Trees

DOI 10.1007/s00468-015-1276-2

ORIGINAL ARTICLE

Mediterranean basin Ficus carica L.: from genetic diversity

and structure to authentication of a Protected Designation
of Origin cultivar using microsatellite markers
Ioannis Ganopoulos1 Aliki Xanthopoulou1,2 Athanasios Molassiotis3
Evangelos Karagiannis3 Theodoros Moysiadis5 Panagiotis Katsaris6
Filippos Aravanopoulos7 Athanasios Tsaftaris1,2 Apostolos Kalivas4
Panagiotis Madesis1
Received: 21 April 2015 / Revised: 5 August 2015 / Accepted: 12 August 2015
Springer-Verlag Berlin Heidelberg 2015

Abstract
Key message The selected germplasm of Ficus carica
established in a gene bank collection will be useful for
conservation and management and important for fig
tree breeding programs.
Abstract Advancement in plant breeding is assisted by
accurate information on genetic diversity and structure. A
collection of ninety fig tree (Ficus carica L.) cultivars
originating from the Mediterranean basin and conserved in
an ex situ gene bank collection was genotyped using seven
microsatellite markers. A total of 91 alleles were detected
presenting an average of 13 alleles per locus. The gene bank
fig tree collection preserved a high level of genetic diversity. The mean expected and observed heterozygosities over

the seven single locus microsatellites averaged 0.747 and

0.784, respectively. The total value of the probability of
identity was 2 9 10-6. The 90 fig tree accessions formed
four clusters in the unweighted pair group method using
arithmetic averages (UPGMA) dendrogram, although the
clustering did not indicate any clear division among the fig
tree accessions based on their geographical origin. Moreover, the 90 fig tree accessions could be divided into two
clusters based on STRUCTURE analysis. Additionally,
microsatellites coupled with high-resolution melting analysis enabled both the distinction, identification and
authentication of the Kymis fig tree Protected Designation
of Origin cultivar and its products. In conclusion, results
presented here are significant for the management of gene
bank collections, breeding programs and authentication of
fig tree cultivars.

Communicated by F. Canovas.
I. Ganopoulos and A. Xanthopoulou contribute equally to this work.

Electronic supplementary material The online version of this

article (doi:10.1007/s00468-015-1276-2) contains supplementary
material, which is available to authorized users.

Keywords Microsatellites
Genetic diversity

Mediterranean basin
High-resolution melting (HRM)
authentication
Protected Designation of Origin (PDO)
cultivar
Ficus carica

& Apostolos Kalivas

kalivasapostolis@yahoo.gr

Cotton and Industrial Plants Institute, Hellenic Agricultural

Organization Demeter, Thermi, 57001 Thessaloniki, Greece

& Panagiotis Madesis

pmadesis@certh.gr

Department of Mathematics and Statistics, Faculty of Pure

and Applied Sciences, University of Cyprus, Nicosia, Cyprus

Institute of Applied Biosciences, CERTH, Thermi,

570 01 Thessaloniki, Greece

National Agricultural Research Foundation, Institute of

Kalamata, Lakonikis 87, 24100 Kalamata, Greece

Laboratory of Genetics and Plant Breeding, Faculty of

Agriculture, Forestry and Natural Environment, School of
Agriculture, Aristotle University of Thessaloniki,
54 124 Thessaloniki, Greece

Forest Genetics and Tree Breeding, Faculty of Agriculture,

Forestry and Natural Environment, Aristotle University of
Thessaloniki, Thessaloniki, Greece

Department of Pomology, Faculty of Agriculture, Forestry

and Natural Environment, School of Agriculture, Aristotle
University of Thessaloniki, 541 24 Thessaloniki, Greece

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Trees

Introduction
Common fig tree (Ficus carica L.) is indigenous to Persia
and Syria (Tous and Ferguson 1996). Theophrastus and
others referred to cultivation of fig fruits in ancient Greece
(Khadari et al. 2005). Nowadays, fig trees are cultivated in
all Mediterranean countries (70 % of the worlds production originates from the Mediterranean basin) as well as in
other temperate countries (Sadder and Ateyyeh 2006). Fig
tree cultivars are of high commercial importance and are
cultivated worldwide for dry and fresh consumption
(Duenas et al. 2008). Ficus spp. are also used as medical
plants in traditional Chinese medicine (Lansky et al. 2008)
and their consumption is affiliated to longevity (Trichopoulou et al. 2006). Traditionally, figs are considered to
have antibacterial properties (Lazreg-Aref et al. 2012) and
their organic extracts are considered to have antiviral and
antioxidant activities (Solomon et al. 2006).
Fig tree domestication had started at 12,000 to 9000 BC
(Kislev et al. 2006). The large number of the existing fig
tree cultivars ([700) may be the result of selection focusing on agronomic characteristics and/or selection and
transportation to distant regions from breeders and growers
(Condit 1955). The identification of commonly used cultivars depends on phenotypic traits. Phenotypic classification results to misassessment of genetic relationships
among different cultivars. Furthermore, plant phenotype
may vary through years and regions due to genotype
environment interactions. Moreover, in many cases, the
same variety may have different names depending on the
region cultivated. For example, the cultivar Sari-Lop
(Smyrna) from Asia has been referred in California as
Calimyrna. Thus, genetic differentiation of morphologically similar cultivars is very crucial for the assessment of
fig tree genetic resources. Molecular markers constitute a
precise and reliable method for the genetic characterization
of germplasm collections. Different marker systems have
been previously applied for the identification of fig tree
cultivars, landraces and population identification and
classification such as: restriction fragment length polymorphisms (RFLPs) (Khadari et al. 2005), random amplified polymorphic DNA (RAPDs) (Cabrita et al. 2001;
Papadopoulou et al. 2002; De Masi et al. 2005), inter-single
sequence repeats (ISSRs) (Amel et al. 2004; Ikegami et al.
2009), amplified fragment length polymorphisms (AFLPs)
(Cabrita et al. 2001; Baraket et al. 2009), and simple
sequence repeats (SSRs) (Khadari et al. 2001; Saddoud
et al. 2007; Giraldo et al. 2008; Ikegami et al. 2009;
Aradhya et al. 2010; Perez-Jimenez et al. 2012). In addition, a novel method has been developed, high-resolution
melting analysis (HRM), that is able to detect polymorphisms even at the single nucleotide level (Wilhelm and

123

Pingoud 2003). HRM measures the rate of double-stranded

DNA dissociation to single-stranded DNA with increasing
temperature (Reed and Wittwer 2004). This method is costeffective and less time-consuming. Moreover, the ability of
HRM to detect a broad range of SNPs and indels can assist
genotypes discrimination and genetic mapping (Chagne
et al. 2008; Lehmensiek et al. 2008). Identification of
cultivars using SSRs and/or SNPs through HRM analysis
has successfully been performed before in other species
(Madesis et al. 2014 and references therein).
The most popular Greek fig tree cultivar, Kymis,
which is widely cultivated in the island of Evia, has a
central market position and high potential, since it is in the
process to be appointed a Protected Designation of Origin
(PDO) EU mark. Currently, it is highly priced, offering a
significant income to local farmers (Vlachos 2013).
Herein, we describe a marker-based characterization of
a wide set of fig tree cultivars established in a gene bank
collection. Using microsatellite markers we have assessed
the genetic diversity and population structure of a fig tree
germplasm collection originating from the Mediterranean
basin. In addition, by applying the SSR markers coupled
with HRM analysis we could distinguish the different fig
tree cultivar and authenticate the PDO Kymis cultivar.

Materials and methods

Plant material and DNA isolation
The F. carica collection of cultivars was established in the
early 80s at the Institute of Olive and Horticultural crops of
Kalamata (ELGO Demeter). The set of 90 entries was
composed of 47 cultivars originating from Greece, 24 from
Italy, 12 from Cyprus, 4 from Spain, 2 from Turkey and 1
from France (Fig. S1; Table 1). Total genomic DNA was
extracted from dry leaves collected from all localities using
the cetyl trimethylammonium bromide (CTAB) method
(Doyle 1987). The quality and concentration of the
extracted DNA was determined by 1 % agarose gel electrophoresis and ultraviolet spectrophotometry.
Genotyping by microsatellite markers
Samples were analyzed by microsatellite molecular markers. Achtak et al. (2009) have studied the application of
microsatellite markers for the genotyping of fig tree populations. Using the data provided we analyzed the best seven
ranked microsatellite loci (Achtak et al. 2009), which represent (at present) the most informative microsatellites for
fig tree cultivar discrimination (Achtak et al. 2009)
(Table 2). The selected molecular markers are able to

Trees
Table 1 List of the 90 fig cultivars analyzed, germplasm collections where cultivars were originated, corresponding accession code and some
morphological characteristics
No

Cultivar

Code

Origin

Leaf shape

Feature

Fruit
weight

Fruit shape

Fruit skin
ground color

Troiana

TRO

Cyprus

Trilobate

Fresh

Medium

Globose

Yellow

Vasilika cyprus

VASC

Cyprus

Trilobate or
pentalobate

Fresh

Big

Kymis-2 cyprus

KYM2C

Cyprus

Trilobate

Fresh

Big

Globose

Prodromi white

PROW

Cyprus

Trilobate or
pentalobate

Fresh

Medium

Globose

Klirou Kiladonika

KLKIL

Cyprus

Pentalobate

Fresh

Medium

Globose

Bluish green

Aheleias

AHEL

Cyprus

Pentalobate

Fresh

Medium

Globose

Violet

Analata

ANAL

Cyprus

Trilobate or
pentalobate

Fresh

Medium

Globose

Violet

Klirou white

KLWH

Cyprus

Pentalobate

Fresh

Medium

Globose

Violet

Klirou black

KLIBL

Cyprus

Pentalobate

Fresh

Medium

Globose

Black violet

10

Psoma

PSO

Cyprus

Trilobate

Fresh

Medium

Globose

Violet

11

Opsima Xirokitias

OPSX

Cyprus

Trilobate

Fresh

Medium

Globose

Violet

12

Klirou Kiladonika-1

KLKIL1

Cyprus

Pentalobate

Fresh

Medium

Globose

Bluish green

13

Fragussano

FRAG

France

Trilobate or
pentalobate

Freshdried

Medium

Pyriform
flattened

Bluish green

14

Vasilika Agsa

VASAG

Greece

Freshdried

15

Livano

LIV

Greece

Pentalobate

Fresh

Medium

Pyriform
flattened

Black violet

16

Agriosykies Likotrafou

AGRLIK

Greece

Fresh

Small

Globose

17

Vasilika honey-white-1

VASHW

Greece

Fresh

Globose

Violet

18

Black difori 1

BLDIF1

Greece

Fresh

Globose

19

Little green fig

LIGRE

Greece

Fresh

Globose

20

Black difori 2

BLDIF2

Greece

Fresh

Globose

21

Achladi

ACHL

Greece

Fresh

Globose

22

Aidinia

AIDI

Greece

Trilobate

Fresh

Medium

Globose

Violet

23

Kymis

KYM

Greece

Trilobate

Freshdried

Big

Globose

Green

24

Greenfig Lesvos

GREL

Greece

Trilobate

Fresh

Medium

Globose

25

Kalamon

KAL

Greece

Trilobate or
pentalobate

Dried

Medium

Globose

Yellow
green
Violet

26

Green difori

GREDIF

Greece

Fresh

27

Kalamatiani Istieas

KALIST

Greece

Trilobate or
pentalobate

Fresh

Medium

Globose

Violet

28

Perkoulia Lesvos

PERL

Greece

Pentalobate

Fresh

Medium

Globose

Black violet

29

Zailata

ZAIL

Greece

Trilobate

Fresh

Medium

Globose

Violet

30

Bianca Al Fiore

BALF

Greece

Fresh

31
32

Vasilika honey-white-3
Ormathosykia

VASHW3
ORMA

Greece
Greece

Pentalobate

Fresh
Fresh

Medium

Pyriform
flattened

Violet

33

Rigota

RIGO

Greece

Fresh

Medium

Globose

34

Kountouris-K1

KOUK1

Greece

Fresh

Medium

Globose

35

Kountouris-K2

KOUK2

Greece

Fresh

Medium

Globose

36

Vasilika honey white2

VASHW2

Greece

Trilobate

Fresh

Medium

Globose

37

Ithakis black

ITHB

Greece

Trilobate

Fresh

Medium

Globose

38

Perdikosyka

PERDIK

Greece

Pentalobate

Fresh

Medium

Pyriform
flattened

Black violet

123

Trees
Table 1 continued
No

Cultivar

Code

Origin

Leaf shape

Feature

Fruit
weight

Fruit shape

Fruit skin
ground color

39

Smyrnaiki

SMAIKI

Greece

Pentalobate

Dried

Medium

Pyriform
flattened

Yellow
green

40

Valosykia

VALO

Greece

Trilobate or
pentalobate

Fresh

Medium

Globose

Black violet

41

White little fthinoporina

WLFTH

Greece

Fresh

Globose

Violet

42

Politika Lesvos

POLLE

Greece

Fresh

Globose

Violet

43

Vardikia

VARDI

Greece

Trilobate or
pentalobate

Fresh dried

Medium

Globose

44

Caprifig-2

CAPRI2

Greece

Fresh

Small

Globose

45

Vasilika honey-medit

VASHME

Greece

Trilobate

Fresh

Big

Globose

46

Vazanata

VAZAN

Greece

Trilobate or
pentalobate

Fresh

Medium

Globose

Violet

47

Vasilika honey-3

VASH3

Greece

Trilobate

Fresh

Medium

Globose

48

Ximoniatiki

XIMON

Greece

Trilobate

Fresh

Medium

Globose

49

Lesvos Long Pedicle

LELP

Greece

Pentalobate

Fresh

Medium

Globose

Violet

50

Kalimirna

KARNA

Greece

Pentalobate

Fresh

Big

Globose

51

Makrotsanata Istieas

MAIST

Greece

Pentalobate

Fresh

Medium

Pyriform
flattened

Black violet

52

Caprifig-1

CAPRI1

Greece

Fresh

53

Large white Androussis

LWAND

Greece

Pentalobate

Fresh

Medium

Pyriform
flattened

54

Fourtzeiki

FOUR

Greece

Fresh

55

Black Sykikis

BLSYK

Greece

Fresh

Globose

56

Vasilika black medit

VASBME

Greece

Trilobate or
pentalobate

Fresh

Big

Globose

Violet

57

Vasilika Sykikis honey

Sykiki

VASHSY

Greece

Trilobate

Freshdried

Big

Globose

Green

58

Kastanosyka Arnas

KASARN

Greece

Trilobate pentalobate

Fresh

Medium

Globose

Violet

59

Stamatina

STAMAT

Greece

Fresh

Globose

60

Bratiliana

BRATIL

Greece

Pentalobate

Fresh

Medium

Globose

61

Lansi Anese

LANE

Italy

Trilobate

Freshdried

Medium

Globose

Violet

62

Pescarola no 319

PESCA

Italy

Trilobate

Fresh

Medium

Globose

63

Brogiotto Nero-1

BRON1

Italy

Pentalobate

Fresh

Medium

Globose

Violet

64

Dottato-1

DOT1

Italy

Trilobate

Fresh

Medium

Globose

65
66

Columbra Nera no 308

Bianca Al Fiore

COLN
BIFIO

Italy
Italy

Trilobate

Fresh
Fresh

Medium

Globose
Globose

Violet

67

Napolitana negra

NAPN

Italy

Trilobate

Fresh

Medium

Globose

Black violet

68

Porto Gallo

POGAL

Italy

Pentalobate

Fresh

Medium

Globose

69

Dotatto-3

DOT3

Italy

Trilobate

Fresh

Medium

Globose

70

Luri Grossa

LUGRO

Italy

Trilobate or
pentalobate

Fresh

Medium

Pyriform
flattened

Violet

71

Murara

MURA

Italy

Trilobate

Fresh

Medium

Globose

Bluish green

72

Briogiotto Nero

BRON1

Italy

Pentalobate

Fresh

Medium

Globose

Violet

73

Brogiotto Nero 2

BRON2

Italy

Pentalobate

Fresh

Medium

Globose

Violet

74

Vertino

VERT

Italy

Trilobate

Fresh

Medium

Globose

Black violet

75

Dottato-2

DOT2

Italy

Trilobate

Fresh

Medium

Globose

76

Paradiso 317

PARAD

Italy

Trilobate or
pentalobate

Fresh or
dried

Medium

Globose

Violet

77

Rosso Dendro

ROSDE

Italy

Trilobate or
pentalobate

Freshdried

Medium

Globose

Bluish green

123

Trees
Table 1 continued
No

Cultivar

Code

Origin

Leaf shape

Feature

Fruit
weight

Fruit shape

Fruit skin
ground color

78

Gentile Bianco

GENBIA

Italy

Trilobate or
pentalobate

Fresh

Medium

Globose

Violet

79

Melo Grano no 286

MELGR286

Italy

Trilobate

Fresh

Medium

Globose

Violet

80

Briogiotto Nero-3

BRON3

Italy

Trilobate

Fresh

Globose

Violet

81

San Pietro

SANP

Italy

Trilobate

Fresh

Medium

Pyriform
flattened

Yellow

82

Diuri

DIU

Italy

Pentalobate

Fresh

Medium

Globose

Bluish green

83
84

Tre Volte L anno

Melo Grano

VANNO
MELGR

Italy
Italy

Trilobate
Pentalobate

Fresh
Freshdried

Medium
Medium

Globose
Globose

Bluish green

85

Julia

JUL

Spain

Trilobate

Fresh

Globose

86

Mission-2

MIS2

Spain

Trilobate

Fresh

Big

Globose

87

Mission-1

MIS1

Spain

Trilobate

Fresh

Big

Globose

88

Mission-3

MIS3

Spain

Trilobate

Fresh

Big

Globose

89

Polis white

POLW

Turkey

Pentalobate

Fresh

Medium

Globose

Violet

90

Smirna

SMI

Turkey

Freshdried

Table 2 Genetic diversity parameters for seven polymorphic microsatellites in 90 fig cultivars
Microsatellite

NA

Allele size range (bp)

Ho

He

PIC

F (null)

PI

MFC02

13

152214

0.900

0.839

0.816

-0.042

0.024

MFC03

17

99147

0.966

0.802

0.778

-0.117

0.061

Fsyc01

18

93169

0.878

0.771

0.741

-0.074

0.079

LMFC19

295331

0.356

0.559

0.537

?0.226

0.215

LMFC28

10

185243

0.610

0.738

0.694

?0.075

0.110

LMFC30

14

200274

0.978

0.885

0.869

-0.053

0.026

LMFC32

11

196248

0.800

0.639

0.591

-0.155

0.176

Mean

13

0.784

0.747

0.718

-0.0200

0.098

Total PI = 2 9 10-6

NA number of alleles, Ho homozygosity, He heterozygosity, PIC polymorphism information content, F fixation index, PI probability of identity

distinguish among more than 99 % of the analyzed cultivars

(Achtak et al. 2009). PCR (polymerase chain reaction)
amplifications were performed in a reaction volume of 20 ll
containing 30 ng of template DNA, 109 PCR buffer,
200 lM of each dNTP, 10 pmol of each primer (forward
primer labeled with FAM, NED, PET and VIC fluorescent
dyes) and 1 U of KAPA Taq DNA Polymerase (KAPA
Biosystems, Woburn, MA, USA). PCR amplifications were
performed according to (Ahmed et al. 2009; Giraldo et al.
2005; Khadari et al. 2001). The resulting PCR products were
first visualized by 2 % agarose gel electrophoresis and then
loaded into an ABI PRISM 3730xl DNA sequencer (Applied
Biosystems). Microsatellites were analyzed with STRand
version 1.2.30 (The Regents of the University of California).
Genetic diversity analysis
Basic statistics were calculated using the PowerMarker
genetic analysis package (version 3.25) (Liu and Muse

2005) to measure the diversity at each SSR locus, including

the total number of alleles (NA), major allele frequency
(MAF), accession-specific alleles, number of genotypes
(NG), polymorphism information content (PIC), observed
(Ho) and expected heterozygosity (He). Wrights fixation
index, F, (Wright 1951) was estimated for each polymorphic locus as F = 1 - Ho/He). The numbers of rare alleles
(RA), common alleles (CA) and abundant alleles (AA) were
calculated using GenAlEx (version 6.5b5) (Peakall and
Smouse 2012). Genetic distances between each pair of
accessions were measured based on shared allele frequencies using PowerMarker (Peakall and Smouse 2012). The
P 4 PP
probability of identity (PI = 1 pi ?
(2pipj),
where pi and pj are the frequency of the ith and jth alleles,
respectively) measures the probability that two randomly
drawn diploid genotypes will be identical assuming
observed allele frequencies and random assortment (Paetkau et al. 1995). The total probability of identity, defined as
the probability of two cultivars sharing the same genetic

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Trees

profile by chance, was also calculated from the individual

PI values. PI was calculated by IDENTITY 1.0 (Centre for
Applied Genetics, University of Agricultural Sciences,
Vienna, Austria). Dendrograms were constructed using
UPGMA based on the similarities between genotypes
estimated by Dices coefficient (Nei and Li 1979). Principal Coordinate Analysis (PCoA) was performed in a multidimensional space with data standardization using
GENALEX version 6.51 (Peakall and Smouse 2012).
Possible population structure was investigated using a
model-based Bayesian procedure implemented in the
software STRUCTURE v2.3.2 (Pritchard et al. 2000). The
analysis was carried out using a burning period of 10,000
iterations and a run length of 200,000 MCMC replications.
We tested a continuous series of K, from 1 to 8, in 10
independent runs. We did not introduce prior knowledge
about the population of origin, and assumed correlated
allele frequencies and admixture (Falush et al. 2003). For
selecting the optimal value of K, DK values (Evanno et al.
2005) were calculated using STRUCTURE HARVESTER
(Earl 2012).
Authentication of PDO Kymis fig tree cultivar
Typing of seven microsatellite regions with HRM analysis
for authentication of PDO Kymis fig tree cultivar was
performed as previously described (Ganopoulos et al.
2011; Xanthopoulou et al. 2014a, b). For genotyping by
HRM analysis, the genotype of each DNA sample was
determined based on the shape of curves depicted by
temperature-shifted melting curves or difference plots.

Results
Microsatellites polymorphism and genetic diversity
The level of polymorphism of the seven microsatellites
applied here was investigated in the 90 fig tree cultivars
(Table 2). The number of alleles per locus ranged from 8 to
18, with an average of 13 alleles per locus. Expected
heterozygosity ranged from 0.559 in LMFC19 to 0.885 in
LMFC30, with a mean of 0.747. Observed heterozygosity
ranged from 0.356 in LMFC19 to 0.978 in LMFC30, with a
mean of 0.784. The values of expected and observed
heterozygosities were compared using the fixation index
(F), which had an average over all the loci of -0.020, with
values from -0.015 for LMFC32 to 0.226 for LMFC19. F
was negative for five microsatellite loci (MFC02, MFC03,
Fsyc01, LMFC30 and LMFC32), indicating an excess of
observed heterozygotes, whereas F was positive for two
loci (LMFC19 and LMFC28), indicating an excess of
observed homozygotes. The maximum probability of

123

identity, 0.215, was detected in LMFC19 that presented

eight alleles, and the minimum, 0.024, in MFC02 that
presented thirteen alleles. The average probability of
identity was 0.098. To test whether the seven microsatellite
loci were informative to distinguish fig tree cultivars, we
performed statistical resampling which showed that these
loci were sufficient. According to the discriminating power
value for each locus, we tested stepwise combinations
starting with the most discriminating locus. The optimal
combination (Fsyc01 ? MFC3 ? MFC2 ? LMFC30 ?
LMFC19 ? LMFC32 ? LMFC28) allowed discrimination
of all genotypes except three pairs. Using this locus combination, we observed a low probability of identity
(PI = 2 9 10-6; Table 2; Table S1).
Allelic diversity of microsatellite loci
Twenty-one genotype-specific alleles (SA) were identified
in all samples using the seven microsatellites (Table 3). A
maximum of six SA were detected in microsatellite marker
Fsyc01. Specific alleles were also found with other markers: allele 152 bp of locus MFC03 was present only in
Ithakis black, allele 185 bp of LMFC28 was present only
in Analata, allele 93 bp of the locus Fsyc01 in Klirou
Kiladonika, 244 bp LMFC32 in Politika Lesvos, allele
200 bp of the LMFC30 locus in Gentile Bianco, allele
115 bp of the locus Fsyc01 in Stamatina, allele 133 bp of
MFC03 in Black Difori 1, and allele 149 bp of the locus
Fsyc01 was found only in Vazanata. Furthermore, allele
205 of the LMFC28 locus was mostly restricted to the
Cyprus germplasm, apart from its presence in two Greek
(Black Sykikis and Vasilika honey Sykiki) and one
Italian (Rosso Dendro) cultivars, and allele 196 bp of the
EMO90 locus was present mostly in the Calabrian germplasm, with the exception of its presence in the Sicilian
cultivar Santagatese and two Campanian cultivars Cornia and Pisciottana. The RA ranged from 5 (LMFC19) to
13 (Fsyc01) with a total of 55 rare alleles, with each allele
having a frequency \5 %. The ratio of cultivars with rare
alleles was the highest in cultivars from Greece, and was
the lowest in cultivars from Spain. No rare alleles were
found in cultivars from Turkey. The CA ranged from three
(LMFC19 and LMFC32) to six (MFC03 and LMFC30),
with a frequency of 550 %, and the total number of AA
alleles with frequency [50 % was two (Table 3).
Genetic relationships among fig tree cultivars
The cluster analysis using UPGMA method showed low to
moderate differentiation within the fig tree gene bank
collection (Fig. 1). Results of the microsatellite analysis
indicated that the 90 cultivars classified into three main
clusters with three major subgroups of twenty, fourteen and

Trees
Table 3 Details of allele types identified in 7 SSR markers among the 90 fig cultivars
Microsatellite

RA

CA

AA

SA (cultivar)

MFC02

12

MFC03

152 (Ithakis black; GR), 178 (Politika Lesvos; GR), 214 (Politika Lesvos; GR)

Fsyc01

113 (Kastanosyka Arnas; GR), 114 (Ithakis black; GR), 117 (Ithakis black; GR), 133 (Black difori 1; GR)

13

93 (Klirou Kiladonika; CY), 113 (Black Sykikis; GR), 115 (Stamatina; GR), 147 (Politika Lesvos; GR), 149
(Vazanata; GR), 151 (Ithakis black; GR)

LMFC19

LMFC28

185 (Analata; CY)

LMFC30

200 (Gentile Bianco; IT), 212 (Gentile Bianco; IT), 266 (Politika Lesvos; GR), 274 (Politika Lesvos; GR)

LMFC32
Total

8
55

3
36

1
2

212 (Gentile Bianco; IT), 244 (Politika Lesvos; GR), 248 (Politika Lesvos; GR)
21

RA number of rare (\5 %) alleles, CA number of common (550 %) alleles, AA number of abundant ([50 %) alleles, SA specific allele code
(specific allele-scored accession number and country of origin)

Fig. 1 UPGMA tree showing

the genetic relationships among
the fig tree cultivars analyzed in
the study

123

Trees
Fig. 2 PCoA analysis of fig
cultivars analyzed

twelve accessions each (Fig. 1, clusters A1, B3 and C1,

respectively), and four smaller clusters comprised of seven
to eleven cultivars (Fig. 1, clusters A2, B1, B2 and C2).
Interestingly, cultivars Kymis and Large White
Androussis have a strong genetic relationship, as revealed
by their position on the dendrogram.
The first two coordinates of the PCoA explained
44.46 % of the total variation among a collection of 90 fig
tree cultivars, while the first coordinate explained 24.82 %
of the variation. Prediction of unique genotypes on a twodimensional multivariate space partially confirmed the
results of the UPGMA method. The fig tree accessions
from the UPGMA clusters likewise tended to form their
own groups in the PCoA, though usually overlapping
(Fig. 2). Overlapping groups of accessions from almost all
UPGMA clusters comprise the central to top quadrants of
the projection. They represent varieties that have various
geographic origins and uses. Interestingly, five fig tree
cultivars originating from Cyprus grouped together in the
third quadrant of scatter plot. Similarly, seven fig tree
cultivars from Greece and one from Cyprus are found at the
second quadrant (Fig. 2).
Population structure of fig tree cultivars
in Mediterranean basin
The genetic structure of the Mediterranean basin fig tree
cultivars was investigated by a Bayesian-based population
assignment analysis using STRUCTURE software
(Pritchard et al. 2000). Our results show a clear maximum
for DK at K = 2 (Fig. 3b), in which all individuals were
classified into two different clusters. As shown in Fig. 3,
most cultivars were divided into two sub-populations
(Cluster 1 and Cluster 2), including 22 and 68 fig tree

123

accessions, respectively (Fig. 3). Cluster 1 consisted of 22

cultivars, originating from three countries mostly from
Greece (12) followed by those from the Cyprus (7) and
Italy (4). Cluster 2 contained in total 68 accessions originating from Cyprus (6), France (1), Greece (34), Italy (20),
Spain (4) and Turkey (3).
Authentication of PDO fig tree cultivar Kymis
with microsatellite HRM analysis
To genotype and distinguish the PDO Kymis cultivar
from closely related varieties, we evaluated the same seven
different microsatellite markers using HRM analysis by
developing melting curves for all varieties. Figure 4a
depicts the normalized HRM melting curves of 11 representative fig tree cultivars using microsatellite marker
Fsyc01 (all varieties have been used in the study but for
simplicity reasons we present only the 11 representative
curves). Using the shape of the melting curves, we could
assign the differences between curves of the different
cultivars under investigation and reveal that most of the
accessions could be easily distinguished visually, for
example Kymis and Kalamon (Fig. 4a). The results
with the other markers used similarly showed a clear discrimination of the cultivars used.
Figure 4b depicts the difference graph of the eleven fig
tree cultivars compared to Kymis, which represents the
baseline. This closer examination of the HRM difference
curve revealed part of the curve sitting outside the 90 % CI
curve, suggesting that all the examined cultivars via the
HRM curves are indeed different. Furthermore, we were
able to estimate the confidence value of similarity between
Kymis and the other fig tree cultivars and show that
Fsyc01 was a sufficient microsatellite region to distinguish

Trees
Fig. 3 Fig tree cultivars
genetic structure. a Inferred
population structure for K = 2
the probable number of
subpopulation with the fig gene
bank collection consisting of 90
fig accessions. Each individual
is represented by a thin tine
partitioning into K colored
segments representing the
membership fraction in
K clusters. b Estimation of the
number of populations for
K ranging from 1 to 8 by
calculating delta K values

Fig. 4 Authentication of PDO Kymis fig tree cultivar with

microsatellite HRM analysis. a Microsatellite typing of 11 representative fig tree cultivars using HRM analysis with microsatellite
marker Fsyc01. b Difference graph of 11 representative fig tree

varieties using Kymis cultivar as reference genotype. Assigned

genotypes using a cutoff confidence value of 90 %. c Closer
examination of the most popular dried figs and its difference graph
using Kymis cultivar as a control genotype

123

Trees

most of the fig tree cultivars analyzed. Hence, further

analysis of the melt profile depicted three clearly distinct
difference curves of the most popular Greek dried figs
(Fig. 4c).

Discussion
Researchers dedicated to the investigation, conservation,
management and use of germplasm maintained in gene
banks, face a number of common problems including, for
example, the development of strategies for sampling representative individuals in natural populations, the
improvement of tools and technology for long-term conservation or high-throughput genetic analysis of an
increasingly high number of stored accessions. Thus, it is
becoming crucial to obtain the information on the genetic
diversity present in a gene bank for the sustainable conservation and increased use of crop genetic resources.
Germplasm characterization of plant accessions deposited
in gene banks has been limited and is probably a major
cause for the limited use of accessions in breeding
programs.
In this study, seven microsatellite loci were used to
investigate the genetic relationships among 90 fig tree
cultivars originating from six different Mediterranean
countries and conserved in the Greek gene bank collection.
Moreover, HRM analysis coupled with SSR markers
was used to authenticate PDO Kymis fig tree cultivar.
Genetic diversity and population structure of fig tree
cultivars in Mediterranean basin
Germplasm collections in gene banks need to be adequately characterized genetically for their efficient conservation, management and utilization. So far, gene banks
have focused mainly on phenotypic characterization, yet
with the advent of genetics and omics technologies this is
no longer sufficient. Genetic characterization allows the
identification of genetic diversity and genetic relationships
among the gene bank accessions, offering unique advantages over strategies for conservation and breeding efforts.
The selected seven microsatellite loci were reported to be
the most highly resolving microsatellites for fig trees and
able to distinguish more than 99 % of the varieties under
study (Achtak et al. 2009). Indeed in this study they have
also proved to be highly polymorphic and generated different amplification patterns for all 90 fig tree cultivars
analyzed.
Although genetic diversity results in gene bank collections depend on sampling and are, therefore, gene bank
specific, they provide comparative information on the
inherent genetic diversity present in different gene banks,

123

archives and collections. In the present study, the average

number of alleles per locus (NA) was 13, higher than the NA
(5.2) obtained by (Ikegami et al. 2009), using 13
microsatellites and a collection of 19 fig tree accessions
originating from Europe and China (Caliskan et al. 2012),
6.8 (NA) with 10 microsatellites screened in 76 Turkish
accessions (Aradhya et al. 2010), 4.9 (NA) analyzing 15
microsatellites in 194 fig tree cultivars from 11 different
countries, (Saddoud et al. 2007) 9.33 (NA) with 6
microsatellites in 124 Tunisian genotypes (Perez-Jimenez
et al. 2012) and 3.6 (NA) with 21 microsatellite markers in
57 Spanish fig tree accessions.
The average expected heterozygosity (He) of 0.747 was
higher in comparison to previously published works, 0.482
in 194 worldwide fig tree accessions (Aradhya et al. 2010),
0.678 in 76 Turkish fig tree varieties (Caliskan et al. 2012),
0.44 in 19 European and Asian fig tree varieties (Ikegami
et al. 2009) and 0.53 in 57 Spanish fig tree accessions
(Perez-Jimenez et al. 2012). This result indicated extremely
large genetic diversity among the germplasm studied from
the Mediterranean basin. For all microsatellites, PIC values
of at least 0.537 (LMFC39) were recorded, showing that all
loci were highly informative and suitable for genotype
identification and were in agreement with Perez-Jimenez
et al. (2012) that found PIC values ranged from 0.239
(MFC2) to 0.798 (LMFC30). The value of the total probability of identity (PI) was very low (2 9 10-6), demonstrating that the seven microsatellites used were
exceedingly powerful at discriminating fig tree cultivars.
The presence of cultivar-specific alleles also revealed vast
genetic diversity within the gene bank collection. The gene
pool examined possesses significant genetic variability and
exhibits differentiation among the three genetic groups
identified by the cluster analysis and PCoA. These results
indicate that the majority of the fig tree genotypes examined had mixed origin and probably share common
ancestry. Furthermore, the cluster analysis and the
UPGMA dendrogram unraveled three cases of synonymy,
i.e. Mission1 and Mission2 synonymous to Rigota,
Brogiotto Nero-1 synonymous to Brogiotto Nero-2 and
Dottato 1 synonymous to Dottato 2. This result was
clear, even though the number of SSR markers was low,
yet they were the most informative (Table S1).
The weak genetic structure observed in the present study
is probably due to the fact that fig tree circulates genetic
variability across different fig tree types through a dynamic
mutationrecombination process, facilitated by a complex
pollination mechanism, involving the symbiotic relationship between the fig tree and its pollinator. Furthermore,
the genetic relationships within and among fig tree groups
observed in the cluster analysis should reflect a complex
combination of natural evolution, genetic drift and founder
events during domestication, historical migration of

Trees

cultivars along human migrations from the center of origin

and diversity to secondary centers and regions of commercial production. Clonally propagated perennial species
such as fig trees are known to carry relatively high genetic
load and tend to exhibit an excess of heterozygotes, as we
have found in this study for the 90 cultivars studied, as a
mechanism to overcome the deleterious effects of recessive
mutations (Klekowski 1988).
Both Bayesian- and distance-based approaches that
were used to characterize the genetic structure and differentiation revealed weak genetic structure, probably due
to inherently narrow genetic base from which the fig tree
was domesticated, the historical migration of germplasm
and the outcrossing mode of pollination, which have
countered human selection in different fig tree-growing
regions of the world (Aradhya et al. 2010). Nevertheless,
the cluster analysis using the UPGMA method identified
three clusters and seven subgroups, some of which were
further confirmed in the PCA analysis. The Bayesian
analysis indicated that most fig tree genotypes have mixed
ancestry, which becomes clear at K = 2. Overall, the gene
pool of cultivated fig trees analyzed, possesses high
genetic diversity and exhibits narrow differentiation. It is
evident that fig tree accessions from Cyprus are genetically different from the rest of the Mediterranean fig trees,
probably due to isolation. However, a long history of
domestication and cultivation with extensive dispersal of
cultivars has often resulted in a great deal of confusion in
the identification and classification of cultivars (Aradhya
et al. 2010).
Herein, we assumed that the fig tree germplasm is characterized by a typical continuous genetic diversity, which is
further supported by the independent clustering of the
studied cultivars in relation to their geographic origin.
Furthermore, grouping of both uniferous and biferous, fig
tree cultivars together in the same cluster might indicate a
possible common origin of these cultivars. This is in
agreement with the monoecious origin of Ficus that has
evolved into two gynodioecious forms as suggested by
Machado et al. (2001). Moreover, we should stretch here
that similar data have been reported in Tunisian fig trees
using different molecular markers (Salhi Hannachi et al.
2006; Chatti et al. 2007; Baraket et al. 2009, 2011). Overall,
the gene pool of cultivated fig trees analyzed possesses
substantial genetic polymorphism but at the same time
shows limited genetic differentiation among the different
geographical groups tested probably due to the common
origin of the cultivars. In addition we could observe that fig
trees accessions from Cyprus are somewhat genetically
different from the rest of the Mediterranean fig trees. Overall
our results show that the long history of domestication and
cultivation with extensive dispersal of cultivars has often
resulted in a great deal of confusion in the identification and

classification of fig tree cultivars and suggest multiple

domestication, migration and cultivation events.
Authentication of PDO fig tree cultivar Kymis
using microsatellite HRM analysis
High-quality products often acquire an EU trademark,
testifying that they are produced from local varieties
(Piergiovanni et al. 2000). It is generally accepted that
local cultivars are well adapted to the local environment
(Villa et al. 2005). Moreover, farmers have developed
traditional growing and processing methods, especially
adapted to their cultivars, which in many cases are fundamental for a high-quality final product. Yet, introduction
of new, usually foreign varieties which are highly productive, often replace or are intermixed with the local ones,
altering the genetic pool and reducing the quality of the end
product (Ganopoulos et al. 2012). Particularly, Kymis
cultivar, which is exclusively produced on Evia Island, is
suggested as one of the tastiest dry fig tree cultivars.
Named after the town of Kymi, this unique, local variety is
acknowledged by the European Union with Protected
Designation of Origin (PDO). Microsatellite genotyping
with HRM analysis has proved to be suitable for distinguishing cultivars that are phenotypically very similar
(Jaakola et al. 2010; Ganopoulos et al. 2011, 2012; Bai
et al. 2012; Bosmali et al. 2012; Distefano et al. 2012;
Xanthopoulou et al. 2014a, b). As depicted in Table 1,
Kymis is phenotypically similar with Vasilika Sykikis.
In the present study, microsatellite genotyping has provided useful information for the authentication of PDO
Kymis fig tree cultivar compared to other cultivars and
also for gaining a molecular fingerprinting that permits
differentiating Kymis from other phenotypically similar
cultivars.

Conclusion
In this study, genetic resources of the F. carica of the
Mediterranean basin have been genetically characterized
using microsatellite markers. Genetic data obtained could
be a useful contribution towards optimizing fig tree
germplasm management, conservation programs and
breeding activities. It was also proved that microsatellite
markers could be applied to authenticate PDO fig tree
cultivar using HRM approach, without the requirement of
post-PCR procedure, as has been required in conventional
microsatellite analysis.
Author contribution statement Conceived and designed the
experiments: IG, AX, AM, AK and PM. Performed the experiments:
IG, AX and EK. Analyzed the data: IG, AX, TM, FA, AM, AT and

123

Trees
PM. Contributed reagents/materials/analysis tools: AK and PM.
Wrote the paper: IG, AX, AM, FA, AT and PM. Commented on
manuscript: AK and EK.
Compliance with ethical standards
Conflict of interest
of interest.

The authors declare that they have no conflict

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