Olive Germplasm - Italian Catalogue of Olive Varieties

1. Introduction

It is of great importance to evaluate and characterize the existing genetic diversity of the crop species, mainly for those, such as the case of olive, which still have a well preserved great cultivar patrimony, in spite of the disturbance of the environments where they are cultivated. This issue is of particular importance in areas where a number of varieties show adaptation to the difficult local environmental conditions.

The genetic patrimony of the Mediterranean Basin’s olive trees are very rich and is characterised by an abundance of varieties. Based on estimates by the FAO Plant Production and Protection Division Olive Germplasm (FAO, 2010), the world’s olive germplasm contains more than 2.629 different varieties, with many local varieties and ecotypes contributing to this richness.

The problem of olive germplasm classification is not only complicated by the richness of its genetic patrimony, but also by the absence of reference standards and by the confusion regarding the cultivar names, with numerous cases of homonymy (one denomination for several genotypes) and synonymy (one genotype with several denominations).

The Italian olive germplasm is estimated to include about 800 cultivars, most of them landraces vegetatively propagated at a farm level since ancient times. The number is probably underestimated because of the scarce information on minor local varieties widespread in the different olive growing areas. The study of these less-common cultivars is important because they may have traits not considered important in the past but necessary to meet the challenges of modern olive growing. Low vigour, resistance to low temperatures, salinity tolerance, adaptability to low pruning systems, late ripening and fatty-acid content are important traits for olive or olive oil quality. Additionally, morphological characters are sometime correlated or associated with disease susceptibility and can be used as markers in breeding.

The largest olive collection (accounting for 17 percent of the total olive trees with more than 500 varieties) is held by Agricultural Research Council - Olive growing and oil industry research centre (CRA-OLI) in Italy, followed by the collections of the Centro de Investigación y FormaciónAgroalimentaria Córdoba (CIFACOR) in Spain. The systematic collection of Italian olive varieties for deposit into specific catalogue fields began in Italy in the 1980s. A similar international collection was begun in 1997 by CRA-OLI of Rende, Italy. Collection entailed the following steps: a survey of the territory, individuation, basic characterization, and introduction into the gene bank field. Material identified by other international scientific institutions (International Treaty on Plant Genetic Resources for Food and Agriculture - Plant Genetic Resources RGV-FAO Projects) was also included. To date, roughly 500 varieties have been introduced into the CRA-OLI collection, and this list has been published (web site http://apps3.fao.org/wiews/olive/oliv.jsp). The goal of such collections is to safeguard all cultivars, and particularly the minor ones, to avoid a loss in genetic diversity and to offer an interesting genetic basis for breeding programs. Knowledge of genetic diversity in a crop species is fundamental to its improvement. A variety of molecular, chemical and morphological descriptors are used to characterize the genetic diversity among and within crop species.

Morphological and biological characters have been widely used for descriptive purposes and are commonly used to distinguish olive cultivars. Agronomic characterization also allowed the classification of different olive cultivars. Different molecular markers have recently been used to characterize and distinguish the olive cultivars.

Management of the CRA-OLI collection includes a description of its genetic diversity for a reliable characterization of all accessions since several cases of mislabelling, homonymy and synonymy could exist.

In the present work, we used morphological characterization and molecular markers and to characterize all accessions present in the CRA-OLI collection, to build a first molecular and morphological data-base and to analyze the genetic relationships between cultivars.

We used SSR markers for genotyping the complete collection of the olive germplasm. The experimental approach was based on using the parameters as recommended by International Olive Council (COI) for characterization and the bio-agronomic observations concerned the morphological characteristic of the trees, leaves, fruit and inflorescence and the flowering period. The fruits were examined for their morphology and oil composition and for endocarp characteristics.

Over 200elaiographic cards with colour photos, graphs and tables and with full details relating to the identification the olive varieties growing in the CRA-OLI olive germplasm collection in Calabria, Italy were reported in annex. Additional information about agronomic behaviour of the plants, and the organoleptic oil values, as determined by a panel test, were also recorded.

2. Materials and methods

The passport data of the olive cultivars has permitted the unequivocal classification and location of all identified genotypes. The information provided in the passport data has the aim of integrating the morphological, agronomic, and molecular data of the olive genetic resources; this information has been complemented by the photographic documentation of each accession recovered.

The elaiographical cards were made up according to the International Union for the Protection of New Varieties of Plants - UPOV method and the biometric and morphological parameters evaluated for each of the accessions were as follows: trees, leaves; inflorescences; fruits and stones.

The agronomic characteristics have been evaluated on the observations of 4-5 trees over a period of 4-5 years. Additional information about cold and agronomic behaviour of the plants, and the organoleptic oil values, as determined by a panel test, were also recorded. All the olives varieties analysed were growing in the CRA-OLI olive germplasm collection at Mirto-Crosia (Cosenza, Italy).

2.1. Morphological characterization

The systematic utilization of descriptive morphological characters of the tree and various tree organs has enabled the characterization and discriminatory identification of varieties. The methodology used for describing the olive biodiversity recovered has considereda set of 24 morphological characters (tree: 3 characters; leaf: 3; inflorescence: 2; fruit: 8; endocarp: 8).

2.1.1. Characters of the tree

Three qualitative characters (vigour, growth habit, and canopy-density) are considered.

2.1.1.1. Plant vigour

This refers to both the size of the tree and the intrinsic ability of the scaffold branches and shoots to grow in length and width. It is divided into the following categories:

1. Weak. Tree whose growth is modest even under optimal agronomic conditions. When mature, the trunk and the area projected by the canopy of the tree are distinctly less than what is expected of a specimen of this species.

2. Medium. Tree which, in each area and when normal cultural practicesareapplied, displays the average development expected of an olive tree.

3. Strong. Tree which, in each area and when normal cultural practicesareapplied, displays strong growth, marked trunk and canopy development in terms of both height and volume, and vigorous, long branches.

2.1.1.2. Growth habit

This character describes the natural distribution of the scaffold branches and shoots before there is interference from the training shape adopted and when vigour exerts little influence. Growth habit is divided into three categories (Figure 1):

1. Drooping. Natural growth habit that can be characterized by plagiotropic branching, i.e. by shoots and limbs which are small in diameter and bend downwards from the outset.

2. Spreading. Characterized by initial orthotropic branching. The weight of the canopy and/or of the crop subsequently forces the limb to bend down and turn in the direction in which the greatest amount of light and space is available. The canopy thus becomes hemispherical in shape (even when the olive has several trunks, they always remain quite distinct from each other).

3. Erect. Habit characteristic of certain cultivars whose branches tend to grow vertically and have a strong apical dominance. The tree acquires a fairly pronounced conical shape which becomes cylindrical on reaching maturity. As a rule, cultivars which have an erect growth habit are also vigorous although there are some major exceptions.

Figure 1.

Categories of growth habit of the olive trees

2.1.1.3. Growth habit

Indicates the extent of canopy vegetation and can be measured by the possibility of light penetration. Result of the interaction between the length of the internodes, the number and vigour of the shoots and the size of the leaves. It is classified into three categories:

1. Sparse. This is normally associated with fast-growing cultivars with long internodes. From any point “space” is observed through which light can penetrate.

2. Medium. Density typical of the species. Vegetation is abundant but internode length and growth always leave internal space which produce a shading effect.

3. Dense. This is characteristic of cultivars with short internodes, abundant branching and heavy foliage. The canopy displays a compact surface, the inner section of which is shaded.

2.1.2. Characters of the leaf

Three quantitative characters (length, width, and shape) are considered. Observed in samples of about 100 adult leaves and taken from the middle section of 8-10 one year old shoots chosen from the most representative shoots on the south facing side of the tree at shoulder level.

2.1.2.1. Blade length

Observed in samples of about 100 adult leaves taken from the middle section of 8-10 one year old shoots chosen from the most representative shoots on the south facing side of the tree at shoulder level.

Blade length (L) is classified into three categories:

1. Short(< at 5 cm)

2. Medium(from 5 to 7 cm)

3. Long(> at 7 cm)

2.1.2.2. Blade width

Observed in samples of about 100 adult leaves taken from the middle section of 8-10 one year old shoots chosen from the most representative shoots on the south facing side of the tree at shoulder level. Blade width (W) is classified into three categories:

1. Narrow(< at 1 cm)

2. Medium(from 1 to 1.5 cm)

3. Broad(> 1.5 cm)

2.1.2.3. Shape

This is determined by the ratio between the length and width. Shape is divided into three categories:

2.1.3. Characters of the inflorescence

Two quantitative characters (length and number of flowers for inflorescence) are considered (Figure 2).

Figure 2.

Two different inflorescences

2.1.3.1. Length

Observed in samples of about 100 inflorescence at bud stage, taken from the middle section of 8-10 fruiting shoots chosen from the most representative shoots on the south facing side of the tree. It is classified into three categories:

1. Short(< at 2.5 cm)

2. Medium(from 2.5 to 3.5 cm)

3. Long(> at 35 cm)

2.1.3.2. Number of flowers for inflorescence

Observed in samples of about 100 inflorescence at bud stage, taken from the middle section of 8-10 fruiting shoots chosen from the most representative shoots on the south facing side of the tree. It is classified into three categories:

1. Low(< at 18 flowers)

2. Medium(from 18 to 25 flowers)

3. High(> 25 flowers)

2.1.4. Characters of the fruits

Two quantitative characters (weight and shape) and six qualitative characters (symmetry, position of maximum transverse diameter, apex, base, nipple, and lenticels) are considered. Some characters refer to two positions:

PositionA in which the fruit generally displays the greatest asymmetry when held by either and between the index finger and thumb.

Position B is reached by turning 90° from position A.

2.1.4.1.Weight

Determined in a sample of 100 fruits taken from the middle section of fruiting shoots chosen from the most representative shoots on the south facing side of the tree.It is classified into four categories:

1. Low(< at 2 g)

2. Medium(from 2 to 4 g)

3. High(from 4 to 6 g)

4. Very high(> at 6)

2.1.4.2. Shape (position A)

Ratio between the length and width of fruits.Determined in a sample of 100 fruits taken from the middle section of fruiting shoots chosen from the most representative shoots on the south facing side of the tree. Shape is classified into three categories:

2.1.4.3. Symmetry (position A)

Extent to which the two longitudinal halves match.Determined in a sample of 100 fruits taken from the middle section of fruiting shoots chosen from the most representative shoots on the south facing side of the tree. It is classified into three categories:

2.1.4.4. Position of maximum transverse diameter (position B)

According to its location.Determined in a sample of 100 fruits taken from the middle section of fruiting shoots chosen from the most representative shoots on the south facing side of the tree. It is classified into three categories:

2.1.4.5. Apex (position A)

Determined in a sample of 100 fruits taken from the middle section of fruiting shoots chosen from the most representative shoots on the south facing side of the tree. It is classified into two categories:

2.1.4.6. Base (position A)

Determined in a sample of 100 fruits taken from the middle section of fruiting shoots chosen from the most representative shoots on the south facing side of the tree. It is classified into two categories:

2.1.4.7. Nipple

Determined in a sample of 100 fruits taken from the middle section of fruiting shoots chosen from the most representative shoots on the south facing side of the tree. This characteristic of the tip of the fruit style is:

2.1.4.8. Lenticelles

When the fruit is still green.Determined in a sample of 100 fruits taken from the middle section of fruiting shoots chosen from the most representative shoots on the south facing side of the tree. It is classified into the following categories:

1. few or many

2. small or large

2.1.5. Characters of the endocarp (stone)

The endocarp is the internal, woody part ofthe fruit that encloses the seed. Three quantitative characters (weight, shape, and number of grooves) and five qualitative characters (symmetry, position of maximum transverse diameter, apex, base, and surface) are considered. These are evaluated in the afore-mentioned sample of 100 fruits. As in the case of the fruit, some characters also refer to two positions. Position A is normally the position of maximum asymmetry and is that in which the carpel suture faces the observer. Position B is reached by turning 90° from position A.

2.1.5.1. Weight

The weight is evaluated on the afore-mentioned sample of 100 fruits. It is classified into four categories:

1. Low(< at 0.3 g)

2. Medium (from 0.3 to 0.45 g)

3. High(from 0.45 to 0.7 g)

4. Very high(> at 0.7)

2.1.5.2. Shape

Ratio between the length and width of endocarps. Shape is classified into four categories:

2.1.5.3.Symmetry. (position A)

Extent to which the two longitudinal halves match. It is classified into three categories:

2.1.5.4. Position of maximum transverse diameter (position B)

According to its location. It is classified into three categories:

2.1.5.5. Apex (position A)

The apex is classified into two categories:

2.1.5.6. Base (position A)

The base is classified into three categories:

2.1.5.7. Surface (position A)

Based on depth and abundance of fibrovascular bundles. It is classified into three categories:

2.1.5.8. Number of grooves

Number of grooves that can be seen from the stalk insertion point. It is classified into three categories:

1. Low(< at 7)

2. Medium(from 7 at 10)

3. High(> at 10)

2.2. Pomological characterization and oil quality

To complete the description of the varieties recovered a methodology has also been adopted for the secondary characterization (pomological and oil quality) of the varieties held in the CRA-OLI collections; this characterization had the aim of providing reliable data on the pomological value of the varieties identified and a common method has been used, the object being to unify the criteria for evaluating these parameters. This will help to minimize the differences that the use of different methods of study could cause in the characterization of world germplasm.

2.2.1. Average fresh weight of fruit

This parameter is calculated from 100 olives harvested at the black stage of maturation. The following categories have been established.

1. Very low(< at 2 g)

2. Low(from 2 at 4 g)

3. Medium(from 4 at 6 g)

4. High(from 6 at 8 g)

5. Very high(> at 8 g)

2.2.2. Average fresh weight of stone

This parameter is calculated from 100 stones obtained from the fruit and after removing the flesh. The following categories have been established.

1. Very low(< at 0.2 g)

2. Low(from 0.2 at 0.4 g)

3. Medium (from 0.4 at 0.6 g)

4. High(from 0.6 at 0.8 g)

5. Very high(> at 0.8 g)

2.2.3. Flesh/Stone ratio of the fruit

This parameter is calculated from the fruit and stone weight data obtained from samples. The following categories have been established.

1. Low(< at 5.0)

2. Medium (from 5.0 at 7.5)

3. High(from 7.5 at 10.0)

4. Very high(> at 10.0)

2.2.4. Percentage of oil in the fruit

This parameter is calculated from 0.3 kg paste olive samples that was milled for 1 min (standardized time to obtain a homogeneous sample and to minimize heating of the olive paste) in a small hammer mill. For oil content determination, the sample was analyzed using a Fourier Transform Near-InfraRed instrument (MPA Multi PurposeAnalyzerFT-NIR spectrometer system (Bruker Optics, Milan,Italy), according to Bendiniet al., 2007. The following categories have been established.

1. Very low(< at 30 %)

2. Low(from 30 at 40 %)

3. Medium(from 40 at 50 %)

4. High(from 50 at 60 %)

5. Very high(> at 60%)

2.2.5. Olive oil extraction

For each sample 5 kg of olives were picked from five trees grown in the CRA-OLI germplasm collection, and then milled in a laboratory scale hammer mill (Oliomio, Toscana Enologica Mori, Tavernelle Val di Pesa, Italy). After 30 minutes of malaxation at room temperature the oil was separated by centrifugation in the same operative conditions (Figure 3). Then the oil was filtered and stored in the dark at 8 °C until analysis.

Figure 3.

Decanter and disc stack centrifuges

2.2.6. Oil determinations

The quality of the oil has been determined according to its fatty acid composition (Muzzalupoet al., 2011). Expressed as a percentage and determined according to the official method of European Community (Commission Regulation No 61/2011).

2.2.7. Sensory analysis

Quantitative descriptive analysis was carried out by a total of 9 assessors, members of staff of the CRA-OLI of Rende (CS), using the procedure reported in a later chapter (“sensory properties of virgin olive oils”). The assessors had experience in Qualitative Data Analysis - QDA (Stone and Sidel, 2004) and virgin olive oil sensory evaluation in accordance with the current UE Regulation (N. 61/2011).

Virgin olive oil samples (15 g) were served to the assessors in clear glass tumblers (100 mL), covered with watch glasses at room temperature (approximately 28 °C). The samples were presented in duplicate, in balanced order to each assessor.

For a description of the virgin olive oils, the following terms were used: yellow, yellow-green, intense green, musk-green to describe the virgin olive oil colour; fruity, cut-grass, floral, hay-like, almond, apple, and artichoke for the virgin olive oil olfactory profile; pungent, bitter, greasy, and sweet to define the virgin olive oil taste notes.

The intensity of those sensations was graded using a line scale and thus converted into a numerical score by measuring the position of the placed mark along a 10 cm line. The results were calculated as the medianamong assessor sensory scores.

2.3. Molecular markers

The total genomic DNA was isolated from fresh, young leaves following a CTAB protocol originally developed by Murry and Tompson (1980) and further modified by Muzzalupo and Perri (2002). The olive trees were genotyped at 11 nuclear microsatellite loci, selected among those available in literature and proved to be suitable for the characterization and identification of olive varieties in previous papers (Baldoniet al., 2009; Muzzalupoet al., 2009). The loci used were four (GAPU59, GAPU71A, GAPU71B and GAPU103A) among those described by Carrieroet al. (2002) five (UDO01, UDO03, UDO12, UDO28 and UDO39) among those described by Ciprianiet al. (2002) and two (DCA9 and DCA18) among those described by Sefcet al. (2000). Four loci of the selected markers (GAPU71B, GAPU103A, DCA9 and DCA18) were chosen from the common list reported by Baldoniet al. (2009) for use in a future comparison of data, while others (Gapu59, GAPU71A, UDO01, UDO03, UDO12, UDO28 and UDO39) were chosen as in other recent studies they were found to be very suitable for Italian intra-cultivars characterization and for characterization of olive germplasm collections (Muzzalupoet al., 2009; Muzzalupoet al., 2010). Electrophoresis and detection of PCR products were carried out according to Muzzalupo et al.(2007).

Figure 4.

SSR profile of olive cultivar. The electropherograms were obtained using the 2100 Bioanalyzer running DNA 500 LabChips. DNA extracted from leaf. GAPU71A (1), UDO03 (2), UDO 39 (3), and Ladder (L). The horizontal axis represents migration time of DNA fragments in seconds, and the vertical axis represents fluorescence. Upper and lower peaks in the electropherogram represent the internal DNA markers at 15 and 600 bp, which were used to size and quantify the PCR products

3. Conclusion

The morphological and molecular characterizations (elaiographic cards) are efficient for olive germplasm management, including the characterisation of varieties and the establishment of relationships between cultivars in the CRA-OLI olive collection. Beyond this identification, we constructed a data base that can be used to make a reference collection of Italian olive germplasm by comparing the morphological and molecular pattern of each identified varieties with samples from different areas.

Introducing new accessions by prospecting in different Mediterranean areas is currently in progress. The choice of which new varieties enter into the CRA-OLI collection can now take into account our results, both by avoiding duplicates and also by maximising genetic diversity.

Acknowledgement

The elaiographic cards annex has been possible thanks to the contribution of time, energy and expertise by many individuals of CRA-OLI. The author would like to take this opportunity to acknowledge their generosity. The author thank the Projects: CERTOLIO, GERMOLI, OLIOPIU’ and RGV-FAO for financial support.

Annex. - Elaiographic Cards

Many CRA-OLI staff members and consultants have provided specific contributions to annexes:

• Alessandrino Maria, Boccuti Alfonso, Briccoli-Bati Caterina, Ciliberti Agostino, Cruceli Giuseppe,Godino Gianluca, Iannotta Nino, Lombardo Nicola, Madeo Alfreo, Muzzalupo Innocenzo,Pellegrino Massimiliano, Toscano Pietro,Turco Domenico, and Zaffina Francesco who contributed to the collection field;

• Alessandrino Maria, Boccuti Alfonso, Ciliberti Agostino, Cruceli Giuseppe, Godino Gianluca, Lombardo Nicola, Madeo Alfreo, Muzzalupo Innocenzo, Pellegrino Massimiliano,Turco Domenico, and Zaffina Francesco who contributed to the morpho-bioagronomic analysis;

• Fiorita Antonio, Longo Domenico, Parise Attilio, Ripoli Antonio, and Tucci Paolo who contributed to the extractionof monovarietal oliveoil;

• Benincasa Cinzia, Muzzalupo Innocenzo, Parise Attilio, Patarino Alba, Pellegrino Massimiliano, and Perri Enzo who contributed to the biochemical analysis;

• Benincasa Cinzia, Godino Gianluca, Miranda Osvaldo, Muzzalupo Innocenzo, Pellegrino Massimiliano, Perri Enzo, Ripoli Antonio, Rizzuti Biagio, and Zaffina Francesco who contributed to the sensory analysisof monovarietal oliveoil;

• Muzzalupo Innocenzo and Pellegrino Massimiliano who contributed to the molecular analysis;

• Alessandrino Maria and MuzzalupoInnocenzo who supported the preparation of design of the elaiografic card.

• Alessandrino Maria, GianlucaGodino, and Muzzalupo Innocenzo who provided photos of the olive varieties.

List of olive varieties