AJCS 7(6):763-769 (2013) ISSN:1835-2707
Comparison of the effectiveness of ISSR and SSR markers in determination of date palm
(Phoenix dactylifera L.) agronomic traits
Hammadi Hamza*, Mohammed Ali Ben Abederrahim, Mokhtar Elbekkay and Ali Ferchichi
Arid and Oases Cropping Laboratory, Arid Area Institute, Medenine 4119, Tunisia
Abstract
Date palm (Phoenix dactyliferaL.) is extensively cultivated in the Middle East and in North Africa. Seven ISSR markers and five
SSR loci were selected and used to evaluate genetic diversity in twenty-six Tunisian cultivars. ISSR primers amplified a total of 43
polymorphic DNA fragments. The average was 6.1 fragments per primer. The microsatellites examined in this study were highly
polymorphic possessing a great number of alleles with an average of 7.2 alleles per locus. Principal component analyses based on Nei
Genetic distances showed groups of cultivars with a common maturity period and a common fruit consistency. SSR markers
discriminate the fruit characteristics subpopulations in a more convincing way than ISSR markers. The Mantel test emphasizes a
significant correlation between genetic distance and fruit consistency. A significant difference was observed between soft and dry
subpopulations using ISSR data and between semisoft and the other fruit consistency subpopulations using SSR data.
Keywords: AMOVA, fruit consistency, Mantel test, maturity period, molecular markers.
Abbreviations: analysis of molecular variance (AMOVA), inter-simple sequence repeat (ISSR), simple sequence repeat (SSR),
random amplified polymorphic DNA (RAPD), principle component analysis (PCA), random amplified microsatellites polymorphism
(RAMPO), amplified fragment length polymorphism (AFLP).
Introduction
The date palm (Phoenix dactylifera L.) is one of the most of these descriptions, it remains difficult to identify cultivars,
important fruit trees in the arid and semi-arid regions especially outside of the fruiting period. In fact, owing to the
(Munier, 1973). It is extensively grown as a food crop and great adaptive flexibility of this genus, many farmers cannot
covers about 3% of the cultivated area of the world (Dowson, recognise cultivars outside their oasis (Munier, 1973).
1982). This species has a great value for populations in the However, Hamza et al. (2009) have selected vegetative traits
Middle East and the north of Africa where it has been that are steady and unaffected by the environment, and these
intensively cultivated. It provides a wide range of products have been very useful in identifying the fruit maturity period
and services, including many necessities of life. Historical and the consistency. Molecular markers may provide a
investigations suggest that date palm domestication has been reliable tool for measuring genetic divergence. Several
practiced for five millennia (Nixon, 1959). The date palm markers have been used, including random amplification
trunk is bounded by leaf bases, and it can grow to 20m in polymorphism DNA (RAPD) (Sedra et al., 1998; Trifi et al.,
many localities (Munier, 1973). The leaves cover the top of 2000; Al-Khalifa and Askari, 2003), inter simple sequence
the plant, and they are pinnate with spines at the base. The repeats (ISSR) (Zehdi et al., 2002), random amplified
fruit is a ‘berry’ with a single seed in each. The fruit is borne microsatellites polymorphism (RAMPO) (Rhouma, 2008)
on a bunch, and a productive palm can support up to 13 and amplified fragment length polymorphism (AFLP)
bunches. The ripening time depends on the cultivar, but (Rhouma et al., 2007). These studies revealed a high
generally it takes up to 200 days from the date of pollination polymorphism among date palm cultivars. Simple sequence
to the Tamer stage (Hamza et al., 2009). The date palm has a repeat (SSR) was very useful to identify date palm cultivars,
wide range of geographical and ecological distribution, which and a high polymorphism has been detected in date palm
is pronounced by its considerable genetic diversity (Elshibli cultivars (Zehdi et al., 2004; Hamza et al., 2011). All these
and Korpelainen, 2008). This diversity is the result of the molecular markers showed a high degree of independence
dioecious nature (separate male and female plants) of the with the geographical origin. The purpose of the present
specie (Munier, 1981). Many cultivars have been study was to examine the genetic diversity in date palms
characterized in several areas. This characterization has using two markers: ISSR and SSR. A comparative study was
involved many morphological and molecular tools. done to search possible relations between the fruit
Concerning the phenotypic markers, many traits were used to characteristics of Phoenix dactylifera L. in Tunisian oases.
characterise the morphology of leaves, spines and fruit
characters. Such morphological features are sensitive to Results
environmental factors (Sedraet al., 1993; 1996), and they can
be observed only in mature trees. In Tunisia many reports, A total of seven ISSR primers were screened for their ability
have described the importance of morphological traits in to generate consistently amplified band patterns and to assess
identifying the Tunisian date palm cultivars (Ben Salah, polymorphism in the tested cultivars. All the primers used
1993; Ben Salah and Hellali, 2004; Rhouma, 2005). In spite have revealed unambiguously scorable polymorphic bands. In
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Table 1. List of the ISSR primers and SSR loci and their characteristics.
Primers / locus Sequence of primer (5'–3') or Repetitive motif A Ap P
ISSR D9 (CT)10G 8 5 62.5%
D12 (GA)6CC 5 4 80%
UBC 888 (GCT)(AGT)(GCT)(CA)7 6 5 83.3%
UBC 890 (AGC)(ACT)(AGC)(GT)7 7 7 100%
UBC 891 (ACT)(AGC)(ACT)(TG)7 6 6 100%
PO6 (AG)10C 5 3 60%
PO7 (AG)10T 6 4 66.6%
SSR mPdCIR10 (GA)22 8 8 100%
mPdCIR15 (GA)15 6 6 100%
mPdCIR32 (GA)19 7 7 100%
mPdCIR70 (GA)17 8 8 100%
mPdCIR93 (GA)16 7 7 100%
A: Total number of Bands or alleles, Ap Number of polymorphic bands or alleles, P: Percentage of polymorphic Bands or
alleles.
Fig 1. Cultivars distribution on the plan 1-2 and 1-3 of PCA based on ISSR-amplified loci. (a) The scores of fruit-
consistency subpopulations (b) The scores of maturity-period subpopulations.
fact, the seven primers amplified a total of 43 polymorphic exhibited 36 alleles with an average of 7.2 alleles per locus.
fragments ranging from 250 to 2,500 bp. The maximum The microsatellite markers were found to be highly
number of fragments was 8 bands that are produced by the polymorphic with the number of alleles ranging from six to
primer D9 with 62.5% polymorphism. The minimum number eight among the 26 cultivar genotypes. All the used loci
of fragments was 5 bands produced by the primers D12 and showed 100% polymorphism (Table 1). Nei genetic distances
PO6 with, respectively, 80 and 60% polymorphism (Table 1). were used to analyse the variability of the studied cultivars by
The average was 6.1 ISSR bands per primer. Trimer primers a principal component analysis. Concerning the ISSR
like UBC ones produced the maximum polymorphism; for markers, the data showed that the first three axes of the PCA
example, UBC 890 and UBC 891 amplified 100% of explained 62.51% of the total variability. The distribution of
polymorphic bands. Concerning SSR profiles, five loci cultivars (Fig. 1) shows a difference between the Dhahbi
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cultivar and the others. This cultivar was noted only in the polymorphic bands (34 bands) and the average number of
Tamerza oasis (Table 2). No other geographical group was fragments produced per primer (6.14) are less than those
distinguished; the different cultivars are classified regardless obtained in previous studies of date palms (Zehdi et al., 2002;
of their oasis. However, we can distinguish clusters of Mitra et al., 2011). On the other hand, our SSR results
cultivars according to their fruit characteristics. In fact, PCA indicate the presence of high genetic diversity in Tunisian
results show a grouping of cultivars with soft or semisoft date palms but less than in Sudanese date palms (Elshibli and
fruits and with early or mid-season maturities (Fig. 1). On the Korpelainen, 2008). This may be explained by intensive
other hand, the cultivar distribution based on SSR data was, selection in Tunisian date palm oases (Zehdi et al., 2004).
also, independent of the geographical origin. In the This result agrees with other reports for Moroccan, Algerian
scattergram, a subpopulation separation can be observed. For and Tunisian date palm cultivars based on analyses using
the fruit consistency, the subpopulations are more easily microsatellite markers (Zehdi et al., 2004) and isozyme
grouped (Fig. 2).Concerning the maturity period, cultivars markers (Bennaceur et al., 1991; Ould Mohamed Salem et al.,
with early and mid-season maturity are associated (Fig. 2). 2001). The numbers of alleles per locus detected in this study
were comparable with those scored by Zehdi et al. (2004): for
Mantel test 46 Tunisian date palm accessions, 100 different alleles were
identified at 14 microsatellite loci with an average of 7.14
The null hypothesis of no correlation between different alleles per locus. In addition, a high degree of independence
matrices was tested (Table 3). The ISSR data showed a between the geographical origin and molecular data was
significant correlation with the fruit consistency matrix (r = - indicated. The only distinction is in the case of ISSR data for
0.120; P = 0.026) but not with the maturity period matrix (r = the Dhahbi cultivar, which is a specific variety of continental
- 0.054; P = 0.225). The same result was found with the SSR mountain oasis (Tamerza), where it is intensively cultivated.
data, in which a significant correlation was found with the The RAMPO and AFLP data applied on Tunisian date palms
consistency matrix (r = 0.110; P = 0.029) but not with the (Rhouma et al., 2007; Rhouma-Chatti et al., 2011) showed
maturity matrix (r = -0.027; P = 0.382). However, no that the studied cultivars clustered independently from their
significant correlation was found between the ISSR and SSR geographic origin. This is in favour of the hypothesis
data (r = -0.108; P = 0.151). proposed by Wrigley (1995), which suggests a common
genetic base of the cultivars in the Tunisian continental oasis.
AMOVA The lack of basic geographical differentiation is explained by
the fact that communication often facilitates the exchange of
According to ISSR data, no significant genetic difference plant material in the studied oases. Molecular studies have
(P > 0.05) among fruit characteristics subpopulations was proved the efficiency of the molecular markers to assess
observed, although 3% of total genetic diversity was detected genetic diversity between date-palm genotypes. However,
among fruit-consistency subpopulations. Pair-wise few studies have shown a correlation between molecular and
comparisons of subpopulations (Table 4) showed that the soft phenotypic markers. Mirta et al. (2011) have underscored
subpopulation and the dry one are significantly different. with RAPD and ISSR markers a discrimination of date palm
AMOVA tests based on SSR data showed no genetic cultivars on the basis of the tree sex. However, Rhouma-
differentiation among maturity-period subpopulations; Chatti et al. (2011) did not show a significant discrimination
however, significant genetic differentiation was observed of male trees using AFLP and RAMPO markers. The current
among the fruit-consistency subpopulations (P < 0.05) with result unveils a significant correlation between the genetic
7% of total genetic diversity detected among fruit- data and the fruit consistency as shown by the Mantel test,
consistency subpopulations. Pair-wise comparisons of which is partly supported by PCA analysis. The correlation
populations (Table 5) show that significant genetic between SSR markers and fruit consistency is more
differences exist between the semi-soft subpopulation and the convincing than the ISSR study. These results allowed us to
semi-dry and soft groups. Another significant genetic compare genetic structure between groups based on fruit
differentiation was detected between semi-soft groups and characteristics. The ISSR and the SSR data showed that no
early-season groups. genetic differentiation was observed among maturity period
subpopulations. However, when the fruit consistency
Discussion subpopulations were compared, a significant differentiation
was detected. Indeed, soft and dry subpopulations were
Molecular markers are efficient tools for cultivar genetically differentiated on the basis of ISSR data (PhipT =
identification and estimation of relatedness through DNA 0.126, P < 0.05). Another differentiation trend is revealed in
fingerprinting. In the present investigation, ISSR and SSR this study using SSR data; the FST values suggest significant
were employed to assess genetic polymorphisms in Tunisian genetic differentiation between subpopulations. The semi-soft
date palm cultivars. ISSR is a dominant marker that has, in subpopulation was significantly differentiated from the other
comparison with RAPD techniques, high reproducibility fruit-consistency subpopulations and from the early-maturity
(Williams et al., 1990). In the field of date palms, ISSR subpopulation. The discrepancy between molecular markers
markers were found more informative than the RAPD and maturity period support the statement that the maturity
markers (Mitra et al., 2011). Many ISSR primers were tested period could be affected by the local environment, whereas
in the literature and applied on date palm genotypes (Zehdi et the used markers are not, and their variation is based directly
al., 2002; Mitra et al., 2011) or on other monocotyledon on DNA sequence variation (Bruschi et al., 2003).
species such as the genus Poa (Arslanet al., 2011) and durum
wheat (Pasqualone et al., 2000). A total of seven primers Material and Methods
were screened for ISSR-PCR analysis, and they were useful
to characterize the samples and produced strongly amplified Plant materials
polymorphic bands. The selected primers generated an
appropriate amplification pattern with clear and consistent Cultivars belong to the continental Tunisian oases were
reproducible bands. In the present study, the number of chosen for their fruit importance (Ferchichi and Hamza,
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Table 2. Name, origin, and main characteristics of date-palm genotypes studied.
Geographical Fruit characteristics (at Tamer stage)
No. Name Code
distribution Colour Consistency Maturity period
1 Alig Alg Nefzoua&Jerid Dark brown Semi-dry Late
2 Ammary Amm Nefzoua&Jerid Black Soft Early
3 Bejjou Bjj Nefzoua&Jerid Brown Dry Late
4 BissrHelou Bsh Nefzoua&Jerid Pale brown Dry Season
5 Choddakh Cdk Nefzoua&Jerid Dark Amber Semi-Soft Season
6 Choddakh Ben Jbir Cbj Nefzoua&Jerid Dark Amber Semi-soft Season
7 Dhahbi Dhb Temerza Amber Semi-soft Late
8 DegletNour Dnr Nefzoua&Jerid Amber Semi-soft Late
9 Fermla Frm Nefzoua Brown Semi-dry Season
10 Fezzani Fez Nefzoua&Jerid Amber Semi-dry Season
11 Gondi Gnd Nefzoua&Jerid Amber Semi-soft Season
12 Gosbi Gsb Nefzoua&Jerid Black Soft Early
13 Gharssouf Gsf Nefzoua&Jerid Dark brown Soft Season
14 Hissa His Nefzoua&Jerid Honey Soft Early
15 Hlwa Hlw Nefzoua Honey Semi-dry Late
16 Hamra Hmr Nefzoua&Jerid Amber Semi-dry Season
17 Horra Hor Nefzoua&Jerid Amber Dry Season
18 Kintichi Knt Jerid Reddish Dry Late
19 Loghrabi Lgr Jerid Dark brown Semi-soft Season
20 Om Leghlez Olg Jerid Amber Soft Early
21 RtobHoudh Rth Nefzoua&Jerid Amber Soft Season
22 Rtotbayetelmansoura Rtm Nefzoua Brouwn Soft Season
23 Rotbayetyagouta Rty Nefzoua Dark amber Soft Early
24 Tronja Trj Nefzoua&Jerid Dark brown Semi-dry Late
25 TezerzayetKahla Tzk Nefzoua&Jerid Black Soft Season
26 TezerzayetSafra Tzs Jerid Dark brown Soft Early
Fig 2. Scattergram showing relative position of date palm cultivars defined by the first three principal components based on the
genetic distance of the five microsatellite loci. (a) The scores of fruit-consistency subpopulations (b) The scores of maturity-period
subpopulations.
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Table 3. Result of Mantel’s test of the pair-wise correlations between dissimilarity matrices.
First matrix Second matrix Mantel’s r Probability
Maturity period ISSR -0.054 0.225
Consistency ISSR -0.120 0.026*
Maturity SSR -0.027 0.382
Fruit consistency SSR 0.110 0.029*
SSR ISSR -0.108 0.151
* Rejection of the null hypothesis of no correlation within a 5% confidence interval.
Table 4. PhiPT values between different subpopulations based on seven ISSR primers.
Subpopulations Early Late Mid-season Dry Semi-dry Semi-soft Soft
Early 0.443 0.460 0.168 0.475 0.417 0.432
Late 0.000 0.443 0.421 0.410 0.493 0.174
Mid-season 0.000 0.000 0.254 0.437 0.419 0.443
Dry 0.057 0.000 0.033 0.081 0.450 0.007*
Semi-dry 0.000 0.000 0.000 0.096 0.496 0.440
Semi-soft 0.000 0.000 0.000 0.000 0.000 0.458
Soft 0.000 0.036 0.000 0.126 0.004 0.001
PhiPTvalues below diagonal. Negative PhiPT values converted to zero. Probability values based on 999
permutations are shown above diagonal.
Table 5. FST values between different subpopulations based on five microsatellite loci.
Subpopulations Early Late Mid-season Dry Semi-dry Semi-soft Soft
Early 0.158 0.261 0.222 0.431 0.011* 0.447
Late 0.026 0.436 0.438 0.421 0.420 0.149
Mid-season 0.012 0.000 0.409 0.379 0.272 0.450
Dry 0.026 0.000 0.000 0.244 0.142 0.104
Semi-dry 0.000 0.000 0.004 0.026 0.016* 0.329
Semi-soft 0.093 0.000 0.011 0.049 0.093 0.013*
Soft 0.000 0.020 0.000 0.042 0.007 0.065
FST values below diagonal. Probability values based on 999 permutations are shown above diagonal. Negative
pair-wise FST converted to zero.
2008) (Fig. 3). These areas represent more than 85% of the
total date palm oases of Tunisia. Analyses were performed on N
Mediterranean Sea
52 individual trees belonging to the 26 cultivars (Table 2) at
the rate of two replications for each cultivar. The replications Tunisia
were done to confirm the intra-cultivar stability underscored
by previous work (Zehdi et al., 2002,2004). According to the
Continental oasis
maturity period, the studied cultivars have three different
maturity periods: Early, Mid-season and Late. The fruit
consistencies were Soft, Semi-soft, Semi-dry and Dry. Total
nuclear DNA was extracted from young leaves according to Littoral oasis
the Invisorb® Spin Plant Mini Kit (Invitek). The
manufacturer’s protocol was followed: 20 μl proteinase K
was added to the mixture in the lysis step. The final DNA
product was eluted in 100 μl of pre-heated Elution Buffer D Algeria
Libya
and incubated for 3 min. This protocol gave a high DNA
purity with a concentrationup to 25 ng/µl. DNA
polymorphisms were detected by the polymerase chain
reaction (PCR).
ISSR amplification Fig 3. Locations of Tunisian oases
Seven ISSR primers were used in this study (Table 1). PCR followed by a primer-specific annealing temperature for 55 s
was carried out in 20-μl final volume using 25 ng of genomic and ended by extension at 72°C for 1 min. A final extension
DNA containing, 4 μl of 5Green GoTaq® (pH 8.5, 7.5 mM cycle was performed at 72°C for 7 min. The PCR machine
MgCl2), 100 µM dNTPs, 150 pmol random primer, and 1.2 was adjusted to hold the product at 4°C. The PCR products
units of Taq DNA polymerase. The mixture was made up to and 1kb DNA ladder (Promega) were electrophoresed on 2%
20 μl by the addition of sterilised distilled water. The agarose gels (stained with EtBr). The separated fragments
mixture was amplified in a thermal cycler (GeneAmp® PCR were visualised with an ultraviolet (UV) transilluminator.
System 9700), which was programmed for one cycle of initial
denaturation at 94°C for 5 min, 35 cycles of 94°C for 1 min,
767
SSR amplification
Five microsatellite loci were used in this study were (GenExpress lab, University of Florence, Italy) for their help
developed for Phoenix dactylifera L. by Billotte et al. (2004) with molecular analyses.
(Table 1). The SSR-PCR was performed in a volume of 12.5
µl containing 50 ng of genomic DNA, 5X Green GoTaq® References
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