Scientia Horticulturae 236 (2018) 127–136

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Scientia Horticulturae
journal homepage: www.elsevier.com/locate/scihorti

Characterization of olive progenies derived from a Tunisian breeding T

program by morphological traits and SSR markers
⁎
Jihène Dridia, , Mahdi Fendrib, Catherine Marie Bretonc, Monji Msallemb
a
National Institute of Agronomy of Tunis, University of Carthage, 43, Avenue Charles Nicolle, 1082 Tunis, Tunisia
b
Olive Tree Institute, 286, El Mahrajene City, 1082 Tunis, Tunisia
c
Institut des Sciences de l’Evolution de Montpellier (ISE-M), UMR CNRS 5554 Place E. Bataillon, cc63,Bât 24, 1er étage, F-34095 Monpellier Cedex 5, France

A R T I C LE I N FO A B S T R A C T

Keywords: An olive breeding program was conducted in Tunisia since 1994 using controlled crosses and open pollination on
Breeding 'Meski' cultivar mainly used for table olives in northern Tunisia. From a total of 200 progenies, thirteen geno-
Genotypes types were subsequently selected and are potentially suitable for both oil processing and table olive uses. The
Identification study aimed to identify the aforementioned genotypes as well as the pollen donors used for cross breeding using
Morphological
morphological and molecular approaches. The morphological study showed high variation between genotypes
Olive
and permitted their repartition into two groups according to the main discriminative parameters, which are the
Paternity
SSR markers fruit and endocarp weights based on principal component analysis. The parentage of thirteen olive progenies and
the molecular identification of 26 olive cultivars, including 8 olive varieties used as the parents in the cross
breeding program, were analyzed using a set of six co-dominant polymorphic Simple Sequence Repeats (SSRs)
selected among the most suitable markers for olive identification. SSR analysis revealed a high polymorphic
information content value and a low probability of identity among genotypes, indicating the efficiency of SSR for
detecting genetic diversity among genotypes and their potential use for olive breeding. Progenies were grouped
into two groups, according to the crosses. Transmission of alleles from parents to offspring was observed, in
which ten of the thirteen progenies sampled were matched consistently to their maternal parent ‘Meski’ and the
paternity was confirmed. Three did not match, but their parents were identified and they appear derivatives
from other breeding crosses. These genotypes remain to be evaluated for their agronomic performances.

1. Introduction uniformity, or a high pulp:stone ratio (Lavee, 1994). Cross-breeding is

Olive trees (Olea europaea subsp. europaea var. europaeaL.) are
grown for oil and table olive production in the Mediterranean basin
since ancient times, an enormous genetic diversity between varieties is
resulting (Breton et al., 2006). The richness of the cultivated olive
germplasm represents an unusual case among horticultural crops, as a
consequence of tree longevity and little turnover with new breeding
genotypes (Belaj et al., 2011). The need for more suitable cultivars
prompted the development of olive breeding programs in the main
olive-producing countries based in intra-specific cross-breeding be-
tween cultivars of known merit aiming at combining the good qualities
of the progenitors in some of the genotypes of the progenies (De Medina
et al., 2014; León et al., 2015). These programs aim at improving oil
yield and quality, resistance to diseases, suitability to mechanical har-
vesting, early and low alternate bearing, high productivity and oil
content, and shortening of the juvenile period (Fabbri et al., 2009). In
the case of table olives, other features include shape, size, ripening time
considered the best strategy in olive breeding programs due to the high
level of heterozygosity of varieties.
Olive trees are propagated by vegetative methods. Clonal propaga-
tion enables to multiply a single tree based on cuttings or suckers. Aims
of clonal propagation is to obtain homogenous cultivars characterized
by interesting features such as high yield, disease resistance and oil
composition (Hannachi et al., 2011). However, Clonal propagation does
not allow genotypes combining new traits. Therefore, this method could
lead to reduction of variability. In this situation, genetic improvement
based on breeding program would be more efficient to obtain new
genotypes having interest characters.
Breeding programs by crossing and selection in the progenies are
reported in Turkey (Arsel and Cirik, 1994), Spain (Rallo, 1995), Tunisia
(Trigui, 1996), Iran (Zeinanloo, 2006), China (Shan-An et al., 1981),
Ukraine (Sholokhova, 1984), Greece (Pritsa et al., 2003), Israel (Lavee
et al., 1999, 2003), and Italy (Bellini et al., 2002; Fontanazza et al.,
1999). However, until recently, very few cultivars have been emerged

⁎
Corresponding author.
E-mail address: drjihene@gmail.com (J. Dridi).

https://doi.org/10.1016/j.scienta.2018.03.042
Received 19 August 2017; Received in revised form 13 March 2018; Accepted 20 March 2018
0304-4238/ © 2018 Elsevier B.V. All rights reserved.
J. Dridi et al. Scientia Horticulturae 236 (2018) 127–136

from formal olive breeding programs and were selected empirically Table 1
within their region of cultivation (Marchese et al., 2016). To date, only List of crosses.
a very small number of new cultivars and advanced selections, coming Code cross Code cross
from different breeding programs, have been described (e.g. ‘Moncita’
in France (Besnard et al., 2000), ‘Askal’ in Israel (Lavee et al., 2003); 17 C ‘Meski’ x ‘Agezzi’ 16I ‘Meski’ x ‘Picholine’
16D ‘Meski’ x’ Chétoui’ 22I ‘Meski’ x ‘Picholine’
‘Arno’ in Italy (Bellini et al., 2000) and ‘Chiquitita’ in Spain (Rallo et al.,
10E ‘Meski’ x ‘Chemlali’ 23I ‘Meski’ x ‘Picholine’
2008). 9F ‘Meski’ x’ Besbessi’ 12 J ‘Meski’ x ‘Picholine’
New cultivar selections largely depend on early identification and 14 F ‘Meski’ x’ Besbessi’ 21 K ‘Meski’ x’ Ascolano tenera’
protection of olive varieties. Breeders have used morphological traits to 8H ‘Meski’ x’ Manzanillo’ IO2 ‘Meski’ x’ Ascolano tenera’
identify hybrid progenies and selfing progenies. However, the use of 22 H ‘Meski’ x ‘Picholine’

morphological traits has limitations to identify varieties because they

are largely affected by plant development and highly influenced by the
12 J (‘Meski’x‘Picholine’) and 21 K and IO2 (‘Meski’x’Ascolano tenera’)
environment. Alternatively, DNA molecular markers have added a new
(Table 1).
and powerful dimension to olive breeding and management. Among the
molecular markers, microsatellites or SSR (Simple Sequence Repeats)
are now widely accepted. Genetic studies using microsatellite markers
2.2. Morphological characterization
have increased rapidly because they have codominant segregation and
a high level of polymorphism in olive (Bracci et al., 2011). In fact,
Morphological description was conducted on eight parent cultivars
microsatellites were previously validated for olives to characterize the
and thirteen olive progenies, according to the methodology for primary
genetic diversity and cultivars identification (De la Rosa et al., 2002;
characterization of olive varieties proposed by the International Olive
Belaj et al., 2004; Díaz et al., 2006; Sarri et al., 2006; Baldoni et al.,
Oil Council (IOOC, 1997) on olive leaves, fruits and stones characters.
2009; Khadari et al., 2010; Arbeiter et al., 2014; Fendri et al., 2014;
The studied olive genotypes were evaluated for 29 morphological traits
Muzzalupo et al., 2014). In an other hand, Simple Sequence Repeats
following the descriptors reported by the IOOC (1997a,b). Four char-
have become the markers of choice for paternity analysis in olive
acters related to the leaf (length, widht, shape and longitudinal cur-
(Mookerjee et al., 2005; Arbeiter et al., 2014). They allow the early
vature of the blade), Eleven related to the fruit (weight, length, width,
identification of true interspecific hybrid progenies for further evalua-
shape, symmetry in position (A), position of maximum transversal
tion. True hybrid progenies are detected by the presence of DNA se-
diameter, apex, base, nipple presence, presence and size of lenticels)
quences corresponding to one of the two alleles contributed by each of
and thirteen related to endocarp (weight, length, width, shape, sym-
the two parents. The effectiveness of microsatellite markers in paternity
metry in position (A), symmetry in position (B), position of maximum
testing of progeny obtained from an olive breeding program has been
transversal diameter, apex, base, surface, number of grooves, distribu-
demonstrated (De la Rosa et al., 2004; Díaz et al., 2007).
tion of grooves and mucro presence (Table 3). The flesh to stone ratio
In Tunisia, the olive tree is widely cultivated and it constitutes one
(FSR) was also determined according to the pomological characteriza-
of the principal economical and agricultural strategic sectors that are
tion procedure (IOOC, 1997b).
known for their richness of varieties (Abaza et al., 2005). A cross-
The investigation included 13 quantitative and 16 qualitative traits.
breeding program has been carried out in 1994 within the context of
All the measurements were evaluated for random samples of 30 leaves
the project “olive breeding” (supported by the IOOC) by crossing two
and fruits from each tree. Endocarps were removed and were subject of
local cultivars ‘Meski’ (table olive) and ‘Chétoui’ (oil olive) with both
characterization.
local and foreign pollen donors in the goal of selecting new dual pur-
All statistical analyses based on quantitative traits were performed
pose, as well as new oil or table olive varieties to meet the requirement
using SPSS® 24.0 (IBM®). Average data, standard deviation, minimum
of the international market. An exhaustive agronomic evaluation of 200
and maximum values and coefficient of variation were calculated.
progenies of the above-mentioned crosses was undertaken and the best
Pearson correlation coefficients were calculated for each trait between
thirteen progenies were selected as those with better performance in
all other traits in order to study the associations among thirteen
terms of either yield or quality in either oil or fruits throughout several
quantitative traits used in the analysis of twenty-one olive genotypes.
years of production.
Principal Component Analysis (PCA) was carried out on quantitative
The present work aimed to study the genetic diversity among the
traits in order to distinguish between groups of olive cultivars and
selections and their parents by using morphological traits and SSR
progenies.
markers, to check the paternity of the selections derived from olive
crossing by using six SSR markers and to discriminate them from the 26
varieties presented in the olive collection studied.
2.3. DNA extraction
2. Materials and methods
Total genomic DNA was extracted according to the CTAB protocol
previously established by Murray and Thompson (1980) with slight
2.1. Plant material
modifications described by De la Rosa et al. (2002), starting from
100 mg fresh leaves for all olive tree and progenies samples, previously
The experimental material used in the present study comprised
ground in liquid nitrogen. Then, 500 μl CTAB/DTT mix was added in
thirteen olive progenies, obtained through genetic controlled olive
addition to 500 μl of chlorophormisoamyl alcohol (24:1). After cen-
crossing using the combinations on ‘Meski’ as mother variety, their
trifuging the suspension, DNA was precipitated by 2/3 vol of iso-
parents (8 varieties), and 18 olive varieties from the collection of ‘Borj
propanol from the aqueous phase at −20 °C. Finally, the dry pellet was
Amri’ (North of Tunisia; 36° 42ˈ 10″ North, 9° 53ˈ 16″ East). The thirteen
re-suspended in 100 μl of TE solution with 5 μl RNAse and incubated at
olive progenies were supposed to be the result of crossing ‘Meski’ with
37 °C for 1 h.
three local varieties: (‘Besbessi’, ‘Chemlali’ and ‘Chétoui’) and four
DNA concentration and purity were evaluated by NanoDrop 2000
foreign varieties (‘Agezzi’, Egypt; ‘Ascolano tenera’, Italy; ‘Manzanillo’,
spectrophotometer (Thermo Scientific). The extracted DNA was stored
Spain; and ‘Picholine’, France). The progenies were obtained from the
at −20 °C until use.
seven following crossings: 17 C (‘Meski’x‘Agezzi’); 16D
(‘Meski’x’Chétoui’); 10E (‘Meski’x ‘Chemlali’); 9 F and 14 F
(‘Meski’x’Besbessi’); 8 H (‘Meski’x’Manzanillo’); 22 H, 16I, 22I, 23I and

128
J. Dridi et al.
Table 2
Source, sequence, repeat motif, fluorescent dye and expected allele range for six micrositellite loci used in molecular analysis.
Source SSR locus Sequence (5'-3')
Sefc et al. (2000) ssrOeUA-DCA3 F-CCCAAGCGGAGGTGTATATTGTTAC
R-TGCTTTTGTCGTGTTTGAGATGTTG
ssrOeUA-DCA18 F-AAGAAAGAAAAAGGCAGAATTAAGC
R-GTTTTCGTCTCTCTACATAAGTGAC
Carriero et al. (2002) GAPU101 F-CATGAAAGGAGGGGGACATA
R-GGCACTTGTTGTGCAGATTG
GAPU103A F-TGAATTTAACTTTAAACCCACACA
R-GCATCGCTCGATTTTTATCC
GAPU71B F-GATCAAAGGAAGAAGGGGATAAA
R-ACAACAAATCCGTACGCCTTG
Cipriani et al. (2002) UDO99-043 F-TCGGCTTTACAACCCATTTC
R-TGCCAATTATGGGGCTAACT
2.4. Microsatellite amplification and genotyping in cultivar collection and
progeny testing
A set of six microsatellite (SSR) primer pairs were used for finger-
printing the olive cultivars and the progenies (Table 2). Two SSRs
(DCA3 and DCA18) have been developed by Sefc et al. (2000), three
(GAPU101, GAPU103A and GAPU71B) by Carriero et al. (2002) and
one (UDO99-043) by Cipriani et al. (2002). PCR amplifications were
carried out in a final volume of 20 μl containing 5 ng of DNA, 1.5 mM
MgCl2, 60 μM dNTPs, 0.028 units of Taq DNA polymerase (PROMEGA),
1 μM of forward primer labeled with a fluorescent dye (FAM, HEX or
TET) and 1 μM of reverse primer. SSR amplifications were performed on
a thermal cycler (Biometra®) under the following conditions : initial
denaturation at 95 °C for 5 min, followed by 35 cycles at 95 °C for 20 s
(denaturation), 52 °C for 30 s (annealing) and 72 °C for 30 s (extension),
and a final extension at 72 °C for 8 min.
After successful amplification, evaluated by electrophoresis in 1.5%
agarose gel, PCR products were combined with Liz 1200 internal size
standards and were separated using an automatic capillary sequencer
(ABI 3130 Genetic Analyser Applied Biosystems). The fragments were
visualized as peaks with the respective size and intensities using Peak
Scanner v.1.0 and GeneMapper v.4.0 softwares from Applied
Biosystems.
2.5. Data analyses
Descriptive statistics data were performed using CERVUS software
v.3.0 (Marshall et al., 1998). The number of alleles, null allele fre-
quency, observed heterozygozity (Ho), expected heterozygosity (He)
(Nei, 1987) for each locus were calculated based on Hardy Weinberg
(HW) as well as the polymorphic informaion content (PIC) (Botstein
et al., 1980) and the probability of identity (PI) which represents the
probability of matching for two individuals randomly selected at all the
six microsatellite loci (Paetkau and Strobeck, 1994).
For each individual, all alleles detected were converted to a binary
data matrix (0/1) using “1″ for the presence and “0″ for the absence for
each amplified fragment. Genetic relationship between the selections
and the cultivars used as parents was analysed with the NTSYS-pc
program v.2.02 (Rohlf, 1998). The similarity matrix analysis was ob-
tained using the Dice Similarity coefficient (Sneath and Sokal, 1973).
Finally, a dendrogram was generated based on the unweighted pair
group method with arithmetic mean (UPGMA).
2.6. Parentage analysis using SSR markers
The set of microsatellites markers were used to verify the paternity
of the progenies from the seven crosses. According to the microsatellite
profile, the offsprings of each crossing were classified as true hybrid or
foreign origin. Parentage was analyzed based on the inheritance of one
129
Scientia Horticulturae 236 (2018) 127–136
Repeat motif Fluorescent Expected allele
dye range
(GA)19 HEX 226–254
(CA)4CT(CA)3(GA)19 TET 162–187
(GA)8(G)3(AG)3 FAM 183–219
(TC)15 HEX 132–220
GA(AG)6(AAG)8 HEX 117–144
(GT)12 FAM 166–218
allele at a single SSR locus transmitted from the parent to the offspring.
If one maternal and one paternal allele were present for each primer,
the progeny was considered ‘true’. In the case where the parentage was
different than that expected, the real parent was estimated (Díaz et al.,
2007
3. Results
3.1. Morphological characterization of olive progenies and their parents
A total of thirteen variables was used for statistical analyses. The
mean range, the standard deviation, the minimum and maximum va-
lues, the coefficient of variation and the degree of significance for the
quantitative traits are shown on Table 4. All the agro-morphological
characteristics showed a significant difference (P ≪ 0.001) between
descendants and progenitors. The highest coefficient of variation was
observed in fruit and stone weight (48%), while a lower variation was
found in fruit shape (fruit length/fruit width) (11%). Fruit weight of
parent cultivars ranged from 1 (cv.‘Chemlali’) to 6.39 g (cv.‘Ascolano
tenera’), and from 1.29 (hybrid ‘21 K’) to 5.77 g (hybrid ‘IO2′) for
descendants. Stone weight varied from 0.21 (cv.‘Chemlali’) to 0.72 g
(cv.‘Meski’) for parent cultivars and from 0.21 (hybrid ‘16D’) to 0.81 g
(hybride ‘9 F’) for descendants. Flesh to stone ratio (FSR) showed also
an important variation (30%) among the analysed genotypes. In olive
parents the highest flesh to stone ratio recorded was 8.32 (cv.‘Picho-
line’) while the lowest was 3.55 (cv. ‘Chétoui’). Among the descendants,
the FSR ranged between 2.21 for hybrid ‘23I’ and 7.04 for hybrid ‘IO2′.
Principal Component Analysis (PCA) was conducted using mor-
phological traits. The first three principal components explained
79.42% of the total morphological variance (45.65%, 18.03% and
15.73% respectively). The first principal component (PC) accounted for
45.65% explaining the largest portion of the variance. It’s correlated
with length, width and weight of fruit and stone (FL, FWI, FW, SL, SWI
and SW), flesh to stone ratio (FSR) and number of grooves of endocarp
(SG). Therefore, theses traits were important attributes for the classi-
fication of olive varieties and descendants. The second PC is correlated
with fruit and stone shape (FS and SS). The third component is de-
termined mainly by the width and the shape of leaves. The first two
components explained the largest fraction of variance, 63.68%.
The projection of progenies and their parents on the plan de-
termined by the first two principal components reveals a consistent
ordination of olive genotypes by fruit and stone weight (Fig. 1). The
olive cultivars with bigger fruit and endocarp and highest flesh to stone
ratio trended to group together on the right corner of graph while
others presenting smallest fruits and endocarps and a lower flesh to
stone ratio were associated on the left apart. The first group included six
cultivars (‘Agezzi’, ‘Ascolano tenera’, ‘Besbessi’, ‘Meski’, ‘Manzanillo’
and ‘Picholine’) and seven hybrid progenies derived from five different
crosses with ‘Meski’ (‘10E’ from ‘Meski’x‘Chemlali’, ‘9 F’ and ‘14 F’ from
 Table 3
Morphological characteristics according to the International Olive Council of olive cultivars and their descendants.
Characteristic
J. Dridi et al.

Cultivar Leaf Fruit

Shape Longitudinal Curvature of the Weight Shape Symmetry (position Position of max. transverse Apex Base Nipple Presence of Size of
blade A) diameter lenticels lenticels

Agezzi Elliptic Epinastic Very high Elongated Symmetric Central Rounded Truncated Present Many Small
Ascolano tenera Elliptic Epinastic Very high Spherical Slightly asymmetric Towards base Pointed Truncated Absent Few Small
Besbessi Elliptic Flat Very high Spherical Symmetric Central Rounded Truncated Absent Many Small
Chemlali Elliptic- Flat Low Ovoid Symmetric Central Rounded Rounded Absent Many Small
lanceolate
Chétoui Elliptic Epinastic Medium Elongated Slightly asymmetric Central Pointed Truncated Present Few Small
Manzanillo Elliptic Epinastic Medium Spherical Symmetric Central Rounded Rounded Absent Many Small
Meski Elliptic Epinastic Very high Spherical Slightly asymmetric Central Rounded Rounded Absent Few Big
Picholine Elliptic- Epinastic Medium Elongated Symmetric Central Pointed Rounded Absent Many Small
lanceolate
17 C (Chétoui x Agezzi) Elliptic- Epinastic Medium Ovoid Symmetric Central Rounded Rounded Absent Many Small
lanceolate
16D (Meski x Chétoui) Elliptic- Flat Low Elongated Asymmetric Towards apex Pointed Rounded Absent Few Small
lanceolate
10E (Meski x Chemlali) Lanceolate Flat High Ovoid Slightly asymmetric Central Rounded Truncated Absent Few Small
9 F (Meski x Besbessi) Elliptic- Flat High Elongated Slightly asymmetric Towards apex Pointed Rounded Present Few Small
lanceolate
14 F (Meski x Besbessi) Elliptic- Epinastic High Ovoid Symmetric Central Rounded Rounded Absent Few Small
lanceolate
8 H (Meski x Elliptic Flat High Spherical Symmetric Central Rounded Rounded Absent Many Small

130
Manzanillo)
22 H (Meski x Picholine) Elliptic- Flat High Ovoid Slightly asymmetric Towards base Pointed Truncated Present Few Small
lanceolate
16I (Meski x Picholine) Elliptic Flat Medium Elongated Slightly asymmetric Central Pointed Rounded Absent Few Big
22I (Meski x Picholine) Elliptic- Helicoid High Ovoid Symmetric Central Pointed Rounded Absent Few Small
lanceolate
23I (Meski x Picholine) Elliptic- Flat Low Elongated Symmetric Central Pointed Rounded Absent Few Small
lanceolate
12 J (Meski x Chétoui) Elliptic- Flat Low Ovoid Slightly asymmetric Towards base Pointed Rounded Absent Many Small
lanceolate
21 K (Chétoui x Elliptic- Flat Low Ovoid Symmetric Central Rounded Rounded Absent Many Small
Ascolano) lanceolate
IO2 (Meski x Ascolano) Elliptic Helicoid Very high Elongated Asymmetric Central Pointed Truncated Present Few Small
Agezzi High Elliptic Symmetric Slightly Central Pointed Pointed Rough High regular Present
asymmetric
Ascolano tenera High Elliptic Symmetric Symmetric Central Pointed Pointed Wrinkled Medium regular Present
Besbessi High Elliptic Symmetric Symmetric Central Rounded Truncated Wrinkled Medium regular Present
Chemlali Low Elongated Symmetric Symmetric Central Pointed Rounded Smooth Low regular Present
Chétoui Low Elongated Symmetric Slightly Central Pointed Pointed Rough Medium regular Present
asymmetric
Manzanillo High Elliptic Symmetric Symmetric Towards apex Rounded Pointed Wrinkled High regular Present
Meski Very high Elliptic Symmetric Symmetric Central Pointed Pointed Wrinkled Medium regular Present
Picholine Medium Elongated Symmetric Symmetric Central Pointed Pointed Rough Medium regular Present
17 C (Chétoui x Agezzi) Medium Ovoid Symmetric Symmetric Central Rounded Rounded Rough Medium regular Absent
16D (Meski x Chétoui) Low Elliptic Symmetric Symmetric Central Pointed Pointed Smooth High regular Present
10E (Meski x Chemlali) High Elliptic Symmetric Symmetric Central Rounded Truncated Wrinkled High regular Present
9 F (Meski x Besbessi) Very high Ovoid Symmetric Symmetric Central Rounded Pointed Wrinkled High regular Absent
14 F (Meski x Besbessi) High Elliptic Symmetric Symmetric Central Rounded Pointed Wrinkled Medium regular Present
(continued on next page)
Scientia Horticulturae 236 (2018) 127–136
J. Dridi et al. Scientia Horticulturae 236 (2018) 127–136

Table 4
Mean, standard deviation (SD), minimum, maximum and the coefficient of
variation of thirteen quantitative agronomic traits evaluated for thirteen olive

lenticels

Present

Present
Present
Present
Present
Present

Present

Present
Size of
progenies and their parents.
Trait name Abbreviation Mean SD Minimum Maximum CV (%)

Leaf length (cm) LL 4.99 0.70 2.92 6.12 14***

Presence of

Leaf width (cm) LW 1.22 0.18 0.78 1.61 15***

lenticels

Leaf shape LS 4.27 0.95 2.19 7.10 22***

regular

regular
regular
regular
regular
regular

regular

regular
Fruit weight (g) FW 3.82 1.82 1.00 6.39 48***
Fruit length FL 21.51 4.16 14.97 28.71 19***
(mm)
Medium
Medium
Medium
Medium
Fruit width (mm) FWI 15.95 3.42 10.03 20.91 21***
Nipple

High

High
Low

Low
fruit shape FS 1.36 0.15 1.14 1.57 11***
Stone weight (g) SW 0.46 0.24 0.21 0.81 48***
Stone length SL 14.95 2.65 10.56 19.36 18***
Wrinkled

Wrinkled

Smooth
Smooth

Smooth (mm)
Rough
Rough

Rough
Base

Stone width SWI 7.17 1.25 5.33 9.99 17***

(mm)
Stone shape SS 2.11 0.31 1.66 2.68 15***
Rounded

Rounded
Rounded

Number of SG 8.94 1.32 6.00 11.00 15***

Pointed

Pointed

Pointed

Pointed

Pointed
Apex

grooves
Fruit flesh to FSR 5.90 1.82 2.21 8.32 31***
stone ratio
Position of max. transverse

:Significant at P ≪ 0.001.
***

‘Meski’x‘Besbessi’, ‘8 H’ from ‘Meski’x‘Manzanillo’, ‘22 H’ and ‘22I’ from

‘Meski’x‘Picholine’ and ‘IO2′ from ‘Meski’x‘Ascolano tenera’). The
second group consisted of two oil olive varieties (‘Chemlali’ and ‘Ché-
Rounded

Rounded

Rounded
diameter

Pointed

Pointed
Pointed
Pointed

Pointed

toui’) and 6 hybrid progenies (‘17 C’ which supposed derived from

‘Meski’x‘Agezzi’, ‘16D’ from ‘Meski’x‘Chétoui’, ‘12 J’, ‘16I’ and ‘23I’
from ‘Meski’x‘Picholine’ and ‘21 K’ from ‘Meski’x‘Ascolano tenera’).
Symmetry (position

Towards apex

3.2. SSR diversity and cultivar identification

Central

Central
Central
Central
Central
Central

Central

A set of six SSR primers was employed to identify 26 olive varieties

A)

present in the olive germplasm collection of Borj Amri and to analyse

the paternity of thirteen progenies derived from cross-breeding pro-
gram. The six microsatellites appertaining to the series OeUA-DCA (Sefc
asymmetric

asymmetric

et al. 2000), GAPU (Carriero et al., 2002) and UDO (Cipriani et al.,
Symmetric

Symmetric
Symmetric
Symmetric
Symmetric

Symmetric

2002) were successfully amplified in all the studied genotypes and only
Slightly

Slightly
Shape

two alleles at each locus were retained. The PCR product size ranged
from 117 to 248pb and a total of 43 alleles were detected, ranging from
Asymmetric

Asymmetric

5 at both GAPU101 et GAPU71b loci to 10 alleles at both UDO43 and

Symmetric

Symmetric
Symmetric

Symmetric
Symmetric

Symmetric

GAPU103A loci with an average of 7.17 (Table 5). The mean value of
Weight
Fruit

observed heterozygosity (Ho = 0.987) was higher than the expected

heterozygosity (He = 0.823), indicating high genetic variability among
Longitudinal Curvature of the

the analysed cultivars (Table 5). Average polymorphic information

content (PIC) was 0.779, and ranged from 0.695 for GAPU101 to 0.853
for GAPU103A, showing that markers are highly polymorphic
(Table 5). The probability of sampling identical genotypes (PI) varied
among loci in a range from 0.031 to 0.111 and the cumulative PI value
calculated for all loci was 5.10−8, indicating acceptable discriminating
Elongated
Elongated
Elongated
Elongated

Elongated
Elliptic

Elliptic

power of the analysed microsatellite markers.

Ovoid
blade

Allele frequencies ranged for 0.013 for the allele 159 of locus
DCA18 and alleles 170 and 216 of locus UDO43 to 0.385 for the allele
235 of locus DCA3 (Table 6).
Medium
Medium

3.3. Parentage analysis and seedling discrimination

Shape

High

High

High
Low
Low

Low
Leaf

The parentage of 13 progenies olive cultivars, using 6 SSR loci, re-

22 H (Meski x Picholine)

IO2 (Meski x Ascolano)

16I (Meski x Picholine)
22I (Meski x Picholine)
23I (Meski x Picholine)
12 J (Meski x Chétoui)

vealed that in 10 of the 13 parent-offspring relationships, both female

Table 3 (continued)

(‘Meski’) and male cultivars were established as parents because alleles

Manzanillo)

21 K (Chétoui x

at all SSR loci of offspring cultivars were inherited from their parents
Ascolano)
Characteristic

8 H (Meski x

(‘16D’, ‘10E’, ‘9 F’, ‘14 F’, ‘8 H’, ‘22 H’, ‘16I’, ‘22I’, ‘23I’ and ‘IO2′). Three
Cultivar

progenies (‘17 C’, ‘12 J’ and ‘21 K’) were not confirmed as offspring of
their cited parents because there was discrepancy in the patterns of SSR
alleles (Tables 7 and 8). At least one of the two maternal alleles should

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Fig. 1. Plot of the first and second principal components illustring the relationships among 13 olive progenies derived from a Tunisian cross-breeding program and
their parents based on Principal Component Analysis using 13 quantitative morphological traits.

be always present in all of the descendance for all the SSR loci analyzed
to confirm the maternity of descendance which ‘Meski’ olive trees acted
as the femele parent in all crosses. However, in our study only in eleven
cases, the female parent (Meski) was confirmed by the presence of one
allele of the two maternal alleles for all the SSR loci analyzed. In two
hybrid progenies (17 C and 21 K), the maternity was erroneous and the
pattern using the six SSR loci suggests that ‘17 C’ is a hybrid between
‘Chétoui’ and ‘Agezzi’ and that ‘21 K’ is hybrid of ‘Chétoui’ and ‘Asco-
lano tenera’. In the case of a progeny ‘12 J’, the paternity was erroneous
and the progeny is a derivative from the cross of ‘Meski’ with ‘Chétoui’
but not with ‘Picholine’. This would indicate that there was con-
tamination by pollen of ‘Chétoui’, the predominant cultivar in the area
in which the crosses were performed. Among these thirteen descen-
dants, all progenies were successfully matched to a mother and the
father (Table 7).
3.4. Genetic relationship between selections and their parents (UPGMA)
A presence-absence matrix for the SSR fragments was constructed,
genetic similarities were calculated among progenies and parents for
each crossing and among all progenies using the Dice coefficient, and
UPGMA grouping was performed. The final dendrogram grouped the 13
progenies and their parents at a similarity coefficient higher than 0.30
(Fig. 2). Cluster analysis showed two main groups corresponding to the
crossings (with Chétoui or Meski as mother parents). The progenies are
clearly associated at least with one of their parent in each cluster. The
first group (I) consisted of two progenies of chétoui (17 C and 21 K)
including the two male parents Agezzi and Ascolano tenera respectively
and the cultivar Chemlali. The second group (II) clustered 10 progenies
with their maternal parent (Meski). Into this group, some hybrids de-
rived from the same cross were closely related and very similar with
Dice coefficient higher than 0.80 such as the progenies of Meski x Pi-
choline (16I and 23I) and Meski x Chétoui (16D and 12 J). The hybrid
9 F was clearly separated from the other groups but clustered with its
male parent (Besbessi). This separation due to the inheritance of the
less-frequent alleles among the progenies analysed. In deed, the unique
profiles in 9 F (238/240) and (119/125) based respectively on DCA3
and GAPU71b loci were strongly determinant of this separation.

Table 5
Studied SSR loci, size range of alleles (pb), number of alleles, observed heterozygosity (Ho), expected heterozygosity (He), polymorphism information content (PIC),
probability of identity (PI) and number of total and unique genotypes in the 26 olive cultivars grown in Borj Amri.
SSR name Size range (pb) No.of alleles Ho He PIC PI No. genotypes

Total Unique

ssrOeUA-DCA3 228–248 6 0.962 0.804 0.758 0.075 12 6

ssrOeUA-DCA18 159–175 7 1.000 0.823 0.78 0.064 11 4
GAPU101 180–214 5 1.000 0.753 0.695 0.111 6 0
GAPU103A 132–220 10 1.000 0.884 0.853 0.031 16 10
UDO99-043 166–216 10 0.962 0.882 0.85 0.033 17 12
GAPU71b 117–138 5 1.000 0.793 0.793 0.086 8 2
Mean – 7.16 0.987 0.823 0.779 5.10−8* ˗ ˗

*
Product of PI values across all 6 loci.

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Table 6
Allele size (pb) and allele frequencies given (in parenthesis), for olive cultivars at 6 loci.
Locus Alleles

1 2 3 4 5 6 7 8 9 10

DCA3 228 (0.205) 235 (0.385) 238 (0.064) 240 (0.128) 244 (0.077) 248 (0.141)
DCA18 159 (0.013) 165 (0.038) 167 (0.128) 169 (0.333) 171 (0.270) 173 (0.128) 175 (0.089)
GAPU101 180 (0.026) 187 (0.372) 195 (0.294) 202 (0.128) 214 (0.179)
GAPU103A 132 (0.128) 147 (0.179) 155 (0.089) 169 (0.154) 172 (0.103) 183 (0.167) 188 (0.038) 192 (0.089) 201 (0.026) 220 (0.026)
UDO43 166 (0.064) 170 (0.013) 172 (0.205) 174 (0.205) 178 (0.038) 207 (0.115) 210 (0.089) 212 (0.128) 214 (0.128) 216 (0.013)
GAPU71b 117 (0.192) 119 (0.192) 122 (0.179) 125 (0.128) 138 (0.308)

4. Discussion
Twenty-one olive samples composed of four local Tunisian cultivars
(‘Besbessi’, ‘Chemlali’, ‘Chétoui’ and ‘Meski’), four introduced
Mediterranean cultivars (‘Agezzi’, ‘Ascolano tenera’, ‘Manzanillo’ and
‘Picholine’) and thirteen progenies obtained from eight genetic combi-
nations, were investigated morphologically, measuring both qualitative
and quantitative traits of leaf, fruit and endocarp, and at the molecular
level by using most of currently recommended SSR markers.
A considerable amount of variation in morphological traits was
found between olive cultivars and hybrid progenies. It has been re-
ported that morphological traits are highly influenced by many en-
vironmental factors. In this work the effect of environment was not
taken into account because all olive trees were conducted in a unique
orchard with identical cultural techniques. Therefore, the observed
morphological variation reflected the genotype effect. High coefficient
of variation and highly significant differences (P ≪ 0.05) were noted
between progenies and genitors for most of fruit and stone characters.
These differences could be attributable to the genetic factors, as all
olive progenies and varieties were grown under the same agro-climatic
conditions. The morphological variability revealed in this study coin-
cide with previous studies carried out for olive cultivars (Caruso et al.,
2014; Marra et al., 2013), wild olive trees (Belaj et al., 2011; Hannachi
et al., 2008) and for other olive cross-breeding programs (Lavee, 1990;
Bellini, 1993; Fontanazza et al., 1999; León et al., 2004; Pannelli et al.,
2006; Laaribi et al., 2014; Guellaoui et al., 2015). In other hand, cat-
alogues for olive cultivars display description and pictures of trees and
fruits of each denomination in different countries. International Plant
Genetic Resources Institute (IPGRI, Roma) with International Olive Oil
Council (IOOC, Madrid) have regrouped most of the morphological
criteria for olive description. In current study, we used quantitative and
qualitative characters of leaf and fruit to describe new olive genotypes
and their parents in order to determine patterns of olive progenies
variations. Thus, these results confirm that cross breeding programs
allow providing new genotypes with wide ranges of variation for any
character due to the high level of heterozygosity in the varieties.
Previous studies explained that the traditional method of agro-
morphological plant characterization is a common step in plant
breeding for selection of parents and it represents a usual methodology
accepted for description and registration of varieties (Badanes et al.,
1998). In this study, both quantitative and qualitative traits of leaf, fruit
and endocarp were analyzed. Concerning quantitative traits, a higher
polymorphism was observed for fruit and stone rather than leaf char-
acters. In fact, the highest variation coefficients were noted for fruit and
stone size. Flesh to stone ratio showed also an important variation
coefficient. A similar result was recorded on the studied ‘Chemlali’
seedlings’ (Laaribi et al., 2014; Guellaoui et al., 2015) and olive culti-
vars (Belaj et al., 2011; Mnasri et al., 2014).
The evaluation of morphological variability should be conducted to
explore the new potentialities found in olive progenies. Consequently,
variation in agronomical character can be used in the selection of new
genotypes for future cultivars. The controlled crossing constitutes the
only method to increase the genetic diversity. Therefore, the recent
olive progenies obtained through controlled crossing broadened mor-
phological diversity. The large variation would be explained by sexual
reproduction.
PCA was useful for identifying the most important traits associated
with variation among the studied olive genotypes. In addition, dis-
tributions of the genotypes in the obtained PCA plots were in agreement
with morphological differences observed. In our study, the PCA analysis

Table 7
Allele combinations obtained at the six microsatellite loci in the parents and progenies tested.
Parent/Offspring DCA3 DCA18 GAPU101 GAPU103A UDO99-043 GAPU71b

Agezzi 228/235 165/171 187/195 132/155 166/207 117/119

Ascolano tenera 235/244 167/169 187/195 147/155 174/210 119/138
Besbessi 238/244 171/173 195/202 147/172 174/210 119/125
Chemlali 228/235 169/173 187/195 155/192 166/214 117/138
Chétoui 228/235 169/173 187/202 132/192 174/212 119/138
Manzanillo 240/248 167/175 195/214 132/147 210/212 117/138
Meski 235/240 169/171 187/214 169/183 172/214 122/125
Picholine 228/248 169/175 187/195 172/188 207/212 117/138
17 C (Chétoui x Agezzi) 235/235 169/171 187/195 155/192 166/174 119/138
16D (Meski x Chétoui) 235/235 169/171 187/214 132/169 174/214 119/122
10E (Meski x Chemlali) 235/240 169/171 187/187 183/192 166/172 117/122
9 F (Meski x Besbessi) 238/240 169/171 187/195 147/183 174/214 119/125
14 F (Meski x Besbessi) 235/244 169/171 187/195 169/172 172/174 119/122
8 H (Meski x Manzanillo) 235/240 171/175 187/195 147/183 172/212 122/138
22 H (Meski x Picholine) 228/240 169/171 187/195 172/183 172/207 122/138
16I (Meski x Picholine) 235/248 169/171 195/214 169/172 172/207 122/138
22I (Meski x Picholine) 235/248 169/171 195/214 169/188 212/214 125/138
23I (Meski x Picholine) 228/235 169/175 195/214 169/172 172/207 122/138
12 J (Meski x Chétoui) 235/235 169/171 187/214 169/192 174/214 122/138
21 K (Chétoui x Ascolano tenera) 228/235 169/173 187/202 132/147 174/212 138/138
IO2 (Meski x Ascolano tenera) 235/240 169/171 195/214 155/169 210/214 122/138

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Table 8
List of crosses with correction after molecular analyzes.
Code cross Code cross

17 C ‘Meski’ x ‘Agezzi’ wrong 16I ‘Meski’ x ‘Picholine’

17 C ‘Chétoui’ x ‘Agezzi’ correct 22I ‘Meski’ x ‘Picholine’
16D ‘Meski’ x’ Chétoui’ 23I ‘unicode-charMeski’unicode-char x ‘unicode-charPicholine’unicode-char
10E ‘Meski’ x ‘Chemlali’ 12 J ‘unicode-charMeski’unicode-char x ‘unicode-charPicholine’unicode-char wrong
9F ‘Meski’ x’ Besbessi’ 12 J ‘Meski’ x ‘Chétoui’ correct
14 F ‘Meski’ x’ Besbessi’ 21 K ‘Meski’ x ‘Ascolano tenera’ wrong
8H ‘Meski’ x’ Manzanillo’ 21 K ‘Chétoui’ x ‘Ascolano tenera’ correct
22 H ‘Meski’ x ‘Picholine’ IO2 ‘Meski’ x’ Ascolano tenera’

Fig. 2. Dendrogram based on the UPGMA method showing the relationships between thirteen progenies of olive and their parents based on 6 SSR markers using Dice
genetic similarity.
Similarity Coefficient.

classified the olive genotypes in two major groups according, mainly, to
fruit and endocarp size and flesh to stone ratio. We found that the first
group included 7 progenies (‘10E’, ‘9 F’, ‘14 F’, ‘8 H’, ‘22 H’, ‘22I’ and
‘IO2′) with 6 parents mainly classified as table olive. Whereas, the
second group integrated 6 hybrid progenies (‘17 C’, ‘16D’, ‘16I’, ‘23I’,
‘12 J’ and ‘21 K’) with the two mainly Tunisian oil olive varieties
(‘Chemlali’ and ‘Chétoui’). This classification revealed that fruit and
stone traits seemed to be the most effective for morphological char-
acterization and discrimination of olive varieties and new selections
which is in agreement with above researchers on olive tree (Idrissi and
Ouazzani, 2006; Hannachi et al., 2007; Klepo et al., 2013; Laaribi et al.,
2014; Mnasri et al., 2014).
Apart from the morphological traits, a high degree of polymorphism
was also found at the molecular level. Baldoni et al. (2009) examined
various previously published SSR markers and proposed 11 of them as
the most effective for olive cultivars characterization. In our study, six
of the 11 most effective markers were chosen for the analysis of parent-
offspring relationships. In fact, the codominant segregation of micro-
satellites allows for more efficient parentage analyses compared with
dominant markers (Gerber et al., 2000).
Studied parameters showed high variability in olive collection and
are common for the majority of outcross species (Table 5). The results
obtained in the present study demonstrate that the microsatellite
markers used were adequate for distinguishing of all the studied
genotypes and were polymorphic and informative (PIC mean = 0.779)
allowing the identification of 43 alleles and 70 profiles with little
probability of sampling identical genotypes (PI = 5.10−8). The average
observed heterozygosity value was higher than the expected hetero-
zygosity value, similar to the finding previously in other microsatellites
developed for olive tree (Díaz et al., 2006a; Poljuha et al., 2008;
Baldoni et al., 2009; Muzaluppo et al., 2014). SSR loci analyzed gave
good discrimination value with the probability of identical genotypes
ranging from 0.031 to 0.111 among loci (Table 5). All these results
confirm the effectiveness of microsatellite markers in the exploration of
the genetic diversity of olive cultivars.
Based on SSR profiles of olive progenies and cultivars we were able
to assess the paternity of the thirteen descendants derived from con-
trolled cross breeding program among which both local and foreign
olive varieties were used as pollen donors of the table olive ‘Meski’. The
putative maternity and paternity of the progenies were tested by the
presence of one maternal and one paternal allele for each of the six
primer pairs used in this study, as previously described in some in-
vestigations for paternity analysis (De la Rosa et al., 2004; Díaz et al.,
2007). Transmission of alleles from parents to progenies was clearly
observed, indicating that the marker set was able to resolve parentage
to two parent combination for thirteen progenies. A further, ten pro-
genies were matched to both maternal and paternal parents and were
identified as true hybrid progenies because alleles at all SSR loci of

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J. Dridi et al.
these descendants were found in putative parents (Table 7). One pro-
geny (‘12 J’, ‘Meski’ x ‘Picholine’) was matched to a maternal parent
only (‘Meski’). This would indicate that there was contamination by
pollen from other olive varieties from the olive collection. Allele data
showed that the progeny ‘12 J’ shared common alleles with ‘Chétoui’ at
all the studied microsatellite markers (Table 7). Thus, data suggested
that ‘Chétoui’ was a probable male parent. Given that ‘Chétoui’ is the
predominant cultivar in the area in which the crosses were performed;
therfore, it is logic to suspect that there was contamination by pollen
from this cultivar. This suggestion is in agreement with previous finding
of paternity testing of olive seedlings (De la Rosa et al., 2004; Díaz
et al., 2007) and self-incompatibility studies (Díaz et al., 2006b). On the
other hand, among the thirteen progenies from crossing, two descen-
dants (‘17 C’, ‘Meski’ x Agezzi’ and ‘21 K’, ‘Meski’ x ‘Ascolano tenera’)
have no maternal match. Indeed, the maternity was not confirmed since
absence of the alleles transmission from ‘Meski’ to these two progenies.
Previous studies of parentage analysis, reported that the maternal
identity was certain and a false paternity can occur in several cross
breeding programs caused by the contamination by foreign pollen. In
our study, the maternity assignment of the ‘17 C’ and ‘21 K’ was wrong
and the allele data showed that the alleles of ‘Chétoui’ were consistently
transmitted to these progenies for all the SSR loci (Table 7). Conse-
quently, we estimated that was confusion in progeny denomination and
some error in labeling might have occurred during the cross-pollination
procedure, especially because two crosses in olive breeding programs
were performed in the same area using the two predominant cultivars
in Tunisia ‘Meski’ and ‘Chétoui’ as mothers. On the basis of the data
obtained in this study and with concordance with previous studies (Díaz
et al., 2006b; Mookerjee et al., 2005) no real self-ferilization event were
found. These results demonstrate that SSR markers can be efficient and
therefore useful for identification of both parents in a true hybrid
progenies and as well as to detect pollen contamination, and thus
identification of the effective pollen donor in olive cross progenies.
5. Conclusion
The morphological traits and SSR markers applied on studied pro-
genies have allowed for the characterization of new selections from an
olive breeding program recombining the diversity found in parents. The
morphological characterization was useful to improve many future
agronomical traits. The SSR markers confirm the morphological varia-
bility and are used for cultivar identification and paternity testing.
Therefore, it is of interest to evaluate and conserve the recent olive
genetic resources of the most interesting new progenies with high
quality features. Further work is required to evaluate the putative
varieties in different environmental conditions and eventually, to reg-
ister the best genotypes as new cultivars.
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