Background: cultivars in terms of their genetic

Background:
Inter-Simple Sequence Repeat (ISSR) and Start Codon
Targeted (SCoT) markers were used to evaluate of genetic diversity in 20
samples of four Iranian pistachio (Pistacia vera L.) cultivars and
relative efficiencies of the marker systems were compared.

Materials and Methods: 15 and 20
primers were utilized in ISSR and SCoT markers, respectively. Effective
Multiplex Ratio (EMR), Marker Index (MI), Resolving Power (RP) and Polymorphic
Information Content (PIC) of the primers were calculated for the two marker
systems. Cluster analysis for
molecular data was performed. Principal Coordinate Analysis (PCoA) was also
done.

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Results: The most of the parameters examined found to be
more suitable in ISSR system. The most remarkable result of the current study
is that cluster analysis on ISSR and SCoT data clearly discriminated the
cultivars in terms of their genetic characterizations. There was a high
similarity between dendrogram derived from ISSR marker and dendrogram derived
from both markers, although some differences were observed.

Conclusions:
The present study showed that there is remarkable
genetic diversity among evaluated samples. ISSR and SCoT analysis produces
sufficient polymorphisms for DNA fingerprinting. This study reports the first
application of the SCoT marker in characterization of Iranian pistachio
cultivars.

 

Keywords:
Genetic
diversity, Pistachio,
SCoT marker,
ISSR marker,

 

1. Introduction

Pistacia vera L. (pistachio) is a diploid (2n=2x=30) member of the
Anacardiaceae family (1). The most widely accepted
classifications divide the family into five tribes: Anacardieae, Rhoeae,
Semecarpeae, Spondiadeae, and Dobineae (2, 3). Pistacia vera belongs to
the Rhoeae tribe. The pistachio genus contains 13 or more species (4). Research
efforts have mainly focused on P. vera, and the other
species have been overlooked due to a large extent to the economic importance
of Pistacia vera (5). There are two main centers of diversity for Pistachio. The
first one comprises the Mediterranean region of Europe, Northern Africa, and
the Middle Eastern countries. The second one is Caucasus regions and the
Eastern part of Zagros Mountains from Crimea to the Caspian
Sea (1). The main cultivars grown in Iran are Fandoghi,Akbari, Kaleh ghochi,
Ahmad Aghai, Badami, and Pustpiazi (6). DNA-based markers provide accurate and useful
information on the extent of diversity and relationships among plant species (4). The classification of Pistachio
species at the molecular level was first done based on chloroplast DNA profiles
by Parfitt and Badenes in 1997 (4). Pistachio has a high genetic diversity
because of its dioecious and heterozygous nature (7). Pistachio breeding programs have recently been initiated to develop
new cultivars (7). DNA markers that are closely linked to important agronomic
traits greatly contribute to practical crop improvement programs (8). Since
1994, a new molecular marker technique called Inter Simple Sequence Repeat
(ISSR) has been available (8). ISSRs are semi-arbitrary markers amplified by
PCR in the presence of one primer complementary to a target microsatellite (9). The technique uses microsatellites, usually 16–25
bp long, as primers in a single primer PCR reaction targeting multiple genomic
loci to amplify mainly the inter- SSR sequences of different sizes (8, 10). In recent
years, many new alternative and promising marker techniques have been developed
in line with the rapid growth of genomic research (11). Due to the tremendous
growth in public biological databases, the development of functional markers
that are located in or near the candidate genes have become considerably easy
(12). Initiating a trend away from random DNA markers towards gene-targeted
markers, a novel marker system called SCoT was developed based on the short
conserved region flanking the ATG start codon in plant genes. SCoT markers are
generally reproducible, and it is suggested that primer length and annealing
temperature are not the only factors that determine reproducibility. They are
dominant markers like RAPDs and could be used for genetic analysis,
quantitative trait loci (QTL) mapping, and bulk segregation analysis (13). In
principle, SCoT is similar to RAPD and ISSR because the same single primer is
used as the forward and reverse primer (13, 14). A limited
number of studies have investigated the genetic diversity of P. vera cultivars.
Therefore, our objectives in this study were to evaluate the genetic diversity
of some Pistacia vera cultivars
grown in Iran and to compare relative efficiencies of ISSR and SCoT markers with
respect to their applicability in genetic diversity studies of Pistacia vera genotypes.

 

2. Materials and Methods

2.1. Plant materials

Fresh
leaf segments of pistachio common cultivars (Akbari,
Ahmad Aghaei, Kaleghouchi, and Fandoghi, five samples for each
one) were randomly harvested from five different regions with a large area under the cultivation of
pistachio of Kerman province that are listed in table 1. In order to prevent deterioration, the samples
with three replications were immediately transferred to liquid nitrogen tanks.
Then Young leaves tissues (100 mg) were ground under liquid nitrogen to obtain a fine powder. Genomic DNA was
extracted from powdered leaves using CTAB (the Doyle and Doyle method) (1987)
with minor modifications (4). Purified total DNA was quantified, and its
quality was verified by spectrophotometer and gel electrophoresis. DNA samples
were diluted to 40 ng/µl with distilled water and stored at -20°C for further use.

 

2.2.
PCR primers, materials, and conditions

In the present study, which was conducted in Graduate University of
Advanced Technology and Vali-e-Asr University of
Rafsanjan in 2016, 35 available ISSR primers were tested for initial screening.
Of the 35 primers, 15 primer which gave the most informative patterns (in terms
of repeatability, scorability, and the ability to distinguish between
cultivars) were selected for identification. Twenty single SCoT primers without any initial screening and 15
ISSR primers were used for fingerprinting. Amplifications of SCoT and ISSR
primers were performed in a 25
?L reaction
volume containing 1 ?L DNA(40 ng), 12.5 ?L  Taq DNA Polymerase, 2x Master Mix Red Ampliqon ( 1.5 mM MgCl2
final concentration), 1.1 ?L of 10 ?M 
primer, and 10.4 ?L of distilled water. Amplification of SCoT primers
was performed in a programmed thermocycler (Biorad Model T100) with an initial
denaturation step at 94°C for 3 min, followed by 35 cycles of 94°C for 1 min,
between 50°C to 57°C
for 1 min, and 72°C for 2 min about denaturation, annealing, and extension steps,
respectively. The final extension at 72°C was held for 5 min. PCR reactions for amplification of ISSR primers were carried out as described in SCoT analysis with
the exception of primer volume (2.4 ?L). The
thermo cycler program for PCR was set to 2 min at 94°C, followed by 35 cycles
of 94°C for 0.5 min, 1 min at between 45°C to 56°C and 2 min at 72°C about
denaturation, annealing, and extension steps, respectively. The final extension
at 72°C was held for 5 min. All PCR amplification products were separated on
1.5% agarose gel in Tris-Borate
Buffer (TBA) stained with Ethidium-Bromide and documented
using gel documentation system (UVTEC, UK). Each experiment was repeated two times with
each primer and only those primers which gave reproducible fingerprints (DNA
bands) were considered for the data analysis. The set of ISSR and SCoT primers used in our
investigation is shown in table 2.

 

2.3.
Data Analysis

Only the consistent reproducible
bands were scored, and data matrix was prepared based on presence (1) and
absence (0). Intensity of the band was not considered while scoring. A dendrogram showing the genetic relationships between
cultivars, based on the unweighted pair group method with arithmetic averages,
was constructed using NTSYS-pc (Numerical Taxonomy and Multivariate
Analysis System) version 2.02 (15). Genetic association
amongst samples was evaluated by the Jaccard’s similarity coefficient (16) with the SIMQUAL (Similarity for qualitative data program in NTSYS)
module of NTSYS-pc software. The similarity matrix was subjected to cluster
analysis of UPGMA method, and a dendrogram was generated by using the SAHN
(sequential, agglomerative, hierarchical, and nested clustering) module in
NTSYSpc program. Principal Coordinate Analysis (PCoA) was also done. Polymorphic Information Content (PIC) of each of the
analyzed SCoT and ISSR was calculated using the formula PIC = 1- p2-q2
(17), where p is frequency of present band, and q is frequency of absent band.
The band in formativeness (Ib) was estimated as Ib = 1 – (2 × |0.5 – p|) (18),
where p is the proportion of the varieties or genotypes containing the band.
The Resolving Power of the primer (RP) was measured in accordance with RP =
?Ib. Marker Index (MI) was computed as EMR × PIC, where EMR (Effective
Multiplex Ratio) was defined as the product of the total number of
fragments per primer (n) and the fraction of polymorphic fragments (?). EMR =
n? : n= total number of bands, ?= total number of polymorphic bands. 18, 19,
20.

 

3. Results

This study reports the first
application of the SCoT technique in pistachio characterization of P. vera
cultivars. The genetic diversity indices of 20 SCoT primers are shown in Table
3. Using 20 selected SCoT primers, 151 bands
were generated, of which 138 bands (91.3%) were polymorphic (Fig. 1). The maximum and lowest numbers of amplified bands
were for SCoT13 with 13 bands and SCoT12 and SCoT20 with 5 bands, respectively.
The values of polymorphism varied from 75% to 100%. The maximum and lowest PIC
values were for SCoT10 (0.407) and SCoT9 (0.211), respectively. The average RP value in SCoT marker was 4.18. The
range of MI (Marker Index) was from 0.953 (SCoT9) to 4.62 (SCoT13). The average
MI value in SCoT marker was 2.335. The UPGMA clustering algorithm from SCoT
marker analysis grouped the 20 samples of four cultivars into three main
clusters (Fig. 2). The first cluster, group ?, consists of two cultivars. The
second major cluster, group ??, consists of 17 cultivars. The third major cluster, group ???, consists of
one cultivar: Akbari Rafsanjan (C1). All of the groups include several samples
except for Akbari Rafsanjan (C1) that builds a separate group in SCoT marker. The
Cultivars Kaleghouchi Ravar (B4) and Fandoghi Ravar (D4), which are genetically
close cultivars, fell in same group in SCoT marker. Other cutting lines (Fig. 2) show subgroups and more details of
classification. In the present study, we investigated the ability of 15 ISSR
primers to generate polymorphic DNA fragments. The genetic diversity indices of
15 ISSR primers are shown in Table 3. 15 selected ISSR primers amplified 131
bands with 124 (94. 6%) being polymorphic (Fig. 3). The maximum and lowest
number of amplified bands was for UBC851 and K13 with 11 bands and UBC808 with
four bands, respectively. The values of polymorphism ranged from 80% to 100%.
The maximum and lowest PIC values were ISSR9 (0.47) and ISSR7 (0.187),
respectively. The average RP value in ISSR marker was 5.13. The maximum and
lowest MI indices were K13 (4.43) and
UBC808 (1.145). The average MI value in ISSR marker was 2.916. The UPGMA
clustering algorithm from ISSR marker analysis grouped the 20 samples of four
cultivar into three main clusters (Fig. 4). The first cluster (group ?) further
divided into two sub-clusters. The first sub-cluster consisted of Ahmad Aghaei
Rafsanjan (A1). The second sub-cluster consisted of seven samples. The second major
cluster (group ??) further divided into two sub-clusters. The first sub-cluster
consisted of seven cultivars. The second sub-cluster consisted of 4 samples.
All of the groups include several samples except for Akbari Rafsanjan (C1) that
builds a separate group in ISSR marker. Other
cutting lines (Fig. 4) show subgroups and more details of classification. A
possible explanation for the difference in the resolution of SCoT and ISSR
primers is that the two-marker techniques target different portions of the
genome. These differences may also be attributed to marker sampling errors
and/or the percent polymorphism detected by different markers, highlighting the
importance of the number of loci and their coverage of the overall genome in
obtaining reliable estimates of genetic relationships among cultivars (21, 22). The UPGMA clustering algorithm from ISSR and
SCoT marker analysis grouped the 20 cultivars into three main clusters (Fig.
5). The first cluster, group ?, further divided into two sub-clusters. The first
sub-cluster consisted of one cultivar, Ahmad Aghaei Rafsanjan (A1). The second
sub-cluster consisted of seven cultivars. The second major cluster, group ??,
further divided into two sub-clusters. The first sub-cluster consisted of seven
cultivars. The second sub-cluster consisted of four cultivars. All of the
groups include several samples except for Akbari Rafsanjan (C1) that builds a
separate group in ISSR and SCoT markers. Other
cutting lines (Fig. 5) show subgroups and more details of classification. The
general UPGMA dendrogram were constructed using the combined data of the two
sets of molecular markers that are approximately similar to ISSR marker
dendrogram (for instance, group of Ahmad Aghaei Ravar and Kaleghouchi Ravar,
Fandoghi Ravar; Kaleghouchi Kerman, Fandoghi Kerman; Fandoghi Rafsanjan,
Kaleghouchi Zarand; Kaleghouchi Rafsanjan, Fandoghi Anar; Fandoghi Zarand,
Kaleghouchi Anar; Akbari Rafsanjan and Ahmad Aghaei Rafsanjan). According to
Fig 5, the strongest similarity was between Kaleghouchi Ravar and Fandoghi
Ravar and the weakest similarity was observed in Akbari and Ahmad Aghaei
Rafsanjan. The cophenetic coefficient was
acceptable in both molecular marker systems, indicating a good fit for
clustering. The values of mantel test correlation showed a positive  and significant
correlation between the SCoT and ISSR. Based on PCoA analysis, all Pistachio
samples fell in three distinct groups (Fig. 6). The results of SCoT and ISSR
markers Dendrogram and PCoA analysis are relatively consistent with each other

 

4.
Discussion

Using
more than one marker has always been recommended for a better analysis of
genetic homogeneity of plants (23). Recently, simple sequence repeat (SSR)
technique has been used to identify 17 pistachio cultivars using their nuts
collected from the markets in the U.S.A and in Europe (24), and in another
study, SSR markers were used to analyze four commercially important pistachio
rootstocks grown in California (25). Genetic relationships among thirty one
samples of
Iranian pistachio were assessed using six inter simple sequence
repeat (ISSR) primers. Good
amplification products were obtained from primers based on GA, CA, and GAA
repeats. The range of genetic similarity was from 0.84 to 1.
The cluster analysis divided the samples into 11 groups (9).  In the present study, we performed a comparative
assessment of SCoT and ISSR markers for the evaluation of Pistachio genetic
diversity. Not all ISSR primers were suitable. 20 out of 35 ISSR primers did
not produce any bands. SCoT and ISSR markers
generated high numbers of polymorphic bands that can be used in diagnostic
fingerprinting of Pistacia vera. Based on the data obtained from
these two molecular markers, i.e., polymorphism percentage, PIC values, RP
values, and marker index, the efficiency of ISSR
for fingerprinting of cultivars was more
than other marker. These two techniques could be used in conjunction with each
other for diagnostic fingerprinting of Pistacia vera
cultivars. Pakseresht et al. (2013) performed a comparative
assessment of ISSR, DAMD, and SCoT markers for the evaluation of genetic
diversity and conservation of landrace chickpea genotypes and reported that the
SCoT and DAMD markers are more effective in fingerprinting of chickpea
genotypes (26). Application of SCoT molecular marker in other plants
such as mango (27), potato (28), and Cicer (29) showed that this marker
is efficient.

 

5.
Conclusions

The
present study showed that there is remarkable genetic diversity among evaluated
samples. ISSR and SCoT analysis produces sufficient polymorphisms for DNA
fingerprinting. This study reports the first application of the SCoT marker in
the characterization of Iranian pistachio cultivars. Generally, ISSR and SCoT markers could be used in
conjunction with each other for diagnostic fingerprinting of Pistacia vera
cultivars.