Research Journal of Biology, 2: 84 - 98 (2014)
www.researchjournalofbiology.weebly.com
RESEARCH ARTICLE
Open Access
Characterization of Aspergillus Isolates from Saudi
Arabia Based on Molecular Genetic Fingerprints
Ehab A. Kamel
Biology Department, University College, Umm Al-Qura University, P. O. Box: 2064, Makkah, Saudi Arabia.
Abstract
In the present study, 20 Aspergillus isolates (from eight Aspergillus species) collected from different habitats in
Jeddah, Saudi Arabia were cultivated on two different media (Czapek Dox and Waksman), and molecular genetic
fingerprints was conducted to elucidate the relationships between these isolates. Extracted DNA from these isolates
was used to identify the molecular fingerprints of Aspergillus genotypes. Only five primers (each consisted of 10
base pairs) successfully generated reproducible polymorphic products to discriminate among the genotypes. The
fingerprints generated by these primers revealed characteristic profiles for each Aspergillus genotype, in terms of
the number and position of RAPD bands. The results revealed that both the number and size of the amplified
products varied considerably with the different primers. A sum of 47 polymorphic bands was generated by these
primers in the Aspergillus genotypes growing on Czapek Dox's medium in this study. Additionally, 58 polymorphic
bands were generated by these primers in the Aspergillus genotypes growing on Waksman's medium. In this study,
two unique bands were identified out of the polymorphic bands in the Aspergillus genotypes growing on Czapek
Dox's medium, whereas, 11 unique bands were detected out of the polymorphic bands in the Aspergillus genotypes
growing on Waksman's medium. Fifteen monomorphic bands were detected in the Aspergillus genotypes growing
on either Czapek Dox's and Waksman's media. These unique bands were used to discriminate among the studied
Aspergillus isolates. The data were analyzed through the clustering method and similarity coefficients using
NTSYSpc version 2.02i. Three different phenograms were produced for the studied Aspergillus species and the
relationships between the species were discussed.
Key Words: Aspergillus spp., DNA, RAPD-PCR technique, genotypic relationship and numerical taxonomy.
(Received: 28/06/2014; Accepted: 22/07/2014; Published: 26/07/2014)
Several methods such as SDS-PAGE, isozymes
and RAPD-PCR banding pattern variations have
been used in the fingerprinting and analysis of the
genetic relationships of many different species. The
study of closely related taxonomical units for a
range of characteristics not only sheds light on the
genetic fingerprints of the genotypes under study
but also illuminates the relationships between
these units (Suh et al., 1997).
The morphological characteristics of microbes may be
influenced by environmental factors, and genomic
mutations cannot be investigated by morphological
markers. One additional step (molecular markers reveal
characterization) has been taken to overcome this
problem characterizing Aspergillus species. Polymorphisms
were studied at the molecular level through random
amplified polymorphic DNA (RAPD) marker technique
(Taylor and Natvig 1987).
DNA (RAPD) markers, which utilize polymerase
chain reaction (PCR) and amplification from single
primers of arbitrary nucleotide sequence, have
emerged
as
powerful
tools
for
genome
fingerprinting and analysis (Williams et al., 1990;
Welsh and Mc-Clelland 1990). The PCR-based
molecular marker method is straightforward, does
not require prior knowledge of the DNA sequence
and can be performed using as little as 1 ng/ l of
template DNA isolated from tissue, according to
simplified procedures (Caetano-Anolles, 1994;
Thomson and Henry, 1995).
Introduction
Aspergillus (Micheli, 1729) are a common group of
filamentous fungi that have universal dissemination and
are readily recovered from soil, decaying vegetation, air
and many other environments. Their conidia become
aerosolized are widely distributed in the environment and
are inhaled by humans and animals. Some members of this
genus (Aspergillus fumigatus, A. flavus and A. niger) can
operate as opportunistic aggressors and cause a group of
diseases known as Aspergillosis, especially in individuals
with weakened immune systems.
Fungal identification and taxonomy is a dynamic
progressive discipline. Due to its dynamic nature, it may
require certain changes in the nomenclature, classification,
or both. Such changes are extremely frustrating and
distracting for non-taxonomists and may lead to some
interruption in the accumulation of knowledge of living
organisms such as fungi among nontaxonomists. Thus, one
major goal should be to elucidate more reliable methods
in the classification of fungi to produce more stable
classification systems that require fewer changes in fungal
nomenclature or classification.
Molecular markers have been developed to solve this
problem. Molecular markers are independent of
environmental conditions and show high levels of
polymorphism.
Corresponding author: [email protected]
84
Copyright © 2014 RJB
Research Journal of Biology, 2: 84 - 98 (2014)
www.researchjournalofbiology.weebly.com
RAPD
markers
are
useful
in
genetic
fingerprinting
and
in
elucidating
genetic
relationships in different genera and species of
fungi. Several studies have been conducted with
the genus Aspergillus: Abdel-Fattah and Hammad
(2002); Aiat (2006); Khan and Anwer (2007); Abed (2008);
Batista et al., (2008); Midorikawa et al., (2008);
Narasimhan and Asokan (2010).
The aim of the present study is to establish genetic
fingerprints to differentiate among isolates (gathered from
different sources) belonging to eight species of the genus
Aspergillus by using RAPD markers and to estimate the
genetic relationships between species of the genus. This
study also addresses the impact of location and source on
genotypic variation. Additionally, data analysis was
performed on the different isolates of Aspergillus species
using NTSYSpc version 2.02i (Rohlf, 1998). The data were
analyzed by the clustering method and similarity
coefficients. Next, three different phenograms were
produced for the studied Aspergillus species, and the
relationships between the species were discussed.
Materials and methods
In the present study, eight species of Aspergillus
comprising 20 isolates were studied. These isolates of
Aspergillus species are Aspergillus japonicas (two isolates),
A. versicolor (two isolates), A. parasiticus (three isolates),
A. niger (three isolates), A. terreus (two isolates); A. flavus
(three isolates); A. carneus (two isolates) and A. tamari
(three isolates). Some of the isolates were collected from
four localities of Jeddah of Saudi Arabia and have been
previously identified by Al-Hazmi (2010) using the
references; Raper and Fennell (1965), von Arx (1974),
Domsch et al. (1988), Klich (2002); Kirk et al. (2008). The
studied isolates and their habitat are presented in Table 1.
The experimental fungi were cultivated on two types
of media for the determination of DNA patterns: Czapek
’
Dox s medium, in which NaNO3 is the source of inorganic
’
nitrogen, and Waksman s medium, in which peptone is the
source of organic nitrogen. For each fungus, a triplicate set
of 250 ml Erlenmeyer conical flasks, each containing 50 ml
of medium, was prepared, sterilized at 121°C for 15
minutes under 1.5 atmospheric pressure, cooled and
inoculated with the experimental fungus. The cultures
were then incubated at 25°C for seven days.
Next, approximately 0.5 g of fungal mat was used for
DNA extraction following the Dellaporta method
(Dellaporta et al., 1983). Fifteen 10-mer random DNA
oligonucleotide primers (UBC) were independently used in
the PCR reactions according to Williams et al. (1990). The
primers were synthesized by the University of British
Colombia. Only five primers generated reproducible
polymorphisms in the DNA profiles. Each experiment was
repeated two times and only stable products were scored.
The code and sequences of these primers are listed in
Table 2.
The data obtained from each isolate from RAPD-PCR
were pooled and coded to create the data matrix of
computation. They were scored for the absence of a band
(0) and the presence of a band (1) in each species. The
relationships between the studied species, expressed by
the similarity coefficient, were presented as a phenogram,
based on the analysis of the recorded characters using
NTSYSpc version 2.02i (Rohlf, 1998).
Table 1. Names and sources of the studied isolates of Aspergillus.
Sample
Sources and
Identification
number
habitat
1
Agricultural
Aspergillus japonicus Saito
soil
2
Agricultural
Aspergillus versicolor (Vuill.) Tirab. str. I
soil
3
Marine fauna
Aspergillus japonicus Saito
4
Aspergillus parasiticus Spear var.
Marine fauna
parasiticus
5
Marine fauna
Aspergillus niger Tiegh.
6
Marine fauna
Aspergillus versicolor (Vuill.) Tirab. str. II
7
Aspergillus parasiticus Speare var.
Sewage dump
parasiticus
8
Sewage dump
Aspergillus terreus Thom
9
Sewage dump
Aspergillus niger Tiegh.
10
Sewage dump
Aspergillus flavus Link
11
Soil around car
Aspergillus tamarii Kita
oil dump
12
Soil around car
Aspergillus tamarii Kita
oil dump
13
Soil around car
Aspergillus terreus Thom
oil dump
14
Agricultural
Aspergillus parasiticus Speare var.
soil
parasiticus
15
Wheat grain
Aspergillus carneus Blochwitz
16
Wheat grain
Aspergillus flavus Link
17
Wheat grain
Aspergillus niger Tiegh
18
Wheat grain
Aspergillus tamarii Kita
19
Wheat grain
Aspergillus flavus Link
20
Wheat grain
Aspergillus carneus Blochwitz
Table 2. List of operon primers (A and O) and their nucleotide
sequence.
No;
1
2
3
4
5
Sequence
OP-A10
5-GTGATCGCAG-3
OP-A15
5-TTCCGAACCC-3
OP-A1
5-CAGGCCCTTC-3
OP-O12
5-CAGTGCTGTG-3
OP-O19
5-GTGAGGCGTC-3
One kilobase DNA Ladder 1μg/μl (Invitrogen) was used.
Results and discussion
In the present study, randomly amplified polymorphic DNA
(RAPD) based polymerase chain reaction (PCR) analysis
was conducted to fingerprint and elucidate similarity
indices among 20 isolates representing eight Aspergillus
species.
Randomly amplified polymorphic DNA (RAPD)
markers are based on the amplification of random
locations in the plant genome by polymerase chain
reaction (PCR). Using this technique, a single
oligonucleotide is used to prime the amplification of the
genomic DNA. Because these primers are 10-mer long,
they have the possibility of annealing at a number of
locations in the genome. To generate amplification
products, the primers must bind to inverted annealing
sites that are generally 150-4000 base apart. The number
of amplification products is directly related to the number
and orientation of the annealing sites in the genome.
Ten random primers were initially tested, but fiverevealed distinct inter- and intra-specific polymorphism
85
Copyright © 2014 RJB
Description
Kamel, 2014
among the 20 Aspergillus isolates that were studied. These
primers are named OP-A10, OP-A15, OP-A1, OP-O12 and
OP-O19. Figure 1 and 2 illustrate the RAPD profiles
generated by these primers. The occurrence of bands was
expressed as (1) and the absence as (0) in Table 3 and 4.
the bands most often observed that were generated by
primer OP-O19 (Table 3).
A total of one monomorphic and seven polymorphic
bands were generated by primer OP-O12 (Table 5). No
unique bands were identified from the polymorphic bands.
The clearest non-unique polymorphic bands generated by
primer OP-O12 are those scored at the approximate
molecular sizes of 1030, 950, 860, 640, 490, 320 and 210
bp (Table 3).
RAPD-PCR Analysis of Fungal Isolates Grown on
Czapek Dox's Medium
In isolates grown on Czapek Dox's medium, 47 DNA bands
were detected. These bands were sorted as 15
monomorphic and 32 polymorphic bands. Of the
polymorphic bands, two unique bands were scored (Table
5). The five primers revealed considerable variation
between the studied species and within genotypes of the
same species from the different sources. The range of
polymorphism for these primers varied from 50.0 %
(primers OP-A10 and OP-A1) to 87.5 % (primer OP-O12).
All of the primers used generated monomorphic bands.
The sizes of the DNA bands ranged from 200 bp (OP-A1
and OP-O19) to 1350 bp (OP-A1). The profiles of the DNA
bands varied with the primer used (Figure 1).
The primer OP-A10 generated eight polymorphic
bands in the studied isolate genotypes (Table 5). No
unique bands were identified from these polymorphic
bands. Four monomorphic bands were detected (at
approximately 1130, 840, 730 and 400 bp) (Table 3). These
bands provided further capability to discriminate among
the studied Aspergillus isolates. Aspergillus terreus and A.
parasiticus isolates, lane 13 & 14 were identified by the
presence of one polymorphic band (940 bp), whereas the
isolates (Aspergillus versicolor str. I and A. japonicas) in
lane 2 & 3 were identified by the presence of different
polymorphic band (200 bp).
The primer OP-A15 generated seven polymorphic
bands. Two monomorphic DNA bands were observed in
the studied isolates genotypes (Table 5). One unique band
was identified out of the polymorphic bands at
approximately 245 bp in the isolate of Aspergillus carneus
(lane 20). This band clearly discriminates between
Aspergillus carneus and the other studied isolate
genotypes (Table 3). The six non-unique polymorphic
bands detected at approximately 1300, 830, 665, 570, 500
and 400 bp represent the most observed bands generated
by primer OP-A15 (Table 3). Similarly, these bands provide
additional ability to discriminate among the studied
genotypes.
Primer OP-A1 generated five monomorphic and five
polymorphic bands. No unique bands were identified from
the polymorphic bands (Table 5). The five monomorphic
bands detected at approximately 1100, 900, 800, 605 and
330 bp represent the bands most often observed that
were generated by primer OP-A1. The clearest non-unique
polymorphic bands generated by primer OP-A1 are those
scored at the approximate molecular sizes of 1350, 680,
645, 500 and 445 bp (Table 3).
In the RAPD profile generated by primer OP-O19
(Figure 1), nine polymorphic DNA bands were scored out
of the total twelve bands (Table 5). One polymorphic band
was identified as a unique band (Table 5). This unique
band was observed in Aspergillus parasiticus at the
apparent molecular size of 800 bp (lane 14). The eight
non-unique polymorphic bands detected at approximately
1090, 1000, 710, 625, 445, 325, 290 and 200 bp represent
RAPD-PCR Analysis of Fungal Isolates Grown on
Waksman's Medium
Of the 58 DNA bands detected, 15 were monomorphic
bands and 43 were polymorphic bands. Out of the
polymorphic bands, eleven unique bands were scored
(Table 6). The five primers revealed considerable variation
between the species and within genotypes of the same
species from different sources. The range of polymorphism
for these primers varied from 63.6 % (primer OP-O19) to
91.9 % (primer OP-O12). All the primers used generated
monomorphic bands. The sizes of DNA bands ranged from
210 bp (OP-O19) to 1350 bp (OP-A10). The profiles of DNA
bands varied with the primer used (Figure 2).
Eleven polymorphic DNA bands were generated by
primer OP-A10. Four unique bands were detected in these
bands (Table 6). The first unique band was identified at the
molecular size of approximately 980 bp in the isolate of
Aspergillus terreus (lane 13). The second and third unique
polymorphic bands were recognized at the molecular sizes
of approximately 680 and 600 bp in Aspergillus versicolor
str. II (lane 6). The fourth band was identified at the
molecular size of approximately 285 bp in the isolate of
Aspergillus tamari (lane 12). The most observable non
unique polymorphic bands generated by primer OP-A10
were those detected at molecular sizes of approximately
1350, 1170, 950, 570, 500, 410 and 370 bp (Table 4).
Primer OP-A15 generated five polymorphic bands in
the studied isolate genotypes (Table 6). One unique band
was identified out of the polymorphic bands at
approximately 550 bp in the isolate of Aspergillus tamaris
(lane 18). The clearest non-unique polymorphic bands
generated were those scored at the approximate
molecular sizes of 850, 600, 330 and 230 bp (Table 4). Two
monomorphic bands were detected (at approximately 680
and 420 bp) (Table 4). These bands provide further
capability to discriminate among the studied Aspergillus
isolates.
Primer OP-A1 generated nine polymorphic bands.
Five monomorphic DNA bands were observed in the
studied isolates genotypes (Table 6). Three unique bands
were identified at approximately 750 bp in the isolate of
Aspergillus niger (lane 9), at approximately 530 bp in the
isolate of Aspergillus terreus (lane 8) and 230 bp in the
isolate of Aspergillus tamari (lane 12). These bands clearly
discriminate Aspergillus niger, A. terreus and A. tamari
from the other studied isolate genotypes (Table 4). The six
non-unique polymorphic bands detected at approximately
1300, 1250, 1180, 1115, 500 and 380 bp represent the
bands most often observed that were generated by primer
OP-A1 (Table 4). Similarly, these bands provide additional
capability to discriminate among the studied genotypes.
Primer OP-O19 generated a total of four
monomorphic and seven polymorphic bands. Two unique
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Res. J. Biol., 2014 [2:84-98]
E-ISSN: 2322-0066
bands with molecular weight of 700 and 360 bp were
identified in the isolate of Aspergillus japonicas (lane 3)
and Aspergillus tamaris (lane 12), respectively (Table 6).
The four monomorphic bands detected at approximately
1050, 990, 830 and 580 bp represent the bands most often
observed that were generated by primer OP-A1. The
clearest non-unique polymorphic bands (five bands)
generated by primer OP-O19 are those scored at the
approximate molecular sizes of 740, 680, 525, 415 and 210
bp (Table 4).
In the RAPD profile generated by primer OP-O12
(Figure 2), eleven polymorphic DNA bands were scored out
of the total twelve bands (Table 6). Only one polymorphic
band was identified as a unique band detected; it was
located in an isolate of Aspergillus terreus at the apparent
molecular size of 1050 bp (lane 8) (Table 6). The ten non
unique polymorphic bands detected at approximately
1300, 1150, 1110, 970, 900, 790, 650, 620, 500 and 240 bp
represent the most observed bands generated by primer
OP-O12 (Table 6).
Although, RAPD analysis was effective in providing
sufficient polymorphism to discriminate among the
studied species, no single primer could differentiate
between all of the studied isolates of Aspergillus
genotypes. Therefore, when the data from the five primers
and from both cultural media were combined complete
identification and discrimination was achieved for all
studied isolate genotypes. Each of the studied isolates was
discriminated by one or more unique bands or a group of
combined class patterns.
In this light, the following isolates of Aspergillus ssp.
’
cultivated on Czapek Dox s medium were identified by a
unique band from the polymorphic bands generated by
the five primers. A. parasiticus was identified by one band
(by OP-O19) at the apparent molecular size of 800 bp
(lane 14), and A. carneus was identified by one band (by
OP-A15) at the apparent molecular size of 245 bp (lane
20) (Table 5).
Additionally, some isolates of Aspergillus ssp. were
identified by a unique band from the polymorphic bands
generated by the five primers when cultivated on
’
Waksman s medium. A. japonicas was identified by one
band (by OP-O19) at apparent molecular size of 700 bp
(lane 3). A. versicolor str. II was identified by two bands (by
OP-A10) at the apparent molecular sizes of 680 and 600
bp (lane 6). A. terreus was identified by two bands (by OPA1 and OP-O12) at the apparent molecular sizes of 530
and 1050 bp, respectively (lane 8). A. niger was identified
by one band (by OP-A1) at the apparent molecular size of
750 bp (lane 9). A. tamari was identified by three bands
(by OP-A10, OP-A1 and OP-O19) at the apparent
molecular sizes of 285, 230 and 360 bp, (lane 12); the
same species (lane 18) was identified by another band (by
OP-A15) at the apparent molecular size of 550 bp. Finally,
A. terreus was identified by one band (by OP-A10) at the
apparent molecular size of 980 bp (lane 13) (Table 6).
More recently, techniques that utilize polymerase
chain reaction have allowed a more representative
assessment of genetic variation in fungi by screening
multiple loci distributed throughout the genome. The
analyses reveal sufficient polymorphism for the
examination of fine-scale genetic differences among
individuals.
In this study, only five of the ten primers were able to
generate polymorphic and reproducible amplification
products. Many authors reported the use of a large
number of primers to identify and characterize many
fungal genotypes, but a limited number of primers
succeeded in generating distinct and reproducible profiles
with sufficient polymorphism.
Similarly, Abdel-Fattah and Hammad (2002) used only
five primers to study the genetic variations between two
species of Aspergillus, A. nigra and A. terreus. Aiat (2006)
used ten random primers to study genetic diversity among
three species of Aspergillus, A. niger, A. flavus and A.
parasiticus, by RAPD analysis, but only four primers
produced clear amplification products. Additionally,
Batista et al. (2008) used five primers out of 29 to analyze
genetic diversity in Aspergillus flavus. In contrast,
Midorikawa et al. (2008) used 11 primers to establish the
genetic variations within the same species. In 2010,
Narasimhan and Asoka used only two primers when they
studied the genetic variation within Aspergillus terreus.
Numerical Analysis of RAPD-PCR Data
The data obtained from RAPD-PCR of each isolate were
pooled together and coded to create a data matrix of
computation; the data were scored for the absence of a
band (0) and for the presence of a band (1) in each
species. The relationships between the studied species,
expressed by a similarity coefficient, were presented as a
phenogram based on the analysis of the recorded
attributes using NTSYSpc version 2.02i, as described by
Rohlf (1998).
For data analysis, the total number of recorded
attributes (105) in each isolate were scored, combined into
three sets of data and coded to create the data matrix of
computation: (1) the first set of data was for the RAPD-PCR
analysis of isolates growing on Czapek Dox’s medium (47
attributes) (Table 7); (2) the second set of data was for the
RAPD-PCR analysis of isolates growing on Waksman’s
medium (58 attributes) (Table 8); and (3) the third set of
data was for all of the characters combined.
The constructed phenogram based on estimated
RAPD-PCR of 20 isolates of fungi belonging to the genus
Aspergillus grown on Czapek Dox's medium is shown in
Figure 3. A simplified phenogram was made (Figure 6) to
construct reasonable species groups; it is of little value to
discuss the results at the OTU level. This phenogram shows
that the examined OTU at a similarity coefficient level of
approximately 1.62 are divided into three GROUPs: I
(comprises six isolates), II (comprises eight isolates) & III
(comprises six isolates). GROUP I included two isolates of
Aspergillus parasiticus (2/3) (7 & 14). While GROUP II (at
the level of 1.24 similarity coefficient) included the three
isolates each of Aspergillus tamari (3/3) (11, 12 & 18) and
Aspergillus flavus (3/3) (10, 16 & 19); this group revealed a
very close relationship between isolates of both A. tamari
and A. flavus. GROUP III (at the level of 1.21 similarity
coefficient) included two isolates of Aspergillus niger (2/3)
(5 & 9).
Regarding the same isolates of Aspergillii grown on
Waksman's medium, the constructed phenogram based on
estimated RAPD-PCR is shown in Figure 4. The simplified
phenogram (Figure 7) shows that the examined OTU at a
similarity coefficient level of approximately 1.96 are also
87
Kamel, 2014
divided into three GROUPs: I (comprises five isolates), II
(comprises seven isolates) & III (comprises eight isolates).
GROUP I included the two isolates of Aspergillus terreus
(2/2) (8 & 13). While GROUP II (at the level of 1.19
similarity coefficient) included three species (Aspergillus
tamari, Aspergillus flavus and Aspergillus carneus): two
isolates of Aspergillus tamari (2/3) (11 & 18), two isolates
of Aspergillus flavus (2/3) (16 & 19) and two isolates of
Aspergillus carneus (2/2) (15 & 20). This group revealed a
very close relationship between isolates of those species.
GROUP III (at the level of 1.23 similarity coefficient)
included the two species (Aspergillus japonicas and
Aspergillus parasiticus): two isolates of Aspergillus
japonicas (2/2) (1 & 3) and three isolates of Aspergillus
parasiticus (3/3) (4, 7 & 14). This also revealed a very close
relationship between isolates of those species.
Finally, the phenogram based on estimated RAPD-PCR
for the analysis of the total number of recorded attributes
(105) of each isolate grown on Czapek Dox's and
Waksman's media is shown in Figure 5. The simplified
phenogram (Figure 8) shows that the examined OTU at a
similarity coefficient level of approximately 1.73 are also
divided into three GROUPs: I (comprises six isolates), II
(comprises seven isolates) & III (comprises seven isolates).
GROUP I included two isolates of Aspergillus terreus (2/2)
(8 & 13). GROUP II (at the level of 1.23 similarity
coefficient) included three species (Aspergillus tamari,
Aspergillus carneus and Aspergillus flavus), two isolates of
Aspergillus tamari (2/3) (11 & 18), two isolates of
Aspergillus carneus (2/2) (15 & 20) and two isolates of
Aspergillus flavus (2/3) (16 & 19). These data revealed a
close relationship between isolates of those three species.
GROUP III (at the level of 1.26 similarity coefficient)
included two species (Aspergillus parasiticus and
Aspergillus niger): two isolates of Aspergillus parasiticus
(2/3) (4 & 7) and two isolates of Aspergillus niger (2/3) (5
& 9). These data, also revealed a close relationship
between isolates of those species.
In their review, Hadrich et al., (2011) reported
different genotyping methods developed and employed to
better understand the genetic and epidemiological
relationships between environmental and clinical isolates
of Aspergillus flavus. In the same species, Rudramurthy et
al. (2011) reported that there is a large genotypic diversity
in clinical A. flavus isolates in India and that microsatellites
are excellent typing targets for discriminating between A.
flavus isolates from various origins.
Hong et al. (2010) reported that the species concept
of Aspergillus fumigatus sensu stricto has recently been
defined by polyphasic taxonomy and species delimitations;
that study reviewed 146 worldwide strains of Aspergillus
fumigatus sensu lato that were re-identified by the use of
random amplification of polymorphic DNA polymerase
chain reaction (RAPD-PCR) with primers PELF and URP1F
techniques.
El Khoury et al. (2011) investigated the differentiation
between Aspergillus flavus and Aspergillus parasiticus
from pure cultures and Aflatoxin contaminated grapes
using PCR-RFLP analysis of the aflR-aflJ intergenic spacer.
Their results showed that both species displayed different
PCR-based RFLP (PCR-RFLP) profiles. PCR products from A.
flavus were cleaved into three fragments of 362, 210 and
102 bp. However, there is only one restriction site for this
enzyme in the sequence of A. parasiticus that produced
two fragments of 363 and 311 bp. The method was
successfully applied to contaminated grape samples. This
approach to differentiate these two species would be
simpler, less costly and quicker than conventional
sequencing of PCR products and/or morphological
identification. Our results confirmed the validity of this
approach, as shown in the simplified phenogram (Figure 8)
of combined data from both media in which both species
(isolate no; 4 and 10) were grouped together in group III.
León et al. (2011) reported on the epidemiology of the
fungus by analyzing the phenotypic variability of the
Aspergillus section Fumigati isolates from different Latin
American countries. They also showed the relationship
between this variability, geographical origin and genotypic
characteristics, isolates of Peru showed specific
phenotypic characteristics; that clearly differentiate them
from the rest of the isolates, which matches the genotypic
data. The correlation between the phenotypic and
genotypic characteristics showed a statistically significant
correlation.
A previous study by Al-Hazmi and Kamel (2012)
on the same fungal isolates used the SDS-PAGE
technique to elucidate the protein profiles and the
impact of the different environmental sources on
protein banding patterns. They confirmed that
fungal growing habitats played a role in fungal
isolates of the genus Aspergillus; therefore, the
present study aims to use the RAPD-PCR technique
and DNA fingerprinting to distinguish between
these isolates.
In conclusion, the RAPD technique is useful in
establishing genetic fingerprints of Aspergillus
genotypes. In addition, RAPD technique could
detect enough polymorphism in the studied
genotypes to distinguish each genotype from the
others by at least one unique band or a group of
combined class patterns. The polymorphism
information obtained through RAPD analysis may
also help to identify polymorphic primers for
further studies in other Aspergillus genotypes.
Additionally, by comparing the two phenograms of each
group, the basic relation between the studied isolates
revealed that there is a remarkable correlation and
variation between the geographical origin (sources) and
the DNA profiles of the studied isolates.
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89
Kamel, 2014
Table 3. The presence (1) and absence (0) of DNA in the generated RAPD profiles of the 20 Aspergillus isolates grown on Czapek
Dox's medium.
Primer
OPA10
OPA15
OPA1
OPO19
OPO12
Molecular
Weight in bp.
1180
1130
940
840
730
640
400
200
1300
830
815
665
570
500
450
400
245
1350
1100
900
800
680
645
605
500
445
330
1090
1000
920
850
800
710
650
625
445
325
290
200
1030
950
860
640
490
425
320
210
1
0
1
0
1
1
1
1
0
0
1
1
1
0
1
1
1
0
1
1
1
1
0
0
1
0
1
1
1
0
1
1
0
0
1
1
0
0
1
0
1
1
0
0
1
1
0
0
2
1
1
0
1
1
0
1
1
1
1
1
0
0
1
1
1
0
1
1
1
1
0
0
1
0
1
1
1
0
1
1
0
0
1
1
0
0
1
0
0
0
0
0
1
1
1
1
3
1
1
0
1
1
0
1
1
0
1
1
0
0
1
1
1
0
0
1
1
1
0
0
1
0
1
1
0
0
1
1
0
0
1
1
0
0
0
0
0
0
0
0
1
1
1
1
4
0
1
0
1
1
1
1
0
0
0
1
1
0
0
1
0
0
1
1
1
1
0
0
1
0
1
1
1
0
1
1
0
0
1
0
1
0
1
0
0
0
0
0
0
1
1
1
5
0
1
0
1
1
1
1
0
0
0
1
1
0
0
1
0
0
1
1
1
1
0
0
1
0
1
1
1
0
1
1
0
0
1
0
1
0
1
0
0
0
0
0
0
1
1
1
6
0
1
0
1
1
1
1
0
0
0
1
1
0
0
1
0
0
1
1
1
1
1
0
1
0
1
1
1
0
1
1
0
0
1
0
1
0
1
1
0
0
0
0
1
1
1
1
7
0
1
0
1
1
1
1
0
1
1
1
0
1
0
1
1
0
1
1
1
1
0
0
1
0
1
1
1
0
1
1
0
0
1
0
1
0
1
1
0
1
0
1
0
1
0
0
8
0
1
0
1
1
1
1
0
0
1
1
1
0
0
1
1
0
0
1
1
1
0
0
1
0
1
1
1
0
1
1
0
0
1
0
1
1
1
1
0
0
0
0
1
1
1
0
90
9
0
1
0
1
1
1
1
0
0
1
1
1
0
0
1
0
0
1
1
1
1
0
0
1
0
0
1
1
0
1
1
0
0
1
1
1
0
1
1
1
1
1
0
1
1
1
0
Aspergillus isolates.
10 11 12 13
0
1
1
1
1
1
1
1
0
0
0
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0
0
0
0
0
0
1
1
0
0
0
0
1
1
1
1
1
1
0
0
0
0
0
1
0
0
0
0
1
1
1
1
1
1
0
0
0
0
0
0
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0
0
0
0
0
0
0
0
1
1
1
1
0
1
0
0
0
0
0
0
1
1
1
1
1
1
1
1
0
0
0
0
1
1
1
1
1
1
1
1
0
0
0
0
0
1
0
0
1
1
1
1
0
0
0
0
0
1
0
1
0
0
0
1
0
1
0
0
0
0
0
0
1
1
1
1
1
0
0
1
0
1
1
1
0
0
0
0
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0
14
1
1
1
1
1
1
1
0
0
0
1
0
1
0
1
0
0
0
1
1
1
0
0
1
1
0
1
1
0
1
1
1
0
1
0
0
1
0
0
1
1
1
0
1
1
1
1
15
1
1
0
1
1
1
1
0
0
1
1
0
0
0
1
0
0
1
1
1
1
0
0
1
0
0
1
1
1
1
1
0
1
1
0
0
0
1
0
1
0
1
0
0
1
1
1
16
1
1
0
1
1
0
1
0
0
0
1
0
0
0
1
0
0
1
1
1
1
0
1
1
1
0
1
1
0
1
1
0
1
1
0
0
0
0
0
0
0
0
0
1
1
1
1
17
1
1
0
1
1
1
1
0
0
1
1
0
0
0
1
1
0
0
1
1
1
1
0
1
0
0
1
1
0
1
1
0
1
1
0
0
0
0
0
0
0
0
0
1
1
1
0
18
1
1
0
1
1
1
1
0
0
1
1
0
0
0
1
0
0
1
1
1
1
0
0
1
0
0
1
1
1
1
1
0
1
1
0
0
0
0
0
1
0
1
1
1
1
1
1
19
1
1
0
1
1
1
1
0
0
1
1
0
0
0
1
0
0
1
1
1
1
0
1
1
0
0
1
1
0
1
1
0
1
1
0
0
0
0
0
1
0
1
1
0
1
0
0
20
1
1
0
1
1
0
1
0
0
1
1
0
0
0
1
1
1
0
1
1
1
0
0
1
0
0
1
1
0
1
1
0
0
1
0
0
0
0
0
0
0
0
0
0
1
1
1
Res. J. Biol., 2014 [2:84-98]
E-ISSN: 2322-0066
Figure 1. RAPD fingerprints of the 20 Aspergillus isolates grown on Czapek Dox's
medium by the primers OPA10, OPA15, OPA1, OPO19 and OPO12.
91
Kamel, 2014
Table 4. The presence (1) and absence (0) of DNA in the generated RAPD profiles of the 20 Aspergillus isolates grown on
Waksman's medium.
Primer
OPA10
OPA15
OPA1
OPO19
OPO12
Molecular
Weight in bp.
1350
1170
160
980
950
830
700
680
600
570
500
410
370
285
850
680
600
550
420
330
230
1300
1250
1180
1115
980
950
830
750
600
530
500
420
380
230
1050
990
830
740
700
680
580
525
415
360
210
1300
1150
1110
1050
970
900
790
650
620
500
410
240
1
0
0
1
0
0
1
1
0
0
0
0
1
1
0
0
1
0
0
1
1
0
0
0
0
0
1
1
1
0
1
0
0
1
1
0
1
1
1
0
0
1
1
1
1
0
1
1
1
0
0
0
1
1
0
1
0
1
0
2
1
0
1
0
0
1
1
0
0
1
1
1
1
0
1
1
0
0
1
0
0
0
0
0
0
1
1
1
0
1
0
1
1
1
0
1
1
1
0
0
1
1
1
1
0
1
1
1
0
0
0
1
1
0
0
1
1
0
3
0
1
1
0
1
1
1
0
0
1
1
1
1
0
1
1
0
0
1
0
0
0
0
0
0
1
1
1
0
1
0
1
1
0
0
1
1
1
0
1
0
1
0
1
0
1
1
1
0
0
0
1
1
0
0
1
1
0
4
0
0
1
0
0
1
1
0
0
1
1
1
1
0
0
1
0
0
1
0
0
0
0
0
1
1
1
1
0
1
0
1
1
0
0
1
1
1
0
0
1
1
0
0
0
0
1
1
0
0
0
1
1
0
0
0
1
0
5
0
0
1
0
0
1
1
0
0
0
1
1
1
0
0
1
0
0
1
0
0
0
1
0
0
1
1
1
0
1
0
1
1
0
0
1
1
1
0
0
0
1
0
0
0
1
1
1
0
0
0
1
1
0
0
0
1
0
6
0
0
1
0
0
1
1
1
1
0
1
1
1
0
1
1
0
0
1
1
1
0
0
0
0
1
1
1
0
1
0
0
1
0
0
1
1
1
0
0
0
1
0
0
0
0
1
1
0
0
0
1
1
0
0
1
1
0
7
0
1
1
0
1
1
1
0
0
1
1
1
1
0
1
1
1
0
1
1
1
0
0
0
0
1
1
1
0
1
0
0
1
0
0
1
1
1
0
0
0
1
0
0
0
0
1
1
1
0
0
1
1
0
0
1
1
0
8
0
1
1
0
0
1
1
0
0
0
0
0
1
0
1
1
0
0
1
1
0
0
0
1
1
1
1
1
0
1
1
0
1
0
0
1
1
1
0
0
1
1
0
0
0
0
0
0
0
1
0
0
0
0
0
0
1
0
92
9
0
0
1
0
0
1
1
0
0
1
1
0
1
0
1
1
1
0
1
1
0
0
1
0
1
1
1
1
1
1
0
1
1
0
0
1
1
1
1
0
0
1
0
0
0
0
1
1
0
0
0
1
1
0
1
1
1
0
Aspergillus isolates.
10 11 12 13
1
0
0
0
0
0
0
0
1
1
1
1
0
0
0
1
0
0
1
0
1
1
1
1
1
1
1
1
0
0
0
0
0
0
0
0
1
1
1
0
1
0
0
1
0
1
1
0
1
0
1
1
0
0
1
0
1
0
0
1
1
1
1
1
0
1
0
1
0
0
0
0
1
1
1
1
1
1
1
0
1
1
1
0
0
0
0
1
1
0
0
1
0
0
0
0
0
0
0
1
1
1
1
1
1
1
1
1
1
1
1
1
0
0
0
0
1
1
1
1
0
0
0
0
1
1
1
1
1
1
1
1
0
1
0
1
0
0
1
0
1
1
1
1
1
1
1
1
1
1
1
1
0
0
0
1
0
0
0
0
0
1
1
1
1
1
1
1
0
0
1
0
0
0
1
0
0
0
1
0
0
1
1
1
1
1
1
1
1
1
1
1
0
1
1
0
0
0
0
0
0
0
0
0
1
0
0
1
1
0
0
1
0
0
0
0
1
1
1
0
1
0
0
0
1
1
1
1
0
1
1
0
14
1
0
1
0
1
1
1
0
0
1
1
1
1
0
1
1
0
0
1
0
0
0
0
0
0
1
1
1
0
1
0
1
1
1
0
1
1
1
0
0
1
1
0
0
0
1
1
1
0
0
0
0
1
1
0
1
1
1
15
1
0
1
0
0
1
1
0
0
1
1
0
1
0
1
1
0
0
1
1
1
1
1
0
1
1
1
1
0
1
0
1
1
1
0
1
1
1
1
0
1
1
0
0
0
1
1
1
1
0
1
0
1
1
0
0
1
0
16
0
0
1
0
0
1
1
0
0
1
1
0
1
0
1
1
0
0
1
1
1
1
1
0
1
1
1
1
0
1
0
1
1
1
0
1
1
1
0
0
1
1
0
0
0
1
1
1
1
0
1
0
1
0
0
1
1
1
17
0
0
1
0
0
1
1
0
0
1
1
0
1
0
1
1
0
0
1
1
0
0
0
1
1
1
1
1
0
1
0
1
1
1
0
1
1
1
0
0
1
1
0
0
0
1
1
1
1
0
1
0
1
0
0
1
1
1
18
0
0
1
0
0
1
1
0
0
1
1
0
0
0
0
1
0
1
1
1
1
0
0
0
0
1
1
1
0
1
0
1
1
0
0
1
1
1
0
0
1
1
1
0
0
1
1
1
1
0
1
0
1
0
0
0
1
1
19
0
0
1
0
0
1
1
0
0
1
1
1
0
0
0
1
0
0
1
1
1
0
0
0
0
1
1
1
0
1
0
1
1
0
0
1
1
1
1
0
1
1
0
0
0
1
1
1
1
0
1
0
1
0
0
1
1
0
20
0
0
1
0
0
1
1
0
0
1
0
0
0
0
1
1
1
0
1
1
1
0
0
0
0
1
1
1
0
1
0
1
1
0
0
1
1
1
0
0
1
1
0
0
0
1
1
1
1
0
0
1
1
0
0
1
1
0
Res. J. Biol., 2014 [2:84-98]
E-ISSN: 2322-0066
Figure 2: RAPD fingerprints of the 20 Aspergillus isolates grown on Waksman's
medium by the primers OPA10, OPA15, OPA1, OPO19 and OPO12.
93
Kamel, 2014
Table 6. Number and types of the amplified DNA bands and the percentage of the total polymorphisms generated by five
primers in Aspergillus isolates grown on Waksman's medium.
Polymorphic
Monomorphic
bands
Unique
Non-unique
Total bands
Polymorphic
%
OP- A10
3
4
7
14
78.5%
OP-A15
2
1
4
7
71.4%
OP-A1
5
3
6
14
64.3%
OP-O19
4
2
5
11
63.6%
OP-O12
1
1
10
12
91.9%
Total
15
11
32
58
------
Primer code
Table 5. Number and types of the amplified DNA bands and the percentage of the total polymorphisms generated by five
primers in Aspergillus isolates grown on Czapek Dox's medium.
Polymorphic
Primer
code
Monomorphic
bands
Unique
Non-unique
Total bands
Polymorphic
%
OP-A10
4
-
4
8
50.0 %
OP-A15
2
1
6
9
77.8 %
OP-A1
5
-
5
10
50.0 %
OP-O19
3
1
8
12
75.0 %
OP-O12
1
-
7
8
87.5 %
Total
15
2
30
47
-------
94
Res. J. Biol., 2014 [2:84-98]
E-ISSN: 2322-0066
Table 7. Amplified fragments obtained from Aspergillus isolates grown on Czapek Dox's medium from all primers.
,
Aspergillus isolates (OTU s)
No;
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
1
0 1 1 0 0 0 0 0 0
0
1
1
1
1
1
1
1
1
1
1
2
1 1 1 1 1 1 1 1 1
1
1
1
1
1
1
1
1
1
1
1
3
0 0 0 0 0 0 0 0 0
0
0
0
1
1
0
0
0
0
0
0
4
1 1 1 1 1 1 1 1 1
1
1
1
1
1
1
1
1
1
1
1
5
1 1 1 1 1 1 1 1 1
1
1
1
1
1
1
1
1
1
1
1
6
1 0 0 1 1 1 1 1 1
1
1
1
1
1
1
0
1
1
1
0
7
1 1 1 1 1 1 1 1 1
1
1
1
1
1
1
1
1
1
1
1
8
0 1 1 0 0 0 0 0 0
0
0
0
0
0
0
0
0
0
0
0
9
0 1 0 0 0 0 1 0 0
0
0
1
1
0
0
0
0
0
0
0
10
1 1 1 0 0 0 1 1 1
0
0
0
0
0
1
0
1
1
1
1
11
1 1 1 1 1 1 1 1 1
1
1
1
1
1
1
1
1
1
1
1
12
1 0 0 1 1 1 0 1 1
1
1
0
0
0
0
0
0
0
0
0
13
0 0 0 0 0 0 1 0 0
0
0
0
1
1
0
0
0
0
0
0
14
1 1 1 0 0 0 0 0 0
0
0
0
0
0
0
0
0
0
0
0
15
1 1 1 1 1 1 1 1 1
1
1
1
1
1
1
1
1
1
1
1
16
1 1 1 0 0 0 1 1 0
1
1
0
0
0
0
0
1
0
0
1
17
0 0 0 0 0 0 0 0 0
0
0
0
0
0
0
0
0
0
0
1
18
1 1 0 1 1 1 1 0 1
1
1
1
1
0
1
1
0
1
1
0
19
1 1 1 1 1 1 1 1 1
1
1
1
1
1
1
1
1
1
1
1
20
1 1 1 1 1 1 1 1 1
1
1
1
1
1
1
1
1
1
1
1
21
1 1 1 1 1 1 1 1 1
1
1
1
1
1
1
1
1
1
1
1
22
0 0 0 0 0 1 0 0 0
0
0
0
0
0
0
0
1
0
0
0
23
0 0 0 0 0 0 0 0 0
0
0
0
0
0
0
1
0
0
1
0
24
1 1 1 1 1 1 1 1 1
1
1
1
1
1
1
1
1
1
1
1
25
0 0 0 0 0 0 0 0 0
0
1
0
0
1
0
1
0
0
0
0
26
1 1 1 1 1 1 1 1 0
0
0
0
0
0
0
0
0
0
0
0
27
1 1 1 1 1 1 1 1 1
1
1
1
1
1
1
1
1
1
1
1
28
1 1 0 1 1 1 1 1 1
1
1
1
1
1
1
1
1
1
1
1
29
0 0 0 0 0 0 0 0 0
0
0
0
0
0
1
0
0
1
0
0
30
1 1 1 1 1 1 1 1 1
1
1
1
1
1
1
1
1
1
1
1
31
1 1 1 1 1 1 1 1 1
1
1
1
1
1
1
1
1
1
1
1
32
0 0 0 0 0 0 0 0 0
0
0
0
0
1
0
0
0
0
0
0
33
0 0 0 0 0 0 0 0 0
0
1
0
0
0
1
1
1
1
1
0
34
1 1 1 1 1 1 1 1 1
1
1
1
1
1
1
1
1
1
1
1
35
1 1 1 0 0 0 0 0 1
0
0
0
0
0
0
0
0
0
0
0
36
0 0 0 1 1 1 1 1 1
0
1
0
1
0
0
0
0
0
0
0
37
0 0 0 0 0 0 0 1 0
0
0
0
1
1
0
0
0
0
0
0
38
1 1 0 1 1 1 1 1 1
0
1
0
0
0
1
0
0
0
0
0
39
0 0 0 0 0 1 1 1 1
0
0
0
0
0
0
0
0
0
0
0
40
1 0 0 0 0 0 0 0 1
1
1
1
1
1
1
0
0
1
1
0
41
1 0 0 0 0 0 1 0 1
1
0
0
1
1
0
0
0
0
0
0
42
0 0 0 0 0 0 0 0 1
0
1
1
1
1
1
0
0
1
1
0
43
0 0 0 0 0 0 1 0 0
0
0
0
0
0
0
0
0
1
1
0
44
1 1 1 0 0 1 0 1 1
1
1
1
1
1
0
1
1
1
0
0
45
1 1 1 1 1 1 1 1 1
1
1
1
1
1
1
1
1
1
1
1
46
0 1 1 1 1 1 0 1 1
1
1
1
1
1
1
1
1
1
0
1
47
0 1 1 1 1 1 0 0 0
1
1
1
0
1
1
1
0
1
0
1
95
Kamel, 2014
Table 8. Amplified fragments obtained from Aspergillus isolates grown on Waksman's medium from all primers.
,
Aspergillus isolates (OTU s)
No;
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20
1
0 1 0 0 0 0 0 0 0
1
0
0
0
1
1
0
0
0
0
0
2
0 0 1 0 0 0 1 1 0
0
0
0
0
0
0
0
0
0
0
0
3
1 1 1 1 1 1 1 1 1
1
1
1
1
1
1
1
1
1
1
1
4
0 0 0 0 0 0 0 0 0
0
0
0
1
0
0
0
0
0
0
0
5
0 0 1 0 0 0 1 0 0
0
0
1
0
1
0
0
0
0
0
0
6
1 1 1 1 1 1 1 1 1
1
1
1
1
1
1
1
1
1
1
1
7
1 1 1 1 1 1 1 1 1
1
1
1
1
1
1
1
1
1
1
1
8
0 0 0 0 0 1 0 0 0
0
0
0
0
0
0
0
0
0
0
0
9
0 0 0 0 0 1 0 0 0
0
0
0
0
0
0
0
0
0
0
0
10
0 1 1 1 0 0 1 0 1
1
1
1
0
1
1
1
1
1
1
1
11
0 1 1 1 1 1 1 0 1
1
0
0
1
1
1
1
1
1
1
0
12
1 1 1 1 1 1 1 0 0
0
1
1
0
1
0
0
0
0
1
0
13
1 1 1 1 1 1 1 1 1
1
0
1
1
1
1
1
1
0
0
0
14
0 0 0 0 0 0 0 0 0
0
0
1
0
0
0
0
0
0
0
0
15
0 1 1 0 0 1 1 1 1
1
0
0
1
1
1
1
1
0
0
1
16
1 1 1 1 1 1 1 1 1
1
1
1
1
1
1
1
1
1
1
1
17
0 0 0 0 0 0 1 0 1
0
1
0
1
0
0
0
0
0
0
1
18
0 0 0 0 0 0 0 0 0
0
0
0
0
0
0
0
0
1
0
0
19
1 1 1 1 1 1 1 1 1
1
1
1
1
1
1
1
1
1
1
1
20
1 0 0 0 0 1 1 1 1
1
1
1
0
0
1
1
1
1
1
1
21
0 0 0 0 0 1 1 0 0
1
1
1
0
0
1
1
0
1
1
1
22
0 0 0 0 0 0 0 0 0
0
0
0
1
0
1
1
0
0
0
0
23
0 0 0 0 1 0 0 0 1
1
0
0
1
0
1
1
0
0
0
0
24
0 0 0 0 0 0 0 1 0
0
0
0
0
0
0
0
1
0
0
0
25
0 0 0 1 0 0 0 1 1
0
0
0
1
0
1
1
1
0
0
0
26
1 1 1 1 1 1 1 1 1
1
1
1
1
1
1
1
1
1
1
1
27
1 1 1 1 1 1 1 1 1
1
1
1
1
1
1
1
1
1
1
1
28
1 1 1 1 1 1 1 1 1
1
1
1
1
1
1
1
1
1
1
1
29
0 0 0 0 0 0 0 0 1
0
0
0
0
0
0
0
0
0
0
0
30
1 1 1 1 1 1 1 1 1
1
1
1
1
1
1
1
1
1
1
1
31
0 0 0 0 0 0 0 1 0
0
0
0
0
0
0
0
0
0
0
0
32
0 1 1 1 1 0 0 0 1
1
1
1
1
1
1
1
1
1
1
1
33
1 1 1 1 1 1 1 1 1
1
1
1
1
1
1
1
1
1
1
1
34
1 1 0 0 0 0 0 0 0
0
1
0
1
1
1
1
1
0
0
0
35
0 0 0 0 0 0 0 0 0
0
0
1
0
0
0
0
0
0
0
0
36
1 1 1 1 1 1 1 1 1
1
1
1
1
1
1
1
1
1
1
1
37
1 1 1 1 1 1 1 1 1
1
1
1
1
1
1
1
1
1
1
1
38
1 1 1 1 1 1 1 1 1
1
1
1
1
1
1
1
1
1
1
1
39
0 0 0 0 0 0 0 0 1
0
0
0
1
0
1
0
0
0
1
0
40
0 0 1 0 0 0 0 0 0
0
0
0
0
0
0
0
0
0
0
0
41
1 1 0 1 0 0 0 1 0
0
1
1
1
1
1
1
1
1
1
1
42
1 1 1 1 1 1 1 1 1
1
1
1
1
1
1
1
1
1
1
1
43
1 1 0 0 0 0 0 0 0
0
0
1
0
0
0
0
0
1
0
0
44
1 1 1 0 0 0 0 0 0
0
0
1
0
0
0
0
0
0
0
0
45
0 0 0 0 0 0 0 0 0
0
0
1
0
0
0
0
0
0
0
0
46
1 1 1 0 1 0 0 0 0
0
1
1
1
1
1
1
1
1
1
1
47
1 1 1 1 1 1 1 0 1
1
1
1
1
1
1
1
1
1
1
1
48
1 1 1 1 1 1 1 0 1
1
1
1
1
1
1
1
1
1
1
1
49
0 0 0 0 0 0 1 0 0
0
1
1
0
0
1
1
1
1
1
1
50
0 0 0 0 0 0 0 1 0
0
0
0
0
0
0
0
0
0
0
0
51
0 0 0 0 0 0 0 0 0
0
0
0
0
0
1
1
1
1
1
0
52
1 1 1 1 1 1 1 0 1
1
0
0
1
0
0
0
0
0
0
1
53
1 1 1 1 1 1 1 0 1
1
0
0
1
1
1
1
1
1
1
1
54
0 0 0 0 0 0 0 0 0
0
0
0
0
1
1
0
0
0
0
0
55
1 0 0 0 0 0 0 0 1
1
1
1
0
0
0
0
0
0
0
0
56
0 1 1 0 0 1 1 0 1
1
0
0
0
1
0
1
1
0
1
1
57
1 1 1 1 1 1 1 1 1
1
1
1
1
1
1
1
1
1
1
1
58
0 0 0 0 0 0 0 0 0
0
1
1
0
1
0
1
1
1
0
0
96
Res. J. Biol., 2014 [2:84-98]
E-ISSN: 2322-0066
97
Kamel, 2014
98