*Evolution of methods to get and to analyze molecular data*

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

evidence for olive oil extraction.

Recently, Terral's team has revealed that wood charcoal could also reveals traces of watering in the Middle Ages [40]. If the reasons people were pruning the oleaster are unknown, the consequences of pruning probably appeared to these peoples by more regular blossoming over years and more fruits. Today olive cultivars display a wide variability in response to pruning methods that raises questions on the origin of the diversity [15, 47, 48, 49] at the Neolithic C site of Atlit-Yam on the Levantine coast (dated to 7100-6300 yrs. BP, uncalibrated C14) found underwater wells constructed of alternating layers of tree branches and stones, stone-installations, some lined with undressed stones and others dug into the clay sediment. Some of the crushing installations contained thousands of crushed olivestones and waste resulting from the extraction of olive oil. So far this is the oldest known

Remains that enabled us to trace the olive tree are more and more numerous from the Mesolithic to the Historical periods. The most informative remains are olive endocarps (stones) that are frequently found in fireplaces (they are charred or carbonized). Terral's team has developed morphometric methods that appeared efficient in analyzing such remains [50]. The main features that result from their analyses are based on the fact that the morphometry of the endocarps has change during the domestication process. Using modern and ancient reference samples they screened and pointed out domesticated remains and unraveled some cultivar relationships. If many stones together in an archaeological site can reveal some transition phase between the wild to the domesticated olive (many broken stones together probably represent oil processing), numerous remains are single or a few stones and consequently these methods are limited on such samples. The accumulation of a few stones probably represents eating olives. However, secondary usage of olive pressing wastes may limit finding traces of olive oil production based on olive remains alone [51].

Pottery types absorb indications of the type of fat they have stored. Pottery types devoted to olive oil as containers for perfumes are aryballos and alabasters, which are widely present throughout the Mediterranean basin due to their diffusion by Greek and Roman cultures [52]. Ceramic chronologies are strict and factories are well recorded, providing a large corpus of data on exchange and trade during the historic periods. Documentation indicates that people used several plant oils (at least flax, saffron, safflower, castor oil and poppy), however, it is possible to differentiate plant oils from animal fats and to identify plant oils

Remains are concrete and their preservation is of importance for future diagnostic methods. The materials from sites studied by all the authors will probably tell more in the future. Furthermore, archaeology continues to uncover new sites and materials and this is likely to

In conclusion, the archaeological materials have enabled researchers from different disciplines to anchor the wild and cultivated olive in regions where they naturally thrived and colonized, respectively. Moreover, biologists and archaeologists have defined the basic elicit statistical differences between the wild and the cultivated olive for key historical

by the fatty acid composition obtained from pottery remains [53, 54, 55, 56].

continue especially for the southern and eastern parts of the Mediterranean coasts.

Evolution of methods is permanent due to progresses in their development. We will not reiterate all methods of studying the history of the olive since the last ice age through domestication, but will try to enable non geneticists to follow our reasoning. The progress in developing molecular markers over the last twenty years has made some techniques lapsed, although they have released plenty of information [4, 57].

Whatever the techniques used to visualize the genetic diversity, the main feature is to aggregate data from the three DNA supports in the olive tree: the mitochondrial DNA (mt, ), the Chloroplast DNA (cp, [58] and the nuclear DNA (nu-, [15, 59, 60, 61]. The information brought by the three compartments is not proportional to the length of the DNA, but by the mode of inheritance and by their mode of evolution. The mt-DNA is maternally inherited as the cp-DNA [62], and evolves by recombination and by mutation and deletion, respectively. These DNA pools are constituted from several copies of the same molecule (they are haploid) and define 'haplotype'. The nuclear DNA is made of two halves from each parent. If the two alleles at one locus are discernible they are said to be codominant, and if only one is discernible due to the other one being absent then the discernible allele is dominant over the hinted allele. If the same dominant allele is found in two different individuals they are said to be similar, whereas if the same two alleles are found in two different individuals they are said to be identical.

Population genetic methods based on similarities (established for dominant alleles) compares at each locus two heterogeneous groups, one homogenous (the double recessive) and one heterogeneous (the double dominant and the heterozygous). All methods used to structure the genetic diversity are based on the allelic frequencies that are firm with co-

dominant markers but are estimated with dominant markers, i.e., Correspondence (FCA) and Hierarchic analyses (leading to dendrograms). Bayesian methods for nuclear DNA (nu-DNA) appeared in 2000 to analyze the data sets and they enable to constitute clusters (based on inferences of allele frequencies) and to check in each individual under examination the proportion of the genome that is coming from the different groups made by the software [63]. These clusters under some hypotheses may correspond to ancestor origins. However, Bayesian method can mix data from nu-, and cp- or mt-DNA. All the methods have contributed in eliciting the olive's origin and they have opened the way to use more adequate methods [64]. Obviously, old data sets could be treated with new methods to get new information.

Origin and History of the Olive 13

structure software [63] by different a priori packages made of oleaster trees, cultivars, oleaster and feral trees. All clusters revealed by this study were checked by other aggregation methods (FCA, Dendrograms) to verify their consistency. However, these methods cannot ensure the biological existence of such groups. [70] have examined about 250 cultivars and two oleaster populations with AFLP markers (there are mostly dominant markers) from the central Mediterranean with similar methods. [71] has examined 171 wild trees with 8 SSR from the north-western Mediterranean [30] has studied about 32 cultivars

and 70 oleaster trees from Tunisia, with morphological and molecular methods.

**Figure 3.** Anti Atlas Morocco elevation 1525masl. © Catherine Breton

**7. Relationships between the oleaster and the olive** 

[70] Baldoni et al. concluded that most cultivars have been introduced into Central Italy regions from the outside and that Umbrian cultivars have originated by selection from local oleaster trees.[71] Belaj et al. concluded that the genetic structure (=density of alleles across the geographic distribution of individuals) is not strong enough to positively establish relationships between true oleaster trees and cultivated varieties. The impact of these studies has probably been limited due to the limited sampling of the wild forms. [30] Hannachi et al. (2009) has revealed that the cultivar sets can be split into those of local

By the year 2000, after several completed projects (European projects and country projects), the molecular diversity in the wild form appeared deeply structured, that means the geographic distribution of the molecular markers in the wild tree was not homogenous [1, 24, 65, 66]. The genetic structure (estimated by the Fst) was stronger with mt- and cp-DNA markers than with the nu-DNA. Moreover, the mt- and cp-DNA distribution in the eastern and the western halves of the Mediterranean Sea appeared strongly structured. Even if sampling problems for all the studies had biased their data, the trend from the whole data supports that clines for allele frequencies do exist in the wild olive diversity. The clines could be due to different causes and as for other tree species the spread of the wild olive at the end of the last ice age may explain its present distribution.
