International Journal of Integrative Biology A journal for biology beyond borders ISSN

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1 Regular International Journal of Integrative Biology A journal for biology beyond borders ISSN Isozymes variation of some species of the genus Vicia (Fabaceae) collected from Tunisia Safia El Bok *, Mohamed El Gazzah * Laboratory of Genetics Populations and Biological Resources, Faculty of Sciences of Tunis, Tunis, Tunisia Submitted: 30 Aug. 2008; Accepted: 25 Nov Abstract Ten spontaneous populations of Vicia genus made the object of enzymatic analysis. The choice of species was based on their possible application in crossbreeding and also for their agricultural value. Polymorphisms of ten spontaneous populations belonging to Vicia genus were investigated by using leaf isozymes of young plants. The differential isoenzymatic patterns of four systems, namely: ICD, 6-PGD, EST and MDH revealed either an intra and/or interpopulations variability. Five loci have been identified with an average number of alleles per locus varying from according to populations. The average proportions of polymorphic loci (58%) demonstrate a wealth of allelic intrapopulation. Interpopulations diversity measured by the mean of the genetic distances of Nei (1978) as well as throughout the factorial analysis of correspondences is comparatively reduced for the majority of the populations but more important for the species Vicia leucantha (Bk) adding a subspecies of Vicia sativa subsp. sativa (Sr) compared to other populations. The use of other molecular markers such as AFLPs, Microsatellites, might provide more specific information into this impasse. Keywords: variability; Vicia; isozymes; Fabaceae ; genetic diversity; spontaneous populations. INTRODUCTION The genus Vicia (Fabaceae) includes 140 species located in Europe, Asia and North America (Kupicha, 1981). In Tunisia, the Vicia genus is represented by 16 annual spontaneous and sub-spontaneous species (Pottier-Alapetite, 1979). Considering the economic and agronomic interest that the culture of Vicia in Tunisia might be a successful endeavour, we aimed at analysing the phytogenetic resources of the natural populations of various species collected in the North of Tunisia. In this work, we were interested in four spontaneous and forage species of the north of Tunisia: Vicia narbonensis, Vicia sativa, Vicia leucantha and Vicia lutea. Our objective in this work is to characterize these resources through an enzymatic evaluation. In addition, isoenzymes constitute good markers to consider the genetic variability of the populations to evaluate gene flows among them and further, to establish a phylogenetic relationship among the species (Morris et al., 2002). * Corresponding author: Safia EL BOK, Ph.D. Laboratory of Genetics Populations and Biological Resources, Faculty of Sciences of Tunis, 2092, El Manar II, Tunis, Tunisia safia.elbok@planet.tn MATERIALS AND METHODS Studied populations Many field trips were carried out to the North of Tunisia which enabled us to locate several natural populations localized in bioclimatic energy stages of wet and semi-arid superior (Fig.1). The collection has interesting samples of pods representative of each population of the Vicia genus. The sites were selected randomly. In each site, a surface of approximately 5 ha was surveyed and more than 20 plants per population were collected. Our study was limited to ten natural populations of Vicia. Four species were analyzed: Vicia sativa is represented by five subspecies: Vcia sativa L. ssp. angustifolia L. (C 1 ), Vcia sativa L. ssp. obovata gaud. (C 2 ), Vcia sativa L. ssp. macrocarpa Moris. (S), Vcia sativa L. ssp. sativa (J and Sr) and Vcia sativa ssp. (C 3 ). Vicia lutea is represented by Vicia lutea L. var. hirta (Balb.) Lois. (C 4 and G).Vicia lencantha Biv. (Bk).Vicia narbonensis is represented by Vicia narbonensis L. var. narbonensis L. (Vn). International Journal of Integrative Biology IJIB, 2008, Vol. 4, No. 2, 81

2 Electrophoresis and isoenzymatic systems The extracts were obtained starting from young sheets, not completely extended, taken near the apexes of the ramifications of the plants. For each individual plant, 50 mg of fresh matter were crushed in 200µl cold extraction buffer (ph 8.2) consisted of 0.42mol/L of sodium ascorbate, sucrose 0.49mol/L, 35 µl of β- mercaptoethanol;qsp 100 ml of distilled water. A spin at 14,000 rpm for 30 min. at 4 C is necessary to separate the supernatant containing the enzymes in solution. The supernatant is immediately absorbed by a paper Wathman n 3 and introduced into a starch gel (13%). To study the already quoted enzymatic systems, we used two types of starch freezing by adapting the techniques Cardy et al [2], Scandalios[ 10 ] Stuber et al. [12], Stuber and Goodman [13].Four enzymatic systems: malate dehydrogenase (MDH; EC ), isocitrate dehydrogenase (ICD; EC ), 6- Phosphogluconate dehydrogenase (6-PGD; EC ) and esterase (EST; EC ) were revealed. The various solutions of revelation used are those described by Shaw and Prassard (Shaw and Prassard, 1970)], Stuber, Cardy, Stuber and Goodman, Goodman. Figure 1: Geographical localisation of 10 population s vetches collected Methods of analysis of the data The Biosy-1 program was applied to evaluate several parameters including: intrapopulation genetic diversity, calculate: allelic frequencies, the average number of alleles per polymorphic loci (Ap), percentage of polymorphic loci (P) and the average heterozygosity observed (Ho) and expected (He) according to the Hardy-Weinberg Law. Interpopulation genetic diversity was estimated by the genetic distances from Nei,[7] as well as the factorial analysis of correspondences (AFC). This last method uses the frequencies of the electrophoretic bands. We selected the value (1) when a band of a given level is present and the value (0) when this same band is missing on another zymogram. RESULTS The revealed zymogram shows a number of variable bands according to the systems and analyzed populations' (Fig.2). For (MDH) system the high number (12 bands) and the revealed large number of zymogram made the interpretation a complicated issue. The profiles were analyzed on a phenotypical level by adopting the annotation: presence (1), absence (0). Allelic frequencies For the three enzymatic systems (ICD, 6-PGD and EST) six loci were identified, and while based on the structure of the enzyme. the analysis of the allelic frequencies were related only to five polymorphic loci (ICD 1, ICD 2, 6-PGD 1, 6-PGD 2 and EST 1 ).The allelic frequency in the population for each isoenzyme system is detailed in Table I. The results show that the various alleles, for the loci considered, present variable frequencies with respect to populations. Four alleles were not detected in many populations. The EST 1b allele was not found in Vicia narbonensis var. narbonensis (Vn), Vicia sativa ssp. angustifolia (C1) as at Vicia sativa ssp. macrocarpa (S). However, ICD 1b allele was missing in Vicia sativa ssp. (C3) and Vicia leucantha (Bk). 6-pgd 1a allele was not revealed in the populations of Vicia narbonensis var. narbonensis (Vn), Vicia lutea var hirta (G and C4), Vicia sativa ssp. (C3), Vicia sativa ssp. macrocarpa (S) and Vicia sativa ssp. sativa (Sr). The populations of Vicia narbonensis var. narbonensis (Vn), Vicia lutea var. hirta (G and C4), Vicia sativa ssp. (C3), Vicia sativa ssp. sativa (J), Vicia sativa ssp. angustifolia (C1) Vicia sativa ssp. obovata (C2) and Vicia sativa macrocarpa (S) did not express the allele 6 PGD 2b. The absence of these alleles indicates that these species are closely related particularly marking the Vicia sativa species as well as the two populations of Vicia lutea (C 4 and G). Vicia narbonensis seems to to be as closely related to Vicia sativa species. The species Vicia leucantha (Bk) is different from the other species by the presence of a greater number of alleles. Percentage of the polymorphic loci This parameter is established for each population belonging to the four studied species, starting from the whole of the polymorphic loci observed. Table II shows that the rate of polymorphisms is variable in line with International Journal of Integrative Biology IJIB, 2008, Vol. 4, No. 2, 82

3 a b c d Figure 2: Zymograms observed for the 4 system analysed. a : 6-Phosphogluconate déshydrogénase (6-PGD) observed into the population of Vicia sativa ssp. macrocarpa (S), b : Estérases (EST) observed into Vicia sativa ssp. obovata (C 2 ), c : Isocitrate déshydrogénase (ICD) observed into Vicia lutea var. hirta (C 4 ), d : Malate déshydrogénase (MDH) into Vicia sativa ssp. macrocarpa (S) populations. They vary between 40% and 80%. The rate of average polymorphism is 58%. The highest percentage (80%) is a characteristic of the Vicia leucantha (Bk) species. The average rate for Vicia sativa (J, C 1, C 2, C 3, S and Sr) is 60%. The relatively low rates (from 40 to 50%) are characteristic of Vicia narbonensis (Vn) and Vicia lutea (C 4 and G), respectively. Figure3: Dendrogram of genetic distances of Nei. The means numbers of polymorphic alleles by locus (Ap) and averages of heterozygosity (Ho and He) The means numbers of alleles by locus (Ap) vary from 1.4 for Vicia narbonensis (Vn), Vicia sativa ssp. (C 3 ), Vicia sativa ssp. macrocarpa (S)) to 1.8 for Vicia sativa ssp. sativa (J), Vcia leucantha (Bk) and Vicia sativa ssp. sativa (Sr)). The highest average heterozygosity observed (H 0 = 0.435) is detected in the population of Vicia lutea var. hirta (C4), yet, the lowest (0.253) was noted at Vicia leucantha (Bk). The average heterozygosity is rather high on the level of each population in spite of the mode of reproduction autogamy of the plant except for the species Vicia leucantha (Bk) which moves away from balance panmictic (Table II). Genetic Distances Nei To consider genetic diversity between the four species studied, we proposed to calculate the genetic distances from Nei (Nei, 1978). The analysis of the results shown in Table III, indicates that the smallest genetic distance observed between the two populations of Vcia lutea (C 4 and G) is high (0.001). This shows that these two populations are genetically close and probably belong to the bioclimatic floor (semi-arid superior). These two populations thus express a great isoenzymatic similarity. The longest genetic distances (D = or D = 0.244) are observed between Vicia leucantha (Bk), Vicia narbonensis (Vn) and Vicia lutea (C 4 and G), respectively. This shows a genetic difference between the populations quoted in spite of their membership on the same bioclimatic floor. The pairs of close populations present weak genetic distances: case of the populations of Vicia sativa between them and also of Vicia narbonensis with the populations of Vicia lutea International Journal of Integrative Biology IJIB, 2008, Vol. 4, No. 2, 83

4 and of Vicia sativa. Analysis of the dendrogram starting from the genetic distances not skewed from Nei (Nei, 1978) (Fig. 3) shows the distance 0.17 three groups of populations: -A third group projected near the origin of the axes characterized by the presence of the species Vicia lutea with an insulation not very distinct from the individuals from Vicia sativa ssp. sativa (Sr). The first group joins together the populations of Vicia narbonensis (Vn), Vicia sativa (J, C1, S and C 2 ) as Vicia lutea (C 4 and G). This aggregate can be subdivided at the distance (0.05) in four subsets: -The first includes the populations of Vicia lutea (C 4 and G) and of Vicia narbonensis (V n) coming from the semi-arid region. -The second subset includes Vicia sativa ssp. sativa (J) originating in the wet inferior. -The third subset joins together Vicia sativa ssp. angustifolia (C 1 ) and Vicia sativa ssp. macrocarpa (S). The first population belongs to the semi-arid stage and the second population on the lower wet floor. -The fourth subset includes Vicia sativa ssp. obovata (C 2 ) bellowing to the semi-arid one. The second group includes Vicia sativa ssp. (C 3 ) pertaining to semi-arid also. The third group joins together Vicia leucantha (B K ) and Vicia sativa ssp. sativa (S R ) respectively originating in sub-wet and the wet inferior. The short distances separating the populations, more particularly to the first group would testify to a genetic diversity inter-populations rather reduced. Genetic diversity would be explained primarily by the component intrapopulation. Salhi- Hannachi. (Salhi-Hannachi, et al., 1998) paid comparable results on species of the genus Medicago, autogamous obtained a genetic variability intrapopulation more significant than that between the populations. The dendogram shows it well for the populations of Vicia sativa. Factorial analysis of the correspondences The factorial analysis of correspondences (AFC) shows that the first three axes of the AFC absorb 72,11% of total inertia ( Table IV). That shows a good structuring of the variability of the species within the prospected surface. The structuring of the populations according to plans' defined by axis 1 defined by the bands P 1, I 1, I 2, M 08, M 09, M 10, M 11, and M 12 and the axis 2 defined by the bands E 1 and E 2 of the system (EST) and by the bands M 11 and M 12 of system (MDH) showed a stretching of cloud on the positive side of axis 1. The differentiation of the populations is done primarily according to bands' of systems ICD and MDH defining this axis (Fig. 4). Some representatives of the species Vicia leucantha (Bk) seem isolated significantly from the individuals of the other populations. However, of the more detailed groupings can be defined: -A group on the positive side of axis 2 including the majority of the populations of Vcia sativa. -A group on positive side of axis 1, clearly showing the insulation of the individuals of Vicia leucantha (Bk). Figure 4: Factorial analysis of correspondences (AFC). CONCLUSION The polymorphic analysis of the loci (ICD, 6-PGD, EST and MDH) for the 10 populations of Vicia which we collected revealed a variable polymorphism according to populations. Five loci on the whole were identified, the average number of alleles by locus varies from 1.4 to 1.8 according to populations'. Intrapopulation diversity estimated through the percentage of polymorphic loci and the rate of heterozygosity is relatively significant. The average percentage of polymorphic loci (58%) would testify to an allelic richness rather significant intrapopulation. Bingham and Ranker (Bingham and Ranker, 2000) reported high rates close as of ours, in autogamous plants. Interpopulation genetic diversity, measured through the genetic distances (Nei, 1978) and through the factorial analysis of correspondences (AFC) is relatively reduced for the majority of the populations but more significant for the species Vicia leucantha compared to the other populations. The dendrogram of the genetic distances revealed a bringing together of the same populations species independently of their bioclimatic stage. Thus, these results, although giving an estimate of interspecific variability remain insufficient as for the emission of assumptions on the phylogeny of the studied species. The analysis of a high number of isozymes could bring more precise information in this field. International Journal of Integrative Biology IJIB, 2008, Vol. 4, No. 2, 84

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