**3.2.2 Gene identification in crop species**

The sequencing and assembly of large and complex crop genomes remains a valuable goal, but at the moment, a significant amount of knowledge can be gained from low coverage shotgun sequencing of these genomes. In this contest, the second generation technologies of sequencing are particularly suitable to know genes and gene promoters in crop plants that are homologous to related species. Therefore, designing polymerase chain reaction (PCR) primers to the read pairs enables the amplification and sequencing of the gene and corresponding genomic region in the target species. This approach to gene discovery offers the potential to identify genes, gene promoters and polymorphisms in a wide range of agronomically important crop species (Bracci *et al.*, 2011).

Microarray represents functional genomic approaches that have revolutionized global gene expression profiling. In fact they allow studying the entire gene complement of the genome in a single experiment (Duggan *et al.*, 1999; Li *et al.*, 2005). At the moment, cDNA and oligonucleotide microarrays have been widely used in plants, such as *Arabidopsis,* rice, maize, strawberry, petunia, ice plants and lima bean, to study and compare global gene expression levels in specific organs and/or tissues under controlled physiological conditions.

In olive, the genomics information present on the international database NCBI, concerning the identification and characterization of functional genes are prevalently based on EST identification and they are predominantly related to pollen allergens and characteristics of olive fruit.

*Olea europaea* trees are widely distributed throughout the Mediterranean basin and therefore their pollen is one of the most prevalent causes of respiratory allergy such as allergic rhinitis and allergic asthma in the Mediterranean region and some other countries between late April and early June (Kalyoncu *et al.*, 1995). Olive pollen is also responible of allergic inflammation of the upper and/or lower airways that may persist after the pollination season is over (Quiralte *et al.*, 2005).


Table 1. The olive pollen allergens (from Villalba *et al.*, 2007)

to survey the genome of Miscanthus (Swaminathan *et al.*, 2009), while Sanger, Illumina Solexa and Roche 454 sequencing are being used to characterize the genome of banana

The sequencing and assembly of large and complex crop genomes remains a valuable goal, but at the moment, a significant amount of knowledge can be gained from low coverage shotgun sequencing of these genomes. In this contest, the second generation technologies of sequencing are particularly suitable to know genes and gene promoters in crop plants that are homologous to related species. Therefore, designing polymerase chain reaction (PCR) primers to the read pairs enables the amplification and sequencing of the gene and corresponding genomic region in the target species. This approach to gene discovery offers the potential to identify genes, gene promoters and polymorphisms in a wide range of

Microarray represents functional genomic approaches that have revolutionized global gene expression profiling. In fact they allow studying the entire gene complement of the genome in a single experiment (Duggan *et al.*, 1999; Li *et al.*, 2005). At the moment, cDNA and oligonucleotide microarrays have been widely used in plants, such as *Arabidopsis,* rice, maize, strawberry, petunia, ice plants and lima bean, to study and compare global gene expression levels in specific organs and/or tissues under controlled physiological

In olive, the genomics information present on the international database NCBI, concerning the identification and characterization of functional genes are prevalently based on EST identification and they are predominantly related to pollen allergens and characteristics of

*Olea europaea* trees are widely distributed throughout the Mediterranean basin and therefore their pollen is one of the most prevalent causes of respiratory allergy such as allergic rhinitis and allergic asthma in the Mediterranean region and some other countries between late April and early June (Kalyoncu *et al.*, 1995). Olive pollen is also responible of allergic inflammation of the upper and/or lower airways that may persist after the pollination

> Ole e 1 ~ 19 Unknown Ole e 2 ~ 15 Profiling Ole e 3 ~ 9 Polcalcin Ole e 4 ~ 32 Unknown

Ole e 6 ~ 6 Unknown Ole e 7 ~ 10 Lipid transfer protein Ole e 8 ~ 19 Ca++ binging protein Ole e 9 ~ 46 1,3 β glucanase Ole e 10 ~ 10 Carbohydrate binding protein

Ole e 5 ~ 16 Cu/Zn superoxide dismutase

**Allergenic proteins name Molecular mass (kDalton) Family** 

Table 1. The olive pollen allergens (from Villalba *et al.*, 2007)

(Hribova *et al.*, 2009).

conditions.

olive fruit.

season is over (Quiralte *et al.*, 2005).

**3.2.2 Gene identification in crop species** 

agronomically important crop species (Bracci *et al.*, 2011).

At the moment 10 olive pollen allergens have been purified and characterized from *Olea europaea* pollen extract (Table 1). Several of these allergenic proteins, eg, Ole e 6, fail to show any homology to known protein sequences and, therefore, the biochemical function of these gene products remains unknown. Many other allergens belong to well-known families of proteins, such as profilin (Ole e 2), superoxide dismutase (Ole e 5), calciumbinding proteins (Ole e 3 and Ole e 8), lipid transfer proteins (Ole e 7) and 1,3-β-glucanases (Ole e 9) (Villalba *et al.*, 2007).

Photo 10. Cross sections of mesocarp olive fruit at level of insect injury (*Bactrocera oleae*, right: sections stained with safranin O/ azur II; left: localization of *OeCHLP* transcripts by *in situ* hybridization with dig-labelled *OeCHLP* antisense probe).

The biochemical composition of olive fruit is variable because it depends on olive variety, soil, climate, and cultivation. The virgin olive oil is overwhelmingly composed of triglycerides (>98%), along with traces of other compounds. The dominant triglyceride fatty acid species are the oleic acids (57-78%) such as palmitic, stearic, linoleic and linolenic acids (Caravita *et al.,* 2007). The other minor constituents such as alcohols, polyphenols, chlorophyll, carotenoids, sterols, tocopherols and flavonoids, contribute to the olive's organoleptic qualities, taste, flavour, and nutritional value (Perri *et al.,* 2002; Servili *et al.*, 2004). These constituents may also serve to distinguish olive oils originating from different regions. Olive oil, especially extra-virgin oil also contains small amounts of hydroxytyrosol, secoiridoids, lignans (Bianco *et al.,* 1999, 2001; De Nino *et al.,* 2005) and other compounds thought to possess anticancer properties (*i.e.*, squalene and terpenoids) (Fabiani *et al.,* 2002; Owen *et al.*, 2004). In spite of its economical importance and metabolic peculiarities, very few data are available on gene sequences controlling the main metabolic pathways. Particular attention has been paid to the genes encoding the key enzymes involved in fatty acid biosynthesis, fatty acid modification, triacylglycerol synthesis, and fat storage (Hatzopoulos *et al.,* 2002; De la Rosa *et al.,* 2003; Banilas *et al.,* 2005).

In recent years, much attention has turned to the olive fruit. In this contest, the parallel sequencing of different fruit cDNA collections has provided large scale information about the structure and putative function of gene transcripts accumulated during fruit development (Alagna *et al.*, 2009).

A nuclear gene, named *OeCHLP* (*Olea europaea* GERANYLGERANYL REDUCTASE was isolated and characterizated by Bruno *et al.,* (2009). This gene encodes a chloroplastic enzyme involved in the formation of phytolic side chain of tocopherols chlorophyll, and plastoquinones. In olive fruits *OeCHLP* gene expression was enhanced in dark fruit very likely in relation to the increase in mature fruits of the level of total tocopherols suggesting a role in the synthesis of the antioxidant. It is noteworthy that the variations in gene transcript levels that occurred during the ripening of olive fruits depend on the genotype analyzed (Muzzalupo *et al.,* 2011). In this contest, in olive fruits tocopherols confer not only nutritional value (Valk and Hornstra, 2000), but also contribute to product stability and post harvesting shelf life (Goffman and Bohme, 2001) by protecting storage oil from oxidative damage (Sattler *et al.,* 2004). *OeCHLP* was also detected in fruits attacked by *Bactrocera oleae*  pathogen as well as in fruits wounded by needle suggesting a role in protection mechanisms related to cell damage and oxidative burst induced by pathogen (photo 8 and 10) (Ebel, 1998; Klessig *et al.,* 2000; Bruno *et al.*, 2009).
