Scientia Horticulturae 120 (2009) 34–40 Contents lists available at ScienceDirect Scientia Horticulturae journal homepage: www.elsevier.com/locate/scihorti Sequence analysis of the internal transcribed spacers (ITSs) region of the nuclear ribosomal DNA (nrDNA) in fig cultivars (Ficus carica L.) Baraket Ghadaa, Saddoud Olfaa, Chatti Khaleda, Mars Messaoudb, Marrakchi Mohameda, Trifi Mokhtara, Salhi-Hannachi Amela,* a b Laboratoire de Génétique Moléculaire, Immunologie & Biotechnologie, Faculté des sciences de Tunis, Campus Universitaire, 2092 El Manar Tunis, Tunisia U. R. Agrobiodiversité, Institut Supérieur Agronomique, 4042 Chott Mariem, Sousse, Tunisia A R T I C L E I N F O A B S T R A C T Article history: Received 18 March 2008 Received in revised form 6 September 2008 Accepted 8 September 2008 Sequences of the internal transcribed spacers (ITSs) of nuclear ribosomal DNA (nrDNA) were analysed in a set of Tunisian fig (Ficus carica L.) cultivars. The size of the spacers sequences ranged from 200 to 279 bases for ITS1 and from 253 to 314 bases for ITS2. Variation of GC contents has been also observed and scored as 59–68% and 55–68% for ITS1 and ITS2, respectively. This data exhibited the presence of polymorphism among cultivars. The intra-specific variability level of the ITS sequences proved a variation both in the length and in the sequences studied. In fact, ITS1 and ITS2 sequences were considered as a useful tool to establish genetic relationships among cultivars. Our results indicate that the diversity detected among closely related genotypes supported strongly the efficiency of ITS sequences for establishing relationships between cultivars. ITS2 seems to be relatively more informative than ITS1 regarding length or GC contents. Considerable genetic diversity was observed among fig at intra and inter-cultivars levels. Two polyclonal varieties were identified. In addition, data proved that a typically continuous genetic diversity characterizes the local fig germplasm. The topology of the derived dendrogram strongly supported this assumption. In fact, genotypes are clustered independently from their geographical origin or the sex of trees suggesting a narrow genetic basis among the ecotypes studied in spite of their phenotypic distinctiveness. Implications of these results for management of fig germplasm collections are discussed. ß 2008 Elsevier B.V. All rights reserved. Keywords: Ficus carica Fig ITS sequences Genetic relationships Nuclear ribosomal DNA (nrDNA) 1. Introduction The common fig (Ficus carica L., 2n = 26 chromosomes) (Weiblen, 2000) belongs to the order of Urticales and the family of Moraceae, with over 1400 species classified into about 40 genera (Watson and Dallwitz, 2004). The Ficus species are gynodioecious, and functionally dioecious. Some of them are functionally female producing only a seed-bearing fruit, whereas others are functionally male and produce only pollen and pollen-carrying wasp progeny (Kjellberg et al., 1987; Janzen, 1979; Weibes, 1979). The symbiosis between figs and their pollinators is a prominent example of coevolution. A specific wasp species belonging to the Agaonidae family (Hymenoptera, chalcidoidea) is required for pollination in each of the 700 Ficus species (Berg, 2003). It should be stressed that, among these species F. carica is one of the oldest known fruit crop and used for fruit production (Beck and Lord, 1988; Kislev et al., 2006). Total * Corresponding author. Tel.: +216 71 87 26 00; fax: +216 70 86 04 32. E-mail address: Amel.SalhiHannachi@fsb.rnu.tn (A. Salhi-Hannachi). 0304-4238/$ – see front matter ß 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.scienta.2008.09.013 world fig production is over 1 million tones. Turkey, Egypt, Greece, Iran, Morocco, Spain and USA yielded about 70% of total production (FAOSTAT, 2004). In Tunisia, fig cultivation consists of diversified and well-adapted ecotypes exhibiting the presence of a large array of genotypes that characterizes the local germplasm. Despite this phenotypic diversity, fig cultivation remains traditional and relatively limited since only a small part of the total area reserved to the fruit trees contains fig plantations yielding about 30,000 tons per year (DGPA, 2006). Fruits are consumed fresh or dried and for industrial production. Figs are also used traditionally for their medicinal properties. Moreover, the local germplasm is threatened by genetic erosion (Urbanization, absence of phytosanitary norms, rainfall irregularities, mono-varietal culture) and some biotic constraints such as the fig mosaic disease. Consequently, a lack of cultivars has occurred and constituted a constraint in the preservation and the improvement of the local fig phytogenetic resources. Therefore, it is imperative to establish strategies for preservation and conservation of local germplasm. For this purpose, studies have reported the use of morphometric, pomological traits and isozymes and permitted to evidence a large phenotypic variability (Mars et al., G. Baraket et al. / Scientia Horticulturae 120 (2009) 34–40 1998; Rhouma, 1996; Hedfi et al., 2003; Salhi-Hannachi et al., 2003; Chatti et al., 2004a). However, these studies are less rewarding since the derived characterization is not suitable to establish reference genotypes for fig breeding programs. To overcome this inconvenience, large scale of PCR-DNA based methods such as random amplified polymorphic DNA (RAPD), inter simple sequence repeats (ISSR), random amplified microsatellite polymorphism (RAMPO) and simple sequence repeats (SSR) have been successfully designed in this crop (Valizadeh et al., 1977; Khadari et al., 1995; Chessa et al., 1998; Elisiario et al., 1998; Cabrita et al., 2001; Chatti et al., 2004b, 2007; Saddoud et al., 2007; Salhi-Hannachi et al., 2004, 2005, 2006). Thus, it was assumed that the local germplasm is characterized by a typically continuous genetic diversity. In addition, despite the suitability of these techniques in the ecotypes’ discrimination, several cases of synonymy have been scored and not well elucidated using the evidenced markers (Chatti et al., 2007; Saddoud et al., 2007). Therefore, we become interested in the development of additional markers in order to precise the genetic diversity organisation and to contribute to the molecular characterization of fig ecotypes. For this purpose, analysis of the internal transcribed spacer (ITS) of nuclear ribosomal DNA was investigated. Note worthy that the nrDNA organisation, high copies of tandem units in a single or multiple loci, offers several advantages over other parts of the genome (Rogers and Bendich, 1987; Hamby and Zimmer, 1992). (i) It is useful for phylogenetic studies at the intra specific level (Jorgensen and Cluster, 1988; Downie et al., 1994; Baldwin et al., 1995; Campbell et al., 1995; Susanna et al., 1995). (ii) This gene family undertakes rapid concerted evolution (Arnheim, 1983; Zimmer et al., 1980; Hillis et al., 1991). (iii) Its detection, amplification and sequencing are made easy by regards to the presence of highly conserved sequences flanking each of the two spacers. Therefore, analysis of the rDNA would provide an attractive approach suitable to generate efficient molecular markers to assess phylogenic relationships as well as the genetic diversity organisation in higher plants. The present study portrays the achievement of nrDNA in a set of fig cultivars to analyse genetic diversity and establish a molecular database for fig breeding programs and the rational management of the local fig germplasm conservation. 2. Materials and methods 2.1. Plant materials Twelve fig cultivars, collected from traditional plantations and ex situ conserved at the ISA (Chott Mariam), were used in this study (Table 1). The experimental material consisted of 11 common fig cultivars and 1 caprifig that represent the main cultivated forms in the Sahel region of Tunisia. These were chosen according to their attractive fruit qualities and their economic importance. Punica granatum is used in this study as an out-group sequence to root the tree. 35 Table 1 Tunisian fig (Ficus carica) cultivars studied with their localities of origin and fruit characteristics. Label Cultivar Geographic origin Variety type Fruit colour 1 2 3 4 5 6 7 8 9 10 11 12 Besbessi 1 Besbessi 2 Besbessi 3 Zidi Soltani Bidhi 1 Hemri Bidh beghal Jrania Bidhi 2 Delgane Bither abiadh Mesjed Aı̂ssa Mesjed Aı̂ssa Mesjed Aı̂ssa Mesjed Aı̂ssa Ouardanine Kalaa Kebira Ghadhabna Mesjed Aı̂ssa Ghadhabna Khmara El Alia Khmara San Pedro San Pedro San Pedro Smyrna Smyrna Smyrna Common Smyrna Caprifig Smyrna Smyrna San Pedro Yellowish green Yellowish green Yellowish green Black Purple White yellow Reddish Dark Purple – White yellow Yellowish Yellowish a Caprifig: male tree. 2.2. DNA purification Total cellular DNA was extracted from 3 g of green tissues (leaves) according to Dellaporta et al. (1983). The DNA concentration was spectrophotometrically estimated and its integrity was performed by 0.8% analytic agarose gel electrophoresis (Sambrook et al., 1989). 2.3. Primers and ITS assays The internal transcribed spacers region was amplified using the polymerase chain reaction (PCR) method (Saiki et al., 1988). For this purpose, the following primers identified as 50 -AAGGTTTCCGTAGGTGAAC-30 for the 30 end of 18S nrDNA and 50 -TATGCT TAAACTCCAGCGGG-30 for the 50 end of 26S nrDNA (Fig. 1) were used as reported by Weiblen (2000). Amplifications were carried out in a 25 mL reaction mixture volume containing 2.5 mL of 10 Taq polymerase reaction buffer, 1 U of Taq DNA polymerase (QBIOgene, Illkirch, France), 200 mM of each dNTP (DNA polymerization mix, Pharmacia), 6 pM of each primer and 20 ng of total cellular DNA. The reaction mixture was overlaid with a drop of mineral oil to prevent evaporation during thermal cycling. PCRs were performed in a DNA thermocycler (Crocodile III QBIOgene, Illkirch, France) as follows: reaction mixtures were heated at 94 8C for 5 min as an initial denaturation step before entering 35 cycles consisting of 45 s at 94 8C for denaturation, 1 min at primer appropriate temperature (45 8C) for annealing and 1 min 30 s at 72 8C for elongation. A final extension step of 10 min at 72 8C was performed at the last cycle. Amplification products were separated using 1.5% agarose gel electrophoresis in 0.5 TBE buffer, stained with ethidium bromide and visualized under UV light (Sambrook et al., 1989). Excess of primers and dNTPs after amplification were removed by purification using the Wizard SV Gel PCR Clean-Up System kit according to the manufacturer’s instructions (Promega, WI, USA). Sequencing Fig. 1. Organisation of the internal transcribed spacer (ITS) region. The orientation and the approximate primer sites are presented according to Baldwin et al. (1995). G. Baraket et al. / Scientia Horticulturae 120 (2009) 34–40 36 was performed, on both strands, by the automated fluorescent cycle sequencing method using the Big Dye Terminator Ready Reaction Kit (Applied Biosystems, Foster City, CA, USA). DNA magnifications were performed by 30 cycles of 30 s at 95 8C, 15 s at 60 8C and 4 min at 72 8C. The primers ITS4 and ITS5 were used for the first and second strand sequencing. The labeled reactions were resolved and the DNA sequence was analysed by an automated DNA sequencer (Applied Biosystems, Foster City, CA, USA) after migration on polyacrylamide gels. The derived ITS sequences identity was attested with the appropriate program Blast (NCBI) to search DNA sequence databases for high similarity with other Ficus species (http://www.ncbi.nlm.nih.gov/BLAST). 2.4. Alignment and sequence analysis Nucleotide sequences were aligned using appropriate programs of the DNAMAN (V.4.0.1.1) software. The 30 end of the 18S gene, both 50 and 30 ends of the 5.8s gene and the 50 end of the 26S gene were used to make the alignment of sequences easier. The alignment was manually checked and pairwise sequence divergence between cultivars in ITS, ITS1, ITS2 was determined according to the Kimura-2 method (Kimura, 1980). The resultant distance matrix was then computed to generate phylogenetic trees according to the Neighbor-joining method (Saitou and Nei, 1987). Internal support for groupings was assessed using 1000 bootstrap replicates (Felsenstein, 1985). The Pearson’s correlation coefficient was estimated between the ITS1 and ITS2 genetic distances matrix using the Statistical Analysis System, V.6.07 (SAS, 1990). 3. Results 3.1. ITS sequence analysis Starting from the total DNAs used as templates, the entire ITS region (ITS1-5.8S-ITS2) was successfully amplified and displayed a single DNA band of about 700 bp. In order to confirm the identity of the derived DNA stretches, a BLAST search was performed (Altschul et al., 1997) and exhibited mostly significant alignments with available ITS sequences from Ficus species. Therefore, the resultant sequences were edited in GenBank under the references: EF579611–EF579622. Analysis of the derived sequences permitted to identify variations both in length and in nucleotides’ content (Table 2). ITS region varied in length from 668 bp in ‘Besbessi 2’ to 682 bp in ‘Soltani’ and ‘Hemri’ cultivars. Similarly, ITS1 and ITS2 spacers are extended between 200 bp in ‘Zidi’ to 279 bp in ‘Jrani’ and from 253 bp in ‘Besbessi 2’ to 314 bp in ‘Zidi’, respectively. Table 2 Length and G + C contents of ITS sequences of Tunisian fig (Ficus carica) cultivars. Cultivar Length (bp) GC content (%) ITS ITS1 ITS2 5.8S ITS ITS1 ITS2 5.8S Besbessi 1 Besbessi 2 Besbessi 3 Zidi Soltani Bidhi 1 Hemri Bidh beghal Jrani Bidhi 2 Delgane Bither abiadh 671 668 676 678 682 671 682 681 671 674 675 677 252 251 278 200 258 252 259 258 279 254 253 254 254 253 256 314 259 258 257 259 256 255 257 259 165 164 142 164 165 161 166 164 136 165 165 164 59 58 58 64 63 62 62 61 65 58 63 58 60 64 61 68 64 66 65 65 65 66 66 61 62 55 59 65 67 64 65 64 68 55 65 60 52 53 52 55 55 52 52 54 56 53 53 51 Mean 675.50 254 261.41 160.08 60.91 64.25 62.41 53.16 In addition, the GC content of the amplified sequences varied either for the entire ITS or the ITS1 and ITS2 spacers. In fact, the percentage of GC varied from 58 to 65 in the ITS region (ITS1 + 5.8S + ITS2), from 60 to 68 for ITS1 and from 55 to 68 for ITS2 (Table 2). 3.2. Inter-cultivars ITS polymorphisms Genetic distances matrix based on ITS sequences was estimated according to the formula of Kimura-2 (Table 3). Genetic distances varied from 0.027 to 0.386 with a mean of 0.216. Thus, the cultivars studied are characterized by significant divergence at the nrDNA ITS sequences. The smallest genetic distance value of 0.027 was registered between ‘Zidi’ and ‘Soltani’ cultivars characterized by great similarities at the ITS sequence. However, ‘Besbessi 1’ and ‘Bither abiadh’ cultivars were the most divergent since they exhibited the highest genetic distance of 0.386. All the remaining cultivars showed intermediate levels of similarities. The derived Neighbor-joining phenogram is illustrated in Fig. 2. This tree supported the fig cultivars organisation in two main clusters. The first group labeled I is composed by ‘Bither abiadh’ cultivar. All the remaining cultivars are ranged in the second cluster (labeled II) that comprises two sub-groups. The first sub-group (II-1) is composed by ‘Besbessi 3’ cultivar; while the remaining cultivars constitute the second sub-cluster with a significant divergence of the ‘Besbessi 2’ and ‘Bidhi 2’ cultivars from all the others. In addition, this tree branching is made independently either from the geographic origin of the cultivars or from their denomination. Table 3 Genetic distances matrix among Tunisian fig (Ficus carica L.) cultivars based on ITS sequences data calculated using the formula of Kimura-2. Bold values correspond to the smallest and the highest genetic distances. Genotype 1 2 3 4 5 6 7 8 9 10 11 12 1. Besbessi 1 2. Besbessi 2 3. Besbessi 3 4. Bidhi 1 5. Bidhi 2 6. Bidh beghal 7. Bither abiadh 8. Zidi 9. Soltani 10. Hemri 11. Jrani 12. Delgane 0 0.296 0.322 0.178 0.316 0.191 0.386 0.182 0.183 0.211 0.177 0.157 0 0.351 0.252 0.154 0.286 0.382 0.289 0.284 0.300 0.239 0.262 0 0.285 0.382 0.315 0.354 0.308 0.312 0.306 0.290 0.290 0 0.268 0.111 0.325 0.081 0.105 0.131 0.089 0.073 0 0.299 0.378 0.289 0.283 0.308 0.236 0.265 0 0.307 0.047 0.056 0.099 0.075 0.074 0 0.331 0.301 0.304 0.313 0.308 0 0.027 0.070 0.034 0.034 0 0.080 0.057 0.056 0 0.081 0.089 0 0.042 0 P. granatum 0.629 0.653 0.629 0.610 0.649 0.620 0.599 0.628 0.622 0.634 0.615 0.623 G. Baraket et al. / Scientia Horticulturae 120 (2009) 34–40 37 Fig. 2. Neighbor-joining phenogram of 12 Tunisian fig (Ficus carica) cultivars based on nrDNA ITS sequences. Numbers at nodes are bootstrap values. In order to precise the contribution of ITS1 and ITS2 in the observed polymorphisms, we examined the inter-cultivars diversity using these sequences. As reported in Table 4, the pairwise sequence divergences in ITS1 are ranged from 0.005 between ‘Zidi’ and ‘Soltani’, ‘Zidi’ and ‘Delgane’ and ‘Zidi’ and ‘Bidhi 1’, to 0.227 between ‘Besbessi 3’ and ‘Hemri’. Consequently, we assume that ‘Besbessi 3’ and ‘Hemri’ are the most divergent, while; ‘Zidi’ presents great similarities with ‘Soltani’, ‘Delgane’ and ‘Bidhi 1’ cultivars. The phenogram based on ITS1 sequence analysis illustrated the divergence described above and allowed cultivars clustering into two main groups (Fig. 3a). The first one labeled (I) is composed of the ‘Besbessi 3’ cultivar. All the remaining accessions are grouped in the second cluster (II) where ‘Bither abiadh’ and ‘Besbessi 1’ are significantly divergent from the others. The genetic distances based on the ITS2 spacer ranged from 0.043 to 0.652. The smallest distance of 0.043 was observed between the caprifig ‘Jrani’ and the female cultivars ‘Hemri’ and ‘Zidi’ cultivars suggesting their great similarities (Table 4). However, ‘Bidhi 2’ and ‘Besbessi 1’ cultivars are characterized by the maximum of divergence since they exhibited the greatest genetic distance value of 0.652. The derived phenogram illustrated divergence between the cultivars studied (Fig. 3b). Two main clusters could be identified. The first one labeled (I), is composed of ‘Bidhi 2’, ‘Bither abiadh’ and ‘Besbessi 2’ that are significantly divergent from all the remaining cultivars ranged in the second cluster labeled (II). Cluster analysis show that the considered cultivars are gathered independently from either geographical origin or the sex of trees. Moreover, some cultivars having the same denomination not cluster together. It is the case of cultivars Besbessi 1, Besbessi 2 and Besbessi 3 originating from Mesjed Aı̂ssa and the two cultivars Bidhi 1 and Bidhi 2 collected from Kalaa Kebira and Khmara in the Centre East Tunisia. These may be considered as homonomies and the hypothesis of polyclonal varieties ‘Besbessi’ and ‘Bidhi’ could be forwarded to explain this result. Pearson’s correlation coefficient was estimated between ITS1 and ITS2 genetic distances. Lower and no significant correlation was scored between ITS1 and ITS2 sequences (0.074, Pr = 0.547). This result confirm the ability of Table 4 Genetic distances matrix among Tunisian fig Ficus carica cultivars based on ITS1 (lower diagonal) and ITS2 (upper diagonal) sequences estimated according to the formula of Kimura-2. Bold values indicate the smallest and the greatest genetic distances. Genotype 1. Besbessi 1 2. Besbessi 2 3. Besbessi 3 4. Bidhi 1 5. Bidhi 2 6. Bither abiadh 7. Bidh beghal 8. Zidi 9. Hemri 10. Jrani 11. Delgane 12. Soltani 1 0.081 0.217 0.072 0.080 0.095 0.115 0.085 0.119 0.087 0.079 0.111 2 3 4 5 6 7 8 9 10 11 12 0.544 0.238 0.547 0.178 0.561 0.200 0.652 0.448 0.633 0.623 0.551 0.218 0.581 0.534 0.511 0.170 0.550 0.272 0.181 0.632 0.515 0.143 0.558 0.276 0.146 0.643 0.541 0.106 0.154 0.545 0.280 0.169 0.636 0.506 0.090 0.071 0.134 0.545 0.263 0.154 0.640 0.506 0.102 0.043 0.043 0.104 0.556 0.225 0.141 0.638 0.523 0.122 0.067 0.090 0.055 0.149 0.537 0.258 0.165 0.636 0.502 0.120 0.063 0.078 0.051 0.078 0.190 0.032 0.040 0.056 0.084 0.040 0.084 0.048 0.040 0.080 0.193 0.203 0.187 0.220 0.162 0.227 0.192 0.196 0.217 0.016 0.060 0.052 0.005 0.056 0.016 0.012 0.048 0.051 0.063 0.015 0.071 0.032 0.028 0.063 0.079 0.045 0.103 0.071 0.059 0.079 0.010 0.086 0.063 0.059 0.019 0.040 0.010 0.005 0.005 0.051 0.051 0.070 0.020 0.051 0.047 38 G. Baraket et al. / Scientia Horticulturae 120 (2009) 34–40 Fig. 3. (a) Neighbor-joining phenogram of 12 Tunisian fig (Ficus carica) genotypes based on ITS1 sequences. Numbers at nodes are bootstrap values. (b) Neighborjoining phenogram of 12 Tunisian fig (Ficus carica) genotypes based on ITS2 sequences. Numbers at nodes are bootstrap values. the ITS sequences to differentiate cultivars and lead to the slight differences between the clustering results obtained with these two types of sequences data considered separately. Furthermore, the present study portrays the achievement and the use ITS sequences variation as a powerful method to discriminate fig genotypes and to assess the genetic diversity in this crop at inter- and intra-cultivar levels. 4. Discussion and conclusion The study herein illustrates the use of ITS sequences variation to generate new molecular markers suitable in the assessment of the genetic diversity within Tunisian fig cultivars. Using a set of local genotypes, the primers tested in this study have permitted to amplify ITS regions. Therefore, compared with data previously reported in Tunisian figs (Chatti et al., 2004a,b, 2007; Hedfi et al., 2003; Saddoud et al., 2007; Salhi-Hannachi et al., 2004, 2005, 2006) we assume that the designed procedure has permitted to detect a high level of polymorphism in this crop. Globally, the scored size of the ITS1 and ITS2 spacers is in agreement with the reported ITS length in other plant species (ITS1: 187–298 bp, ITS2: 187–252 bp) (Baldwin et al., 1995; Campbell et al., 1995; Cerbah et al., 1998; Kollibara et al., 1997). Moreover, data proved that ITS2 spacer is larger than ITS1 in F. carica. Except for ‘Jrani’, ‘Besbessi 3’ and ‘Hemri’ cultivars, in which the ITS1 sequence is consistently longer than ITS2. Similar results have been reported in other crop families such as Betulaceae, Cucurbitaceae, Fabaceae, Poaceae, Scrophulariaceae and Viscaceae (Baldwin et al., 1995). The 5.8S coding region exhibited a relatively homogenous size in the studied cultivars (164–166 bp) except for ‘Jrani’ (136 bp), ‘Besbessi 3’ (142 bp) cultivars. This result supports the hypothesis of invariant length, mostly 163 or 164 bp, of this subunit in angiosperm species (Baldwin et al., 1995). The smallest length of the 5.8S coding region obtained for ‘Jrani’ and ‘Besbassi 3’ cultivars could be attributed to the smallest length of the entire ITS region. Variation in length and nucleotide content confirm the genetic diversity and the variability inter- and intra-cultivar variability. It should be stressed that the scored GC contents are nearly similar to those reported for other plant species such as Quercus spp. (Baldwin et al., 1995; Bellarosa et al., 2005). Moreover, the high similarity level of the GC contents scored in the ITS1 and ITS2 has been also reported in angiosperms and mostly in other eukaryotes reflecting their co-evolution (Torres et al., 1990; Van der Sande et al., 1992). In addition, data proved that a typically continuous genetic diversity characterizes the local fig germplasm. The topology of the derived dendrograms strongly supported this assumption. In fact, genotypes are clustered independently either from their geographical origin or the sex of trees suggesting a narrow genetic basis among the ecotypes studied in spite of their phenotypic distinctiveness. In fact, ecotypes are selected by farmers mainly for the fruit traits that are encoded by a small part of the genome. This is well exemplified in the case of the three ‘Besbessi’ and two ‘Bidhi’ cultivars, which are located in divergent clusters. In addition, the caprifig ‘Jrani’, did not significantly diverge from the female trees. Thus, our present data as our previous studies (Chatti et al., 2004b, 2007; Saddoud et al., 2007; Salhi-Hannachi et al., 2004, 2005, 2006) corroborate with the hypothesis of a monoecious origin of the common fig (F. carica L.) that evolved later into a dioecious plant (caprifig and edible fig) (Machado et al., 2001). It should be stressed that little differences are scored in the derived phenogram’s topology based on ITS, ITS1 and ITS2 sequences. This result suggests that the analysed sequences contribute differently in the discrimination of fig genotypes. This assumption is strongly supported since polymorphisms obtained with ITS1 and ITS2 spacers have different underlying sources at the molecular level and may differ in their usefulness for the exploration of genetic diversity and the establishment of relationships among genotypes. Therefore, it is of a great interest to determine whatever these sequences contribute the mostly in the observed diversity. This was made possible throughout estimation of the Pearson’s coefficient between the ITS1 and ITS2 genetic distances matrix. Lower and no significant correlation was scored between ITS1 and ITS2 sequences (0.074, Pr = 0.547). This result could be explained by the relatively more heterogeneous sequence of the ITS2 compared to the ITS1. In fact, analysis of these sequences showed that the ITS1 and ITS2 differed either in length or in the GC content. Moreover, ITS2 sequence exhibited more nucleotide substitutions among genotypes than the ITS1 (data not shown) suggesting the presence of different evolution events in these regions. As reported in other plant species, the present data proved that ITS2 seems to evolve much faster than ITS1 (Baldwin, 1993; Chennaoui et al., 2007). It should be noted, that in F. carica L., G. Baraket et al. / Scientia Horticulturae 120 (2009) 34–40 the ITS region has evolved mainly by single mutation events. The sequence alignment required the inclusion of indel zones (insertion/deletion). Substitutions (transversion and transition) are also registered. Results from the present study indicate that the variation detected among closely related genotypes supported strongly the efficiency of ITS sequences for the phylogeny reconstruction in fig germplasm. In our study, complementarity of ITS1 and ITS2 data sets was further indicated by more complete and robust resolution in tree based on combined spacer data than in trees based on ITS1 or ITS2 alone. This is indicated by the topology of the phenogram based on the entire ITS sequence and robustly supported clusters (98–100%). A previous study based on the use morphometric parameters related to the tree vegetative development of the tested cultivars has been reported (Chatti et al., 2004a). The parameters measured could be used as descriptors for identifying fig varieties. Therefore, it has been assumed that the studied cultivars are characterized by a large phenotypic diversity; and hypothesis of synonymy and/or homonymy were forwarded to explain cultivars clustering, since cultivars are locally named according to their origin and/or some fruit traits such as colour (Chatti et al., 2004a). Thus, cultivars Besbessi (1, 2 and 3) may be considered as a polyclonal variety ‘Besbessi’. Cultivars Bidhi (1 and 2) may be grouped in a multiclonal variety ‘Bidi’. This is in agreement with the result founded by Chatti et al. (2004a) using morphological characters. Variation in length of the ITS1 and ITS2 and the entire ITS region as well as the nucleotide content (GC %) confirm this assumption. Oukabli and Khadari (2005) have reported similar results for Moroccan genotypes. These authors have described the use of pomological and molecular (SSR and ISSR) markers to survey polymorphism among fig genotypes. Studies conducted on Tunisian fig cultivars indicate that no significant clustering has been observed according to the morphological traits and the meaning of accession’s names (Hedfi et al., 2003; Chatti et al., 2004b; Salhi-Hannachi et al., 2004, 2005, 2006). The analysis of the genetic diversity mediated by molecular markers, RAPD and RAMPO, shows that the resultant patterns strongly supported the suitability of these markers as a powerful tool to distinguish fig cultivars (Chatti et al., 2007; Salhi-Hannachi et al., 2005, 2006). In fact, an important variability has been evidenced in this collection. These authors have reported that the inter-cultivars variability, but not structured, has been evidenced in fig collections (Chatti et al., 2004b). Despite the low level of genetic distances between cultivars, the ITS sequence data were useful to clarify more the relationships at this lower taxonomic level. The dendrograms resulting from morphological and molecular analyses (RAPD, RAMPO and ITS) were similar in their overall topology. They differed slightly in the support of some clades. On the other hand, we may assume that the ITS dendrograms are suitable to demonstrate level of genetic diversity since cultivars similarly named are clustered in different groups and varieties closely grouped although they have different appellations. The apparent variation between the considered cultivars is supported by higher variation within cultivars. Thus, our data strongly support the use of the ITS sequences together with the other revealed molecular markers revealed in the cultivars of this collection as powerful tools to discriminate fig accessions. This is in agreement with other studies describing the use of this gene family to assess intra specific variation in plants. Opportunely, the present study provides molecular markers reliable for genetic diversity surveying or for the differentiation among cultivars and/or polyclonal accessions. ITS sequences constitute an attractive approach to generate a useful tool genetic diversity study. The enlargement of the number of cultivars would supply more useful information to assist 39 selection and conservation for a rationally management of the local fig germplasm and allowed us to build a molecular database for the fig genetic resources. Acknowledgement Financial support for this work was provided by the Tunisian Ministère de l’enseignement Supérieur de la Recherche Scientifique et de la Technologie. References Altschul, S.F., Madden, T., Schaffer, A.A., Zhang, J., Zhang, Z., Miller, W., Lipman, D.J., 1997. Gapped BLAST and PSI-BLAST A new generation of protein data base search programs. Nucleic Acids Res. 25, 3389–3402. Arnheim, N., 1983. Concerted evolution in multigene families. In: Nei, M., Koehn, R. (Eds.), Evolution of Genes and Proteins. Sinauer Associates, Sunderland, Massachusetts, pp. 38–61. Baldwin, B.G., Sanderson, M.J., Porter, J.M., Wojciechowski, M.F., Campbell, C.S., Donoghue, M.J., 1995. The ITS of nuclear ribosomal DNA: a valuable source of evidence on angiosperm pylogeny. Ann. Missouri Bot. Gard. 82, 247–277. Baldwin, B.G., 1993. Molecular phylogenetics of calycadenia (compositae) based on ITS sequences of nuclear ribosomal DNA: chromosomal and morphological evolution re-examined. Am. J. Bot. 80 (2), 222–238. Beck, N.G., Lord, E.M., 1988. Breeding System in Ficus carica, the Common Fig II Pollination Events. Am. J. Bot. 75, 1913–1922. Bellarosa, R., Simeone, M.C., Papini, A., Schirone, B., 2005. Utility of ITS sequence data for phylogenetic reconstruction of Italian Quercus spp. Mol. Phylogenet. Evol. 34, 355–370. Berg, C.C., 2003. Flora malesiana precursor for the treatment of Moracea 1: the main subdivision of Ficus: the subgenera. Blumea 48, 167–178. Cabrita, L.F., Aksoy, U., Hepaksoy, S., Leitao, J.M., 2001. Suitability of isozyme, RAPD and AFLP markers to assess genetic differences and relatedness among fig (Ficus carica L.) clones. Sci. Hortic. 87, 261–273. Campbell, C.S., Donoghue, M.J., Baldwin, B.G., Wojciechowski, M.F., 1995. Phylogenetic relationships in maloideae (Rosaceae): evidence from sequences of the internal transcribed spacers of nuclear ribosomal DNA and its congruence with morphology. Am. J. Bot. 82 (7), 903–918. Cerbah, M., Souza-Chies, T., Jubier, M.F., Lejeune, B., Siljak-Yakovlev, S., 1998. Molecular phylogeny of the genus Hypochaeris using internal transcribed spacers of nuclear rDNA: inference for chromosomal evolution. Mol. Biol. Evol. 3, 345–354. Chatti, K., Salhi-Hannachi, A., Mars, M., Marrakchi, M., Trifi, M., 2004a. Analyse de la diversité génétique de cultivars tunisiens de figuier (Ficus carica L.) à l’aide de caractères morphologiques. Fruits 59 (1), 49–61. Chatti, K., Salhi-Hannachi, A., Mars, M., Marrakchi, M., Trifi, M., 2004b. Genetic diversity and phylogenic relationships in Tunisian fig (Ficus carica L.) cultivars mediated by RAPD. Biologia Tunisie 1 (2), 1–4. Chatti, K., Saddoud, O., Salhi-Hannachi, A., Mars, M., Marrakchi, M., Trifi, M., 2007. Inferring of genetic diversity and relationships in a Tunisian fig (Ficus carica L.) germplasm collection by random amplified microsatellite polymorphisms. J. Int. Plant Biol. 49 (3), 386–391. Chennaoui, H., Marghali, M., Marrakchi, M., Trifi, N., 2007. Phylogenetic relationships in the north African genus Hedysarum as inferred from ITS sequences of nuclear ribosomal DNA. Genet. Resour. Crop Evol. 54, 389–397. Chessa, I., Neidu, G., Serra, P., 1998. Fig germplasm characterization using isosymes analysis. Acta Hortic. 480, 43–148. Dellaporta, S.L., Wood, J., Hicks, J.B., 1983. A plant DNA minipreparation: version II. Plant Mol. Biol. Rep. 1, 19–21. DGPA, 2006. Direction Générale de la Production Agricole, Ministère de l’Agriculture. Tunisie. Downie, S.R., Ramanath, S., Katz-downiie, S., 1994. A molecular phylogeny of Apiaceae subfamily Apiodeae: evidence from nuclear ribosomal DNA internal transcribed spacer sequences and chloroplast DNA restriction site polymorphisms. Am. J. Bot 81 (Suppl.), 153 (abstract). Elisiario, P.J., Neto, M.C., Cabrita, L.F., Leitao, J.M., 1998. Isozyme and RAPDs characterisation of a collection of fig (Ficus carica L.) traditional varieties. Acta Hortic. 480, 149–154. FAOSTAT, 2004. FAO statistics database on the World Wide Web. http://www.apps.fao.org (accessed June, 2004). Felsenstein, J., 1985. Confidence limits on phylogenies: an approach using the bootstrap. Evolution 39, 783–791. Hamby, R.K., Zimmer, E.A., 1992. Ribosomal RNA as a phylogenetic tool in plant systematics. In: Soltis, P.S., Doyle, J.J. (Eds.), Mol Syst Pl. Chapman and Hall, New York, pp. 50–91. Hedfi, J., Trifi, M., Salhi-Hannachi, A., Ould Mohamed Salem, A., Marrakchi, M., 2003. Morphological and isoenzymatic polymorphism in Tunisian fig (Ficus carica L.) collection. Acta Hortic. 605, 319–325. Hillis, D.M., Moritz, C., Porter, C.A., Baker, R.J., 1991. Evidence for biased gene conversion in concerted evolution of ribosomal DNA. Science 251, 308–310. Janzen, D.H., 1979. How to be a fig. Ann. Rev. Ecol. Syst. 10, 13–51. 40 G. Baraket et al. / Scientia Horticulturae 120 (2009) 34–40 Jorgensen, R.A., Cluster, P.D., 1988. Modes and tempos in the evolution of nuclear ribosomal DNA: new characters for evolutionary studies and new markers for genetic and population studies. Ann. Missouri Bot. Gard. 75, 1238–1247. Khadari, B., Lashermes, P.H., Kjellberg, F., 1995. RAPD fingerprints for identification and genetic characterization of fig (Ficus carica L.) genotypes. J. Genet. Breed. 49, 77–86. Kimura, M., 1980. A simple method for estimating evolutionary rate of base substitution through comparative studies of nucleotide sequences. J. Mol. Evol. 16, 111–120. Kislev, E.M., Hartmann, A., Bar-yosef, O., 2006. Early domesticated fig in the Jordan Valley. Science 312, 1372–1374. Kjellberg, F., Gouyon, P.H., Ibrahim, M., Raumond, M., Valdeyron, G., 1987. The stability of the symbiosis between dioecious figs and theirs pollinators: a study of Ficus carica L. and Blastophaga Psenes L. Int. J. Org. Evol. 41 (4), 693–704. Kollibara, K.P., Singh, R.J., Hymowitz, T., 1997. Phylogenetic and genomic relationships in the genus glycine wild based on sequences from the ITS region of nuclear rDNA. Genome 40, 57–68. Machado, C.A., Jousselin, E., Kjellberg, F., Compton, S.G., Herre, E.A., 2001. Phylogenetic relationships, historical biogeography and character evolution of fig pollinating wasps. Proc. R. Soc. Lond. Biol. 268, 685–694. Mars, M., Marrakchi, M., Chebli, T., 1998. Multivariate analysis of fig (Ficus carica L.) germplasm in southern Tunisia. Acta Hortic. 480, 75–81. Rhouma, A., 1996. Les ressources phylogénétiques oasiennes: le figuier (Ficus carica L.). Actes des 3èmes journées nationales sur les acquis de la recherche agronomique, vétérinaire et halieutique. Oukabli, A., Khadari, B., 2005. Caractérisation des variétés polyclonales marocaines de figuiers, Ficus carica L. Fruits 60, 47–54. Rogers, S.O., Bendich, A.J., 1987. Ribosomal RNA genes in plants: variability in copy number and in the intergenic spacer. Plant Mol. Biol. 9, 509–520. Saddoud, O., Chatti, K., Salhi-Hannachi, A., Mars, M., Rhouma, A., Marrakchi, M., Trifi, M., 2007. Genetic diversity of Tunisian figs (Ficus carica L.) as revealed by nuclear microsatellites. Hereditas 144, 149–157. Saiki, R.K., Gelfand, D.H., Stoffel, S., Scharf, S.J., Higuchi, R., Horn, G.T., Mullis, K.B., Erlich, H.A., 1988. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science 239, 487–491. Saitou, N., Nei, M., 1987. The Neighbor-joining method A new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4, 406–425. Salhi-Hannachi, A., Mars, M., Chatti, K., Marrakchi, M., Trifi, M., 2003. Specific genetic markers for Tunisian fig germplasm: evidence of morphological traits, random amplified polymorphic DNA and Inter simple sequence repeats markers. J. Genet. Breed. 5, 125–136. Salhi-Hannachi, A., Trifi, M., Zehdi, S., Mars, M., Rhouma, A., Marrakchi, M., 2004. Inter simple sequence repeat finger printings to assess genetic diversity in Tunisian fig (Ficus carica L.). Genet. Resour. Crop Evol. 51 (3), 269–275. Salhi-Hannachi, A., Chatti, K., Mars, M., Marrakchi, M., Trifi, M., 2005. Comparative analysis of genetic diversity in two collections figs cultivars based on random amplified polymorphic DNA and Inter Simple Sequence repeats fingerprints. Genet. Resour. Crop Evol. 52 (5), 563–573. Salhi-Hannachi, A., Chatti, K., Saddoud, O., Mars, M., Rhouma, A., Marrakchi, M., Trifi, M., 2006. Genetic diversity of different Tunisian fig (Ficus carica L.) collections revealed by RAPD fingerprints. Hereditas 143, 15–22. Sambrook, J., Frithsch, E.F., Maniatis, T., 1989. Molecular Cloning: A Laboratory Manual, 2nd ed. Cold spring Harbor Laboratory, Cold Spring Harbor, New York. SAS, 1990. SAS User’s Guide: SAS/STAT, SAS BASIC. Version 6.07, 4th ed. SAS Inc., Box 8000 cary, NC 27512-8000. Susanna, A., Jacas, N.G., Soltis, D.E., Soltis, Ps., 1995. Phylogenetic relationships in tribe Cardueae (Asteraceae) based on ITS sequences. Am. J. Bot. 82, 1056– 1086. Torres, R.A., Ganal, M., Hemleben, V., 1990. GC balance in the internal transcribed spacers ITS1 and ITS2 of nuclear ribosomal RNA genes. J. Mol. Evol. 30, 170–181. Valizadeh, M., Rivals, P., Valdeyron, G., 1977. Utilisation du polymorphisme protéique des variétés de figuier (Ficus carica L.) Extrait du procès verbal de la séance du 1er juin 1977. Académie d’Agriculture de France. Van der Sande, C.A., Kwa, M., Van Nues, R.W., Van Heerikhuizen, H., Raué, H.A., Planta, R.J., 1992. Functional analysis of internal transcribed spacer 2 of Saccharomyces cerevisiae ribosomal DNA. J. Mol. Biol. 223, 899–910. Watson, L., Dallwitz, M.J., 2004. The Families of Flowering Plants: Description, Illustration, Identification, and Information Retrieval. http://biodiversity.uno.edu/delta/ (accessed June 2004). Weibes, J.T., 1979. Co-evolution of figs and their insect pollinators. Ann. Rev. Ecol. Syst. 10, 1–12. Weiblen, G.D., 2000. Phylogenetic relationships of functionally diocious Ficus (Moraceae) based on ribosomal DNA sequences and morphology. Am. J. Bot. 87 (9), 1342–1357. Zimmer, E.A., Martin, S.L., Beverley, S.M., Kan, Y.W., Wilson, A.C., 1980. Rapid duplication and loss of genes coding for the alpha chains of hemoglobine. Proc. Natl. Acad. Sci. U.S.A. 77, 2158–2162.