Studying the Genetic Variation among Clones of Kalamon and Koroneiki Using Molecular Techniques

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1 Studying the Genetic Variation among Clones of Kalamon and Koroneiki Using Molecular Techniques E. Despotaki, A. Linos and M. Hagidimitriou Pomology Laboratory Crop Science Department Agricultural University of Athens Iera Odos 75, 11855, Athens Greece Keywords: Olea europea L., RAPD, ISSRs, genetic similarity, geographical clustering Abstract The olive tree (Olea europea L.) is cultivated in the Mediterranean Basin since 5800 BC. Its socio-economic impact is very important for the countries in the area. Greece occupies the third place in the world rank of olive oil producers and the second place in the European Union as a table olive producer. Koroneiki is an olive oil cultivar while Kalamon is a table olive cultivar. Both of them are the most wellknown worldwide Greek olive cultivars. In this study, healthy, young leaves of both cultivars were collected from seven different regions in Greece and Cyprus in order to study the intra-varietal variability. DNA extraction was performed according to Doyle and Doyle protocol. Markers originating from two different molecular techniques, Randomly Amplified Polymorphic DNA (RAPD) and Inter Simple Sequence Repeat (ISSR), were used for investigating the germplasm variability. In order to establish the genetic relationships among the clones of Kalamon and Koroneiki, 30 RAPD primers were tested and 10 were used while for ISSR 10 primers were tested and 6 of them were used. Based on the results from both methods, but primarily from ISSR, intra-varietal variability was present in both cultivars. INTRODUCTION The olive tree (Olea europea L.) is cultivated in the Mediterranean Basin since 5800 BC. (Zohary and Hopf, 1994). Its socio-economic impact is very important for the countries in the area. During the last decade non-mediterranean countries are also trying to establish olive cultivation, mainly due to the unique nutritional value of the olive oil. Greece is considered as a secondary center of diversity of olive (Damania, 1995) while the oldest evidence for olive tree cultivation in Greece was in Crete during the middle- Minoan period, BC, where olive seeds were found to be crashed. Koroneiki is an olive oil cultivar while Kalamon is a table olive cultivar. Both of them are the most well-known worldwide Greek olive cultivars. Many researchers used the PCR technology to evaluate the genetic variation in olive (Rallo et al., 2000; Hagidimitriou et al., 2005; Martins-Lopes et al., 2009). In this study, healthy, young leaves of two Greek cultivars, Kalamon and Koroneiki, were collected from 7 different regions in Greece and Cyprus in order to study the intra-varietal variability using two different molecular techniques, the randomly amplified polymorphic DNA (RAPDs) and the inter simple sequence repeats (ISSRs). Also, the two techniques were compared according to their discrimination capacity. MATERIALS AND METHODS Olive trees were chosen from farms or collections located in 5 Greek regions (Fthiotida, Agricultural University of Athens collection, Poros, Kalamata-NAGREF, Chania NAGREF) and one region in Cyprus (National collection). These 6 regions covered the main areas of cultivation of the two Greek cultivars Kalamon and Koroneiki. Young, healthy leaves were collected randomly from a total of 27 individuals, 13 of which belonged to Koroneiki cultivar and 14 to Kalamon cultivar. Leaves were stored at -20 C. Genomic DNA was extracted using a modified C-TAB method following Proc. XXVIII th IHC Olive Trends Symposium Eds.: J. Tous et al. Acta Hort. 924, ISHS

2 the procedure described by Doyle and Doyle (1987). DNA quality and concentration were determined spectrophotometrically. From the 30 RAPD primers tested ten were selected (Table 1). The 30 μl volume PCR reactions contained 1x PCR buffer, 270 μm dntps, 300 μm MgCl 2, 1 μm primer, 1 U Taq DNA polymerase (PROMEGA) and 25 ng of total genomic DNA. The amplification was performed in a Sensoquest Labcycler Standard thermocycler. The temperature profile consisted of an initial 2 min denaturation step at 94 C, followed by 30 cycles of: denaturation at 94 C for 45 s, primer annealing at 38 C for 60 s and extension step at 72 C for 120 s. The final elongation step was at 72 C for 7 min. Amplification products were separated in 2.5% agarose gel by electrophoresis stained with ethidium bromide in 1x TAE buffer. The RAPD bands were visualized under UV and photographed with a digital camera. The DNA Molecular Weight Marker 100 bp (New England BioLabs, USA) was used as a standard molecular weight size marker. From the 10 ISSRs primers tested 6 were selected (Table 1). The 30-μl volume PCR reactions contained 1x PCR buffer, 270 μm dntps, 300 μm MgCl 2, 1 μm primer, 1 U Taq DNA polymerase (PROMEGA) and 25 ng of total genomic DNA. The temperature profile consisted of an initial 5 min denaturation step at 94 C, followed by 30 cycles of: denaturation at 94 C for 30 s, primer annealing at 52 C for 45 s and extension step at 72 C for 120 s. The final elongation step was at 72 C for 7 min. Amplification products were separated in 2.5% agarose gel and treated similarly to the RAPD reactions. RAPD and ISSR products were scored as present (1)/absent (0) for each entry. Very faint bands were omitted from scoring. Genetic similarities for the RAPD and ISSR data were calculated using the Jaccard (Sneath and Sokal, 1973) similarity coefficient. Phylogenetic trees were created using the UPGMA (Unweighted Pair Group Method with Arithmetic mean) and N.J. (Neighbor-Joining) methods. The correlation among all genetic similarity matrices was checked using the Mantel (Mantel, 1967) test. The analysis was performed using the NTSYS pc 2.02i (Rohlf, 1998). RESULTS AND DISCUSSION A total of 188 RAPD bands were scored, with 81 being polymorphic (43%). The total number of bands per RAPD primer ranged from 16 (RAPD-3) to 21 (OPAH-17) with an average of 18.8 bands per primer. Primer RI-5 yielded the highest percentage of polymorphic bands (67%), while primer RI-4 (16%) yielded the lowest percentage of polymorphic bands. Similarly, the ISSRs primers amplified 104 fragments, of which 72 were polymorphic (69%). The total number of bands per ISSRs primer ranged from 10 (UBC-818) to 21 (UBC-842, UBC-826 and UBC-856) with an average of 17.3 bands per primer. Primer UBC-825 yielded the highest percentage of polymorphic bands (100%), while primer UBC-826 (43%) yielded the lowest percentage of polymorphic bands. According to Jaccard s coefficient of similarity, the genetic similarities for the combination of the two methods, RAPD and ISSRs (Fig. 1) ranged from 0.64 to Within the Koroneiki cultivar the Jaccard s coefficient of similarity ranged from 0.83 ( Koroneiki-Cyprus and Koroneiki-Kriti-2, Koroneiki-Cyprus and Koroneiki-Kriti- 4 ) to 1.00 ( Koroneiki-Fthiotida-1 and Koroneiki-Fthiotida-2 ) while for Kalamon, from 0.80 ( Kalamon-Poros-Nana and Kalamon-Kalamata-1, Kalamon-Poros-Nana and Kalamon-Kalamata-2, Kalamon-Poros-Nana and Kalamon-Cyprus-1, Kalamon- Poros-Koini and Kalamon-Cyprus-1, Kalamon-Poros-Nana and Kalamon-Cyprus-2, Kalamon-Poros-Nana and Kalamon-Cyprus-3 ) to 0.99 ( Kalamon-A.U.A.-3 and Kalamon-A.U.A.-2, Kalamon-Kriti-1 and Kalamon-Kriti-2, Kalamon-Kalamata-2 and Kalamon-Kalamata-1 ). In an intra-varietal study of 120 clones of the Portuguese cultivar Cobrançosa (Martins-Lopes et al., 2009), the Jaccard s coefficient ranged from , and for the RAPD, ISSRs and their combination respectively. In another study (Sensi et al., 2003) of intra-varietal variability of 12 genotypes, belonging to three Italian cultivars, the Jaccard s coefficient ranged between , and for Mignolo, Moraiolo and Leccino respectively. It seems that the two cultivars examined showed lower intra-varietal variability than other foreign cultivars. 336

3 Within the RAPD-ISSRs UPGMA compined dendrogram (Fig. 1), Branch 1 contains Koroneiki clones while Branch 2 contains Kalamon clones. The same clustering for the clones of the two cultivars can be found in the RAPD and ISSRs UPGMA dendrograms. Also, in all dendrograms clones are clustered according to their provenance. More particularly, clones from A.U.A and Fthiotida, for both cultivars, are clustered together. Finally, for Koroneiki, clones from Kriti and Kalamata seem to be genetically close. The cophenetic correlation coefficient was high for RAPD-UPGMA (r=0.97), ISSR-UPGMA (r=0.97) and RAPD and ISSR-UPGMA (r=0.99) revealing a good fitness of the genetic similarity matrices to the obtained phenograms. Literature Cited Barranco, D., Cimato, A., Fiorino, P., Rallo, L., Touzani, A., Castaneda, C., Serafin, F. and Trujillo I World catalogue of olive varieties. International Olive Oil Council, Madrid. Ciferri, R Datied ipotesi sull origine e l evoluzione dell olivo. Olearia 1: Damania, A.B Olive, the plant of peace, reigns throughout Mediterranean. Diversity 11: Doyle, J.J. and Doyle, J.L A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochemical Bulletin 19: Hagidimitriou, M., Katsiotis, A., Menexes, G., Pontikis, C. and Loukas, M Genetic diversity of major Greek olive cultivars using molecular (AFLP and RAPDs) markers and morphological traits. J. Amer. Soc. Hort. Sci. 130(2): Loukas, M. and Krimbas, C.B History of olive cultivars based on their genetic distances. J. Hort. Sci. 58: Mantel, N The detection of disease clustering and a generalized regression approach. Cancer Res. 27: Martins-Lopes, P., Gomes, S., Lima-Brito, J., Lopes, J. and Guedes-Pinto, H Assessment of clonal genetic variability in Olea europaea L. Cobrançosa by molecular markers. Sc. Hort. 123: Pontikis, C., Loukas, M. and Kousounis, G The use of biochemical markers to distinguish olive cultivars. J. Hort. Sci. 54: Rallo, P., Dorado, G. and Martin, A Development of simple sequence repeats (SSRs) in olive tree (Olea europaea L.). Theor. Appl. Genet. 101: Rohlf, M NTSYS-PC. Numerical Taxonomy and Multivariate Analysis System, version 2.02i. Department of Ecology and Evolution. State University of New York, Setauket, NY. Sensi, E., Vignani, R., Scali, M., Masi, E. and Cresti, M DNA fingerprinting and genetic relatedness among cultivated varieties of Olea europaea L. estimated by AFLP analysis. Scientia Horticulturae 97: Zohary, D. and Hopf, M Domestication of Plants in the Old World, second edition. Clarendon Press, Oxford. Zohary, D. and Spiegel-Roy, P Beginning of fruits growing in the Old World. Science 187:

4 Tables Table 1. Primers used for RAPD and ISSR analyses: total number, polymorphic bands and % of polymorphism obtained. Primer Sequence 5 3 Total bands Polymorphic bands % polymorphism RAPD RI - 4 ATACACCAGC RAPD - 1 TCCGCAACCA OPAH - 17 CAGTGGGGAG OPB - 11 GTAGACCCGT RAPD - 13 CACCACCACC OPG - 5 CTGAGACGGA OPA - 9 GGGTAACGCC RI - 5 TTGCGTCATG RAPD - 3 GTAGACCCGT OPB - 1 GTTTCGCTCC Total: ISSR UBC (GA) 8 G UBC (AC) 8 C UBC (GGAGA) UBC (CT) 8 RC UBC (AC) 8 T UBC (CA) 8 G Total:

5 Figurese 1α 1 1β 2α 2 2β Fig. 1. Relationships among 27 olive entries of the two Greek cultivars using RAPD-ISSR primers. 339

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