**1. Introduction**

132 Olive Germplasm – The Olive Cultivation, Table Olive and Olive Oil Industry in Italy

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The cultivation of olive trees dates back to ancient time. Mythology ascribes to the divine will the domestication of this species; the goddess Athena taught the people of the city of Athens, as a gift, the cultivation of the tree and the treatment of the drupe (Kakridis, 1986). Despite the economic, cultural and ecological importance of olive groves in the Mediterranean area, now extending to other regions, olive has been a poorly characterized species at genetic and genomic level among other fruit tree crops. Therefore, still remains unknown the inheritance of most genes controlling the agronomical performance and quality traits, even though in the last thirty years a wide molecular survey has been performed on the olive germplasm (Rugini et al., 2011). In the Mediterranean Basin, in fact, is conserved the majority of a large number of olive cultivars estimated in more than 1,200 (Bartolini et al., 2004).

*Olea europaea* subsp. *europaea* is present in two forms, namely wild (*Olea europaea* subsp. *europaea* var. *sylvestris*) and cultivated (*Olea europaea* subsp. *europaea* var. *europaea*); it is a diploid species (2*n* = 2*x* = 46), and the genome size range between 2.90 pg/2C and 3.07 pg/2C, with 1C = 1,400- 1,500 Mbp (Loureiro et al., 2007). Crosses with other subspecies and with the wild plants are possible and may produce fertile offsprings, providing access to an enormous pool of genetic variability. Over the last two decades, new knowledge on olive genetics has been produced, with the development of nuclear and plastidial molecular markers and linkage maps.

The long generation time of the species has severely restricted breeding strategies to clonal or varietal selection and, in a very few cases, to inter-varietal crosses. Approaches of marker assisted selection could speed up the cross breeding programs but QTL markers are not yet available. The first linkage map of *Olea europaea* was constructed by de La Rosa and coworkers (2003), through the use of dominant PCR markers, such as RAPDs and AFLPs, and codominant marker as RFLPs and SSRs on a cross progeny between two highly heterozygous cultivars. Other maps have been constructed by the use of RAPDs,

© 2012 Muleo et al., licensee InTech. This is an open access chapter distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 Muleo et al., licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

microsatellites and SCAR markers on a Frantoio x Kalamata progeny (Wu et al., 2004) and, more recently, a new maps has been derived through SSR, AFLP, ISSR, RAPD and SCAR marker, scored on a 140 F1 progeny from a Picholine Marocaine x Picholine du Languedoc cultivars cross (El Aabidine et al., 2010). In any case, no QTLs of agronomical interest for olive breeding have been detected.

Olive Tree Genomic 135

from one paired-end library with 1000 bp inserts that was sequenced on the Illumina HiSeq2000 system producing 100 bp reads for the remaining 17X coverage (25 Gbp). Finally two mate-pair libraries with 3 Kbp inserts were constructed and sequenced on the HiSeq2000

We have produced approximately 18 Gbp of Roche-454 sequence data, corresponding to 12X coverage approximately. 12 Gbp were obtained as long single reads of which approximately one third were 400 bp long reads (FLX TITANIUM technology) and two thirds were 700 bp long reads (FLX XL PLUS technology). Additionally 6.2 Gbp of sequence data were obtained as paired end reads from 3 libraries with 3 Kbp inserts (3.8 Gbp) and 10

The 454 single reds and the Illumina paired-end reads are being used in a traditional WGS assembly. The Illumina mate-pair and the 454 paired end sequenced, i.e. all those sequences that have been obtained from inserts of larger size, will be utilised in order to scaffold into larger assemblies the contigs obtained from the assembly of the reads from the shorter inserts and try to overcome the assembly problems posed by the occurrence of repetitive elements. Since many of the transposable elements in plant genomes are larger than 3 Kbp

We have performed a number of assemblies to test different strategies and to obtain a first rough draft of the olive genome. We tested assemblies both using the Illumina data only, as well as using Illumina and 454 data. All data sets have been initially filtered for low quality sequences and for chloroplast DNA contamination and then subject to assembly using the CLCBio assembler. When only the Illumina data were used (53X coverage after filtering), we produced an assembly of total size of 1.1 Gbp and N50 size of 1.7 Kbp. The scaffolding using the mate pair and paired end information on the same assembly using the SSPACE tool increased the N50 size to 2.3 Kbp. The addition of an initial set of 454 data (3.5 genome equivalents after filtering, single reads only) increased the total assembly size to 1.5 Gbp and the N50 size of contigs and scaffolds to 2.8 and 3.7 Kbp, respectively. We expect that the addition of the remaining 454 sequenced from the large insert libraries (3 and 8 Kbp inserts) should greatly improve the assembly by increasing considerably the N50 size of the scaffolds. However, due to the problems posed by the high levels of sequence heterozygosity present in the olive genome of cultivar Leccino, we consider the sequencing of the pools of BACs a necessary component of our strategy in order to obtain a satisfactory assembly. The problems here are represented by the difficulties in obtaining BAC libraries with large insert sizes (>100 Kbp) from cultivar Leccino. Should this not prove feasible we

Some of the biggest technical challenges in sequencing eukaryotic genomes are caused by repetitive DNA (Alkan et al., 2011): that is, sequences that are similar or identical to

to produce 100 bp reads and reach a coverage of 4 genome equivalents (6 Gbp).

libraries with 8 Kbp inserts (4.4 Gbp).

the larger inserts are going to be of crucial importance.

will anyhow resort to using a fosmid library (40 Kbp inserts).

**3.1. Assembly of olive repetitive sequences** 

sequences elsewhere in the genome.

**3. Analysis of the repetitive component of the genome** 

The Italian project, OLEA, is an initiative, mainly supported by Italian Minister of Agricultural, Food and Forestry Policies, dedicated toward the development genomic resources of olive, and it aims to identify, isolate and determine the function of genes that are associated with both vegetative and reproductive phenotype. Therefore, the knowledge of the genetic structural basis is the first step to identify the relevant differences in the control of gene expression of the same sets of genes that exist among different genotypes. The development of new molecular tools through approaches of structural and functional genomics, together with those from proteomics, metabolomics, mapping and genotyping, will allow to advance in molecular breeding of olive, pull out under-exploited natural diversity that is present in the *Olea* complex and in olive germplasm, dissect the molecular mechanisms underlying traits related to high valued compounds and those involved in plant-environment interactions, establish a platform for a rapid and cost-effective transfer of knowledge and technologies.
