Simple Sequence Repeat Identification and Endocarp Characterization of Olive Tree Accessions in a Tunisian Germplasm

Most traditional olive-producing countries possess a diversified genetic patrimony in Olea europaea L. Since the emergence of modern olive growing system, the identification, classification, and conservation of autochthonous olive cultivars is a priority for these countries. In this work, a total of 84 accessions belonging to the ‘‘Boughrara’’-Sfax olive germplasm collection located in Tunisia have been screened using a powerful set of eight simple sequence repeat markers (SSRs). The study revealed a high genetic variability among the collection and detected a total of 64 alleles. For better management of the mentioned germplasm bank, an improved classification of the entries, including new denominations, has been proposed. In addition, several cases of mislabeling, synonymy, and homonymy have been clarified. Genetic relationships among cultivars have been analyzed showing four major clusters. Finally, a correspondence factor analysis demonstrated that cultivars tend to cluster depending on their main use as oil or table olives. No clear clustering tendencies were observed when the geographical origin of cultivars was used as the criteria for the analysis. All results obtained by SSR screening and classification were in accordance with classification based on morphological traits of fruit endocarps. Olive (Olea europaea L.) is one of the oldest cultivated species in the Mediterranean basin. Tunisia has played an important role in the establishment of the crop in this area as a result of its dynamic history and geographical location (Trigui et al., 2006). Olive still plays a significant role in both the economy and society of Tunisia today. It occupies onethird of the cultivated land (1,667,000 ha), represents the predominant activity of 29% of farmers, and affects one million Tunisian people’s revenues (Jardak, 2006). Tunisia is considered the second producer and exporter of olive oil in the world, only preceded by the European Union. Tunisian production and exporting averages reach 185,000 tons and 139,600 tons, respectively (averages 2004 to 2009; International Olive Council, 2010). Like most of traditional olive-producing countries (Rallo et al., 2005), Tunisia includes diversified genetic resources, which are mainly formed by small distributed autochthonous cultivars. The characterization of this diversity is essential for the better management of the national germplasm and for genetic breeding programs. The classical approach in the identification of olive cultivars has been based on agronomical and/or morphological studies. It consists of measures such as production average, precocity, vigor, etc. and the direct observation of several distinctive traits on branches, fruits, endocarps, leaves, and inflorescences (Barranco and Rallo, 1984; Barranco et al., 2005). Among the aforementioned morphological characters, endocarps are considered one of the most discriminative traits. These traits are highly inheritable, have low sensitivity to environmental conditions, and are easily observed on collected and stored material (Rallo et al., 2005). A number of analyses has been conducted on Tunisian germplasm material from different regions of the country using morphological and/or agronomical characters (Mehri et al., 1997; Taamalli et al., 2006; Trigui et al., 2002). Several collections of olive cultivars have been created in Tunisia. Among them, the ‘‘Boughrara’’-Sfax olive germplasm collection is considered the most important in terms of cultivar numbers. Although the morphological and agronomical studies are very useful for both exploration surveys (Barranco et al., 2000, 2005; Barranco and Rallo, 1984; Cimato et al., 1993) and germplasm bank characterization (Caballero et al., 2006), results can be influenced by several non-genetic factors. As a consequence, molecular methods are very suitable to reach a better understanding of the material’s genetic diversity. Molecular studies have started with the use of isoenzyme markers (Ouazzani et al., 1993; Trujillo et al., 1995) and at a later stage have been carried out using DNA markers as restriction fragment length polymorphisms (Besnard et al., 2001a), random amplified polymorphic DNAs (Belaj et al., 2001; Fabbri et al., 1995), amplified fragment length polymorphisms (Angiolillo et al., 1999) and microsatellite markers [simple sequence repeats (SSRs)] (Baldoni et al., 2009; La Mantia et al., 2005). The microsatellite technique is one of the most reliable methods used in olive cultivar characterization. It has revealed a high informative level because the markers are polymorphic, multiallelic, and codominant. Moreover, it consists of a relatively simple methodology that permits an easy interpretation of results (Rafalski et al., 1996). SSR markers have been successfully used in germplasm bank classification and contributed to better management of several olive collections around the world (Khadari et al., 2003; Muzzalupo et al., 2006). To provide a better worldwide applicable tool for olive DNA typing, a list of 11 SSR markers has been recently selected among 37 microsatellites available for olive cultivar characterization. The mentioned list has been based on the reproducibility, quality of scoring, information content, independent segregation, Received for publication 22 Apr. 2010. Accepted for publication 10 Aug. 2010. This research has been supported by FEDER cofunded Projects BFU 2004-00601/BFI, BFU200800629, and Junta de Andalucı́a P06-AGR-01791. Research carried out by M. Fendri was funded by AECID. Contribution from the Department of Biochemistry, Cell and Molecular Biology of Plants, ‘‘Estación Experimental del Zaidı́n’’ Granada (Spain) and the Department of Agronomy, University of Córdoba, Córdoba (Spain). We are grateful to the ‘‘Institut de l’olivier’’ Sfax (Tunisia) for providing the plant material used in the study. Special thanks are due to Drs. C. Muñoz Dı́ez, R. De la Herrán, and A. Hamman-Khalifa for their help analyzing simple sequence repeats. To whom reprint requests should be addressed; e-mail juandedios.alche@eez.csic. es. HORTSCIENCE VOL. 45(10) OCTOBER 201