Genet Resour Crop Evol
DOI 10.1007/s10722-006-9202-5
RESEARCH ARTICLE
Genetic diversity of Syrian pistachio (Pistacia vera L.)
varieties evaluated by AFLP markers
Amer Ibrahim Basha Æ Stefano Padulosi Æ Kamel Chabane Æ Adnan Hadj-Hassan Æ
Ehsan Dulloo Æ Mario Augusto Pagnotta Æ Enrico Porceddu
Received: 6 July 2006 / Accepted: 11 December 2006
Ó Springer Science+Business Media B.V. 2007
Abstract Pistachio (Pistacia vera L.) is a strategic
nut tree species in the Middle East which holds
comparative advantage over other fruit trees in view
of its hardiness, income generation opportunities and
benefits for the ecosystem. Yet pistachio cultivation
depends on a very narrow genetic base, in spite of the
existence of many varieties still marginally exploited.
Syria is an important center of diversity for pistachio.
A country wide ecogeographic survey in this country
was carried out to determine the extent of pistachio
A. Ibrahim Basha (&) A. Hadj-Hassan
Department of Horticulture, Faculty of Agriculture,
University of Aleppo, P.O. Box 8660, Aleppo, Syria
e-mail: a.ibrahimbasha@cgiar.org
S. Padulosi
Regional office of Central & West Asia and North Africa
IPGRI-CWANA, International Plant Genetic Resources
Institute, Aleppo, Syria
K. Chabane
Integrate Gene Management, ICARDA, International
Center for Agricultural Research in the Dry Areas,
Aleppo, Syria
E. Dulloo
International Plant Genetic Resources Institute (IPGRI),
Maccarese, Fiumicino, Italy
M. A. Pagnotta E. Porceddu
Department of Agrobiology and Agrochemistry,
University of Tuscia, Viterbo, Italy
genetic diversity and its use. As a whole, 114
accessions were collected from 37 farms to assess
diversity at morphological and molecular level.
Molecular evaluation was carried out using Amplified
Fragment Length Polymorphism (AFLP) technique
and performed using seven primer pair combinations.
Results from the studies allowed the identification of
25 pistachio female varieties in Syria, some of which
unique and described for the first time. Three groups
of pistachio diversity were identified by cluster
analysis which provides useful information about
the distribution of genetic diversity in Syria for
enhanced use and sustainable conservation.
Keywords AFLP Genetic diversity Pistacia vera
L. Syrian pistachio
Introduction
Pistachio is cultivated over large areas around the
world. According to FAO more than 400,000 ha
are cultivated. Major producers include Iran, United
States, Syria and Turkey (FAO 2006). Such cultivations are also speculated to be based on a very
narrow genetic base (Maggs 1973; Tous and
Ferguson 1996), making them vulnerable to pest
or disease attacks and hence putting at risk the
well-being of thousands of people who depend on
this crop as a source of livelihood. It has been
suggested that the reasons for low genetic diversity
123
Genet Resour Crop Evol
among cultivated varieties are due to the long
juvenile period of pistachio (10–12 years) and its
long life-span (Whitehouse and Stone 1941). Today
traditional cultivars are still being replaced by a
few improved types in areas where greatest diversity is found. The destruction of natural habitats is
also contributing to the loss of wild species. Yet
today, most research efforts are being directed
mainly towards crop breeding (Parfitt 1995) rather
than sustainable deployment of existing diversity
available either in the wild or under cultivation.
Pistacia (Anacardiaceae) includes 11 species,
grouped into four taxonomic Sections; Pistacia vera
L., being the most commercially utilized species, is
native to Central Asia (Zohary 1952). Syria is
considered the center of origin of some wild Pistacia
species, viz. P. atlantica Desf., P. palaestina Boiss.,
P. khinjuk Stocks, and P. terebinthus L. (Post 1932;
Khalife 1958; Mouterde 1966). In addition, many
locally cultivated varieties of Pistacia vera are also
recorded from this country (Hadj-Hassan 1988; Hadj
Ibrahim et al. 1998). Several investigations have been
conducted to study the genetic relationship among
these species, looking at xenia and metaxenia (Crane
and Iwakiri 1980; Riazi and Rahemi 1994) and
rootstock compatibility (Kaska et al. 1996; Kafkas
and Kaska 1998; Alti and Kaska 2002).
Molecular methods are now increasingly being
used to study diversity in crops. The use of molecular
techniques in order to assess at the DNA level, the
genetic variation and genetic distance among germplasm accessions is widely used. The AFLP technique (Vos et al. 1995) provides a useful tool to
visualize polymorphic bands and study the genetic
relatedness within several members of the plant
kingdom (Hartl and Seefelder 1998; Manubens et al.
1999; Angiolillo et al. 1999), as well as for its
efficiency in diversity studies, phylogeny, genomic
linkage mapping, and identification of varieties
(Sensi et al. 1996; Maughan et al. 1996; Wang
et al. 1997; Xu et al. 2000; Katsiotisl 2003; GolanGoldhirsh 2004). This has also been recognized as a
reliable DNA marker system when compared with
RFLP, RAPD, or microsatellites techniques (Powell
et al. 1996; Winfield et al. 1998; Russell et al. 1999).
In this study, AFLP markers have been used to
investigate the extent of diversity in cultivated
varieties of Pistacia vera L. gathered across Syria
123
and to assess the level of diversity maintained onfarm.
Material and methods
Plant material
The material deployed in this study originated from a
previous in situ agro-morphological characterization
of 114 accessions randomly cultivated over 37
different farms as specified in Fig. 1 (Ibrahim Basha
et al. 2003). According to farmers knowledge and
morphological investigations, 100 accessions represented 20 varieties and other 14 were unknown. Each
variety is represented by five accessions as indicated
in Table 1, which have been recognized as being the
full spectrum of pistachio diversity within the country. Number of samples per farm varied according to
farm size and phenotypes observed.
Upon sampling, each accession was then introduced into a field gene bank by budding (using T-bud
technique applied on 3 years-old pistachio rootstocks). 114 young leaf-samples were later collected
from the gene bank and kept in liquid nitrogen tanks
for the DNA extraction and AFLP analyses.
Molecular analysis
Total genomic DNA was extracted from freeze-dried
leaf-tissue by using Cetyl Trimethyl Ammonium
Bromide (2X CTAB) mini-extraction protocol based
on Sasanuma et al. (2002) with minor modifications.
The extraction was done at the Genetic Resources
Unit molecular laboratory of ICARDA (Syria).
Molecular evaluation was performed at the Department of Agrobiology and Agrochemistry, University
of Tuscia (Viterbo) in collaboration with the Agroenvironmental and Forest Biology Institute, National
Research Council (CNR) of Porano (Orvieto), Italy.
The AFLP protocol was as Vos et al. (1995).
Genomic DNA (100–400 ng) was double digested
with EcoRI and MseI enzymes and ligated to the
specific adapters. Primers with no selected nucleotide
were used first for the pre-amplification. The preamplification products were diluted 1:20 and amplified with seven primer combinations with three
selective nucleotides (i.e. EcoRI + ACT/
Genet Resour Crop Evol
Fig. 1 Collection sites in
Syria where the analyzed
pistachio accessions were
sampled
MseI + GGC, EcoRI + ACT/MseI + GGG,
EcoRI + ACT/MseI + GTA, EcoRI + ACC/
MseI + GAG, EcoRI + ACG/MseI + CTC,
EcoRI + AAG/MseI + CTA, EcoRI + AGG/
MseI + CTG). The amplified products were separated
on a denaturing 6% polyacrylamide sequencing gel.
After electrophoresis, the gel was scanned with
automated scanner connected to the computer and
the images were acquired.
Gel interpretation
AFLP bands were visually scored with the aid of
digital pictures of the gels and adobe Photoshop
computer software. AFLP possible bands, ranging in
size from 150–500bp, across all 114 DNA samples
including 20 varieties of each of the 7 primer pair
combinations were scored (Table 1; Fig. 2) as present
(1) and absent (0). Only the bands showing unambiguous polymorphism were considered in the statistical analysis.
Data analyses
Given that farmers recognized 20 varieties among
114 accessions, the analyses of genetic similarity was
carried out among and within accessions and groups
in order to confirm farmers’ indications that accessions belonged to a named variety. To this aim the
genetic similarity was calculated using simple matching coefficients (Sneath and Sokal 1973) by NTSYSpc version 2.02i computer software (Rohlf 2000).
Then the similarity matrices were subjected to cluster
analysis by the un-weighted pair-group method with
the arithmetic averages (UPGMA) cluster method.
Principle Component Analysis (PCA) based on
Similarity matrix was calculated by using NTSYSPC-2.02i computer software (Rohlf 2000).
Analysis of Molecular Variance (AMOVA) was
also conducted by using Genetic Analysis in Excel
(GenALEx) (Peakall and Smouse 2001) based on
Euclidean Distance matrix which provides information on the extent of diversity at the farm level as well
at the regional level:
Eij ¼
qX
ffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
2ffi
k xki xkj
Because the diversity of pistachio is uneven across
the country, we also used the Shannon index of
diversity (Magurran 1988) to study the geographical
pattern of diversity, calculated using DIVA-GIS
software:
H0 = R Piln Pi
Pi – Proportional abundance of the ith class = ni/N;
N – Number of observations per cell; ni – Number of
individuals in the i-th class
123
Genet Resour Crop Evol
Table 1 Studied varieties of pistachio cultivated in Syria and their main morphological features
No. Variety
No. of
accessions
Growth
habit
Terminal
leaflet shape
Nut shape
Hull Color
Suture opening
position
Kernel
color
5
Erect
Ovate
Ovoid or
Narrowly
Cordate
Red–Purple
group
Mainly dorsal side
Greenish
yellow
Ovoid
Mainly dorsal side
Ovoid
Red–Purple
group
Reddish–White
Greenish
yellow
Greenish
yellow
1
Ashoury
2
Red oleimy 5
3
Baidy
5
SemiRound ovate
erect
Spreading Round ovate
4
Red jalab
5
Spreading Elliptic
Ovoid or
Elongate
Red group with
dark spots
Mainly dorsal side
Yellowish
5
5
Drooping Elliptic
Spreading Broad
lanceolate
Yellow– White
group
Light red group
Greenish
5
Ovoid or
Elongate
Ovoid
Dorsal side only
6
Nab al
dajaml
Marawhy
Mainly dorsal side
Greenish
yellow
7
Boundouky 5
Semierect
Elliptic
Ovoid
Light red group
Dorsal and ventral
side completely
Greenish
yellow
8
5
Erect
Elliptic
Ovoid
5
Semierect
Round ovate
Elongate
Yellow– white
group
Red group
Mainly dorsal side
9
White
ashoury
Entaby
Greenish
yellow
Greenish
10
Lazwardy
5
Spreading Round ovate
Ovoid
Light red group
Mainly dorsal side
Greenish
yellow
11
Batoury
5
Drooping Ovate
Light cream
12
Ajamy
5
Spreading Ovate
Ovoid or
Elongate
Ovoid
Dorsal and ventral
side completely
Dorsal and ventral
side completely
Greenish
yellow
Greenish
yellow
13
Ain al arab 5
Semierect
14
Sen al feel 5
15
Wardany
16
Elliptic
Red–Purple
group
Mainly ventral side
Mainly ventral side
Elongate
Red-Purple
group
Mainly dorsal side
Greenish
yellow
Spreading Ovate
Ovoid
Red group
Mainly dorsal side
5
Spreading Round ovate
Elongate
Light Red group Mainly ventral side
Greenish
yellow
Greenish
yellow
White
oleimy
5
Semierect
Cordate
Light cream
Mainly dorsal side
Yellowish
17
Zaroory
5
Spreading Ovate
Ovoid
Not opened
Green
18
Lesan al
tair
5
Semierect
Broad
lanceolate
Ovoid
Red-Purple
group
Red-Purple
group
Dorsal side only
Green
19
White
turkey
5
Semierect
Ovate
Elongate
Light cream
Mainly dorsal side
Greenish
yellow
20
Ain al
tinaha
5
Spreading Roundish
Ovoid
Reddish white
Dorsal and ventral
side completely
Greenish
yellow
Roundish
Results and Discussion
Each primer pair produced polymorphic AFLPs
bands with an average of 24 per primer pair
combinations. Associations among the varieties were
investigated by UPGMA cluster analysis based on
123
simple match similarity index coefficient using
NTSYS software (Table 2; Fig. 3).
The cluster analysis revealed three main groups
among the pistachio varieties studied, as follows: (i)
Group I (containing vars. Ashoury, Red Jalab, Entaby
2, Red Oleimy, Batoury, Nab Al-Djamal, White
Genet Resour Crop Evol
Fig. 2 AFLP gel of primer combination E38-M73
Ashoury, Ajamy and Sen Al feel) and having greatest
genetic similarity among them (78–95% similarity
index/SI ); (ii) Group II (containing vars. Baidy,
White Turkey, Marawhy, Boundouky, Entaby 1,
Bayd Al-Tair, Wardany and Ain Al-Arab (68–86%
SI); (iii) Group III (containing vars. Lazwardy,
Botmy, Abu–Rieha, Al-Grahy, White Oleimy Zaroory and Ain Al-Tinaha (70–86% SI)); and lastly a
minor cluster with one individual variety Lesan AlTair. The results show that var Entaby is characterized by high diversity, which spread across two
groups (Group I and II). The accessions of this
variety were thus split into two varieties, named
Entaby 1 and Entaby 2.
Principle component analysis (PCA) confirmed
clearly the three main groups mentioned above based
on genetic relatedness (Fig. 4).
Analysis of molecular variance (AMOVA) showed
that the genetic diversity between the varieties within
an orchard was very high (86%) compared to the
diversity between regions (8%) and the one across the
whole country (6%) (Table 3). This analysis indicates
that a higher genetic diversity of Pistacia vera is
found in the older orchards. This fact confirms our
speculation that pistachio cultivations have increas-
ingly led to the reduction of their genetic variation
due to deployment of improved varieties and to the
availability of private or public grafted seedling
nurseries for pistachio, as well as the changing
livelihood conditions (e.g. in the past exchange of
local cultivars among farmers was very popular along
with the consumption of nuts from wild taxa which
were also object of exchange based on a 50% ratio
with cultivated material). Recently the method of
pistachio cultivation is changing leading towards an
increased reduction of crop diversity deployed on
farm. In the past (until app. 25 years ago) pistachio
diversity was maintained high in the field through a
number of cultivation practices, s.a. use of male
varieties derived from seed, use of wild Pistacia
species to boost pollination and hence the fruit
setting, use of natural populations of wild Pistacia (P.
atlantica) as rootstock due to their well known
resistance to stony and calcareous soils.
The Shannon index that combines both richness
and evenness, (Fig. 5) shows that the richest parts in
diversity are in sites belonging to the Aleppo, Idleb
and Hama provinces. These areas coincide with the
main pistachio cultivating land in Syria. All other
areas, show comparably lower diversity and it is
interesting to note that they include new cultivations,
where monoculture systems are being preferred to
traditional ones based on larger diversity.
This study has successfully determined that pistachio cultivated diversity in Syria can be grouped into
25 varieties, 20 of which have been previously
studied albeit using limited morpho-agronomic traits
and genetic relationship observations (Hadj-Hassan
1988; Ibrahim Basha et al. 2003; Riaz et al 2003). In
addition of the confirmation of varieties previously
recorded, this study is proposing 5 new ones, which
have not been described before (Table 4).
The geographic origin and morphological characteristics (Fig. 1; Table 1) of samples for each of
AFLP groups were considered. The varieties of group
(I), having the greatest genetic similarity among them
(Table 2; Fig. 3), could be divided into two subgroups
described as ‘‘red variety subgroup’’ (90% similarity
index) s.a. Ashoury, Red Oleimy, Entaby2 and Red
Jalab, also ‘‘white variety subgroup’’ (86–90% SI)
contains all those varieties with white hull s.a.
Batoury, Nab Al-Djamal, White Ashoury and Ajamy.
Red Jalab and Ashoury are the closest varieties
genetically and morphologically (95% SI.). Group II
123
123
Table 2 The similarity coefficient matrix of studied pistachio varieties and clones from Syria
Varieties
Ashoury
Ashoury
1.00
Red
Oliemy
0.91
1.00
Baidy
0.58
0.67
1.00
Jalab
0.95
0.91
0.62
1.00
Nab Al
Dajaml
0.84
0.89
0.65
0.83
1.00
Marawhy
0.64
0.68
0.79
0.63
0.69
1.00
Boundouky
0.69
0.77
0.80
0.69
0.80
0.83
1.00
White
Ashoury
0.89
0.90
0.66
0.89
0.92
0.69
0.78
1.00
Entaby1
0.76
0.80
0.77
0.80
0.76
0.77
0.81
0.80
1.00
Entaby2
0.92
0.88
0.65
0.94
0.83
0.63
0.72
0.86
0.78
1.00
Lazwardy
0.43
0.50
0.70
0.48
0.54
0.74
0.67
0.53
0.66
0.48
1.00
Batoury
0.87
0.92
0.67
0.87
0.89
0.69
0.76
0.91
0.79
0.85
0.54
1.00
Ajamy
0.83
0.87
0.63
0.88
0.83
0.66
0.73
0.88
0.77
0.85
0.55
0.88
1.00
Bayd
Al Tair
0.64
0.66
0.69
0.68
0.63
0.74
0.71
0.66
0.79
0.70
0.75
0.67
0.66
1.00
Ain
Al Arab
0.51
0.52
0.66
0.55
0.55
0.60
0.66
0.54
0.64
0.58
0.73
0.54
0.54
0.74
1.00
Sen
Al Feel
0.75
0.76
0.67
0.79
0.77
0.68
0.69
0.79
0.71
0.80
0.60
0.79
0.85
0.68
0.59
1.00
Wardany
0.69
0.71
0.73
0.71
0.66
0.66
0.73
0.68
0.74
0.74
0.65
0.65
0.66
0.76
0.70
0.64
1.00
White
Oliemy
0.44
0.53
0.69
0.46
0.57
0.69
0.67
0.55
0.64
0.51
0.76
0.57
0.55
0.69
0.66
0.58
0.62
1.00
Zaroory
0.44
0.53
0.69
0.46
0.56
0.71
0.71
0.54
0.63
0.49
0.76
0.55
0.58
0.69
0.70
0.54
0.60
0.77
1.00
Abou
Rieha
0.46
0.55
0.78
0.49
0.62
0.82
0.76
0.56
0.66
0.51
0.83
0.57
0.55
0.69
0.69
0.67
0.66
0.75
0.74
1.00
Lesan
Al Tair
0.58
0.62
0.67
0.61
0.70
0.64
0.68
0.70
0.67
0.65
0.68
0.68
0.70
0.63
0.61
0.76
0.57
0.77
0.64
0.71
1.00
Al Grahy
0.39
0.46
0.73
0.44
0.52
0.72
0.66
0.50
0.60
0.45
0.86
0.49
0.53
0.67
0.67
0.63
0.64
0.73
0.69
0.87
0.72
Red
Oliemy
Baidy
Jalab
Nab Al
Dajaml
Marawhy
Boundouky
White
Ashoury
Entaby 1
Entaby 2
Lazwardy
Batoury
Ajamy
Bayd
Al Tair
Ain
Al Arab
Sen
Al Feel
Wardany
White
Oliemy
Zaroory
Abou
Rieha
Lesan Al
Tair
Al
Grahy
Botmy
White
Turkey
Ain Al
Tinaha
1.00
0.41
0.48
0.70
0.42
0.55
0.70
0.66
0.50
0.61
0.46
0.85
0.52
0.51
0.72
0.74
0.61
0.63
0.74
0.74
0.80
0.63
0.77
1.00
White
Turkey
0.50
0.60
0.80
0.52
0.57
0.70
0.75
0.57
0.68
0.58
0.69
0.61
0.60
0.64
0.69
0.62
0.77
0.71
0.73
0.73
0.63
0.71
0.70
1.00
Ain Al
Tinaha
0.43
0.51
0.67
0.43
0.58
0.68
0.69
0.52
0.57
0.46
0.69
0.54
0.52
0.62
0.58
0.54
0.61
0.66
0.72
0.76
0.58
0.74
0.67
0.71
1.00
Genet Resour Crop Evol
Botmy
Genet Resour Crop Evol
Ashoury
Red Jalab
Entaby 2
Red Oliemy
1
Batoury
Nab Al Dajaml
White Ashoury
Ajamy
Sen Al-Feel
Lesan Al Tair
Baidy
White Turkey
Marawhy
Boundouky
2
Entaby1
Bayd AlTair
Wardany
Ain AlA rab
Lazwardy
Botmy
Abou Rieha
Al Grahy
3
White Oliemy
Zaroory
Ain AlTinaha
0.598
0.631
0.663
0.695
0.727
0.759
0.792
0.824
0.856
0.888
0.920
0.952
Coefficient
Fig. 3 Dendrogram based on simple matching coefficient for pistachio varieties using seven primer combinations.
Fig. 4 Principle component
analysis for pistachio
varieties
Lesan Al Tair
Sen Al Feel
Al Grahy
Nab Al Dajaml
Ajamy
White Oliemy
Abou Rieha
Coord. 2
White Ashoury
Batoury
Lazwardy
Botmy
Ain Al Tinaha
Zaroory
Red Oliemy
Marawhy
Ashoury
Jalab
Boundouky
Entaby2
Baidy
Entaby1
White Turkey
Ain Al Arab
Bayd Al Tair
Wardany
Coord. 1
Table 3 Analysis of molecular variance (AMOVA)
Level of variation
Across the country
df
5
SS
42.184
MS
Variance
8.437
0.209
Percentage (%)
6
Between regions
17
74.822
4.402
0.280
8
Between varieties within same orchard
91
280.175
3.079
3.079
86
123
Genet Resour Crop Evol
Fig. 5 Shannon Index of
the female pistachio
varieties in Syria
Table 4 Some morphological characters of the new five varieties
No. Variety
Growth
habit
Terminal leaflet
shape
Nut shape
Hull Color
Suture opening position
Kernel
color
1
Entaby2
Semierect
Elliptic
Elongate
Dark red (violet)
Mainly dorsal side
Greenish
yellow
2
Bayd Al
Tair
Drooping
Round ovate
Ovoid or
Elongate
Yellow - white group
Dorsal and ventral side
completely
Yellowish
3
Abou
Rieha
Spreading Ovate
Ovoid
Light cream with
orange lines
Dorsal side only
Greenish
yellow
4
Al Grahy
Drooping
Ovate
Ovoid
Light cream with
orange lines
Mainly dorsal side
Greenish
5
Botmy
Semierect
Roundish
Roundish
Oily green
Non
Green
(68–86% SI) contains varieties cultivated in a well
defined large growing area of Syria, especially in the
northernmost region and close to the Turkish border
viz. Entaby 1, Bayd Al-Tair, Wardany, Ain Al-Arab
and Sen Al feel. Group III could be divided according
to the principle component analysis into two subgroups (Fig. 4). Subgroup I, (70% SI) has varieties
Lazwardy, Botmy, Zaroory, Ain Al-Tinaha, which
are described as relative small nuts and hybrid
varieties due to the open pollination in Pistacia gene
pool. Subgroup II, (77% SI) has varieties Al-Grahy,
Abu–Rieha and White Oleimy, distinguished by
large, white hulls with red tips. In addition to this
grouping, this result does also suggest that var.
Entaby could be considered as made of two distinct
123
varieties with 78% of similarity index. Nevertheless,
they are morphologically quite different from each
other though being cultivated in the same environment and called with same name by farmers.
In this investigation, the AFLP technique was able
to successfully assess the genetic variation among
Syrian pistachio varieties and showing a clear
relationship with the morphological characters. For
instance, red hulls or white hulls were found in most
varieties of groups I and II. Ashoury and Red Jalab,
the closest varieties, detected the highest genetic
similarity index 95%. Similar results were obtained
by Tignon et al. (2001) using AFLP technique
applied to better classify apple varieties. Federici
et al. (2001) also reported that the genetic relationship
Genet Resour Crop Evol
among Oryza spp. which was evaluated by AFLP
markers, had matched the morphological characterization of these varieties.
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