Plant Biosystems, Vol. 140, No. 1, March 2006, pp. 17 – 20
BIODIVERSITY AND CONSERVATION
Molecular diversity in Ligurian local races of common bean (Phaseolus vulgaris L.)
F. GRASSI1, M. LABRA2 & L. MINUTO3 1
Orto Botanico dell’ Universita` di Milano, Italy, 2Dipartimento di Scienze dell’Ambiente e del Territorio, Universita` degli Studi di Milano-Bicocca, Milano, Italy, and 3DIP.TE.RIS, Universita` di Genova, Italy
Abstract Eight varieties of Ligurian common beans (Phaseolus vulgaris L.) were analysed using molecular approaches. Results were compared with two commercial cultivars (‘Cannellino’ and ‘Borlotto’). Data suggest that all Ligurian bean varieties have a low genetic variability and are very close to the commercial varieties. In particular, the three ‘Bianco’ varieties showed a molecular affinity, probably due to their common genomic origin.
Key words: DNA fingerprinting, genetic variability, Phaseolus vulgaris L., RAPD
Introduction Bean (Phaseolus vulgaris L.) is an ancient crop, which was independently domesticated in Central America (small-seeded forms) and in the Andes (large-seeded forms) with an additional smaller centre in the north-west of South America (Gepts & Debouck, 1991). The germplasm from both centres was introduced into the Old World (Sonnate et al., 1994; Gepts, 1998; Piergiovanni et al., 2000). The species exhibits a wide range of phenotypical characters (growth form, leaf, flower, pod and seed traits). Thousands of land-races, old and modern cultivars are cultivated and maintained in the gene banks of various countries. The European Phaseolus Database, was established in 1995 by the European Cooperative Programme for Crop Genetic Resources (ECP/GR) following the principles of the International Plant Genetic Resources Institute (IPGRI)/FAO Multicrop descriptors list. For centuries, Italian farmers selected and maintained several local common bean populations. Local constraints to production and various consumer requirements in different Italian regions has resulted in a great differentiation between these populations. Due to the diffusion of commercial cultivars however, an unknown number of local populations disappeared, and the future of those
presently under cultivation is unpredictable. The aim of our work is to preserve genetic resources and safeguard the biodiversity of the local bean. Ligury is a small region in northern Italy where many different bean cultivars were introduced by the French, the Spanish, and by the intense commercial activity throughout the Mediterranean area by the Maritime Republic of Genoa, since the beginning of the 16th century. During the following centuries these varieties were preserved in the inland valleys while new entities were selected and cultivated locally. Relations between the different Phaseolus species have been investigated locally using biochemical approaches (Jaaska, 1996). Recent advances in molecular biology have favoured the development of screening methods to study genetic diversity and relations between individuals (Llaca et al., 1994). RAPD markers (Williams et al., 1990; Johns et al., 1997; Duarte et al., 1999; Me´tais et al., 2000; Maciel et al., 2001; Nowosielski et al., 2002) facilitate the acquisition of a large amount of genetic information aimed at ‘fingerprinting’ and characterizing germplasm collections. Estimates of inter- and intra-genetic diversity can also provide important information for plant breeders, such as relevant background data that can be utilized in the development of new cultivars with a broader genetic base.
Correspondence: Dr. Fabrizio Grassi, Orto Botanico di Milano, Dipartimento di Biologia, Universita` di Milano, Via Celoria 26, 20133 Milano, Italy. Tel.: þ 39.02.50314818. Fax: þ 39.02.50314764. E-mail:
[email protected] ISSN 1126-3504 print/ISSN 1724-5575 online ª 2006 Societa` Botanica Italiana DOI: 10.1080/11263500500500747
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Materials and methods Plant samples Eight local races, actually cultivated in small farms and sometimes traded on the local market and two common commercial cultivars were analysed and used for comparative purposes: (a) ‘Bianco di Pigna’ from Pigna, Val Nervia (Imperia), 300 m a.s.l.; (b) ‘Bianco di Badalucco’ from Badalucco, Valle Argentina (Imperia), 200 m a.s.l.; (c) ‘Bianco di Conio’ from Conio/Borgomaro, Valle Impero (Imperia), 600 m a.s.l; (d) ‘Reale’ from Molini di Triora, Valle Argentina (Imperia), 450 m a.s.l.; (e) Borlotto; (f) Cannellino; (g) ‘Mascherin’ from Campovecchio tre Fontane, Montaggio (Genoa), 700 m a.s.l.; (h) ‘Albenghin’ from Avosso (Genoa), 400 m a.s.l.; (i) ‘Gianetto’ from Nasino and Arnasco (Savona), 300 m a.s.l.; (j) ‘De Milan’ from Sanguineto Montaggio (Genoa), 700 m a.s.l. ‘Borlotto’ and ‘Canellino’ supplied by Blumen Ltd. (The Netherlands). DNA extraction and RAPD analysis For each variety, 3 – 5 individuals were sampled from local races and commercial bean cultivars. DNA extraction was performed on young leaves. Genomic DNA was extracted using the CTAB method according to Doyle & Doyle (1990). RAPD analysis was performed using random decamer oligonucleotides (Table I) as single primer for the DNA amplifications. The reactions were performed according to Williams et al. (1990) with slight changes: each reaction contained 25 mL consisting of 10 mM TRIS-HCl pH 9.0, 50 mM KCl, 2 mM MgCl2, 100 mM of each dNTP (New England Biolabs), 20 nM primer, 50 ng of genomic DNA and 0.5 U Taq DNA polymerase (Dynazyme, Finzyme, Finland). The reaction mixture was overlaid with 20 mL of mineral oil. Amplification was
Table I. Descriptive statistics of RAPD markers, estimated using data from the common bean variety. Name of primer, sequence of primer, number of amplified loci, gene diversity values (G.D.). Primer OPAA-01 OPAA-04 OPAA-06 OPAA-14 OPAA-15 OPAA-17 OPAA2* OPEE7* OPAA11* OPAA13*
Sequence
No. loci
G.D.
50 -CAGGCCCTTC-30 50 -AGGACTGCTC-30 50 -GTGGGTGCCA-30 50 -AACGGGCCAA-30 50 -ACGGAAGCCC-30 50 -GAGCCCGACT-30 50 -GAGACCAGAC-30 50 -AGATGCAGCC-30 50 -ACCCGACCTG-30 50 -GAGCGTCGCT-30
6 8 6 5 9 6 5 6 4 5
0.056 0.252 0.128 0.362 0.227 0.217 – – – –
*To confirm the genetic identity of the ‘Bianco di Pigna’, ‘Bianco di Conio’ and ‘Bianco di Badalucco’ varieties.
performed in an Eppendorf thermal cycler programmed for 45 cycles of 1 min at 948C, 1 min at 368C and 105 s at 728C, with a precycle of 4 min at 948C, 1 min at 368C, 2 min at 728C and a final cycle of 1 min at 948C, 1 min at 368C and 20 min at 728C. All the amplification products were analysed by electrophoresis on 1.4% agarose gels and visualized after ethidium bromide staining. Each band was considered as a separate putative locus, and scored as present (1) or absent (0) for each sample. Genetic distances were computed using the formula proposed by Nei & Li (1979): 1 – 2xy/ x þ y, where xy is the number of shared bands by the pair and x þ y is the number of total bands of the pair. All RAPD markers, both mono- and polymorphic, were used for data analysis. The phenograms were computed on the basis of the unweighted pair-group method using arithmetic means (UPGMA) (Sneath & Sokal, 1973). The degree of RAPD polymorphism was also quantified using the total number, polymorphic bands, the number of alleles and gene diversity value (G.D.). Gene diversity was evaluated as follows: X n G:D: ¼ 1 pi 2 ðn 1Þ where pi is the frequency, in each group, of the i-th allele at each RAPD locus; and n is the number of individuals in each group of samples (Nei, 1987).
Results RAPD analysis A phenetic analysis was conducted to analyse the genetic relationship between eight Ligurian common bean landraces and two commercial cultivars. The RAPD primers (OPAA1, OPAA4, OPAA6 OPAA14, OPAA15, OPAA17) tested produced a total of 40 reproducibile bands and of these 24 (60%) were polymorphic. Data were computed on a genetic distance matrix and the resulting UPGMA phenogram (Figure 1A) shows genetic relationships among varieties. A sufficient level of genetic variability was detected by all tested primers (Table I). All Ligurian varieties showed a low genetic variability and proved to be very closely related to commercial varieties. In particular, the three ‘Bianco’ varieties, ‘Mascherin’ and ‘Reale’ were very similar from a genetic point of view, with a genetic distance value about 0.07. To confirm the genetic similarity of the three ‘Bianco’ varieties, the DNA of these accessions were analysed using a total of 10 primers (OPAA1, OPAA2, OPAA4, OPAA6, OPAA7, OPAA11, OPAA13, OPAA14, OPAA15, OPAA17). Electrophoretic analysis of the amplified products revealed 61 bands, of which only 9 (14.75%) were polymorphic. The
Molecular diversity in common bean
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A high level of genetic variability was detected in commercial accessions of the common bean. On the other hand, Ligurian varieties show a low nuclear DNA variability. This supports the existence of a few common genetic ancestors introduced in Ligury by trade routes. In addition, the scarce exchange of varieties among local farmers, and reproduction by seedling selection may be the main causes for low variability among cultivars. Molecular analysis showed that differences occur in these accessions, with the presence of a private allele in the ‘Bianco di Badalucco’ variety that allows it to be distinguished from the others. Based on these molecular analyses and on morphological traits (data not show) we could distinguish the three ‘Bianchi’ varieties from Imperia, but, due to their genetic similarity, we cannot exclude their common genomic origin. Acknowledgements
Figure 1. (A) UPGMA dendrograms obtained from RAPD analysis (data obtained from six primers). The numbers reported on the branches are bootstrap values (%) obtained from 1,000 replicate analyses. Only values showing 440% support were reported. B. Badalucco: ‘Bianco di Badalucco’; B. Pigna: ‘Bianco di Pigna’; B. Conio: ‘Bianco di Conio’. (B) Unrooted UPGMA tree obtained from RAPD analysis (data obtained from 10 primers) on three ‘Bianco’ varieties: B.C: ‘Bianco di Conio’; B.P: ‘Bianco di Pigna’; B.B: ‘Bianco di Badalucco’. The numbers on the branches are bootstrap values (%) obtained from 1,000 replicate analyses. Only values showing 440% support were reported.
unrooted phenogram thus obtained showed a high genetic similarity between the ‘Bianco di Pigna’ and ‘Bianco di Conio’ varieties (Figure 1B). On the other hand, the ‘Bianco di Badalucco’ variety showed evident molecular differences with a private allele (detected with the OPAA14 primer) present in all samples. Discussion The purpose of this study was to investigate the genetic variability among Ligurian and commercial bean cultivars. Knowledge of the genetic distance between accessions is essential for breeders in order to select the genotypes to cross and control, and to preserve local germplasm resources (Briand et al., 1998; Maciel et al., 2001; Labra et al., 2002; Grassi et al., 2003). Results show that, RAPD data could be useful for DNA fingerprinting of the common bean, in agreement with a previous study conducted by Nowosielski et al. (2002) and Galva`n et al. (2001).
This research was supported by FIRB-RNBE01SF project, MIUR, Italy. We thank Roberto Brontini and Fabrizio Celia for technical support and molecular analyses.
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