Differences between native and introduced olive cultivars as revealed by morphology of drupes, oil composition and SSR polymorphisms: A case study in Tunisia He ´dia Hannachi a , Catherine Breton b , Monji Msallem c , Salem Ben El Hadj d , Mohamed El Gazzah a , Andre ´ Berville ´ e, * a Faculte ´ des Sciences de Tunis, De ´partement de Biologie, Campus Universitaire 2092, Tunis, Tunisia b AFIDOL, Maison des agriculteurs, 22 Avenue Henri Pontier, F-13626 Aix-en-Provence cedex 1, France c Institut de l’Olivier, BP 208, 1082 Tunis, Tunisia d Institut National Agronomique de Tunisie, 43 avenue Charles Nicolle, 1082 Tunis, Mahraje `ne, Tunisia e INRA, UMR DIA PC, Ba ˆt 33, 2 Place Viala, F-34060 Montpellier cedex 1, France Received 14 September 2007; received in revised form 3 January 2008; accepted 10 January 2008 Abstract Olive cultivars are diversified but nothing is known on their origins and if they are local or introduced in any regions. The study aims to determine which traits may help to identify native from introduced cultivars and oleaster trees. We compared cultivars and oleasters from North Tunisia to determine their relationships based on morphological traits, oil composition and SSR genotyping at seven loci. We used those parameters to examine 32 cultivar trees from 17 denominations and 70 oleaster trees sampled. We used multivariate analysis, enabling to retain the best variables, to establish relationships among trees based on morphological and pomological parameters. Gas chromatography was used to determine fatty acid composition of 30 cultivar trees and 13 oleaster trees. We determined for one cultivar Gerboui the steady drupe, pit morphological and oil composition variation ranges in six different contrasted agro-systems. SSR genotyping was performed in polyacrylamide gels after fluorescent labelling. Based on morphology, oleaster trees from agro-ecosystems clustered broadly in an intermediate position between cultivars and oleasters from natural ecosystems. SSR revealed that the feral and genuine oleasters plus cultivars are always overlapping. Relationships between cultivars are displayed in two dendrograms. They revealed six and three main clusters based on Unweighted Pair Group Method (UPGMA) and Ward algorithm, respectively. They mix olive cultivar and oleaster trees suggesting kinship relationships between some cultivar and some oleaster trees. In contrast, on PCA, some morphological parameters split our sample approximately between olive and oleaster trees. Oil composition was similar between cultivar and oleaster trees. Kinship relationships between cultivar and oleaster trees based on molecular polymorphisms suggested that olive cultivars may have origin in local oleasters. Oil composition as fruit descriptors and drupe size appeared inefficient to discriminate between olive and oleaster trees, in comparison to SSR. Our results suggested several domestication events for the olive. It is important to know which cultivars have local origin to promote and sale products from Tunisia as from all around the Mediterranean basin. # 2008 Elsevier B.V. All rights reserved. Keywords: Domestication; Cultivar origins; Drupe morphology; Oil composition; Oil content; SSR; Olea europaea 1. Introduction Olive growers question all around the Mediterranean basin whether their olive cultivars have been introduced or are derived from local oleasters. The olive and the wild olive or oleaster border in many places, but little is known on their relationships, and whether in each place oleaster are feral, i.e., they derived from the olive, or are genuine or natural (Lumaret and Ouazzani, 2001) and the source of cultivars. Distinction of a crop from its wild relatives is based on several morphological traits and Botanists have usually made distinct species of the two taxa (Gressel, 2005). There are always ambiguities in the distinction, particularly when gene flow between crop and wild is recurrent. Indeed, gene flow leads to feral forms escaped from culture and established in agro- and natural ecosystems. The olive, the feral olive and the www.elsevier.com/locate/scihorti Available online at www.sciencedirect.com Scientia Horticulturae 116 (2008) 280–290 * Corresponding author. Tel.: +33499612233; fax: +33467045415. E-mail address: [email protected](A. Berville ´). 0304-4238/$ – see front matter # 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.scienta.2008.01.004
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www.elsevier.com/locate/scihorti
Available online at www.sciencedirect.com
16 (2008) 280–290
Scientia Horticulturae 1
Differences between native and introduced olive cultivars as revealed
by morphology of drupes, oil composition and SSR polymorphisms:
A case study in Tunisia
Hedia Hannachi a, Catherine Breton b, Monji Msallem c, Salem Ben El Hadj d,Mohamed El Gazzah a, Andre Berville e,*
a Faculte des Sciences de Tunis, Departement de Biologie, Campus Universitaire 2092, Tunis, Tunisiab AFIDOL, Maison des agriculteurs, 22 Avenue Henri Pontier, F-13626 Aix-en-Provence cedex 1, France
c Institut de l’Olivier, BP 208, 1082 Tunis, Tunisiad Institut National Agronomique de Tunisie, 43 avenue Charles Nicolle, 1082 Tunis, Mahrajene, Tunisia
e INRA, UMR DIA PC, Bat 33, 2 Place Viala, F-34060 Montpellier cedex 1, France
Received 14 September 2007; received in revised form 3 January 2008; accepted 10 January 2008
Abstract
Olive cultivars are diversified but nothing is known on their origins and if they are local or introduced in any regions. The study aims to
determine which traits may help to identify native from introduced cultivars and oleaster trees. We compared cultivars and oleasters from North
Tunisia to determine their relationships based on morphological traits, oil composition and SSR genotyping at seven loci. We used those parameters
to examine 32 cultivar trees from 17 denominations and 70 oleaster trees sampled. We used multivariate analysis, enabling to retain the best
variables, to establish relationships among trees based on morphological and pomological parameters. Gas chromatography was used to determine
fatty acid composition of 30 cultivar trees and 13 oleaster trees. We determined for one cultivar Gerboui the steady drupe, pit morphological and oil
composition variation ranges in six different contrasted agro-systems. SSR genotyping was performed in polyacrylamide gels after fluorescent
labelling. Based on morphology, oleaster trees from agro-ecosystems clustered broadly in an intermediate position between cultivars and oleasters
from natural ecosystems. SSR revealed that the feral and genuine oleasters plus cultivars are always overlapping. Relationships between cultivars
are displayed in two dendrograms. They revealed six and three main clusters based on Unweighted Pair Group Method (UPGMA) and Ward
algorithm, respectively. They mix olive cultivar and oleaster trees suggesting kinship relationships between some cultivar and some oleaster trees.
In contrast, on PCA, some morphological parameters split our sample approximately between olive and oleaster trees. Oil composition was similar
between cultivar and oleaster trees. Kinship relationships between cultivar and oleaster trees based on molecular polymorphisms suggested that
olive cultivars may have origin in local oleasters. Oil composition as fruit descriptors and drupe size appeared inefficient to discriminate between
olive and oleaster trees, in comparison to SSR. Our results suggested several domestication events for the olive. It is important to know which
cultivars have local origin to promote and sale products from Tunisia as from all around the Mediterranean basin.
(+) For columns D, O, M, mean DNA, oil and the morphology were studied, respectively; (�) means not determined.a Governorates are BA: Beja; BZ: Bizerte; EF: El Kef; JB: Jendouba; NL: Nabeul; SA: Seliana; TN: Tunis; ZN: Zaghouan.b Cultivated geographical area were determined by several authors (Minangoin, 1901; Decrully, 1907; Valdeyron and Crossa-Raynaud, 1950; Mehri and Hellali,
1995; Mehri et al., 1997; Trigui and Msallem, 2002).c Oleaster ecosystems.d This cultivar (Nib) was not previously identified.e This cultivar (Awam) was only indicated by Hannachi et al. (2006).
Table 2
The different locations and agro-ecosystems with climate characteristics according to Mtimet (1999) where the Gerboui cultivar was sampled
Code for Gerboui
locations
Location Elevation
(m)
Soil Annual means
raining (mm)
Annual means
temperature
Bioclimatic stage
1 Slouguia 112 Mediterranean red soil 597 17.8 8C Sub-humid at mild winters
Fig. 1. (a) Principal Component Analysis on morphological parameters determined on 40 oleasters (16 oleasters from natural ecosystem (o) and 24 oleasters from
agro-ecosystem (O) and 33 cultivars (C) on 7 locations). The fleshes show the cultivars C32 and C33 located in oleasters group. (b) Variables of PCA on
morphological parameters ((+) active variables; (*) additional variables).
Fig. 3. Discriminate analysis on morphological variables determined in 40
oleasters (16 oleasters (O) from natural ecosystem and 24 oleasters from agro-
H. Hannachi et al. / Scientia Horticulturae 116 (2008) 280–290286
length (Vr6), and width (Vr7) of leaves; drupe weight (Vr8),
drupe shape bottom (Vr13) and presence or absence of knoll
(Vr14), pit weight (Vr17), diameter position (Vr21). Axis 2
explained 10.95% of the inertia and is made by tree habit (Vr2),
C22). The group-2 contains nine oleaster and two olive trees
(C8 Chemlali and C27 Roumi). The group-3 contains two
cultivar (Chetoui C2 and Nib C31) and six oleaster trees. The
groups 4 and 5 contain only oleasters. O11 is single. Three
groups contain olive and oleaster trees and two groups contain
only oleaster trees.
Clustering analysis using NJ method (Fig. 6b) shows three
groups. Each group was composed with several clusters. The
group-1 contains 17 oleaster and 2 olive trees (Sayali C1,
Chemlali C8). It was composed with two clusters: cluster-1
contains two olive trees (C1, C8) and cluster-2 contains only
oleasters. The group-2 contains 13 oleaster and 4 olive trees. It
was composed by two clusters that contain olive and oleaster
trees. The group-3 contains 22 oleaster and 9 olive trees. It
divided on seven clusters. Three clusters of this group contain
only oleaster and four clusters contain olive and oleaster trees.
The distribution in mixture of the two olive taxa shows that
probably tightly relationships do exist between some oleasters
and olive cultivar independently of locations.
We examined the clusters supposing that feral oleaster trees
have escaped from cultivars. In Fig. 6a, group 1 is composed of
natural and feral oleasters and cultivars. Sayali C1 is very
closely related to O6 from Beja and Chemlali C8 (group 2) is
related to O61 and O19 from Tunis and Nabeul. Roumi (group
2) is close to Chemlali and to natural oleasters from Tunis. In
Fig. 6b Chemlali group1 and Roumi are in different clusters but
closely related to the same oleasters from natural sites. Chetoui
C2 (Fig. 6b, group 3) is also closely related to two oleasters
from natural sites (O11 Ichkeul – Bizerte, O29 Jendouba). In
Fig. 6a groups 4 and 5 do not include cultivars and In Fig. 6b,
groups 2 and 4 mix natural and feral oleaster and oleasters plus
cultivars, but less consistently than proceeding (Fig. 5).
4. Discussion
We sampled all trees recording ecosystem data, expecting
that oleaster trees will split in genuine and feral in the natural-
and agro-ecosystem, respectively. Oleasters were broadly split
mainly based on morphological parameters declared between
trees from natural ecosystems versus those from the agro-
ecosystems (Table 3).
Cultivars showed a continuous variation broadly by drupe
sizes. The morphological parameters show only partial
separation between oleaster and cultivar trees (Fig. 1a,
Table 1). The class of cultivars with small fruit was close to
oleasters. Because of the vegetative propagation, an oleaster
propagated in an orchard will conserve more or less small fruits.
The later class was intermediate between cultivars with small
fruits and oleaster trees. Once eliminated the class of feral olive
keeping all cultivars together, some cultivated olive trees with
the smallest fruits were still mixed with oleasters (Fig. 2). This
suggests that they may belong to the oleaster pool. Fatty acid
composition was similar between oleaster and cultivated olive
trees, but inefficient to define relationships between them
(Fig. 4) in contrast to the oil content used on PCA analysis
(Fig. 5). Oil content is the most efficient parameter to split
between oleaster and cultivar trees.
The SSR markers revealed that cultivar and oleaster trees
from North Tunisia cluster together in the dendrograms. Based
on similarities we can conclude that they shared close
relationships (Fig. 6a and b).
Based on SSR markers, oleaster and olive trees shared close
relationships. Oleaster forests are natural in Tunisia and several
historical reports (Camps-Fabrer, 1953, 1997) have pointed out
that the oleaster is native in Tunisia. Breton et al. (2006b) have
pointed out that modern Tunisian oleasters are issued from one
refuge area. Moreover, the molecular diversity revealed in
oleasters and a few of Tunisian cultivars (Breton et al., 2006a)
has suggested that a few of cultivars are issued from the
Tunisian oleaster based on assignation and admixture analyses.
It is logic to suppose that cultivars that share tight similarities
with natural oleasters may have they origin in the correspond-
ing oleaster population. However, we have noticed that feral
oleasters are frequent and some cannot be distinguished from
native. Consequently, we cannot exclude that some natural
oleaster could be feral.
Domestication had taken place 5800–5900 years ago for
olive (Zohary and Spiegel Roy, 1975; Galili et al., 1988).
Moreover, cultivars have been further differentiated for
adaptation and uses. At the morphological level, it is likely
Fig. 6. Dendrograms of 52 oleaster (O) and 15 olive cultivated trees (C), based on SSR data, using (a) UPGMA algorithm; (b) Ward algorithm. The arrows indicate the
genuine oleasters and some related cultivars.
H. Hannachi et al. / Scientia Horticulturae 116 (2008) 280–290288
that drupe size has been increased for table uses, but it is also
conceivable that for oil extraction the size of the drupes is not
important to high oil yield. Consequently, this suggests
expecting a genetic structure in cultivars due to drupe size.
However, selection pressures exerted for increasing fruit size
and probably also oil content have diversified the cultivars that
now, based on fruit morphology did not still resemble to
oleaster fruits.
It is now documented that several domestication events
occurred according to pit morphology (Terral et al., 2004)
H. Hannachi et al. / Scientia Horticulturae 116 (2008) 280–290 289
molecular markers such as RAPD, SSRs and cytoplasmic
DNAs (Besnard and Berville, 2002; Breton et al., 2005). Terral
and Arnold-Simard (1996), Terral (1997b), and Terral et al.
(2004) have proved such events in different regions of the
Mediterranean basin. Probably, they occurred concomitantly
with domestication in the Near East (Lanfranchi and Bui, 1995;
Terral, 2000). Thus, present olive cultivars may display
diversity according to domestication origins (Breton et al.,
2006a). The effect of cultivation on wild trees was evaluated by
Terral and Arnold-Simard (1996) on wood anatomy, sap
conduction, growth and development. Terral (1997a) has
carefully shown that after pruning, oleaster trees produced
larger fruits but the ratio length/diameter was not modified.
This suggests that cultivation caused swollen of the fruits and
olive growers consider that cares on such trees, increase
significantly fruit size. However, the design of a rational
experiment to check parameters on olive-oleaster is too long
and expensive.
Seed gene flow is locally detected when in a region cultivars
have been introduced and local oleasters do not carry the same
cytoplasm (Besnard and Berville, 2002). Pollen gene flow is
recurrent in both direction but we do not know their
evolutionary consequence. However, in case of drupe dispersal
they progenies are hybrid olive–oleaster and may blur the
distinction. We effectively obtained a better distinction when
we eliminated feral trees from analyses (Fig. 2). Cultivars that
bear large fruits (>2 cm) are usually used for table (canning)
and cultivars with small fruits (<2 cm) are mainly for
producing oil. However, there are many exceptions, i.e.,
Cailletier producing small fruits is used for canning but only the
big fruits are used for canning, the remaining olives are used for
oil. Fruit size is influenced by fertilization, watering and the
load of each tree. The average size of drupes on a tree is
therefore due to complex genetic and environment parameters
interfering with the year for observation. Domestication has
always been based on a narrow genetic basis and consequently
the reduction in polymorphisms is mainly due to genetic drift.
A priori, the probability to find a locus under selection is weak,
whereas genetic drift affects all the genome of the crop. This
explains that molecular markers such as Isozymes, RAPD, and
SSRs, are efficient to distinguish crop from wild in most of
examined loci (Lumaret et al., 1997; Besnard and Berville,
2000; Breton et al., 2005).
Olive cultivars appeared diversified in Tunisia. One group of
cultivars appeared in tight relationships with local oleasters that
suggest cultivars Chetoui, Chemlali and Roumi could derive
from domestication of oleasters. However, oleasters could also
derive from introduced cultivars. Historical and archaeological
data are scarce. They could say only whether cultivars have been
domesticated locally or have been introduced from elsewhere.
In comparison with other crop/wild couples, we look for
explanations to this situation in the olive/oleaster couple. A
priori we expected morphological differences between olive
cultivar and oleaster trees due to the differences in genotypes
and because they grow in agro- and natural ecosystems,
respectively. Such expectations are reported for plenty of
crop—wild couples (Gressel, 2005). The effect of domestica-
tion in olive is still unclear and if some authors considered fruit
size as main modification (Lumaret and Ouazzani, 2001) other
authors considered rather fruit oil content (Breton et al., 2006b).
Consequently, the olive/oleaster couple is an original example
of crop/wild complex because they are not fully separated.
Olive oil composition displays a range of composition for fatty
acids (saturated, mono-unsaturated, and poly-unsaturated)
according to cultivars and environment that leads to olive oil
appellations, but little attention has been brought to oil
composition in oleaster trees. Surprisingly, oleaster and cultivar
trees display the same range of variation.
The morphological parameters showed a continuous
variation between cultivated olive tree and oleaster, but they
defined two extreme classes based on drupe size (small and big
fruits). These parameters appeared more efficient when they
were associated with ecosystems for the oleaster trees. In
contrast, the oil content and fatty acid composition used
together define oleaster and cultivar relationships. Moreover,
SSR are the most efficient to establish relationships between
oleasters and cultivated olive trees.
Chemlali has unambiguously conserved the fruit morphol-
ogy of an oleaster. The oil content is much high than in other
cultivars and Chemlali could be due to local domestication.
Standards for olive oil as for Protected Geographical Indication
(PGI) appellations fix the lower value for oleic acid (55%).
Variation in oil composition enabled to distinguish cultivars
(Ollivier et al., 2003), but was never used to study oil cultivar
and oleaster trees. A local origin of a cultivar, as Chemlali, may
document geographic labels to valorise products.
Acknowledgements
Thanks are due to Abderazzek Bousselmi & Samira Melek
(Institut de l’Olivier Tunis) for helpful technical assistance.
