Genetic Resources and Crop Evolution45: 243–251, 1998. 243c 1998Kluwer Academic Publishers. Printed in the Netherlands.

Genetic variation in common and runner bean of the Northern Meseta inSpain

M.T. Alvarez, L.E. Saenz de Miera & M. Perez de la Vega�Area de Genetica, Facultad de Biologia, Universidad de Leon, 24071 Leon, Spain (� author for correspondence)

Received 25 July 1997; accepted in revised form 21 October 1997

Key words: Phaseolus, genetic variability, amino acids, isozymes, proteins, RAPD

Summary

The genetic variation within and between Spanish landraces or varieties ofPhaseolus vulgarisL. (common bean)andP. coccineusL. (runner bean) has been estimated by means of isozymes and random amplified polymorphicDNA (RAPD) analyses. Likewise, storage protein and amino acid content in dry seeds have been estimated.Fifteen landraces (60 accessions) ofP. vulgarisand six ofP. coccineus(six accessions) have been studied. Of theseven isozymatic systems analyzed only three systems and three loci showed variability in each species. Isozymeanalyses revealed that genetic variability within and between landraces exist in both species. Even variabilitywithin accession was detected in someP. vulgarislandraces. Comparison of isozyme data indicated that Spanishlandraces have a lower level of genetic variability than wild American materials and probably also lower thanAmerican landraces. RAPD analysis allowed for the uniquely distinguishing of all landraces. Genetic similarityamong landraces, estimated by both isozymes and RAPDs, were not related with the seed morphological characters(color, size and shape) which define each variety or landrace. Variation in protein and amino acid content amonglandraces was also detected. The average protein content in common bean (20.48%) was similar to values previouslyreported in this species and higher than the average in the runner bean landraces (16.33%). In relation to the aminoacid content methionine and cysteine showed the lowest values in all samples, although the content of these twoamino acids varied widely among landraces.

Introduction

The genusPhaseolusincludes several wild and cul-tivates species such asP. vulgaris L. and P. coc-cineus L., common and runner bean, respectively.Phaseolusspecies were domesticated in two Americanareas, Mesoamerica and the Andean Region (BecerraVelasquez & Gepts, 1994; Kaplan & Kaplan, 1988)and were introduced into Spain during the early col-onization period, where the common bean in particu-lar became a widely used staple crop in many areas,and currently is one of the two most widely cultivatedgrain legumes. Although new cultivars are displacinglandraces in Spain, it is still possible to find farmerswho grow bean for self-consumption and for sale atfarmer-markets. The variability of Spanish commonbean landraces in relation to seed color, size (although,in general, large seeded varieties are preferred) and

shape and other plant quantitative characters is veryhigh (Gil & Ron, 1992).

As in other crop species, isozymes and DNA mark-ers have been widely used to evaluate germplasmgenetic diversity in and betweenPhaseolusspecies,within both cultivated and wild materials, to analyzethe crop evolution and the effect of domestication ongenetic diversity (Becerra Velasquez & Gepts, 1994;Gepts et al., 1992; Koenig & Gepts, 1989; Singh etal., 1991; Skroch & Nienhuis, 1995a). Likewise thesegenetic markers have been used as tools for breedingpurposes, such as genetically linked tags to quantita-tive or qualitative genes (Haley et al., 1993; Vallejos &Chase, 1991).

Pulse seeds are characterized by a relatively highcontent of proteins, which in dry seeds ofPhaseolusspecies ranges from 17% to 32% (Baldi & Salamini,1973; Salunkhe et al., 1985). This fact make beans

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a cheap source of proteins, although their nutritionalvalue is limited due the relatively low amounts of someessential amino acids such as methionine.A wide rangeof variation in the content of some amino acids has alsobeen reported inPhaseolusspecies (Baldi & Salamini,1973).

The aim of this work was to estimate the genet-ic variation existing within and between landraces ofP. vulgarisand P. coccineuscultivated in one of themain areas of bean cultivation in Spain, the NorthernMeseta, as a preliminary evaluation of their potentialsfor further breeding programs. Variation of isozymes,RAPD markers, protein and amino acids content wasanalyzed. Variation between accessions within vari-eties (only isozymes were analyzed at that level) andbetween varieties was observed. Protein and aminoacid contents have also been estimated.

Materials and methods

The materials used were 60 samples (hereafter desig-nated as accessions) ofPhaseolus vulgarisL. corre-sponding to 15 local Spanish varieties and six samplesfrom six local varieties ofP. coccineusL. (Tables 1 and2). Most of the accessions were collected directly fromfarmers who cultivated these varieties of dry beans fortheir own consumption and to sell on a small scale inopen air markets. Some accessions were from selectedlines obtained from these varieties at the Servicio deInvestigacion Agraria, Zamaduenas, Valladolid, andkindly supplied by Dr. C. Asensio and J. A. Ponga.

Sample sizes of 20 to 54 seedlings per accessionwere analyzed to determine isozyme variability. Seedswere germinated in a growth chamber under controlledconditions of temperature and photoperiod on wet cot-ton. After 10–15 days, a leaf and roots for each seedlingwere crushed separately in a mortar and the crudeextracts were absorbed in paper wicks (Whatman 3MM0.4� 1 cm) and inserted in 10% horizontal starch gels.Each plant extract was electrophoresed in two differentbuffer systems: 1) Leaf extracts in Tris – borate acid pH8.9 (Tris 0.15 M, boric acid 0.036 M, EDTA Na2 0.002M) as the gel buffer and pH 8.5 (Tris 0.2 M, boric acid0.15 M, EDTA Na2 0.002 M) as the electrode buffer,a modification of Wendel and Weeden (1989); and 2)root extracts in histidine 0.005 M adjusted to pH 7.0with NaOH as the gel buffer and Tris - citrate pH 7.0(Tris 0.135 M, citric acid 0.043 M) as the electrodebuffer (Hutchinson et al., 1983). After electrophoresiseach gel, 1 cm thick, was sliced into 2 mm thick slabs

and each slab was stained for a different enzymaticsystem. The slabs from the Tris - borate buffer sys-tem (leaves) were stained for shikimate dehydrogenase(SKD, EC 1.1.1.25), aspartate aminotransferase (AAT,EC 2.6.1.1, also known as glutamate-oxaloacetatetransaminase or GOT), glucose-6-phosphate isomerase(GPI, EC 5.3.1.9), and phosphoglucomutase (PGM,EC 2.7.5.1); the slabs from the histidine buffer sys-tem (roots) were stained for malate dehydrogenase(MDH, EC 1.1.1.37), 6-phosphogluconate dehydro-genase (6PGD, EC 1.1.1.44), and alcohol dehydroge-nase (ADH, EC 1.1.1.1). The staining procedures were,with minor modifications, those described by Vallejos(1983).

Genomic DNA was extracted from five dry seedsper variety (P. vulgaris) or accession (P. coccineus).Seeds were crushed together in a micro-mill and DNAwas extracted by adding 750�l of CTAB buffer for 30min at 65�C, then adding 500�l of chloroform-octanol24:1, and two extractions in phenol-chloroform. DNAmarkers were obtained by polymerase chain reaction(PCR) from approximately 20 ng of genomic DNAper reaction. Random amplified polymorphic DNA(RAPD) was studied in the 15 varieties ofP. vulgarisand the six accessions ofP. coccineususing as singleprimers 10-mer oligonucleotides (sets OPA, OPB andOPD, 60 primers) obtained from Operon Technolo-gies Inc (Alameda, California) andTaq polymerase(Promega). Samples were incubated in a thermal cycler(Perkin Elmer 480) programmed as follows: initialstrand separation at 94�C (1 min), 45 cycles of 36�C(1 min)/72 �C (2 min)/94 �C (1 min), final exten-sion at 72�C (10 min). Amplification products wereelectrophoresed in 2% agarose gels and detected bystaining with ethidium bromide. Bands were designat-ed by the name of the primer used and a correspondingnumber, in decreasing order of size (#1 the largest).The ability of primers to produce repeatable RAPDpatterns was assayed by analyzing two DNA samplesper extraction. Each PCR-RAPD band was treated asa single unit character with two phenotypes: presencevs. absence. Similarities were estimated by the Jaccardindex, and phenograms from both isozyme and RAPDdata were obtained by the U.P.G.M.A. method.

In order to analyze protein and amino acid con-tent an accession per variety ofP. vulgarisand threeaccessions ofP. coccineuswere chosen (those morewidely cultivated and consumed). To minimize envi-ronmental effects, new seeds were obtained by cul-turing all the varieties together. Three rows of seeds(25 per row) per variety were planted in each of

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Table 1. Allelic frequencies of variable isozyme loci inP. vulgaris

Variety N S P Gpi-2 1 Gpi-2 2 Mdh-3 1 Mdh-3 2 Skd-1 1 Skd-1 2

Aceite 100 2 2 0.410 0.590 1.000 0.000 1.000 0.000

Arroz 199 4 3 0.769 0.231 1.000 0.000 0.131 0.869

Canela 420 9 4 0.283 0.717 1.000 0.000 1.000 0.000

Canellini 202 4 1 0.970 0.030 1.000 0.000 0.515 0.485

Cara Virgen 125 3 0 1.000 0.000 1.000 0.000 1.000 0.000

Carota 149 3 1 0.725 0.275 1.000 0.000 0.000 1.000

Fabada 160 4 2 0.619 0.381 0.444 0.556 0.906 0.094

M. Virgen 112 3 3 1.000 0.000 0.893 0.107 0.795 0.205

Morada 89 2 1 0.663 0.337 0.000 1.000 1.000 0.000

Palmena 123 3 1 1.000 0.000 1.000 0.000 0.959 0.041

Pinta 376 8 6 0.559 0.441 0.782 0.218 1.000 0.000

Planchada 137 3 3 0.109 0.891 1.000 0.000 0.971 0.029

Plancheta 80 2 1 0.038 0.962 1.000 0.000 1.000 0.000

Redonda 177 4 0 0.678 0.322 1.000 0.000 1.000 0.000

Rinon 261 6 4 0.946 0.054 0.559 0.441 0.682 0.318

P. vulgaris 2710 60 32 0.638 0.362 0.857 0.143 0.797 0.203

N = number of individuals, S = number of accessions. The average of accesssion sample size was 45.17(range from 20 to 54 seeds). P = Number of polymorphic accessions.

Table 2. Allelic frequencies of variable isozyme loci inP. coccineus

Accession N Gpi-2 1 Gpi-2 2 Skd-1 1 Skd-1 2 Skd-1 3 Adh-1 1 Adh-1 2

61 24 0.292 0.708 1.000 0.000 0.000 0.917 0.083

62 32 0.609 0.391 0.844 0.062 0.094 0.516 0.484

63 26 0.519 0.481 0.538 0.192 0.269 0.808 0.192

64 22 0.614 0.386 0.591 0.000 0.409 0.409 0.591

65 26 0.558 0.442 0.462 0.000 0.538 0.865 0.135

66 26 0.519 0.481 0.615 0.269 0.115 0.923 0.077

P. coccineus 156 0.522 0.478 0.679 0.090 0.231 0.737 0.263

N = Sample size.

three replicated plots. Discarding the plants at theextremes, the seeds from the plants of the central rowof each variety and replica were separately harvest-ed; thus, each variety was represented by three seedsamples. Seeds were homogenized in a micro mill,and extracts for protein and for amino acid analyseswere obtained and analyzed in three different sets ofassays. Therefore, from each variety nine measureswere obtained in each case. The analysis of proteincontent was carried out by means of the bicinchonicacid method (Smith et al., 1985). Amino acids wereanalyzed by means of the PICO-TAG method (Mil-lipore): protein extracts were hydrolyzed and aminoacid derivatized with phenylisothiocyanate (Heinrik-son and Meredith, 1984) and analyzed by means of

reversed-phase high performance liquid chromatogra-phy (Chang et al., 1989).

Results

Isozyme variation

Of the seven isoenzymatic systems analyzed only threesystems and three loci, among 17 loci, showed vari-ability in P. vulgaris (Gpi-2, Mdh-3 and Skd-1) andin P. coccineus(Gpi-2, Skd-1 and Adh-1). Isozymepatterns were similar to those previously reported byother authors (Gepts et al., 1993; Weeden et al., 1989).Some differences are probably due to the use of dif-ferent buffer systems and/or seedling tissue. The most

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Table 3. Examples of allelic frequencies in some varieties ofP. vulgaris

Variety N Gpi-2 1 Gpi-2 2 Mdh-3 1 Mdh-3 2 Skd-1 1 Skd-1 2

and accession

Canela 420 0.283 0.717 1.000 0.000 1.000 0.000

1 50 1.000 0.000 1.000 0.000 1.000 0.000

2 42 0.000 1.000 1.000 0.000 1.000 0.000

3 50 1.000 0.000 1.000 0.000 1.000 0.000

4 29 0.000 1.000 1.000 0.000 1.000 0.000

5 50 0.060 0.940 1.000 0.000 1.000 0.000

6 50 0.100 0.900 1.000 0.000 1.000 0.000

7 49 0.122 0.878 1.000 0.000 1.000 0.000

8 50 0.100 0.900 1.000 0.000 1.000 0.000

9 50 0.000 1.000 1.000 0.000 1.000 0.000

Pinta 376 0.559 0.441 0.782 0.218 1.000 0.000

1 50 1.000 0.000 0.800 0.200 1.000 0.000

2 50 0.200 0.800 0.000 1.000 1.000 0.000

3 38 0.895 0.105 0.895 0.105 1.000 0.000

4 50 0.920 0.080 0.740 0.260 1.000 0.000

5 46 0.435 0.565 1.000 0.000 1.000 0.000

6 38 0.000 1.000 1.000 0.000 1.000 0.000

7 50 1.000 0.000 0.900 0.100 1.000 0.000

8 54 0.000 1.000 1.000 0.000 1.000 0.000

Rinon 261 0.946 0.054 0.559 0.441 0.682 0.318

1 50 1.000 0.000 0.920 0.080 1.000 0.000

2 51 0.941 0.059 0.118 0.882 1.000 0.000

3 50 0.840 0.160 0.960 0.040 0.260 0.740

4 41 1.000 0.000 0.000 1.000 1.000 0.000

5 46 0.935 0.065 1.000 0.000 0.000 1.000

6 23 1.000 0.000 0.000 1.000 1.000 0.000

noticeable observation was in MDH. The faster MDHzone, MDH-1, always showed a three-banded patterncharacteristic of a dimeric enzyme, while MDH-2 wasmonomorphic for a single band. However, a mobili-ty shift of the MDH-1 three-band set was observed inrelation to MDH-2. This mobility shift was deducedto be under the control of locusMdh-3and the sloweralternative was related to alleleMdh-3 1and the fasterto Mdh-3 2. Tables 1 and 2 show the allelic frequenciesof variable loci found in the varieties and accessions ofboth species. To reduce the set of data, Table 1 presentsthe allelic frequencies ofP. vulgarisvarieties, whichwere calculated by pooling all individuals of each vari-ety, although some varieties presented variation withinand between accessions. Table 3 shows some examplesof common bean varieties with diversity within andbetween accessions. Most varieties were polymorphic,with the exception of two varieties, Cara de la Virgenand Palmena, which can be considered monomorphic

at the level of 95%. Some polymorphic varieties, suchas Redonda, however, consisted of a combination ofaccessions fixed for different alleles. Of the 60 acces-sions, 28 did not show variability, 26 were polymorphicat one locus, five at two loci and one was polymorphicat three loci. Variability in common bean accessionswas due to the presence of mixtures of homozygousgenotypes as no heterozygotes were observed. In thepartially outcrossingP. coccineus,genetic variationin accessions was higher than in the predominantlyself-pollinating common bean. Thus, all six accessionswere polymorphic, and heterozygous individuals wereobserved. The higher variation inP. coccineusthan inP. vulgariscan be also observed in Table 4. The averagenumber of alleles per locus and the genetic diversityindices are higher in the former species. The propor-tion of variation residing among varieties or accessions(GST ) is higher in common bean (0.834) than inP.coccineusin which most of the total variation is due

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Table 4. Levels of isozyme variation inPhaseolusvarietiesincluding monomorphic and polymorphic loci

Variety P A HS HT DST GST

Aceite 1 1.059 0.018 0.028 0.010 0.357

Arroz 2 1.118 0.014 0.034 0.020 0.588

Canela 1 1.059 0.005 0.024 0.019 0.792

Canellini 1 1.118 0.003 0.033 0.030 0.909

Cara Virgen 0 1.000 0.000 0.000 0.000 –

Carota 1 1.059 0.006 0.025 0.019 0.760

Fabada 3 1.176 0.018 0.067 0.049 0.731

Manto Virgen 2 1.118 0.021 0.030 0.009 0.300

Morada 1 1.059 0.014 0.026 0.012 0.462

Palmena 1 1.059 0.004 0.005 0.001 0.200

Pinta 2 1.118 0.016 0.049 0.033 0.673

Planchada 2 1.118 0.015 0.015 0.000 –

Plancheta 1 1.059 0.004 0.004 0.000 –

Redonda 1 1.059 0.000 0.026 0.026 1.000

Rinon 2 1.176 0.013 0.061 0.048 0.787

P. vulgaris1 2 1.090 0.028 0.061 0.033 0.541

P. vulgaris2 2 1.038 0.010 0.061 0.051 0.834

P. coccineus 3 1.270 0.069 0.089 0.020 0.225

P = Number of polymorphic loci per variety; A = average num-ber of alelles per locus;. HS = genetic diversity within acces-sions; HT = total genetic diversity in varieties, in the threebotton rows inP. vulgarisor P. coccineus.1 A and HS are average values within accessions.2 A and HS are average values within varieties considering allaccessions of each variety as a single population.

to variation within accessions (GST = 0.225). Whenthe variation of accessions withinP. vulgarisvarietiesis considered the total genetic variation is distributedalmost equally within and between accessions (GST =0.541).

The low number of variable isozyme loci andalleles, and the variation between accession withinvarieties prevented discrimination between varietiesin common bean. Only the two species were clear-ly differentiated, mainly due to the differences in thespecies-specific polymorphism ofAdh-1andMdh-3. Infact, when Nei’s genetic distance and the U.P.G.M.A.clustering methods were used, accessions of the samecommon bean varieties were scattered in several clus-ters, many distances between accessions of the same ordifferent variety were zero and the highest distance wasas low as 0.1. Runner bean accessions were grouped ina separte cluster (data not shown).

Fig 1. Phenogram of thePhaseolusvarieties based on RAPD pat-terns. Similarities were estimated by the Jaccard index and thephenogram obtained by the U.P.G.M.A. method.

RAPD analysis

The RAPD analysis was carried out with a mixture ofDNA from five seeds per variety of common bean oraccession of runner bean. Thus, the band patterns ofP.vulgariswere representative of varieties and only dif-ferences between them, not between accessions, wereobserved. Of the 60 primers tested, 35 amplified DNAconsistently generating a total of 120 clear and repeat-able bands, of which 70 were observed inP. vulgarisand 73 inP. coccineus. Only 23 bands were shared bythe two species. Of the 35 positive primers, 12 generat-ed bands only inP. vulgarisand 9 only inP. coccineus.Thirty-two amplified at least one PCR-band which wasobserved in accessions of only one species but not inthose of the other species, thus discriminating betweenthe two species (Fig. 1). The average Jaccard similari-ty index between common and runner bean was 0.170� 0.010 (range from 0.150 to 0.198), whereas within

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Table 5. Proteina and amino acidb content in somePhase-olusvarieties and accessions.

Variety Protein Met Cys Lys

P. vulgaris

Canela 21.823 1.266 0.302 7.543

Canellini 17.655 1.485 0.371 7.359

Fabada 22.314 1.398 0.413 7.243

Morada 19.578 1.434 0.314 7.153

Palmena 19.771 0.822 0.208 7.556

Pinta 20.356 1.375 0.316 7.487

Planchada 15.825 1.610 0.389 7.260

Plancheta 24.701 1.823 0.425 7.787

Redonda 22.625 0.867 0.190 7.299

Rinon 20.101 0.953 0.323 7.130

Mean (S. D.) 20.481 1.303 0.325 7.440

(2.42) (0.31) (0.07) (0.20)

P. coccineus

61 15.176 0.939 0.396 8.785

62 15.562 1.350 0.701 8.037

63 18.259 0.973 0.476 7.973

Mean (S. D.) 16.333 1.087 0.524 8.265

(1.37) (0.19) (0.13) (0.37)

a Percentage of protein in seed dry matter.b Percentage of the total content in amino acids. Onlylimiting amino acids in legumes and lysine have beenincluded. The complete set of data can be obtained onrequest from the authors.

species this index was 0.832� 0.007 (0.776 to 0.984)for common bean varieties and 0.926� 0.008 (0.875to 0.971) forP. coccineus. Each variety could be dis-tinguished by the RAPD patterns. As for isozymes,clusters based on RAPD similarity (Fig. 1) were notrelated with seed morphological characters (color, sizeand shape). Thus, Morada is a dark-red-seeded varietywhile Rinon is white-seeded, or Aceite is one of thesmallest-seeded while Plancheta is one of the largest-seeded varieties.

Protein and amino acid variation

The protein and amino acid content analysis was car-ried out in 10 varieties of common bean and threeaccessions of runner bean (Table 5). They were select-ed to represent the most commonly planted varieties inthe Spanish Northern Meseta. Table 5 shows that pro-tein content varied between approximately 16% and25% and that, on average, common bean presentedhigher contents of protein (20.48%) than runner bean(16.33%). An ANOVA analysis of the protein content

of the common bean varieties indicated that there wasa highly significant difference due to the variety, a sig-nificant difference due to field plot, but no significantdifferences due to the remaining causes of variance,including the set of analyses (data not shown). In factit was possible to distinguish three groups of varietieswhich showed no significant differences in protein con-tent within the group but, in most cases, significant tohighly significant differences between varieties of dif-ferent groups. Plancheta, Redonda, Fabada and Canelaconstituted the group with the highest protein content;Canellini and Planchada constituted the low protein-content group; and Morada, Palmena, Pinta and Rinonrepresented the middle protein-content group.

Seventeen amino acids were analyzed. Due to themethod of analysis, tryptophan was hydrolyzed andasparagine and glutamine were included as asparticand glutamic acids, respectively. Of these amino acids,aspartic and glutamic acids were always the mostabundant, representing together on average 28.5% and26.5% inP. vulgarisandP. coccineus, respectively. Onthe other hand, sulfur-containing amino acids showedthe lowest values in all varieties (Table 5). No oth-er amino acid was present in a percentage lower than3% in any estimate. Likewise, sulfur-containing aminoacids showed the greatest relative variation among vari-eties since the highest percentage was approximatelytwice the lowest one (1.610% vs. 0.822% for methio-nine, 0.425% vs. 0.190% for cysteine), whereas otheramino acids showed a proportionally narrower vari-ation among varieties. In general, the relative differ-ences between the lowest and the highest concentra-tions were lower than 25% of the lowest concentrations(e.g., for serine the lowest was 5.6% and the highest6.2%). The lysine content ranged between 7.1% and8.8% approximately, always being higher in runnerbean accessions (Table 5).

Discussion

The two species,P. vulgarisandP. coccineusdiffer,among other characteristics, in their mating system.It is known that the levels of genetic variation andthe form variation is distributed correlating positive-ly with the levels of outbreeding in the populationsor species (Hamrick & Godt, 1990). The commonbean is a predominantly self-pollinating species where-as cross-pollination frequencies as high as 0.698 havebeen estimated by multilocus analyses in runner bean(Escalante et al., 1994). This fact agrees with the genet-

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ic structure observed in the accessions. Heterozygousplants were observed inP. coccineusaccessions butwere absent inP. vulgarisones. However, the levelof genetic diversity (HS) within accesions was low inboth species (Table 4) since most isozyme loci werefixed. A characteristic of the common bean varietieswas the isozyme variability within and between acces-sions in most varieties. This fact indicates that thereis genetic variation in these varieties, although theylook uniform on the basis of seed characteristics, thosethat farmers use to recognize and designate landracesand varieties. Likewise, some variation in plant growthhabits have been observed in these varieties (data notshown). This result agrees with the landrace nature ofthese materials, while modern improved varieties ofinbreeding crops are genetically uniform, landraces ofthese species are usually mixtures of genotypes.

Koenig and Gepts (1989), analyzing 83 wild acces-sions ofP. vulgarisfrom the two major center of geneticdiversity by means of 14 enzyme systems and 22 loci,described a total genetic diversity (HT ) value of 0.132,higher than the value observed in our cultivated acces-sions (0.061, Table 4). Other parameters reported inthat work were DST = 0.126 and GST = 0.952. Like-wise, genetic variability of cultivated American mate-rials seem to be higher than that of the Spanish onesanalyzed here (Singh et al., 1991; genetic diversityparameters are not explicit in this paper). Therefore,cultivated Spanish varieties showed a lower level ofgenetic variability than wild American materials andprobably also lower than American landraces. Thisresult agrees with the known reduction of genetic diver-sity caused by common bean domestication, althoughit was not as pronounced for isozymes as for phase-olin (Singh et al., 1991), and the fact that only part ofthe American germplasm is represented in Spain since,according to phaseolin patterns (Gepts & Bliss, 1988)and quantitative characters (Gil & Ron, 1992), most ofthe Spanish varieties are of Andean origin. The GST

value estimated in our materials was lower than thatreported previously, this fact means that genetic vari-ation within varieties and accessions is proportionallyhigher in Spanish landraces than in other common beanmaterials previously studied. This result must be partlycaused by the relative small sample size used in previ-ous works (Koenig & Gepts, 1978; Singh at al., 1991),which makes the detection of within-sample variationmore difficult.

With regard toP. coccineus, Escalante et al. (1994)reported the variability of seven variable isozyme lociin four Mexican accessions. Although the number of

accessions and loci were low in both cases, the data ongenetic variability for variable loci from Escalante etal. (1994) and from our work suggest that genetic vari-ability was similar in both set of samples. Escalanteet al. (1994) found an average number of alleles perlocus of 2.7 and an average of heterozygosity of 0.31,while in our case were 2.33 and 0.40, respectively,considering only variable loci. Escalante et al. (1994)pointed out that apparently the domestication processhad not eroded the levels of genetic variation ofP. coc-cineusand that the similar levels of genetic variationamong wild and cultivated materials was mainly dueto the outcrossing rates in this species. Unfortunately,data on SpanishP. coccineusvarieties are too scarce toconclude on the genetic variation maintained after itsintroduction into Spain, although our results suggestthat it can be high and somewhat higher than inP. vul-garis. On the other hand, it is clear that outbreeding inrunner bean has maintained most of the genetic varia-tion distributed within accessions, as indicated by theGST value obtained from isozyme data (Table 4) andthe higher average of genetic similarity obtained fromRAPD data.

Because of the low variability observed withisozymes in and between accessions of each varietywe decided to carry out a first screening of RAPD vari-ability obtaining band patterns characteristic of eachvariety using a DNA pool of five seeds from differ-ent accessions (Yu & Pauls, 1993). Serious concernsregarding the reliability of RAPD markers in determin-ing genetic relationships have been raised. However,other works indicated that RAPDs provide a level ofresolution equivalent to RFLPs for determining genet-ic relationships among genotypes and lines (Halldenet al., 1994; dos Santos et al., 1994), and may be use-ful for the unique identification of varieties, for theorganization of bean germplasm and for applicationsto bean breeding (Skroch & Nienhuis, 1995a, 1995b).Our RAPD results clearly discriminate the two speciesunder study although, within each species, the inclu-sion of varieties into clusters was unrelated with thevariety seed type (size, shape, color and color pattern).RAPD markers have also proved to be useful in dis-criminating betweenCentrosemaspecies (Phaseoleae)(Penteado et al., 1996). Weeden (1984) indicated that10 isozyme loci and 72 allozyme combinations wereable to uniquely distinguish 58% of a total of 90 com-mon bean cultivars. The number of RAPD bands gen-erated allowed us to uniquely distinguish all the vari-eties used, and since the number of available primersis much higher, it is probable that RAPD multiband

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patterns will allow for the discrimination of most, ifnot all, common bean genotypes.

As previously mentioned, both isozymes andRAPD markers were not related to seed type. A sim-ilar result with isozymes was reported by Singh et al.(1991) in 227 American landraces. Isozymes and sizecharacteristics, except seed size, have a simple geneticcontrol (Singh et al., 1991). These authors stated thata given allozyme genotype could be present in mor-phologically different American landraces, and theysuggested that most of the cultivars with the sameallozyme genotype, following their origination froma common ancestor, have undergone further diversi-fication for morphological traits but not for molecu-lar markers. We found a complementary situation, inthat allozyme genotypes are shared by morphologi-cally different varieties but also inside them, or eveninside accessions with clearly defined seed morphol-ogy, color, and color pattern. It is relatively frequentto find two allozyme genotypes differing at one ortwo loci (Table 3). Our results suggest a differenthypothesis. Occasional outcrosses primarily generat-ed some variability among neutral or nearly neutralallozymes, and further mixtures of seeds, due to seedinterchanges among farmers, maintain this invisiblewithin-variety isozyme variability, while farmers suc-cessfully maintain the seed characteristics which allowfor their recognition.

The average protein content estimated in the com-mon bean varieties (20.48%) is similar to the officialaverage value of Spanish varieties of 20.75% (Minis-terio de Agricultura, 1984) and the value of 21.90%reported in another set of Spanish varieties (Escribanoet al., 1990), and slightly lower than values reportedin materials from other origins (Baldi and Salamini,1973; Mendoza de Bosque et al., 1990) although aver-age contents as high as 28% have been reported inPhaseolus vulgarissamples (Salunkhe et al., 1985).The fact that our results agree with most of the previ-ous results points out that the bicinchonic acid methodto evaluate protein content of legume seeds is a reliableone. Baldi and Salamini (1973) also reported a lowerprotein content inP. coccineusthan inP. vulgaris.

High performance liquid chromatography has beendescribed as a suitable method to evaluate amino acidcontent in legume seeds (Chang et al., 1989). The val-ues obtained by this method in our samples are in gen-eral agreement with previous values analyzed in otherbean materials and by using the same and other meth-ods (Chang et al., 1989; Kelly & Bliss, 1975; Ma &Bliss, 1978), although it has been reported that differ-

ent analytical methods can determine significant differ-ences in estimations. In our comparative study betweenvarieties and accessions, the proportionally more sig-nificant differences were observed for sulfur-containigamino acids. No significant correlation between pro-tein content and sulfur-containing amino acids wasobserved.

The analyses performed in this work indicate thatSpanish traditional varieties of common and runnerbean maintain relatively high levels of genetic vari-ation, and that variation exist even inside samples.This germplasm represent a valuable source of genet-ic diversity that probably would be highly useful forfuture breeding programs.

Acknowledgements

We are grateful to Dr. C. Asensio and J. A. Pongawho supplied us with some materials. This work waspartially supported by research grants (AGF92-0816)and a doctoral grant to M.T. Alvarez also from theSpanish D.G.I.C.Y.T.

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