Using RAPD and AFLP Markers to Distinguish Individuals Obtained by Clonal Selection of Arbequina and. Manzanilla de Sevilla

HORTSCIENCE 39(7):1566–1570. 2004. Using RAPD and AFLP Markers to Distinguish Individuals Obtained by Clonal Selection of ‘Arbequina’ and ‘Manzanilla...
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HORTSCIENCE 39(7):1566–1570. 2004.

Using RAPD and AFLP Markers to Distinguish Individuals Obtained by Clonal Selection of ‘Arbequina’ and ‘Manzanilla de Sevilla’ Olive A. Belaj,1 L.Rallo, and I.Trujillo Departamento de Agronomía. ETSIAM. Universidad de Córdoba. Avenida Ménendez Pídal s/n, Apdo 3048, 14080 Córdoba, Spain L. Baldoni Istituto di Genetica Vegetale, Sezione di Perugia, CNR, Via Madonna Alta 130, 06128 Perugia, Italy Additional index words. clonal selection, intracultivar variability, DNA fingerprinting, Olea europaea L. Abstract. Eight and seven clones, respectively selected within the olive cultivars ‘Arbequina’ and ‘Manzanilla de Sevilla’, were studied by means of randomly amplified polymorphic DNA (RAPD) and amplified fragment-length polymorphism (AFLP) markers. Two clones of ‘Arbequina’, C3 and C12, showed polymorphism with respect to the standard cultivar by means of both markers. In fact, about 33.6% RAPD bands and 9.2% AFLP bands were polymorphic for these clones. This high level of polymorphism and the presence of a high percentage of bands absent in ‘Arbequina’ suggest their possible origin as ‘Arbequina’ seedlings. The dendrogram obtained by both molecular markers also supports the hypothesis of a seedling origin of these clones as they clustered separately from the original cultivar and the rest of monomorphic clones at low values of similarity. Also within the ‘Manzanilla de Sevilla’ group, two clones (31 and 44) showed diversity with respect to the standard cultivar; 4.5% RAPD and 6.3% AFLP markers were polymorphic for these genotypes while all the other clones didn’t show any difference with the standard ‘Manzanilla de Sevilla’. RAPD and AFLP markers effectively revealed intracultivar variability due to gametic or multiple mutational events, while the detection of other kind of differences such as eventual single mutations remains uncertain and requires further investigation. Olive (Olea europaea L.) is one of the oldest agricultural tree crops in the Mediterranean basin, which lasts about 5,000 years (Zohary and Hopf, 1994). Most of the olive cultivars are very old, vegetatively propagated, and distributed within restricted areas (Barranco, 1997). The origin of these cultivars is still uncertain but biochemical (Massei and Hartley, 2000) and molecular (Besnard et al., 2001a) evidence demonstrates their origin from the wild Mediterranean olive and their further spreading by humans. The cross between cultivated olives or between cultivated and wild plants has also occurred at a local level during the long history of olive cultivation. Received for publication 5 Aug. 2003. Accepted for publication 1 Mar. 2004. Contribution from the Department of Agronomy, University of Cordoba, Cordoba, Spain and the Institute of Plant Genetics, Research Division of Perugia, CNR, Via Madonna Alta 130, 06128 Perugia, Italy. We are in debt with J. Tous and M. P. Suárez for the supply of the plant material and helpful information and suggestions. Thanks are due to L.M. Martín and R. FernándezEscobar for their helpful advices, and to I. García for her assistance in the laboratory. This research was funded by the INIA (Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria del MAPA), Project CAO 98-001-C3-1. A. Belaj has a PhD grant from the Agencia Española de Cooperación Internacional (AECI), Spain. 1 To whom reprint requests may be adressed; e.mail [email protected].

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In long-living plants such as olive, natural mutations may occur, causing alterations of some traits of agronomic interest, and individuals carrying the improved traits may have been selected and vegetatively propagated, originating clonal populations different from the original cultivar (Rallo, 1995). Continuous vegetative propagation has probably extended the natural lifetime of many olive genotypes. The mechanisms responsible for clonal differences may include changes due to the presence of viruses or phytoplasms (Kenyon et al., 1998; Kovacs et al., 2001), epigenetic mutations (Cubas et al., 1999), somatic spontaneous mutations (Asins et al., 1999; Thompson et al., 1999), limb sport occurrence (Crist, 2000), or, for those olives grafted on seedling plants, some differences may be the consequence of the rootstock effect (Sugar et al., 1999). Clonal selection has been carried out in the main olive cultivars of various Mediterranean countries (Arsel and Cirik, 1994; Bartolini et al., 1994; Boulouha, 1986; Grati-Kamoun et al., 2000; Lavee et al., 1995; Martins et al., 1997; Suárez et al., 1990; Tous et al., 1998). The exploration of the phenotypic variability for either agronomical or technological characters and the identification of outstanding trees for these traits have been the first steps in the above reported works of clonal selection. In spite of the efforts made using clonal collection in olive, only very few clones have

been obtained and commercialised (Bartolini et al., 2002; Loussert and Berrichi, 1995; Tous et al., 1998). The possibility of identifying the products of clonal selection may have important implications in the protection and management of these plants. Morphological characterization does not generally help to distinguish very close genotypes. Molecular markers should be more effective in detecting genetic differences. Their use would help characterize and identify in a quick and reliable way the selected material as well as to indicate if the phenotypic variability observed among individuals of the same cultivar could have a genetic basis. However, in many cases these methods have shown a low resolution to distinguish clones originated by somatic mutation (Cervera et al., 1998). The low rate of mutational events with phenotypical effects may imply that a very large number of markers might have to be scored for each system to estimate genetic distances within olive cultivars. Some recent studies with molecular markers (Cipriani et al., 2002; Gemas et al., 2000) have successfully discriminated among different individuals within the same olive cultivar. Vergari et al. (1999) studied different cultivars with the same general name ‘Manzanilla’ (Barranco et al., 2000) as well as different individuals of ‘Manzanilla de Sevilla’ collected in different olive producing areas. To our knowledge, identification studies of individuals obtained from clonal selection works have not been reported yet in olive. ‘Manzanilla de Sevilla’ and ‘Arbequina’ are two main Spanish cultivars, widely distributed in Andalusia and Catalonia, respectively, but also in other olive producing countries such as Argentina, Australia, and California, due to their high agronomical and technological value. The present study aims to determine if presumed clones of these cultivars can be distinguished among themselves and from the original cultivars by means of randomly amplified polymorphic DNA (RAPD) and amplified fragment-length polymorphism (AFLP) fingerprints. Material and Methods Plant material and DNA extraction. Plant material (Table 1) consisted of eight individuals obtained from the clonal selection carried out on ‘Arbequina’ (Tous et al., 1998) and other Table 1. Plant material included in the study: Cultivars and the clones obtained by clonal selection of these cultivars. ‘Arbequina’ group C3 C6 C12 C15 C18 C25 C28 F79 Standard ‘Arbequina’

‘Manzanilla de Sevilla’ group 44 31 42 13 37 41 5 Standard ‘Manzanilla de Sevilla’

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Table 2. Polymorphisms obtained by the RAPD analysis. ‘Arbequina’ ‘Manzanilla de Sevilla’ Primer Total bands Polymorphic bands Total bands Polymorphic bands OPA-01 4 0 ----OPA-02 6 2 ----z OPA-03 7 0 5 0 OPA-08y ----8 0 OPA-19y ----6 0 OPF-06y --7 0 OPI-06 6 0 ----OPI-12 4 0 ----OPJ-18z 8 2 8 0 OPK-16z 7 3 5 0 y OPK-17 ----6 1 OPP-19z 6 3 4 0 OPQ-12z 6 1 8 2 OPQ-15 8 3 ----OPR-03z 5 1 6 0 OPX-01z 8 3 6 1 OPX-03z 7 3 8 0 OPX-09 6 2 ----z OPX-13 8 5 4 0 OPX-14 8 4 ----OPX-15 4 1 ----OPZ-07z 7 3 7 0 OPZ-10 5 4 ----OPZ-11 5 2 ----Total 125 42 88 4 z RAPD primers selected for both ‘Manzanilla de Sevilla’ and ‘Arbequina’ as well as their respective clones. y RAPD primers selected only for ‘Manzanilla de Sevilla’ and its clones.

seven from ‘Manzanilla de Sevilla’ (Suárez et al., 1990). The ‘Arbequina’ clones were selected for high and regular cropping in Lleida and Tarragona, northeast of Spain (Tous et al., 1998). The ‘Manzanilla de Sevilla’ clones were selected in Andalusia (southern Spain) for their precocity, productivity, and regular cropping (Suárez et al., 1990). The ‘Arbequina’ clone C18 is actually marketed under the name ‘Irta-I-18’ (Tous et al., 1998), while clone 44 of ‘Manzanilla de Sevilla’ is sold under the name ‘Cortijo del Cuarto’ (M.P. Suárez, personal communication). Plant material was supplied by the Institut de Recerca i Tecnologia Agroalimentàries (IRTA),

Centre de Mas Bové, Tarragona, Spain and by EUITA, Cortijo de Cuarto, Seville, Spain. For RAPD and AFLP analysis, total DNA was isolated from fresh leaf material following the procedure of Belaj et al. (2001). Marker analysis. RAPD amplifications were performed as described by Belaj et al. (2001). All the reactions were conducted three times using DNA of various extractions and different lots of the AmpliTaq DNA polymerase. The amplification products were separated on polyacrylamide gels containing 10% acrylamide, 0.126% piperazine diacrylamide crosslinker in 375 mM Tris-HCl, pH 8.8, using Tris glycine (25 mM Tris, and 192 mM glycine) and were

visualized by silver staining as described by Bassam et al. (1991). Thirty primers from kits A, F, I, J, K, P, Q, X, and Z (Operon Technologies, Alameda, Calif.) were screened in the study (data not shown). RAPD bands were scored as 1 (present) or 0 (absent) in a binary matrix for each primer following a conservative criterion for their selection (Belaj et al., 2001) and each gel was scored independently. AFLP analysis was performed as described by Angiolillo et al. (1999). Seven primer combinations with three selective nucleotides were used (Table 3): four Mse I primers (M-CAC, M-CAA, M-CTG and M-CTT) and three Eco RI primers (E-AGC, E-ACT and E-AAC). Eco RI primers were end-labelled with y-[33P]-ATP. The amplified products were separated by denaturing 6% polyacrilamide gel electrophoresis and visualized by autoradiography. The reproducibility of AFLP fingerprints was assessed on three DNA samples by replicating the entire procedure for all the primer combinations. Data analysis. Jaccard’s similarity coefficients (Jaccard, 1908) were calculated for the AFLP and RAPD markers obtained for the clones and the respective original cultivars. The clones and their original cultivars were grouped by cluster analysis using the unweighted pair-group method with arithmetic averages (UPGMA). The computer program used was NTSYS-pc version 2.02 (Rohlf, 1998). The cophenetic correlation coefficient was calculated, and Mantel’s test (Mantel, 1967) was performed to check the goodness of fit of a cluster analysis to the matrix on which it was based. Results Clones of ‘Arbequina’. Twenty RAPD primers were selected to analyze the clones of ‘Arbequina’ (Table 2). All the primers produced well-separated and reproducible amplification fragments. RAPD reactions yielded a total of 125 bands for the group ‘Arbequina’, 6.3 bands

A

Fig. 1. (A) RAPD amplification products obtained by the primers OPQ-15 and OPR-03 and (B) AFLP patterns obtained with the primer combination E-AGC/M-CAC with the ‘Arbequina’ clones. The polymorphic markers are indicated by arrows. Legend: M.W: Molecular weight marker; A = ‘Abequina’; 3 (C3); 6 (C6); 12 (C12); 15 (C15); 18 (C18); 25 (C25); 28 (C28); F79.

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A

B

Fig. 2. Dendrogram of ‘Arbequina’s clones obtained by RAPD (A) and AFLP (B) markers using UPGMA clustering method and Jaccard’s index of similarity.

per primer on average. Two of the eight clones studied, C3 and C12 (Table 1) showed polymorphisms with the original ‘Arbequina’. Thirty-three polymorphic bands (26.4%) were detected between the clone C3 and the standard ‘Arbequina’, and 21 bands (16.8%) were polymorphic for C12. Most polymorphisms were due to lack of amplification products in the clones, but 19 bands (45% of the total polymorphic bands) were lacking in the original cultivar. Both these clones shared a total of 12 polymorphic fragments. An example of the patterns obtained with primers OPQ-15 and OPR-03 is given in Fig. 1A. The similarity value (similarity matrix not shown) between the clone C3 and the standard ‘Arbequina’ was 0.73. The similarity value obtained between this clone and the rest of the other monomorphic clones ranged from 0.71 (F79) to 0.73. The similarity value between the clone C12 and the standard ‘Arbequina’ was 0.80 as with the other clones. The similarity value obtained between the two polymorphic clones was 0.74. The cophenetic correlation coefficient between the dendrogram and the original distance matrix was high: r = 0.98. The dendrogram obtained by the UPGMA (Fig. 2A) shows two different clusters. The first group includes a branch formed by the standard ‘Arbequina’ and all the non-polymorphic clones, and a second branch includes the clone C12. Only the clone C3 represents the second clustering group. An example of the patterns obtained with the AFLP primer combination E-AGC/M-CAC with the ‘Arbequina’ group is given in Fig. 1B. A total of 584 bands was obtained and 37 resulted polymorphic with a percentage of 6.3% (Table 3). As in the case of RAPD markers, polymorphisms to the standard cultivar ‘Arbequina’ were found only for the clones C3 (4.4%) and C12 (6.1%). About 50% of the polymorphic bands found for the clones C3 and C12 were present in one of the two clones and absent in ‘Arbequina’. For the clones C3 and C12 the values of similarity with ‘Arbequina’ were 0.32 and 0.19,

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respectively and the similarity value between them was 0.11. As expected, all the nonpolymorphic clones showed the similarity value of 1 with ‘Arbequina’ while, when compared to the polymorphic clones, their similarity values ranged from 0.32 to 0.54 for the clone C3 and from 0.06 to 0.19 for the clone C12. The cophenetic correlation coefficient between the dendrogram and the original distance matrix was high: r = 0.98. The dendrogram obtained by means of AFLP markers for the ‘Arbequina’ group (Fig. 2B) was very similar to that obtained by RAPD markers, with the exception that the first group of ‘Arbequina’ included the clone C3 instead of C12 as it was in the RAPD dendrogram. Clones of ‘Manzanilla de Sevilla’. Fourteen primers were selected for the RAPD analysis of the ‘Manzanilla de Sevilla’ clones (Table 2). Eighty-eight fragments, with an average of 6.3 fragments per primer, were obtained. Polymorphisms were only detected between this cultivar and two of its clones, namely 31 and 44. On 4 polymorphic bands, 2 were due to presence of amplification fragments absent in the standard cultivar. Three polymorphic bands (3.4%) were detected between the clone 31 and the standard ‘Manzanilla’, and 2 bands (2.2%) were polymorphic for the clone 44. The similarity value of the clone 31 with ‘Manzanilla de Sevilla’ and the rest of monomorphic clones was 0.97. That of clone 44 with the standard cultivar and the monomorphic clones was 0.95. The two polymorphic clones shared only one polymorphic band. Cophenetic correlation coefficient between the dendrogram and the

original distance matrix was high: r = 0.92. Applying the UPGMA, only two groups were obtained in the dendrogram (Fig. 3A). The first group was divided into two different branches, the first one formed by ‘Manzanilla de Sevilla’ and all the non polymorphic clones and the second branch included only the clone 44. The second group was represented only by the clone 31. The results of the AFLP analysis for ‘Manzanilla de Sevilla’ clones are shown in Table 3. On a total of 580 bands 21 resulted polymorphic for the clone 31 (3.6%) and 22 for the clone 44 (3.8%). Of these polymorphic bands only 6 and 7, respectively,were due to presence of amplification fragments absent in the original cultivar. The similarity values of clones 31 and 44 with ‘Manzanilla de Sevilla’ were 0.25 and 0.44, respectively. The similiarity between the two plymorphic clones was 0.50. That of clone 31 with the monomorphic clones ranged from 0.08 (5) to 0. 20 (41). The similarity value obtained between the clone 44 and the monomorphic clones ranged from 0.11 (42) to 0.42 (41). Clones 13 and 14 clustered separately but their similarity value with ‘Manzanilla de Sevilla’ was 1, and they did not show genetic differences at banding profiles with neither the original cultivar nor the rest of monomorphic clones. Cophenetic correlation coefficient between the dendrogram and the original distance matrix was high: r = 0.97. The dendrogram obtained with the AFLP markers (Fig. 3.B) was very similar to that obtained by means of RAPD markers, but the

Table 3. Primer combinations used for AFLP analysis and polymorphisms obtained. Primer combination E-AAC/M-CAG E-AGC/M-CAC E-ACT/M-CAC E-ACT/M-CAA E-AGC/M-CTG E-AAC/M-CTT E-AAG/M-CAA Total

‘Arbequina’ Total bands Polymorphic bands 110 8 87 12 63 6 81 7 69 8 114 7 60 5 584 53

‘Manzanilla de Sevilla’ Total bands Polymorphic bands 106 3 67 14 78 3 88 5 61 1 112 10 68 1 580 37

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A

B

Fig. 3. Dendrogram of ‘Manzanilla de Sevilla’’s clones obtained by RAPD (A) and AFLP (B) markers using UPGMA clustering method and Jaccard’s index of similarity.

two polymorphic clones clustered together in a separate group. Discussion The work was mainly aimed at verifying if RAPD and AFLP markers were able to distinguish among the presumed olive clones and their original cultivars ‘Arbequina’ and ‘Manzanilla de Sevilla’ as well as among themselves. These markers, which have proved to be useful in genetic variability and identification studies in olive (Angiolillo et al., 1999; Belaj et al., 2001; Fabbri et al., 1995), have numerous advantages, such as the multiple loci nature and the large number of bands able to produce. Arbitrarily primed markers may sample the whole genome, ranging from conserved functionally important sequences to middle and highly repetitive sequences, but the chance to detect differences among clones of the same cultivar due to mutational events by means of these molecular markers is considered very low (Weising et al., 1995), taking into account the size of the olive genome of about 2,200 Mb (de la Rosa et al., 2003) and the low rate of mutations, around 10–6 per genome and generation (Debener et al., 2000). In most cases, no differences have been found between sport mutants and the original cultivar from which they were derived. Markers uniquely characterizing sports were found only in few cases (Trigiano et al., 1998). However, in the present work the levels of polymorphism detected in each cultivar group, even though lower than that usually found among different olive cultivars, which may range from 63% to 93% on average for both markers (Angiolillo et al., 1999; Belaj et al., 2002), were very high among individuals considered as clones of a given cultivar. These differences were obtained exclusively for two individuals in each cultivar group (C3 and C12 for ‘Arbequina’ and 31 and 44 for ‘Manzanilla de Sevilla’), while all the other genotypes were not distinguished. HORTSCIENCE VOL. 39(7) DECEMBER 2004

Among the clones that didn’t show any difference with the standard ‘Arbequina’ with both kind of markers it is also included C18, which is actually marketed under the name ‘Irta-I-18’ (Tous et al., 1998). This result does not exclude the possibility that this and the other non polymorphic clones may derive from mutations that were not revealed by the markers used in this work. It is underlined that in the ‘Arbequina’ clones the RAPD polymorphism shown by C3 and C12 was significantly higher than that found in the 31 and 44 clones of ‘Manzanilla de Sevilla’ with the same markers. Furthermore, the ‘Arbequina’ clones show morphological differences not visible for the clones of ‘Manzanilla de Sevilla’. The high polymorphism observed for the clones C3 and C12 with ‘Arbequina’ are compatible with that observed between a parent and each cross seedling, which is around 25% for RAPD markers (R. de la Rosa, personal communication) and about 20% for AFLPs (L. Baldoni, personal communication) and it decreases at 10% in the case of self-pollination. Taking into account that about 45% and 50% of the RAPD and AFLP polymorphic bands, respectively, were present in one of the two clones and absent in ‘Arbequina’, it is possible that they can derive from the cross of Arbequina with other cultivars acting as pollinators. The clones C3 and C12 were selected within ‘Arbequina’ plantations in Lleida and Tarragona districts, respectively and they show some differences from the standard ‘Arbequina’ on the fruit and stone shape and their origin as ‘Arbequina’ seedlings can not be excluded (Tous, personal communication). Similar conclusions may be reported for the clones of ‘Manzanilla de Sevilla’: the high differences with the standard cultivar of the two genotypes 31 and 44 and the existence of bands present in one of the two clones and absent in the original cultivar may be attributed to their origin as seedlings. In this case, however, the percentage of AFLP bands present in the clones and absent in ‘Manzanilla de Sevilla’

was lower (around 28% in both clones) but their possible origin from self-pollination has to be excluded. Another possible explanation of the polymorphic clones should be attributed to multiple mutations occurring in both long lived cultivars. It is known, in fact, that both ‘Arbequina’ and ‘Manzanilla de Sevilla’ are very ancient cultivars eventhough does not exist any document or accurate data to demontrate their real origin (Barranco, personal communication). However, the high level of the polymorphism found in these cases and the absence of polymorphism in the other clones of the same cultivars or in other ancient cultivars (Rotondi et al., 2003) reduces this possibility. Also in the research carried out by Cipriani et al. (2002) with SSRs and by Gemas et al. (2000) with RAPD markers it was possible to find genetic variability within olive cultivars by screening a few markers, whereas other studies in olive (Besnard et al., 2001b; Khadari et al., 2001) have not detected any RAPD variability at the intracultivar level. These contrasting results may depend on numerous reasons, such as the type of cultivar analysed, the confusion within the olive genetic resources as well as the sensibility of the markers under use. Vergari et al (1999), analysisng with RAPD markers a group of cultivars with a common root name ‘Manzanilla’, which includes different cultivars with a common apple fruit shape, were able to distinguish different genotypes. In other species contrasting results have also been obtained, such as the case of pear (Oliveira et al., 1999), grape (Gogorcena et al., 1993; Loureiro et al., 1998) and apple (Mulcahy et al., 1993), where RAPD variability at intracultivar level has not been found, while in grape and rose RAPD markers were able to differentiate certain clones (Debener et al., 2000; Moreno et al., 1995). Clonal variations have been found in grape only after analysing several primer pairs of AFLP or hundreds of ISTR markers (Scott et al., 2000; Sensi et al., 1996). Up to now, the AFLP markers have shown good efficiency for the distinction of

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different clones within a given cultivar (Cervera et al., 1998; Scott et al., 2000), most likely due to their high multiplex ratio (Powell et al., 1996) which allows the rapid screening of thousands of loci. AFLP markers were able to distinguish 16 out of 24 biotypes of the ‘Traminer’ grape cultivar (Imazio et al., 2002). Our results support the view that in olive the use of the term population cultivar, which refers to local cultivars that conserve through successive sexual generations some relevant agronomical traits and show variability for others, may be misleading (Rallo, 1999). Population cultivars are common in autogamous species, such as peach and apricot, but in olive, as a partial outcrossing plant, this term may more probably indicate a generic denomination including different genotypes. It can be concluded that both RAPD and AFLP markers may be useful to detect intracultivar variability when it derives from gamic events or involves important changes in the genome, easily detectable also with random primed markers, while the detection of other kind of differences remains still uncertain and should probably require further investigations. In the case of few polymorphisms for example, it should be necessary the screening of a larger number of primer combinations or the use of other molecular systems. Literature Cited Angiolillo, A., M. Mencuccini, and L. Baldoni. 1999. Olive genetic diversity assessed using amplified fragment length polymorphisms. Theor. Appl. Genet. 98:411–421. Arsel, H., and N. Cirik. 1994. Panorama general sobre las actividades de mejora de olivo en Turquía. Olivae 52:25–27. Asins M.J., A.J. Monforte, P.F. Mestre, and E.A. Carbonell. 1999. Citrus and Prunus copia-like retrotransposons. Theor. Appl. Genet. 99:503–510. Barranco, D. 1997. Variedades y patrones, p. 59–80. In: D. Barranco, R. Fernández-Escobar, and L. Rallo (eds.). El cultivo del olivo. Mundiprensa y Junta de Andalucía, Madrid, Spain. Barranco, D., A. Cimato, P. Fiorino, L. Rallo, A. Touzani, C. Castañeda, F. Serafíni, and I. Trujillo. 2000. World catalogue of olive varieties. International Olive Oil Council, Madrid. España. Bartolini, G., R. Petruccelli, M. Panicucci, M.A. Topón, and G. Di Monte. 1994. Morphological and biochemical evaluation of Olea europaea L., cv. “Leccino”. Acta Hort. 356:78–81. Bartolini, S., R. Guerreiro, and F. Loreti. 2002. Two new clones of cv. Leccino. Acta Hort. 586:225–228. Bassam, B.J., G. Caetano-Anollés, and P.M. Gresshoff. 1991. Fast and sensitive silver staining of DNA in polyacrylamide gels. Anal. Biochem. 80:81–84. Belaj A., I. Trujillo, R. de la Rosa, L. Rallo, and M.J. Giménez. 2001. Polymorphism and discriminating capacity of randomly amplified polymorphic markers in an olive germplasm bank. J. Amer. Soc. Hort. Sci. 126:64-71. Belaj A., Z. Satovic, L. Rallo and I. Trujillo. 2002. Genetic diversity and relationships in olive (Olea europaea L.) germplasm collections as determined by randomly amplified polymorphic DNA. Theor. Appl. Genet. 105:638-644. Besnard, G., P. Baradat, and A. Bervillé. 2001a. Genetic relationships in the olive (Olea europaea L.) reflect multilocal selection of cultivars. Theor. Appl. Genet. 102:251–258. Besnard, G., C. Breton, P. Baradat, B. Khadari, and

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