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Molecular Characterization of Moroccan Fig Germplasm Using Intersimple Sequence Repeat and Simple Sequence Repeat Markers To Establish A Reference Collection

A study was conducted to identify genotypes present in a Moroccan fig germplasm collection and provide the first database for a reference collection in northern Morocco. In total, 75 fig samples were analyzed using 8 intersimple sequence repeat primers and 6 simple sequence repeat loci. From these samples, we identified 72 fig genotypes. In genetically heterogeneous cultivars, genotypes under the same denomination were distinguished by both molecular markers and pomological traits. Molecular analysis was used to classify the germplasm into 46 well-defined cultivars and 6 caprifig trees. The remaining genotypes were not clearly identified due to three cases of mislabeling and four cases of homonymy. No evidence was found for the occurrence of geographically widespread genotypes. Common fig, Ficus carica L., is one of the most ancient cultivated Mediterranean fruit (Zohary and Hopf, 2000). In northern Morocco, fig cultivars are highly diversified and provide a large array of genotypes (Oukabli et al., 2003). However, due to numerous cases of synonymy (several denominations for the same genotype) and homonymy (several genotypes are highly polymorphic and are not readily influenced by environmental conditions (Geuna et al., 2003; Hokanson et al., 2001; Oraguzie et al., 2001; see also review by Wünsch and Hormaza, 2002). RAPD markers have been previously used for cultivar characterization in fig (De Masi et al., 2003; Elisiario et al., 1998; Khadari et al., 1995), but because of the use of arbitrary short sequences as primers and the relatively low annealing temperature, these markers cannot be exchanged among laboratories according to standardized protocols (Jones et al., 1997). To overcome this limitation, we previously compared data obtained by RAPD, intersimple sequence repeat (ISSR), and SSR analyses of 30 fig cultivars and showed that SSR and ISSR markers are complementary tools for a reliable fig characterization (Khadari et al., 2003c). In the present study, ISSR and SSR markers were used to characterize local accessions in a Moroccan fig collection and provide a molecular database for fig breeding. Material and Methods Plant material. A Moroccan fig collection located at the Aïn Taoujdate experimental station (INRA Meknès, Morocco) was used for study. The collection contained 72 local accessions and consisted of 54 cultivars and 8 caprifig (male) trees collected from northern Morocco and Algeria (Table 1). We defined an accession as one or several fig trees under the same denomination and registration number. In some cases, several accessions were classified under the same denomination (e.g., see ‘Bioudi’ denomination in Table 1). Molecular characterization was performed on one tree per accession except for three accessions, ‘Abiarous’ (65-3015), ‘Bouankirh’ (98-2397) and ‘El Khal’ (84-2283), in which two trees were analysed because distinct pomological traits were noted among trees within these accessions. Molecular analyses. DNA was extracted from 100 mg of frozen leaf material using the Dneasy Plant Mini Kit (Qiagen) following manufacturer recommendations with the exception that 1% polyvinylpyrrolidone (PVP 40 000) was added to the AP1 buffer. For ISSR analysis, four previously selected primers (IMA5, IMA8, IMA9, and IMA12; Table 2; Khadari et al., 2003c) and four new primers were used (IMA303, IMA834, UBC818, and UBC841; Table2). Following a test for polymorphism and for consistent and reproducible DNA amplification on five fig cultivars, these new primers were selected from seven primers obtained from MGW. Reproducibility in ISSR patterns was verified by three different DNA amplifications from a set of 10 accessions. The PCR amplification was performed in 10 mM Tris-HCl (pH 9.0), 50 mM KCl, 1.5 mM MgCl 2, 0.1% TritonX100, 0.02% gelatin, with 0.2 mM of each dNTP, 10 pmol of primer, 2 units of Taq DNA polymerase (Appligene-Oncor), and 50 ng of DNA in a final volume of 25 μL. Amplifications were carried out in a 96-well thermocycler (model PTC100;MJ Research) using the following conditions: initial denaturation at 94 °C for 5 under the same denomination), pomological characterization is insufficient to establish reference genotypes for fig breeding programs in the region. Molecular markers are needed to accurately identify fig genotypes and define a complete reference collection. Isozymes were the first genetic markers used in genotype fingerprinting and have been applied in several fruit species including fig (Chessa et al., 1998; Elisiario et al., 1998; Uzun et al., 2003; Valizadeh et al., 1977). However, their usefulness has been limited due to the small number of isozyme systems available, the low level of polymorphism obtained, and the influence of environmental factors. The emergence of new polymerase chain reaction (PCR)-based molecular markers, such as randomly amplified polymorphic DNA (RAPD), amplified fragment-length polymorphisms (AFLPs), and simple sequence repeats (SSR), has created the opportunity for fine-scale genetic characterization of germplasm collections because they HORTSCIENCE 40(1):29–32. 2004. Received for publication 5 Aug. 2003. Accepted for publication 17 Mar. 2004. We thank I. Hochu and S. Santoni for technical support, M. Lahlou for tending the fig collection, and F. Boillot for his encouragement. We also thank F. Dosba and I.J. Hormaza for comments on the manuscript. The work was supported by funding from the BRG (Bureau des Ressources Génétiques, Paris) and by French-Moroccan cooperation program (PRAD 00-12). To whom correspondence should be addressed. Current address: UMR 1098 Biologie du Développement des Espèces Pérennes Cultivées (BEPC), Bat. 33, INRA, 2, place Viala, 34 060 Montpellier cedex 1, France; e-mail khadari@ensam.inra.fr.

are highly polymorphic and are not readily influenced by environmental conditions (Geuna et al., 2003;Hokanson et al., 2001;Oraguzie et al., 2001; see also review by Wünsch and Hormaza, 2002). RAPD markers have been previously used for cultivar characterization in fig (De Masi et al., 2003;Elisiario et al., 1998;Khadari et al., 1995), but because of the use of arbitrary short sequences as primers and the relatively low annealing temperature, these markers cannot be exchanged among laboratories according to standardized protocols (Jones et al., 1997). To overcome this limitation, we previously compared data obtained by RAPD, intersimple sequence repeat (ISSR), and SSR analyses of 30 fig cultivars and showed that SSR and ISSR markers are complementary tools for a reliable fig characterization (Khadari et al., 2003c).
In the present study, ISSR and SSR markers were used to characterize local accessions in a  (Table 1). We defined an accession as one or several fig trees under the same denomination and registration number. In some cases, several accessions were classified under the same denomination (e.g., see 'Bioudi' denomination in Table 1). Molecular characterization was performed on one tree per accession except for three accessions, 'Abiarous' (65-3015), 'Bouankirh' (98-2397) and 'El Khal' (84-2283), in which two trees were analysed because distinct pomological traits were noted among trees within these accessions.
Molecular analyses. DNA was extracted from 100 mg of frozen leaf material using the Dneasy Plant Mini Kit (Qiagen) following manufacturer recommendations with the exception that 1% polyvinylpyrrolidone (PVP 40 000) was added to the AP1 buffer.
For ISSR analysis, four previously selected primers (IMA5, IMA8, IMA9, and IMA12; Table 2; Khadari et al., 2003c) and four new primers were used (IMA303, IMA834, UBC818, and UBC841; Table2). Following a test for polymorphism and for consistent and reproducible DNA amplification on five fig cultivars, these new primers were selected from seven primers obtained from MGW. Reproducibility in ISSR patterns was verified by three different DNA amplifications from a set of 10 accessions. The PCR amplification was performed in 10 mM Tris-HCl (pH 9.0), 50 mM KCl, 1.5 mM MgCl 2, 0.1% TritonX100, 0.02% gelatin, with 0.2 mM of each dNTP, 10 pmol of primer, 2 units of Taq DNA polymerase (Appligene-Oncor), and 50 ng of DNA in a final volume of 25 µL. Amplifications were carried out in a 96-well thermocycler (model PTC100;MJ Research) using the following conditions: initial denaturation at 94 °C for 5 under the same denomination), pomological characterization is insufficient to establish reference genotypes for fig breeding programs in the region. Molecular markers are needed to accurately identify fig genotypes and define a complete reference collection.
Isozymes were the first genetic markers used in genotype fingerprinting and have been applied in several fruit species including fig (Chessa et al., 1998;Elisiario et al., 1998;Uzun et al., 2003;Valizadeh et al., 1977). However, their usefulness has been limited due to the small number of isozyme systems available, the low level of polymorphism obtained, and the influence of environmental factors. The emergence of new polymerase chain reaction (PCR)-based molecular markers, such as randomly amplified polymorphic DNA (RAPD), amplified fragment-length polymorphisms (AFLPs), and simple sequence repeats (SSR), has created the opportunity for fine-scale genetic characterization of germplasm collections because they min, followed by 35 cycles (94 °C 30 s, 50 to 56 °C 1 min, 72 °C 1 min) and a fi nal extension at 72 °C for 7 min. The amplifi cation products were electrophoresed in 2% agarose gels at 120 volts followed by staining with ethidium bromide and photographed on Polaroïd 667 fi lm under ultra-violet light.
For SSR analysis, six loci out of eight developed by Khadari et al. (2001) were used (Table  2). Analysis was performed following a protocol described previously (Khadari et al., 2001).
Data analysis. For ISSR data, polymorphic bands were scored as present (1) or absent (0) based on products consistently amplifi ed for each primer. We assumed each band position corresponded to a locus with two alleles. For SSR analysis, one or two bands were scored for each sample corresponding to homozygotic and heterozygotic individuals, respectively. Each polymorphic band was named by the primer code followed by the size of the amplifi ed fragment in base pairs. ISSR and SSR markers were scored in a binary data matrix.
For each SSR loci, expected heterozygosity values were computed under the Hardy-Weinberg hypothesis according to the no-bias model (Nei, 1978) using Genetics 4.0 software (Belkhir et al., 1999). Signifi cance of heterozygote defi ciency was assessed using exact tests and Genepop 3.1b software (Raymond and Rousset, 1995). Probability of obtaining a given genotype was calculated under the hypothesis of independence between markers as the product of the corresponding allele frequency for each locus of the pattern i: Pi = ∏ fj. Discriminating power of each ISSR primer and each SSR locus was calculated as follows: Dj = ∑p i (Np i -1) /(N -1) where p i was the frequency of the i-th molecular pattern revealed by primer or locus j, and N was the number of genotypes (Tessier et al., 1999).
Relationships between fi g genotypes were based on a binary matrix and estimated according to the Jaccard similarity coeffi cient, taking the presence of bands into account only. A phenogram was drawn using the UPGMA algorithm. Phenetic analysis was performed using a program developed by J. Brzustowski (http://www.biology.ualberta.ca/jbrzusto/cluster.ph).

Results
Polymorphism and fi g genotyping. In total, 38 alleles were obtained using 6 SSR loci. The number of alleles per locus varied from 3 (loci MFC4 and MFC7) to 14 (MFC6 locus) with an average of 6.3 alleles per locus (Table 2). Observed heterozygosity ranged from 0.175 for the MFC7 locus to 0.900 for the MFC6 locus with an average for all loci of 0.557. Compared to the expected heterozygosity value, a signifi cant heterozygote defi ciency was observed for MFC1, MFC2, MFC3 and MFC7 loci, probably due to a selection effect that would have occurred within a limited gene pool ( Table 2). The 8 ISSR primers selected for polymorphism and for clear bands revealed a total of 37 markers ranging from 2 (primer IMA303) to 8 (primer IMA5), with a mean value of 4.6 markers per primer ( Table 2). The highest frequencies were obtained for the UBC841-350 ISSR marker (97%) and the MFC4-219 SSR allele (89%). The lowest frequencies (about 1%) were observed for 7 alleles belonging to the MFC2, MFC3, and MFC6 SSR loci and for the IMA303-900 ISSR marker.
Analysis of 75 fi g samples using 75 polymorphic bands (38 SSR alleles and 37 ISSR bands) revealed 72 different molecular patterns. Of the genotype pairs, 98% were differentiated by 10 to 33 polymorphic bands; among 2,556 pairwise comparisons, only 5, 6, and 7 molecular profi le pairs were distinguished by 1, 2, and 3 polymorphic bands, respectively (Fig. 1). The distribution of genetic differences in pairwise comparisons showed a bimodal distribution with a main mode centered at about 21 to 22 differences and a minor mode centered at about 2 to 3 differences (Fig. 1). Under the hypothesis of non-linkage between markers, the probability of obtaining a given molecular pattern was very low and ranged from 1.03 × 10 -12 for 'Filalia' (34-2211) to 2.87 × 10 -5 for 'Amzin' (caprifi g 18-6). The use of ISSR markers alone enabled us to identify 72 molecular profi les, although the probability of obtaining a given pattern was much higher than when using all markers (ranging from 3.90 × 10 -7 to 0.060). In contrast, 18 genotype pairs were not distinguished using SSR markers alone.
The discriminating power of each ISSR primer or SSR locus used in this study varied from 0.944 to 0.516 (Table 2). The value obtained for the ISSR primer IMA303 was close to null because the markers were detected in only three genotypes. Discriminating power variation among primers or loci was due to either the number of markers per primer or the number of alleles per locus, and also to marker frequencies. Primer IMA5 was more discriminating than the MFC6 and MFC3 loci because the frequency of IMA5 markers varied from 10% to 86% while most of the MFC6 and MFC3 alleles (14 among 22 alleles) were <6%. Seven combinations of primers and/or loci were tested according to their discriminating power in order to identify the most effi cient combination for genotype discrimination. Starting with the most discriminating, one or several primers were added for each successive combination. Combination n ° 5 (IMA5 + MFC6 + IMA9 + UBC841 + MFC3) discriminated all genotypes except two [IMA8-350 for 'Bioudi' (64-2218)/'Ournakssi' (5-2282) and IMA12-580 for 'Bousbati' (2-2880)/ 'Ournakssi' (6-2214)] with the probability of obtaining a given genotype <10 -2 .
SSR and ISSR analyses enabled us to classify the germplasm into 52 molecular profiles corresponding to 46 well-defined cultivars and 6 caprifigs (Table 1). Variety assignation was unclear in the 20 remaining due to either mislabelling or homonymy.

SSR and ISSR markers are reliable and complementary tools for fig characterization
With the exception of three pairs of acces-sions, distinct fig genotypes under the same denomination were clearly distinguished in the present study. For example, four of five accessions with 'Bioudi' denomination were differentiated by 12 to 28 markers and belonged to different genetic clusters. These four accessions were also identified as different genotypes using pomological descriptors such as skin color, fruit and fruit stalk shape, ostiole size, and skin cracks (Oukabli et al., 2003). In contrast, no pomological differences were observed among the following three pairs of accessions distinguished by only 1 to 2 ISSR markers: 'Bioudi' (64-2218)/ 'Bioudi' (1-2222), 'Ournakssi' (6-2214)/ 'Ournakssi' (3-2280), and 'Rhoudane' (24-2223)/ 'Rhoudane' (25-2227) ( Table 3). Validity of discriminating ISSR markers, which were present in several other fig genotypes, was verified by repeating the amplifications and molecular analysis three times. These results strongly suggest that each of these pairs corresponds to a single original clone and that 1 or 2 distinct ISSR bands correspond to somatic mutations, which may indicate ancient cultivars. Indeed, the minor mode observed in the distribution of genetic differences in pair-wise comparisons would also indicate somatic variation within clones (Fig. 1). This explanation may also apply to the 11 accession pairs distinguished by only 1 to 3 ISSR bands, which led to misclassification of 12 out of 46 previously defined cultivars (Tables 1 and 3). However, due to distinctive pomological characteristics (Oukabli et al., 2003), characterization must be validated with more SSR loci in order to verify that each pair of accessions originate from a single clone. The original sampling scheme of the Moroccan germplasm appears to have followed a policy of a single introduction for highly localized, apparently homogeneous, cultivar denominations, as well as several introductions for more widespread denominations. Our results show that 1) large diversity in cultivar names corresponds to large genetic diversity and 2) variety names that have broader diffusion correspond to several unrelated and pomologically-distinct genotypes. Thus, it appears that Moroccan cultivars are generally highly local.
To choose a genotype as a reference genotype, we propose the following rule for monoclonal cultivars: when several fig trees grouped under the same denomination have similar morphological characteristics, originate from different collections, nurseries, and orchards, and display the same molecular pattern, they shsould be considered as a reference genotype for the cultivar. This rule was proposed by Khadari et al. (2003a) to establish reference genotypes for olive germplasm in France. Conversely, choosing one or several genotypes as reference for a genetically heterogeneous cultivar should also take into account discriminative pomological traits, as well as the relative importance of cultivation among the clones.
The present study provides the first molecular database for