Random amplified polymorphic DNA analysis of olive (Olea europaea L.) cultivars

Seventeen olive (Olea europaea L.) cultivars, including oil and table olive cultivars originating from throughout the Mediterranean area, were screened using random amplified polymorphic DNA (RAPD) markers. The results indicate that a high degree of polymorphism is evident in the olive germplasm reexamined. Forty random decamer primers were screened; seventeen of these produced 47 reproducible amplification fragments useful as polymorphic markers. Each of the 17 cultivars can be discriminated with a few primers. Results were analyzed for similarity among the cultivars and a cluster analysis was performed. These analyses revealed two main groups: one comprising primarily small-fruited cultivars grown mainly for oil production, and the other characterized by having large fruit. There was no apparent clustering of olive cultivars according to their geographic origins. Olive (Olea europaea) is one of the most ancient cultivated fruit tree species in the Mediterranean basin. It is the only Mediterranean representative of the genus Olea, which includes 35-40 species distributed over tropical and southern Africa, south Asia, eastern Australia, New Caledonia, and New Zealand (Zohary and Hopf 1993). Traditionally, morphological and phonological traits are used to identify olive cultivars. More recently, workers have found isozymes to be useful for cultivar identification and determining patterns of relatedness among cultivars. Pontikis et al. (1980) identified 27 olive cultivars, mostly of Greek origin, using 16 enzyme systems in pollen. Trujillo et al. (1990) found that by using five pollen enzyme systems they could distinguish 134 of 155 cultivars. Ouazzani et al. (1993) distinguished 33 of 44 cultivars using 9 enzyme systems in leaf tissue. Although isozyme analysis has proved useful in olive, mainly due to the high level of isozyme polymorphism present in the species, direct analysis of DNA could increase considerably the number of markers produced. Furthermore, because isozymes are products of gene expression, differential expression by environment, tissue-specificit y, and other factors is common and may make interpretation of results difficult. Random amplified polymorphic DNA (RAPD) analysis, first described by Williams et al. (1990) and Welsh and McClelland (1990), has proven to be a useful tool for genetic typing and mapping. Bogani et al. (1994) described preliminary results of RAPD analyses of olive cultivars. They used 5 decamer nucleotide primers to generate RAPD polymorphisms among 11 olive cultivars, but found no consistent relationships. In this paper, we report the use of RAPD markers to distinguish among 17 olive cultivars, and we discuss the use of RAPDs to study relationships among cultivars of this species. Materials and Methods Plant materials. Seventeen Olea europaea cultivars were used in this study (Table 1). These were obtained from a collection mainor publication 26 Sept. 94. Accepted for publication 11 Jan 1995. We acknowledge K. Pinney for her capable technical assistance and S. for his advice on technique, J. Hormaza was supported by an INIA rom the Spanish Ministry of Agriculture, The cost of publishing this paper d in part by the payment of page charges. Under postal regulations, this fore must be hereby marked advertisement solely to indicate this fact. om Universita degli Studi di Catania, Istituto di Coltivazioni Arboree, Via 5, Catania Italy 95123. To whom reprint requests should be addressed. dress: Dept. Fruticultura, S.I. A. -D.G. A. , Apartado 727, Campus de 0080 Zaragoza, Spain. tained in experimental orchards at the Univ. of California, Davis. DNA extraction and RAPD analysis. Young olive leaves were collected in Spring 1993 and stored at –70C before DNA extraction. Total DNA was extracted from leaf tissue following the CTAB method of Doyle and Doyle (1987) with minor modifications. Young leaves (5.0 g) were ground in liquid nitrogen and mixed with 20 ml of CTAB buffer (100 mM Tris-HCl pH 8, 1.4 M NaCl, 20 mM EDTA, 2% CTAB, 1% PVP, 0.2% β− mercaptoethanol, 0.1 % NaHSO4). Samples were incubated at 65C for 1 h, mixed with an equal volume of chloroform–isoamyl alcohol (24:1), and centrifuged at 2000× g for 15 min. The aqueous phase was recovered and mixed with two-thirds volume of isopropanol. The nucleic acid precipitate was recovered with a glass hook, washed with 10 ml of 10 mM ammonium acetate in 76% ethanol, dried overnight, and resuspended in 1 ml of modified TE buffer (10 mM Tris-HCl pH 8, 0.1 mM EDTA). Extracted DNA was diluted to 10 ng·μl and used for PCR amplification. Forty decamer oligonucleotides (Operon Technologies, Alameda, Calif.) were used for PCR amplifications following the procedure of Williams et al. (1990), with some modifications. Amplification reactions were carried out in 25-μl volumes containing 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.9 mM MgCl2, 0.00 1% gelatin, 100 μM each of dATP, dGTP, dCTP, and dTTP (Promega, Madison, Wis.), 0.4 μM primer, 0.75 units of AmpliTaq DNA polymerase (Perkin-Elmer-Cetus, Norwolk, Conn.) and 50 ng of genomic DNA. Each reaction mixture was overlaid with 25 μl of mineral oil to prevent evaporation. To destroy putative carryover products, reaction mixtures and mineral oil were placed on a UV (300 nm) transilluminator before addition of template DNA and DNA polymerase (Sarkar and Sommer, 1993). DNA amplification reactions were performed in a thermal cycler (PTC100; MJ Research, Watertown, Mass.) programmed for 1 cycle of 2 min at 94C followed by 40 cycles of 45 sec at 92C, 1 min at 36C, 2 min at 72C, for denaturing, annealing and primer extension, respectively. The last cycle was followed by incubation for 5 min at 72C; PCR products were stored at 4C before analysis. Amplification products were analyzed by gel electrophoresis in 2910 agarose (SeaKem; FMC, Rockland, Maine) in 1 x TBE buffer, stained with ethidium bromide, and photographed under UV light using Type 57 Polaroid film. Molecular sizes of amplification products were estimated using a 123 bp DNA ladder (Sigma Chemical Company, St. Louis). All reactions were repeated at least three times using different lots of Taq polymerase and only reproducible bands were used in further analyses. Each amplification fragment J. AMER. SOC. HORT. SCI. 120(3):538–542. 1995. Table 1. Olive cultivars included in this study and their country of origin.