Potato morphotypes and intermediate Sieva-Potato and Sieva-Big Lima ... geographic distribution: Big Lima with big flat .... Big Lima type show a pattern (Fig .
Genetic Resources and Crop Evolution 41 : 8 1-85, 1994 . © 1994 Kluwer Academic Publishers. Printed in The Netherlands .
Morphotype relationships in Lima bean (Phaseolus lunatus L.) deduced from variation of the evolutionary marker phaseolin L . Lioi Germplasm Institute, CNR, Via Amendola 165/A, 70126 Bari, Italy Received 11 May 1993 ; 28 September 1993
Key words :
electrophoresis, Lima bean, phaseolin,
Phaseolus lunatus,
variation
Summary Variation of the seed storage protein phaseolin was analysed in a collection of 100 accessions of Phaseolus lunatus L . using one-dimensional SDS/PAGE . Cultivated small-seeded genotypes belonging to Sieva and Potato morphotypes and intermediate Sieva-Potato and Sieva-Big Lima morphotypes showed the M (Mesoamerican) pattern, confirming their origin in the same gene pool . Cultivated Big Lima morphotypes showed the A (Andean) pattern, confirming that they belong to a distinct gene pool .
Introduction The Lima bean (Phaseolus lunatus L .), one of the major cultivated species of the genus Phaseolus L ., is highly polymorphic . Three morphotypes are usually recognized according to seed characteristics (Baudet, 1977 ; Baudoin, 1988) . More recently, Debouck and co-workers showed that each different morphotype had a particular geographic distribution : Big Lima with big flat seeds was distributed in Peru, the highlands of Colombia, Ecuador, and Bolivia ; the Sieva type with medium flat seeds was distributed in Mexico, Guatemala, and Colombia at mid and low altitudes, and the Potato type with small rounded seeds was distributed in the Caribbean area, coastal Colombia, and Yucatan (Debouck et al ., 1989) . Intermediate Potato-Sieva and Sieva-Big Lima types have recently been described by Esquivel et al . (1990) among Cuban germplasm . Introgressive hybridization, in one case, and selection toward larger seeds, in the other case, have been used to explain the presence of intermediate forms . Electrophoretic analysis of seed storage proteins on the same
materials supported that hypothesis (Lioi et al ., 1991) . The importance of phaseolin, the major storage protein in P. vulgaris L ., as a genetic marker for evolutionary studies has been stressed by Gepts (1988) . Through the analysis of phaseolin from different wild and cultivated accessions of common bean, multiple domestication centers have been suggested, the two major ones being located in Middle America and the Southern Andes (Debouck & Tohme, 1989 ; Gepts et al ., 1986; Koenig et al ., 1990) . Based on electrophoretic analysis of seed storage proteins, Debouck et al . (1989) and Maquet et al . (1990) reported the existence, for P. lunatus, of two gene pools, a Mesoamerican and an Andean one . The recent identification and characterization of phaseolin among seed storage proteins in P . lunatus (Lioi et al ., 1993) allowed better evaluation of variability among different morphotypes . This study compares electrophoretic patterns in some cultivated accessions of P . lunatus from localities distributed all over the world to assess phaseolin variation and the possible relationships among different morphotypes .
82 Statistical analysis . Data on seed size were statistically analysed using the SAS program package . Mean values were compared by Duncan's multiple range test .
Fig . 1 . One-dimensional SDS/PAGE of total seed proteins of Phaseolus lunatus L . accessions. Lanes 1 and 12-Molecular weight markers; lanes 2 and 7-M (Mesoamerican) reference pattern; lanes 3 to 6-Variation in M type banding pattern ; lanes 8 and 11-A (Andean) reference pattern ; lanes 9 and 10 Variation in A type banding pattern . Asterisk = phaseolin polypeptides ; head of arrow = a very common band in the A pattern .
Protein extraction and SDS/PAGE. The non-germ end of each seed was finely ground and proteins extracted for two hours by adding 10 volumes of 0 .02 M borate buffer pH 9 .0 . After centrifugation at 10,000 rpm for 15 minutes, the supernatant was used for electrophoresis . Proteins were dissociated by heating to 90°C for two minutes in the presence of the denaturating buffer (20 mM Tris-HC1 pH 8 .6 containing 1 % SDS, 2% 2-mercaptoethanol and 8 .3% glycerol) . One-dimensional sodium dodecyl sulphate (SDS)/PAGE was performed following the procedure described by Laemmli (1970), using 15% polyacrylamide gel slabs overlaid with a 4 .5% stacking gel . The electrophoresis was run at 10 mA for about 20 hours . Gel slabs were stained with Coomassie Brilliant Blue R-250 .
Material and methods Results and discussion Seed material . A total of 100 accessions of P . lunatus, belonging to the groups Big Lima, Sieva-Big Lima, Sieva, Potato, and Potato-Sieva, were investigated . Part of the materials analysed were obtained from the Germplasm Institute of the National Research Council in Bari (Italy) . The other accessions were obtained from : Centro Internacional de Agricultura Tropical (CIAT), Cali, Colombia ; Empresa Brasileira de Pesquisas Agropecuarias (EMBRAPA), Brazil ; Zentralinstitut fur Genetik and Kulturpflanzenforschung, Gatersleben, Germany ; Jardin Botanique National de Belgique, Meise, Belgium .
The number of accessions analysed for each morphotype and seed size comparisons are shown in Table 1 . Seed length and width were measured as reported by Gepts et al . (1986) . Seed thickness was not measured since Esquivel et al . (1990) reported that in P . lunatus this value cannot distinguish among different morphotypes . Electrophoretic analysis of seed storage proteins confirmed that all investigated accessions could be attributed to one of the two gene pools, the Mesoamerican (M) and the Andean (A) one, described by Debouck et al . (1989) and by Maquet
Table 1 . Distribution of Lima bean accessions in morphotypes, electrophoretic patterns, and comparison among seed sizes Morphotype Big Lima Sieva-Big Lima Sieva Potato-Sieva Potato Total
No . Access 14 15 41 6 24
Electroph . pattern
100/Seed weight (g)
'Seed length (mm)
'Seed width (mm)
A M M M M
108 .9A 77 .5B 45 .3C 36 .3C 34 .5C
19A 17B 13C 11 C 9D
12A 11A 9B 8 BC 8C
100
Mean values have been statistically compared by Duncan's multiple range test . Values followed by the same letter are not significantly different at the p = 0 .005 level .
83
Fig. 2. Some representative seed types of Phaseolus lunatus L . Typical morphotypes-Big Lima, Sieva, and Potato ; Panel A-Lima bean seed with A (Andean) pattern, morphologically similar to P . vulgaris ; Panel B-Intermediate Sieva-Big Lima seeds ; Panel C-Intermediate Sieva-Potato seeds .
84 et al . (1990) . The same denomination of A and M patterns attributed by Maquet et al . (1990) to total protein pattern will be used for phaseolin variants, as discriminating protein bands were actually phaseolin polypeptides . The major phaseolin bands, marked by asterisks in Fig . 1, show notable differences in molecular weight between M (lanes 2), and A (lane 8) phaseolin pattern types . In particular, polypeptides with molecular weights between 31 and 21 .5 Kd in the A pattern are faster than the corresponding polypeptides in the M pattern . Small-seeded morphotypes, Sieva and Potato, both show the M pattern (Fig . 1, lane 2, 7), confirmaing that they belong to the same gene pool, and that diversification for seed type probably followed domestication . Intermediate Sieva-Potato seeds (Fig . 2, panel C) show an M type phaseolin pattern and could either be the result of gene flow between the Sieva and the Potato morphogroups or the result of natural variation within the small-seeded cultivars . Big Lima genotypes (A pattern) (Fig . 1, lane 8) had larger seeds (Fig . 2), but often intermediate SievaBig Lima types (Fig . 2, panel B) were misclassified as Big Lima, though their sizes were somewhat smaller than those of Big Lima (Table 1) . Intermediate Sieva-Big Lima morphotypes appeared to be the result of a selection towards large-seeded genotypes within the Mesoamerican gene pool, rather than a genic exchange between the Mesoamerican and the Andean gene pools . Phaseolin variation is narrow inside the same gene pool . Nine accessions of intermediate SievaBig Lima type show a pattern (Fig . 1, lane 3) with a slightly heavier phaseolin band (around 36 Kd) . Seed materials were collected in South America and in Africa and we could suppose that diversification occurred before dissemination . One accession from Costa Rica showed the pattern presented in lane 4 (Fig . 1) with a doublet of heavier phaseolin bands . Patterns in lanes 5 and 6 (Fig . 1) were from seeds of the same accession, and show a band with a molecular weight around 40 Kd (head of arrow), very common in the Andean gene pool (compare with Andean reference pattern in lane 8, Fig . 1) . An introgression between the two gene pools could be suggested . In the Andean gene pool, 5 accessions apparently lacking a band around 40 Kd (head of
arrow) and one Peruvian accession showing the pattern in lane 10 (Fig . 1) were observed . In this study, small- and large-seeded cultivars generally exhibited Mesoamerican and Andean phaseolin patterns, respectively . This situation agrees with that already described for P . vulgaris (Gepts, 1988) . Moreover, in P. vulgaris, wild forms exhibit the same relationships between phaseolin and seed size as the cultivated ones (Gepts & Debouck, 1991) . Also for P . lunatus, Debouck et al . (1987) described smaller seeds in Mesoamerican than in Peruvian wild forms . According to these authors, large- and small-seeded cultivated genotypes, both in P. vulgaris and in P . lunatus, originated from large and small-seeded wild forms respectively . These results thus confirm the existence of two gene pools in P . lunatus, and at least two separate domestication events, possibly originating from two different groups of wild ancestral forms (Debouck et al ., 1987, 1989 ; Maquet et al ., 1990) . Noteworthy is the finding among Lima bean accessions from Brazil of two particular landraces, one with the Andean (Fig . 2, panel A) and the other with the Mesoamerican pattern (Fig . 2, panel Sieva, the eighth seed); both are morphologically more similar to P . vulgaris than to P . lunatus . They appear to be the result of selection towards forms morphologically similar to the common bean, rather than crosses between the two species, which show strong incompatibility (Leonard et al ., 1987 ; Mok et al., 1978) .
Acknowledgements The author wishes to thank Sig G . Scippa for assistance in the statistical analysis of the data .
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