Aspergillus Dna (RAPD) Markers for Genetic Analysis Nahid Aiat

Journal of Applied Sciences Research, 2(10): 709-713, 2006
© 2006, INSInet Publication
Genetic Variability among Three Species of Aspergillus 2. Random Amplified Polymorphic
Dna (RAPD) Markers for Genetic Analysis
Nahid Aiat
Department of Botany, Faculty of Science, University of Banha, Egypt.
Abstract: Randomly amplified polymorphic DNA (RAPD) fingerprints were used to analysis genetic
relationships amongA. niger, A. flavus and A. parasiticus. Four arbitrary 5-base primers weresuccessfully used
to amplify DNA extracted from mycelium of these primers showed characteristic. RAPD fingerprinting of the
different Aspergillus species can be used to gain rapid and precise information about genetic similarities and
dissimilarities of different Aspergillus species. RAPD fingerprints of A. niger, A. flavus and S. parasiticus
revealed polymorphism in 37, 59, 51% of the analyzed Aspergillus sp. The frequency of genetic variability was
detected in three isolates of Aspergillus dependant, variations in threadlike structure (TLS) chromosomes,
conidial spores and aflatoxins type production. RAPD analysis appeared genetic variability in approximately
5% of the analyzed conidial dimensions, TLS and 50% of the aflatoxin typing between A. flavus and A.
parasiticus or A. niger. It was observed also that all morphologically abnormal growth on PDA medium.RAPD
fingerprints analysis appeared genetic variability between A. niger, A. flavus, A. parasiticus. The similarity
percent was 37% in A. niger, 58% in A. flavus and 51.5% in A. parasiticus according to the percent of PAF
(10/27; 16/27 and 14/27 respectively).
Key words: Aspergillus sp., PCR, RAPD
INTRODUCTION
Therefore this study was conducted to employ RAPD
analysis as sample molecular marker tool for the analysis
of A. niger, A. flavus and A. paeasiticus variations
molecular marker(s). Such marker(s) could be used in
genetic relationships of three isolates of Aspergillus.
Williams et al. [16] and Welsh and McClelland[15]
demonstrated the utility of single short oligonucleotide
primers of arbitrary sequence for the amplification of
DNA segments distributed randomly throughout the
genome. Welshand McClelland [15] showed that the pattern
of amplified bonds could be used for genome
fingerprinting and Williams et al. [16] showed that the
differences (polymorphisms) in the pattern of bands
amplifiedfrom genetically distinct individualsbehaved as
mendelian genetic markers (named RAPDs, for Random
Amplified Polymorphic DNA). Most of the published
studies on genetic characterization, detection of genetic
variations and gene mutations were concentrated on the
variations in chromosomes, isozyme polymorphism and
biochemical diversity. A single set of arbitrary-sequence
10 mers may be used for fingerprinting any species. The
many advantages of RAPD markers over RFLDs or
isozyme markers accelerated the adoption of RAPD
technology for the construction of genetic maps,
fingerprinting and population genetic studies[6]. Current
reviews of the applications of RAPD technology are
available[14].
Wöstemeyer and Kreibich[17], Sharma et al. [11],
Sharma[10] and Swelim (2005 a,b) recorded that the utility
of DNA markers as RAPD-DNA in detecting genetic
variability is well established for many phytopathogenic
fungi.
MATRIALS AND METHODS
Three species of Aspergillus (A. niger, A. flavus and
A. parasiticus) were isolated from seeds of Phaseolus
vulgaris cv. Kontender collected from market using agar
plate methods. Three species of Aspergillus were
identified in plant pathology Dep. Fac. of Agric., Ain
Shams Univ. (in the previous research No. 1). These
species differ in morphologically growth on PDA
medium, conidial dimensions, chromosome threadlike
structure (TLS) and Aflatoxin typing production. These
species of Aspergillus were used for RAPD PCR
analysis[7].
Isolation of genomic DNA: DNA isolation was
performed using theCTAB method of Doyle and Doyle[5].
The spores were collected from colony by pipeting 50 µl
of triton-X 100 up and down several times over the some
spot on the plate. The spore/triton-X100 mixed to 500 µl
of CTA buffer to 1.5 ml tubes using vortex (Disruptor
Genie) for 2 mins and incubated at 65ºC for 15 mins. The
suspension was then mixed with 237 µl of isopropanol
and 7.5 M NH 2 OAC into a new 1.5 ml tube using vortex
CorrespondingAuthor: Nahid Aiat, Department of Botany, Faculty of Science, University of Banha, Egypt.
709
J. Appl. Sci. Res., 2(10): 709-713, 2006
electrophoreis apparatus 1% agarose gel in TAE buffer
was prepared and a total sample volume of 6 µl (1 µl of
miniprep, 4 µl d-H 2O and 1 µl 6X loading dye) of each
nucleic acid extract was loaded in each well. The gel was
electrophoresed in 65 V for 1.5 hour and them stained
with ethidium bromide solution (10 mg/ml) for around
10-15 minutes. DNA was visualized on a UV
transilluminator (l= 254 nm) and photographed with on
UVP laboratory products Epichemi 11 Dark room, 3 UV
transilluminator pharmacia.
Table 1: Oligonucleotide sequences of the primers used.
Primer
Primers sequence
OPB2
TGATCCCTGG
OPB3
FATCCCCCTG
OPB6
TGCTCTGCCC
OPD5
TGAGCGGACA
1 min and thenincubated at 65ºC for 15 mins. Then added
500 µl of chloroform: 150 amyl alcohol 24:1 and mixed
by shaking and centrifuged 5 min at maximum speed and
theupper aqueous layer was transferred to a new sterilized
tube, 2/3 volume of 150 propanol and NH4OAC was
added and mixed by shaking the centrifugated for 5 mins
at maximumspeed. The pellet was washed carefully twice
with 500 µl of cold 70% ethanol, dried at room
temperature and resuspended in 20 µl distilled water.
DNA was purified by incubation the resuspended sample
at 37ºC for 30 mins with RNase (Boehringer Mannheim),
DNAconcentration was determined using electrophoresis
of 5 µl of sample along water serial dilutions to Lambda
DNA in 0.8 agarose[3].
RESULTS AND DISCUSSIONS
DNA samples preparation before RAPD-PCR
amplification was found crucial for fingerprint of three
species of Aspergillus. The DNA were extracted by using
CTAB extraction method. The yields of DNA was
determined spectrophotometrically as 12 µg/0.05 g of
tissues. The purity of DNA genome samples as indicated
by A max/Amin ratio was 1-8 and DNA quantity was
evaluated by agarose gel electrophoresis (Fig. 1).
The reproducibility of RAPD analysisis knownto be
highly influenced by experimental conditions. It is
therefore essential to optimize the PCR conditions to
obtain reproducible and interpretable results before going
on routine analysis. The PCR conditions for RAPD
analysis were optimized by investigating each factor
individually. This included genomic DNA quality and
concentration, prime r annealing and extension
temperature as well as denaturation time and temperature.
The optimized conditions were detailed in materials and
methods section. It was found that quality of genomic
DNA extracted as described here was a good template for
PCR amplification. However, treatments of DNA
with RNase gave sharp and clear amplification
products compared with untreated DNA. This may be a
result of inactivation of endogenous endonucleases.
Castiglione et al.[3] also reported similar observations.
PCR amplification: Amplification was performed in 10
µl react mixture containing2.0 µl template DNA (25 mg);
0.2 µl tag DNA polymerase (unit); 3.0 µl DNTPs (25 mol
of each dATP, dCTP, dGTP, dTTP), 3.0 µl HgCl2
(25 mM), 3.0 µl PCR buffer (10X), and 2.0 µl random
primer (10 p mole) (Table 1) and 16.8 µl H2O (d.w.). The
mixture was assembled on ice, overlaid with a drop of
mineral oil. The amplification was carried out in DNA
thermal cycler (MWG-BIOTECH Primuse) programmed
as follows. One cycle at 94ºC for 4 min and then 40 cycles
at 94ºC for 30 sec., 35ºC for 1 min and 72ºC for 2 min
(for dernaturation, annealing and extension respectively).
One cycle at 72ºC for 5 min, then 40ºC for 10 min
infinitive.
Gel electrophoresis analysis: All electrophoresis was
carried out using a pharmacia GN-100 submorine gel
Fig.1: Agarose gel electrophoresis 1% showing the integrity of DNA genome extracted from mycellium tissues using
CTAB method for RABD-PCR amplification
(A) DNA genome isolated form A. niger (1); A. Flavus (2) and A. paraiticus (3).
(B) RNase treated DNA (purified DNA).
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J. Appl. Sci. Res., 2(10): 709-713, 2006
Table 2: The four random primers that produced polymorphic bands useful for distinguish genetic variability among three species of Aspergillus.
Sequence of
DNA bonds
Size of
A. niger
A. flavus
A. parasiticus
Random primers
------------------PAF
--------------------------------- --------------------------------- -------------------------------------5`-3`
TAF
PAF
(bp)
PAF
APF%
MAF PAF
APF% MAF
PAF
APF%
MAF
OPB2
3
1350
+
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------TGATCCCTGG
9
750
3-Jan
8
3-Jan
8
+
3-Jan
8
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------480
+
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------OPB3
6
1625
+
7
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------GATCCCCCTG
11
850
+
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------650
6-Jan
10
+
6-Apr
+
6-May
6
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------565
+
+
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------315
+
+
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------150
+
+
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------OPB6
14
11
1725
+
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------TGCTCTGCCC
1350
+
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------1675
+
11-May 9
+
11-Jun 8
11-May 9
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------850
+
+
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------800
+
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------850
+
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------500
+
+
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------450
+
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------400
+
+
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------350
+
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------200
+
+
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------OPD5
9
7
1925
+
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------TGAGCGGACA
1675
+
7-Mar
6
+
7-May 4
7-Mar
6
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------800
+
+
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------650
+
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------630
-+
+
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------575
+
+
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------450
+
--------------------------------------------------------------------------------------------------------------------------------------------------------------------------------Total
43
27
10
27-Oct
33
16
16/27
27
14
14/27
29
TAF = Total amplification fragments
MAF = Monomorphic amplification fragments
PAF = Polymorphic amplification fragments or specific amplification fragments
Annealing temperature lower than 35ºC led to
generation of very crowded RAPD patterns, while
higher annea ling temperature gave insufficie nt
amplification products.
After optimization of the reaction conditions,
polymorphismamong the different species of Aspergillus
were detected using different random primers. RAPD
analysiswhich was performed as detailed in materials and
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J. Appl. Sci. Res., 2(10): 709-713, 2006
Fig. 2: RAPD products obtained by PCR amplification of DNA isolated A. niger; A. flavus, A. parasiticus (Lones
1,2,3) using to met random primers (OPB2, OPB3m OPB 3, OPD5) respectively. Marker is shown to the
left of figure
methods section, gave the best results of amplification,
expressedas average number of bandsper primer.Twenty
random primers screened (operon random primer) were
surveyed. For the reproducibility of RAPD patterns, two
independentexperiments were performed for each primer.
Repetition of the experiments using different DNA
samplesconfirmed the stability and reproducibility of the
results. Of the twenty random primers that were screened
in RAPD analysis for their ability to produce sufficient
amplification products, 4 random primers namely OPB2,
OPB3, OPB 6 and OPD5 weremore stable and reproducible
and gave sufficient polymorphism among A. niger,
A. flavus and A. parasitiens. Therefore we focused our
efforts on these primers. The distribution of the
polymorphic bandswhich were generated using 4 selected
random primers, among different species of Aspergillus
are summarized in Table (2) and Fig. (2). The results
revealed that, by using theprimer OPB 2 one-PAF band for
each species, 1350, 750 and 480 pb were detected in
A. niger, A. flavus and A. parasiticus respectively. With
the primer OPB 3 one-PAF specific bond (1625) was
detected in A. flavus and (850) was detected in
A. parasiticus. Meanwhile, three PAF bands (650, 315,
150, 150 bp) were detected in A. flavus and A. parasiticus
and 565 bp was detected in A. niger and A. parasiticus.
With the primer OPB 6 five PAF specific band, 1725,
1350, 650, 450 and 350 only were detected in
A. parasiticus, while one specific (PAF) band was found
in A. flavus. Meanwhile, five PAF bands 1675, 850, 500,
400 and 200 were found in A. niger and A. flavus. With
the primer OPD5 was found two specific bands (650 and
450 bp) in A. niger and one band (1925 bp) only in
A. flavus, one PAF band (1675 bp) in A. niger and
A. flavus while found three DAF bands (800, 630 and
575 bp) were found in both A. flavus and A. parasiticus.
The results of the present study gave preliminary
informative DNA-based makers for 3 species of
Aspergilus identification. Also optimization of physical
experimental conditions of PCR amplification are a
prerequisite for the performance of RAPD analysis.
This increases the reproducibility and efficiency of
RAPD as a molecular marker technique. The three
species of Aspergillus analyzed were selected from
different species isolated from bean seeds. However, only
random primer OPB 2 gave reproducible and very stable
results peculiar to the same specie from different
accessions. The other primers sometimes did not give
the exact fingerprints for cultivars from different
seeds. Accordingly, it may be suggested to use bulked
DNA samples of different species to eliminate
intraspecific variations.
Itis concluded thatdistinct RAPD fingerprints among
the different species were obtained when suitable primers
were used and PCR conditions were optimized. During
the past few years, numerous publications demonstrated
theutility of RAPD markers for the analysis of the genetic
diversity among species and within fungi populations
and plant populations, Bagheri et al.; Debener et al.;
Sedra et al. and Saker et al.; Sharma et al. and
Swelim[2,4,9,8,11,12,13].
The frequency of genetic variability was detected
in three isolates of Aspergillus dependant, variations
in threadlike structure (TLS) chromosomes, conidial
spores and aflatoxins type production. RAPD analysis
appeared genetic variability in approximately 5% of the
analyzed conidial dimensions, TLS and 50% of the
aflatoxin typing between A. flavus and A. parasiticus or
A. niger. It was observed also that all morphologically
abnormal growth on PDA medium.
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J. Appl. Sci. Res., 2(10): 709-713, 2006
9.
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