Conserv Genet (2010) 11:311318

DOI 10.1007/s10592-009-9812-5

RESEARCH ARTICLE

Genetic diversity and relationships among Pistacia species

and cultivars
Leila Pazouki Mohsen Mardi Parvin Salehi Shanjani Marianna Hagidimitriou
Seyed M. Pirseyedi Mohammad R. Naghavi Damiano Avanzato
Elisa Vendramin Salih Kafkas Behzad Ghareyazie M. R. Ghaffari
S. M. Khayam Nekoui

Received: 2 June 2008 / Accepted: 18 January 2009 / Published online: 12 February 2009
Springer Science+Business Media B.V. 2009

Abstract Iran is one of the two major centres of Pistacia

diversity and the main producer of pistachios in the world.
About 282 Iranian pistachio genotypes (Pistacia spp.),
together with 22 foreign cultivars (P. vera), were genotyped using 10 simple sequence repeat (SSR) markers to
analyse the genetic diversity and relationships among
Pistacia species and cultivars. The results revealed that the
genetic diversity within P. atlantica subsp. kurdica was
considerably lower than in P. vera or P. khinjuk. Principal
coordinate analysis revealed a clear separation between the
different Pistacia spices, as well as between the Iranian and
foreign cultivars. AMOVA analysis showed that the variation between the species, between different populations,
and within populations accounted for 41, 9, and 50% of the

total variation, respectively. The results demonstrated that

the study of genetic diversity and relationships among
Pistacia species and cultivars using SSR markers provides
important information for the collection and conservation
of pistachio germplasm. In addition, the Iranian cultivars
had a broader genetic background than that of the foreign
cultivars. Thus, they are very important for genetic conservation and the planning of future breeding programmes.
We also determined the different levels of genetic diversity
that exist between and within the species and populations
and showed that gene flow occurs between the Iranian
cultivars and wild-type P. vera populations. The study
provides practical information that policy-makers and
scientists can apply to the conservation and sustainable use
of all the species studied.

L. Pazouki
M. Mardi (&)
S. M. Pirseyedi
B. Ghareyazie

M. R. Ghaffari
S. M. Khayam Nekoui
Department of Genomics, Agricultural Biotechnology Research
Institute of Iran, Mahdasht Road, Karaj, Iran
e-mail: mardi@abrii.ac.ir

Keywords Pistachio
Microsatellite
Phylogenetics

Pistacia
Population genetics
SSR
Genetic diversity

P. S. Shanjani
Natural Resources Gene Bank, Research Institute of Forests and
Rangelands, P.O. Box 13185-116, Tehran, Iran
M. Hagidimitriou
Pomology Laboratory, Department of Crop Science,
Agricultural University of Athens, Athens, Greece
M. R. Naghavi
Department of Agronomy, Faculty of Agriculture,
University of Tehran, Karaj, Iran
D. Avanzato
E. Vendramin
CRA Centro di Ricerca per la Frutticoltura, Rome, Italy
S. Kafkas
Department of Horticulture, Faculty of Agriculture,
University of Cukurova, Adana, Turkey

Introduction
The genus Pistacia is a member of the Anacardiaceae
family. It consists of 11 species (Zohary 1952). Pistacia
vera L. is the only cultivated and commercially-grown
species in the genus (Zohary 1996). Iran, which has
350,000 ha that are devoted to pistachio cultivation and
produces more than 300,000 tons/year, is one of the two
main centers of Pistacia diversity (Maggs 1973; Hormaza
et al. 1994, 1998; Sheibani 1995). In addition to the cultivated species (P. vera L.), Iran is also home to three wild
species of pistachio: P. vera, P. khinjuk and P. atlantica.
The latter has three subspecies (mutica, kurdica, and
cabolica) (Khatamsaz 1988). Although Iran is the major
pistachio producer in the world, the Iranian pistachio

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industry relies mainly upon a limited number of cultivars,

which include Ohadi, Ahmad Aghaei, Fandoghi, Akbari,
and Kalle Ghoochi and cover more than 95% of the cultivation area. Previous research indicates that these kind of
cultivation have a very narrow genetic base (Maggs 1973;
Tous and Fergusen 1996), which makes them vulnerable to
attack by pests or diseases, and hence threatens the livelihood of thousands of people who depend on this crop. The
destruction of natural habitats in central and southwestern
Asia, and the change from traditional to modern methods
of cultivation, may cause genetic erosion (Hormaza et al.
1998) and the loss of important germplasm. Therefore, it is
important to preserve the genetic variability of natural
pistachio populations.
Studies that have investigated genetic variability in
pistachio used a variety of morphological, physiological,
and biochemical methods (Zohary 1952; Barone et al.
1993; Dollo 1993). Researchers are now using molecular
methods, i.e. at the DNA level, increasingly to study
genetic diversity. Many studies of pistachio have used
these methods (Ahmad et al. 2005; Ahmad et al. 2003a, b;
Cipriani et al. 1999; Golan-Goldhirsh et al. 2004; Guilford
et al. 1997; Kafkas 2006; Kafkas et al. 2006a, b; Katsiotis
et al. 2003; Struss et al. 2003). Although previous studies
have characterized pistachio diversity in Iran partially, they
did not conduct a full analysis. The study reported herein
constitutes the first comprehensive analysis of diversity in
the Iranian pistachio germplasm using simple sequence
repeat (SSR) markers which is the most reliable and
reproducible molecular marker for genetic diversity studies. In the study, the authors used SSR markers to genotype
282 Iranian pistachio genotypes (Pistacia spp.) and 22
foreign cultivars in order to study the genetic diversity and
relationships within and between pistachio species. The
results of the study should provide relevant information for
the genetic conservation and management of Iranian pistachio orchards/forests.

Conserv Genet (2010) 11:311318

Microsatellite analysis
About ten SSR primer pairs, which were based on Ahmad
et al. (2003a), were used (Table 3). PCR was amplified
using a Bio-Rad thermocycler (Bio-Rad Laboratories Inc.,
Hercules, CA, USA). Amplification reaction products were
separated on a 5% denaturing polyacrylamide gel using a
50 cm Sequi-Gen GT sequencing cell gel apparatus (BioRad Laboratories Inc.). The resulting images were scored
manually.
Data analysis
Each SSR fragment was scored as either present (1) or
absent (0) across all genotypes. The mean number of
alleles across all loci (na), [the effective number of alleles
(ne), and the average expected heterozygosity (He), calculated according to Nei (1978)], were calculated using the
GenAlEx software package version 6 (Peakall and Smouse
2006). An analysis of molecular variance (AMOVA) was
performed using the GenAlEx 6 software (Peakall and
Smouse 2006) in order to partition the genetic variation
among species, among populations within species, and
among individuals within populations (Schneider et al.
2000). The significance of each variance component was
tested with permutation tests (Excoffier et al. 1992).
Genetic distances were estimated according to Nei (1978),
and principal coordinate analysis (PCO); (Gower 1966) and
neighbour-joining (NJ) analysis were performed. The NJ
dendrogram was constructed with the MEGA4 software
(Tamura et al. 2007). Wrights Fst was used to estimate
population differentiation. The rate of gene flow was estimated indirectly from the proportion of total diversity that
was found among populations (Wright 1931, 1951). An
assignment test (Paetkau et al. 1995) was used to obtain the
observed distributions of alleles among predefined species
and to assign each individual to the species to which its
genotype frequency corresponded most closely.

Materials and methods

Results
Plant material
Diversity measures
A total of 304 pistachio (Pistacia spp.) genotypes were
analysed: 131 local cultivars from six Iranian pistachio
collections and orchards; 22 foreign cultivars from the
CRAfruit tree research centre, Rome, and 151 individuals that represented one, two, and four populations of
P. khinjuk, P. atlantica subsp. kurdica, and P. vera (wildtype), respectively (Fig. 1; Tables 1, 2). Samples of
young leaves were collected and DNA was extracted
using the commercial kit DNeasy Plant Mini Kit (Qiagen
Inc.).

123

The 10 pistachio SSR loci that were examined produced a

total of 45 alleles across all the genotypes. The number
of alleles per locus ranged from 2 to 5, with an average of
1.96 alleles per locus (Table 3). The average number of
alleles across all loci (na) was slightly higher in the Iranian
cultivars than in the wild-type samples of P. vera
(Table 4). The values of the parameters ne and He were
similar for P. vera (wild-type) and P. khinjuk, but were
lower for the two P. atlantica subsp. kurdica samples. The

Conserv Genet (2010) 11:311318

313

Fig. 1 The distribution of

pistachio species in Iran
according to Esmailpour (2001).
Collecting sites for the wildtype species and cultivars are
marked by squares and
triangles, respectively

wild samples of P. vera and P. atlantica subsp. kurdica had

the highest and the lowest Heand ne, values, respectively
(Table 4).
Genetic differentiation
The pairwise values for Neis genetic distances between
the analysed datasets of the pistachios (the Iranian and
foreign cultivars were pooled as two distinct groups) ranged from 0.03 (P. vera population Kh1 and P. vera
population S) to 0.8 (P. khinjukforeign cultivars)
(Table 5). Pairwise values for Fst (P \ 0.01) ranged from
0.03 (P. vera populations Kh1P. vera population S and
P. vera populations Kh1P. vera population Kh2) to 0.52
(P. khinjukP. atlantica subsp. kurdica K2) (Table 5). All
P. vera populations were related, with Fst values that ranged from 0.034 to 0.37, whereas, the values between P.
vera and the other Pistacia species were higher (Table 5).
The results of the PCO showed that the three pistachio
species are clearly separated from each other (Fig. 2). The

first three principal coordinates accounted for 87% of the

total variation among the populations or species. Overall
patterns of genetic differentiation were also examined
using NJ analysis (Fig. 3). The resulting tree had long
terminal branches, which suggested that the populations,
species, and cultivar groups were well differentiated.
AMOVA analysis showed that the variation between the
species, between the populations, and within the populations accounted for 41, 9, and 50% of the total variation,
respectively (Table 6). The strength of the study was
revealed by the high percentage of pistachio cultivars that
were assigned correctly into their corresponding populations. The assignment tests revealed that all the foreign P.
vera cultivars and P. khinjuk samples were classified within
the correct group. Ten of the 136 Iranian cultivars were
classified as wild-type P. vera, and one of the 99 wild-type
P. vera individuals was classified within the Iranian cultivars. Furthermore, there was an intersection between the
Iranian cultivars and the wild-type P. vera populations, and
their separation from the foreign cultivars (Fig. 4).

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Conserv Genet (2010) 11:311318

Table 1 Iranian and foreign pistachio cultivars included in the study

Iranian pistachio cultivars

Foreign pistachio cultivars

No.

Cultivar name

No.

Cultivar name

No.

Cultivar name

No.

Cultivar name

RaOhadi

45

NOhadi

89

KRokn Abadi

RoAeginis

RaGholamrezaei

46

NMomtaz

90

KPoost Kaghazi

RoRed Aleppo

RaBadami Zarand

47

NKalle Ghoochi

91

KAhmad Aghaei

RoNaz

RaSirizi

48

NAmiri

92

KFrootani

RoEnk

RaPooste Piazi

49

NJandaghi

93

KMohyeddini

Ro40

RaKalle Ghoochi

50

NVahedi

94

KHeidar Abadi

RoGreco

7
8

RaSabz Peste Nooghi

RKhanjari Damghan

51
52

NGhafoori
NSoltani

95
96

KKarim Abadi
KAbdollahi

7
8

RoAchourI
RoSfax

RaHassani

53

NRezaei

97

KKhandani

RoLarnaka

10

RaRavar N0: 1

54

NJabbari

98

KGhazvini

10

RoBaglico

11

RaFandoghi Ghafoori

55

NGholamrezaei

99

KShasti

11

Ro502

12

RaBadamie Zoodras

56

NHarati

100

KJabbari

12

RoChico

13

RaRezaei

57

NSeifoddini

101

KSirizi

13

RoIraq

14

RaBadami Ravar

58

NShasti

102

KGhafoori

14

RoInzolia

15

RaRavar N0: 2

59

NKhandani

103

KVahedi

15

RoCerasuola

16

RaSeifoddini

60

NSefidPeste Nooghi

104

KJandaghi

16

RoP. vera Selection

17

RaBadami NisheKalaghi

61

NSirizi

105

KAmiri

17

RoBronte

18

RaHarati

62

NGhazvini

106

KKalle Ghoochi

18

RoBianca

19

RaMoosa Abadi

63

NEbrahimi

107

KMomtaz

19

RoTignusa

20

RaRavar No: 3

64

NMoosa Abadi

108

KOhadi

20

RoNapoletana

21

RaEbrahim Abadi

65

NBadami Nishe Kalaghi

109

FDaneshmandi

21

RoAsk

22
23

RaFandoghi 48
RaGhazvini

66
67

NLahijani
NEbrahim Abadi

110
111

FBarg Siah
FOhadi

22

RoSelvatico

24

RaAmiri

68

NChorook Khorde

112

FAkbari

25

RaGhafoori

69

NJavad Aghaei

113

FPeste Garme

26

RaAkbari

70

NSaeid Abadi

114

FPeste Ghermez

27

RaJandaghi

71

NAmeri

115

FBadami Sefid

28

RaKhanjari Ravar

72

NBadami Kaj

116

FKalle Ghoochi

29

RaMomtaz

73

NHassan Zadeh

117

GhGhazvini (Kalle Bozi)

30

RaVahedi

74

NKarim Abadi

118

GhOhadi

31

RAhmad Aghaei

75

NSabz Peste Nooghi

119

GhBadami

32

RFandoghi Riz

76

NBehesht Abadi

120

GhKal Khandan

33

RaSefidPeste Nooghi

77

NLak Sirizi

121

GhPeste Sefid

34

RaJavad Aghaei

78

NFandoghi Ghafoori

122

GhPeste Ghermez

35

RaHassanzade

79

NPoost Khormaei Ghafoori

123

DKhanjani

36

RaShahpasand

80

NMohseni

124

DShahpasand

37

RaMomtaz Tajabadi

81

NFandoghi 48

125

DAbbas Ali

38
39

RaShasti
RaPoost Khormaei

82
83

KPoost Piazi
KSeifoddini

126
127

DAhmad Aghaei
D-1

40

RaMohseni

84

KHarati

128

D-2

41

RaEbrahimi

85

KGholamrezaei

129

D-3

42

RaKelasi Rizi

86

KSefid Peste Nooghi

130

D-4

43

RaBehesht Abadi

87

KMoosa Abadi

131

D-5

44

RaLahijani

88

KNish Kalaghi

The collection sites are abbreviated as follows: Ra Rafsanjan, Iran; N Naserieh, Iran; K Kerman, Iran; F Feiz Abad, Iran; G Gazvin, Iran;
D Damghan, Iran; and Ro Rome, Italy

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Conserv Genet (2010) 11:311318

Table 2 Information on the
collection of the populations of
Iranian pistachio species

315

Species

Pop. code

Locality

Longitude

Latitude

Sample size

P. vera

Sarakhs

36170 N

61120 E

25

P. vera

Kh1

Khaje kalat

3701 N

59400 E

25

P. vera

Kh2

Khaje kalat

37030 N

60020 E

25

P. vera

Kh3

Khaje kalat

37070 N

60220 E

24

P. khinjuk

Damghan

35410 N

54210 E

12

K1

Kermanshah

3345 N

4746 E

20

K2

Kermanshah

33440 N

46300 E

20

Repeat motif

Loci No.

Alleles No.

Ptms-3

(CA)16

Ptms-7

(CA)15

4
4
5

Ptms-9

(CA)7

Ptms-10

(CT)15

Ptms-14

(CA)46

Ptms-31

(CA)20

Ptms-40

(CTTT)4

Ptms-41

(CA)11

Ptms-45

(CAAA)3(CA)4

Ptms-47

(CTT)13

Genetic differentiation
In order to utilize different genotypes of pistachio efficiently, it is important to know the genetic variation and
genetic relationships that exist between them. The 10 SSR
primer pairs used in our study revealed moderate levels of
polymorphism in Iranian pistachios. In addition, the

Standard error

number of alleles that we detected was relatively more than

from those recorded previously for Syrian, Turkish, and
American pistachios (Ahmad et al. 2003b, 2005). AMOVA
analysis indicated that most of the variation (50%) occurred within pistachio populations. Therefore, a substantial
proportion of the total diversity in pistachios, which are
long-lived dioecious angiosperms, may occur within populations rather than between populations (Hamrick and
Godt 1996). In comparison with other species, the genetic
diversity of P. atlantica subsp. kurdica was relatively low.
This may be due to a severe decline in the habitat for P.
atlantica, in addition to over-collection and -exploitation,
which result in genetic erosion (Esmailpour 2001). The low
genetic diversity of P. atlantica subsp. kurdica suggests
that it could be susceptible to environmental change and
attack by diseases. Protection against the loss of genetic
diversity is required urgently.
Genetic diversity and relationships among
and within species

Discussion

For population codes, see

Table 2

P. atlantica subsp. kurdica

Marker

P. atlantica subsp. kurdica

Table 3 Description of pistachio simple sequence repeat (SSR)

markers developed by Ahmad et al. (2003a), repeat motifs and
number of observed loci and alleles

Table 4 The mean number of

alleles across all loci (na), the
effective number of alleles (ne),
the average expected
heterozygosity (He) and
Percentage of polymorphic loci
across the different pistachio
populations, species, and
cultivars

Our results showed that the three pistachio species are

clearly separated from each other. However, different
populations of each species (the two populations of P. atlantica subsp. kurdica and the four wild populations of P.
vera), with diverse geographical backgrounds, were related
closely and we confirmed that P. khinjuk and P. atlantica
subsp. kurdica might have originated from P. vera (Fig. 3).

Populationa

na

P. vera (Iranian cultivars)

2.77 0.36b

1.48 0.15

0.25 0.06

76.92

P. vera (Foreign cultivars)

1.92 0.40

1.23 0.25

0.22 0.07

61.54

ne

He

Percentage of
polymorphic loci

P. vera (S)

2.08 0.24

1.69 0.21

0.32 0.07

76.92

P. vera (Kh1)

2.46 0.31

1.76 0.16

0.37 0.06

76.92

P. vera (Kh2)

2.39 0.33

1.79 0.18

0.36 0.07

76.92

P. vera (Kh3)
P. khinjuk

1.92 0.31
2.08 0.40

1.56 0.22
1.66 0.32

0.30 0.07
0.31 0.08

61.54
61.5

P. atlantica subsp. kurdica (K1)

1.46 0.43

1.00 0.29

0.17 0.07

38.46

P. atlantica subsp. kurdica (K2)

1.36 0.40

1.09 0.31

0.20 0.07

46.15

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Table 5 Pairwise values for Neis genetic distances (below the diagonal) and Fst (above the diagonal) of the pistachio populations, species, and
cultivars studied
P. vera
(Iranian
cultivars)
P. vera (Iranian cultivars)
P. vera (Foreign cultivars)

0.34

P. vera (S)a

0.12

P. vera
(Foreign
cultivars)

P. vera
(S)

P. vera
(Kh1)

P. vera
(Kh2)

P. vera
(Kh3)

P. khinjuk

P. atlantica
subsp. kurdica
(K 1)

P. atlantica
subsp. kurdica
(K 2)

0.33

0.12

0.11

0.14

0.17

0.44

0.50

0.48

0.34
0.42

0.33

0.31

0.37

0.51

0.46

0.45

0.03

0.05

0.14

0.36

0.50

0.50

P. vera (Kh1)

0.13

0.44

0.03

P. vera (Kh2)

0.17

0.37

0.06

0.04

P. vera (Kh3)

0.14

0.41

0.10

0.06

0.06

P. khinjuk

0.77

0.80

0.57

0.57

0.50

0.52

P. atlantica subsp. kurdica (K 1)

0.50

0.39

0.56

0.49

0.41

0.36

0.58

P. atlantica subsp. kurdica (K 2)

0.48

0.36

0.61

0.51

0.43

0.39

0.65

0.10

0.35

0.46

0.46

0.10

0.33

0.43

0.43

0.37

0.46

0.47

0.51

0.52
0.48

0.04

For population codes, see Table 2

aic
rd
ku
p.
bs
su
a-

P.
in

kh

Foreign
cultivars

ic

Iranian
cultivars

nt

P.vera
(wildtype)

la

P. atlantica
subsp.
kurdica

P.

ju

0.05

at

Coord. 2

P. khinjuk

K2

P.atlantica-subsp.kurdica-K1

0.03

Coord. 1

Fig. 2 Two-dimensional graph based on the ordination scores of the

principal coordinate analysis

-Kh2

h1

-K

ra
.ve

Ira

-S

nia

n-c

ult

iva

P.v
era

-Kh
era

123

ar

-cultiv

foreign

P.vera

P.v

Parfitt and Badenes (1997) confirmed that the closest species to P. vera is P. khinjuk, followed by P. atlantica.
All the foreign cultivars and P. khinjuk samples were
assigned correctly. This may have been due to the distinct
origin of the foreign cultivars as compared to the Iranian
germplasm, and the topological/geographical isolation of
P. khinjuk. The results of the PCO showed that the Iranian
cultivars were related closely to the wild-type P. vera
populations, whereas, the foreign cultivars were related
more closely to the P. atlantica subsp. kurdica populations.
The Iranian cultivars were assigned to groups that contained geographically-neighbouring wild populations. This
suggested that gene flow might occur between the Iranian
cultivars and wild P. vera populations. However, calculation of the effective number of migrants, based on
estimates of Fst, indicated low levels of gene flow between
the wild species and the cultivars. As a result of its
domestication and the spread of pistachio cultivation far
beyond the natural range of the wild progenitor, P. vera
came into contact with several different Pistacia species. In
traditional areas of pistachio cultivation, contact between
the cultivated clones and wild species is quite common and

Fig. 3 Dendrogram of different populations/species/cultivar groups

produced by the neighbour-joining clustering method

Table 6 Analysis of molecular variance (AMOVA) of the pistachio

species and populations
Source

df

SS

MS

Est.
var.

Among
spices

2 1,253.538 313.385

5.033

Among pops.

Total
(%)

P
value

41

0.001

123.724

30.931

1.055

0.001

Within pops. 303 1,843.549

6.084

6.084

50

0.001

Total

311 3,220.811 350.400 12.172

Conserv Genet (2010) 11:311318

Assignment index in Pop 1

-20.000

-15.000

-10.000

-5.000

0.000
0.000

-5.000

-10.000

-15.000

-20.000

-25.000

Assignment index in Pop 2

Fig. 4 Logarithms of the

expected frequencies
(assignment indices) of
genotypes drawn from Iranian
cultivars, foreign cultivars, and
wild-type populations of P. vera

317

-30.000

Iranian cultivars

has existed for hundreds or even thousands of years

(Zohary 1996). As a result, interspecific hybridization of
P. vera with other Pistacia species led to the development
of various hybrids with different backgrounds (Maggs
1973; Kafkas and Perl-Treves 2001; Barazani et al. 2003).

Conclusions
Although Iran is one of the two major centres of Pistacia
diversity and the main pistachio producer in the world, the
Iranian pistachio industry has a very narrow genetic base.
Our results demonstrated that the study of genetic diversity
and relationships among Pistacia species and cultivars
using SSR markers provides information that is relevant
for the conservation of pistachio germplasm. Our study
showed that Iranian cultivars have a different genetic
background from foreign cultivars and therefore are very
important for genetic conservation and the planning of
future breeding programmes. We also determined that
different levels of genetic diversity exist between and
within the different species and populations and showed
that gene flow occurs between the Iranian cultivars and
wild type P. vera populations. The present study provides
practical information for policy-makers and scientists for
the conservation and sustainable use of all the species
studied.
Acknowledgments The Agricultural Biotechnology Research
Institute of Iran supported this study. The authors would like to thank
Dr. A. A. Javanshah (Director General of the Iranian Pistachio
Research Institute), in particular, for his contribution to the collection
of plant materials.

Foregin cultivars

Wildtype

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