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Advances in Environmental Biology, 11(5) May 2017, Pages: 95-109
AENSI Journals
Advances in Environmental Biology
ISSN-1995-0756 EISSN-1998-1066
Journal home page: http://www.aensiweb.com/AEB/
Copyright © 2017 by authors and Copyright, American-Eurasian Network for Scientific Information (AENSI Publication).
Molecular characterization of genetic variation in algeria durum wheat accessions (Triticum durum desf.) Using rapd and issr markers
1Belattar. R, 2Chaib. G, 2Boudour.L, 2Bouchteb K.
1Department of Biology and Plant Ecology, Faculty of Natural Sciences and Life, University Ferhat Abbas Setif, Algeria..
2Department of Biology and Plant Ecology, Faculty of Natural Sciences and Life, Brothers University Mentouri Constantine 1 Algeria.
Address For Correspondence: Belattar. R, Department of Biology and Plant Ecology, Faculty of Natural Sciences and Life, University Ferhat Abbas Setif, Algeria. This work is licensed under the Creative Commons Attribution International License (CC BY). http://creativecommons.org/licenses/by/4.0/
Received 8 January 2017; Accepted 28 April 2017; Available online 24 May 2017
ABSTRACT Durum wheat (Triticum turgidum L. var. durum) is an important crop with high protein content and superior cooking quality which used for
Pasta production. To estimate genetic the relationships of 50 Algerian durum wheat accessions, RAPD and ISSR analysis was performed
with 10 primers for both RAPD and ISSR. The most discriminating primers were ISSR M9, ISSR F2, ISSRM1, RAPDL2, RAPD B10, RAPD b5, RAPD B13 CRAPD1, CRAPD2, and RAPDb3 which showed the highest values of the polymorphism percentage. in RAPD
analyses, 35 out of 38 bands (92%) were polymorphic. The different primers produce a number of amplifications ranging from 2 to 7, the
size of the amplified fragments ranging from 100 to 1600 bp. in ISSR analyses, a total of 32 alleles were detected, among which 22 alleles (68,7%) were polymorphic. Cluster analyses indicated that both RAPD and ISSR markers could distinguish all 50 wheat accessions.
although the analysis of RAPD and ISSR markers could successfully be used to investigate the genetic diversity of the wheat accessions.
The classification of the two markers RAPD and ISSR of the studied 50 genotypes made it possible to distinguish many groups. These results indicate a high level of polymorphism in an accession of durum wheat . We found that the genotypes were grouped according to their
botanical varieties and, in some cases, their namesake. In addition, ISSR analyses are more specifi than RAPD analyses,due to the longer
SSR-based primers with higher primer annealing temperature, which enable higher-stringency and greater band reproducibility amplifiations. The present study has provided the genetic data that diagnose the level of polymorphisms between 50 genotypes of durum
wheats.
KEYWORDS: Durum wheat accessions, genetic diversity, ISSR, RAPD
INTRODUCTION
Modern wheat cultivars usually refer to two species: hexaploid bread wheat, Triticum aestivumand
tetraploid, hard or durum-type wheat, T. durum [26]. Durum wheat is traditionally grown around the
Mediterranean Sea and it is the most commoncultivated form of allotetraploid wheat. Currently,more than half
of the durum wheat is still grown in the Mediterranean basin, mainly in Italy, Spain, France, Greece, West
Asian, and North African countries [27].
In the last 50 years, wheat persisted as one of the most used and stable food grain cereals with 1% yearly
gain due to the implementation of advanced agricultural techniques and involvement of productive cultivars.
However, to cover up the population needs, it is necessary to raise the annual productivity gains to 2.5% up to
2025[12].
96 Belattar. R et al, 2017
Advances in Environmental Biology, 11(5) May 2017, Pages: 95-109
The availability of the information on the genetic variation within samples and the differentiation between
samples plays a significant role in the formulation of appropriate management strategies for conservation of
genetic resources .
Molecular markers are powerful tools in the study of genetic diversity, genotype description and genetic
structure of wheat populations [1].
Random Amplified Polymorphic DNA (RAPD) molecular marker systems used in this study are
significantly quicker and simpler in comparison to few other molecular strategies, making the technique famous
for evaluating genetic polymorphism in wheat species [11,25]. RAPD has several advantages, such as low cost,
and the use of small amount of plant material. RAPD’s were proved to be useful as genetic markers in the case
of self-pollinating species with a relatively low level of intraspecific polymorphism, such as hexaploidy wheat.
Due to the distinguishing features like abundance, good reproducibility, high polymorphism, vastly informative
and quick to use, Inter Simple Sequence Repeats (ISSR) markers can be supportive to RAPD markers. ISSR are
a new kind of molecular markers involving PCR amplification of DNA by a single primer 16-18 bp. long
composed of a repeated sequence anchored at the 3' or 5' end by 2-4 arbitrary nucleotides. [21],revealed that the
ISSR markers provided sufficient polymorphism and reproducible fingerprinting profiles for evaluating genetic
diversity of wheat genotypes. Similar to RAPD primers, no aforementioned sequence information is necessitated
for the genetic studies. High annealing temperature contributes significantly towards the elevated reproducibility
among ISSR markers as compared to RAPD markers. A number of researchers all over the world estimated
genetic diversity/genetic similarity among bread wheat varieties using ISSR markers [8,21,17].
Molecular variation evaluated in their study in combination with agronomic and morphological characters
of wheat can be useful in traditional and molecular breeding programs.
Our objectives in the present study were: to determine the genetic diversity in durum wheat genotypes
using ISSR and RAPD markers, and to assess the suitability of the ISSR and RAPD markers for detecting
molecular variation. in addition, we aim to report the usefulness of RAPD and ISSR for the assessment of
genetic diversity and relationships among wheat accessions.
MATERIALS AND METHODS
Plant materials:
The plant material is composed of Algerian durum wheat accession, containing 50 genotypes belonging to
reichenbachi and leucomelan varieties [7]. This collection comes from different regions of Algeria and stored at
the ITGC "El Khroub Constantine" (Tab.1).
Table 1: Characteristics of the varieties and number of individuals per variety [7].
variety Characteristics Number of
genotypes
Ear glume Seed Straw precocity
White, compact, glabrous
or smooth, pyramidal, triangular flattened
White,
elongated
Shaded bright,
big, elongated
and hunchbacked
Hollow or
half-hollow
to ¾ hollow.
Half early
25
White, glabrous, compact,
long spindle-shaped
pyramidal.
White, weakly
diverging black
beards.
Dark shaded
(red), medium-
sized and
hunchbacked.
Hollow or
half-hollow
to ¾
hollow..
early
25
Methodology adopted for DNA marker analysis:
DNA Isolation:
Seed samples grown in Petri dishes and then transferred to hydroponic system to get the fresh seedling leaf
tissue for DNA extraction. The DNA Isolation was carried out following CTAB method of [13], with
modifications. Approximately 0.1 to 0.2 g of fresh seedling leaf tissue was crushed in mortar and pestle using
liquid nitrogen to obtain fine powder. The powder was added to 750 µl CTAB and 7.5 µl of β-mercaptoethanol
leu
com
ela
n
reic
hen
ba
ch
i
97 Belattar. R et al, 2017
Advances in Environmental Biology, 11(5) May 2017, Pages: 95-109
present in Eppendorf tube. Later, 10 µl RNase A was added to the mixture and kept on a hot block at 65⁰C for
incubation for 30 minutes. Following incubation, 750 µl of Phenol: chloroform: isoamyl alcohol reagent
(25:24:1) was supplemented to the mixture containing tissue sample further centrifuged at 7000 rpm for 5
minutes at 25⁰C and supernatant was collected in a fresh vial. To the remaining mixture, 300µl CTAB with β-
mercaptoethanol was included and again centrifuged at 15000 rpm for 5 minutes at 25⁰C. The supernatant
obtained was mixed with the previous one. On adding and gently shaking, 0.6 volume of chilled isopropanol to
the total collected supernatant, DNA strands were observed. Again, the mixture was revolved at 15000 rpm for 5
minutes at 25⁰C so that the pellet was collected at the bottom and the remaining isopropanol portion was
removed. The DNA pellet was washed using 1000 µl of 70% ethanol and spanned at 15000 rpm at 25⁰C for 5
minutes. The DNA Pellet was dried and 100µl DNase RNase free water was added to dissolve and later stored
in the deep freezer at -20⁰C.
DNA Quantification:
The quantity and clarity of the extracted DNA was measured with the help of Nanodrop spectrophotometer
by measuring absorbance at 260, 280 and 230 nm. Purity was checked at 260 / 280 and 260 / 230 nm and pure
DNA solutions were diluted to make up 50 ng/µl concentration for PCR reactions.
DNA Quality Analysis:
The 50 ng/µl DNA was loaded on 1% (w/v) agarose gel in 1x TBE buffer having 10 µg/ml ethidium
bromides. Gene Ruler DNA ladder 100 bp was employed for estimating the rough DNA size range.
Optimization of RAPD and ISSR PCR volumes and conditions:
The optimization of RAPD and ISSR reactions were executed by following the set protocols of [31] and
[32] respectively. Other protocols including [5,6], (for RAPD) and [23], (for ISSR) were also tried for
optimizing the concentrations of various chemical components of PCR, like primers, magnesium chloride,
dNTPs, Taq DNA polymerase and DNA template. The annealing temperatures and number of cycles for each
individual primer were optimized. For PCR reactions, three types of Taq buffers were tried, one with
ammonium sulfate, the second one with potassium chloride and in the third one both ammonium sulfate and
potassium chloride were included.
DNA Amplification using RAPD primers:
RAPD analysis is performed by the use of 10 random primers (Table 1). For each primer, a 15 µl
amplification reaction mixture was prepared containing 1.5 µl of 10 X Taq buffer with ammonium sulfate, 2.5
µl of 25 mM MgCl2, 3 µl of 1 mM dNTP, 3 units Taq DNA polymerase (Thermoscientific), 1.5 µl of 5 µM
RAPD primer and 50 ng of template DNA. Eppendorf Master Cycler was utilized to carry out PCR reactions
with initial denaturation at 94⁰C for 3 minutes, trailed by repeated cycles of denaturation at 94⁰C for 45
seconds, annealing as per the primer’s melting temperature for 1 min and primer extension at 72⁰C for 1 min.
On the completion of repeated number of cycles, final extension was performed at 72⁰C for 10 min.
DNA Amplification using ISSR primers:
For detecting the polymorphism, we used ten (10) ISSR primers (Table 2). For every reaction, 25µl reaction
mixture was prepared with 2.5µl of 10X Taq Buffer containing ammonium sulfate (except ISSR F3 where KCl
was used), 3µl of 25mM MgCl2, 0.4µl of 25mM dNTP, 0.5µl of 10µM primer, 1.5 units of Taq Polymerase and
100ng of template DNA. The 2-step ISSR PCR reactions were performed in Eppendorf Master Cycler. The
physical reaction conditions and the number of initial and final PCR cycles were optimized for each individual
ISSR primer.
Identification of reproducible amplified bands:
All the RAPD and ISSR reactions were repeated thrice to confirm the total reproducible bands count. Only
the reliable bands in all the repeated amplifications were scored for the final analysis.
Gel Electrophoresis:
After PCR reactions, 3 µl and 5 µl of 6X loading dye has been added to 15 µl RAPD and 25 µl ISSR
reaction products respectively and electrophoresed on 1.5% agarose gel with 10µg/ml ethidium bromide at 80 V
for 5-6 hours in Gel Documentation System. Thermoscientific Gene ruler 100 bp Plus DNA ladder was
employed for estimating the size and weight of bands scored in the gel.
Data Scoring and Genetic Diversity Analysis:
Ethidium bromide staining of agarose gels generally showed several bands. The size of the most intensively
amplified band for each microsatellite marker was determined based on its electrophoretic mobility relative to
98 Belattar. R et al, 2017
Advances in Environmental Biology, 11(5) May 2017, Pages: 95-109
the molecular weight markers. Amplified products from ISSR/RAPD analysis were scored qualitatively for
presence and absence of each marker allele-genotype combination (0 for absence and 1 for presence). Each
ISSR band amplified by a given primer was treated as a unit character. Data was entered into a binary matrix as
discrete variables, 1 for presence and 0 for absence of the character.
The most informative primers were selected based on the extent of polymorphism. The polymorphic
information content (PIC) value of a marker was calculated according to [4]. Average allele numbers, PIC
values, and genetic similarities were calculated on the basis of different wheat genotypes, chromosomes and
microsatellite classes. Pair-wise comparisons of the genotypes based on the proportion of unique and shared
amplification products (alleles) were used to measure the genetic similarity by Dice coefficients using PAST
program [15]. Genetic similarities (F) between all genotypes were calculated according to [22]. A dendrogram
was constructed using pair-group method to get genetic relationships among genotypes.
RESULTS AND DISCUSSIONS
RAPD -ISSR anlyses for fivty (50) genotyps:
The quality of the samples of purified diluted genomic DNA is checked by electrophoresis on 1% agarose
gels [18] (fig. 1).
Fig. 1: The ADN of the 50 genotyps electrophoresis ,L; leucomelan, R;rechenbachi
1. RAPD analyses:
The RAPD markers are dispersed in the non-coding regions of the genome and are more likely to mutate in
comparison with the coding genes. Also, they have become indicators of genetic variation in phylogenetic
studies. The difference in density of the strips reflects the number of copies for each amplified sequence. The
number of fragments differs according to the primers used and which have responded positively in numbers of
bands / primer and have proved polymorphic (Fig. 2). Each band corresponds to the amplification of the same
amplified DNA sequence.
Fig. 2: 50 genotypes RAPD electrophoretic Diagram (tow varieties rechenbachi et leucomelan) of durum wheat
generated by the ten (10) RAPD primers: RAPDL6 (1), RAPDL4 (2), RAPDL2 (3), RAPDB10 (4),
RAPDB13 (5), CRAPD1 (6), CRAPD2 (7), RAPDB3 (8), RAPDB5 (9), RAPDB4 (10).
The ten primers represent a high level of polymorphism equivalent to 91.60%. While all primers reached a
maximum polymorphism of 100%, with the exception, the two primers RAPD L4 and RAPD L6 differed from a
polymorphism equal to 66% and 50% respectively (Tab. 2)
1 2 3 4 5
6 7 8 9 10
0
99 Belattar. R et al, 2017
Advances in Environmental Biology, 11(5) May 2017, Pages: 95-109
Table 2: Number’s and types of amplified DNA and the polymorphism percentage issued from ten (10) RAPD primer’s in the durum
wheat 50 genotypes accession.
Primer
RAPD
Total Band Monomorphic Band Polymorphic Band % Polymorphism
unique non unique
RAPD L6 4 2 0 2 50%
RAPD L4 3 1 0 2 66%
RAPD L2 4 0 0 4 100%
RAPD B10 7 0 0 7 100%
RAPD B13 3 0 0 3 100%
CRAPD 1 3 0 0 3 100%
CRAPD 2 3 0 1 2 100%
RAPD b3 2 0 1 1 100%
RAPD b4 4 0 0 4 100%
RAPD b5 5 0 0 5 100%
The element of the amplified DNA fragments is between 0 and 6 bands. The different primers produce an
amplification number ranging from 2 to 7 bands in RAPD b3 and RAPD B10 respectively (Tab. 3). The size of
the amplified fragments ranges from 100 to 1600 bp. The primers RAPD L4, RAPD B13, CRAPD 1 and
CRAPD 2 reveal the same number of bands which is 3 bands. The three primers RAPD L6, RAPD L2 and
RAPD b4 also have 4 bands. The RAPD b5 primer reveals 5 bands.
A total of 38 fragments are obtained. The two primers CRAPD2, RAPD b3 revealed two specific markers
for the two genotypes L132 [220 (+) bp] and L15 [110 (+) bp]. Thus, it appears that of the 38 fragments, 36 are
polymorphic.
All the bands are convergent in all genotypes for the ten primers. It varies from 14 bands to 25 bands. The
genotype R68 shows the highest number of bands of 25 bands, 22 of which are polymorphic, genotypes R19,
L119, L113 and L45 show only 14 bands followed by the L88, L20, R76 and R75 genotypes (15 bands). As for
the other genotypes, they have a number of intermediate bands (Tab. 3).
Table 3: The durum wheat 50 genotypes RAPD-PCR analysis generated by ten primers
P: polymorphic band, U +: specific band, M: mono morphic band
Amor
ces
Géno
types
Amor
cePM
(Pb)
L95
L28
L61
L138
L93
L139
L17
L99
L74
L111
L136
L137
L20
L88
L14
L45
L72
L132
L113
L92
L120
L15
L26
L57
L119
R76
R68
R26
R36
R31
R4R1
5R1
9R1
8R1
4R1
1R3
2R5
9R8
R35
R34
R38
R57
R63
R41
R67
R53
R52
R75
R72
RAPD
L650
01
10
00
00
00
00
00
11
01
10
11
11
01
00
00
00
00
00
00
10
00
00
10
00
00
0P
300
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
M
220
11
11
10
11
11
11
00
00
00
01
11
11
10
00
00
00
00
00
01
01
01
01
11
11
11
P
100
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
M
Total
44
33
32
33
33
33
23
32
33
24
44
43
42
22
22
22
22
22
24
23
23
24
33
33
33
RAPD
L460
01
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
1M
350
10
11
11
11
11
11
11
11
11
11
11
11
11
10
11
10
11
11
11
11
11
11
11
11
01
P
150
10
11
11
00
11
11
11
10
11
11
01
11
11
10
11
10
10
11
11
11
11
11
11
11
01
P
Total
31
33
33
22
33
33
33
32
33
33
23
33
33
31
33
31
32
33
33
33
33
33
33
33
13
RAPD
L268
00
00
01
00
00
00
00
00
00
00
00
00
11
00
00
01
00
00
00
00
00
00
01
10
00
0P
400
11
11
11
11
11
11
11
11
11
11
11
11
11
10
00
01
01
11
11
11
11
11
11
11
10
P
380
00
00
10
00
00
00
00
01
11
00
11
11
10
00
00
00
00
00
00
00
00
00
00
10
00
P
125
00
00
00
00
00
00
00
01
11
00
11
11
10
00
00
00
00
00
00
00
00
00
00
00
00
P
Total
11
11
31
11
11
11
11
13
33
11
33
34
41
10
00
11
01
11
11
11
11
11
22
21
10
RAPD
B10
1600
00
00
00
00
00
00
00
00
00
00
00
00
00
11
00
00
01
00
00
10
00
01
00
01
00
P
1500
10
00
00
00
00
00
00
10
01
00
00
00
00
11
00
11
01
01
00
00
10
01
00
01
00
P
800
00
00
00
00
00
00
00
00
00
00
00
00
00
00
10
00
00
00
00
00
00
01
00
00
00
P
660
01
00
01
11
00
10
00
11
11
10
10
00
00
11
01
11
01
01
11
11
10
10
10
01
11
P
455
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
01
11
11
11
11
11
11
11
11
P
359
11
00
01
11
00
11
10
11
11
10
11
00
10
11
11
11
01
01
11
11
10
11
00
01
11
P
255
00
00
00
01
00
00
00
00
00
00
00
00
00
11
00
01
01
00
00
01
00
00
00
01
00
P
Total
33
11
13
34
11
32
21
43
34
31
32
11
21
56
23
45
06
14
33
44
41
35
21
16
33
RAPD
B13
550
01
11
01
11
01
00
10
10
11
01
11
11
00
11
11
11
01
11
11
11
11
11
11
11
10
P
390
00
11
01
01
00
00
00
00
10
01
11
11
01
11
11
11
00
01
10
11
11
11
11
11
10
P
245
00
00
00
00
00
00
00
00
00
01
00
10
00
11
10
11
00
01
00
11
11
10
11
01
00
P
Total
01
22
02
12
01
00
10
10
21
03
22
32
01
33
32
33
01
13
21
33
33
32
33
23
20
C RAP
D 115
500
11
00
01
01
01
01
10
00
11
00
00
00
11
11
11
11
11
00
00
00
00
00
00
00
1P
1100
00
00
00
10
00
10
00
00
00
10
00
00
00
00
00
00
00
11
00
00
00
00
00
00
00
P
560
00
00
00
10
00
00
00
00
00
00
00
00
01
11
10
01
11
11
00
00
00
00
00
01
01
P
Total
01
10
00
30
10
20
11
00
01
20
00
00
02
22
21
12
22
32
00
00
00
00
00
01
02
C RAP
D 212
001
00
01
11
01
11
10
00
00
00
00
00
00
01
10
11
11
11
01
01
01
01
10
01
11
1P
645
10
01
11
11
11
11
00
00
00
00
00
00
01
11
01
11
11
10
11
10
10
11
00
11
11
P
220
00
00
00
00
00
00
00
00
01
00
00
00
00
00
00
00
00
00
00
00
00
00
00
00
00
U+
Total
20
01
22
21
22
22
00
00
01
00
00
00
01
22
02
22
22
20
21
20
20
22
00
22
22
RAPD
b325
40
00
00
00
00
00
00
00
10
00
00
00
00
00
00
00
00
11
00
00
00
00
11
00
01
0P
110
00
00
00
00
00
00
00
00
00
00
01
00
00
00
00
00
00
00
00
00
00
00
00
00
00
U+
Total
00
00
00
00
00
00
00
01
00
00
01
00
00
00
00
00
01
10
00
00
00
01
10
00
10
RAPD
b490
01
11
11
11
11
10
11
11
11
11
11
11
10
11
11
11
11
11
11
11
11
11
11
11
11
1P
669
10
10
11
00
11
01
11
11
11
11
11
11
01
11
11
11
11
11
01
11
11
11
11
11
01
P
280
00
00
11
00
00
00
00
00
00
00
00
00
00
00
00
00
10
10
00
00
00
00
00
00
00
P
165
10
11
11
11
11
01
11
11
11
11
11
11
01
11
11
11
11
11
11
11
11
11
11
10
01
P
Total
31
32
44
22
33
03
33
33
33
33
33
33
03
33
33
33
43
43
23
33
33
33
33
32
13
RAPD
b510
000
11
10
10
01
11
11
10
00
00
00
00
00
00
00
00
00
00
00
00
00
00
00
00
00
0P
850
01
11
11
11
11
11
10
00
00
00
00
00
00
00
00
00
00
00
00
00
00
00
00
00
00
P
700
01
11
01
00
01
10
01
10
10
01
11
00
11
11
11
11
11
11
01
11
11
11
11
11
11
P
529
01
11
01
00
01
11
00
00
00
00
00
00
00
10
00
00
00
00
10
11
00
11
00
00
01
P
235
11
11
11
10
11
11
01
10
10
01
01
11
00
11
11
11
00
00
00
11
11
11
10
11
00
P
Total
15
55
25
21
35
54
23
20
20
02
12
11
11
32
22
22
11
11
11
33
22
33
21
22
12
Nomb
re tot
al17
1719
1818
2219
1616
1919
1715
1517
1419
1914
1718
2018
1714
1525
2118
1821
2114
2119
1916
1721
2020
1620
2419
1618
2315
18
100 Belattar. R et al, 2017
Advances in Environmental Biology, 11(5) May 2017, Pages: 95-109
These results were compared with other reports on wheat; [24], determined 273 polymorphic bands from
102 doubled haploid wheat accessions, while [3],detected 89.96% of the polymorphic bands in local varieties of
Turkish durum wheat, which is Close and consistent with our results. In 2012, in a study by [11]. of 16 varieties
of soft wheat using RAPD primers, genetic similarity ranged from 0.316 to 0.860. [29], used twenty-two RAPD
primers of the size of the amplification products selected for statistical analysis ranging from 160 to 2800 bp.
DNA PCR amplification isolated from thirty-three wheat genotypes yielded a total of 374 amplified products,
315 of which were polymorphic. For each primer, the number of bands varied from 8 to 30, with an average of
17. The hierarchical classification of the RAPD molecular markers divides the different genotypes into eight
large distinct clusters of equivalent similarity of 100%. The first class contains the genotype R67 which differs
from the other genotypes of two varieties leucomelan and rechenbachi with a similarity of 0%. The second
cluster encompasses the L57 and L119 genotypes with a strong similarity of 72.7%. The third cluster includes
the four genotypes L14, L45, L120 and L92, the first two genotypes of which are identical with a similarity of
71.2%, the L120 and L92 genotypes have a similarity of 76.9%. While the L45 genotype correlates with a
strong similarity equivalent to 92.3% with the L120 genotype. The fourth major cluster is divided into two sub-
groups (Tab.4). The first subgroup represents the five identical genotypes L93, L26, L137, L20 and L88 with a
maximum similarity of 100%, the L93 genotype is correlated to a similarity of 83.3% with the L88 genotype.
The second subgroup groups together the five genotypes L138, L28, L61, R38 and R36 with a similarity ranging
from 73.3% to 81.5%. The fifth Cluster consists of three subgroups. The first subgroup includes nine L139,
L111, L74, R32, R8, R38, R35, R34 and L95 genotypes with a maximum similarity of 100% between L139 and
L111 and strong similarity between the nine genotypes around 90%. The second subgroup is distinguished by
the six genotypes L72, R53, L63, R41, L26 and R52 with a similarity of 70% to 93.37% simultaneously. The
third subgroup comprises the two genotypes L132, L113 with a maximum similarity of 100% and the genotype
R59 which differs from 80.3% with the first two genotypes. The sixth group is divided into two subgroups: One
combines the four genotypes R68, R36, R11 and R4 which have a level between 84.7% and 94.7%. The other
subgroup consists of four genotypes R26, R15, R18 and R72, the first three of which appear to be very similar
with a similarity rate of about 88%. The seventh Cluster encompasses the three genotypes R76, R14 and R19,
the latter two appear to be very similar with a similarity rate of approximately 85%. The last Cluster regroups
the four L17, L136, L99 and R75 genotypes with a similarity of 87, 5%, 73% and 75% correlated successively
(Fig 3).
Fig. 3: Dendrogram of RAPD markers based on the Euclidean distance of the fifty genotypes of durum wheat.
101 Belattar. R et al, 2017
Advances in Environmental Biology, 11(5) May 2017, Pages: 95-109
Table 4: Matrix of the genetic similarity of five genotypes of the two varieties rechenbachi and leucomelan based on PCR-RAPD variation
L95
L28
L61
L13
8L
93L
139
L17
L99
L74
L11
1L
136
L13
7L
20L
88L
14L
45L
72L
132
L11
3L
92L
120
L15
L26
L57
L11
9R
76R
68R
26R
36R
31R
4R
15R
19R
18R
14R
11R
32R
59R
8R
35R
34R
38R
57R
63R
41R
67R
53R
52R
75R
76
L95
1
L28
0,63
1
L61
0,75
0,86
1
L13
80,
80,
790,
81
L93
0,79
0,64
0,79
0,71
1
L13
90,
880,
650,
760,
810,
691
L17
0,61
0,69
0,61
0,65
0,44
0,72
1
L99
0,56
0,44
0,39
0,5
0,47
0,59
0,73
1
L74
0,82
0,71
0,82
0,76
0,65
0,94
0,78
0,56
1
L11
10,
880,
650,
760,
810,
691
0,72
0,59
0,94
1
L13
60,
610,
590,
610,
650,
440,
720,
880,
630,
780,
721
L13
70,
730,
60,
730,
670,
770,
750,
50,
530,
710,
750,
51
L20
0,63
0,6
0,73
0,56
0,77
0,65
0,5
0,53
0,71
0,65
0,5
0,85
1
L88
0,67
0,64
0,79
0,6
0,83
0,59
0,44
0,47
0,65
0,59
0,44
0,77
0,92
1
L14
0,53
0,5
0,63
0,56
0,64
0,65
0,5
0,53
0,61
0,65
0,5
0,71
0,71
0,64
1
L45
0,63
0,6
0,63
0,67
0,64
0,75
0,59
0,64
0,71
0,75
0,5
0,71
0,71
0,64
0,71
1
L72
0,87
0,63
0,75
0,8
0,79
0,88
0,61
0,56
0,82
0,88
0,61
0,63
0,63
0,67
0,63
0,73
1
L13
20,
710,
590,
710,
560,
730,
720,
670,
630,
780,
720,
670,
690,
80,
730,
690,
590,
711
L11
30,
710,
590,
710,
560,
730,
720,
670,
630,
780,
720,
670,
690,
80,
730,
690,
590,
711
1
L92
0,6
0,69
0,71
0,64
0,75
0,63
0,47
0,4
0,59
0,63
0,39
0,57
0,57
0,62
0,69
0,69
0,71
0,56
0,56
1
L12
00,
690,
670,
690,
730,
710,
810,
650,
60,
760,
810,
560,
670,
670,
60,
670,
920,
80,
650,
650,
771
L15
0,81
0,59
0,71
0,75
0,73
0,94
0,67
0,63
0,88
0,94
0,67
0,69
0,69
0,63
0,69
0,8
0,93
0,76
0,76
0,67
0,87
1
L26
0,79
0,64
0,79
0,71
10,
690,
440,
470,
650,
690,
440,
770,
770,
830,
640,
640,
790,
730,
730,
750,
710,
731
L57
0,53
0,5
0,53
0,57
0,67
0,56
0,5
0,54
0,53
0,56
0,6
0,62
0,62
0,54
0,5
0,5
0,53
0,6
0,6
0,46
0,57
0,6
0,67
1
L11
90,
60,
570,
60,
770,
620,
630,
560,
50,
590,
630,
670,
470,
470,
50,
470,
570,
710,
470,
470,
540,
640,
670,
620,
731
R76
0,63
0,6
0,73
0,56
0,77
0,56
0,42
0,35
0,61
0,56
0,42
0,6
0,71
0,77
0,5
0,5
0,63
0,69
0,69
0,57
0,56
0,59
0,77
0,5
0,47
1
R68
0,63
0,61
0,72
0,58
0,56
0,74
0,68
0,47
0,79
0,74
0,68
0,61
0,61
0,56
0,61
0,53
0,63
0,78
0,78
0,5
0,58
0,68
0,56
0,44
0,42
0,71
1
R26
0,47
0,53
0,56
0,42
0,47
0,58
0,61
0,47
0,63
0,58
0,53
0,44
0,53
0,47
0,63
0,53
0,56
0,71
0,71
0,6
0,59
0,61
0,47
0,35
0,33
0,63
0,82
1
R36
0,61
0,69
0,81
0,65
0,63
0,72
0,58
0,37
0,78
0,72
0,58
0,69
0,69
0,63
0,59
0,59
0,61
0,67
0,67
0,56
0,65
0,67
0,63
0,5
0,47
0,8
0,88
0,71
1
R31
0,68
0,58
0,68
0,63
0,53
0,79
0,74
0,53
0,84
0,79
0,74
0,58
0,58
0,53
0,58
0,58
0,68
0,74
0,74
0,47
0,63
0,74
0,53
0,42
0,47
0,67
0,94
0,78
0,83
1
R4
0,78
0,58
0,68
0,63
0,61
0,79
0,74
0,61
0,84
0,79
0,74
0,67
0,67
0,61
0,58
0,58
0,68
0,83
0,83
0,47
0,63
0,74
0,61
0,5
0,47
0,67
0,84
0,68
0,74
0,89
1
R15
0,58
0,56
0,58
0,53
0,42
0,68
0,72
0,5
0,74
0,68
0,63
0,47
0,47
0,42
0,56
0,56
0,58
0,63
0,63
0,53
0,61
0,63
0,42
0,32
0,37
0,56
0,83
0,88
0,72
0,89
0,79
1
R19
0,59
0,47
0,59
0,44
0,6
0,53
0,4
0,41
0,58
0,53
0,4
0,56
0,67
0,71
0,47
0,47
0,59
0,65
0,65
0,44
0,44
0,56
0,6
0,38
0,35
0,67
0,58
0,5
0,56
0,55
0,63
0,45
1
R18
0,5
0,47
0,5
0,45
0,42
0,6
0,63
0,5
0,65
0,6
0,55
0,4
0,47
0,42
0,56
0,56
0,58
0,63
0,63
0,53
0,61
0,63
0,42
0,32
0,37
0,56
0,74
0,88
0,63
0,79
0,7
0,88
0,53
1
R14
0,75
0,53
0,65
0,59
0,67
0,67
0,53
0,47
0,72
0,67
0,53
0,53
0,63
0,67
0,44
0,53
0,75
0,71
0,71
0,5
0,59
0,71
0,67
0,44
0,5
0,86
0,72
0,65
0,71
0,78
0,78
0,67
0,69
0,67
1
R11
0,74
0,63
0,74
0,68
0,58
0,84
0,79
0,58
0,89
0,84
0,79
0,63
0,63
0,58
0,63
0,63
0,74
0,79
0,79
0,53
0,68
0,79
0,58
0,47
0,53
0,63
0,89
0,74
0,79
0,95
0,95
0,84
0,6
0,75
0,74
1
R32
0,88
0,56
0,67
0,71
0,69
0,88
0,72
0,69
0,83
0,88
0,72
0,75
0,65
0,59
0,65
0,65
0,76
0,82
0,82
0,53
0,71
0,82
0,69
0,56
0,53
0,56
0,74
0,58
0,63
0,79
0,89
0,68
0,53
0,6
0,67
0,84
1
R59
0,75
0,44
0,56
0,59
0,67
0,76
0,61
0,79
0,72
0,76
0,61
0,73
0,73
0,67
0,73
0,73
0,75
0,81
0,81
0,5
0,69
0,81
0,67
0,53
0,5
0,53
0,63
0,56
0,53
0,68
0,78
0,58
0,59
0,58
0,65
0,74
0,88
1
R8
0,82
0,61
0,72
0,76
0,65
0,94
0,78
0,65
0,89
0,94
0,78
0,71
0,61
0,56
0,71
0,71
0,82
0,78
0,78
0,59
0,76
0,88
0,65
0,53
0,59
0,53
0,79
0,63
0,68
0,84
0,84
0,74
0,5
0,65
0,63
0,89
0,94
0,82
1
R35
0,76
0,65
0,76
0,71
0,69
0,88
0,72
0,59
0,83
0,88
0,72
0,75
0,65
0,59
0,75
0,65
0,76
0,82
0,82
0,63
0,71
0,82
0,69
0,56
0,53
0,56
0,83
0,67
0,72
0,79
0,79
0,68
0,53
0,6
0,58
0,84
0,88
0,76
0,94
1
R34
0,88
0,65
0,76
0,81
0,69
0,88
0,72
0,59
0,83
0,88
0,72
0,65
0,56
0,59
0,65
0,65
0,88
0,72
0,72
0,63
0,71
0,82
0,69
0,47
0,63
0,56
0,74
0,58
0,63
0,79
0,79
0,68
0,53
0,6
0,67
0,84
0,88
0,76
0,94
0,88
1
R38
0,75
0,73
0,87
0,8
0,79
0,88
0,61
0,47
0,82
0,88
0,61
0,86
0,73
0,67
0,73
0,73
0,75
0,71
0,71
0,71
0,8
0,81
0,79
0,64
0,6
0,63
0,72
0,56
0,81
0,68
0,68
0,58
0,5
0,5
0,56
0,74
0,76
0,65
0,82
0,88
0,76
1
R57
0,82
0,61
0,72
0,76
0,65
0,94
0,78
0,65
0,89
0,94
0,78
0,71
0,61
0,56
0,71
0,71
0,82
0,78
0,78
0,59
0,76
0,88
0,65
0,53
0,59
0,53
0,79
0,63
0,68
0,84
0,84
0,74
0,5
0,65
0,63
0,89
0,94
0,82
10,
940,
940,
821
R63
0,76
0,56
0,67
0,71
0,69
0,88
0,63
0,59
0,83
0,88
0,63
0,65
0,65
0,59
0,65
0,75
0,88
0,72
0,72
0,63
0,81
0,94
0,69
0,56
0,63
0,56
0,65
0,58
0,63
0,7
0,7
0,6
0,61
0,68
0,67
0,75
0,78
0,76
0,83
0,78
0,78
0,76
0,83
1
R41
0,76
0,56
0,67
0,71
0,69
0,88
0,63
0,59
0,83
0,88
0,63
0,65
0,65
0,59
0,65
0,75
0,88
0,72
0,72
0,63
0,81
0,94
0,69
0,56
0,63
0,56
0,65
0,58
0,63
0,7
0,7
0,6
0,61
0,68
0,67
0,75
0,78
0,76
0,83
0,78
0,78
0,76
0,83
11
R67
00
00
00
00
00
00
00
00
00
00
00
00
00
00
00
00
00
00
00
00
00
00
01
R53
0,81
0,59
0,71
0,75
0,73
0,82
0,58
0,53
0,78
0,82
0,58
0,59
0,59
0,63
0,59
0,69
0,93
0,67
0,67
0,67
0,75
0,88
0,73
0,5
0,67
0,59
0,6
0,53
0,58
0,65
0,65
0,55
0,65
0,63
0,71
0,7
0,72
0,71
0,78
0,72
0,82
0,71
0,78
0,94
0,94
01
R52
0,68
0,58
0,68
0,63
0,61
0,79
0,74
0,61
0,84
0,79
0,74
0,58
0,67
0,61
0,67
0,67
0,78
0,83
0,83
0,56
0,72
0,83
0,61
0,5
0,56
0,58
0,75
0,68
0,65
0,8
0,8
0,7
0,63
0,79
0,68
0,85
0,79
0,78
0,84
0,79
0,79
0,68
0,84
0,89
0,89
00,
831
R75
0,5
0,47
0,42
0,53
0,41
0,61
0,75
0,71
0,58
0,61
0,65
0,39
0,39
0,33
0,56
0,56
0,59
0,56
0,56
0,53
0,63
0,65
0,41
0,47
0,53
0,32
0,5
0,59
0,4
0,55
0,55
0,61
0,37
0,71
0,42
0,6
0,61
0,59
0,67
0,61
0,61
0,5
0,67
0,71
0,71
00,
650,
721
R72
0,61
0,59
0,61
0,56
0,53
0,72
0,76
0,63
0,78
0,72
0,67
0,5
0,59
0,53
0,69
0,69
0,71
0,76
0,76
0,67
0,75
0,76
0,53
0,41
0,47
0,5
0,68
0,81
0,58
0,74
0,74
0,82
0,47
0,82
0,61
0,79
0,72
0,71
0,78
0,72
0,72
0,61
0,78
0,72
0,72
00,
670,
830,
751
102 Belattar. R et al, 2017
Advances in Environmental Biology, 11(5) May 2017, Pages: 95-109
2. The ISSR analysis:
The use of ISSR primers, led to DNA amplification of the 50 genotypes of durum wheat. Indeed, each
primer showed reproducible and readable genomic profiles, as evidenced by the clarity of the markers and the
diversity revealed by the primers (Fig. 4).
Fig. 4: ISSR electrophoretic diagram of the 50 genotypes (two varieties reichenbachi (R) and leucomelan (L))
of durum wheat generated by ten ISSR primers: ISSR F2 (1), ISSR F4 (2), ISSR F9 (3), ISSR M3 (4),
ISSR M1 (5), ISSR M2 (6), ISSR M8 (7), ISSR M9 (8), ISSR 17(9), ISSR 12(10).
The percentage polymorphism of the ten ISSR primers used is less than the percentage of RAPD
polymorphism. It is 68.7%. On the other hand, [16], explained that the number of polymorphic bands (58.62%)
detected by ISSR markers was much higher than that of the RAPD marker (46.02%). The polymorphic
percentage of ISSR primers M9, ISSR F2 and ISSR F9 is the highest and equal to 100%. While the ISSR M12
primer showed no equivalent polymorphism of 0%. The ISSR M1 primer revealed 75% polymorphism. The
lowest percentage of polymorphism at 33% is recorded by the ISSR M17 primer. The rest of the primers (ISSR
M2, ISSR M3, ISSR M8 and ISSR F4) detected 50% polymorphism (Tab.5).
Table 5: of Number and type of amplified DNA and percentage of polymorphism generated by ten ISSR primers in an accession of fifty
genotypes durum wheat
Primer ISSR Total Band Monomorphic Band Polymorphic Band % Polymorphism
unique non unique
ISSR M1 4 1 0 3 75%
ISSR M2 4 2 0 2 50%
ISSR M3 2 1 0 1 50%
ISSR M8 2 1 0 1 50%
ISSR M9 3 0 0 3 100%
ISSR M12 1 1 0 0 0%
ISSR M17 3 2 0 1 33%
ISSR F2 5 0 0 5 100%
ISSR F4 4 2 0 2 50%
ISSR F9 4 0 0 4 100%
The element of the amplified DNA fragments is between 0 and 4 bands whose size varies from 230 bp to
about 1500 bp. A total of 32 amplified DNA fragments were produced, in the fifty genotypes, by the ten ISSR
primers used. Among which 22 bands are non-single polymorphs (68.7%), of which 5 bands belong to the ISSR
F2 primer, 4 bands to the ISSR F9 primer, 3 bands to the ISSR M1 primer with a mono morphic band, 3 bands
at the M 9 primer, the ISSR primer M2 and F4 with 2 mono-morphic bands and 1 band at the ISSR M3, M8 and
M17 primer with molecular weights of 550 bp, 260 bp and 745 bp respectively. The number of DNA fragments
obtained varies from 1 for the primer M12 to 5 fragments for the primer F2. The two genotypes L137 and R36
reveal the highest number of bands, 24. Unlike the previous one in variety L113, the lowest number of bands is
13 bands. The genotypes L92, L111, L15, L119, R11, R32, R57, R67, R53, R52, R75 and R72, L88, L57, R68,
R26, R31, R4, R15, R19 and R14 genotypes and L61, L132, R76, R35, R38 and R63 are characterized by the
same number of bands equal to 19.22 and 21 successively between monomorphic and polymorphic. The rest of
the genotypes amplified an intermediate number of bands between 18 and 14 bands. The L99 genotype only
amplifies 14 bands (Tab. 6).
Our results conclude with the studies of [20],who studied the genetic diversity of wheat varieties (Triticum
aestivum L.) released for high yield, quality and abiotic stress in India and found 68.42% polymorphism using
1 2 3 4
5 6 7 8
9 10
103 Belattar. R et al, 2017
Advances in Environmental Biology, 11(5) May 2017, Pages: 95-109
20 UBC series of ISSR markers. [21], used 10 ISSR primers which generated 80.2% polymorphism of wheat
accessions. Similarly, [30], used fifteen ISSR primers amplified a total of 221 groups throughout the forty-one-
durum accession, of which 163 bands were polymorphic. The number of bands varies from ten to nineteen. The
percentage of polymorphic bands (PBP) varied between 61.5 and 82.3 with an average of 73.6%. It is generally
accepted that the genetic diversity of the plant is abundant when the percentage of polymorphism in bands
reaches about 50% at the population level [19,28]. The dendrogram of the ISSR molecular markers revealed
four distinct heterogeneous groups with a similarity slightly less than 68%. The first distinct cluster of similarity
greater than 76% is composed of the four subgroup (Tab. 7, Fig.5).
Table 6: ISSR-PCR analysis of the 50 genotypes of durum wheat generated by ten primers
P: polymorphic band, M: mono morphic band.
Gén
otyp
es
Am
orce
PM(p
b)L9
5L2
8L6
1L1
38L9
3L1
39L1
7L9
9L7
4L1
11L1
36L1
37L2
0L8
8L1
4L4
5L7
2L1
32L1
13L9
2L1
20L1
5L2
6L5
7L1
19R7
6R6
8R2
6R3
6R3
1R4
R15
R19
R18
R14
R11
R32
R59
R8R3
5R3
4R3
8R5
7R6
3R4
1R6
7R5
3R5
2R7
5R7
2
M1
1000
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
M
780
00
00
00
01
11
11
11
11
11
01
01
11
01
11
11
11
11
11
11
11
11
11
11
11
11
P
500
11
11
10
11
11
11
11
11
11
01
01
11
11
00
00
00
00
00
00
01
00
00
00
00
00
P
250
00
00
10
00
00
11
00
10
11
00
01
10
00
00
00
00
00
00
00
01
01
00
00
00
00
P
Tot
al2
22
23
12
33
34
43
34
34
41
31
44
32
32
22
22
22
22
22
22
42
32
22
22
22
2
M2
1500
11
10
11
10
11
01
01
11
11
00
10
01
00
00
00
00
00
00
00
00
00
00
00
00
00
P
750
11
10
11
10
00
01
01
11
11
00
10
01
00
00
00
00
00
00
00
00
00
00
00
00
00
P
600
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
M
255
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
M
Tot
al4
44
24
44
23
32
42
44
44
44
24
22
42
22
22
22
22
22
22
22
22
22
22
22
22
2
M3
750
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
M
550
10
10
00
00
00
00
11
01
00
00
00
11
10
00
00
00
00
00
00
00
00
00
00
00
00
P
Tot
al2
12
11
11
11
11
12
21
21
11
11
12
22
11
11
11
11
11
11
11
11
11
11
11
11
1
M8
770
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
M
260
00
00
00
00
00
00
00
00
00
00
00
00
01
11
11
11
11
11
10
11
11
10
01
11
11
P
Tot
al1
11
11
11
11
11
11
11
11
11
11
11
11
22
22
22
22
22
22
12
22
22
11
22
22
2
M9
700
10
10
10
00
01
11
00
11
11
00
11
01
11
11
11
11
10
10
10
11
11
11
11
11
11
P
450
11
10
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
P
270
01
01
01
10
00
00
11
11
10
11
01
01
10
01
11
10
10
01
01
10
11
01
00
00
00
P
Tot
al2
22
12
22
11
22
22
23
33
22
22
31
33
22
33
33
23
12
22
23
23
32
32
22
22
2
M12
580
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
M
Tot
al1
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
1
M17
745
10
10
00
00
00
01
10
00
00
00
00
00
00
00
00
00
00
00
00
00
00
00
00
00
00
P
630
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
M
230
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
M
Tot
al3
23
22
22
22
22
33
22
22
22
22
22
22
22
22
22
22
22
22
22
22
22
22
22
22
2
F2
1100
00
00
00
00
00
00
00
00
00
00
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
P
764
01
11
11
00
01
01
01
01
01
01
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
P
555
01
11
11
00
01
01
01
01
01
01
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
P
300
01
00
10
10
01
01
01
01
01
01
10
11
11
11
10
00
00
00
00
00
00
01
00
00
00
P
280
01
00
10
00
01
01
01
01
00
11
00
00
00
00
00
00
00
00
00
00
00
00
00
00
00
P
Tot
al0
42
24
21
00
40
40
40
40
31
44
34
44
44
44
33
33
33
33
33
33
33
43
33
33
3
F4
5000
00
00
00
00
00
00
00
00
00
00
00
00
00
00
00
00
01
10
00
00
00
00
00
00
00
P
785
00
01
10
00
00
00
00
00
00
00
00
00
00
01
10
00
00
00
00
00
00
01
00
00
00
P
552
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
M
245
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
11
M
Tot
al2
22
33
22
22
22
22
22
22
22
22
22
22
22
33
22
22
22
22
22
22
22
32
22
22
2
F9
1000
00
00
00
00
00
00
00
00
00
00
00
00
00
01
01
11
11
01
00
00
00
00
00
00
00
0P
5000
00
00
00
00
00
00
00
00
00
00
00
00
00
10
11
11
10
10
00
00
00
00
00
00
00
P
785
01
11
00
11
10
01
01
01
01
01
00
10
01
11
11
11
10
11
11
11
11
11
11
11
11
P
599
00
11
00
00
10
01
00
00
00
00
00
00
01
11
11
11
10
11
11
11
11
11
11
11
11
P
Tot
al0
12
20
01
12
00
20
10
10
10
10
01
00
24
24
44
44
04
22
22
22
22
22
22
22
2
Nom
bre
tota
l17
2021
1721
1617
1416
1915
2416
2218
2318
2113
1917
1920
2219
2122
2224
2222
2222
1722
1919
1820
2120
2119
2118
1919
1919
19
104 Belattar. R et al, 2017
Advances in Environmental Biology, 11(5) May 2017, Pages: 95-109
Fig. 5: Dendrogram of the ISSR markers based on the Euclidean distance of the fifty genotypes of durum wheat.
The first subgroup encompasses the 11 genotypes of the rechenbachi (R76, R35, R15, R14, R32, R57, R41,
R67, R53, R52 and R75) varieties that show 94% strong similarity and 100% maximum similarity between the
genotype And the one that is followed. The second subgroup contains the four genotypes of the rechenbachi
variety (R38, R63, R11 and R59) with a rate between 94.4% and 100%. Intermediate genotypes (R8, R72, R31,
R4, R19 and R34) are very similar genotypes with the highest 100% similarity linked to previous genotypes
with a maximum similarity of 100%. The third subgroup consists of three genotypes (R68, R26 and R36) appear
to be very similar with a similarity rate of about 94.7% and 100%. The genotype R18 is linked to the other
genotypes of the reichenbachi variety by a similarity of 77.8% and 82.4%. The fourth subgroup includes
genotypes L61 and L138, genotypes L111, R87 and L125 with similarity around 82.4%. The second class
comprises 14 genotypes and subdivides into three subgroups. The first one encompasses the six genotypes (L93,
L111, L132, L137, L45 and L26) with 94% similarity between the first five genotypes, a maximum 100%
similarity between the two L111 and L132 genotypes. These genotypes are related to L26 genotypes with a
similarity of 77.8%, 83.3%, 68.4%, 88.9% and 84.2%, respectively. The second subgroup contains the four
genotypes of the leucomelan variety (L28, L88, L92 and L17), the first two appear to be very similar with a
maximum similarity rate of 100%. which are correlated by 94% similarity with the other two genotypes. The
third subgroup includes genotypes (L120, L15, L57, L119), the two genotypes L15 and L57 are identical with a
very high 100% similarity, the L120 genotype is correlated by 77.8% and 88. 2% similarity with each of them.
The third cluster is composed of the two subgroups, the first containing the four genotypes (L99, L74, L136 and
L95), with a similarity of 93.3%, 81.3% and 86.7%, 87.5% correlated seccusively. The second subgroup
consists of three genotypes (L20, L14, L72), with the last two genotypes L14 and L72 presenting a maximum
similarity of 100%. Whereas the L20 genotype is correlated by a 93.3% similarity with the last two genotypes.
The last cluster unites the two genotypes L139 and L113 with a similarity of 92.3% (Table 7). [8],used ISSR
markers for fingerprints and to estimate genetic diversity in a set of 27 genotypes that included Indian varieties
of bread wheat released for high yield. Quality, abiotic stress and specific traces of traits with known pedigrees.
They found that dendrogram analysis placed these genotypes into six groups and is consistent with their known
origin. [2],analyzed genetic diversity and relationships between wheat genotypes including Hexaploid,
Tetraploid and Diploid varieties. The dendrogram indicates that ISSR markers have been able to distinguish
most of the 20 varieties from their genetic and geographic origins, and that the tetraploid varieties have been
grouped together as well as the diploid varieties.
105 Belattar. R et al, 2017
Advances in Environmental Biology, 11(5) May 2017, Pages: 95-109
Table 7: Matrix of the genetic similarity of the two varieties rechenbachi and leucomelan five genotypes based on the PCR-ISSR variation
L95
L28
L61
L13
8L
93L
139
L17
L99
L74
L11
1L
136
L13
7L
20L
88L
14L
45L
72L
132
L11
3L
92L
120
L15
L26
L57
L11
9R
76R
68R
26R
36R
31R
4R
15R
19R
18R
14R
11R
32R
59R
8R
35R
34R
38R
57R
63R
41R
67R
53R
52R
75R
72
L95
1
L28
0,71
1
L61
0,81
0,78
1
L13
80,
650,
820,
821
L93
0,87
0,82
0,82
0,67
1
L13
90,
730,
810,
710,
750,
751
L17
0,75
0,94
0,72
0,76
0,76
0,75
1
L99
0,75
0,72
0,82
0,76
0,67
0,65
0,76
1
L74
0,8
0,76
0,76
0,71
0,71
0,69
0,81
0,93
1
L11
10,
810,
780,
780,
630,
940,
710,
720,
720,
761
L13
60,
870,
630,
820,
670,
760,
650,
670,
880,
810,
821
L13
70,
760,
830,
830,
680,
880,
670,
780,
780,
820,
940,
781
L20
0,8
0,76
0,67
0,71
0,71
0,8
0,81
0,81
0,87
0,76
0,81
0,72
1
L88
0,71
10,
780,
820,
820,
810,
940,
720,
760,
780,
630,
830,
761
L14
0,87
0,72
0,72
0,67
0,76
0,75
0,76
0,76
0,81
0,82
0,88
0,78
0,93
0,72
1
L45
0,72
0,89
0,79
0,74
0,83
0,72
0,83
0,74
0,78
0,89
0,74
0,94
0,78
0,89
0,83
1
L72
0,87
0,72
0,72
0,67
0,76
0,75
0,76
0,76
0,81
0,82
0,88
0,78
0,93
0,72
10,
831
L13
20,
810,
780,
780,
630,
940,
710,
720,
720,
761
0,82
0,94
0,76
0,78
0,82
0,89
0,82
1
L11
30,
790,
750,
650,
690,
690,
920,
80,
690,
730,
650,
690,
610,
860,
750,
80,
670,
80,
651
L92
0,67
0,94
0,74
0,78
0,78
0,76
0,88
0,78
0,82
0,83
0,68
0,89
0,82
0,94
0,78
0,94
0,78
0,83
0,71
1
L12
00,
750,
720,
720,
580,
880,
750,
670,
580,
610,
820,
670,
780,
610,
720,
670,
740,
670,
820,
690,
681
L15
0,76
0,74
0,74
0,68
0,78
0,76
0,68
0,68
0,72
0,83
0,78
0,79
0,82
0,74
0,88
0,84
0,88
0,83
0,71
0,79
0,78
1
L26
0,67
0,83
0,74
0,68
0,78
0,67
0,78
0,78
0,82
0,83
0,68
0,89
0,72
0,83
0,68
0,84
0,68
0,83
0,61
0,89
0,78
0,79
1
L57
0,76
0,74
0,74
0,68
0,78
0,76
0,68
0,68
0,72
0,83
0,78
0,79
0,82
0,74
0,88
0,84
0,88
0,83
0,71
0,79
0,78
10,
791
L11
90,
760,
830,
740,
680,
880,
760,
780,
60,
630,
830,
680,
790,
720,
830,
780,
840,
780,
830,
710,
790,
880,
890,
790,
891
R76
0,72
0,7
0,89
0,74
0,74
0,63
0,65
0,83
0,78
0,79
0,83
0,84
0,68
0,7
0,74
0,8
0,74
0,79
0,58
0,75
0,74
0,84
0,84
0,84
0,75
1
R68
0,63
0,7
0,79
0,65
0,74
0,63
0,65
0,74
0,68
0,79
0,74
0,84
0,6
0,7
0,65
0,8
0,65
0,79
0,58
0,75
0,83
0,75
0,84
0,75
0,75
0,89
1
R26
0,6
0,75
0,75
0,7
0,7
0,68
0,7
0,7
0,65
0,75
0,7
0,8
0,65
0,75
0,7
0,85
0,7
0,75
0,63
0,8
0,79
0,8
0,8
0,8
0,8
0,85
0,95
1
R36
0,6
0,75
0,75
0,7
0,7
0,68
0,7
0,7
0,65
0,75
0,7
0,8
0,65
0,75
0,7
0,85
0,7
0,75
0,63
0,8
0,79
0,8
0,8
0,8
0,8
0,85
0,95
11
R31
0,63
0,7
0,79
0,74
0,65
0,72
0,65
0,74
0,68
0,7
0,74
0,75
0,68
0,7
0,74
0,8
0,74
0,7
0,67
0,75
0,74
0,84
0,75
0,84
0,75
0,89
0,89
0,95
0,95
1
R4
0,63
0,7
0,79
0,74
0,65
0,72
0,65
0,74
0,68
0,7
0,74
0,75
0,68
0,7
0,74
0,8
0,74
0,7
0,67
0,75
0,74
0,84
0,75
0,84
0,75
0,89
0,89
0,95
0,95
11
R15
0,67
0,65
0,83
0,68
0,68
0,67
0,6
0,78
0,72
0,74
0,78
0,79
0,63
0,65
0,68
0,75
0,68
0,74
0,61
0,7
0,78
0,79
0,79
0,79
0,7
0,94
0,94
0,89
0,89
0,94
0,94
1
R19
0,63
0,7
0,79
0,74
0,65
0,72
0,65
0,74
0,68
0,7
0,74
0,75
0,68
0,7
0,74
0,8
0,74
0,7
0,67
0,75
0,74
0,84
0,75
0,84
0,75
0,89
0,89
0,95
0,95
11
0,94
1
R18
0,69
0,67
0,67
0,61
0,71
0,8
0,61
0,71
0,75
0,76
0,71
0,72
0,75
0,67
0,71
0,68
0,71
0,76
0,73
0,72
0,81
0,82
0,82
0,82
0,72
0,78
0,78
0,74
0,74
0,78
0,78
0,82
0,78
1
R14
0,67
0,65
0,83
0,68
0,68
0,67
0,6
0,78
0,72
0,74
0,78
0,79
0,63
0,65
0,68
0,75
0,68
0,74
0,61
0,7
0,78
0,79
0,79
0,79
0,7
0,94
0,94
0,89
0,89
0,94
0,94
10,
940,
821
R11
0,58
0,74
0,74
0,78
0,6
0,76
0,68
0,78
0,72
0,65
0,68
0,7
0,72
0,74
0,68
0,75
0,68
0,65
0,71
0,79
0,68
0,79
0,79
0,79
0,7
0,84
0,84
0,89
0,89
0,94
0,94
0,89
0,94
0,82
0,89
1
R32
0,67
0,65
0,83
0,68
0,68
0,67
0,6
0,78
0,72
0,74
0,78
0,79
0,63
0,65
0,68
0,75
0,68
0,74
0,61
0,7
0,78
0,79
0,79
0,79
0,7
0,94
0,94
0,89
0,89
0,94
0,94
10,
940,
821
0,89
1
R59
0,58
0,74
0,74
0,78
0,6
0,76
0,68
0,78
0,72
0,65
0,68
0,7
0,72
0,74
0,68
0,75
0,68
0,65
0,71
0,79
0,68
0,79
0,79
0,79
0,7
0,84
0,84
0,89
0,89
0,94
0,94
0,89
0,94
0,82
0,89
10,
891
R8
0,63
0,7
0,79
0,74
0,65
0,72
0,65
0,74
0,68
0,7
0,74
0,75
0,68
0,7
0,74
0,8
0,74
0,7
0,67
0,75
0,74
0,84
0,75
0,84
0,75
0,89
0,89
0,95
0,95
11
0,94
10,
780,
940,
940,
940,
941
R35
0,72
0,7
0,89
0,74
0,74
0,63
0,65
0,83
0,78
0,79
0,83
0,84
0,68
0,7
0,74
0,8
0,74
0,79
0,58
0,75
0,74
0,84
0,84
0,84
0,75
10,
890,
850,
850,
890,
890,
940,
890,
780,
940,
840,
940,
840,
891
R34
0,67
0,74
0,83
0,78
0,68
0,76
0,68
0,72
0,67
0,68
0,72
0,74
0,67
0,74
0,72
0,79
0,72
0,68
0,71
0,74
0,78
0,83
0,74
0,83
0,79
0,89
0,89
0,94
0,94
11
0,94
10,
760,
940,
940,
940,
941
0,89
1
R38
0,63
0,7
0,79
0,74
0,65
0,72
0,65
0,74
0,68
0,7
0,74
0,75
0,68
0,7
0,74
0,8
0,74
0,7
0,67
0,75
0,74
0,84
0,75
0,84
0,75
0,89
0,89
0,95
0,95
11
0,94
10,
780,
940,
940,
940,
941
0,89
11
R57
0,67
0,65
0,83
0,68
0,68
0,67
0,6
0,78
0,72
0,74
0,78
0,79
0,63
0,65
0,68
0,75
0,68
0,74
0,61
0,7
0,78
0,79
0,79
0,79
0,7
0,94
0,94
0,89
0,89
0,94
0,94
10,
940,
821
0,89
10,
890,
940,
940,
940,
941
R63
0,63
0,7
0,79
0,74
0,65
0,72
0,65
0,74
0,68
0,7
0,74
0,75
0,68
0,7
0,74
0,8
0,74
0,7
0,67
0,75
0,74
0,84
0,75
0,84
0,75
0,89
0,89
0,95
0,95
11
0,94
10,
780,
940,
940,
940,
941
0,89
11
0,94
1
R41
0,67
0,65
0,83
0,68
0,68
0,67
0,6
0,78
0,72
0,74
0,78
0,79
0,63
0,65
0,68
0,75
0,68
0,74
0,61
0,7
0,78
0,79
0,79
0,79
0,7
0,94
0,94
0,89
0,89
0,94
0,94
10,
940,
821
0,89
10,
890,
940,
940,
940,
941
0,94
1
R67
0,67
0,65
0,83
0,68
0,68
0,67
0,6
0,78
0,72
0,74
0,78
0,79
0,63
0,65
0,68
0,75
0,68
0,74
0,61
0,7
0,78
0,79
0,79
0,79
0,7
0,94
0,94
0,89
0,89
0,94
0,94
10,
940,
821
0,89
10,
890,
940,
940,
940,
941
0,94
11
R53
0,67
0,65
0,83
0,68
0,68
0,67
0,6
0,78
0,72
0,74
0,78
0,79
0,63
0,65
0,68
0,75
0,68
0,74
0,61
0,7
0,78
0,79
0,79
0,79
0,7
0,94
0,94
0,89
0,89
0,94
0,94
10,
940,
821
0,89
10,
890,
940,
940,
940,
941
0,94
11
1
R52
0,67
0,65
0,83
0,68
0,68
0,67
0,6
0,78
0,72
0,74
0,78
0,79
0,63
0,65
0,68
0,75
0,68
0,74
0,61
0,7
0,78
0,79
0,79
0,79
0,7
0,94
0,94
0,89
0,89
0,94
0,94
10,
940,
821
0,89
10,
890,
940,
940,
940,
941
0,94
11
11
R75
0,67
0,65
0,83
0,68
0,68
0,67
0,6
0,78
0,72
0,74
0,78
0,79
0,63
0,65
0,68
0,75
0,68
0,74
0,61
0,7
0,78
0,79
0,79
0,79
0,7
0,94
0,94
0,89
0,89
0,94
0,94
10,
940,
821
0,89
10,
890,
940,
940,
940,
941
0,94
11
11
1
R72
0,67
0,65
0,83
0,68
0,68
0,67
0,6
0,78
0,72
0,74
0,78
0,79
0,63
0,65
0,68
0,75
0,68
0,74
0,61
0,7
0,78
0,79
0,79
0,79
0,7
0,94
0,94
0,89
0,89
0,94
0,94
10,
940,
821
0,89
10,
890,
940,
940,
940,
941
0,94
11
11
11
106 Belattar. R et al, 2017
Advances in Environmental Biology, 11(5) May 2017, Pages: 95-109
3. The hierarchical classification of the two markers:
The classification of the two markers RAPD and ISSR of the 50 genotypes studied allowed distinguishing
five main groups (Table 8, FIG. 6). The first cluster presents the late genotype R67, which differs from the other
genotypes of the two leucomelan and reichenbachi varieties with a similarity ranging from 33% to 50% and
which is characterized by a length of ear, water content and a rate of chlorophylls. The second major cluster is
distinguished by two early genotypes L57 and L119 with a similarity of 82.8%. The third major cluster is
divided into four subgroups: the first cluster consists of the three genotypes L14, L20 and L113 with 82.8%,
82.8% and 74.2% similarities correlated successively. The second subgroup consists of the eight L93, L26,
L132, L137, L45, L28, L88 and L92 genotypes with a similarity between 74.2% and 83.9%. The third subgroup
consisted of eleven L139, L111, L72, L15, L95, L74 and L120 genotypes, which ranged between 78% and 85%.
The fourth subgroup encompasses the two L61 and L138 genotypes with a very high similarity of 81.3%. The
fourth major cluster is divided into four subgroups. The first group is distinguished by three genotypes R76, R14
and R19, with a similarity of 90.6%, 82.3% and 79.4% respectively. The second subgroup contains three
genotypes R68, R36 and R26, the first two genotypes show a similar similarity of 91.6%, linked to R26
genotypes with a similarity of 88.8% and 86.7% respectively. The third subgroup groups the seventeen
genotypes R31, R4, R11, R32, R8, R53, R59, R53, R52, R59, R15, R72 and R75 with very high similarity
between R57, R8 and R32 (97.4%, 97.4 and 97.06% successively and96.9% between R41 and R53).
Finally, the genotype R18 in a single subgroup differs from the other genotypes of the reichenbachi variety
with a similarity that varies from 64.8% to 82.3% with the R72 genotype. The fifth major cluster presents the
three genotypes L17, L136 and L99 with a similarity between 75% and 76.5%. The results obtained show that
most of the genotypes belonging to the same botanical variety were grouped in the same main group, but an
ISSR - RAPD intra-variety polymorphism was also observed. This is in agreement with the work of [8,9]. [14],
studied the discriminating capacity of RAPD and ISSR markers and their effectiveness in establishing the
genetic relationship and diversity between eleven wheat cultivars and local breeds collected in Egypt and Saudi
Arabia. The dendrogram classified the genotypes evaluated in three main clusters corresponding to crop regions.
[23],reported that the genetic relationships of wheat accessions estimated by the ISSR marker polymorphism
were identical to those determined by the RFLP and RAPD markers, indicating the reliability of the ISSR
markers for the estimation of genotypes.
Fig. 6: Dendrogram of the RAPD-ISSR markers based on the Euclidean distance of the fifty genotypes of
durum wheat.
107 Belattar. R et al, 2017
Advances in Environmental Biology, 11(5) May 2017, Pages: 95-109
Table 8: Matrix of the genetic similarity of five genotypes of the two varieties rechenbachi and leucomelan based on variation PCR-RAPD /
ISSR
L95
L28
L61
L13
8L
93L
139
L17
L99
L74
L11
1L
136
L13
7L
20L
88L
14L
45L
72L
132
L11
3L
92L
120
L15
L26
L57
L11
9R
76R
68R
26R
36R
31R
4R
15R
19R
18R
14R
11R
32R
59R
8R
35R
34R
38R
57R
63R
41R
67R
53R
52R
75R
72
L95
1
L28
0,67
1
L61
0,78
0,81
1
L13
80,
720,
810,
811
L93
0,83
0,74
0,81
0,69
1
L13
90,
810,
730,
740,
780,
721
L17
0,68
0,81
0,67
0,71
0,6
0,74
1
L99
0,66
0,59
0,6
0,64
0,58
0,62
0,75
1
L74
0,81
0,74
0,79
0,74
0,68
0,82
0,79
0,73
1
L11
10,
840,
710,
770,
710,
810,
850,
720,
660,
851
L13
60,
730,
610,
710,
660,
60,
690,
760,
750,
790,
771
L13
70,
750,
730,
790,
680,
830,
710,
640,
670,
760,
850,
641
L20
0,71
0,69
0,7
0,64
0,73
0,72
0,65
0,68
0,78
0,71
0,65
0,77
1
L88
0,69
0,83
0,78
0,72
0,83
0,7
0,68
0,61
0,71
0,69
0,54
0,81
0,83
1
L14
0,69
0,62
0,68
0,62
0,71
0,7
0,63
0,66
0,71
0,74
0,68
0,75
0,83
0,69
1
L45
0,68
0,76
0,71
0,71
0,75
0,74
0,71
0,7
0,74
0,82
0,62
0,84
0,75
0,78
0,78
1
L72
0,87
0,68
0,74
0,73
0,77
0,81
0,69
0,67
0,82
0,85
0,74
0,71
0,77
0,7
0,81
0,79
1
L13
20,
760,
690,
740,
590,
840,
710,
690,
680,
770,
850,
740,
820,
780,
760,
760,
740,
761
L11
30,
740,
670,
680,
620,
710,
810,
730,
660,
760,
690,
680,
650,
830,
740,
740,
630,
750,
811
L92
0,64
0,83
0,73
0,72
0,77
0,7
0,68
0,61
0,71
0,74
0,54
0,75
0,71
0,8
0,74
0,84
0,75
0,71
0,64
1
L12
00,
720,
70,
710,
650,
80,
780,
660,
590,
690,
820,
610,
730,
640,
670,
670,
810,
730,
740,
670,
721
L15
0,79
0,67
0,72
0,71
0,76
0,85
0,68
0,66
0,8
0,88
0,72
0,74
0,76
0,69
0,79
0,82
0,91
0,8
0,74
0,74
0,82
1
L26
0,72
0,75
0,76
0,7
0,86
0,68
0,61
0,64
0,74
0,76
0,57
0,84
0,74
0,83
0,67
0,76
0,73
0,79
0,67
0,83
0,75
0,76
1
L57
0,66
0,64
0,65
0,64
0,73
0,67
0,6
0,63
0,63
0,71
0,7
0,72
0,73
0,66
0,71
0,7
0,72
0,73
0,66
0,66
0,69
0,81
0,74
1
L11
90,
690,
720,
680,
720,
770,
70,
680,
560,
610,
740,
680,
650,
610,
690,
640,
730,
750,
660,
590,
690,
770,
790,
720,
831
R76
0,68
0,66
0,82
0,66
0,75
0,59
0,54
0,6
0,69
0,68
0,62
0,74
0,7
0,73
0,63
0,67
0,69
0,74
0,63
0,68
0,66
0,72
0,81
0,7
0,63
1
R68
0,63
0,66
0,76
0,62
0,65
0,68
0,67
0,61
0,74
0,76
0,71
0,73
0,61
0,63
0,63
0,67
0,64
0,78
0,68
0,63
0,7
0,72
0,7
0,61
0,59
0,81
1
R26
0,54
0,65
0,66
0,56
0,59
0,63
0,66
0,59
0,64
0,67
0,62
0,63
0,59
0,62
0,67
0,7
0,63
0,73
0,67
0,71
0,69
0,71
0,65
0,59
0,58
0,75
0,89
1
R36
0,61
0,72
0,78
0,68
0,67
0,7
0,64
0,54
0,71
0,74
0,64
0,75
0,67
0,69
0,65
0,73
0,66
0,71
0,65
0,69
0,72
0,74
0,72
0,67
0,65
0,83
0,92
0,86
1
R31
0,66
0,64
0,74
0,68
0,59
0,76
0,69
0,63
0,76
0,74
0,74
0,67
0,63
0,62
0,66
0,69
0,71
0,72
0,7
0,62
0,68
0,79
0,64
0,63
0,62
0,78
0,92
0,86
0,89
1
R4
0,7
0,64
0,74
0,68
0,63
0,76
0,69
0,68
0,76
0,74
0,74
0,71
0,68
0,66
0,66
0,69
0,71
0,76
0,75
0,62
0,68
0,79
0,68
0,68
0,62
0,78
0,87
0,82
0,84
0,95
1
R15
0,62
0,61
0,7
0,61
0,55
0,68
0,66
0,64
0,73
0,71
0,7
0,63
0,55
0,54
0,62
0,66
0,63
0,68
0,62
0,62
0,69
0,71
0,61
0,55
0,54
0,75
0,89
0,89
0,81
0,92
0,86
1
R19
0,61
0,59
0,69
0,59
0,63
0,62
0,53
0,58
0,63
0,62
0,56
0,67
0,68
0,71
0,61
0,65
0,67
0,68
0,66
0,61
0,59
0,7
0,69
0,63
0,57
0,79
0,74
0,73
0,76
0,76
0,81
0,68
1
R18
0,58
0,57
0,58
0,53
0,56
0,69
0,62
0,6
0,69
0,68
0,62
0,55
0,6
0,54
0,63
0,62
0,64
0,69
0,68
0,63
0,71
0,72
0,61
0,56
0,54
0,67
0,76
0,8
0,68
0,78
0,74
0,85
0,65
1
R14
0,71
0,59
0,74
0,64
0,68
0,67
0,56
0,63
0,72
0,7
0,65
0,67
0,63
0,66
0,57
0,65
0,71
0,72
0,66
0,61
0,69
0,75
0,74
0,63
0,61
0,91
0,83
0,78
0,81
0,86
0,86
0,83
0,82
0,74
1
R11
0,66
0,68
0,74
0,73
0,59
0,81
0,74
0,68
0,81
0,74
0,74
0,67
0,68
0,66
0,66
0,69
0,71
0,72
0,75
0,66
0,68
0,79
0,68
0,63
0,62
0,74
0,87
0,82
0,84
0,95
0,95
0,86
0,76
0,78
0,81
1
R32
0,76
0,61
0,75
0,69
0,69
0,77
0,66
0,74
0,78
0,81
0,75
0,77
0,64
0,62
0,67
0,7
0,72
0,78
0,71
0,62
0,74
0,81
0,74
0,69
0,62
0,75
0,84
0,74
0,76
0,86
0,92
0,83
0,73
0,7
0,83
0,86
1
R59
0,66
0,59
0,65
0,69
0,63
0,76
0,65
0,78
0,72
0,7
0,65
0,71
0,73
0,71
0,71
0,74
0,71
0,72
0,76
0,66
0,69
0,8
0,74
0,68
0,61
0,69
0,74
0,73
0,71
0,81
0,86
0,73
0,77
0,69
0,77
0,86
0,88
1
R8
0,72
0,66
0,76
0,75
0,65
0,83
0,71
0,69
0,78
0,81
0,76
0,73
0,65
0,63
0,72
0,76
0,78
0,74
0,72
0,68
0,75
0,86
0,7
0,69
0,68
0,71
0,84
0,79
0,82
0,92
0,92
0,84
0,74
0,71
0,78
0,92
0,94
0,89
1
R35
0,74
0,68
0,83
0,72
0,71
0,75
0,68
0,71
0,81
0,83
0,78
0,8
0,67
0,65
0,74
0,73
0,75
0,81
0,69
0,69
0,72
0,83
0,77
0,71
0,65
0,78
0,86
0,76
0,79
0,84
0,84
0,81
0,71
0,68
0,76
0,84
0,91
0,81
0,92
1
R34
0,76
0,69
0,8
0,79
0,69
0,82
0,7
0,66
0,75
0,78
0,72
0,69
0,61
0,67
0,69
0,72
0,79
0,7
0,71
0,69
0,74
0,83
0,71
0,66
0,71
0,72
0,81
0,76
0,78
0,89
0,89
0,81
0,75
0,68
0,8
0,89
0,91
0,85
0,97
0,89
1
R38
0,69
0,71
0,82
0,76
0,71
0,79
0,63
0,61
0,75
0,78
0,68
0,79
0,71
0,69
0,74
0,77
0,74
0,7
0,69
0,74
0,76
0,83
0,76
0,76
0,69
0,77
0,81
0,76
0,89
0,84
0,84
0,76
0,75
0,63
0,75
0,84
0,86
0,8
0,91
0,89
0,88
1
R57
0,74
0,63
0,78
0,72
0,67
0,8
0,68
0,71
0,81
0,83
0,78
0,75
0,62
0,61
0,69
0,73
0,75
0,76
0,69
0,65
0,77
0,83
0,72
0,67
0,65
0,73
0,86
0,76
0,79
0,89
0,89
0,86
0,71
0,73
0,81
0,89
0,97
0,86
0,97
0,94
0,94
0,89
1
R63
0,69
0,63
0,73
0,72
0,67
0,8
0,64
0,67
0,76
0,78
0,68
0,7
0,67
0,65
0,69
0,78
0,8
0,71
0,69
0,69
0,77
0,89
0,72
0,71
0,69
0,73
0,77
0,76
0,79
0,84
0,84
0,76
0,81
0,73
0,81
0,84
0,86
0,86
0,92
0,84
0,89
0,89
0,89
1
R41
0,71
0,61
0,75
0,69
0,69
0,77
0,62
0,69
0,78
0,81
0,7
0,72
0,64
0,62
0,67
0,75
0,77
0,73
0,67
0,67
0,79
0,86
0,74
0,69
0,67
0,75
0,79
0,74
0,76
0,82
0,82
0,78
0,78
0,75
0,83
0,82
0,89
0,83
0,89
0,86
0,86
0,86
0,91
0,97
1
R67
0,38
0,41
0,47
0,41
0,43
0,35
0,34
0,48
0,37
0,4
0,42
0,48
0,39
0,42
0,42
0,47
0,39
0,41
0,33
0,47
0,45
0,44
0,5
0,54
0,47
0,57
0,49
0,52
0,5
0,47
0,47
0,52
0,55
0,42
0,55
0,43
0,52
0,5
0,49
0,5
0,48
0,53
0,5
0,5
0,52
1
R53
0,74
0,62
0,77
0,71
0,71
0,74
0,59
0,66
0,75
0,78
0,68
0,69
0,61
0,64
0,64
0,72
0,79
0,7
0,64
0,69
0,76
0,83
0,76
0,66
0,69
0,77
0,76
0,71
0,74
0,79
0,79
0,76
0,8
0,72
0,85
0,79
0,86
0,8
0,86
0,83
0,88
0,83
0,89
0,94
0,97
0,53
1
R52
0,68
0,62
0,76
0,66
0,65
0,73
0,67
0,69
0,78
0,76
0,76
0,68
0,65
0,63
0,68
0,71
0,73
0,78
0,72
0,63
0,75
0,81
0,7
0,65
0,63
0,76
0,84
0,79
0,77
0,87
0,87
0,84
0,78
0,81
0,83
0,87
0,89
0,83
0,89
0,86
0,86
0,81
0,92
0,92
0,94
0,49
0,91
1
R75
0,58
0,57
0,62
0,61
0,56
0,64
0,67
0,75
0,65
0,68
0,71
0,59
0,51
0,5
0,63
0,67
0,64
0,65
0,58
0,63
0,71
0,72
0,61
0,65
0,63
0,62
0,71
0,75
0,64
0,74
0,74
0,8
0,65
0,76
0,69
0,74
0,8
0,74
0,81
0,78
0,77
0,72
0,83
0,83
0,85
0,57
0,82
0,86
1
R72
0,64
0,62
0,72
0,62
0,61
0,69
0,68
0,71
0,75
0,73
0,72
0,65
0,61
0,59
0,69
0,72
0,69
0,75
0,69
0,69
0,76
0,78
0,67
0,61
0,59
0,72
0,81
0,86
0,74
0,84
0,84
0,91
0,7
0,82
0,8
0,84
0,86
0,8
0,86
0,83
0,83
0,78
0,89
0,83
0,86
0,53
0,83
0,91
0,88
1
108 Belattar. R et al, 2017
Advances in Environmental Biology, 11(5) May 2017, Pages: 95-109
Conclusion:
This study demonstrates the utility of RAPD and ISSR markers for the estimation of genetic relationships
between 50 genotypes belonging to two varieties of durum wheat (Leucomelan and Rechenbachi). The results
showed that the choice of random marker techniques is always appropriate and attractive due to low cost
analysis and fast acquisition of data. RAPD markers reported 92.10% for mean polymorphism compared with
ISSR markers (68.7%). The classification of the two RAPD and ISSR markers of the 50 genotypes studied
revealed interesting results. We found that the genotypes were grouped according to their botanical varieties
and, in some cases, their namesake (s); First group (R67), Second group (L57 and L119), Third group (L14,
L193, L113, L93, L123, L123, L137, L45, L28, L88, L92, L139, L111, L72, L15, L95, L74, L120, L61 and
L138), Fourth group (R14, R15, R8, R7, R7, R8, R7, R7, R7, R9, and R18) and Fifth group (L17, L136 and
L99). The polymorphism detected among the durum accessions studied can be used in breeding programs to
maximize the use of genetic resources. The results obtained in the study enrich the previous data obtained by the
morphological analyzes.
Appendix:
CTAB: Cetyl trimethylammonium bromide
DNA: Acide désoxyribonucléique
TBE: Tris, Borate, EDTA
RAPD: Random Amplification of Polymorphic DNA
ISSR: inter simple sequence repeat
dNTPs: Deoxynucleotide
PCR: polymerase chain reaction
KCl: Chlorure de potassium
PAST program: PAleontological STatistics
ACKNOWLEDGEMENTS
I thank Mr. Bouteldjoune Imed Eddine chemical engineer, Quality control service chief in Hamma
Bouziane’s cement factory, Constantine, Algeria, for his contribution in the English translation.
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