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Genetic Resources and Crop Evolution 51: 269–275, 2004.  2004 Kluwer Academic Publishers. Printed in the Netherlands.

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Inter-Simple Sequence Repeat fingerprints to assess genetic diversity in Tunisian fig (Ficus carica L.) germplasm ` 1 , Mars Messaoud 1 , Salhi-Hannachi Amel 1, *, Trifi Mokhtar 1 , Zehdi Salwa 1 , Hedfi Jihene 1 1 Rhouma Abdelmajid and Marrakchi Mohamed 1

´ ´ ´ Faculte´ des Sciences de Tunis, Laboratoire de Genetique Moleculaire , Immunologie et Biotechnologie, Campus Universitaire El Manar, 2092, Tunisie; 2 ESHE Chatt Mariem, Tunisie; * Author for correspondence ( phone: 1216 71 87 26 00 fax: 1216 71 88 54 80; e-mail: hsalhi@ evdoramail.com) Received 28 January 2002; accepted in revised form 18 October 2002

Key words: Ficus carica L., Genetic diversity, Germplasm collection, ISSR

Abstract The genetic diversity of 18 Tunisian fig cultivars was investigated at the DNA level using the Inter Simple Sequence Repeat (ISSR) associated with the Polymerase Chain Reaction (PCR). Using a set of primers, the most informative ones were selected that were characterized by an important Resolving power value of 29.6. A total of 47 discernible fragments were scored from samples, with a mean of 11.7 fragments per primer. The 90.4% of sample that were polymorphic were scored as molecular markers to examine the Tunisian fig germplasm polymorphism at DNA level. A large genetic diversity as related to ISSR patterns was found within the local Tunisian fig germplasm. An UPGMA dendrogram exhibits the unstructured variability in this crop. Moreover, the principal component analysis shows that the observed diversity was typically continuous. Our data provide a large number of ISSR markers that are useful in the fingerprinting of Ficus carica L. cultivars, and in the understanding of the genetic relationships among these accessions.

Introduction The genus Ficus (Moraceae) includes more than 750 species occurring throughout tropical and sub-tropical regions all over the world (Berg 1989). Each species is dependent on a specific mutualistic wasp species belonging to the Agonideae family (Hymenoptera, Chlacidoideae) for pollination (Wiebes 1963). The common fig Ficus carica L. (2n 5 2x 5 26) is a gynodioecious species (Valdeyron and Lloyd 1979). It is functionally dioecious, with the separation of sexual function resulting from the interaction of pollinator wasps (Blastophaga psenes L.) with florets in two separate fig plants: caprifig and edible fig (Kjellberg et al. 1987). Condit (1995) classified more than 600 fig varieties clonally propagated and widely spread throughout a wide range of soils and bioclimatic stages. Ficus carica L. occurring in Mediterranean basin (Kjellberg et al. 1987) is widely cultivated in Tunisia.

Fig cultivars are particularly suitable for fruit production (figs are consumed fresh or dried) but also for industry (jam and spirit beverage production). Moreover, some by-products are used for animal feeding (Mars et al. 1998). Tunisian fig germplasm has great phenotypic diversity. Unfortunately, it is threatened by severe genetic erosion due to biotic and abiotic pressures (urbanization, intensive cultivation extension, absence of caprification, fertilization and phytosanitary norms). In addition, traditional fig cultivation areas have significantly decreased and genetic variability was reduced due to the disappearance of many cultivars selected in the past (Mars et al. 1998). In order to preserve and improve local fig germplasm a research program was initiated that included prospection, collection and evaluation of genetic diversity. Areas in central and south Tunisia were the first regions to be explored (Mars et al. 1994; Rhouma 1996). More than 50 cultivars had already been collected and three collections were established.

270 These consisted of common type and caprifigs. In addition, it was assumed that the actual number of Tunisian fig cultivars is difficult to estimate since problems of synonymy and homonymy have impeded varietal identification in the established collections. Hence, it is imperative to develop a relevant method suitable for cultivar characterization and polymorphism evaluation. On the other hand, few genetic studies have been carried out on cultivated fig to assess either the genetic variation or the varietal classification. Fig genetic resources have been traditionally characterized using morphological and agronomic traits (Ben Salah et al. 1995; Mars et al. 1994; Mars 2001). Other studies have described the use of isozymes as reliable biochemical markers for fig cultivar identification (Hedfi et al. 2001). It has been assumed that these reports are reliable with regard to cultivar classification. However, lack of apparent diversity may be due to the decreased number of cultivars and / or because morphological parameters are highly influenced by either the environmental conditions or the development of the tree stage. Moreover, genetic variability and homonymy resolution among fig accessions were successfully performed by the help of isozyme markers (Valizadeh et al. 1977; Chessa et al. 1998; Elisario et al. 1998). Therefore, a search for other markers is required to obtain a deeper understanding of the genetic organization in this germplasm. More recently, molecular technologies such as RAPD and microsatellites have been applied to fig germplasm and have permitted the study of the genetic relationships among cultivars (Khadari et al. 1995; Weiblen 2000; De Masi et al. 2001; Khadari et al. 2001). To date, such markers have not been used to study the Tunisian fig’s genetic diversity in terms of molecular polymorphism. Hence, we became interested in the exploration of the Tunisian fig diversity at DNA level by the help of molecular procedures. Among these, Inter Simple Sequence Repeat (ISSR)based PCR is highly informative. This technology has been used to DNA fingerprint a wide range of crops (Zietkiewicz et al. 1994; Gilbert et al. 1999; Reddy et al. 1999; Khadari et al. 2001; Lai et al. 2001; Ajibade et al. 2000; Joshi et al. 2000; Zehdi et al. 2002). In the present study we report the feasibility of the ISSR-based PCR (Polymerase Chain Reaction) as an attractive approach providing molecular markers reliable in the exploration of the genetic diversity among Tunisian fig varieties.

Material and methods Plant material This study was performed using a set of 18 local Tunisian fig varieties listed in Table 1. The plant material consisted of young leaves that were sampled from adult trees at the fig germplasm collections. ´ These are maintained at the IRA (Institut des Regions Arides, Medenine) and the CRP (Centre de Recher´ ches Phoenicicoles. Degache) stations located in the south of Tunisia. Genomic DNA isolation The DNA was extracted from fresh leaves according to the method of Dellaporta et al. (1983). The resultant DNAs’ integrity and quantity were estimated spectrophotometrically as well as visually by ethidium bromide staining on 0.8 % agarose gels (Sambrook et al. 1989). Primers and PCR-ISSR assays The detection of cultivar polymorphism has been performed using a total of 13 primers. These were based either on di-or-multi-nucleotide repeats that were complementary to microsatellites (Table 2). The di-nucleotide repeats were anchored at 3’ ends by a single degenerant nucleotide. DNA amplification was performed in 25 mL of final volume reaction mixture containing 2.5 mL of enzyme buffer (103), 50 mM of dNTPs, 60 pg of primer, 2 U of Taq DNA polymerase (QBIOgene, France) and 20 ng of DNA. Amplifications were carried out in a Crocodile III thermocycler (QBIOgene, France) according to the following conditions: a 5 min initial denaturation cycle at 94 8C is already programmed before entering 35 cycles each one including a denaturation step of 30 s at 94 8C, a second step of 1 min 30 s for annealing at the primer-specific Tm and an extension step of 1 min 30 s at 72 8C with a final extension for 5 min at 72 8C at the end of the last cycle. The standardization between enzyme batches was ensured by preparing a master mix and the use of controls (reaction mixture without any DNA or any enzyme). Each experiment was performed at least twice to obtain reproducible patterns. The amplification products were separated on a 1.4% agarose gel electrophoresis in 0.5 3 TBE buffer

271 Table 1. Tunisian Ficus carica L. accessions with their localities of origin Collection site

´ IRA Medenine

CRP Degache

Accession name

Label

Botanical variety

Geographical origin

Bither Abiadh Dchiche Assal Dhokkar Zarzis Hammouri Kahli Makhbech Rogaby Sawoudi Tayouri Asfar Widlani Zaghoubi Zidi

BA DA DZ HR KH MK RB SD TA WD ZH ZD

Common type ’’ Caprifig Common type ’’ ’’ ’’ ’’ ’’ ’’ ’’ ’’

Tataouine Ghadabna Zarzis Beni Khedache Ennfidha Zarzis Beni Khedache Beni Khedache Douiret Beni Khedache Beni Khedache Ghadabna

Dhokkar Grichy Hamri Khalt Khzami Tounsi

DK GR HM KL KZ TS

Caprifig Common type ’’ ’’ ’’ ’’

Tozeur Tozeur Tozeur Tozeur Tozeur Tozeur

for 2 h 30 min at 50 mA and detected by an ethidium bromide staining method (Sambrook et al. 1998).

Data analysis The ability of the most informative primers to differentiate between accessions was assessed by calculating their Resolving power (Rp) (Prevost and Wilkinson 1999) that has been described to correlate

Table 2. Summary of DNA patterns amplified by ISSR primers from Tunisian fig (Ficus carica L.) accessions. Tm 5 melting temperature in 8C; S 5 smear; MB 5 multiband pattern; P 5 polymorphic; FB 5 few band pattern; * 5 weak band pattern. Primer code

Primer sequence

Tm 8C

Patterns

P01 P02 P03 P04 P05 P06 P12 P13 P07 P08 P09 P10 P11

(AG) 10 G (AG) 10 T (AG) 10 C (CT) 10 A (CT) 10 G (CT) 10 T (TC) 10 A (TC) 10 G (AGG) 6 (TGGA) 5 (ACTG) 4 (GACA) 4 (GACAC) 4

60 57 60 57 60 57 55 60 55 55 45 45 55

MBS MBP MBP BS* MBS MBS* MB* MBS* FBS FBP* FBP* MBP BS

strongly with the ability to distinguish between accessions according to the Gilbert et al. (1999) Rp 5 SIb, where Ib 5 12 (2 3 uO.5 2 pu), p is the proportion of accessions containing the I band. Banding profiles generated by PCR were compiled into a data binary matrix on the basis of the presence (1) or absence (0) of the selected band. This matrix was used to calculate genetic distance according to the Nei and Li’s formula (1979). The generated distance matrix was computed to construct an UPGMA phenogram and to infer genetic relationships using the Phylip software version 3.572 C (Felsenstein 1993). In addition, the genetic diversity was measured by the percentage of polymorphic bands (PPB) calculated by dividing the number of polymorphic bands by the total number of bands surveyed. The population structure and the variability among clones within and between regions were well described after a principal component analysis (PCA) using the Statistical Analysis System Version 6.07 (SAS 1990).

Results

Primers resolving power The 13 primers were screened for their ability to

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Figure 1. ISSR PCR fingerprints of 18 accessions of Ficus carica L. using the 3’-anchored primer (AG) 10 T. M: Standard molecular size (1kp ladder); C: control; lanes 1–18: DNA templates.

generate ISSR polymorphic DNA bands using the accessions’ total cellular DNAs. The resultant multiple band patterns are summarized in Table 2. The data exhibit that the di-nucleotide repeats (i.e.: (AG) 10 T and (AG) 10 C) are more informative than tetra-nucleotide and hexa-nucleotide ones. The latter produced only a few bands often accompanied with background smears. Figure 1 illustrates typical examples of the amplified ISSR banding patterns with DNA stretches ranging from 200 to 2000 bp. In this case, (AG) 10 T oligonucleotide was applied using all the sampled DNAs. On the other hand, we assume that among the 13 tested primers only (AG) 10 T, (AG) 10 C, (ACTG) 4 and (GACA) 4 examined the diversity in the Tunisian fig germplasm on the basis of their resolving power (Rp). The Rp values with a collective rate of 29.4 varied from 4.3 for the (ACTG) 4 primer to 11.4 for the (AG) 10 C primer (Table 3). Similar Rp values have been reported in a lupin germplasm collection and in potato cultivars (Gilbert et al. 1999; Prevost and Wilkinson 1999). Hence, since the (AG) 10 T and (AG) 10 C primers presented the highest Rp rates, we assume that these oligonucleotides mainly contribute in the accession characterization and to examine the genetic diversity in this crop. Genetic diversity and cultivars relationships A total of 52 ISSR bands were amplified using the

four available primers with 18 fig cultivars. Among these bands 47 (90.4 %) are unambiguously reproducible and polymorphic. Depending on the primer, 7 to 18 polymorphic bands were generated with a mean of 11.7. The percentage of polymorphic bands (PPB) was estimated separately for the cultivars of the two collections. This was 84.6 % in Medenine’s varieties and only 67.3 % for Degache’s ones, suggesting that the Medenine collection is characterized by a larger genetic diversity at the DNA level. Such a great variability range may due to small insertion / deletion at the amplified genomic sites located between the microsatellites anchored or unanchored regions (Reddy et al. 1999). On the other hand, the data binary matrix was computed with the genedis program according to Nei and Li’s formula in order to estimate the genetic distances among the accessions. The obtained matrix exhibits genetic distances ranging from 0.16 to 0.95 with a mean of 0.54. These distances express high genetic diversity within local fig germplasm at the DNA level. The smallest genetic distance of 0.16 is observed between Grichy (GR) and Rogaby (RB) cultivars, suggesting that these varieties are characterized by a maximum of similarities. However, Khalt (KL) appears to be the most dissimilar from Tayouri Asfar (TA) and Mkhabech (MK) accessions, since they presented the highest genetic distance of 0.95. All the remaining ones present intermediate genetic distances.

Table 3. Characteristics of ISSR banding profiles produced in Tunisian figs. ISSR primers Code P02 P03 P09 P10 Total

Sequence (AG) 10 T (AG) 10 C (ACTG) 4 (GACA) 4

ISSR-PCR bands Scored

Polymorphic

Resolving power (Rp)

18 15 8 11 52

18 13 7 9 47

11.41 8.40 4.30 5.54 29.65

273 In order to draw the relationships among accessions, the genetic distance matrix was computed with the Neighbour program. Figure 2 shows the derived UPGMA phenogram that exhibits two main clustering groups. The first one is composed of six cultivars of the Medenine collection (i.e. Dchiche Assal (DA), Dhokkar Zarzis (DZ), Zaghoubi (ZH), Kahlt (KH), Tayouri Asfar (TA), Makhbech (MK) varieties) but originated from different regions. The second cluster is divided in two sub-clusters and includes all the remaining varieties that originated either from the Medenine or the Degache collection. In addition, the caprifig cultivars are unlikely clustered with the common fig varieties. These results suggest a common genetic basis in the implied varieties. This consideration is strongly supported by, firstly, the closely groupings of Degache’s and Medenine’s accessions and, secondly by the great similarities registered between Widlani (WD), Hamouri (HR) and Bither Abiadh (BA) clones at the DNA level in spite of their distinctive morphology. On the other hand, this phenogram typology was similar to those obtained on the basis of morphometric and / or analytic parameters, particularly those related to the fruit characteristics. This is well exemplified by Makhbech (MK) and Tayouri Asfar (TA) that are characterized by a similar fruit colour (Mars et al. 1998), or by Khzami (KZ) and Tounsi (TS) Degache’s cultivars that are the most morphologically similar with regard to vegetative development (second internode length and second internode / diameter) (Hedfi et al. 2001). However, the dissimilarity registered between Hammouri (HR) and Hamri (HM) cultivars may suggest a case of homonymy in the nomenclature based on the red fruit skin colour. Furthermore, the PCA confirmed information derived from the UPGMA clustering since varieties are similarly regrouped to axis 1 of this multivariate analysis. Indeed, the distribution of the variability showed in the plan 1–2 of the PCA was typical continuous (Figure 3). The following hypothesis can be made to explain the unstructured genetic diversity that characterizes Tunisian fig genotypes: each variety maintains its independent status because an important gene flow has occurred in the natural populations from which cultivars are originated. Consequently, the agreement between both analyses strongly suggests that the ISSR technology is a powerful and attractive approach that is informative about the genetic diversity at the fig germplasm’s DNA level. In addition, our results concur with those describing the genetic di-

Figure 2. UPGMA phenogram of 18 Tunisian fig accessions constructed from Nei and Li genetic distance estimated from 47 ISSR markers. See Table 1 for cultivars label. Medenine’s and Degache’s accessions are identified by (♦) and (d) respectively

versity either in French or Sardinian fig cultivars using isoenzymes (Chessa et al. 1998) and RAPD markers (Khadari et al. 1995).

Conclusion and discussion Results showed that cultivar clustering was not correlated with geographic origin, suggesting either a common genetic basis among the cultivars or gene flow that has occurred between fig groves. Moreover, the convergence in clustering derived from the morphological traits and ISSR markers analyses suggest that all fig ecotypes are interrelated in spite of their phenotypic divergence. A common and narrow genetic basis is strongly supported by selection applied

274

Figure 3. Plot of principal component analysis of 18 Tunisian fig accessions

by farmers on the basis of either the fruit quality or the accessions adaptation to local conditions. Consequently, only a small part of the fig genome that encodes interesting agronomic parameters is affected by this selection in the natural populations from which clones originated. This study provides evidence that the ISSR procedure is an informative and suitable approach to the examination of the molecular polymorphism and the phylogenic relationships in the fig germplasm. Work is currently in progress to enlarge the number of markers by the use of other molecular technologies in order to have a deeper insight into the molecular polymorphisms and to establish a varietal identification key in this crop.

Acknowledgements This research was supported by grants from the ` de l’Enseignement Superieur ´ ‘‘Ministere de la Recherche Scientifique et de la Technologie’’

References Ajibade S.R., Weeden N.F. and Chite S.M. 2000. Inter simple sequence repeat analysis of genetic relationships in the genus Vigna. Euphytica 111: 47–55. Ben Salah M., Ancilotti M. and Loumeren M. 1995. Etude ´ ´ de figuier (Ficus carica L.) typiques pomologique de six varietes de Beni Khedache. Plant Genet. Res. Newsletter 104: 16–20. Berg C.C. 1989. Classification and distribution of Ficus. Experientia 45: 605–611. Chessa I., Neiddu G. and Serra P. 1998. Fig germplasm characterization using isozyme analysis. Acta Hort. 480: 143–148. Condit I.J. 1955. Fig varieties. A monograph. Hilgardia, Berkeley, 23: 323–538. Dellaporta S.L., Wood J. and Hicks J.B. 1983. A plant DNA preparation. Version II. Plant Mol. Biol. Reporter 4: 19–21. De Masi L., Cipollaros M., Bernardo G., Galderisi U., Galano G., Grassi G. et al. 2001. Clonal selection and molecular characterization by RAPD analysis of the fig (Ficus carica L.) ‘‘dottato’’ and ‘‘bianco del cilento’’ cultivars in Italy. Second international symposium on fig. Caceres (Spain), 23 p. Elisario P.J., Neto M.C., Cabrita L.F. and Leitao J.M. 1998. Isozyme and RAPDs characterization of a collection of fig (Ficus carica L.) traditional varieties. Acta Hort. 480: 149–154. Felsenstein, J., 1993. Phylogenetic Interference Package (PHYLIP). Version 3.5. Departement of Genetics, University of Washington, Seattle, W. A. 98195, USA

275 Gilbert J.E., Lewis R.V., Wilkinson M.J. and Caligari P.D.S. 1999. Devoloping an appropriate strategy to assess genetic variability in plant germplasm collections. Theor. Appl. Genet. 98: 1125– 1131. Hedfi J., Trifi M., Hannachi Salhi A., Ould Mohamed Salem A., Rhouma A. and Marrakchi M. 2001. Morphological and isoenzymatic polymorphism in Tunisian fig collection. Second international symposium on fig. Caceres (Spain), p. 20. Joshi S.P., Gupta V.S., Aggarwal R.K., Ranjkar P.K. and Brar D.S. 2000. Genetic diversity and phylogenetic relationship as revealed by inter simple sequence repeat (ISSR) polymorphism in the genus Oryza. Theor. Appl. Genet. 100: 1311–1320. Khadari B., Lashermes Ph. and Kjellberg F. 1995. RAPD fingerprints for identification and genetic characterization of fig (Ficus carica L.) genotypes. J. Genet. & Breed 49: 77–86. Khadari B., Hochu I., Santoni S., Oukabli A., Ater M., Roger J.P. et al. 2001. Which molecular markers are best suited to identify fig cultivars. A comparison of RAPD, ISSR and microsatellite markers. Second international symposium on fig. Caceres (Spain), p. 22. Kjellberg F., Gouyon P.H., Ibrahim M. and Raymond M. 1987. The stability of the symbiosis between dioecious figs and their pollinators. a study of Ficus carica L. and Blastophaga psenes L. Evolution 41: 603–704. Lai J.A., Yang W.C. and Hsiao J.Y. 2001. An assessment of genetic relationships in cultivated teas clones and native wild tea in Taiwan using RAPD and ISSR markers. Bot. Bull. Acad. Sc. 42: 93–100. Mars M., Carraut A., Marrakchi M., Gouiaa M. and Gaaliche F. ´ ´ ` 1994. Ressources genetiques fruitieres en Tunisie (poirier, oranger, figuier, grenadier). Plant Genet. Res. Newsletter 100: 14–17. Mars M., Chebli T. and Marrakchi M. 1998. Multivariate analysis of fig (Ficus carica L.) germplasm in southern Tunisia. Acta Hort. 480: 75–81. Mars M. 2001. Fig (Ficus carica L.) genetic resources and breeding, a review. Second international symposium on fig. Caceres (Spain) p. 19. Nei M. and Li W.H. 1979. Mathematical model for studying genetical variation in terms of restriction endonucleases. Proc. Natl. Acad. Sci. 74 USA: 5267–5273.

Prevost A. and Wilkinson M.J. 1999. A new system of comparing PCR primers applied to ISSR fingerprinting of potato cultivars. Theor Appl Genet 98: 107–112. Reddy K.D., Nagaraju J. and Abraham E.G. 1999. Genetic characterization of the silkworm Bomyx mori by simple sequence repeats (SSR)-anchored PCR. Heredity 83: 681–687. ´ ´ Rhouma A. 1996. Les ressources phytogenetiques oasiennes. le ` ´ figuier (Ficus carica L.). In: Proceedings of the 3eme Journees ´´ Nationales sur les Acquis de la Recherche Agronomique, Veterinaire et Halieutique, Nabeul (Tunisie), p. 29. Sambrook J., Fritch E.F., Maniatis T., 1998. Molecular cloning. A laboratory manual, second Edition SAS, 1990. SAS user’s guide: SAS / STAT, SAS BASIC. Version 6.07, fourth edition. SAS incl, Box 8000. Cary, NC 27512-8000, Cary, NC: SAS institute Inc. Valdeyron G. and Lloyd D.G. 1979. Sex differences and flowering phenology in the common fig, Ficus carica L. Evolution 33: 673–685. Valizadeh G. and Valdeyron G. 1979. Importance adaptative du polymorphisme enzymatique chez le figuier (Ficus carica L.). Ann. Amelior. Plantes 29: 213–225. Valizadeh M., Rivals P. and valdeyron G. 1977. Utilisation du ´ ´ ´ ´ de figuier polymorphisme proteique pour l’etude des varietes (Ficus carica L.). CR Acad. Agri. 63: 647–655. Weiblen G.D. 2000. Phylogenetic relationships of functionally diocious Ficus (Moraceae) based on ribosomal DNA sequences and morphology. American Journal of Botany 87: 1357–1357. Wiebes J.T. 1963. Taxonomy and host preferences of Indo-Australian fig wasps of the genus Ceratosolen (Agaonidae). Tijdschrift voor Entomologie 106: 1–112. Zehdi S., Trifi M., Ould Mohamed Salem A., Rhouma A. and Marrakchi M. 2002. Survey of inter simple sequence repeat polymorphisms in Tunisian date palms (Phoenix dactylifera L.). J. Genet. & Breeding 56: 77–83. Zietkiewicz E., Rafalski A. and Labuda D. 1994. Genome fingerprinting by simple sequence repeats (SSR)-anchored polymerase chain reaction amplification. Genomics 20: 176–183.