American Journal of Plant Sciences, 2011, 2, 527-534
doi:10.4236/ajps.2011.24062 Published Online October 2011 (http://www.SciRP.org/journal/ajps)
527
DNA Finger Printing of Prosopis cineraria and
Prosopis juliflora Using ISSR and RAPD
Techniques
Khaled Elmeer, Ameena Almalki
Genetic Engineering Department, Biotechnology Center, Doha, Qatar.
Received July 17th, 2011; revised August 16th, 2011; accepted September 5th, 2011.
ABSTRACT
Diversity within and among the populations of Prosopis cineraria and Prosopis juliflora collected from different loca-
tion of Qatar were explored using Inter Simple Sequence Repeat (ISSR) and Random Amplified Polymorphic DNA
(RAPD) markers. A total of one hundred and nine bands were generated from twenty nine ISSR and nineteen bands
from seven RAPD primers with an average polymorphism of more than ninety nine percent across all the genotypes.
ISSR techniques were capable o f distinguishing between P. cineraria and P. juliflora, through twen ty one bands. How-
ever, of the seven RAPD markers, only three bands were able to distinguish between Prosopis species. The dendro-
grams for the analysis of genetic similarity show that the individuals from both species can be separated in two highly
related groups. Our observations suggest that genetic variations among different accessions of Prosopis are identified
using ISSR and RAPD analysis.
Keywords: Ghaf, Ghawiaf, Polymorphism, Dendrogram and Genetic Diversity
1. Introduction
The genus Prosopis is one of the commercially important
genera of legumes in arid climatic zones with multifari-
ous benefits for mankind. Prosopis species are among the
most important multipurpose leguminous trees, and are
used for revegetation, agroforestry, apiculture, fodder
timber, fuel, shade, firewood as well as affecting soil
improvement and sand dune stabilization [1,2]. The ge-
nus Prosopis includes about 44 species that have been
described using morphological criteria and these are, in
turn, grouped into five sections. Further, these five sec-
tions include eight series. However, this classification
does not seem to be rigid [3,4]. At the morphological
level, leaves and flowers are more or less similar while in
case of fruit there is a development from straight to
curved and spirally coiled loments. There is also a
marked vegetative diversification in the presence or ab-
sence of spines, which also provides the foundation for a
sectional subdivision of the genus [3]. Similarly, varia-
tion of characters such as seed lipids [5], and mineral,
crude protein and structural carbohydrate [6], have also
been studied for distinguishing species, at least within a
section and series.
The accelerated and uncontrolled harvesting of this
important natural resource has led to land degradation
and desertification, as well as the loss of genetic diversity
within and acro ss the Prosopis population. Due to multi-
faceted utility of the species, the pressure for it and its
diverse products is mounting. There is a vital need for
thorough exploration and exploitation of all the available
natural variation in Prosopis species so as to manage the
available meager resources for sustainable utilization and
to establish appropriate reforestation programs for the
optimal exploitation of these natural resources [2,7].
Most of the approaches used to determine genetic di-
versity in this genus have involved isozyme analysis.
However, this method has methodological limitations
because of restrictions in the number of loci examined
and the possible tissue, developmental stage, or envi-
ronmental specificity of gene expression [8,9]. Yet there
were some concerted attempts to record natural genetic
variation in various populations of P. cineraria using
cytogenetical [10] and molecular markers [2]. Other than
the above reports of interspecies variations, not much is
known about intraspecific genetic variability in Prosopis
species, especially at the molecular level. Molecular

DNA Finger Printing of Prosopis cineraria and Prosopis juliflora Using ISSR and RAPD Techniques
528
techniques have been found to be more useful and accu-
rate for determination of both interspecies and intraspe-
cies genetic variation in plants. Sharma et al., [2] have
suggested that ISSR (Inter Simple Sequence Repeat)
markers are the best suited DNA markers for genetic
variation analysis in tree species like P. cineraria.
ISSR-PCR is a technique, which involves the use of
microsatellite sequences as primers in a polymerase
chain reaction to generate multilocus markers. ISSR
markers are highly polymorphic and are useful in studies
on genetic diversity, phylogeny, gene tagging, genome
mapping and evolutionary biology [11]. ISSR is a tech-
nique offering the simplicity of RAPDs with an increased
level of reliability. It involves PCR amplification of re-
gions between adjacent, inversely oriented microsatel-
lites using a single simple sequence repeat primer. The
use of the tandem repeat motifs of di-, tri-, or tetra nu-
cleotides that are abundant in all eukaryotic genomes
produces a high number of polymorphic fragments, es-
pecially in plants [12]. Since it is simple, fast, cost-ef-
fective, highly discriminant and highly reliable, in recent
studies, it is widely applied in plant genetic analyses [11,
13-16].
During the last decade, molecular markers such as
Random Amplified Polymorphic DNA (RAPD) have
been widely used to detect polymorphism [17], analyze
phylogenetic relations [18,19], identify cultivars [20,21],
discriminate between wild and cultivated species [2 1,22],
and detect agronomic traits [23,24]. The RAPD markers
in particular, have been successfully used to determine
intraspecies genetic diversity in tropical and se mitropical
forest plants. These include Cedrus [25] and Pinus [26,
27]. In contrast, fewer reports are available on the distri-
bution of forest trees in arid and semiarid regions. In the
Prosopis genus, recent RAPD studies have shown dif-
ferences in allele frequencies among species of section
Algarobia and it has identified species from South
America, Africa and Asia [9,28,29]. These markers have
been used to differentiate accession levels of the Pro-
sopis genus [30]. Randomly amplified polymorphic DNA
(RAPD) markers, in particular, have been successfully
employed for determination of intra-species genetic di-
versity in several plants. These include date palm [31],
papaya [32], poplars [33] and amaranths [34]. The RAPD
technique was also used for detecting inter- and intra-
species variation amongst Prosopis accessions and spe-
cies [35]. The present paper describes the RAPD and
ISSR profile variation between P. cineraria and P. juli-
flora as well as variation within species of Prosopis and
determination of the relative heterogeneity of the species.
2. Materials and Methods
Twelve samples, including six P. cineraria (Arabic name:
Ghaf) and six P. juliflora (Arabic name: Ghawiaf) were
chosen randomly from different locations in Qatar. The
frozen young leaf tissues of Prosopis were first cleaned
carefully with distilled water. Then, one gram of leaf
sample was cut into small pieces and ground into fine
powder using liquid nitrogen. The DNeasy Plant Maxi kit
protocol (QIAGEN) was used to extract DNA by fol-
lowing the manual instructions of the kit (DNeasy Plant
Handbook). The obtain ed DNA wa s quantif ied an d quail-
fied using a Nanodrop Spectrophotometer. For further
estimation of the DNA quantity 2 µl was loaded on
0.85% agarose gel at 100 Volts for thirty min. The gels
mixture were stained with ethidium bromide and visual-
ized under UV light.
Thirty ISSR and fifteen RAPD primers were custom
synthesized from Integrated DNA Technologies (IDT),
Inc. USA. These primers were screened using poly-
merase chain reaction (PCR) in a total reaction mixture
of 20 µl containing 2 µl (20 - 30 ng) of total genomic
DNA, 10µl of AmpliTaq Gold 360 Mastermix (Applied
Biosystems), 1 µl (5 pmol/µl) of primers each and 7 µl of
nuclease free water. Amplification was carried out in a
Veriti 96 Well Fast Thermal cycler (made by Applied
Biosystems) under the following conditions: initial de-
naturation 95˚C for 10 min, 35 cycles (denaturation 95˚C
for 30 s, annealing temperature depending on primer for
30 s, extension 72˚C for one min), final extension 72˚C
for 10 mi n.
The amplified DNA fragments, 5 µl and 2 µl of load-
ing dye (making a total volume of 7 µl), were loaded on
to the gel using the 1.5% agarose at 30 Volts for 180 min.
in 1X TAE buffer (30 mM), the gels were stained in
ethidium bromide and visualized on a UV transillumina-
tor and documented using gel documentation system Al-
phImager EC by Alpha View software V.3.0.0.0.
ISSR and RAPD bands were precisely measured by
the gel documentation system software and scored for
each genotype. Each reproducible polymorphic DNA
band at a particular position on the gel was treated as a
sepa rate character and scored as presen t (1) or absent ( 0)
to generate a binary data matrix. The data was then co m-
puted with the PowerMarker software V 3.0 [36] to de-
tect the major allele frequency, number of alleles, gene
diversity and polymorphism information content (PIC)
value (which is commonly used in genetics as a measure
of polymorphism) for a marker locus used in linkage.
The phylogenetic relationship among the genotypes was
drawn by Past software V 1.91 [37] on the basis of
Hamming similarity index with 100 bootstraps.
3. Results and Discussion
Among thirty ISSR and fifteen RAPD primers tested for
their ability to generate bands patterns in Prosopis geno-

DNA Finger Printing of Prosopis cineraria and Prosopis juliflora Using ISSR and RAPD Techniques
529
types, twenty nine ISSR and seven RAPD primers suc-
cessfully produced clear bands in most of the studied
genotypes. So far, one ISSR primer namely BT26
(GGAT)4, and eight RAPD primers (A12, D10, OPBO4,
OPA12, OPA19, OPC-06, OPC-10 and OPC-15) did not
amplify clear bands in our genetic materials, even when
different PCR conditions were used.
The twenty nine ISSR primers used in this study pro-
vided a total of 109 bands, 108 of which were polymor-
phic (99% polymorphism). The percentage of polymor-
phism produced by each primer differed from one primer
to another. The maximum value of polymorphism was
100%, produced by all primers except BT25, which pro-
duced the minimum value of polymorphism, 50%. There
was an average polymorphism of 99.1% across all the
genotypes.
A total number of 109 amplified DNA bands were gen-
erated across the studied genotypes with average of 3.76
bands per primer. The ISSR profiles of the amplified
products of each primer are shown in (Table 1). A maxi-
mum number of eight bands were amplified with primer
BT10 and a minimum of one band with primer BT3. The
number of monomorphic bands was primer dependent
and only one of these bands was generated by primer
BT25. The Hamming genetic similarity coefficient, rec-
ognized the two studied Prosopis species (Figure 1).
Table 1. Code and sequence of the thirty DNA ISSR primers used for identifying the Prosopis species and types of the ampli-
fied DNA bands.
ISSR No. of bands
Primers Sequence Total Polymorphic Monomorphic % Polymorphic
BT1 (AC) 8T 6 6 0 100
BT2 (ACC) 6 3 3 0 100
BT3 (ACTG) 4 1 1 0 100
BT4 (AG) 10C 3 3 0 1 00
BT5 (AG) 10T 7 7 0 100
BT6 (AG) 8T 5 5 0 100
BT7 (AGG) 6 6 6 0 100
BT8 (ATG) 6 4 4 0 100
BT9 (CA) 6AC 7 7 0 100
BT10 (CA) 6GG 8 8 0 100
BT11 (CA) 6GT 3 3 0 100
BT12 (CA) 8A 4 4 0 100
BT13 (CAC) 3GC 2 2 0 100
BT14 (CT) 10A 4 4 0 100
BT15 (CT) 10G 4 4 0 100
BT16 (CT) 10T 4 4 0 100
BT17 (CT) 8AC 2 2 0 100
BT18 (CT) 8GC 3 3 0 100
BT19 (CT) 8TG 2 2 0 100
BT20 (CTC) 3GC 3 3 0 1 00
BT21 (GA) 6CC 3 3 0 100
BT22 (GA) 6GG 4 4 0 1 00
BT23 (GA) 8C 3 3 0 100
BT24 (GACA) 4 3 3 0 100
BT25 (GAG) 3GC 2 1 1 50
BT27 (GT) 6CC 2 2 0 100
BT28 (GT) 6GG 5 5 0 100
BT29 (G TG) 3GC 2 2 0 100
BT30 (TC) 10C 4 4 0 100
Total 109 108 1 99
Average 3.76

DNA Finger Printing of Prosopis cineraria and Prosopis juliflora Using ISSR and RAPD Techniques
530
Figure 1. Dendrogram of Qatari Prosopis species based on ISSR technique according to hamming similarity coefficient.
The highest similarity value was 0.02 which was re-
corded between P. juliflora 5 and P. juliflo ra 6. The next
highest similarity value was 0.03 between P. cineraria 1
and P. cineraria 3 and between P. cineraria 3 and P.
cineraria 6. The lowest similarity value was 0.82 be-
tween P. cineraria 1 and P. juliflora 5 and 6, P. cineraria
3 and P. juliflora 5 and 6 and between P. cineraria 6 and
P. juliflora 5 and 6. The next lowest similarity value was
0.81 between P. cineraria 4 and P. juliflora 5 and 6.
Similarity coefficient matrices were used to generate a
dendrogram of Prosopis genotypes based on Hamming
analysis (Figure 1). The analysis divided the twelve
genotypes into two distinct clusters. The first cluster in-
cluded P. cineraria, while the second cluster contained P.
juliflora genotypes.
The identification and characterization of species be-
come possible through fingerprinting of each species
since DNA is a source of informative polymorphism [38].
Based on twenty nine ISSR markers twenty one bands
are capable of to distinguishing between P. cineraria and
P. juliflora. Those bands were 2, 1, 1, 6, 2, 2, 1, 1, 1, 1, 2
and 1 represented in BT1, BT4, BT5, BT9, BT10, BT11,
BT12, BT14, BT15, BT18, BT23 and BT30 respectively,
whereas of the seven RAPD markers, only three bands
are distinguishable between P. cineraria and P. juliflora.
However those bands were only one band each in DO5,
OPC02 and D20. The distinguishable bands in all species
appe ared in P. cineraria but not in P. juliflora.
Consequently, techniques of molecular genetic mark-
ers have an important potential for the detection of ge-
netic differences among species [39].
At the same time, it is necessary to develop better
methods of characterization and evaluation of germplasm
collections, to improve strategies for conservation and
collection of germplasm, and to increase the utilization of
plant genetic resource s.
A detailed account of molecular phylogeny and the
diversification history of thirty species of Prosopis were
previous ly done by Catalano et al. [40]. It suggested that
Prosopis is not a natural group, but one that adopted an
ancient occupation of arid environments. This study in-
dicated that old world species of Prosopis are not closely

DNA Finger Printing of Prosopis cineraria and Prosopis juliflora Using ISSR and RAPD Techniques531
related with the new world (American) species. The pre-
sent investigation of P. cineraria revealed that there is a
greater genetic variation of 88 .2% within the populations,
whereas the variance among populations was only 11.8%
only. This is in support of observations by Sharma et al.
(2010) [2], who reported high natural genetic variation at
the DNA level in thirty accessions of P. cineraria col-
lected from different districts of Rajasthan.
During recent years, different studies have supported
the hypothesis that dry-adapted taxa in different regions
of the world diverged concomitantly with the expansion
of arid environments. That is the view of Phylica [41],
Ruchoideae [42], Tiquillia [43] and Agave [44] in North
America, and Rheum [45] in East Asia.
Genetic differentiation depends on the scale of envi-
ronmental heterogeneity and the balance of selection and
higher gene flow especially amongst trees [46]. This fact
supports our study that Prosopis is not a natural group,
but a species that in ancient times, adopted and occupied
arid environments in which its populations were adapt-
able, and where they had the highest level of fitness The
study of sequence variation also provides means to detect
loci responsible for local adaptation [47]. When popula-
tions become locally adapted to contrasting environments,
alleles entering a new population may be eliminated by
selection, so that the gene flow is partly restricted. The
stronger the selection, the more rapid ly immigrant alleles
of lower fitness will be eliminated from the population
thereby reducing effective migration rates and increasing
the time to coalescence [48].
The seven RAPD primers used in this study provided a
total of nineteen bands; all of them were polymorphic
(100% polymorphism). The total number of nineteen
amplified DNA bands was generated across the studied
genotypes with an average of 2.7 bands per primer. The
RAPD profiles of the amplified products of each primer
are shown in Table 2 . A maximum number of five bands
were amplified with primer OPC11 and a minimum of
two bands were amplified with primers A13, D20, A10
and D07.
Using the Hamming genetic similarity coefficient,
recognized the Prosopis species, the highest similarity
value was 0.0, which is identically recorded between (P.
cineraria 1 and P. cineraria 4) and (P. juliflora 4, 5 and
6 were identical as will), the next highe st similarity value
was 0.05 between (P. cineraria 1 and P. cineraria 3)
which was the same result with ISSR markers and (P.
cineraria 3 and P. cineraria 4) while the lowest similar-
ity value was 0.95 between (P. cineraria 1 and P. juli-
flora 4, 5 and 6), (P. cineraria 4 and P. juliflora 4, 5 and
6), the next lowest similarity value was 0.89 between (P.
cineraria 3 and P. juliflora 4, 5 and 6) .
Similarity coefficient matrices were used to generate a
dendrogram of Prosopis genotypes based on Hamming
analysis (Figure 2), the analysis divided the twelve
genotypes into two distinct clusters, the first cluster in-
cludes P. cineraria, while the second cluster contains P.
juliflora genotypes.
The molecular markers obtained by the ISSR tech-
nique revealed a remarkable molecular discrimination
among Prosopis species more than RAPD technique. The
phylogenetic analysis on the basis of ISSR (Figure 1)
discriminate among P jouliflora 4, 5 and 6, while RAPD
markers u sed (Figure 2) cannot revealed almost the same
pattern. Also ISSR technique (Figure 1) discriminate
among P cineraria 1 and 4, while RAPD markers (Fig-
ure 2) did not. However, Ajibade et al. [49] and Galvan
et al. [15] concluded that ISSR would be a better tool
than RAPD for phylogenetic studies. Nagaoka and Ogi-
hara [50] have also reported that the ISSR primers pro-
duced several times more information than RAPD mark-
ers in wheat.
Table 2. Code and sequence of the seven DNA RAPD primers used for identifying the Prosopis species and types of the am-
plified DNA bands.
RPAD No. of bands
Primers Sequence Total Polymorphic Monomorphic % Polymorphic
DO5* TGAGCGGACA 3 3 0 100%
OPC11 AAAGCTGCGG 5 5 0 100%
OPC02* GTGAGGCGTC 3 3 0 100%
A13 CAGCACCCAC 2 2 0 100%
D20* ACCCGGTCAC 2 2 0 100%
A10 GTGATCGCAG 2 2 0 100%
DO7 TTGGCACGGG 2 2 0 100%
Total 19 19 0
Average 2.714 100%
*Band distinguish bet w e en species.

DNA Finger Printing of Prosopis cineraria and Prosopis juliflora Using ISSR and RAPD Techniques
532
Figure 2. Dendrogram of Qatari Prosopis species based on RAPD technique according to hamming similarity coefficient.
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