreeclusterswas observedbyusingtheTochermethod(Fig.1).Ninety-sixaccessions wereallocatedinthefirstcluster.Thetenmostsimilaraccessions were:MerckerS.E.A,ElefanteCachoeiroItapemirim,TaiwanA-25, HíbridoGigantedaColômbia,IJ7125,Mercker86México,IJ7126, 11 AD IR, Gramafante, and 05 AD IRI. According totheTocher clusteringmethod,themeanintraclusterdistancemustbelower thanthemeaninterclusterdistance.Theclustercriterionisgiven bythemaximumvalue()ofthedissimilaritymeasurefoundin theclusterofsmallerdistancesthatinvolveeachpairof individu-als(Rao,1952).Thus,accessionswhichwereextremelydivergent fromtheothers,suchasMott(Fig.1),affecttheTocherclustering method.Thisaccessionshowedgreaterrelativedistancethanthe otheraccessions.Therefore,thereunificationoftheaccessions clas-sifiedinclusterIenabledtheformationof19sub-clusters,which indicateshighvariabilityoftheseaccessions.
ItisworthnotingthattheaccessionMott,uniquelyassignedto clusterII(Fig.1),presentsreducedmeanplantheight(dwarfing genes),lowbiomassproduction,andconsequentlylowpotential forbioenergyproduction.
Inastudywithmolecularmarkers,Struwigetal.(2009)reported thattheRAPD(RandomAmplificationofPolymorphicDNA)markers wereincapableofseparatingelephantgrassaccessionsinregard tomeanplantheight.Likewise,Azevedoetal.(2012)notedthat microsatellites(SSR)markerswerealsoinefficientindetermininga clusteringstandardwithlowheightaccessions.Thishighlightsthe importanceofusingmorpho-agronomictraitsforthegenetic diver-sityofelephantgrass.Nevertheless,wasreportedthatSSRmarkers maybeimportanttothedevelopmentoftoolsforelephantgrass geneticbreeding(Azevedoetal.,2012),andformanagingthe inva-sionriskofelephantgrass(Lópezetal.,2014).TheclonesBAGCE 2,KingGrassandBRSCapiac¸u(clusterIII)havehighertotaldry biomassproduction,stalkdiameter,meanplantheight,phenotypic vigor,andlateflowering.BRSCapiac¸uismoredivergentthanthe cloneMott.Recently,thecloneBRSCapiac¸uhasbeenregistered andprotectedascultivarbytheMinistryofAgriculture,Livestock andFoodSupply(MAPA).Thus,EmbrapaDairyCattlehas recom-mendedBRSCapiac¸utofarmers.
3.3. Biomassqualityclustering
Whenconsideringthevariabilityofthetraitsofbiomassquality evaluatedin100accessions,itwaspossibletodistinguish11 clus-tersbytheTocheroptimizationmethod.Sixtyfivepercentofthe accessionswereconcentratedinthefirstcluster,amongwhichthe tenmostsimilarwere:Kizozi,Ibitinema,IJ7126,IJ7139,Cameroon, ElefanteCachoeiroItapemirim,Guaco,Guac¸u,TaiwanA-121and BRSCanará. High geneticvariability wasalsoobserved (Fig. 2). Therefore,biomassqualityanalysesroutinelyusedinbreeding pro-gramsandintheevaluationofnutritivevalueofforageassistinthe selectionanddistinctionofdivergentclusters,andcancontribute tothecharacterizationofelephantgrassbiomassforenergeticuse (Ampong-NyarkoandMurray,2011).
3.4. Morpho-agronomicandbiomassqualityclustering
Thegeneticdiversityanalysisamongthe100accessionsbased onboth morpho-agronomicandbiomass qualitytraitsrevealed theformationofsixclusters,and91accessionswereallocatedin thefirstcluster(Fig.3).Azevedoetal.(2012),whilestudyingthe geneticdiversityoftheseaccessions,bymeansof microsatellite molecularmarkers,obtainedonlyoneclusterofgenetic similar-ity amongtheevaluatedP.purpureumaccessions.These results
Fig.1.Dendrogramof100accessionsofP.purpureumoftheBAGCE,basedontheneighborjoiningmethod,obtainedfromBLUPmeansofelephantgrassmorpho-agronomic traits.DifferentcolorsrepresenttheclustersofsimilaraccessionsformedusingtheTocherclusteringmethod.
Fig.2.Dendrogramof100accessionsofP.purpureumoftheBAGCE,basedontheneighborjoiningmethod,obtainedfromBLUPmeansofelephantgrassbiomassquality traits.ThedifferentcolorsrepresenttheclustersofsimilaraccessionsformedusingtheTocherclusteringmethod.
Fig.3.Dendrogramof100accessionsofP.purpureumoftheBAGCE,basedontheneighborjoiningmethod,obtainedfromBLUPmeansofelephantgrassmorpho-agronomic andbiomassqualitytraits.ThedifferentcolorsrepresenttheclustersofsimilaraccessionsformedusingtheTocherclusteringmethod.
indicatetheimportanceofmorpho-agronomicandbiomassquality traitsinthequantificationofthegeneticdiversityofelephantgrass, sincetheyenabletheefficientseparationofthegenetic similar-ityclusters.Itisworthmentioningthatthepropertiesofbiomass qualities(moisturecontent,calorificvalue,andlevelsofcellulose, ligninandnitrogen)caninfluencetheentireprocessofconversion intoethanolandthethermalutilizationofthebiomass(McKendry, 2002;GaniandNaruse,2007;Prochnowetal.,2009;Jaradat,2010; Knolletal.,2012;Naetal.,2015,2016a).
Thetenmostsimilaraccessionsintermsofmorpho-agronomic andbiomassqualitytraitswere:Mineiro,Australiano,05ADIRI, 11ADIRI,Napier,Gramafante,10ADIRI,MineirãoIPEACO,BAGCE 50andSemPelo.Azevedoetal.(2012),whenusing microsatel-litemarkersinthegeneticdiversityanalysisofthe100accessions usedinthiswork,reportedthataccessions05ADIRI,Cubanode Pinda,10ADIRIandBAGCE50presentedsimilaritycoefficientof 0.98,indicatingthattheseaccessionscouldhavethesamegenetic ancestors,andthatsomeofthemcouldbediscardedwithoutloss ofgeneticdiversity.
3.5. Relativeimportanceanddiscardoftraits
Theevaluationoftherelativeimportanceofthetraitsinthe geneticdiversityofthe100accessionsoftheBAGCE,usingtheSingh method(1981),indicatedthatthefivemaintraitswere: flower-ing,drymatterconcentration,stalkdiameter,calorificvalue,and nitrogenlevel(Table2).Therestofthetraitshavelowerrelative importance,butshouldnotbediscarded,sincethewithdrawalof
thelessimportanttrait(averageheight)alterstheclustering pat-ternof the accessions.Thus, allof the traitsused in this work were considered for the quantification of thegenetic diversity of the accessions. When the relative importance of morpho-agronomictraitswascontrastedwiththebiomassqualitytraits, it was observed that thelatter contributed with54.10% of the totalaccessionsdiscrimination.Therefore,itcanbeinferredthat theaccessionsoftheBAGCEpresentedgreatergeneticvariability intermsofbiomassqualitytraits,whencomparedwith morpho-agronomictraits.
3.6. Choiceoftheaccessionsfordifferentfinaluses
Table3presentsthegenotypicvaluesofelephantgrass morpho-agronomicandbiomassqualitytraitsusedintheTocherclustering method.Itwaspossibletoidentifysixclusters,whicharedescribed below,basedonthefiveprincipletraits(flowering,drymatter con-centration,stalkdiameter,calorificvalueandnitrogenlevel),which candirecttheaccessionstodifferentfinaluses(e.g.forage,second generationethanolproduction,anddirectbiomasscombustion).
ClusterI:Accessionsarecharacterizedbypresenting interme-diatevaluesforthetraits:flowering,stalk diameter,dry matter concentration,nitrogenlevel,andcalorificvalue.Itisthecluster thatpresentsthegreatestnumberofaccessionsandthegreatest intracluster diversity.Theaccessionsof this clusteraresuitable forbothcombustionandcellulosicethanolproduction,especially thosewiththegreatestpotentialforbiomassproduction.
Table2
Relativecontributionofelephantgrassmorpho-agronomicandbiomassqualitytraits,evaluatedin100accessionsoftheBAGCE,basedontheSighmethod.
Traits S,j Relativeimportance(%)
Flowering(days) 771.83 17.88
Drymatterconcentration(gkg−1) 538.50 12.47
Stalkdiameter(mm) 495.32 11.47
Calorificvalue(MJkg−1) 430.59 9.97
Nitrogen(gkg−1) 324.30 7.51
Invitrodigestibilityofthedrybiomass(gkg−1) 298.57 6.92
Phenotypicvigor(1–5) 297.04 6.88
Cellulose(gkg−1) 252.65 5.85
Lignin(gkg−1) 252.31 5.84
Ash(gkg−1) 239.07 5.54
Totaldrybiomass(Mgha−1) 221.34 5.13
Averageheight(m) 195.66 4.53
S.j,Singhstatistic(1981).
Table3
SummaryofTocherclustering,meangenotypicvaluesforthemorpho-agronomictraits(flowering–days;meanheight–m;phenotypicvigor–1to5;stalkdiameter–mm; totaldrybiomass–Mgha−1)andbiomassqualitytraits(drymatterconcentration–gkg−1;cellulose–gkg−1;lignin–gkg−1;hemicellulose–gkg−1;invitrodigestibilityof thedrybiomass–gkg−1;nitrogen–gkg−1;ash–gkg−1;calorificvalue–MJkg−1)ofelephantgrassindifferentclusters,andcharacterizationoftheclustersforfiveprincipal
traits.
Traits ClusterI ClusterII ClusterIII ClusterIV ClusterV ClusterVI
Floweringa 181g(223h–154i) 158(168–152) 204(210–197) 200(200–200) 168(168–168) 217(217–217) Averageheight 3.63(4.08–3.27) 3.43(3.61–3.18) 4.06(4.11–4.01) 4.16(4.16–4.16) 3.56(3.56–3.56) 2.54(2.54–2.54) Phenotypicvigor 3.20(3.85–2.76) 2.97(3.17–2.84) 3.82(3.94–3.70) 3.22(3.22–3.22) 3.07(3.07–3.07) 2.58(2.58–2.58) Stalkdiameter 18.06 (22.77–14.68) 16.98 (19.99–13.98) 21.96 (22.72–21.20) 20.07 (20.07–20.07) 19.18 (19.18–19.18) 14.29(14.29–14.29) Totaldrybiomassb 21.34
(34.60–14.22) 15.72 (19.17–13.26) 37.23 (41.37–33.09) 15.63 (15.63–15.63) 17.38 (17.38–17.38) 11.53(11.53–11.53) Drymatter concetrationc 407.212 (445.500–360.800) 397.525 (416.800–370.300) 406.600 (427.200–386.000) 419.100 (419.100–419.100) 370.700 (370.700–370.700) 391.500 (391.500–391.500) Cellulose 412.276 (420.059–403.203) 402.262 (404.066–398.420) 414.858 (417.149–412.566) 425.563 (425.563–425.563) 414.123 (414.123–414.123) 402.352 (402.352–402.352) Lignin 102.598 (109.223–95.623) 97.321 (101.111–93.555) 104.036 (104.958–103.114) 105.179 (105.179–105.179) 112.677 (112.677–112.677) 96.415 (96.415–96.415) Hemicellulose 270.909 (272.376–268.404) 271.740 (272.441–271.174) 269.990 (270.548–269.432) 271.787 (271.787–271.787) 267.628 (267.628–267.628) 272.508 (272.508–272.508) Invitrodigestibility
ofthedrybiomass
332.564 (349.380–309.004) 344.607 (355.377–332.133) 331.907 (331.995–331.818) 322.772 (322.772–322.772) 308.329 (308.329–308.329) 347.514 (347.514–347.514) Nitrogen 4.501 (5.244–4.128) 4.799 (4.916–4.674) 4.374(4.413 4–334) 4.270 (4.270–4.270) 4.253 (4.253–4.253) 5.148(5.148–5.148) Ash 66.427 (74.939–59.243) 72.113 (77.756–65.010) 62.332 (63.143–61.521) 60.615 (60.615–60.615) 60.928 (60.928–60.928) 83.407 (83.407–83.407) Calorificvalued 18.145 (18.320–17.945) 18.026 (18.099–17.945) 18.225 (18.261–18.189) 18.223 (18.223–18.223) 18.322 (18.322–18.322) 17.938 (17.938–17.938)
Floweringe Intermediate Early Late Late Premature Late
Stalkdiameter Intermediate Thin Thick Thick Intermediate Verythin
Drymatter concentration
Intermediate Intermediate Intermediate High Low Intermediate
Nitrogen Intermediate High Intermediate Low Verylow Veryhigh
Calorificvaluef Intermediate Low Intermediate Intermediate High Verylow
aandbMorpho-agronomictraits. canddBiomassqualitytraits.
eandfCharacterizationofthesixclustersaccordingtothefirstfiverelativeimportanttraits. g,h,eandiAverage,maximum,andminimumvalues,respectively,ofthetraitwithinthecluster.
ClusterII:Accessionsarecharacterizedbypresentingearly
flow-ering,thinstalkdiameter,highnitrogenlevels,andlowcalorific
value.It isrepresentedbyaccessionsIJ7125,Pioneiro,Banhado
andPCM0701.Theaccessionsofthisclusterhavelowaptitudefor
energeticbiomassproduction,andshouldbedirected,speciallyfor
useasforage.
ClusterIII:Theaccessionsarecharacterizedbypresentinglate
flowering,thickstalkdiameter,andhighheight.Itstandsoutforthe
largetotaldrybiomassproduction.Theclusterincludesthe
acces-sionsBAGCE2andBRSCapiac¸u.Asaresultofthehighpotential
forbiomassproduction,accessionsofthisclusterhavetheability
ofdirectcombustionandcellulosicethanolproduction.
ClusterIV:Theclusteriscomposedofonlyonegenotype,Pusa
Napier N◦ 2, and is characterizedby late flowering,thick stalk
diameter,highdrymatterconcentration,lownitrogenlevels,and
intermediatecalorificvalue.PusaNapierN◦2,canbeusedforthe
productionofcellulosicethanolinviewofitshighcelluloselevel.
ClusterV:Theclusteriscomposedofoneaccession–13AD,and
ischaracterizedbyprematureflowering,lowdrymatter
concen-tration,andverylownitrogenlevel.However,ithasthehighest
calorificvalueandligninlevel,andthelowestinvitro
digestibil-ityofthedrybiomass.Theaccessionofthisclusterisspecifically
adaptedtotheprocessofbiomasscombustion.
ClusterVI:Theclusterisformedbyoneindividual–Mott,and
ischaracterizedbypresentinglateflowering,thinstalkdiameter,
veryhighnitrogenlevel.However,thecalorificvalueisthelowest
amongalloftheaccessions.Mottisspecificallyadaptedtobeused
asforage,inpasture,duetothehighnutritionalvalueandreduced
Late flowering trait is advantageous since it increases the
periodofvegetativegrowth;decreasesthelikelihoodofseedset
beforeharvest,which is animportantmeansof spread for
ele-phantgrass,andincreasesharvestflexibility(Sollenberger etal.,
2014).Moreover, it has beenreported that early flowering for giant miscanthus is related to the low dry matter production (Fedenko etal.,2013).Plantmaturitymaycausechemical com-positionchanges,suchasthereductionofashcontent,nitrogen anddry matterconcentration,andtheincreaseof celluloseand lignin;ingeneral,itimprovesthebiomassqualitytraitsfor com-bustion and production of cellulosic ethanol (Naet al., 2016a, 2016b).
Thebiomassfeedstocktousefor combustionshouldpresent somequalityattributes,suchashighconcentrationsofligninand cellulose(Gani andNaruse,2007),highcalorificvalue,high car-bon/nitrogenratio,inadditiontolowlevelsofmoisture,ash,and nitrogen(McKendry,2002;Longetal.,2006;Jaradat,2010).When thefocusistheproductionofcellulosicethanol,itisrequiredhigh cellulose/ligninratioand hemicellulosetoprovidehighethanol productionbybiomassweight(Porteretal.,2007).
Forbothpurposes(combustionandethanol),biomassshould presentlowmoisturecontent(highlevelsofdrymatter concen-tration)atharvestinorder toobtainhighefficiencyof thermal conversion technologies (McKendry, 2002; Jaradat, 2010). In a study which compared energycane, elephantgrass, giant reed andswitchgrass asbiomass yieldsunderlow-input,Knoll etal. (2012) found that elephantgrass and energycane were those with the lowest dry matter concentration. The high moisture is one of the challenges for efficient processing and trans-portation of the postharvest biomass (Knoll et al., 2012; Na et al., 2015). Results of the current study indicate that there is genetic variability for this dry matter concentration, and therefore it can achieve reduced water content in elephant-grass.
Thecalorificvalue istheenergy releasedwhen thebiomass isburned.Therefore,thehigherthecalorificvalue,thebetteris thefeedstockforcombustion(Jaradat,2010).Stalkwasthemajor contributorofsolublesugarsfortotalshootbiomassin elephant-grass.However,degradationoffreesugarsmaydecreasefeedstock qualityastheyproportionallyreduceconcentrationofstructural carbohydrates(Naetal.,2016a).Lowrequirementofnitrogenis desirablenotonlybecauseitisavaluedconstituentintermsof conversiontoenergy,butforNfertilizerisacostlyinput(Naetal., 2016b).
Theresultsobtainedinthisworkindicatethatitispossibletouse andbreedingelephantgrassasbioenergeticplant.Elephantgrass accessionspresenthighgeneticdiversitydistributedindifferent clusters,whichcanbeexploitedfortheproductionofsecond gen-erationethanolandfordirectbiomasscombustion.
Thus, crosses among divergentindividuals belongingto dis-tinctclustersisrecommendedfortheobtainmentofpopulations withhighpotentialfortheextractionofsuperiorclones. There-fore,the intercrossesof the accessionsof cluster I(accessions: Pusa Gigante Napier, BAGCE 59, Cuba-116 and Pasto Panamá), III and V are promising for the improvement of the potential ofelephantgrassforbiomasscombustion.Forsecondgeneration ethanol production (cellulosic ethanol), the accessions of clus-ters I (accessions: Mercker S.E.A., Taiwan A-25, Mole de Volta GrandeeCNPGL96-25-3),IIIandIVshouldpreferablybe inter-crossed.
Thisstudyisapioneerinthequantificationofgenetic diver-sityaimingattheuseofelephantgrassasbioenergeticfeedstock, andreinforcestheimportanceofpre-breedingactionstorise ele-phantgrasscultivationtoarelevantlevelforasustainableenergetic matrixdiversification.
4. Conclusions
TheActiveElephantgrassGermplasmBank(BAGCE)presented potentialtobeusedwithbioenergypurpose,withgreatergenetic variabilityintermsofbiomassqualitytraits,whencomparedwith morpho-agronomictraits.
The100accessionsoftheBAGCEweredividedintosixgenetic dissimilarityclusters, withpotentialdifferentiated tobeusedin theproductionofsecondgenerationethanolanddirectbiomass combustion.
Itispossibletoimproveelephantgrassasbioenergeticplant. Acknowledgements
TheauthorsacknowledgeCAPES,FAPEMIGandUNIPASTOfor providingscholarshipsforthestudents,andfortheresearch sup-port.
References
AssociationofOfficialAnalyticalChemical(AOAC),1975.OfficialMethodsof AnalysisoftheAssociationofAnalyticalChemists,12thed.AOAC,Washington, D.C,pp.1094.
Ampong-Nyarko,K.,Murray,C.L.,2011.Utilityofforagegrassnutrientcomposition databasesinpredictingethanolproductionpotential.J.BiobasedMater. Bioenergy5,295–305,http://dx.doi.org/10.1166/jbmb.2011.1167. Anderson,W.,Casler,M.,Baldwin,B.,2008.Improvementofperennialforage
speciesasfeedstockforbioenergy.In:Vermerris,W.(Ed.),Genetic ImprovementofBioenergyCrops.Springer,pp.308–345.
Azevedo,A.L.S.,Costa,P.P.,Machado,J.C.,Machado,M.A.,Pereira,A.V.,Lédo,F.J.S., 2012.Cross-speciesamplificationofPennisetumglaucummicrosatellite markersinPennisetumpurpureumandgeneticdiversityofNapiergrass accessions.CropSci.52,1776–1785.
Cruz,C.D.,2013.GENES–asoftwarepackageforanalysisinexperimentalstatistics andquantitativegenetics.ActaSci.Agron.5,271–276.
Fedenko,J.R.,Erickson,J.E.,Woodard,K.R.,Sollenberger,L.E.,Vendramini,J.M.B., Gilbert,R.A.,Helsel,Z.R.,Peter,G.F.,2013.Biomassproductionand
compositionofperennialgrassesgrownforbioenergyinasubtropicalclimate acrossFlorida,USA.BioenergyRes.6,1082–1093,http://dx.doi.org/10.1007/ s12155-013-9342-3.
Fontoura,C.F.,Brandão,L.E.,Gomes,L.L.,2015.Elephantgrassbiorefineries: towardsacleanerBrazilianenergymatrix?J.CleanProd.96,85–93http://dx. doi.org/10.1016/j.jclepro.2014.02.062.
Gani,A.,Naruse,I.,2007.Effectofcelluloseandlignincontentonpyrolysisand combustioncharacteristicsforseveraltypesofbiomass.Renew.Energy32, 649–661.
Goering,H.K.,VanSoest,P.J.,1967.ForageFiberAnalysis:Apparatus,Reagents, ProceduresandSomeApplications.AgriculturalHandbook,Washington,D.C, pp.379.
Harris,K.,Anderson,W.,Malik,R.,2009.Geneticrelationshipsamongnapiergrass (PennisetumpurpureumSchum.)nurseryaccessionsusingAFLPmarkers. PlantGenet.Resour.8,63–70,http://dx.doi.org/10.1017/S1479262109990165. Henderson,C.R.,1975.Bestlinearunbiasedestimationandpredictionundera
selectionmodel.Biometrics31,423–447.
Jaradat,A.A.,2010.Geneticresourcesofenergycrops:biologicalsystemsto combatclimatechange.Aust.J.CropSci.4,309–323.
Knoll,J.E.,Anderson,W.F.,Strickland,T.C.,Hubbard,R.K.,Malik,R.,2012. Low-inputproductionofbiomassfromperennialgrassesinthecoastalplainof Georgia,USA.BioEnergyRes.5,206–214, http://dx.doi.org/10.1007/s12155-011-9122-x.
López,Y.,Woodard,K.R.,Seib,J.C.,Chamusco,K.,Sollenberger,L.E.,Gallo,M.,Flory, S.L.,Chase,C.D.,2014.Geneticdiversityofbiofuelandnaturalizednapiergrass (Pennisetumpurpureum).InvasivePlantSci.Manag.7,229–236,http://dx.doi. org/10.1614/IPSM-D-13-00085.1.
Long,S.P.,Zhu,X.G.,Naidu,S.L.,Ort,D.R.,2006.Canimprovementin photosynthesisincreasecropyields?PlantCellEnviron.29,315–330.
McKendry,P.,2002.Energyproductionfrombiomass(part1):overviewof biomass.Bioresour.Technol.83,37–46.
Morais,R.F.,Souza,J.B.,Leite,J.M.,Soares,L.H.B.,Alves,B.J.R.,Boddey,R.M., Urquiaga,S.,2009.Elephantgrassgenotypesforbioenergyproductionby directbiomasscombustion.Pesquis.Agropecu.Bras.44,133–140.
Morais,R.F.,Quesada,D.M.,Reis,V.M.,Urquiaga,S.,Alves,B.J.R.,Boddey,R.M., 2012.ContributionofbiologicalnitrogenfixationtoElephantgrass (PennisetumpurpureumSchum.).PlantSoil356,23–34,http://dx.doi.org/10. 1007/s11104-011-0944-2.
Na,C.,Sollenberger,L.E.,Erickson,J.E.,Woodard,K.R.,Vendramini,J.M.B.,Silveira, M.L.,2015.ManagementofperennialwarmseasonbioenergygrassesI. Biomassharvested,nutrientremoval,andpersistenceresponsesof
elephantgrassandenergycanetoharvestfrequencyandtiming.BioenergyRes. 8,581589,http://dx.doi.org/10.1007/s1215501495416.
Na,C.,Fedenko,J.R.,Sollenberger,L.E.,Erickson,J.E.,2016a.Harvestmanagement affectsbiomasscompositionresponsesofC4perennialbioenergygrassesin thehumidsubtropicalUSA.Glob.ChangeBiol.Bioenergy,http://dx.doi.org/10. 1111/gcbb.12319.
Na,C.,Sollenberger,L.E.,Fedenko,J.R.,Erickson,J.E.,Woodard,K.R.,2016b. Seasonalchangesinchemicalcompositionandleafproportionof
elephantgrassandenergycanebiomass.Ind.CropsProd.94,107–116,http:// dx.doi.org/10.1016/j.indcrop.2016.07.009.
Naik,S.N.,Goud,V.V.,Rout,P.K.,Dalai,A.K.,2010.Productionoffirstandsecond generationbiofuels:acomprehensivereview.Renew.Sustain.EnergyRev.14, 578–597,http://dx.doi.org/10.1016/j.rser.2009.10.003.
Patterson,H.D.,Thompson,R.,1971.Recoveryofinter-blockinformationwhen blockssizesareunequal.Biometrika58,545–554.
Porter,J.R.,Kirsch,M.M.N.,Streibig,J.,Felby,C.,2007.Choosingcropsasenergy feedstocks.Nat.Biotechnol.25,716–717.
Prochnow,A.,Heiermann,M.,Plöchl,M.,Amon,T.,Hobbs,P.J.,2009.Bioenergy frompermanentgrassland–areview:2.Combustion.Bioresour.Technol.100, 4945–4954,http://dx.doi.org/10.1016/j.biortech.2009.05.069.
RDevelopmentCoreTeam,2015.R:ALanguageandEnvironmentforStatistical Computing.RFoundationforStatisticalComputing,Vienna,Austria(ISBN 3-900051-07-0)http://www.R-project.org.
Ra,K.,Shiotsu,F.,Abe,J.,Morita,S.,2012.Biomassyieldandnitrogenuseefficiency ofcellulosicenergycropsforethanolproduction.BiomassBioenergy37, 330–334.
Rao,C.R.,1952.AdvancedStatisticalMethodsinBiometricResearch.JohnWiley& Sons,NewYork,pp.390.
Rengsirikul,K.,Ishii,Y.,Kangvansaichol,K.,Sripichitt,P.,Punsuvon,V., Vaithanomsat,P.,Nakamanee,G.,Tudsri,S.,2013.Biomassyield:chemical compositionandpotentialethanolyieldsof8cultivarsofNapiergrass (PennisetumpurpureumSchumach.)harvested3-monthlyincentralThailand.J. Sustain.BioenergySyst.3,107–112.
Resende,M.D.V.,2007.Selegen-REML/BLUP:Sistemaestatísticoeselec¸ãogenética computadorizadaviamodeloslinearesmistos.EmbrapaFlorestas,Colombo, pp.359.
Shimoya,A.,Ferreira,R.P.,Pereira,A.V.,Cruz,C.D.,Carneiro,P.C.S.,2001.
Comportamentomorfo-agronômicodegenótiposdecapim-elefante.Rev. Ceres48,141–158(InPortuguesewithEnglishabstract.).
Shimoya,A.,Cruz,C.D.,Ferreira,R.P.,Pereira,A.V.,Carneiro,P.C.S.,2002. Divergênciagenéticaentreacessosdeumbancodegermoplasmadecapim elefante.Pesquis.Agropecu.Bras.37,971–980,http://dx.doi.org/10.1590/ S0100-204X2002000700011(InPortuguese,withEnglishabstract.). Silva,D.J.,Queiroz,A.C.,2002.Análisesdealimentos(métodosquímicose
biológicos),3aed.EditoraUFV,Vic¸osa,MG,pp.235.
Singh,D.,1981.Therelativeimportanceofcharactersaffectinggeneticdivergence. IndianJ.Genet.PlantBreed.41,237–245.
Sollenberger,L.,Woodard,K.,Vendramini,J.M.B.,Erickson,J.,Langeland,K., Mullenix,M.K.,Na,C.,Castillo,M.,Gallo,M.,Chase,C.,López,Y.,2014.Invasive populationsofelephantgrassdifferinmorphologicalandgrowth
characteristicsfromclonesselectedforbiomassproduction.BioenergyRes.7, 1382–1391,http://dx.doi.org/10.1007/s12155-014-9478-9.
Strezov,V.,Evans,T.J.,Hayman,C.,2008.Thermalconversionofelephantgrass (PennisetumPurpureumSchum)tobio-gas,bio-oilandcharcoal.Bioresour. Technol.99,8394–8399,http://dx.doi.org/10.1016/j.biortech.2008.02.039. Struwig,M.,Mienie,C.M.S.,VanDenBerg,J.,Mucina,L.,Buys,M.H.,2009.AFLPsare
incompatiblewithRAPDandmorphologicaldatainPennisetumpurpureum (Napiergrass).Biochem.Syst.Ecol.37,645–652,http://dx.doi.org/10.1016/j. bse.2009.09.010.
Techio,V.H.,Davide,L.C.,Pereira,A.V.,Bearzoti,E.,2002.Cytotaxonomyofsome speciesandofinterspecifichybridsofPennisetum(Poaceae,Poales).Genet. Mol.Biol.25,203–209,http://dx.doi.org/10.1590/S1415-47572002000200014. Tilley,J.H.A.,Terry,R.A.,1963.Atwostagetechniqueforinvitrodigestionofforage
crops.J.Br.Grassl.Soc.18,104–111.
VandeWouw,M.,Hanson,J.,Luethi,S.,1999.Morphologicalandagronomic characterisationofacollectionofnapiergrass(Pennisetumpurpureum)andP: purpureum×P.glaucum.Trop.Grassl.33,150–158.
Wanjala,B.W.,Obonyo,M.,Wachira,F.N.,Muchugi,A.,Mulaa,M.,Harvey,J., Skilton,R.A.,Proud,J.,Hanson,J.,2013.GeneticdiversityinNapiergrass (Pennisetumpurpureum)cultivars:implicationsforbreedingand conservation.AoBPlants5,1–10,http://dx.doi.org/10.1093/aobpla/plt022. Zeng-Hui,L.,Hong-Bo,S.,2010.Maindevelopmentsandtrendsofinternational
