Olive is the main cultivated species belonging to the monophyletic Oleaceae family, within the clade of Asterids, in which the majority of nuclear and organellar genomic sequences are unknown. The Olea genus includes two sections, Olea and Ligustroides. The former comprises the six recognised subspecies of the olive complex, which can be found throughout the Mediterranean area as well as the temperate and subtropical regions of Africa and Asia. The Mediterranean form (Olea europaea, subspecies europaea) includes the wild (var. sylvestris) and cultivated (var. europaea) olives .
Recently, chloroplast genome sequencing of species belonging to this family from the tribe of Jasmineae revealed that two genera, Jasminum and Menodora, carry several distinctive rearrangements, including inversions, gene duplications, insertions, inverted repeat expansions and gene/intron losses . One of these genomic features involves the duplication of the rpl23 protein-coding gene in Jasminum. A similar duplication has also been detected in the Poaceae, and in both Oleaceae and Poaceae, the duplicated copy has been inserted into the intergenic region between rbcL and psaI . By comparative gene mapping and sequencing, Lee and co-workers also demonstrated that all other Oleaceae genera, including Olea, have an identical gene content and order as Nicotiana tabacum. A phylogenetic reconstruction of the entire family, based upon the sequences of the ndhF and rbcL genes, partially confirmed previous results obtained by the analysis of the trnL-F and rps16 chloroplast regions .
Intraspecies variation within other Oleaceae genera, such as Syringa , Forsythia , Ligustrum  and Fraxinus [8, 9] has also been examined.
Different chlorotypes have been identified among the six subspecies of O. europaea. Lumaret et al.  identified 12 distinct chlorotypes by RFLP analysis of DNA isolated from the purified chloroplasts of a wide set of O. europaea taxa. In other O. europaea subspecies Baldoni et al.  identified nine nucleotide substitutions, one insertion-deletion (indel) and a polymorphic poly-T SSR in the trnT-L region. Besnard et al.  in the O. europaea complex identified fourteen polymorphisms in three chloroplast regions (trnT-L, trnQ-R and matK), including five microsatellite motifs, two indels and eight nucleotide substitution sites. Recently, the analysis of four regions (trnL-F, trnT-L, trnS-G and matK) was used to demonstrate the polyphyletic origin of the Olea genus and estimate the divergence times for the major groups of Olea species and subspecies during the Tertiary period .
In cultivated olives chloroplasts are maternally inherited  and, in contrast to that seen at the subspecies level, a low plastidial variability was detected. A strong linkage disequilibrium between the chloroplast and mitochondrial genomes has been demonstrated, particularly for the Mediterranean cultivated and wild olives (subspecies europaea), suggesting that a low level of recurrent mutations occurs in both organellar genomes of the olive .
In particular, RFLP analysis of chloroplast DNA isolated from 72 cultivars revealed that most cultivars have a common chlorotype . Besnard et al. , using two microsatellites and 13 RFLPs on more than 140 olive cultivars, were able to distinguish only four chlorotypes. The majority of cultivars was characterised by the chlorotype CE1, which likely originated from the wild olive populations of the Eastern Mediterranean and was spread to the Western part through cultivar dispersal by humans. Polymorphisms at the varietal level have been detected in the trnD-T locus , but only one polymorphism in this locus was found within a set of 12 cultivars .
Chloroplast DNA represents an ideal system for plant species DNA barcoding, and some chloroplast regions have been indicated as ideal for use in tests that discriminate between different land plants. Based on assessments of recoverability, sequence quality and discriminatory abilities at the species level, the two-locus combination of rbcL-matK has been recommended as a universal framework for plant barcoding . The combination of trnH-psbA coupled with rbcL has been recommended for DNA barcoding to discriminate between lower taxonomic ranks such as genera or related species . In highly valuable crop species, such as the olive, that have a variety of cultivars available in the market, however, typing at the species level is not sufficient. Thus, the development of reliable methods to rapidly and efficiently discriminate between cultivars has become a pressing need. In addition, DNA barcoding may have useful applications to tracking food products  and the analysis of archaeological remains .
In this respect, the availability of complete chloroplast genome sequences from a growing number of species offers the opportunity to evaluate many potentially polymorphic sites and identify new regions that could be used to define cultivar DNA barcodes.
There are numerous approaches to sequence chloroplast genomes: traditional sequence analysis of highly purified chloroplast DNA, as applied for Solanum lycopersicum , Lolium perenne , Trachelium caeruleum , Jasminum nudiflorum  and Parthenium argentatum ; Rolling Circle Amplification (RCA) of high-purity chloroplast DNA, as demonstrated in Cicer arietinum , Platanus occidentalis  and Welwitschia mirabilis ; shot gun sequence analysis of BAC clones containing chloroplast genomic inserts, as demonstrated in Vitis vinifera , Hordeum vulgare  and Brachypodium distachyon ; and the use of universal primers based on chloroplast sequences highly conserved among most Angiosperm species to amplify overlapping fragments [34–36], as demonstrated in Cycas taitungensis  and two Bambusa species . For this study, the last approach was used to sequence the entire chloroplast genome of the O. europaea subsp. europaea cv. Frantoio. The resulting availability of the entire plastome allowed to evaluate the sequence arrangement of the plastid genome in O. europaea and to identify new organellar polymorphisms that could discriminate between cultivated olive varieties.