Theoretical and Applied Genetics

, Volume 107, Issue 4, pp 736–744 | Cite as

Comparative study of the discriminating capacity of RAPD, AFLP and SSR markers and of their effectiveness in establishing genetic relationships in olive

  • A. Belaj
  • Z. Satovic
  • G. Cipriani
  • L. Baldoni
  • R. Testolin
  • L. Rallo
  • I. Trujillo


RAPDs, AFLPs and SSRs were compared in terms of their informativeness and efficiency in a study of genetic diversity and relationships among 32 olive cultivars cultivated in Italy and Spain. SSRs presented a higher level of polymorphism and a greater information content, as assessed by the expected heterozygosity, than AFLPs and RAPDs. The lowest values of expected heterozygosity were obtained for AFLPs, which, nevertheless were the most efficient marker system due to their capacity to reveal the highest number of bands per reaction and because of the high values achieved for a considerable number of indexes. All three techniques discriminated the genotypes very effectively, but only SSRs were able to discriminate the cultivars Frantoio and Cellina. The correlation coefficients of similarity were statistically significant for all three marker systems used but were lower for the SSR data than for RAPDs and AFLPs. For all markers a high similarity in dendrogram topologies was obtained although some differences were observed. All the dendrograms, including that obtained by the combined use of all the marker data, reflect some relationships for most of the cultivars according to their geographic diffusion. AMOVA analysis detected greater genetic differentiation among cultivars within each country than it did between the two countries.


AFLPs RAPDs SSRs Olea europaea L. Genetic relationships 



The authors thank the Dipartimento di Produzione Vegetale e Tecnologie Agrarie, University of Udine (Italy) and the Istituto di Ricerche sul Miglioramento Genetico delle Piante Foraggere, CNR, Perugia (Italy), for the facilities made available to perform the study. Special thanks are due to Dr. L. Zulini for the excellent technical assistance in the laboratory and to Dr. T. Marrazzo and G. di Gaspero for their help at SSRs analyses of data. A. Belaj is in debt to the "Agencia Española de Cooperación Internacional" for the PhD grant.


  1. Angiolillo A, Mencuccini M, Baldoni L (1999) Olive genetic diversity assessed using amplified fragment length polymorphisms. Theor Appl Genet 98:411–421CrossRefGoogle Scholar
  2. Baldoni L, Pellegrini M, Mencuccini M, Angiolillo A, Mulas M (2000) Genetic relationships among cultivated and wild olives revealed by AFLP markers. Acta Hortic 521:275–284Google Scholar
  3. Barlett MS (1937) Some examples of statistical methods of research in agriculture and applied biology. J R Stat Soc [Suppl] 4:137–170Google Scholar
  4. Barranco D (1997) Variedades y patrones. In: Barranco D, Fernández-Escobar R, Rallo L (eds) El cultivo del olivo. Mundiprensa y Junta de Andalucía, Madrid, pp 59–80Google Scholar
  5. Barranco D, Cimato A, Fiorino P, Rallo L, Touzani A, Castañeda C, Serafín F, Trujillo I (2000) World catalogue of olive varieties. Consejo Oleícola Internacional, MadridGoogle Scholar
  6. Bartish IV, Grakava LP, Rumpunen K, Nybom H (2000) Phyologenetic relationships and differentiation among and within populations of Chaenomeles Lindl. (Rosaceae) estimated with RAPDs and isozymes. Theor Appl Genet 101:554–563Google Scholar
  7. Bartolini G, Prevost G, Messeri C, Carignani G (1998) Olive germplasm: cultivars and world-wide collections. FAO, RomeGoogle Scholar
  8. Bassam BJ, Caetano-Anollés G, Gresshoff PM (1991) Fast and sensitive silver staining of DNA in polyacrylamide gels. Anal Biochem 80:81–84Google Scholar
  9. Belaj A, Trujillo I, de la Rosa R, Rallo L, Giménez MJ (2001) Polymorphism and discriminating capacity of randomly amplified polymorphic markers in an olive germplasm bank. J Am Soc Hortic Sci 126:64–71Google Scholar
  10. Belaj A, Satovic Z, Rallo L, Trujillo I (2002) Genetic diversity and relationships in olive (Olea europaea L.) germplasm collections as determined by randomly amplified polymorphic DNA. Theor Appl Genet (online DOI 10.1007/s00122-002-0981-6)Google Scholar
  11. Besnard G, Baradat P, Bervillé A (2001) Genetic relationships in the olive (Olea europaea L.) reflect multilocal selection of cultivars. Theor Appl Genet 102:251–258CrossRefGoogle Scholar
  12. Buteler MI, Jarret RL, LaBonte DR (1999) Sequence characterization of microsatellites in diploid and poliploid Ipomoea. Theor Appl Genet 99:123–132Google Scholar
  13. Cipriani G, Marrazzo MT, Marconi R, Cimato A, Testolin R (2002) Microsatellite markers isolated in olive are suitable for individual fingerprinting and reveal polymorphism within ancient cultivars (Olea europaea L.). Theor Appl Genet 104:223–228Google Scholar
  14. Crouch JH, Crouch HK, Constandt H, Van Gysel A, Breyne P, Van Montagu M, Jarret RL, Ortiz R (2000) Comparison of PCR based molecular markers analyses of Musa breeding populations. Mol Breed 5:233–244CrossRefGoogle Scholar
  15. Dice LR (1945) Measures of the amount of ecologic association between species. Ecology 26:297–302Google Scholar
  16. Excoffier L (1992) winamova ver 1.55-analysis of molecular variance-graphical windows 3.x program for the analysis of population structure from molecular or conventional genetic data. Scholar
  17. Excoffier L, Smouse PE, Quattro JM (1992) Analysis of molecular variance inferred from metric distances among DNA haplotypes: application to human mitochondrial restriction sites. Genetics 131:479–491PubMedGoogle Scholar
  18. Fabbri A, Hormaza JI, Polito VS (1995) Random amplified polymorphic DNA analysis of olive (Olea europaea L.) cultivars. J Am Soc Hortic Sci 120:538–542Google Scholar
  19. Fuentes JL, Escobar F, Alvarez A, Gallego G, Duque MC, Ferrer M, Deus JE, Tohme JM (1999) Analyses of genetic diversity in Cuban rice varieties using isozyme, RAPD and AFLP markers. Euphytica 109:107–115CrossRefGoogle Scholar
  20. Garcia-Mas J, Oliver M, Gómez-Paniagua H, de Vicente MC (2000) Comparing AFLP, RAPD and RFLP markers for measuring genetic diversity in melon. Theor Appl Genet 101:860–864CrossRefGoogle Scholar
  21. Gauer L, Cavalli-Molina S (2000) Genetic variation in natural populations of maté (Illex paraguariensis A. St. -Hil., Aquifoliaceae) using RAPD markers. Heredity 84:647–656PubMedGoogle Scholar
  22. Gemas VJ, Rijo-Johansen MJ, Tenreiro R, Fevereiro P (2000) Inter- and Intra-varietal analysis of three Olea europaea L. cultivars using the RAPD techniques. J Hortic Sci Biotechnol 75:312–319Google Scholar
  23. Gonzalo-Claros M, Crespillos R, Aguilar ML, Canovas FM (2000) DNA fingerprinting and classification of geographically related genotypes of olive-tree (Olea europaea L.). Euphytica 116:131–142CrossRefGoogle Scholar
  24. Hess J, Kadereit W, Vargas P (2000) The colonization history of Olea europaea L. in Macaronesia based on internal transcribed spacer 1 (ITS-1) sequences, randomly amplified polymorphic DNAs (RAPD), and intersimple sequence repeats (ISSR). Mol Ecol 9:857–868CrossRefPubMedGoogle Scholar
  25. Jones CJ, Edwards KJ, Castaglione S, Winfield MO, Sala F, van de Wiel C, Bredemeijer G, Vosman B, Matthes M, Daly A, Brettschneider R, Bettini P, Buiatti M, Maestri E, Malcevschi A, Marmiroli N, Aert R, Volckaert G, Rueda J, Linacero R, Vazquez A, Karp A (1997) Reproducibility testing of RAPD, AFLP and SSR markers in plants by a network of European laboratories. Mol Breed 3:381–390Google Scholar
  26. Lamboy WF, Yu J, Forsline PL, Weeden NF (1996) Partitioning of allozyme diversity in wild populations of Malus sieversii L. and implications for germplasm collection. J Am Soc Hortic Sci 121:982–987Google Scholar
  27. Loukas M, Krimbas CB (1983) History of olive cultivars based on their genetic distances. J Hortic Sci 58:121–127Google Scholar
  28. Mantel N (1967) The detection of disease clustering and a generalized regression approach. Cancer Res 27:209–220PubMedGoogle Scholar
  29. McGregor CE, Lambert CA, Greyling MM, Louw JH, Warnich L (2000) A comparative assessment of DNA fingerprinting techniques (RAPD, ISSR, AFLP and SSR) in tetraploid potato (Solanum tuberosum L.) germplasm. Euphytica 113:135–144CrossRefGoogle Scholar
  30. Mekuria GT, Collins GG, Sedgley M (1999) Genetic variability between different accessions of some common commercial olive cultivars. J Hortic Sci Biotechnol 74:309–314Google Scholar
  31. Milbourne D, Meyer R, Bradshaw JE, Baird E, Bonar N, Provan J, Powell W, Waugh R (1997) Comparison of PCR-based marker systems for the analysis of genetic relationships in cultivated potato. Mol Breed 3:127–136Google Scholar
  32. Minelli S, Maggini F, Gelati MT, Angiolillo A, Cionini PG (2000) The chromosome complement of Olea europaea L.: characterization by differential staining of the chromatin and in situ hybridization of highly repeated DNA sequences. Chromosome Res 8:615–619CrossRefPubMedGoogle Scholar
  33. Morgante M, Rafalski A, Biddle P, Tingey S, Olivieri AM (1994) Genetic mapping and variability of seven soybean sample sequence repeat loci. Genome 37:763–769PubMedGoogle Scholar
  34. Morgante M, Pfeiffer A, Jurman I, Paglia G, Olivieri AM (1998) Isolation of microsatellite markers in plants. In: Karp A, Isaac PG, Ingram DS (eds) Molecular tools for screening biodiversity. Plants and animals. Chapman and Hall, London, pp 75–134Google Scholar
  35. Oraguzie NC, Gardiner SE, Basset CM, Stefanati M, Ball RD, Bus VGM, White AG (2001) Genetic diversity and relationships in Malus sp. germplasm collections as determined by randomly amplified polymorphic DNA. J Am Soc Hortic Sci 126:318–328Google Scholar
  36. Ouazzani N, Lumaret R, Villemur P, di Guisto F (1993) Leaf allozyme variation in cultivated and wild olive trees (Olea europaea L.). J Hered 84:34–42Google Scholar
  37. Ouazzani N, Lumaret R, Villemur P (1996) Genetic variation in the olive tree (Olea europaea L.) cultivated in Morocco. Euphytica 91:9–20Google Scholar
  38. Pejic I, Ajmone-Marsan P, Morgante M, Kozumplik V, Castiglioni P, Taramino G, Motto M (1998) Comparative analysis of genetic similarity among maize inbred lines detected by RFLPs, RAPDs, SSRs and AFLPs. Theor Appl Genet 97:1248–1255CrossRefGoogle Scholar
  39. Powell W, Morgante M, Andre C, Hanafey M, Vogel J, Tingey S, Rafalski A (1996) The comparison of RFLP, RAPD, AFLP and SSR (microsatellite) markers for germplasm analysis. Mol Breed 2:225–238Google Scholar
  40. Rafalski JA, Vogel JM, Morgante M, Powell W, Andre C, Tingey SV (1996) Generating and using DNA markers in plants. In: Birren B, Lai E (eds) Nonmammalian genomic analysis. A practical guide. Academic Press, San Diego, pp 75–134Google Scholar
  41. Rallo P, Dorado G, Martin A (2000) Development of simple sequence repeats (SSRs) in olive tree (Olea europaea L.). Theor Appl Genet 101:984–989CrossRefGoogle Scholar
  42. Rohlf FJ (1998) ntsys-pc. Numerical taxonomy and multivariate analysis system, version 2.00. Exeter Software, Setauket, NYGoogle Scholar
  43. Russell JR, Fuller JD, Macaulay M, Hatz BG, Jahoor A, Powell W, Waugh R (1997) Direct comparison of levels of genetic variation among barley accessions detected by RFLPs, AFLPs, SSRs and RAPDs. Theor Appl Genet 95:714–722CrossRefGoogle Scholar
  44. Sanz-Cortés F, Badenes ML, Paz S, Iñiguez A, Llácer G (2001) Molecular characterization of olive cultivars using RAPD markers. J Am Soc Hort Sci 126:7–12Google Scholar
  45. Sefc KM, Lopes MS, Mendonça D, Rodrigues Dos Santos M, Laimer Da Câmara Machado M, Da Câmara Machado A (2000) Identification of microsatellite loci in olive (Olea europaea) and their characterization in Italian and Iberian olive trees. Mol Ecol 9:1171–1173Google Scholar
  46. Staub JE, Danin-Poleg Y, Fazio G, Horejsi T, Reis N, Katzir N (2000) Comparative analysis of cultivated melon groups (Cucumis melo L.) using random amplified polymorphic DNA and simple sequence repeat markers. Euphytica 115:225–241CrossRefGoogle Scholar
  47. Stewart CN, Excoffier L (1996) Assessing population structure and variability with RAPD data: application to Vaccinium macrocarpon (American cranberry). J Evol Biol 9:153–171Google Scholar
  48. Tessier C, David J, This P, Boursiquot JM, Charrier A (1999) Optimization of the choice of molecular markers for varietal identification in Vitis vinifera L. Theor Appl Genet 98:171–177Google Scholar
  49. Trujillo I, Rallo L, Arus P (1995) Identifying olive cultivars by isozyme analysis. J Am Soc Hortic Sci 120:318–324Google Scholar
  50. Vos P, Hogers R, Bleeker M, Reijans M, Van de Lee T, Hornes M, Frijters A, Pot J, Peleman J, Kuiper M, Zabeau M (1995) AFLP: A new technique for DNA fingerprinting. Nucleic Acids Res 23:4407–4414PubMedGoogle Scholar
  51. Welsh J, McClelland M (1990) Fingerprinting genomes using PCR with arbitrary primers. Nucleic Acids Res 18:7213–7218PubMedGoogle Scholar
  52. Wiesman Z, Avidan N, Lavee S, Quebedeaux B (1998) Molecular characterization of common olive varieties in Israel and the West bank using randomly amplified polymorphic DNA (RAPD) markers. J Am Soc Hortic Sci 123:837–841Google Scholar
  53. Williams JK, Kubelik AR, Livak KJ, Rafalski JA, Tingey SV (1990) DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucleic Acids Res 18:6531–6535PubMedGoogle Scholar

Copyright information

© Springer-Verlag 2003

Authors and Affiliations

  • A. Belaj
    • 1
  • Z. Satovic
    • 2
  • G. Cipriani
    • 3
  • L. Baldoni
    • 4
  • R. Testolin
    • 3
  • L. Rallo
    • 1
  • I. Trujillo
    • 1
  1. 1.Departamento de Agronomía, ETSIAM, Universidad de Córdoba. Ave. Ménendez Pídal s/n, Apdo 3048, 14080 Córdoba, Spain
  2. 2.Faculty of Agriculture, University of Zagreb, Department of Seed Science and Technology, Svetosimunska 25, 10000 Zagreb, Croatia
  3. 3.Dipartimento di Produzione Vegetale e Tecnologie Agrarie, Universita degli Studi di Udine, Via delle Scienze 208, 33100 Udine, Italy
  4. 4.Istituto di Ricerche sul Miglioramento Genetico delle Piante Foraggere, CNR, Via Madonna Alta 130, 06128 Perugia, Italy

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