Genetics Diversity of Some Egyptian Durum Wheat Cultivars

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Genetic Diversity of Some Egyptian Durum Wheat Cultivars
Fouda, A. H.
*1
; Gad, Khaled. I. M.
2
; Diab, A. A.
1,3
; Safwat, G.
1,4
and Hussein, M. H
5
.
1
Faculty of Biotechnology, October University for Modern Sciences and Arts, (MSA), Egypt
2
Wheat Department, Field Crops Research Institute (ARC), Egypt
3
Agricultural Genetic Engineering Research Institute (AGERI), Egypt
4
Horticulture Research Institute, Agriculture Research Centre, Egypt
5
Department of Genetics, Faculty of Agriculture, Cairo University, Egypt
*monahuss@yahoo.com
Abstract: The objective of this investigation was to assess the genetic diversity among three Egyptian durum wheat i.e.
Beni Suif 4, Beni Suif 5 and Beni Suif 6 and one bread wheat i.e. Sids 12 cultivars using sodium dodecyle sulfate
polyacrylamide gel electrophoresis (SDS-PAGE) and randomly amplified polymorphic (RAPD) markers. Protein
electrophoreses showed that Beni Suif 4 was characterized by the absence of band-3 with 41.56 kDa. RAPD analysis
showed that the number of polymorphic amplicons was 56 out of a total of 93 amplicons, thus revealing a level of 60.0
% polymorphism. The highest genetic similarity revealed by RAPD analysis (95.0%) was between Beni Suif 5 and
Beni suif 6. While, lowest similarity (65 %) was found between Beni Suif 4 and Beni Suif 5. The dendrogram
separated Sids 12 from all the other genotypes, thus demonstrating the distinctiveness of the genetic background of
this genotype from all the other genotypes. The three genotypes constituted a subcluster divided into two groups,
one group composed of Beni Suif 5 and Beni Suif 6 , while the second group comprised Beni Suif 4.
[Fouda, A. H.; Gad, Khaled. I. M.; Diab, A. A.; Safwat, G. and Hussein, M. H. Genetic Diversity of Some Egyptian
Durum Wheat Cultivars. Journal of American Science 2011; 7(7):214-221].(ISSN: 1545-1003).
http://www.americanscience.org
.
Key words: Durum Wheat, RAPD, Dendrogram, Dice coefficient, Polymorphism, Turgidum.
1. Introduction:
Morphological characters have been used to
identify plant species, families and varsities.
However, they have many disadvantages, since they
are influenced by the environment and scoring is a
time-consuming process. The use of genetic
molecular markers (protein and DNA-based) have
become widely accepted valuable tools (Cooke,
1999). SDS-PAGE is considered as a low cost,
reproducible and rapid approach (Laemmli, 1970). In
the last decade, molecular markers such as RFLP,
RAPD, ISSR, AFLP have been used to assess genetic
variation at the DNA level, allowing an estimation of
degree of relatedness between individuals without the
influence of environmental variation (Gupta et al.,
1999). DNA RAPD is a useful method for generating
molecular markers (Welsh and McClelland, 1990)
that can be used to construct linkage maps, to identify
varieties (He et al.1992) and to assess genetic
diversity (Koller et al., 1993). It is characterized by
its low technical input and small quantity of plant
DNA needed for the analysis (Hernandez et al., 1999
and Manabe et al., 1999). Also, RAPD based
fingerprinting was used successfully in wheat to
assess genetic diversity (He et al.,1992, Dhaliwal et
al.,1993; Cao et al.,1999 Kudriavtsev et al.,2003;
Munshi et al.,2003 ; Maric et al., 2004 and Abd-El-
Haleem et al., 2009). The aim of this work were to:
(1) characterize three durum and one bread Egyptian
wheat cultivars at the DNA level using RAPD
markers and at protein level using SDS-PAGE and (2)
determine the genetic relationships among these
genotypes.
2. Materials and Methods
Germplasm material
In the present study three Egyptian durum
wheat (Triticum turgidum L.) cultivars (Beni Suif 4,
Beni Suif 5, Beni Suif 6) and one bread wheat ( T.
aestivum) cultivar (Sids 12) provided by Wheat
Research Dept. of the ARC, Egypt were used.
SDS-PAGE
Sodium dodecylsulfate polyacrylamide gel
electrophoresis (SDS-PAGE) was used to study the
banding patterns of the studied genotypes, Protein
fractionation was performed on vertical slab (16.5 cm
x 18.5 cm x 0.2 cm) Hoefer E600, Amersham
Pharmacia biotech. According to the method of
Laemmli (1970) as modified by Studier (1973).
Extraction and purification of genomic DNA
A modified CTAB (hexadecyl trimethyl
ammonium bromide) procedure based on the protocol
of Porebski et al. (1997) was adopted for obtaining
good quality total genomic DNA.
Half gram of fresh leaves from each
genotype were collected from one week old seedlings

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germinated from seeds of each genotype and quickly
frozen in liquid nitrogen and then ground using
mortar and pestle. Five ml of CTAB extraction buffer
(60ºC), 50 mg PVP (polyvinyl pyrolidone) and 15 μl
β-mercaptoethanol (0.3%) were added to leaf powder.
The tubes were mixed by inversion and incubated at
65ºC for one hour. Then, 6 ml of chloroform :
isoamyl alcohol (24:1) was added and contents were
mixed by inversion to form an emulsion. The tubes
were centrifuged at 5000 rpm for 20 min at room
temperature. The top aqueous layer was further
centrifuged at 5000 rpm after addition of 6 ml of
chloroform: isoamyl (24:1). Half-volume of 5 M
NaCl and two volumes of cold isopropanol were
added to the supernatant and mixed well. The tubes
were incubated at –20ºC overnight, then centrifuged
at 8000 rpm for 15 min. The supernatant was
discarded, the pellet washed with 70% cold ethanol,
and dried in speed vacuum for 10 min. The pellet was
dissolved in 300 μl TE buffer (pH 8.0) overnight at 4-
6ºC, then transferred to 1.5 ml epindorfe tube. To
remove RNA contamination, 4 μl (10 mg/ml) RNase
A (Sigma Co., USA) were added to the DNA solution
and incubated at 37ºC for 2 hours. The extracted
DNA was deproteinized by adding 4 μl (1mg/ml)
proteinase K (Sigma Co., USA) and incubating at
37ºC for 2 hours. Three hundred μl of Tris-saturated
phenol-chloroform were added, and mixed by
inversion. Tubes were centrifuged at 14000 rpm for
15 min in a microfuge (Eppendorf, USA). The upper
layer was transferred to new tubes using wide bore
pipette tip and 150 μl of TE buffer was added to the
phenol phase, mixed and spun for 10 min. Then the
upper layer containing the DNA was removed and
added to the sample. DNA was precipitated overnight
at –20ºC using 0.1 volume 3 M sodium acetate (pH
8.0) and two volumes of chilled absolute ethanol. The
samples were centrifuged at 14000 rpm at 4ºC for 15
min. The DNA was washed with 70 % ethanol,
briefly air-dried and re-dissolved in TE buffer.
Estimation of DNA concentration
DNA concentration was determined by
diluting the DNA 1:5 in dH
2
O. The DNA samples
were electrophoresed in 0.7% agarose gel against
10ug of a DNA size marker (Lambda DNA digested
with HindIII and Phi x 174 DNA digested with
HaeIII). This marker covers a range of DNA
fragments size between 23130bp and 310bp, and a
range of concentrations between 95 ng and 11 ng.
Thus, estimation of the DNA concentration in a given
sample was achieved by comparing the degree of
fluorescence of the unknown DNA band with the
different bands in the DNA size marker.
RAPD analysis
A set of nine random 10-mer arbitrary
primers (Table 1) was used in the detection of
polymorphism among the four wheat genotypes.
These primers were synthesized on an ABI 392
DNA/RNA synthesizer (Applied Biosystems) at
AGERI. RAPD assay was preformed as described by
Williams et al. (1990) with some modifications. The
amplifications reactions were carried out in a volume
of 25 μl containing 20ng genomic DNA, 25 pmoles
primer, 2mM dNTPs, 2mM MgCl
2
and 2 U Taq
polymerase (Fermentas) with, 1 x PCR buffer.
Table 1. Sequence of the nine ten-decamer arbitrary primers used in RAPD analysis to detect polymorphism
among four wheat genotypes.
No. Name Sequence
1 OP-R14 5´ TCCGCTCTGG 3`
2 OP-R17 5´ AGGGAACGAG 3`
3 OP-R20 5´ GGACCCTTAC 3`
4 OP-F11 5´ ACGGATCCTG 3`
5 OP-F14 5´ GGTGATCAGG 3`
6 OP-F15 5´ CCGAATTCCC 3`
7 OP-F16 5´ GGGAATTCGG 3`
8 OP-F18 5´ GGGATATCGG 3`
9 OP-F19 5` CCAAGCTTCC 3`
Thermocyling profile and detection of the PCR
products
PCR amplification was performed in a
Perkin-Elmer/GeneAmp® PCR System 9700 (PE
Applied Biosystems) programmed to fulfill 40 cycles
after an initial denaturation cycle for 5 min at 94ºC.
Each cycle consisted of a denaturation step at 94ºC
for 1 min, an annealing step at 36ºC for 1 min and an
elongation step at 72ºC for 1.5 min. The primer

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216
extension segment was extended to 7 min at 72ºC in
the final cycle.
The amplification products were resolved by
electrophoresis in a 1.5% agarose gel containing
ethidium bromide (0.5ug/ml) in 1X TBE buffer at 95
volts. PCR products were visualized on UV light and
photographed using a Polaroid camera. Amplified
products were visually examined and the presence or
absence of each size class was scored as 1 or 0,
respectively.
RAPD data Analysis
The banding patterns generated by RAPD-
PCR markers analyses were compared to determine
the genetic relatedness of the genotypes. Clear and
distinct amplification products were scored as ‘1’ for
presence and ‘0’ for absence of bands. Bands of the
same mobility were scored as identical.
The genetic similarity coefficient (GS)
between two genotypes was estimated according to
Dice coefficient (Sneath and Sokal, 1973).
Dice formula: GSij = 2a/(2a+b+c)
Where GSij is the measure of genetic similarity
between individuals i and j, a is the number of bands
shared by i and j, b is the number of bands present in
i and absent in j, and c is the number of bands present
in j and absent in i.The similarity matrix was used in
the cluster analysis. The cluster analysis was
employed to organize the observed data into
meaningful structures to develop taxonomies. At the
first step, when each accession represents its own
cluster, the distances between these accessions are
defined by the chosen distance measure (Dice
coefficient). However, once several accessions have
been linked together, the distance between two
clusters is calculated as the average distance between
all pairs of accessions in the two different clusters.
This method is called unweighted pair group method
using arithmetic average (UPGMA) according to
(Sneath and Sokal, 1973).
3. Results and Discussion
Genetic diversity based on SDS- PAGE
SDS banding patterns was used to
fingerprint three durum and one bread wheat cultivars.
Dry wheat seeds were ground into soft flour and
water soluble protein fraction was extracted. The
total number of bands ranged from eight to seven
bands (Figure 1). Sex bands were monomorphic
while the others were polymorphic. Beni Suif 4 was
characterized by the absence of band -3 with 41.56
kDa. Because of the very low level of a
polymorphism a dendrogram for genetic distance and
similarity matrix based on SDS-PAGE could not be
performed.
RAPD analysis for genetic diversity among wheat
genotypes
Different methods are available for analysis
of genetic diversity among germplasm accessions.
These methods have relied on morphological,
agronomic and biochemical data and recently on
DNA-based marker data that allow more reliable
differentiation of genotypes. In the present study,
twenty ten-mer arbitrary primers were initially
screened for PCR amplification of the genomic DNA
for the four wheat genotypes. Only nine primers
generated reproducible and easily scorable RAPD
profiles.
The number of amplified fragments from
the genomic DNA of each of the five wheat
genotypes generated by the different primers is
presented in Table (2). Each of the nine primers
produced multiple band profiles with the five wheat
genotypes. The highest number of amplicons (14
amplicons) was generated by the primer OPR17 in
the genomic DNA of the genotype Beni Suif 4, while
the primer OPF15 exhibited the only 2 amplicons,
which were monomorphic across the four wheat
genotypes.
As shown in Table (3) the total number of
DNA fragments amplified by the nine primers was 93
with an average of 10.33 amplicons per primer. The
number of polymorphic amplicons ranged from 0 to
15. Primer OPR16 amplified the highest number of
polymorphic amplicons, while, the primer OPR14
revealed a total of 6 amplicons which were all
monomorphic across the four wheat genotypes.
Figure 1. SDS- PAGE for four wheat
genotypes

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Table 2. Number of amplified DNA fragments produced by each RAPD primer for the four wheat genotypes.
Primer Beni .4 Beni.5 Beni.6 Sids12 Total Mean
OPR14
OPR17
OPR20
OPF11
OPF14
OPF15
OPF16
OPF18
OPF19
8
14
8
6
3
2
9
5
3
11
11
9
4
3
3
4
3
3
12
15
9
4
3
3
9
4
3
12
12
8
8
4
4
7
5
3
43
52
34
22
13
14
29
17
12
10.7
13
8.5
5.5
3.3
3
7.25
4.25
3
Total
Mean
58
6.4
51
5.7
62
6.9
63
7
234
26
58.6
6.5
Therefore, the different primers expressed
different levels of polymorphism, ranging from 0.0%
with primer OPF19 to 78.9 % with primer OPR16.
The total number of polymorphic bands revealed by
the nine primers was 56 and the average number of
polymorphic fragments/ primer was 6.2. Thus, the
average level of polymorphism was 60 %. The size of
amplified fragments varied with the different primers,
ranging from 250 to 4000 bp (Figures 2 and 3). In
this respect, Joshi and Nguyen (1993) investigated
the genetic diversity among 15 wheat varieties (T.
aestivum) using RAPD analysis. Out of 109 amplified
DNA fragments, 41 were polymorphic, representing a
level of polymorphism of 65%. Perenzin et al. (1997)
utilized 87 RAPD primers to assay the genetic
diversity among wheat genotypes. They reported that
304 polymorphic bands were generated with an
average of 3.49 polymorphic amplicon / primer.
Table 3. Total number of amplicons, number of monomorphic and polymorphic amplicons and percentage of
polymorphism, as revealed by RAPD primers
Primer Total # of
amplicons
#of mono
amplicons
# of Poly
amplicons
Polymorphism (%)
OPR14
OPR17
OPR20
OPF11
OPF14
OPF15
OPF16
OPF18
OPF19
15
19
15
11
6
5
12
6
4
6
4
4
4
5
3
4
3
4
9
15
11
7
1
2
8
3
0
60.0
78.9
73.3
63.6
16.6
40.0
66.6
50.0
0.00
Total
Average
93
10.33
37
4.11
56
6.2
60.0
Sun et al. (1998) used 32 arbitrary primers for
RAPD analysis of 46 wheat genotypes, among which
26 primers (81.3%) revealed polymorphism. A total
of 279 amplicons were generated and 182 (65.2%)
were polymorphic.
The number of polymorphic amplicons ranged
from 2 to 20 with an average of 7 polymorphic
amplicons per primer. Zheng et al. (2001) used 55
arbitrary primers in the RAPD analysis of 40 wheat
cultivars. Out of 183 amplified fragments, 93
amplicons 50.8% were polymorphic, this represented
an average of 1.7 polymorphic amplicons per primer.
Moreover, Cao et al. (2002) screened 235 random
primers against four wheat cultivars to detect RAPD
polymorphism. Only, 31 (13.20%) primers produced
polymorphism these 31 primers generated a total of
214 reproducible amplified fragments when used
with 29 common wheat cultivars. The number of
amplified fragments produced by each primer varied
from 3 to 12 with an average of 6.9 and an average of
3.10 polymorphic bands per primer. Al-Naggar et al.
(2004) used 17 arbitrary primers for RAPD analysis
of six bread wheat genotypes. Twelve primers
(70.60%) generated polymorphic profiles. The total
number of amplicons was 98, of which 34 (34.69%)
showed polymorphism. Also, Wjhani (2004) studied
the genetic variability among 14 wheat accessions
using 39 RAPD primers. The total number of
amplicons was 117, including 108 polymorphic
amplicons. This represented a level of polymorphism
of 92.3 % and an average number of 9 polymorphic
bands per primer.

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Figure 2. RAPD profile of the three bread wheat genotypes (Beni Suif 4, Beni Suif 5 and Beni Suif 6) and bread wheat genotype
Sids 12 amplified with RAPD primers,OPR14,OPR17, OPR20, OPF11and OPF14 : MW : 100 bp ladder.
Fig 3. RAPD profile of the three bread wheat genotypes (Beni Suif 4, Beni Suif 5 and Beni Suif 6) and bread wheat genotype
Sids 12 amplified with RAPD primers,OPR14,OPR17, OPR20, OPF11and OPF14 : MW : 100 bp ladder.

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The results of the present study are in good
agreement with those reported in the literature, and
confirm that polymorphism is a general phenomenon
in in good agreement with those reported in the
literature, and confirm that polymorphism is a general
phenomenon in wheat although it is a self- fertilizing
species.
Genetic relationships among the wheat genotypes
Knowledge of the genetic relationships
among genotypes has several important applications
in plant breeding programs and plant improvement. It
permits the organization of germplasm, including
elite lines and provides for more efficient parental
selection.
To determine the genetic relationships
among the four wheat genotypes, the scoring data (1
for presence and 0 for absence) resulting from the
nine tested RAPD primers were used to compute the
similarity matrices according to Dice coefficient
(Sneath and Sokal 1973). These similarity matrices
were then used in the cluster analysis to generate a
dendrogram using the UPGMA method. As shown in
Table (4) the genetic similarity among the four wheat
genotypes ranged from 68.0 to 95.0%.
Table (4). Genetic similarity (GS) matrices among the four wheat genotypes as omputed according to Dice
coefficient from RAPDs.
Beni S.4 Beni S.5 Beni S.6 Sids 12
Beni S.4
Beni S.5 93.1
Beni S.6 92.6 95.0
Sids 12 68.0 65.0 70.0
The highest genetic similarity revealed by
RAPD analysis (95.0%) was between Beni Suif 5 and
Beni suif 6 genotypes and followed by (93.1 %)
between Beni Suif 4 and Beni suif 6, while the lowest
similarity (65 %) was between Beni Suif 4 and Beni
Suif 5. This reveals a great concordance between the
data deduced from the RAPD analysis and the
pedigree of these genotypes.
Cluster analysis as revealed by RAPDs
The Dice RAPD–based coefficients of
genetic similarity among the four wheat genotypes
were employed to develop a dendrogram using the
UPGMA method (Fig.4).The dendrogram separated
Sids 12 from all the other genotypes, thus
demonstrating the distinctiveness of the genetic
background of this genotype from all the other
genotypes. The three genotypes constituted a
subcluster divided into two groups, one group
composed of Beni Suif 5 and Beni Suif 6, while the
second group comprised Beni Suif 4. Thus, the
dendrogram deduced from the RAPD data
corresponded well with the pedigree of the studied
wheat genotypes.
The results of the present study revealed,
therefore that RAPD analysis is an effective tool for
detecting polymorphism, distinguishing between
wheat genotypes and assessing their phylogenetic
relationships. These results agree with Joshi and
Nguyen (1993) who found that analysis of the genetic
relationships among wheat varieties could distinguish
most of the spring and winter wheat cultivars into
different clusters in the dendrogram. Sivolap et al.
(1999) reported that RAPD analysis proved to be one
of the most powerful methods of discriminating
cultivars. The dendrograms based on RAPD markers
most closely conform to the pedigree data. Cao et al.
(2000) used RAPD marker to assess phylogenetic
relationships between 15 wheat accessions. Clusters
analysis classified these accessions into five groups
in agreement with morphological classification. Sun
et al. (2003) found that the dendrogram prepared on
the basis of RAPD data corresponded well with the
pedigree of two groups of wheat genotypes. Shehata
et al (2004) reported that SDS-PAG and RAPD-PCR
were successfully used to construct dendrograms to
arate the wheat cultivars into two main groups.
Corresponding author
Fouda, A. H.
Faculty of Biotechnology, October University for
Modern Sciences and Arts, (MSA), Egypt
monahuss@yahoo.com

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