Euphytica 35 (1986) 201 209

PREDICTING IN B E A N S

INTERSPECIFIC

COMPATIBILITIES

(PHASEOLUS)

BY S E E D P R O T E I N
1

ELECTROPHOREStS
J.G. S U L L I V A N 2andG. F R E Y T A G

University of Puerto Rico, and the Tropical Agricultural Research Station, Mayaguez, Puerto Rico
Received 7 August 1985

INDEX

WORDS

Phaseolus sp., interspecific hybridization, electrophoresis, germplasm, genetic distances, cluster analysis,

similarity indiex. SUMMARY Seed proteins of 17 wild species of Phaseolus were separated by electrophoresis on SDS polyacrylamide gels. There was very little variation of the protein pattern within most species, while considerable variation among species was evident. Relative interspecific similarities of protein patterns were estimated using Jaccard's similarity index, and a cluster analysis was performed on these values. The resultant dendrogram generally agreed with previous research on interspecific relationships in Phaseolus based on morphological characteristics, and also generally agrees with current information on interspecific similarities based on hybridization studies. Suggestions are made regarding interspecific hybridization among Phaseolus species which have not been attempted, but which may be possible based on cluster analysis. These hybrids include
P. vulgaris x P. polystachyus, P. vulgaris x (P. polystachyus x P. meteal/bi) and P. anisotrichos x P. angustissimus.

INTRODUCTION

According to the most recent taxonomic study of beans (MARI~CHALet al., 1978), 31 species have been placed in the genus Phaseolus. The interspecific hybrids reported to date represent less than 2 percent of all possible crosses among Phaseolus species (Table 1). MARECHALet al. (1978) presented dendrograms of phylogenic relationships among Phaseolus species based on morphological and pollen characteristics. Those dendrograms agreed well with genetic similarities among the species, as measured by relative ease of interspecific hybridization. Based on electrophoretic, immunological and agglutination analyses, J. W. S. Brown and F. A. Bliss (University of Wisconsin, Madison, personal communication) found that P. vulgaris was very similar to P. coccineus and somewhat similar to P. acutifolius, while P. lunatus was very different from the other species. These results were in general agreement with morphological similarities
1Supported by the USDA Specific Cooperative Agreement to the University of Puerto Rico 4 58-7B30-0-203 'Bean Improvement by Interspecific Crosses'. 2 Current address: 101A Horticulture Field Lab, 1707 S. Orchard St., University of Illinois, Urbana, IL 61801, USA

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J. G. SULLIVAN AND G. FREYTAG

Table 1. Interspecific hybrids within the genus Phaseolus.

Hybrid
P. acutifolius x P. coccineus P. acutifolius x [P. vulgaris (P. vulgaris P. coccineus)] P. coccineus P. lunatus P. lunatus x P. polystachyus P. lunatus P. ritensis P. ritensis P. lunatus P. vulgaris x P. acutifolius P. vulgaris x P. coccineus P. coccineus P. vulgaris P. vulgaris P. filiformis P. vulgaris x P. lunatus P. vulgaris x P. ritensis

Author
COYNE(1964) COYNE(1964) HONMA& HEECKT(1958) LORZ (1952) LE MARCHAND& MARt~CHAL(1977) LE MARCHAND& MARECHAL(1977) HONMA(1955) MENDEL(1866) TSCHERMAK-SEYSENEGG(1942) MARIE~CHAL 8~.BAUDOVIN(1978) HONMA& HEECKT(1959) BRAAK& KOOISTRA(1975)

reported by MARI~CHALet al. (1978). The usefulness and validity of electrophoresis in taxonomic research has been summarized by BOULTER& TURNER(1966). The purpose of the present study was to utilize the electrophoretic variation of the seed proteins of wild Phaseolus species to suggest genetic relationships which might be useful for predicting successful interspecific crosses. The assumption is that if two species carry similar genes for a wide range of storage proteins, they are probably phylogenetically similar. Therefore, problems associated with cross compatiility, such as lack of chromosomal homology, should be minimal. The major flaw in using SDS (Sodium Dodecyl Sulfate) electrophoresis for systematic studies is that there is an inherent assumption that proteins which migrate to the same point on a gel are identical. This is not necessarily true. This problem could be minimized by using isoenzyme analysis. However, this technique has shown so much variation within species (BASSIRI& ADAMS, 1978), that it would be difficult to detect the differences among species. Another approach would be to separate out specific protein fractions for electrophoresis (Fox et al., 1964). Unfortunately, this technique requires large amounts of seed,which is generally not available for many species of Phaseolus. Despite it flaws, SDS electrophoresis is a powerful tool for analyzing species relationships (LADIZINSKY& HYMOWITZ, 1979). The technique is relatively simple, and there is very little environmental effect on the presence or absence of specific bands of seed proteins.
MATERIALS AND METHODS

A total of 120 accessions of beans were studied (Table 2). The plants were grown in the greenhouses of the Tropical Agricultural Research Station (TARS) in Mayaguez, PR to verify species identify and for preliminary selection and study. All accessions were verified as wild germplasm except one accession of P. coccineus subsp, polyanthus (accession no. 71), which had been found in a truly wild habitat near Panajachel, Gueatemala, but expressed many of the characteristics of cultivated material. Part of the germplasm used in this study had been collected during a plant exploration expedition by the authors in Mexico in 1981. The rest of the germplasm was provided
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PREDICTING INTERSPECIFIC COMPATIBILIT1ES 1N PHASEOLUS Table 2. Identify of accessions used in experiment. Species
P. acutifolius subsp, acutifolius P. acutifolius subsp, tenuifolius P. angustisismus P. anisotrichos P. brevicalyx P. coccineus subsp, coccineus P. coccineus subsp, formosus P. coccineus subsp, polyanthus P. esperanzae P. filiformis P. galactioides P. lunatus P. metcalfei P. microcarpus P. par vulus P. polystachyus P, ritensis P. tuerckheimii P. vulgaris P. wrightii

Number of accessions
7 6 2 7 1 11 2 2 4 8 1 8 5 9 2 1 8 1

27
7

by various bean researchers throughout the world (see Acknowledgements). Seeds from each accession were ground with a mortar and pestle. The protein from a 3-rag sample of the resultant flour was extracted in 100 ~tl of 0.5 M NaCI buffered to pH 7.0. The extract was electrophoresed on a 0.75-mm thick SDS-PAGE gel, using a 14~o polyacrylamide running gel, and a 5~o polyacrylamide stacking gel. The details of the electrophoresis procedures are described by BROWN et al. (1981). Each sample was run in duplicate, and if there was poor agreement between samples, an additional sample was run. Marker proteins of known molecular weight were run each gel as a check. Comparisons ofelectrophoretic patterns are most reliable when the accessions being compared are on the same gel. Therefore, all accessions of a given species were grouped on the same gel in order to measure within species variation (Fig. 1), differences among species were measured by running one representative accession from each species on the same gel (Fig. 2). The number of bands which each pair of species had in common, and the number of bands which were present in only one of the pair of species were recorded. The staining intensity of bands was not considered, since the accurate quantification of protein concentration on SDS electrophoretic gels is difficult (LADIZINSKY & HYMOWITZ, 1979). Jaccard's similarity index (SNEATH & SOKAL, 1973) was calculated for each pair of species by the formula: Number of bands in common Number of different bands + number of bands in common In those cases where intraspecific variation did exist, the similarity index was calculated
as;

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J. G. SULLIVAN AND G. FREYTAG

Fig. 1. Electrophoretic gel of species of Phaseolus. Lanes 1-7 are various accessions of P. an&otrichos, Lanes 8, 10 and 11 are P. coccineus. Lane 9 is marker proteins. Lanes 12-18 are subsamples of a mixed collection of P. vulgaris and P. coccineus. Subsampling was based on seed type. Lanes 12 and 14 are P. coccineus, and the rest of this collection is P. vulgaris. The arrow points to a band, present in P. coccineus and absent in P. vulgaris. 'P' refers to phaseolin, and 'L' refers to lectin.

n i ~ 1 Piqi n i ~ l (Pi + q i Piqi)

where p~ and qi represent the frequencies of the ith band in a pair of species, and n is the total number of band sites. This formula is the same as Jaccard's index when all p's and q's equal 1 or 0. A dendrogram was produced from the matrix of similarity indices using the weighted pair-group of cluster analysis (SNEATH 8 SOKAL, 1973).
RESULTS AND DISCUSSION

Very little within species variation for electrophoretic banding pattern was observed in most of the species (Fig. 1), except in P. vulgaris, where variation in the banding patterns of phaseolin and lectins was evident. The location of these proteins on the gel was inferred from previous reports (MA & BLISS, 1978). This variation in wild P. vulgaris is in contrast to cultivated P. vulgaris in which only a limited number phaseolin banding patterns are known (BROWN et al., 1981). In P. coccineus, there was some variation in the lectin pattern, but little in the phaseolin pattern. The cultivated P. coccineus has also been reported to have variation in the phaseolin pattern (BROWN et al., 1987). This is suprising since P. coccineus is the only outcrossing species in the 204
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PREDICTING INTERSPECIFIC COMPAT1BILITIES IN PHASEOLUS

Fig. 2. Electrophoretic gel of species of Phaseolus. The lanes are: 1. P. acutifolius subsp, acutiJolius, 2. P. acutiJolius subsp, tenuifolius, 3. P. angustissimus, 4. P. anisotrichos, 5. P. coccineus subsp, coccineus, 6. P. filiformis, 7. P. lunatus, 8. P. microcarpus, 9. P. tuerckheimi, 10. P. vulgaris. 11. P. esperanzae, 12. P. polystachyus, 13. P. brevicalyx, 14. P. metcalfei, 15. P. parvulus.

genus, and therefore it might be expected to be more variable than its close relative P. vulgaris. By far the most variable species was P. acutifoius (Fig. 3). Unlike other species, P. acutifolius did not have a single easily recognizable protein pattern. One might speculate that this large variation is due to the fact P. acutifolius is a desert species which tends to grow in isolated pockets, thereby producing discrete gene pools. However, P. filiformis, P. wrightii, P. metcalfei and P. ritensis are also desert species, and these showed little within species variation. Because of the great variation in P. acutifolius, its relative position in the dendrogram produced by cluster analysis should be interpreted with caution (Fig. 4). The errors in measurement of the electrophoretic patterns of this species are perhaps greater than for other species, and small sample size may also be a problem, given the large variation. Although the dendrogram indicates that the two subspecies of P. acutifolius are not very closely related, other methods suggest this is not the case. Furthermore, P. acutifolius is shown as not being very similar to P. vulgaris and P. coccineus, although crossing data indicated otherwise. The cluster analysis indicated that P. vulgaris is closely related to P. eoccineus. This agrees with the conclususions of MAR~CHALet al. (1978), and the hybridization studies of SMARTT(1970). Although the banding patterns of P. coccineus and P. vulgaris were very similar, there was one band of low molecular weight (approximately 22Kdal) which was present in all accessions of all subspecies of P. coccineus and in none of the accessions of P. vulgaris. This was true also in the mixed collection of P. vulgaris and P. coccineus in which there was evidence of natural introgression between the two species (Fig. 1). This hybrid swarm showed no segregation for phaseolin pattern,
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J. G. SULLIVANAND G. FREYTAG

Fig. 3. Electrophoretic gel of various accessions of P. acutifolius, lane 8 is marker proteins.

although there is a large amount of variation in the phaseolin pattern of other wild P. vulgaris. There is evidence that similarity of storage proteins represents a precise evolutionary record in wheat (JOHNSON, 1968, 1972). Diploid species of wheat show considerable variation of storage protein profiles, while more recently evolved hexaploids show more homogeneous profiles. Apparently, as protein profiles become more homogeneous, genetic similarities and interspecific fertilities also increase. It may be that similarity ofphaseolin pattern increases the probability ofinterspecific hybridization of Phaseolus. Cluster analysis points to P. filiformis and P. wrightii as being very closely related (Fig. 4), and, in fact, their zymograms are indistinguishable. The difference between these two species is that the former is annual and the latter perennial (MARI~CHAL et al., 1978). However, when these species were grown in experimental plots in Puerto Rico, no perennials were found in either species. These species names are probably synonymous, the differences being entirely attributable to environmental factors. P. metcalfei and P. ritensis appeared to be closely related from the dendrogram (Fig. 3). These taxons can be separated morphologically (NABHANet al., 1980), but given their electrophoretic similarity, perhaps they should be classified as 2 subspecies of the same species (LE MARCHAND MARI~CHAL, 1977). They show roughly the same degree of similarity as the subspecies of P. coccineus. One would expect that P. metcalfei can cross with P. vulgaris and P. lunatus, since comparable crosses have been made with P. ritensis (BRAAK & KOOISTRA, 1975; LE MARCHAND& MARI~CHAL, 1977). Neither P. ritensis nor P. metcalfei are likely to be useful as bridges between P. vulgaris and P. lunatus, since the Fl ofP. vulgaris and P. ritensis is highly sterile. The dendrogram indicates that P. polystachyus is closely related to the P. metcalfeiP. ritensis complex. Like P. ritensis, P. polystachyus can be hybridized with P. lunatus.
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P R E D I C T I N G I N T E R S P E C I F I C C O M P A T I B I L I T 1 E S IN P H A S E O L U S

.9 P. acutifolius sbsp. acut. P. acutifolius sbsp. lat P. a n g u s t i s s m u s

. . . . .

.8

.7

.6

.5

.4

.3

.2

.1

. . . . . . . . . . . . . .

P. anisotrichos ................. P. c o c c i n e u s sbsp. c o c c . . P. c o c c i n e u s sbsp. form. P. c o c c i n e u s sbsp. poly.

P. v u l g a r i s
P. metcalfeii

......................

P. ritensis .........................
P. polystachyus_ ................ P. filiformis ....................... P. wrightii .......................... P. parvulus ................ P. e s p e r a n z a e ..................... P. brevicalyx ..................... P. tuerckheimii
. . . . . . . . . . . . . . . . .

P. galactioides ................. P. Iunatus ......................... P. m i c r o c a r p u s ................

Fig. 4. Dendrogram resulting from cluster analysisof similarities of electrophoreticpatterns of various bean species. However, there have been no reports in the literature of attempted crosses between P. polystachyus and P. vulgaris. This would be an important hybrid to attempt. Firstly, the P. polystachyus germplasm itself is useful. Its natural habitat stretches well into the temperate latitudes of the United States, whereas all other Phaseolus species are tropical or subtropical. This species would be expected to be an excellent source of genes for cold tolerance. And secondly, if P. polystachyus did hybridize with P. vulgaris, it could be used as a bridge between P. vulgaris and P. lunatus, and quite possibly between P. vulgaris and the P. metealfei-P, ritensis complex. One surprising result from this study was the electrophoretic similarity between P. angustissimus and P. anisotrichos. Morphologically, these two species are very distinct, and MAP,~CHALet al. (1978) placed them relatively far apart. The cross P. angustissimus x P. anisotrichos would be a test of the use of electrophoretic protein separation to indicate relationships which may not be obvious morphologically. Neither species is important economically, but there is interest in incorporating P. anisotrichos germplasm into P. vulgaris, since P. anisotrichos has a long tap root that makes it well adapted to arid regions. To date, all attempts at crossing P. anisotrichos and P. vulgaris have failed. IfP. angustissimus is cross compatible with P. vulgaris, it could be used as a bridge between P. anisotrichos and P. vulgaris. One problem is that P. anisotrichos reportedly has 20 chromosomes, whereas most Phaseolus species have 22 (MARI~CHALet al., 1978). The chromosome number of P. angustissimus is not

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known. P. lunatus and P. microcarpus are distantly related to all other species of Phaseolus (Fig. 4). P. lunatus is also morphologically very different from the other species of Phaseolus (MARI~CHAL et ak., 1978). The fact that it can cross with P. ritensis and P. polystachyus (LORz, 1952; LE MARCHANDt~ MARI~CHAL, 1977) attests to the fact wide crossing within Phaseolus is relatively easy compared with other genera. There was one unidentified species in this study. This was an accession from our plant exploration trip. Morphologically it is similar to P. esperanzae, and the dendrogram indicates that it is related to this species, yet it is different enough to deserve a separate species classification (Fig. 4). This species has tentatively been identified as P. brevicalyx. It is not our intention to imply that electrophoretic data should be used in lieu of morphological and hybridization data. Rather, this should be seen as another piece of the evidence. Where electrophoretic data agree with previous findings the information can be used with more confidence. In cases of conflicting reports, further investigation is necessary.
ACKNOWLEDGEMENTS

The authors are deeply indebted to the following people for their generous donation of germplasm: Dr R. Mar6chal (Facult6 des Sciences Agronomiques de l'Et/tt, Belgium), Dr Adrian Shirlin (University of Cambridge, England), Mr Russ Buhrow (Univeristy of Arizona, USA), Dr Richard Manshardt (University of California at Riverside, USA), Dr Rigoberto Hildalgo (CIAT, Colombia), Dr Gilberto Pfiez (CATIE, Costa Rica), Dr Hugh Iltis (University of Wisconsin at Madison, USA), Dr Mark Bassett (University of Florida at Gainesville, USA), Dr Leland Hudson (USDA/ Washington State University, USA), and Dr Eelco Drijfhout (Institute for Horticultural Plant Breeding, the Netherlands).
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PREDICTING INTERSPECIFIC COMPATIBIL1T1ES IN PHASEOLUS JOHNSON, B. L., 1972. Seed protein profiles and the origin of the hexploid wheats. Amer. J. Bot. 59: 952-960. LADIZINSKY,G. & T. HYMOWITZ, 1979. Seed protein electrophoresis in taxonomic and evolutionary studies. Theor. Appl. Genet. 54:145 151. LE MARCHAND,G. & R. MARECHAL, 1977. Wide crossing in Lima beans. Trop. Grain Leg. Bull. 8: 7-11. LoRz, A. P., 1952. An interspecific cross involving the Lima bean Phaseolus lunatus L. Science 115: 702-703. MA, Y. & F. A. BLISS, 1978. Seed proteins of common bean. Crop Sci. 17:431 437. MARI~CHAL, R. & J. P. BAUDOVIN, 1978. Observations sur quelques hybrides dans le genre Phaseolus. IV. L'hybride Phaseolus vulgaris PhaseolusfiliJbrmis. Bull. Rech. Agron. Gembloux 13: 233-240. MAR~CHAL, R., J. M. MASHERRA& F. STAINER. 1978. i~tude taxonomique d'un groupe complexe d'esp6ce des genres Phaseolus et Vigna (Papilionaceae) sur la base de donn6es morphologiques et pollinique, trait6es para l'analyse inforrnatique. Boissiers, Geneve. Vol. 28,273 p. MENDEL. G., 1866. Versuche fiber Pflanzenhybriden. Verhandlungen des Naturforschenden Vereins in Brfinn. 4:3~47 (English translation in Experiments in plant hybridization. Oliver and Boyd, Edinburgh, 1965, 95 pp.) NABHAN, G. P., J. W. BERRY& C. W. WEBER, 1980. Wild beans of the greater Southwest: Phaseolus metcalfei and P. ritensis. Economic Botany 34:68 85. SMARTT, J., 1970. Interspecific hybridization between cultivated American species of the genus Phaseolus. Euphytica 19: 489-489. SNEATH, P. H. A. & R. R. SOKAL, 1973. Numerical taxonomy. W. H. Freeman, San Francisco. 573 pp. TSCHERMAK-SEYSENEGG, E., 1942. r0ber Bastarde zwischen Fisole (Phaseolus vulgaris L.) und Feuerbone (Phaseolusmultiflorus Lam.) und ihre eventuell praktische Verwertbarkeit. Zuchter 14: 153-164.

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