Electrophoretic analysis of seed proteins in Argentinean species of Phaseolinae (Fabaceae) - Sociedad Argentina de Botánica

(1)

ISSN0373-580 X Bol. Soc. Argent. Bot. 38 (3-4): 311

-

317.2003

ELECTROPHORETIC

ANALYSIS

OF

SEED

PROTEINS

IN

ARGENTINEAN

SPECIES

OF

PHASEOLINAE (FABACEAE)

SHIRLEY MARY ESPERT1andALICIA DIANA BURGHARDT1

Summary: Therelationships among Argentinean speciesof four generaofthe subtribe Phaseolinae

(Phaseoleae,Fabaceae): Phaseolus, Vigna, DolichopsisandMacroptilium, hadnever beenanalyzed

together using molecular markers. Inthis paper, twentyspecies of these generawerestudied bymeans of electrophoresis of seedstorage proteinson aSDS-PAGEsystem. Seedproteinelectrophoretic banding patterns allowed the recognition of a genetic basis of the generic divisions,whichwere

proposed using morphologicalandpalinologicaldata.Polypeptidic profiles provided interestingdatafor the analysis the subgeneric divisions.Moreover,the presence of marker bandsallowstheunequivocal characterization of mostoftheArgentineanspecieshere studied.

KeyWords: Systematics, Phaseolinae, Fabaceae, seedprotein electrophoresis

Resumen:Análisis electroforéticode lasespecies argentinasdePhaseolinae(Fabaceae).Lasrelaciones

entrelas especies argentinasde cuatro génerosde la subtribuPhaseolinae(Phaseoleae,Fabaceae):

Phaseolus, Vigna,Dolichopsisy Macroptilium,fueronanalizadas por primeravez enconjuntousando

marcadoresmoleculares. Seestudiaronveinte especiesde estos géneros utilizandoelectroforeslsdé

proteínasseminales,en unsistemaSDS-PAGE. Los patronesdebandas proteicas evidenciaronla

existenciadeuna basegenética paralasdivisiones genéricas, quéfueronpropuestasmedianteeluso

dedatosmorfológicosypalinológicos.Losperfiles polipeptídicosproveeninformacióninteresantepara

analizarlas divisiones subgenéricas. Además,lapresencia de bandas marcadorespermite la

caracterización inequívocade lamayoría de las especies argentinas aquí estudiadas.

Palabrasclave:Sistemática, Phaseolinae, Fabaceae,electroforesisde proteínasseminales.

taxonomicsystemacceptedatpresent,including delimita¬ tions of thegeneraand theirsubdivisions in subtribe Phaseolinae. Thisclassificationisbasedonmorphological

andpalinological data. These authorsemphasizedthe fact that therearestilltoomanyproblemstobesolved dueto thegreatamountoftaxainvolved.

All of the Phaseolinaespeciesgrowing in Argen¬ tinahadneverbeen analyzed together.

Phaseolus L. has three sections:sect.Phaseolus,

the only section with species in Argentina, Alepidocalyx (Piper) Marèchalelal. andMinkelersia (MartetGall.)Marèchaletal. The “commonbean”P. vulgaris is very important in the economy of Northwestern Argentina region. The study of the naturalpopulations of P. vulgarisvar.aborigineus, the wild relativeofthecommonbean, is crucial for cropbreeding (Palacios & Hoc, 2001).

Manyspecies of Vignaareusedasfoodand for¬ age(Smartt, 1990),sotheknowledge of thegroupis important for their biotechnological improvement. Resultsreportedby Delgado Salinasetal. (1993)are consistent with the hypothesis ofapolyphyletic

ori-INTRODUCTION

TheSubtribe Phaseolinae Benth. (Phaseoleae, Fabaceae)has23 genera, four of them growing in Argentina: Phaseolus L. emendVerde., Vigna Savi, Macroptilium (Benth.) Urb., and Dolichopsis Hassl. Thereareseveral morphological(Verdcourt,1970; Marèchaletal. 1978; Drewes, 1995; Palacios & Hoc, 2001),biochemical (Zallocchi,1992)and molecular studies (Becerra Velasquez & Gepts, 1994; Fofana, 1999;Ruffini Castiglione etal.1998; Goel etal. 2002), which have attemptedtosolve taxonomic problems in thisgroup.However, only partial analyses have beenmade andmostofthestudies have exclusively involved wild species of Mesoamerican origin (Maquetetal. 1999).

Marèchaletal. {op. cit.)developed the morphological

'Lab.Plantas Vasculares,Depto.BiodiversidadyBiología Experimental,Facultad de CienciasExactasyNaturales,Uni¬ versidad de Buenos Aires. Cantilo y Costanera Norte, 4o piso,

Pabellón 2, CiudadUniversitaria(C1428EHA), BuenosAi¬

(2)

Seedprotein analysis. Storageproteinswere ex¬ tractedbygrindingoneor morematureseeds,and mixing 100mgofthepowderwith0.4mlof0.5MNaCl. Thesuspensionwas rest atroom temperaturefortwo hours and centrifugedat10000 rpm for 10min.The supernatantwasmixedwithanequalvolumeofcrack¬

ingbuffer (0.125M Tris-HCl pH 6.8; 4% w/v SDS;

20%. w/vGlycerol; 10% w/v 2-mercaptoethanol;

0.001%w/vbromophenolblue)andboiled for 2min. One-dimensionalsodium dodecyl sulfatepolyacryla¬

mide gelelectrophoresis (SDS-PAGE)

wás

carriedout ac-cordingtoLaemmli(1970).Therunninggelcontained15% ofpolyacrylamidein1.5MTris-HClpH8.8buffer,with10% w/v SDS, 0.05% w/v ammoniumpersulfate and 0.0005% TEMED.Thestackinggelhad4.5%ofpolyacrylamideina 0.5MTris-HClpH6.8buffer,with10%w/vSDS,0.005%w/ vammoniumpersulfate and 0.0001% TEMED. The elec¬ trode buffer had 0.025MTris,0.192M GlycineatpH 8.3, and0.1%w/vSDS.

The runningwasconducted

for

3-5hoursat40 mAand 120 V inaBioRadProteanIIapparatus.The gelwasstained with 0.05% w/v Coomasie Blue and 12% w/v trichloroacetic acid, overnight,and then

washedwith 7%v/Vacetic acid solution.

Numerical analysis. Basic data matrixwasmade consideringproteinbandsintheelectrophoreticrun¬ ningsasabsence/presence characters.UPGMA,Mini¬ mumSpanning Tree andPrincipalCoordinates Analy¬ sis methods were applied.The analysis wasper¬ formedusingtheNTSYS-pc 1.7

(Numerical

Taxonomy andMultivariate Analysis System)computerprogram designedby F. James Rohlf(1988)

ginofthe genus Vigna. They divided this genusin

twosubclades:oneincluding the Old Worldsubgen¬ eraand the American subgenusLasiospron, and the other subcladestrictlyofthe New World. Here, spe¬

cies of the subgeneraSigmoidotropis, Lasiospron and Vignagrowing inArgentina have beenanalyzed.

Only V.hookeri and V.caracalla havenotbeenin¬

cludedinthe analysis. The latter becausewecouldn’t collectmaturelegumes, and V. hookeri because it is believed that isextinct,for the lastcollectionwasin the1950's(TroncosodeBurkart&Bacigalupo, 1987). The third genus here studiedisMacroptilium,

with ninespecies in Argentina,all of them included in the analysis. Cladistic analysis using morphologi¬ cal characterssuggeststhe monophyly of bothsec¬ tions of the genera: Macroptilium andMicrocochle

(Drewes, 1995).Studiesusingmoleculartechniques

hadneverbeencarriedoutinthis group.

Dolichopsis

isamonotypic genus.The onlyspe¬ cies D. paraguariensis Hassler is endemictoPara¬ guay andArgentina (Palacios & Hoc, 2001).

In thepresentwork,speciesofPhaseolus,Vigna,

MacroptiliumandDolichopsishave been studied bymeansofelectrophoresisofseedstorageproteins.

This techniquehasprovedtobe usefulinlegume systematics (Burghardt &Palacios,1997;Burghardt, 2000aandb;Fotsoetal. 1994;Przybylska, 1995;

Przybylska & Zimniak-Przybylska, 1997; Maquetet al. 1999)Ithasbeenusedtoclarifyspecies delimitations,

andtoperformmanysupra-and intraspecificsystematic

studies(DeBustosetal. 1999;Sammour, 1994).

The main objective of this work istofinduseful

molecular markers of theArgentineanspecies of the genera Vigna, Phaseolus, Dolichopsis and

Macroptilium,whichwillallow the identification of each species, andto analyze thepossible genetic variabilitybetween and within them. Many of the species of these generaareeconomically important, so a greaterknowledge of theirvariabilityand rela¬

tionshipsiscrucial inordertoestablishstrategiesfor plantbreedingandgermplasmmanagement.

RESULTS

A hundreadantthirty fiveproteinbandsarefound inthe 20taxaanalyzed. Figure 1showssomeofthe proteinpatternsobtained inthisstudy.Within each species, all seeds had thesame’proteinpattern.The

onlyexceptionisPhaseolusvulgaris:P.vulgarisvar. vulgarisandP.vulgaris

var.

aborigineushaveincom¬ mon16oftheir 17 bands.

Threemarkerbands(i.e.,exclusiveandconstantbands) arefoundinMacroptiliumand fiveinPhaseolus,whileno marker bandsaredetectedinVignaas awhole.Speciesof the fourgenerashareonebandat66kDa. Species of Phaseolus, Vignaand Macroptilium share two bandsat 116and21kDa.AllthespeciesofMacroptiliumand Vigna sharethreebands,twoofthemaround200 andonearound 18kDa(Fig.l).

MATERIALS

AND

METHODS

Plant Material. Sixty-one accessions oftwenty Argentinean

species

of Phaseolus, Vigna,

DolichopsisandMacroptiliumwerestudied."Collec¬

tion data of the material examined in this studyare indicated in Table 1. Theyarealldeposited in the

Herbarium oftheFacultadde CienciasExactasyNatu¬

rales(University ofBuenos Aires),BAFC(abbrevia¬ tionaccordingtoHolmgrenetal. 1981).

(3)

S.M.Espert& A.Burghardt, Electrophoretic Analysisof Seed Proteins inPhaseolinae

TABLE 1. Taxonomic classification (sensu Marèchaletat.1978) of thespecies analyzed. RP=RamónPalacios,HOC=Patricia

Hoc, SD= Susana Drewes, MMS=M.del Carmen Menéndez Sevillano.

Sectionor subgenus

Genus Species Id. Number and Origin

RP769:Capital,Salta;RP1275&SD503: Concordia, Entre Ríos.

M.bmcteatum (Nees et Mart.)

Marèchal etBaudet

RP1357: Guemes, Salta; HOC341: Chicoana,

Salta

M.panduratum (Mart,ex Benth.)

Marèchal etBaudet

Section

Macroptilium

RP891: San Fernando,Chaco

M.lathyroides(L.) Urban

M.erythroloma (Mart,ex

Benth.) Urban

RP892:San Fernando,Chaco; RP878,

RP1100 & RP1284:SanIgnacio,Misiones;

RP1286:Candelaria,Misiones

M.longepedunculatum (Benth.)

Urban RP902, RP908 & RP910: Esquina, Corrientes

Macroptilium

RP 767, RP773 &RP775:Capital, Salta;

RP781:La Caldera,Jujuy;RP1014: Jujuy,

Jujuy

M.fraternum(Piper) Juárez

M.arenarium(Bacigalupo)

Drewes etPalacios

RP1299: Gualeguaychu, EntreRios;HOC373

& HOC374:Concepcion,Entre Rios Section

Microcochle RP1090&RP1280:Gral.Alvear,Corrientes;

RP1279:SanMartin,Corrientes;RP1282:

Capital,Misiones

M.psammodes(Lindm.)

DrewesetPalacios

RP844&RP1205:Colon, EntreRíos;RP856:

Concordia,Entre Ríos;RP1073;SantoTome,

Corrientes;RP1287: San Javier,Misiones

M. prostratum(Benth.)Urban

HOC281 &PH282:Rosario de Lerma,Salta P augusti Harms

P.lunatusLvar. sylvester

(Burk.)Baudet RP1210: Ledesma,Jujuy

Section Phaseolus

Phaseolus _{HOC347, HOC349, HOC351, HOC353, HOC}

354, HOC359 & HOC360:Rosariode Lerma,

Salta; RP1010:Capital, Jujuy P. vulgaris Lvar.aborigineus

(Burk.) Baudet

P vuigaris L var. vulgaris MMS110: Iruya,Salta

RP896: SanFemando, Chaco;RP1048: Famaillá, Tucumán; RP1092: Gral. Alvear, Corrientes;RP1209: Ledesma, Jujuy; SD507:

IslaTalayera,Buenos Aires

V.adenantha (G. Mey)

Marèchal etal. Subgenus

Sigmoidotropis

RP880: San Ignacio, Misiones; RP1288 & RP1289: SanJavier, Misiones

V.candida (Veil.)

Marèchaletal.

RP881&RP1296:SanIgnacio,Misiones;

RP1294: San Javier,Misiones V.peduncularis(Kúnth)

Faluc.etRendle

Vigna

RP956: Delta,Buenos Aires; RP1300:

Gualeguaychu, EntreRíos;RP1378: Predelta,

Entre Ríos Subgenus

Vigna V.luteola(Jacq.)Benth.

V. longifolia (Benth.)Verdcourt RP1297:Paso de los libres,Corrientes

Subgenus

Lasiospron _{V. lasiocarpa (Benth.)}_Verdcourt _{RP1285 &}_RP1295:_San_Ignacio,_Misiones

RP1513: Asunción,Paraguay

Dolichopsis D.paraguariensisHassler

GenusMacroptilium has three exclusive andcon- (sect.Microcochle). stantbands:onearound 116kDa,oneat25andan¬

otherat19kDa. There aren’tanymarker bandschar- ternsofPhaseolusaugustiandP.lunatus;twelve of these acterizing each section. Only four of the nine species bandsareshared by both species. P.augustihasseven ofMacroptilium here analyzed have marker bands: markerbands,andP lunatus has three.Pvulgarishas four M.bracteatum and M.panduratum (section exclusive andconstantbands, three of these conforms the Macroptilium),and

_M.fraternum

and M.prostratum phaseolinprotein placed around 45kDa.Accessionsof Twenty-eight bandsareidentifiedinpolypeptidic

(4)

pat-mm

Sri,

116HD

66HDtt

>

••

i

‘

*

45HDH1

r

₁₁

,

H

31HD

I

£

m

21HDBC=¡>

<=>

<=l

<=3

M Pvv Pv3 PI P3 M Mb Me Ml Mf'MprMpi M V3 Ve Vp VI Vio VI3

Fig.1.Seed proteinpatterns(SDS-PAGE)ofsomespecies of Phaseolus, Vignaand Macroptilium:MProteinladder, Pvv P.

vulgarisvarvulgaris,Pva P.vulgarisvaraborigineus,PIP.lunatus,Pa P. augusti,Mb M.bracteatum, MeM.erythroloma,Ml

M.lathyyroides,MfM.fratemum,Mp M.prostratum, Mps M.psammodes, Va Vadenantha, Vc V.candida,Vp Kpeduncularis,

VIV.luteola,Vlo V.longifolia, ViaV.lasiocarpa.Thearrowsindicate: Phaseolin; Phaseolus marker bands;!•*ÿI'

markerbands; Bandsshared by Macroptiliumand Vigna, Bands sharedbyMacroptilium,Vignaand Phaseolus Bands

shared by the four genera.

Macroptilium

P.vulgarishere studied showaphaseolinpattern typeT, tributewith evidencetoestablish theirrelationships.

accordingtothephaseolinclassification of Brownet The results herereportedareinagreementwith mor-al.(\981).This fraction doesn't appear inanyoftheacces- phological observationspreviouslyperformed sions ofP.augustiorP.lunatus (Fig. 1).

Electrophoregramsofthespecies of Vigna show thirty-fourbands.Subgenus Sigmoidotropis has only onemarkerband,and subgenus Vigna hasfour.These

subgenerahavetwobands incommon,while Vigna identification bydifferencesinthepatternsof pres¬ andLasiospron share three peptidic fractions. In sub- ence/absence of their polypeptidic bands. The genusLasiospron, sixexclusiveandconstantbands phenogram shows the species of eachgenusincluded areobserved in all theirspecies. Each Vigna species inunique clusters.Nevertheless,species of Vigna have marker bands by which itcanbe distinguished, are includedin two different clusters in the

Dolichopsispresents12exclusiveproteinbands, phenogram (Fig. 2). The identity of thegenera can sharinganotheronewithMacroptilium and Vigna. also be inferred viewing theMinimum Spanning Tree

The result of the UPGMA, basedonthe Jaccard coefficient, is showninFigure2.Theclusteringpro¬

ducesaverylow distortion (cophenetic correlation value of 0.95). A minimum spanningtree(Fig. 3) has beenderived fromamatrix oftaxonomic distances. Figure 4shows the distribution of the species ina three-dimensional space, delimitedby thefirstthree

principal coordinates derived from aPrincipal Coordinates

Analysis.

(Verdcourt,1971;Marèchal etal. 1978).

Generic

_{differentiation}

andrelationships.

Proteinprofilesof four generaallow their clear

D. paraguariensis M. erythroloma Mbracteatum Mlathyroides M. longepedunculatum

M.'arenarium

M.fratemum M,prostratum M. Psarmodes M.panduratum Vadenantha V.candida.

V.peduncularis V.luteola V.longlfolla Vlasiocarpa P.v.vulgaris Iv.aborigineus P. augusti P.lunatus

-c

d

R

DISCUSSION

_Q

Proteinpatternscorroborate genetic and’taxo¬

(5)

S. M. Espert & A.Burghardt, Electrophoretic Analysis of SeedProteinsin Phaseolinae

Numericalanalysis showsagreateraffinitybetween

Macroptiliumand Vigna. This fact is inagreementwith

morphological(Marèchaletal.op. cit.;Verdcourt 1970) andmolecular data (Caicedoetal. 1999).

InUPGMAandMinimumSpanningTreediagrams

(Fig. 2and 3), the species of Vigna appear inseparate groups,showingthat individuals ofsubgenera Vigna and Sigmoidotropisare moresimilartoMacroptilium thantosubgeneraLasiospron;as waspointedoutby

Marèchaletal. (1978) basedonmorphologicaldata.

Dolichopsis isthemoredistantgenus.Previous worksplace D.paraguariensis closerto Vignasub¬ genus Vigna (Marèchaletal. op.cit.) or to Vigna

subgenusSigmoidotropis and another genus of

Phaseolinae,StrophostylesElliot,notanalyzed here (DelgadoSalinasetal.1999;Goeletal. 2002). The inclusion of genera andspecies that don’t grow in Argentina will improvethe understanding of the placement ofthis monotypicgenus.

(Figure 3).The position of the speciesonthe space

delimitedby the three firstPrincipalCoordinates (Fig. 4) shows that each genusconstitutesadefinedgroup. Thespecies belongingtoaparticulargenusareposi¬ tionedas awell-definedconstellation in differentre¬ gions ofthe diagram. This indicatesa greatersimilar¬ ity

between

species from thesame genusthan be¬ tweenspecies of different genera.

Phaseolus species composeawell-definedgroup inall the analysis here performed. Ordination analy¬ ses show that Phaseoluslunatus is the Phaseolus

species thatseemstobemostclosely related with theother genera (Fig. 3 and 4). This result isinagree¬ mentwiththeobservationsonflowers: P. lunatusones aresmaller than those of other species ofPhaseolus, beingits size similartothe flowers of M. lathyroides andV,peduncularis(Marèchaletal.op.cit).

P.augusti

oogP.v.vulgaris _{°. lunatus} V.longifolia

. .:e:

P.v.aborigineus

Infrageneric

_{differentiations}

andrelationships.

Phaseolus L. emend Verdcourt.

Thisgenusis the focus of many works because ofits economicimportance. This isincontrast tothe other threegenera

analyzed

here, whichareless stud¬ ied than Phaseolus.

The phenogram basedonseedprotèin analysis

shows that P. lunatus constitutesacluster with P. augusti, distant from P.vulgaris(Fig. 2). One of the greatestdifferences ofP.vulgaris profilesis the pres¬ ence ofaconspicuousgroup ofbands (phaseolin protein),which is absent in the other Phaseolusspe¬ cies(seeblackarrowinFig.1). Otherstudies also showed that Andean wildspeciesofPhaseolus (like P.augusti), don’tpresentthisphaseolin (Maquet et al. 1999).Also,Maquet (op. cit.)reportedthat these species are moreclosely relatedtoP.lunatus,asis shown here. This result agrees with other molecular data (Caicedo etal.1999;Delgado Salinasetal. 1999). Thetwovarieties ofP. vulgaris (varvulgarisand varaborigineus) differ only inonepolypeptidicband. Thishighaffinitysupportsthe inclusion of

both

taxa

inthe same species, inagreementwiththe criteria adopted byBaudet,who studied morphological data (Palacios&Vilela,1993).

Onlycultivated forms ofP.vulgarisvarvulgaris areknown.These cultivated formsofbeansarethe result ofalongdomestication process.Thispractice could leadtoareduction of thevariabilitybecauseof geneticdrift, andanincreaseinthedifferentiation,so one can expect adifferentpattern between cultivated

V. adenantha

M.arenarium

*V. candida

V.peduncularis

D.paraguariensis

.M.bracteatum *M. panduratum

M.lathyroides _{M. fratemum}

M.erythroloma _{M. psammodes}

M.prostratum

Fig. 3.MinimumSpanning Treederivedfromamatrix of

taxonomic distances.

-*7/8

Vigna

•t

9 10

Dolichopsis

C2>

%

Phaseolus

3 _l4

»

lacroptilium

Fig.4.Distribution of thespecies inathree-dimensional space,

delimited bythe first threeprincipal .coordinates,derived from

aPrincipal Coordinates Analysis.(1 P.vulgarisvar vulgaris, 2 P.vulgarisvar aborigineus,3 P. augusti, 4P. lunatus,5 D.

paraguariensis, 6 V. luteola,7 V.longifolia, 8 V.lasiocarpa,9

V.candida, 10 V. adenantha,

.

11 V.peduncularis, 12M.

panduratum, 13 Merythroloma,14M.bracteatum, 15 M.

arenarium, 16M. longepedunculatum, 17 M. lathyroides, 18

(6)

formsand related wildspecies.This would be the ing to four genera ofPhaseolinae (Phaseolus, reasonofthegreat differences observed between Dolichopsis, Vigna andMacroptilium),which had Phaseolus species, whicharesimilartothosefound neverbeen analyzed together byusingmolecular between sectionsand subgenerainMacroptilium markers. Seedprotein electrophoreticbandingpat¬

ternsallowed therecognitionofageneticbasis of the genericdivisions, whichwereproposed using and Vigna (Fig.2)

Macroptilium(Bentham) Urban.

Therearevery fewprevious studiesonspecies morphologicalandpalinologicaldata. This work sup-ffornthis genus, and almostnoneusingmolecular portsthe subgeneric divisionsinPhaseolus and techniques. Drewes (1995 & 1996) pointedoutthe

ViSna

(sensu Marèchal et al.), but not in

separation of Macroptilium in the sections Macroptilium.Besides,the identification of all spe-Microcochle andMacroptilium,asproposedby ciesofthe8enera

nSna-

DolichopsisandPhaseolus, Lackey (1983) basedonmorphological observations. andmostofMacroptiliumispossible using marker The phenogram hereperformeddonotshow clearly bands- An electrophoretic analysis includingspecies thisseparation, neither do theMinimumValue Tree ofother Americancountries is inprogressinour

labo-andthe Principal CoordinatesAnalysis.

ratorT’

and wiU be veiTimportantinordertogeta SpeciesofsectionMacroptiliumarehighlydif-

ireater

knowledgeof the genetics of the group, which

ferentbetweeneachother,regardingmorphological 1Sessential foritsbiotechnologicalimprovementand data. Phenogram basedonelectrophoretic resultsin germplasmmanagement.

particular shows that M.lathyroides and M.longepedunculatum belonging to sect. Macroptiliumclearly havemorecloselyrelationships withspeciesofsect.Microcochle. It isnotpossible tomakecomparisonsabout infrageneric data with

ACKNOWLEDGEMENTS

Financial support from ConsejoNacional de previous works based onDNA sequence data. Investigaciones Científicas y Técnicas and Delgado Salinasetal. 1999 and Goeletal. 2002are Universidad de BuenosAires,both of Argentina,aré theonlytwomolecularsystematicresearchers who gratefully acknowledged. Shirley M. Espert enjoyed included species ofMacroptilium,but they onlyana- ascholarshipfromthe Universidad de BuenosAires,

lyzedtwospecies. Vigna Savi.

Electrophoretic results

show

that V.luteolaformsa

group withspecies of subgenus Sigmoidotropis, and

BIBLIOGRAPHY

presents low affinity with species of subgenus

Lasiospron(Fig.2,3and4).Inallthe analyses, Vluteola is well separated from the otherspecies.This result is inagreementwiththesubdivision of the genusper¬

formedby Marèchaletal.(1978),who placed Vluteola BROWN,j.w._S,_Y._{MA, F. A. BLISS & T.c.}_HALL._1981.

inadifferent subgenus (Vigna), while it doesn’t sup¬ portLackey’ssystem(1983),that groups Vignaluteola,

V.longifolia and V.lasiocarpa inthesamesubgenus. BURGHARDT, A. D.& R.A.PALACIOS.1997. Electro-Thereisagreatdifferentiationbetweenproteinprofiles

ofLasiospron andSigmoidotropisspecies.This featureis

inagreementwith Vigna polyphyletic origin hypothesis, proposed by Delgado Salinasetal. (1993) using cpDNA sequences. Theabsence ofmarker bands that characterize

whosesupportis acknowledged.

BECERRAVELASQUEZ,V. & P.GEPTS.1994. RFLP

diversityof commonbean (Phaseolus vulgaris)in its

centersoforigin.Genome37: 256-263

Genetic variation inthesubunits of globulin1 storage

protein of French bean.Theor.Appl. Genet. 59: 83-88

phoretic characterizationofthe Americansections of

ProsopisL.(Leguminosae: Mimosoideae).Bull.Int.Group

StudyMimosoideae20: 71-83.

BURGHARDT,A.D. 2000a. Estudioelectroforético de

proteínasde semillaenProsopis(Leguminosae) I:Sección Algarabía.Bol.Soc.Argent.Bot. 35: 137-147.

BURGHARDT,A.D.2000b.Estudioelectroforéticode proteínasdesemillaenProsopis(Leguminosae) II: Sección

Strombocarpa.Bol. Soc.Argent.Bot. 35: 149-156.

CAICEDO, A. L.,e.GAITÁN,M. C.DUQUE,O.TORO

CHICA,D. G. DEBOUCK &J. TOHME. 1999. AFLP

Fingerprinting OfPhaseolus lunatus L.and Related Wild

Speciesfrom SouthAmerica.CropScience39: 1497-1507

the genusalsosupportthis hypothesis.

CONCLUSIONS

Inthis work,it has been possibleto studythe

(7)

belong-S.M.Espert&A.Burghardt,Electrophoretic Analysisof SeedProteinsinPhaseolinae

DE BUSTOS,A., C.CASANOVA,N. JOUVE & C. SOLER.

1999.Analysisofthe genetic diversity ofwild Spanish

populationsof the genus Hordeum through thestudyof theirendosperm proteins.Plant Syst. Evol. 214:1-4

DELGADO SALINAS, A., A. BRUNEAU & J. J. DOYLE.

1993.ChloroplastDNAphylogeneticstudies inNew

World Phaseolinae, Leguminosae, Papilionoideae,

Phaseoleae. Syst. Bot. 18: 6-17

DELGADOSALINASA.,T.TURLEY, A. RICHMAN & M.

LAVIN.1999.Phylogeneticanalysis ofthe cultivated and wild species of Phaseolus (Fabaceae). Syst. Bot. 24:

438-460

PALACIOS, R. A. & A. E. VILELA. 1993. Lasespecies

argentinasde Phaseolus (unrecursogenético que necesita protección). Actas 11 Simposio Latinoamericano sobre

recursosgenéticosde especies hortícolas: 123-139.

PALACIOS, R. A.&P.S. HOC. 2001.Phaseolinae. Tribu XV

Phaseoleae.Fabaceae,parte12. Florafanerogámica

argentina._Proflora,Fascículo 75. CONICET.

PRZYBYLSKA, J. 1995. Someexamplesoftheuseofelec¬

trophoreticprotein analysis intaxonomic investigations of leguminousplants.J.Appl. Genet.36: 255-271.

PRZYBYLSKA, J. & Z. ZIMNIAK-PRZYBYLSKA.1997.

Electrophoreticseedalbuminpatternsin Vicia species of

sect.Hypechusaand Peregrinae (Fabaceae). Plant Syst. Evol. 208: 3-4

DREWES, S. I.1995.Revisiónde lasespeciesargentinasdel

géneroMacroptilium (Benth.)Urban

(Leguminosae-Phaseolinae). Tesis Doctoral. Facultad de Ciencias Exactas y Naturales.Universidad de Buenos Aires.

DREWES,S. I. 1996. Análisis cladísticodeMacroptilium (Fabaceae).Libro deresúmenes.XXV Jornadas Argentinas de Botánica: 165.

ROHLF,F.J.1988Numerical Taxonomy and Multivariate Analysis System,Version 1.70.AppliedBiostatistics Inc.,

NewYork.

RUFFINICASTIGLIONE,M.,L. BINI,P.PELOSI,R.

MARROCCO, A. SANTUCCI, F. RUBERTI,F.MAGGINI

& S. AVANZI. 1998. Ribosomal RNA genes of Phaseolus

coccineusV. Relationship between rDNAphenotypeand

somatic differentiation. Protoplasma 203:1-2.

FOFANA, B. 1999. MolecularevidenceforanAndean origin

and a secondary gene pool for the Lima bean using

chloroplast DNA. Theor. Appl.Gen. 98: 202-212

FOTSO, M.,J.L.AZANÀ,R.PASQUET&J. RAYMOND.

1994. Molecular heterogeneity of Cowpea (Vigna

unguiculata, Fabaceae)seed storageproteins.Plant Syst.

Evol.191: 39-56 SAMMOUR, R. H. 1994.Seed protein homologyand species

relationshipinthe tribe Vicieae. Feddes Repert. 105: 191-196.

GOEL,S.,S.N.RAINA & Y. OGIHARA.2002.Molecular

evolution and phylogeneticimplicationsofinternaltran¬ scribed spacer sequences ofnuclear ribosomal DNAinthe

Phaseolus-Vignacomplex.Molec.Phyl.Evol. 22: 1-19

HOLMGREN, P. K„ W. KEUKEN & E. SCHOFIELD. 1981.

IndexHerbariorum. Part 1. The HerbariaoftheWorld.

InRegnum VegetabileVol. 106.Utrecht,Netherlands. 7°

SMARTT, J. 1990. Grainlegumes: evolutionandgenetic

resources.Cambridge Univ. Press, Cambridge, UK.

TRONCOSODEBURKART, N. S. & N. M. BACIGALUPO.

1987. Flora ilustrada de Entre Ríos (Argentina).

Colección científicadelI.N.T.A.Tomo 6, Parte 3. Ed.

VERDCOURT, B. 1970. Studies in the Leguminosae

Papilionoideae forthe “Flora ofTropicalEastAfrica”

IV. Kew Bull. 24: 507-570 LACKEY, J. A. 1983.- A review of geneticconceptsinAmeri¬

canPhaseolinae(Fabaceae, Faboideae).Iselya2: 21-64

LAEMMLI,U. K. 1970.Cleavage of structural proteins dur-'

ing the assembly of the head ofbacteriophage T4.Nature

227: 680-685

MAQUETA., X. VEKEMANS & J. P. BAÚDOIN. 1999. Phylogenetic studyonwildalliesofLima bean, Phaseolus lunatus (Fabaceae), andimplicationsonits origin. Plant Syst.Evol 218:1-2.

MARÈCHALR„ J. M. MASCHERPA & F. STAINER. 1978.

Etude taxonomique d'un groupe complexed'especesdes

genresPhaseoluset Vigna (Papilionaceae)surla base de

donneés morphológiques etpolliniques, traiteés par

l’analyseinformatique.Boissera28: 1-273.

VERDCOURT, B.1971.Phaseoleae. In: E. Milne-Redhead & R.M.Polhill,Flora_ofTropical East_Africa, Leguminosae (Part IV),Papilionoideae(2).London.

ZALLOCCHI,E. 1992. Estudio quimiotaxonómico de las especies argentinas dela subtribuPhaseolinae:géneros

Vigna, Phaseolus, Macroptilium y.Dolichopsis

(Phaseoleae- Papilionoideae -Leguminosae).Tesis

Doctoral. Facultad de Ciencias ExactasyNaturales,

Universidad de Buenos Aires.