Pl. Syst. Evol. 213:153-164 (1998)
PlantSystematics and Evolution © Springer-Verlag 1998 Printed in Austria
Isozyme diversity, RFLP of the rDNA and phylogenetic affinities among cultivated Lima beans, Phaseolus lunatus (Fabaceae) LUCIA LIOI, CONCETTALOTTI, and INCORONATAGALASSO
Received April 7, 1997; in revised version July 17, 1997
Key words" Leguminosae, Phaseolus. - Isozymes, genetic distance, Lima bean, rDNA, RFLE Abstraet: Genetic variation in Phaseolus lunatus (Lima bean) was investigated at isozyme and DNA levels. Sixty cultivated accessions, including representatives of the Mesoamerican and Andean gene pools and intermediate types, were analyzed for variability at 17 isozyme loci. Some accessions were also examined for restriction fragment length polymorphism (RFLP) at the rDNA level. These data were used to constmct two dendrograms showing clear separation in two distinct groups corresponding to each of the gene pools and an intermediate one probably representing a transitional group.
Lima bean (Phaseolus lunatus L.), one of the major cultivated species of the genus Phaseolus, is an important source of proteins for rural populations in South America and Africa. The first hypothesis proposed by MACKm (1943) indicated a single centre of origin for this species in Guatemala, with three main routes of dispersion. However, archaeological (KAPLAN Æ KAPLAN 1988), biochemical (GUTIERREZ SALGADO• al. 1995) and molecular data (JAcoB & al. 1995, NIEN~UIS& al. 1995) do not confirm this area as the possible place of origin of Lima beans, rather suggesting the idea of separate domestications for the small- and largeseeded forms. The existence of two gene pools resulting from independent domestication centres, a Mesoamerican (M) and an Andean (A) one, was first established for Lima bean by MAQUET& al. (1990) and was later confirmed by the subsequent studies of LIoI (1994) and GUTmRREZSALGADO& al. (1995) on the basis of electrophoretic analysis of seed storage proteins. Phaseolin, the major storage protein in Phaseolus, has been successfully used as a biochemical marker in P. vulgaris (GEPTS & al. 1986). Recently, purification and characterization of phaseolin in P. lunatus (SPARVOLI& al. 1996) allowed a better understanding of the relationships among morphotypes. Small-seeded genotypes, Sieva and Potato, with flat or rounded seeds, respectively, and M (Mesoamerican) phaseolin pattern type belong to the Mesoamerican gene pool. The Andean gene pool is characterized by big flat seeds, the Big Lima morphotype, and A (Andean) phaseolin pattern type. A
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third group with intermediate Sieva-Big Lima seed type and MA phaseolin is found among materials stored in different gene banks (LioI 1994). At the same time, on the basis of their rDNA specific hybridization patterns (JACOB & al. 1995) and of RAPD analysis (NmNmJ~s & al. 1995), two major clusters corresponding to the Mesoamerican and Andean gene pools were observed in cultivated Lima bean. The genetic relationships among genotypes established on the basis of RAPDs markers (NTzNHUIS & al. 1995) and RFLP of phytohemagglutinin genes (Z~NK & al. 1994) indicated a separate third cluster formed by Fordhook cultivars, a class of commercial Lima bean cultivated for harvesting at immature seed stage and eaten as vegetables. A good understanding of the domestication pattern and genetic relationships among cultivars in a crop species enables a more efficient organization of germplasm resources and a better evaluation of available genetic diversity. Biochemical and molecular markers have been good tools for studying the domestication pattern in common bean (KozNi~ & GEPTS 1989, BECERRAVELASQUEZ & GEPTS 1994). Recent investigations in Lima bean using isozymes (MAQUZT& al. 1996) and RFLP data from rDNA loci (JACOß & al. 1995) showed that both markers are suitable for evaluating genetic variation. In particular, the intergenic spacer (IGS), a region of rDNA separating the genes encoding the 25S-28S and 18S ribosomal subunits, composed of arrays of short repetitive sequences in which the length, methylation status, composition, and the copy number can vary among plant species, subspecies and varieties (LEITCrI & HESLOP-HARRISON 1993), is a valuable tool for phylogenetic analysis. To achieve a better understanding of genetic relationships among small-, intermediate-, and large-seeded genotypes, we investigated allozymic and rDNA polymorphisms among cultivated Lima bean genotypes. Materials and methods Plant material. The sixty accessions analyzed in this study were obtained from: Centro
International de Agricultura Tropical (CIAT), Cali, Colombia (G accessions); Germplasm Institute, Bari, Italy (MG accessions); Jardin Botanique National de Belgique, Meise, Belgium (NI accessions); Institute of Plant Genetics and Plant Research, Gatersleben, Germany (PHA accessions); Empresa Brasileira de Pesquisas Agropecuarias (EMBRAPA), Brasil (GL accessions). Table 1 lists the accessions analyzed by means of isozymes including 45 small- (32 Sieva and 13 Potato), 7 intermediate- and 8 large-seeded (Big Lima) morphotypes, and those chosen for RFLP analysis. Isozymes. Five seeds from each accession were imbibed for three days in Petri dishes. Cotyledons were ground in a medium containing 0.1 M potassium phosphate buffer pH 7.0 as described by H~SSAIN& al. (1988). After centrifugation at 12000rpm for 10min, the homogenate was stored at - 8 0 °C until required. Assayed isoenzyme systems are listed in Table 2. Eleetrophoresis was performed in 10.5% starch with 1.2% urea. Electrode and gel buffers, 0.135 M and 0.1 M Tris-citrate pH 7.0, respectively, were the same for all analyzed enzyme systems. Staining procedures were those used for forest tree species (MÜELLERSTARCK1997). Locus and alMe designation were assigned as follows: loci were labelled sequentially, with those migrating closest to the anodal end designated as number 1. Accession MG 133931 was considered as a standard and the allelozymes from this genotype were designated as 100. The other allozymes were measured as the relative distance from the standard. Isozyme phenotypes were interpreted genetically on the basis of information
Isozyme diversity and RFLP in Lima bean
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available on isozyme structure and genetic control as reported in KOEMG& GEPTS (1989), JAASKA(1996) and MAQUET& al. (1996). DNA extraction and Southern hybridization. Total genomic DNA was extracted from young leaves using the "maize DNA miniprep" method according to DELLAPORTA& al. (1983). Five or six gg of DNA from each sample (see Table 1) were digested with EcoRI, BamHI, HaeIII, DraI according the supplier's recommendations. The resulting fragments were separated electrophoretically on a 1% agarose gel and transferred onto positively charged nylon membrane using a standard technique. Probe labelling, hybridization and detection were carried out by the non-radioactive chemoluminescence method (ECL) following the manufactures's instructions (Amersham). Source of probes. The DNA probes used were (i) pTa71, a 9kb EcoRI-fragment of rDNA isolated from a common wheat, Triticum aestivum L., containing the coding sequences for the 5.8S, 18S, 25S ribosomal subunits and intergenic spacer regions (GERLACH & BEDBROOK1989); (ii) the EcoRI-BamHI fragment of the pPH5 clone (2.8kb), mostly constituted by the intergenic spacer (IGS) region of Phaseolus coccineus (MAGoINI& al. 1992). Cluster analysis and estimation of genetic distances. Based on allozyme frequencies, NE~'S (1978) genetic diversity statistics were used to measure total gene diversity (Ht) as well as within-(Hs) and among-morphogroups (Dst) differences. NEI'S distance (D) was used to construct a dendrogram by the UPGMA method by means of computer program BIOSYS-1 developed by D. L. SWOFFORD& R. B. SELANDER,Illinois, 1989. Specific restriction fragments were scored for presence (1) or absence (0) and a 0/1 matrix was prepared. This matrix was used to compute a distance matrix based on Jaccard index. NTSYS-pc statistic software, version 1.70, developed by F. J. ROHLF, New York, 1992, was utilized both for generating the Jaccard indices and for UPGMA clusterings of these index values.
Table 1. Identification, morphotype and allozyme constitution in cultivated Phaseolus lunatus Accession no a
Morphotype
Aco-2
Adh-2
Dia-2
Idh-2
Mdh- 1
Pgi-2
Skdh- 1
GL92 GL100 GL145 GL476 MG113217 MG113930 MG113931 b MG115402 NI0003 NI0549 PHA8067 PHA8068 PHA8069 b PHA8070 b PHA8071 PHA8073 PHA8074 PHA8075
Sieva Sieva Sieva Sieva Sieva Sieva Sieva Sieva Sieva Sieva Sieva Sieva Sieva Sieva Sieva Sieva Sieva Sieva
100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100
100 100 100 80, 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100
100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 80, 100
100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100
100 100, 115 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100
100 85, 100 100 85, 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100
82, 100 82, 100 82, 100 100 82, 100 82, 100 82 100 100 82, 100 82, 100 82, 100 82, 100 82, 100 82, 100 82, 100 82, 100 82, 100
L. Lioi & al.:
156 Table 1 (continued) Accession
Morphotype
Aco-2
Adh-2
Dia-2
Idh-2
Mdh-1
Pgi-2
Skdh-1
Sieva Sleva Sieva Sieva Sieva Sleva Sleva Sieva Sieva Sieva Sieva Sieva Sieva Sieva Potato Potato Potato Potato Potato
100 100 100 100 100 100 100 100 100 100 100, 115 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 115 115 115 115 115 115 115 115
100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 90 90 90 90 90 100 100 100 100 100 100 100 100
100 100 100 100 100 100 100 100 100 100 100 100, 100 100 100 100 100 100 100 100 100 100 100 100 100, 100, 100 100 100 100, 100, 100, 100 100, 100 100 100 100 100 100 100 100
100 100 100 100 100 100 100 100 100 100 85, 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 112 112 112 112 112 112 112 85 85 85, 100 85 85 85 85 85
100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100, 115 100 100 115 115 115 115 100 115 100 115 115 115 115 115 115 115 115
100 100 100 100 100 100 100 100 100 100 85, 100 85, 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 100 85 85 85 85 85 85 85 85 85 85 85 85 85 85 85
82, 100 82, 100 100 82, 100 82, 100 82, 100 82, 100 82, 100 82, 100 100 82, 100 82, 100 82, 100 82, 100 82, 100 100 100 100 100 82, 100 100 82, 100 82, 100 82, 100 100 82, 100 82, 100 100 100 100 100 100 82, 100 100 100 100 100 100 100 100 100 100
no a
PHA8076 PHA8077 PHA8079 PHA8109 PHA8111 PHA8128 PHA8151 PHA8153 b G25004 G25129 G25139 G25276Ab G25408A G25743 GL136b GL146 GL181 b NI0153 b NI0283 G25001 b G25100 b G25109 G25135 G25190 G25314 G25250 G25770 GL183 GL189 GL487 b GL491 b NI0157 b G25108 b G25355 b NI0018 PHA8078 b PHA8092b PHA8110 b PHA8152 b G25167 b G25579 G25832
Potato Potato
Potato Potato Potato Potato Potato Potato Intermediate Intermediate Intermediate Intermediate Intermediate Intermediate Intermediate Big Lima Big Lima Big Lima Big Lima Big Lima Big Lima Big Lima Big Lima
145
145 145
145 145 145 145
a The accession numbers preceded by the letters G, GL, MG, NI, and PHA are from Ciat, Cali, Colombia, EMBRAPA, Brasil, Germplasm Institute, Bari, Italy, Jardin Botanique National, Meise Belgium, and Institute of Plant Genetics and Crop Plant Research, Gatersleben, Germany, respectively. bAccessions analyzed for RFLP variation.
Isozyme diversity and RFLP in Lima bean
157
Results Isozyme analysis. Cultivated accessions of Lima bean showed variation in seven of the eight enzyme systems (Table 2) with 17 bands of activity, seven (41%) of which correspond to polymorphic loci. Mean number of alleles per locus (A) was 1.53. At species level, mean genetic diversity, or expected heterozygosity according to HARDY-WEINBERG (H), was 0.13. The genetic diversity among cultivated P. lunatus morphogroups was calculated with three parameters (Ht, Hs and Dst) considering both mono and polymorphic loci. Total genetic diversity (Ht) was 0.168. There was little within-morphogroups gene diversity (Hs = 0.04), but between-morphogroups genetic diversity (Dst) increased to 0.128. The obtained coefficient of gene differentiation (Gst) was 0.29. The isozyme systems analyzed in this work are listed in Table 2. The only monomorphic system, 6PGDH, was visualized as two bands of activity (6Pgdh-1, 6Pgdh-2). Two bands of activity were also described in wild Lima bean (MAQUET& al. 1996), P. acutifolius (GARVIN& al. 1989), and Vigna (PANELLA& GEPTS 1992). Only the SKDH enzyme system had a single well-resolved activity region, with single- and double-banded variants. Data on common bean (WEEDEN1984) indicate that SKDH is a monomeric enzyme coded by a single gene. DIA exhibited a fivebanded pattern, similar to the one described by SPRECHER(1988) in common bean. DIA is described as a tetrameric enzyme coded by two linked genes, Dia-1 and Dia-2, with five bands representing homomeric and heteromeric tetramers (SPRECHER 1988). The same results are found by SC~tlN~L & GEprs (1989) in tepary bean. ADH and PGI, having two loci each and a dimeric structure, produced intergenic hybrid bands as they do in other Phaseolus species (WEEDEN 1986, GARVIN& al. 1989, JAASga 1996). Aconitase was visualized as two bands of activity (Aco-1, Aco-2) appearing close together. This agrees with data reported for P. acutifolius and Vigna (GARVIN& WEEDEN 1990, PANELLA& GEPTS 1992). IDH and Table 2. Assayed enzyme systems, loci and alleles Aconitase (ACO)
E.C.4.2.1.3
Alcohol dehydrogenase (ADH)
E.C.1.1.1.1
Diaphorase (DIA)
E.C.1.6.4.3
Isocitrate dehydrogenase (IDH)
E.C.1.1.1.42
Malate dehydrogenase (MDH)
E.C.1.1.1.37
6-Phosphogluconate dehydrogenase (6PGDH) Phosphoglucoisomerase (PGI)
E.C.1.1.1.43
Shikimate dehydrogenase (SKDH)
E.C.1.1.1.25
E.C.5.3.1.9
Aco-1, Aco-2 Adh-1, Adh-2 Dia-l, Dia-2 ldh-1, ldh-2 ldh-3 Mdh-1, Mdh-2 Mdh-3 6Pgdh-1 6Pgdh-2 Pgi-1, Pgi-2 Skdh-1
100 100, 115 100 80, 90, 100 100 80, 100, 145 100 85, 100, 112 100 100, 115 100 100 100 100 100 85, 100 82, 100
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Fig. 1. Dendrogram of 60 cultivated Phaseolus lunatus accessions based on frequency data at 17 isozyme loci. For details on accessions see Table 1. Letters M, A and I indicate the Mesoamerican, Andean and intermediate groups, respectively
Isozyme diversity and RFLP in Lima bean
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MDH zymograms revealed three bands of activity which were attributed to three different loci. Three bands of independent variation pattern were found for MDH in a number of Phaseolus species (JAASKA 1996). Allelic frequency differences are maximum between the Mesoamerican and the Andean groups, otherwise accessions from intermediate group show some unique alleles (Adh-29°, Idh-21~2), and some alleles similar to the Mesoamerican (Dia2 ~45, Aco-21°°) or to the Andean (Mdh-11~5, Pgi-285) materials (Table 1). The dendrogram constructed by pairwise genetic distances (Fig. 1) revealed the genetic divergence of two groups of accessions. The first group, including Mesoamerican small-seeded accessions, consists of Sieva and Potato morphotypes. The second group consists of large-seeded Big Lima accessions. Between these groups, we could distinguish another one, constituted by intermediate Sieva/Big Lima morphotypes. The latter group occupies an intermediate location in the dendrogram. R F L P patterns, A total of 20 accessions, ten small-seeded (5 Sieva and 5 Potato), plus five for intermediate and Big Lima respectively, were chosen to L~. I
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160
L. LIoI & al.:
compare RFLP patterns (see Table 1). When the pTa71 clone and the 2.8 kb pPH5 fragment (IGS) were used as probes on Southern blots of genomic DNA DraI digests, a single restriction fragment of about 9.4 kb, with slight differences in size among the samples, was detected (data not shown and not considered in subsequent cluster analysis). Southern hybridization to BamHI blots with pTa71 showed constant bands at 0.5 kb, 1 kb, and 2.7 kb (Fig. 2a, b), not observed in membranes probed with IGS (Fig. 3a, b), while polymorphic fragments from 3 kb to 9.4kb among the three groups were observed with both probes. The Mesoamerican group (Fig. 2a) showed five different patterns based on number and length of fragments, the Andean group (Fig. 2b, left) showed two major fragments at 6 kb and 7 kb, while several bands with a maximum of four were visible in the intermediate group (Fig. 2b right). EcoRI blots probed with pTa71 showed two intense bands (Fig. 2c, d), a constant one (4 kb long) in all genotypes, and a variable one among the three
kb
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Fig. 3. Southern hybridization of 2.8 kb pPH5 fragment (IGS) to BamHI (a, b), EcoRI (c, d) and HaeIII (e, f ) -digested DNA of different accessions of Phaseolus lunatus. Letters M, A and I indicate the Mesoamerican, Andean and intermediate groups, respectively. Lambda Hind III-digest DNA was used as DNA size marker (L)
Isozyme diversity and RFLP in Lima bean
161
groups (from 6.5 kb to 9.4 kb). Only the variable band was observed in blots probed with IGS (Fig. 3c, d). The Andean gene pool showed a band at about 9.4kb (Fig. 2d, left), the intermediate group at about 6.5 kb (Fig. 2d, right), while EcoRI rDNA restriction site polymorphisms were observed among the different accessions belonging to the Mesoamerican gene pool (from 6.5kb to 9.4kb, Fig. 2c). Digestion with HaeIII yielded three constant bands between 0.4 kb and 1 kb in all the accessions analyzed after hybridization with pTa71 (Fig. 2e, f). When the pPH5 fragment (IGS) was used as a probe on HaeIII-blots, a more complex hybridization pattern was visible, indicating a high number of HaeIII sites in the intergenic spacer of P. lunatus rDNA. The range between 0.4 kb and 1 kb appeared regularly among all the 20 accessions analyzed for band number and fragment length (Fig. 3e, f), while the range between 1.5 kb and 4.8 kb seemed to be more variable. In the Mesoamerican group, at least three fragments from 2.3 kb to 4 kb were visible (Fig. 3e); the Andean group showed a higher fragment number (about eight) in the range from 1.5kb to 4.8kb in all accessions, and the intermediate group showed fewer fragments than the Andean group, consistent of repeat units of different lengths in the range 2 kb to 4 kb. The results obtained show that polymorphic fragments are strictly groupspecific for the Andean material, whereas intermediate accessions showed some group-specific fragments and some that were common to the Mesoamerican group. On the basis of the hybridization patterns obtained, the observed polymorphism within the rDNA is evidently located in the intergenic spacer; this polymorphism was used for to construct a 0/1 matrix. The dendrogram calculated by the NTSYS program based on JACCARD'Ssimilarity coefficient clearly grouped genotypes into two clusters corresponding to the Mesoamerican (Sieva and Potato) and the Andean (Big Lima) gene pools (Fig. 4). A third distinct cluster, termed inter-
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Fig. 4. UPGMA dendrogram based on JACCARD'S sirnilarity coefficient of rDNA RFLP diversity among cultivated Phaseolus lunatus. For details on accessions see Table 1. Letters M, A and I indicate the Mesoamerican, Andean and intermediate group, respectively
162
L. Lioi & al.:
mediate, corresponding to SievadBig Lima intermediate accessions, was slightly more closely related to the Mesoamerican group. Discussion
Allozyme and RFLP diversity data confirm clearly that the Mesoamerican and the Andean genotypes diverge in two gene pools. At species level, the parameters of genetic diversity in Lima bean are similar to the values found in wild populations of the same species (MAQUET• al. 1996) and in cultivated common bean (KoENIG & GEPTS 1989). Allozyme data confirm the information provided by phaseolin diversity (GUTIERREZSALGADO& al. 1995), indicating that Lima bean cultivars belong to two major gene pools distinguished by contrasting alleles. Genetic variability at these loci is small both in relation to the number of polymorphic loci and to the number of alleles per locus. The selfing mating system and geographic isolation are probably two major causes of gene differentiation between the Mesoamerican and the Andean groups. RFLP data grouping accessions is consistent with isozyme and seed storage protein groupings. Hybridization with the ribosomal DNA probe, pTa 71, show that the regions containing structural genes encoding for the ribosomal subunits are strongly conserved. In fact, most of the observed polymorphisms within the rDNA are evidently located in the intergenic spacers (IGS) separating the coding regions. Differences in copy numbers, length, and sequence of IGS subrepeats generate different sizes of restriction fragments and enable phylogenetic classification among and within plant species (LEITCH& HESLOP-HARRISON1993). In our case, variation of IGS enables discrimination between Mesoamerican and Andean genotypes. At intragroup level, the distribution of small-seeded Sieva and Potato accessions in the Mesoamerican cluster confirms that these morphotypes are not clearly separated from each other. Moreover, a higher genetic variation is detected in accessions from the Mesoamerican as against the Andean gene pool. Although scarce sampling of Andean genotypes many have led to lower estimates of genetic diversity, higher genetic variability in accessions from the Mesoamerican gene pool has already been reported in Lima (NmNHUIS& al. 1995) and common (KOENIG& GEPTS 1989) beans. Additional variability is represented by a third distinct cluster constituted of intermediate accessions. This cluster, which includes phenotypically intermediate SievaJBig Lima seeds (LIOI 1994), represents a genetically distinct group. First, accessions are stored independently in different gene banks, although four of them (GL accessions) originate from Brazil, and the other three from Africa (Ghana, Nigeria). Second, intermediate accessions show the MA phaseolin pattern found also in wild Lima beans from the Mesoamerican gene pool (Lioi 1996). Third, our present allozyme and RFLP data suggest that intermediate types possess unique alleles. So, the intermediate cluster takes up an intermediate and separate position in the dendrograms constructed using isozymes and rDNA polymorphism as markers, Minor centres of genetic diversity have been described in common bean (FREYRE& al. 1996), and also a Lima bean cluster other than the major gene pools has been reported (NIENHUIS& al. 1995). Biochemically and phenotypically the
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intermediate nature of this group could be explained by assuming that the intermediate types originated separately in a geographical transition zone between the two major gene pools. The authors wish to thank Prof. J. S. HESLOP-HARRISON(John Innes Centre, Norwich, U.K.) and Prof. F. MAGGINI(University of Viterbo, Italy) for providing the pTa71 and pPH5 fragment probes respectively; Prof. S. BENEDETTELH(University of Florence, Italy) and Dr S. PUGLISI(Germplasm Institute, CNR, Bari, Italy) for helping with computer programs.
References
BECERRAVELASQUEZ,V., GEvrs, P., 1994: RFLP diversity of common bean (Phaseolus vulgaris) in its centres of origin. - Genome 37: 256-263. DELLAPORTA,S. L., WOOD,J., HICKS,J. B., 1983: A plant DNA minipreparation: version II. Pl. Molec. Biol. Reporter. 1: 19-21. FREYRE, R., RIos, R., GUZMAN, L., DEBOUCK, D. G., GEPTS, P., 1996: Ecogeographic distribution of PhaseoIus spp. (Fabaceae) in Bolivia. - Econ. Bot. 50: 195-215. GARVIN, D. E, ROOSE, M. L., WAINES,J. G., 1989: Isozyme genetics and linkage in tepary bean, Phaseolus acutifolius A. GRAY.- J. Heredity 80: 373-376. - WEEDEN, N. E, 1990: Aconitase variation in tepary bean suggests a common origin for domesticated landraces. - Report (Annual) Bean Improv. Coop. 33: 132-133. GEPTS, P., OSBORN,T. C., RASHKA,K., BLISS, F. A., 1986: Phaseolin protein variability in wild forms and landraces of common bean (Phaseolus vulgaris): evidence for multiple centers of domestication. - Econ. Bot. 40:451-468. GERLACH, W. L., BEDBROOK,J. R., 1989: Cloning and characterization of ribosomal RNA genes from wheat and barley. - Nucl. Acids Res. 7: 1869-1885. GUTmRREZSALGADO,A., GEvrs, E, DEBOUCK,D. G., 1995: Evidence for two gene pools of the Lima bean, PhaseoIus lunatus L., in the Americas. - Genet. Res. Crop Evol. 42: 1528. HUSSMN, A., BUSHUK, W., RAMIREZ, H., ROCA, W., 1988: A practical guide for electrophoretic analysis of isoenzymes and proteins in cassava, field beans and forage legumes. - Working document no. 40 Centro International de Agricultura Tropical (CIAT), Cali, Colombia. JAASKA, V., 1996: Isoenzyme diversity and phylogenetic affinities among the PhaseoIus beans (Fabaceae). - Pl. Syst. Evol. 200: 233-252. JACOB, M., ZINK, D., NAGL, W., 1995: RFLPs of the rDNA genes in the genus Phaseolus. Genet. Res. Crop Evol. 42: 97-106. KAPLAN, L., KAPLAN, N., 1988: Phaseolus in archaeology. - In GepTS, R, (Ed.): Genetic resources of PhaseoIus beans, pp. 125-142. - Dordrecht: Kluwer. KoENm, R., GEPTS, R, 1989: Allozyme diversity in wild Phaseolus vulgaris: further evidence for two major centers of diversity. - Theor. AppL Genet. 78: 809-817. LEITCH, A. R., HESLoP-HARRISON, J. S., 1993: Ribosomal RNA gene expression and localization in cereals. - In SUM~R, A. T., CHANDLEY,A. C., (Eds): Chromosomes today 11, pp. 91-100. - London: Chapmann & Hall. L~o~, L., 1994: Morphotype relationships in Lima bean (Phaseolus lunatus L.) deduced from variation of the evolutionary marker phaseolin. - Genet. Res. Crop Evol. 41: 8185. - 1996: Phaseolin diversity in wild Lima bean (Phaseolus lunatus L.) in American centres of origin. - Genet. Res. Crop Evol. 43: 575-580. MACKm, W. W., 1943: Origin, dispersal and variability of the Lima bean, Phaseolus lunatus. - Hilgardia 15: 1-24.
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Address of the authors: LucIA LIol (correspondence; e-mail:
[email protected]), CONCETTALOTTI, INCORONATAGALASSO,Germplasm Institute, CNR, Via Amendola 165/A, 1-70126 Bari, Italy. Accepted July 19, 1997 by I. KRISAI-GREILttUBER
