1 Journal of Horticultural Science & Biotechnology (2006) 81 (3) Review Article The classification of olive germplasm A review By T. GANINO 1 *, G. BARTOLINI 2 and A. FABBRI 1 1 Dipartimento di Biologia Evolutiva e Funzionale, Università degli Studi di Parma, Parco Area delle Scienze, 11/A Parma, Italy 2 Istituto per la Valorizzazione del Legno e delle Specie Arboree, CNR-IVALSA, Via Madonna del Piano Sesto Fiorentino (FI), Italy ( (Accepted 6 February 2006) SUMMARY Olive cultivar classification has been a primary concern for olive growers since the earliest times of olive cultivation. As one of the earliest fruit crops to be domesticated, the number of olive genotypes cultivated in the Mediterranean region and elsewhere is high, and the assessment of an easy and undisputed system of classification has become increasingly urgent. This is also because of the high number of synonyms and homonyms in existence. The present review examines the history of attempts, in all olive-producing countries and by many researchers, to achieve an ultimate and exhaustive descriptor list that is valid for all past, present and future genotypes, to support technicians, growers and scholars. After a thorough review of the numerous elaiographical lists proposed, the modern research tools that have been applied to olive (i.e., ultrastructural, biochemical, DNA and molecular markers) are examined critically. A balance sheet has been prepared, taking into account initiatives by major international and national institutions. In Roman times, the need to identify and classify cultivated olive plants present in each area was already clear. Agronomists of the time summarily described all those varieties they could distinguish which had interesting agronomic features. Descriptions exist by Cato, Varro, Virgil and other scholars. However it is difficult to find any correspondence between those varieties and varieties cultivated today. Many of the former are likely to have disappeared due to abiotic and biotic environmental stresses (e.g., frost, drought, parasites), or may have altered due to outcrossing, mutation and/or changes in selection pressure, including grower requirements. Over time, a large number of olive varieties have been identified by numerous scholars who described them by taking into consideration their most varied characters, with remarkable descriptive meticulousness. This led to a large amount of data as the description often concerned an individual tree, irrespective of whether or not it belonged to a known cultivar. The result was to confuse the mind of even the most intelligent agronomists and of the sharpest growers (Caruso, 1883). One of the main difficulties was understanding the names of all these varieties, often described in local dialect or vernacular. Another problem was to relate results from different regions. The first result was an elevated homonymy (i.e., when different olive cultivars have the same name in different zones) and synonymy (i.e., a given variety is named in one area, and recorded with another name in a different area) within a given olive district and between different districts. *Author for correspondence. The same problem exists for scientists and growers today. Determination of characters that can be observed and expressed in all environments (i.e., varietal identification) is the first objective in order to: a) classify and describe the existing varietal heritage; b) describe and isolate all genotypes with valuable characteristics; and c) overcome existing confusion concerning varietal names. Hence the importance of distinguishing and identifying olive genotypes. Knowledge of the morphological, ecological and agronomical characteristics of all available germplasm is indispensable for selection of the most promising individuals, best-suited to modern cultivation techniques. At the same time, commercial requirements can be satisfied, particularly those of nurserymen who need to know the main characters of a genotype to be introduced onto the market, to be able to certify it and, at times, patent it. TAONOMIC ENTITIES THAT ARE THE OBJECT OF CLASSIFICATION The smallest taxonomic entity that can usually be defined with distinction is universally termed a variety. In botany, variety implies some morphological criterion of differentiation, while among agricultural varieties we may have identical morphologies alongside marked distinctions in other characters such as bearing precocity, productivity, or resistance to biotic and/or abiotic factors (Trehane et al., 1995). The term cultivar (cultivated variety) is also commonly used in agricultural terminology. Cultivar is defined by the International Code of Nomenclature for
2 320 History of cultivated olive classification and description Cultivated Plants as an ensemble of cultivated plants, clearly distinguished by any character (morphological, physiological, cytological, chemical, etc) which, when reproduced (sexually or asexually), preserves its distinctive features (Trehane et al., 1995). A cultivar may consist of a single clone, or a number of very similar clones (polyclonal variety), where clone is meant as an ensemble of genetically uniform individuals, derived originally from a single individual by asexual propagation, i.e., by cutting, division, grafting or obligate apomixis (Roselli and Scaramuzzi, 1974). Morphological and biological characters may not always appear constant in all areas and for all clones, due to the influence of the particular pedoclimatic conditions in which they may be cultivated. It is therefore necessary to introduce the concept of ecotype ; that is, the peculiar biological, morphological and/or agronomic appearance that cultivars and clones may assume in different environments, usually over a limited geographical range (Lorenzetti et al., 1996). In perennial plants, the concept of a variety is often undetermined, as varieties are often grouped with reference to a number of common characters, allowing a degree of variation for other phenological features. However, it is precisely in perennial plants, including grapevines, olives and the most important fruit crops, that we have devised an empirical selective method, vegetative propagation. This method permits the preservation of genetic inheritance in the progeny, unchanged and easily usable. The most ancient domesticatied fruit crops in the Mediterranean Basin have therefore been those that could be easily propagated from plant parts (e.g., fig, date palm, olive, pomegranate, grape). The number of olive cultivars described has been increasing since ancient times due to progress in taxonomy and plant breeding techniques. To comply with the need for a practical classification of cultivated plants, Marinucci (1908) suggested substitution of the term variety with agrarian race. For olive, this term has not been further explained or adopted. For Marinucci (1908) it applied to groups of individuals, agamically propagated over several generations, and therefore each group should be termed a clone (Baldini and Scaramuzzi, 1952), or a set of clones. The term race was eventually replaced by cultivar, although perhaps too quickly. In fact, all known cultivars should still be considered as wide and heterogeneous populations of clones, separated by a number of characters. CHRONOLOGY OF THE PROPOSED CLASSIFICATION SYSTEMS We owe the first olive classification system to the botanist Pitton de Tournefort (1719). He was soon followed by numerous other botanists, who suggested or adapted several systems to describe large number of olive varieties, but they never achieved an accurate or practical classification (Prevost and Mostardini, 1999; Table I). In Spain, the first to attempt an olive classification in 1815 was Simòn de Rojas Clemente (Barranco et al., 1984), based on features of leaves and fruits in a number of varieties. The proposal to take the olive stone into consideration as a discriminating character in the classification of Italian germplasm was made by Tavanti (1819). He was convinced that endocarp characters had fundamental importance in the study he was to undertake on olive races. The stone was considered both in its whole shape, and in its separate and minute aspects: the base, the apex, the valves and the course of the suture lines of the valves that form the stone. Tavanti (1819) based the distinction he made of Tuscan cultivated olive varieties on these five characters, and accordingly divided them into 21 groups. Analogous classification systems were eventually proposed by Marinucci (1908), who reduced the number of groups listed by Tavanti to five, by Zito (1932), by Frezzotti (1937) and by Savastano (1939). But endocarp characters were no longer considered sufficient, and other characters (morphological, biological and ecological) were added, in order to satisfy technical requirements and to take into account the influence of environment. With this in mind Ciferri and Breviglieri (1942) made the first attempt at a morpho-ecological classification by enforcing the criteria adopted by the Russian school of Vavilov. In all papers of the time, the use of morphological characters was aimed at achieving a sub-division of cultivars according to morphological TABLE I Comparison of different classification systems in the history of olive identification from 1719 to the present-day in various olive-producing countries Year Authors Country Discriminant characters 1719 Pitton de Tournefort France Botanic characters 1815 Simón de Rojas Clemente Spain Leaf and fruit 1819 Tavanti Italy Endocarp 1908 Marinucci Italy Endocarp 1917 Ruby France Leaf, fruit and endocarp 1932 Miliani Italy Ecological characters 1932 Zito Italy Endocarp 1937 Bracci Italy Leaf, inflorescence, fruit, endocarp and ecological characters 1937 Frezzotti Italy Endocarp 1939 Savastano Italy Endocarp 1942 Ciferri et al. Italy Morphological, biological and agronomic characters 1954 Patac Spain Morphological, botanical and agronomic characters 1984 Barranco and Rallo Spain Morphological and agronomic characters 1985 UPOV World Morphological and agronomic characters 1986 Leitão et al. Portugal Morphological and agronomic characters 1998 Bartolini et al. World Passport, agronomic characters, biochemical and molecular markers, collection, etc Barranco et al. World Passport and agronomic characters 2000 Pannelli et al. Italy Morphological, agronomic and commercial characters 2005 Rallo et al. Spain Passport, morphological, agronomic and commercial characters
3 T. GANINO,G.BARTOLINI and A. FABBRI 321 variations in fruits, leaves, inflorescences, endocarp and other organs. Progress lay in increased numbers of characters taken into account. In 1917, in France, Ruby proposed a classification system in which three characters were considered: fruits, stones and leaves. With these, and with the ratios between maximum transverse and longitudinal lengths, data were obtained which were typical of groups of plants, and which enabled differences between varieties existing in the territory to be distinguished and evaluated. Another tentative classification, this time based on ecological grounds, was that of Miliani (1932), who distinguished some Italian olive races with reference to their resistance to low temperatures and arid conditions, to hardiness, to productivity and to the quality of the product. According to the considerations of Ciferri and Breviglieri (1942), and because of the inadequacy and limited usefulness of schemes suggested in previous years, Ciferri and other researchers (Ciferri et al., 1942) elaborated more acceptable solutions. In the classification then put forward, numerous morphological and biological characters were considered, as well as criteria adopted to evaluate the importance of each character. Based on these elements, a very detailed elaiographic (from ancient Greek elaion, pertaining to olive oil) descriptor list was prepared, and used for data collection on approx. 70 cultivars grown in Central Italy (Tuscany, Latium, Marches, Umbria). Where data collection was carried out with care, in a circumscribed environment and in such conditions as to exclude environmental variables, this list allowed varietal identification with an acceptable degree of reliability. The introduction of agronomic and morphological-ecological characters had therefore become necessary, as had been proposed by Caruso in his olive monograph (1883). Caruso had stressed the fact that an ordering of races must be clear and able to put anyone in the condition of being able to appreciate and choose the olive trees according to their agrarian prerequisites (Morettini, 1972). Zonal limitations, however, do not allow the identification of synonyms (i.e., the various names by which the same cultivar is identified in different environments) which is precisely one of the main tasks of any classification. However, a study in 1942 by Ciferri et al. is still considered today when studying new classification systems. Ciferri et al. (1942) acknowledged that their system had some limitations, largely because olive is a very homogeneous species, and consequently subdivision of Italian proles into sub-proles is difficult. In addition, the values considered were so variable, due to different cultivation techniques and environmental conditions, that their exact definition is impossible, including the classification of the same cultivars into proles and sub-proles (Zito, 1942). Further discussion was on the terminology adopted. The word average, as used to describe characters, has a different meaning for statisticians, defines nothing for the practical technician (grower), and is never sufficient to define a given character. Despite criticisms raised at the time of its publication, the 1942 Descriptor List eventually became the starting point and a fundamental reference for all researchers in the field. Towards the end of the 1940 s, Patac propounded a system for the identification of cultivars existing in Spain (Patac et al., 1954). The assumption that morphological characters were highly influenced by environmental conditions and by human activities led Patac to consider as important, all characters that may give information concerning olive transformation, without omitting the description of characters deemed to be more strictly botanical. This innovation consisted of introducing a hierarchy of importance among the characters used. In decreasing order of importance, therefore, were the shape and size of the endocarp, the shape and size of fruits, features of the tree, shoots, inflorescences and seed, and organoleptic characters of the oil. For each character, coefficients and biometric indices were developed and included. Thus, the descriptor aimed to satisfy classification, agronomic and industrial needs. The system, and the problems related to it, were discussed at the International Olive Congress in Seville, in 1950 (Patac et al., 1954). Over the next 30 years, no major innovations were introduced into olive classification. The innovative proposal of Patac s descriptor was not well-received, and the reference document remained the pomological list suggested by Ciferri et al. (1942). The need to understand the complexity of olive germplasm was particularly strong in countries in which the olive industry was most developed. In fact, in the 1980 s interesting proposals were made in Spain, Portugal and by UPOV (Union Internationale pour la Protection des Obtentions Végétales) in Geneva. In 1984, Barranco et al. put forward pomological lists in which the description of the tree and of its fruiting branches, leaves, inflorescences, fruits and endocarps were considered fundamental characters, to catalogue and identify all Andalusian olive cultivars. This descriptive scheme, though, was confined by taking into account that of Ciferri et al. (1942), with a few changes introduced by Bottari and Spina (1953) and by the authors. The changes introduced included using the largest possible amount of information, eliminating characters that were most difficult to obtain and those most influenced by the environment, as well as others considered irrelevant and/or subjective. In addition, for each character, Barranco et al. (1984) tried to use as few categories as possible, to separate cultivars without creating too many classes. To make interpretation and reading easier, each file was accompanied by photographs of those organs important for the description. In 1985, UPOV proposed a specific descriptor list for olive. In that list, whole plant characters, fruiting shoots, leaves, inflorescences, flowers, fruits and endocarp were taken into consideration. The aim was to standardise the methodology of data collection, and to indicate a leader list usable by all researchers who seek to describe olive cultivars. This initiative represented the second most significant attempt, after Ciferri et al. (1942), at classifying all olive germplasm existing in various countries with an homogeneous method. In 1986, Leitâo described 22 Portuguese olive cultivars. For each cultivar, tree, shoot, leaves, flowers and inflorescences, endocarp and fruit characters were considered, together with agronomic characters. For the
4 322 History of cultivated olive classification and description author, this technique had the merit of allowing an easy and fast plant description, without resorting to sophisticated and difficult techniques. The aim was to provide a summary description of the plant, while focussing interest on some characters (leaf, inflorescence, fruit and endocarp) that were considered interesting for the identification and characterisation of cultivars. Leitâo also took general and agronomic aspects, such as rooting ability, disease resistances and susceptibilities into account. The whole description of each cultivar was accompanied by photographs of plant organs. In 1993, in Tuscany, the problem of describing and identifying olive germplasm was again on the agenda, this time involving plants kept in collections in the Provinces of Pistoia, Grosseto and Firenze. The descriptor list proposed by Cimato et al. (1993) included descriptions of the tree, shoots, leaves, inflorescences, fruits and endocarp. For a description of the different characters, reference was made to the UPOV descriptor. By applying a few changes, Cimato et al. (1993) deemed it indispensable to reduce environmental effects on the phenotypic expression of characters. Such changes were not clear, and no adequate explanation was given. In the same year, an elaiographic descriptor was produced for olive germplasm in Catalonia (Tous Martì and Romero Aroca, 1993). No major methodological innovations were adopted. Cristoferi et al. (1997) propounded a list for the identification and classification of Romagna olive cultivars. For each cultivar, the list contained information on its diffusion, synonyms, elaiographic characters (e.g., tree, leaf, inflorescence, fruit, endocarp, etc.), biological and agronomic characters. In the following year, Bartolini and collaborators (1998) advocated the use of a simplified pomological descriptor for the World s olive germplasm. Such a list had plant passport data, tree data and agronomic characters (e.g., productivity, oil yield, tolerance to biotic and abiotic stresses, etc.). For the first time, important information was added such as the existence of patents, any presence in germplasm collections, and where, and data concerning biochemical and molecular characterisations. Soon a scheme was elaborated for the identification of Marches olive cultivars (Pannelli et al., 1998), which was also eventually used for Umbrian cultivars (Pannelli et al., 2000). The first scheme was generated by describing the tree, shoots, leaves, inflorescences, fruits and agronomic behaviour. For the description of morphological characters, reference was made to the terminology adopted by UPOV, with a few minor changes concerning the elimination of some characters that were hard to detect, and the addition or modification of others. As regards agronomic characters, for each cultivar judgments were expressed according to results obtained over several years and in different situations. The work was thus simplified, as only essential characters were used. Practical considerations were also expressed on harvest time, taking into account oil accumulation in the drupes, and judgments on oil quality characteristics and plant productivity; information was also given on the suitability of fruits for milling and extraction with a pressure system. For the first time, technical information was introduced for the producer, relating to the milling and extraction process. A World Catalogue of Olive Varieties (Barranco et al., 2000a) has recently been published by The International Olive Oil Council (IOOC), with a classification system based on descriptor lists which are valid for all olive producing areas. Once more, the need to use a unified and valid classification system was stressed, to guarantee the conservation of olive germplasm. The file consists of a list of descriptive characters, accompanied by a synthetic bio-agronomic evaluation. The information is divided into three groups: plant passport data, morphological characters, and agronomic and commercial considerations. The first group contains the most common name of the cultivar, its synonyms, its country of origin [i.e., the country of most probable cultivar provenance, or in which it has reached the largest distribution, using International Standards Organisation (ISO) codes], the main growing areas, the importance of the cultivar in these areas, and the purpose(s) of production. The morphological characters are structured as primary descriptors used for the identification and characterisation of each cultivar. The characters have been structured according to qualitative (shapes, expression of morphological features) and quantitative (biometric indices) descriptors. The agronomic and commercial considerations contain data that are useful for growers, scientists and technicians interested in the olive industry. The IOOC proposal, which was the result of a joint study by experts and scientists throughout the olive world, is the latest attempt to unify the methodologies of olive classification. In recent years, others have suggested other descriptor lists, starting from existing ones. Again Cimato et al. (2001) published elaiographic lists of Tuscan olive germplasm, relying on former work elaborated by the same authors in Several other elaiographic lists have been elaborated recently (Cicoria et al., 2000; Pugliano et al., 2000; Rotundo and Marone, 2002; Trigui et al., 2002; Parlati and Pandolfi, 2003; Lombardo et al., 2003; 2004; Bassi, 2003). That of Rotondi et al. (2004) appears to be quite interesting. Besides the characters formerly listed in previous work (Cristoferi et al., 1997), detailed information is added on the organoleptic and chemical properties of the oil and, for the first time, on the phytosanitary situation of each cultivar. A monograph by Rallo et al. (2005) has very recently been published in which the elaiographic lists of all Spanish cultivars are collected together (including plant passport, morphological, agronomic and commercial characters). The starting point for this descriptor was that of Barranco et al. (2000a) which also contained information on plant passport, tree, leaf, fruit, endocarp and agronomic features. The distinctive features of the most recent elaiographic lists undoubtedly lie in the introduction of more refined methods of study such as molecular markers and the chemical and organoleptic analyses of oil. Varietal identification by such methods is made difficult by the nature of the species, which is often characterised by the presence of cultivar-populations or populations of clones, rather than by clear cultivars (Bartolini et al., 1992; 1994a). Thanks to statistical analyses of the variability of the least variable individual characters, most suitable
5 T. GANINO,G.BARTOLINI and A. FABBRI 323 characters were identified: leaves, drupes, stones (Baldini and Scaramuzzi, 1952; 1955; Damigella, 1960). More recently, several methods of multivariate statistical analysis have shown the existence of numerous biometric variables related to the fruit and to the plant, or to the acid composition of the oil, which have a satisfactory discriminating power (Barone et al., 1994a,b; Perri et al., 1995; Cantini et al., 1999; Barranco et al., 2000a). Notwithstanding, morphological markers remain poorly discriminating for varietal identification, as they are characterised by high environmental variability. Their study must therefore be integrated by recent ultrastructural, artificial neural networks (ANN), biochemical and molecular genetic techniques. These new research tools represent a turning point to resolve problems in olive taxonomy. Ultrastructural analyses: Some authors (Quiros, 1975; Maas, 1977) proposed pollen morphology (section, shape, etc.) as a useful tool for varietal identification. Pollen features, although part of the morphological characters, are considered intermediate between fully morphological characters and biochemical ones (Petruccelli, 1992; Bartolini et al., 1994b). In particular, exine structure (i.e., the outer coating of the cell wall surrounding the pollen grain at maturity) is considered, as its characteristics (grain diameter, net mesh size and shape, etc.) are under the control of the sporophyte (pollen mother cells; Pandey and Troughton, 1974). Wall structure is therefore a more stable character compared to other morphological characters, as it is specifically genetic, with little or no influence from the environment. Roselli (1979) was the first to use pollen ultrastructural analysis for olive cultivar identification. He studied 13 table olive cultivars; however, the research did not produce sufficient information for the identification of cultivated varieties. In 1995, Lanza et al., used scanning electron microscopy to study the morphology of exine patterns on pollen grains of four olive cultivars. The data obtained allowed complete differentiation of the four genotypes (Lanza et al., 1995). These analyses, however, give little information for the identification of the individual cultivar (Bartolini and Petruccelli, 1994). Further studies on several other anatomical and ultrastructural aspects have also not yielded encouraging results. ANN: Interesting results were obtained by applying the technique of backpropagation neural networks (BPNN) to ten olive cultivars. Sets of phyllometric parameters were used for cultivar identification and the results, albeit preliminary, could be considered encouraging (Mancuso and Nicese, 1999). Biochemical markers: Total and storage proteins, isozymes and molecular markers (RFLPs, RAPDs, AFLPs, micro- and mini-satellites sequences) permit the elimination of two negative aspects of phenotypic analyses: the use of polygenic (quantitative) characters and genotype environment interactions. The basic concept of biochemical and molecular methodologies has been presented by Larsen (1969). He maintained that All morphological displays of varieties must, in the end, have a biochemical differentiation, but not all biochemical characterisations necessarily have a morphological display; hence, biochemical characters will be more numerous than morphological characters. For example, the genotypes of several individuals might be distinguished by their phenolic content, as shown for a number of species by several authors (Singh and Thompson, 1961; Brown et al., 1971). In olive, these compounds have been used to separate ten cultivars, but the results did not prove to be sufficient to discriminate among plants (Heimler et al., 1994). Since the 1970s, protein analyses (total proteins and/or storage proteins) have been as a method to identify and distinguish species and cultivars. Storage proteins are well-suited for use as genome markers. Protein analysis, compared to other biochemical markers, has the advantage of simplicity of extraction and separation technique, although the information it provides on genetic variability is less exhaustive. In olive, seed storage proteins (Durante et al., 1992) and total leaf proteins (Petruccelli, 1992) have been studied. Enzymes are specialised proteins that catalyse the numerous chemical reactions that occurr in living beings. The term isoenzyme (or isozyme) was first proposed by Markert and Moller (1959) to indicate multiple molecular forms of an enzyme, catalysing the same biochemical reaction. Isoenzymatic electrophoresis permits the detection of genetic variability in several enzymatic proteins. Varietal differences are shown by means of enzymic polymorphism on activity gels.therefore the analysis of a protein structure, by means of electrophoresis, is approximately an analysis of the gene (Gottlieb, 1977). Isozyme polymorphism became a simple marker to analyse genetic relationships within a population (Gottlieb, 1981). However, isoenzymes suffer several limitations: the genetic (isoenzymatic) loci tested represent a very small portion of the structural genes in the whole genome; the variation present in a population can not be expressed in full; only some nucleotide substitutions that occur at the DNA level can be identified because variations in amino acid sequence do not always take place. Nevertheless, numerous authors (Pontikis et al., 1980; Trujillo et al., 1989; Roselli et al., 1990; Petruccelli et al., 1992; Seker et al., 2005) employed isozymes to identify olive cultivars. In recent years, in parallel with progress in biochemical and bio-molecular marker techniques (Pontikis et al., 1980; Ouazzani et al., 1993; Trujillo et al., 1995; Hatzopoulos et al., 2002), molecular fingerprinting methodologies have been established. In particular, the introduction of DNA markers and of DNA fingerprinting techniques provide a high resolution discrimination system that is independent of environmental conditions and able to contribute to the resolution of problems such as mis-identification in germplasm collections, nursery certification, varietal protection, and/or guaranteed labelling for certification of geographical origin (Powell et al., 1996; Testolin, 2000). The application of these technologies has already permitted several advances: i) taxonomical revision within the genus Olea and forms connected to Olea europaea L.; ii) clarification of the relationships between wild and cultivated forms; and iii) contributions to the study of variability within the olive genome, by
6 324 History of cultivated olive classification and description identifying markers associated with specific characters of agronomic interest (Rugini and Baldoni, 2003). The first molecular marker technique employed on olive was Restriction Fragment Length Polymorphisms (RFLPs; Gallitelli et al., 1991). RFLP markers confirmed that the Mediterranean Basin was the site of domestication of this fruit crop (Besnard et al., 2001). This technique has also been used by De la Rosa et al. (2003), when 95 plants obtained by crossing two cultivars ( Leccino and Dolce Agogia ) were analysed. Randomly Amplified Polymorphic DNA (RAPD) has been applied successfully in many investigations aimed at the study of polymorphism in olive cultivars, and at ways to distinguish them (Bogani et al. 1994; Fabbri et al., 1995; Cresti et al., 1996; Mekuria et al., 1999; Nikoloudakis et al., 2003; Perri et al., 1999; Gonzalo Claros et al., 2000; Russo et al., 2001; Bronzini de Caraffa et al., 2002; Guerin et al., 2002; Mailer and May, 2002; Fabbri and Ganino, 2003; Belaj et al., 2001; 2002; 2003a; 2004a; Mir Ali and Nabulsi, 2004; Ganino and Fabbri, 2005; Hossein-Mazinani, 2005). The technique was also employed by Gemas et al. (2000) and by Roselli et al. (2002) to evaluate intra- and inter-cultivar variability. Both RAPD and RFLP have contributed greatly to the taxonomic classification within the genus Olea (Besnard et al., 2002). Mini-satellites, or Variable Number of Tandem Repeats (VNTR), are based on the study of small genome fragments which can be isolated due to their specific length. They consist of tandem repeats of a short DNA sequence, and polymorphism is due to the number of times the element is repeated in the genome (Soller and Beekman, 1983). To use mini-satellites, it is not necessary to create genomic libraries, but the technique is expensive. Contento et al. (2002) carried out a study based on tandem repeats in three Olea europaea L. nuclear DNA sequences. Amplified Fragment Length Polymorphisms (AFLPs) are very sensitive when seeking polymorphism in the whole genome (Ranamukraarachchi et al., 2000). The use of AFLP markers made it possible to evaluate the genetic relations between different forms of Olea and the cultivated olive, thus contributing to our understanding of the relative genetic distances separating forms that belong to different geographic areas (Angiolillo et al., 1999; Ambrosino and Rao, 2001; Ambrosino et al., 2002; Labombarda and Fontanazza, 2002; Ricciolini et al., 2003; Sanz-Cortes et al., 2003; Grati Kamoun et al., 2005; Kalaitzis et al., 2005; Owen et al., 2005; Strikic et al., 2005). By employing AFLPs, genetic affinity was also analysed, in an attempt to clarify situations of synonymy and homonymy in a number of cultivars from several Italian regions: Sicily (Angiolillo et al., 1999; Baldoni et al., 2002; 2003a; Labombarda and Fontanazza, 2004); Sardinia (Angiolillo et al., 1998); Umbria (Marchionni et al., 1999; Labombarda et al., 2002; Baldoni et al., 2003b); Apulia (Resta et al., 2002); Marche (Baldoni et al., 2001); Molise (Pilla et al., 1999); Campania (Ambrosino et al., 2003); and Colli Euganei (Ziliotto et al., 2002). The simultaneous use of AFLPs, RAPDs and morphological characters made discrimination possible between olive cultivars (Hagidimitriou et al., 2005) Simple Sequence Polymorphisms, Simple Sequence Repeats (SSR) or microsatellites locate the DNA regions that are characterised by a tandem repeat of only 1 6 bp in a sequence. Microsatellites are widespread and dispersed in the genomes of all plants, and display an elevated level of hypervariability within each species. These features makes them excellent both for gene mapping and fingerprinting. Presently, the SSR technique has provided reliable markers to solve the problems of varietal identification and for genetic characterisation of olive because of their high transferability, elevated polymorphism and codominance, which is particularly interesting for olive (Sefc et al., 2000; Bandelj et al., 2002; Carriero et al., 2002; Marrazzo et al., 2002; Belaj et al., 2004b; 2005a; Bracci et al., 2005; Diaz et al., 2005a, b; Lopes et al., 2005). Further research using microsatellites to identify olive cultivars was carried out by Rallo et al. (2000) who analysed 46 genotypes from several countries. In 2001, the SSR technique was also used to characterise ancient olive cultivars from Central Italy and Lake Garda (Cipriani et al., 2001). Morphological and phenological aspects, as well as molecular investigations (SSR markers) were used in Sicily to characterise germplasm existing in the region (Caruso et al., 2005). With the combined use of RAPDs and SSRs, the solution to some cases of synonymy was made possible (Barranco et al., 2000b). The same combination of markers led to the identification and characterisation of Iranian olive cultivars (Shahriari et al., 2005). Simultaneous use of AFLPs and SSRs clarified relationships between some Slovenian and Italian olive cultivars (Bandelj et al., 2004), within Italian, Spanish, French and Greek cultivars (Montemurro et al., 2005), and within Emilia-Romagna germplasm (Rotondi et al., 2003). RAPDs,AFLPs and SSRs were used by Belaj et al. (2003b) to evaluate the genetic relationships between Italian and Spanish cultivars. Later, Belaj et al. (2005b) used isozymes, RAPDs and SSRs to investigate the relationships between olive cultivars conserved in the World Olive Collection at Córdoba. Single nucleotide polymorphisms (SNPs) represent a new and alternative technique for molecular identification. By this type of analysis it is possible to identify, in a very short time, differences between genomes due to a single base-pair (Cooper et al., 1985). These markers have been shown to be highly discriminatory both in the human genome (Collins et al., 1998) and with plants, particularly maize (Rafalski et al., 2001) and soybean (Rafalski, 2002). In olive, SNPs were used for the first time to classify 51 olive cultivars, with satisfactory results (Diaz Bermudez, 2005). The integration between statistical methods applied to biometry and to bio-molecular analyses is less welldeveloped. Some recent applications of both methodologies to the same genetic pool (Bassi et al., 2002; Rotondi et al., 2003) have demonstrated that the two procedures do not always give matching representations of similarity relations between cultivars. Although, in some cases, only molecular analyses were able to characterise specific olive cultivars and to identify erroneous classifications and synonyms, there have also been cases in which molecular analysis alone
7 T. GANINO,G.BARTOLINI and A. FABBRI 325 did not allow the separation and distinction of presumed clones which, however, presented visible differences in agronomic and morphological characteristics, and in the properties of their oils (Perri et al., 1995). To conclude, improvements in biochemical and molecular techniques are proving to be valid instruments for research in plant taxonomy, and for the identification and characterisation of varieties. The improvement of such techniques is still in progress and, although an increasing number of papers are being published in which these are employed, the old variety descriptor list, with its indications of morphological and agronomic traits, retains its importance and usefulness, and is far from having become obsolete. THE IMPORTANCE OF A CLASSIFICATION A journey through successive classifications since 1900 (Table II) shows a profound change in the choice of characters adopted for cultivar identification. In the beginning, botanical characters were of greatest interest, with some agronomic indications. A typical example is the famous descriptor of Ciferri et al. (1942) which, in its discrimination into families, merely returns to the systematic concept of likeness between individuals. Next, a change occurred as to choice of characters to describe morphological and agronomic features. This caused the loss of the systematic aspect in all subsequently proposed descriptors. By the end of the 1990s, it was felt necessary to introduce more technological and scientific characters, such as accurate chemical and organoleptic oil analyses and molecular characterisation. It is also true that adoption of a new description procedure was not universal. On the contrary, each author tended to represent and satisfy the needs of the context in which s/he operated. This led to the publication of descriptor lists which were similar but different, useful and interesting for one sector of the industry, but less so for another. For example, a descriptor list that focusses on morphological characters is seldom used by geneticists. Geneticists aim to separate genotypes that differ in a number of characters by eliminating, or at least reducing, the environmental component, and this is possible only within a common environment, or by using methodologies that are not influenced by the environment. An elaiographic methodology for olive is important for, and must satisfy the needs of: a) the botanist, to obtain valid information to begin any classification; b) the agronomist, to optimise cultivation techniques; c) the oil mill operator, to know the characteristics of the fruit and its oil content; d) the geneticist, as a starting point for genetic analyses and various breeding techniques; and e) the nurseryman, who must be certain he is propagating the most suitable material to market with due guarantees. The need to construct an elaiographic descriptor that includes all characters useful for the complete description of genotypes, therefore has the purpose of supporting both the production sector and scientific research. The industry-oriented role is clear, there is a perpetual need to improve production, both quantitatively and qualitatively. This can only be achieved if the characteristics of cultivars from various regions are assessed, and there is easy access to certified propagation material. These requirements can only be satisfied using identification tools of undisputed efficacy. We have seen that a consideration of morphological characters alone is not sufficient to completely identify cultivars, although it represents an important advance in knowledge. For a better, more definitive description it is necessary to grow and study plants in the same environment, in which the features of each genotype can be evaluated and compared according to their intrinsic characters. Such evaluation is more accurate if more than one environment is used. Hence the usefulness of reuniting those genotypes to be studied in the same collection fields, possibly covering national or regional germplasm. A collection field, in order to deserve such a definition, must contain plants of indisputed origin, with sufficient numbers of each accession to allow correct evaluation and to guarantee survival. In addition, the collection must be accessible to all scholars, to facilitate comparisons between germplasm, and to start breeding programmes. From a scientific point of view, individual cultivars can be regarded as potential sources of genetic material, to be used to transmit characters concerning productivity, disease resistance or tolerance, etc., to new genotypes produced through breeding programmes. Thus, the protection of olive germplasm is particularly important, as it includes the gene pools of both domestic varieties and related wild species, amenable to cross-breeding. The need to safeguard our existing genetic heritage therefore appears increasingly urgent. But this is only feasible insofar as it is possible to identify, catalogue, and eliminate synonyms and homonyms still present in germplasm in all olive-producing countries. From the above, the need to establish a varietal standard (as has been done with other fruit species) becomes apparent. This requires the creation of a reference point, obtained by using averages of all known characters, so that all fluctuations in a population can serve to define the cultivar more precisely. The more we can eliminate environment-related variables by creating collections and by working in a restricted environment with a given number of trees per variety, the more reliable will be this reference. At the same time, it is extremely important to describe each plant in its original environment, where it grew and developed, and at different sites. The IOOC acknowledged the need to have the largest number of olive genotypes in the same environment, and advocated the organisation of three World germplasm collections located in Spain (Cordoba), Italy (Cosenza) and Morocco (Marrakech). The Spanish collection is already established. The other two are now being realised. Today, although all published monographs on olive classification refer to clearly defined areas, their reliability is fairly low. This is because all authors tend to describe only those cultivars present in their own territory, and disregard those present in adjacent areas and/or in other olive-producing countries, with their frequent homonyms, synonyms and discrepancies as to the classification of the same variety. In addition, this
8 326 History of cultivated olive classification and description TABLE II Comparison of descriptor lists established since 1942 according to the characters they describe and olive growing areas LIST A PLANT PASSPORT DATA Cultivar name Synonyms Origin Diffusion Area of major diffusion Frequency of cultivation % Presence of the cultivar Value Fruit purpose Biochemical characterisation Molecular characterisation Patents Existence of collections LIST B CHARACTERS OF THE TREE Average height Vigour Growth habit Canopy colour Canopy width Canopy shape Canopy section Canopy volume Canopy vigour Canopy density Inflorescence emission Fruit set Trunk size Trunk shape Trunk surface Bark appearance and colour Trunk section LIST C CHARACTERS OF MAIN SCAFFOLDS Average size Shape Surface Colour Growth habit LIST D CHARACTER OF FRUITING SHOOTS Abundance Size Shape Surface Growth habit Length Section Internode length Colour Shoot Feathers *Ciferri et al., 1942, Italy (A); Patac et al., 1954, Spain (B); D Amore et al., 1977, Calabria (Italy) (C); Loussert et al., 1978, World (D); Barranco et al., 1984, Andalusia (Spain) (E); UPOV, 1985, World (F); Leitão et al., 1986, Portugal (G); Cimato et al., 1993, Tuscany (Italy) (H); Tous Martí and Romero Aroca, 1993, Catalonia (Spain) (I); Cristoferi et al., 1997, Emilia Romagna, (Italy) (J); Bartolini et al., 1998, World, (K); Pannelli et al., 1998, Abruzzo (Italy) (L); Cicoria et al., 2000, Molise (Italy) (M); Pannelli et al., 2000, Umbria (Italy) (N); Barranco et al., 2000a, World (O); Pugliano et al., 2000, Campania (Italy) (P); Cimato et al., 2001, Tuscany, (Italy) (Q); Rotundo and Marone, 2002, Lucania (Italy)(R); Trigui et al., 2002, Tunisia (S); Parlati and Pandolfi, 2003, Lazio (Italy)(T); Lombardo et al., 2003, Calabria (Italy)(U); Bassi, 2003, Lombardy (Italy) (V); Lombardo et al., 2004, Apulia (Italy)(W); Rallo et al. 2005, Spain (Y). The characters column carries as accurate as possible a translation of the original terms. When terminology was similar and obviously indicated the same character, the terms were unified in English and listed in the same row; otherwise the character was given a separate row, especially if, in the original publication, no clear indications were given as to the meaning of the character.
9 T. GANINO,G.BARTOLINI and A. FABBRI 327 LIST E CHARACTERS OF THE LEAF Abundance Shape Length/ Width ratio Size Length Width Symmetry Tile curvature Veins Midrib Secondary veins Mucro Petiole Transverse section Leaf bearing % Stomata open in the hottest hours Margins Blade Longitudinal curvature of the blade Surface Profile of blade Torsion Apex angle Base angle Position of maximum width Consistency Colour Dorsal colour Ventral colour Brightness Abnormal leaves Shape of abnormal leaves LIST F CHARACTERS OF THE INFLORESCENCE Time of development Position Shape Rachis length Peduncle length Maximum length Branching type Colour at anthesis Presence of axillary flowers Density Arrangement Number of inflorescence per branch Total number and position of flowers Flower number/inflorescence % Terminal inflorescences % Simple and compound inflorescences Size of swollen buds Frequency of supernumerary flowers Rachis structure Rachis branching Time of full bloom Flower diameter Corolla colour Petal length Stylus length Shape and size of stigma % Abortive flowers Number of fertile flowers % Fertilisation Self-pollination or heterogamy Level of incompatibility Incidence and localisation Axillary flowers Pollen
10 328 History of cultivated olive classification and description LIST G CHARACTERS OF THE FRUIT Fruit number per infructescence Size Weight Volume and weight: averages and upper values Length Symmetry Shape Diameter medium Average length/average diameter ratio Maximum diameter Shape of transverse section Symmetry in position A Symmetry in position B Section Shape maximum transverse section Position maximum transverse diameter Shape of apex in position A Shape of apex in position B Shape of base in position A Shape of base in position B Pedicel insertion Pedicel length Pedicel diameter Shape of pedicel cavity Size of pedicel cavity Depth of pedicel cavity Umbo Mucro Pulp/endocarp ratio Sarcocarp type (at maturity) Sarcocarp consistency (at maturity) Sarcocarp (%) Epicarp surface Epicuticular wax coating Lenticels, coloration, frequency Lenticels size Perceptibility of lenticels at maturity Epicarp Fruit distribution in 2 3 year-old plants Colour uniformity at maturity Colour before maturity Colour at maturity Colour at harvest Colour evolution Pulp consistency Adhesion pulp to epidermis Mesocarp colour Cuticle thickness Position of pistil scar Resistance to abscission Time of maximum oil content Mesocarp clinginess Turning to dark colour (Veraison) Biometrics indices Average weight/single fruit weight ratio Average volume/volume 100 olive fruits Sarcocarp/endocarp ratio Sarcocarp weight/weight of 100 olive fruits Average fruit volume/volume of single fruit ratio Fruit drop (after fruit set) Amount of olive drop (% total olive fruits at end season) Time of onset of olive drop *Ciferri et al., 1942, Italy (A); Patac et al., 1954, Spain (B); D Amore et al., 1977, Calabria (Italy) (C); Loussert et al., 1978, World (D); Barranco et al., 1984, Andalusia (Spain) (E); UPOV, 1985, World (F); Leitão et al., 1986, Portugal (G); Cimato et al., 1993, Tuscany (Italy) (H); Tous Martí and Romero Aroca, 1993, Catalonia (Spain) (I); Cristoferi et al., 1997, Emilia Romagna, (Italy) (J); Bartolini et al., 1998, World, (K); Pannelli et al., 1998, Abruzzo (Italy) (L); Cicoria et al., 2000, Molise (Italy) (M); Pannelli et al., 2000, Umbria (Italy) (N); Barranco et al., 2000a, World (O); Pugliano et al., 2000, Campania (Italy) (P); Cimato et al., 2001, Tuscany, (Italy) (Q); Rotundo and Marone, 2002, Lucania (Italy)(R); Trigui et al., 2002, Tunisia (S); Parlati and Pandolfi, 2003, Lazio (Italy)(T); Lombardo et al., 2003, Calabria (Italy)(U); Bassi, 2003, Lombardy (Italy) (V); Lombardo et al., 2004, Apulia (Italy)(W); Rallo et al. 2005, Spain (Y). The characters column carries as accurate as possible a translation of the original terms. When terminology was similar and obviously indicated the same character, the terms were unified in English and listed in the same row; otherwise the character was given a separate row, especially if, in the original publication, no clear indications were given as to the meaning of the character.
11 T. GANINO,G.BARTOLINI and A. FABBRI 329 LIST H CHARACTERS OF THE ENDOCARP Weight Shape (positions A and B) Thickness Thickness/weight ratio Length Average diameter Length/diameter ratio Size Symmetry (positions A and/or B) Volume and weight: averages and upper values Diameters ratio (longitudinal and transverse) Section Biometrics indices Maximum diameter Shape of largest transverse section Position of longest transverse diameter Apex (positions A and B) Base (positions A and B) Colour Surface: colour, roughness and groove number Surface Groove type Groove number Groove gait Groove distribution Groove depth Suture line Suture curvature Apex termination LIST I CHARACTERS OF THE SEED Shape Colour Size Flavour Average weight Number of seeds per fruit Seedless fruits LIST J AGRONOMIC AND COMMERCIAL CHARACTERS Precocity Onset of bearing Productivity Production regularity Drupe oil content Oil yield Oil quality Course of oil accumulation Mesocarp clinginess Rooting ability Bloom time Time of recovery of vegetative activity and inflorescence emission Bloom duration Days from full bloom to fruit ripening Pollinators Compatibility Flowers/ripe fruits ratio Fruit-set after free pollination Fruit-set after self pollination Average no of seeds per drupe (%) Fruit ripening time Susceptibility to abiotic stress Susceptibility to biotic stress Hardiness Suitability to mechanical harvest Particular conditions for variety identification Influence on the plant of environment and cultivation techniques Additional information
12 330 History of cultivated olive classification and description makes it particularly difficult to transfer data obtained in a given environment to other environments, due to a lack of common references. As a consequence of these considerations, the proposed classification methods, even the most recent ones, suffer grave limitations due to: a) polygenic control of morphological characters and the strong influence of environment and cultivation techniques on their expression; b) the absence of identical collections in different environments, which would be extremely useful in the study of genotype environment interactions; c) the need to consider a large number of characters to draw up a suitable descriptor list; and d) the need to gather data concerning the required characters for a minimum number of years. In conclusion, we must acknowledge that we do not yet possess an accurate methodology to identify olive varieties. This causes a delay in olive breeding, and consequently a delay in improvements in olive production. The unification of all methodologies, by creating a common pomological descriptor list for all scholars and scientists in the field, consisting of a series of characters useful both for genotype description and scientific research, would be a significant advance. In addition, any new list should include information obtained by new research methods (physiological data and ultrastructural analysis, serological, biochemical and molecular marker analyses). We must admit that the use of morphological, biological and agronomic characters alone does not permit an accurate varietal identification of olive cultivars. The sole use of molecular markers, which are receiving increasing interest and credit, does not necessarily provide the accuracy and reliability they seem to inspire. Although DNA marker techniques are theoretically valid, in practice the methodology needs to be better tested and improved. Actually, there are limits in the application of the new techniques. It must be remembered that: a) even reproducible differences in DNA profiles only offer the certainty of the negative result, and never genetic identity; consequently, b) when the molecular profiles between two plants are identical, we still do not have absolute certainty that we are dealing with the same genotype. We can only reasonably deduce that two individuals can be referred to the same genotype, with very low or no significant ( 1%) margin for error, provided the numerical acceptability thresholds of the molecular markers being tested, which vary according to species and type of marker, are passed. The problem of olive classification is therefore of prime importance. Equally apparent is the need for the construction of an univocal descriptor list, the establishment of collection fields, and the creation of a complete data-bank. Nevertheless, this review can close on an optimistic note. The Italian Accademia Nazionale dell Olivo e dell Olio of Spoleto has sponsored a Committee of the most eminent Italian olive scientists to prepare a descriptor list (plant passport data, morphological, biological, agronomic and commercial characters, biochemical and molecular markers, collections, etc.), at different levels, and valid for all olive-producing countries, which will soon be submitted to the international research community. The new descriptor list, once accepted by all those involved in the olive industry, will represent a model that can eliminate, or at least contain the confusion accumulated so far in attempts to describe the cultivated olive. REFERENCES AMBROSINO, O. and RAO, R. (2001). 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