Oyster Mushrooms Species Differentiation Through Molecular Markers RAPD
Gustavo Graciano Fonseca,
Eliezer Avila Gandra,
Liana Fossati Sclowitz,
Ana Paula Antunes Correa,
Jorge Alberto Vieira Costa
Jose Alberto Levy
A RAPD methodology for differentiation and characterization
of the fungal edible species Pleurotus ostreatus and Pleurotus
sajor-caju was developed. Two of the most usual methods of DNA extraction
were studied. The first was based in the simultaneous precipitation of
proteins and polysaccharides by the presence of SDS (sodium dodecyl sulphate)
and sodium acetate and the second by the use of cetyltrimethylammonium
bromide (CTAB). Both methods presented satisfactory results and the best
extraction products were obtained by the one that used SDS and sodium
acetate. The study with RAPDs produced consistent DNA fragments, reproducible
in different gels, serving as good markers for characterization and genetic
differentiation of the studied species of mushrooms. The PCR conditions
for obtaining RAPD markers were optimized with good interpretation.
to cite this article:
Gustavo Graciano Fonseca, Eliezer Avila Gandra, Liana Fossati Sclowitz, Ana Paula Antunes Correa, Jorge Alberto Vieira Costa and Jose Alberto Levy, 2008. Oyster Mushrooms Species Differentiation Through Molecular Markers RAPD
. International Journal of Plant Breeding and Genetics, 2: 13-18.
The genus Pleurotus (Jacq.: Fr.) Kumm. (Pleurotaceae, higher
Basidiomycetes) is one of the most diverse groups of cultivated mushrooms
with high nutritional value, therapeutic properties and various environmental
and biotechnological applications. However evolutionary connection among
species in the genus Pleurotus is still not clear and many taxonomic
issues remain controversial (Cohen et al., 2000).
There are significant problems in classifying Pleurotus isolates using
only morphological characters (which are often unreliable or inconclusive
mainly due to the large influence exerted by environmental factors) or
compatibility experiments (which are based on the application of the controversial
biological species concept) (Zervakis et al., 2001). Moreover,
considering that starting point in the cultivation of an edible mushroom
is usually a pure culture or spores, the use of molecular tools is almost
essential to ensure that the inoculum used is from the correct species.
Molecular tools provide more accurate methods for identification than
the few characters afforded by traditional morphological features. These
tools will soon become widespread in following the survival of inoculant
fungi, detecting contaminant species, the degree of competition from contaminant
and presence of hyperparasites (Amicucci et al., 2001; Hall et
The random amplified polymorphic DNA technique (RAPD) (Williams et
al., 1990; Welsh and Mclelland, 1990; Manaf et al., 2006) has
some advantages, as the efficiency to generate a large number of markers
for genomic mapping without any previous knowledge about the organism
genetics, the requirement of small amount of DNA, the quickness, simplicity
and reproducibility in the data acquisition, the low cost and accessibility
of this technology and the potential automation, with the possibility
of being used in programs of quality control as HACCP (Hazard Analysis
Critical Control Point) for controlling and monitoring of critical points
of contamination in a process. The aim of this study was to optimize conditions,
in order to obtain high quality DNA extraction and develop the RAPD methodology
for differentiation and characterization of the edible mushrooms species
Pleurotus ostreatus and Pleurotus sajor-caju.
MATERIALS AND METHODS
The present research was conducted in the Laboratory of Marine Biochemistry,
from Federal University of Rio Grande, Brazil, during the year of 2007.
Pleurotus ostreatus and Pleurotus sajor-caju strains were
obtained from EMBRAPA (BrasÃlia, DF, Brazil) and UNESP (Botucatu,
SP, Brazil) in lyophilized form, preserved in malt extract agar at 4Â°C
and cultivated through culture methods in plate.
Methods of DNA Extraction
Two methods of DNA extraction were studied. The first, based on the method
proposed by Delidow et al. (1993) and Makimura (1994) (method 1),
is based on the simultaneous precipitation of proteins and polysaccharides
in the presence of SDS and high concentrations of sodium acetate. The
other method was proposed by Weising et al. (1995) and GonzÃ¡lez
and LabarÃ¨re (2000) (method 2) and is based on the use of the detergent
Molecular Markers RAPDs
Pleurotus ostreatus and Pleurotus sajor-caju were characterized
genetically by differentiation through the method RAPD consisting basically
of 3 stages: DNA extraction, PCR and PCR analysis of the products in electrophoresis
The PCR protocols also proceeded also proceeded, with certain modifications,
according to Delidow et al. (1993) and Cushwa and Medrano (1996).
The amplifications were carried out in a thermal cycler PTC-100/110V (MJ
Research, Inc.) adjusted for the program PCR-1 to 40 cycles for the complete
DNA synthesis. The method consisted of 3 stages: denaturation of the DNA
chain in simpler sequences of 92Â°C for 1 min, during regular cycles
(the initial denaturation was longer: 5 min); hybridization of the primers
to complemental sequence sites in the denatured DNA chain at 35Â°C
for 1 min and primers extension for the synthesis of the DNA complemental
sequences at 72Â°C for 2 min. The PCR reaction took 5 h and the reagents
concentrations, the temperature profiles in the thermal cycler and the
electrophoresis gel conditions according to Cushwa and Medrano (1996).
The tubes can be left at 4Â°C in the thermal cycler for indefinite
time or stored in freezer at -20Â°C, before the electrophoresis gel
analysis, being added shipment lid with EDTA (loading dye) to inhibit
any action of DNAses on the PCR products (Ferreira and Grattapaglia, 1996).
RAPD-PCR products were analyzed through electrophoresis driven in gel
of agarose 1.5%. The bands were visualized with ethidium bromide in ultra-violet
light, interpreted and transformed into molecular diagnoses data among
individuals (Cushwa and Medrano, 1996). The banding profiles generated
by RAPD-PCR were analyzed with the RAPDistance program (Armstrong et
al., 1996). The analysis of the diagnoses data was made starting from
the presence or absence of bands at a specific molecular weight.
RESULTS AND DISCUSSION
The DNA extracted from dried materials, such as root, stem or fruit is
often contaminated with proteins, polysaccharides and secondary metabolites,
which decrease the reproducibility of method RAPD. Figure 1 shows P.
ostreatus and P. sajor-caju DNA extraction results for the
two tested methods. The method 1 allowed a more efficient DNA isolation
of the fungal species. Although the good quality extraction presented
in the method 2, it was not possible for this method to eliminate the
RNA, as verified in the inferior part of gel (Fig. 1).
In this study, a combination of methods reported by Delidow et al.
(1993) and Makimura (1994), based in the simultaneous precipitation of
proteins and polysaccharide in the presence of SDS and high concentrations
of sodium acetate, allowed a more efficient DNA extraction (method 1).
It was possible to isolate a high purified DNA that certainly permitted
a better analysis of the RAPD-PCR products.
DNA fingerprint patterns might be useful in identifying the species and
as an aid to quality control (Cheng et al., 2000). Capelari and
Fungaro (2003) analyzed the genetic variability by RAPD of isolates of
Pleurotus cystidiosus and Pleurotus smithii which indicated
that the criteria used to separate the two species are unsatisfactory
and that P. smithii should be considered a synonym of P. cystidiosus.
Shnyreva et al. (2003) studied P. ostreatus and Agaricus
bisporus, using molecular markers for differentiation, which allowed
differentiating groups of genetically similar and distant strains. P.
ostreatus strains showed a higher genetic variation while A. bisporus
strains showed a higher level of homology.
In this study, utilizing the RAPD methodology, 19 different primers were
tested for the two species of mushrooms in study, being obtained a large
number of DNA fragments (bands) consistent and reproduced in several gels.
Among the tested primers, 6 (AC-04, AD-08, AD-09, B-14, D-20, G-02) produced
amplifications for both species allowing the differentiation of the same
ones, 5 (S-03, D-06, G-07, G-08, B-15) produced amplifications only for
Pleurotus ostreatus, the primer S-17 produced amplifications only
for Pleurotus sajor-caju and 7 primers (AC-09, B-02, B-05, B-07,
D-05, G-06, G-09) did not amplify any fragment.
The number of amplification products for each primer varied from 1 to
12, totaling 87, being that 77 were polymorphic and 10 were monomorphic
(present in both species) (Table 1). The analysis of
the data obtained by RAPD methodology was based on the absence or presence
of bands from the electrophoresis gel in ultra-violet light. The size
of the amplified product ranged from 154 to 4072 bp.
||Results of the extractions of DNA
Results of the RAPD methodology in terms of presence
or absence of amplification, polymorphism and number of detected
bands for each tested primer and oyster mushroom species
Representation of products of PCR-RAPDs visualized
in ultra-violet light
The percentages of GC in the utilized primers (60% and 70%) did not influence
the amplification patterns. The high polymorphism with markers RAPD is
probably due to the preferential amplification of repetitive areas of
the genome (Fig. 2).
The methodology RAPD produced consistent fragments of DNA, which were
reproducible in different gels. However, the fragments amplification from
different similar sizes that occupy the same position in a gel after electrophoresis
can happen, but the risk of erroneous interpretations of these fragments
with similar size is minimized by the use of several primers, such a way
that the genetic analysis is based on a high number of markers RAPD. The
analysis based on RAPD-PCR fingerprinting obtained in this study was clear
enough to allow discrimination, characterization and differentiation of
the fungal species P. ostreatus and P. sajor-caju.
The authors thank Maria de Jesus Pinto Lamego for the helpful technical
Amicucci, A., A. Zambonelli, C. Guidi and V. Stocchi, 2001. Morphological and molecular characterisation of Pulvinula constellatio ectomycorrhizae. FEMS Microbiol. Lett., 194: 121-125.
Direct Link |
Armstrong, J., A. Gibbs, R. Peakall and G. Weiller, 1996. RAPDistance Programs, Version 1.04 for the Analysis of Patterns of RAPD Fragments. 1st Edn., Australia National University, Canberra.
Capelari, M. and M.H. Fungaro, 2003. Determination of biological species and analysis of genetic variability by RAPD of isolates of Pleurotus subgenus Coremiopleurotus. Mycol. Res., 107: 1050-1054.
Direct Link |
Cheng, K.T., H.C. Chang, H. Huang and C.T. Lin, 2000. RAPD analysis of Lycium barbarum medicine in Taiwan market. Bot. Bull. Acad. Sin., 41: 11-14.
Direct Link |
Cohen, R., L. Persky and Y. Hadar, 2002. Biotechnological applications and potential of wood-degrading mushrooms of the genus Pleurotus. Applied Microbiol. Biotechnol., 58: 582-594.
CrossRef | Direct Link |
Cushwa, W.T. and J.F. Medrano, 1996. Applications of the Random Amplified Polymorphic DNA (RAPD) assay for genetic analysis of livestock species. Anim. Biotechnol., 7: 11-31.
Delidow, B.C., J.P. Linch, J.J. Peluso and B.A. White, 1993. Polymerase Chain Reaction-Basics Protocols. In: Methods in Molecular Biology, PCR Protocols: Currents Methods and Applications, White, B.A. (Ed.). Humana Press Inc., Totowa, NJ.
Duman, M., B. Patir, E. Duman and O.I. Ilhak, 2007. The effects of salt and storage temperature on Microbiological changes in hot-smoked mirror carp (Cyprinus carpio L.). Pakistan J. Biol. Sci., 10: 3002-3005.
CrossRef | PubMed | Direct Link |
Ferreira, M.E. and D. Grattapaglia, 1996. Ao Uso De Marcadores Moleculares Em Análise Genética. 2nd Edn., EMBRAPA-CENARGEN, Brasília, pp: 220.
Gonzalez, P. and J. Labarere, 2000. Phylogenetic relationships of Pleurotus species according to the sequence and secondary structure of the mitochondrial small-subunit rRNA V4, V6 and V9 domains. Microbiology, 146: 209-221.
Hall, I.R., W. Yun and A. Amicucci, 2003. Cultivation of edible ectomycorrhizal mushrooms. Trends Biotechnol., 21: 433-438.
Makimura, J., 1994. II. Rapid extraction of DNA from mold. J. Med. Microbiol., 40: 358-364.
Manaf, S.R.A., M. Mustafa, N.M. Amin and A.M. Ali, 2006. Genetic relatedness among isolates of Acanthamoeba based on RAPD analysis. J. Applied Sci., 6: 15-19.
CrossRef | Direct Link |
Shnyreva, A.V., I.S. Belokon' and M.M. Belokon', 2003. Use of molecular markers for differentiation of cultivated strains of oyster and button mushrooms. Genetika, 39: 1461-1469.
Direct Link |
Weising, K., H. Nybom, K. Wolff and W. Meyer, 1995. DNA Fingerprinting in Plants and Fungi. 1st Edn., CRC Press, Boca Raton, FL., USA., ISBN: 0-8493-8920-8.
Welsh, J. and M. McClelland, 1990. Fingerprinting genomes using PCR with arbitrary primers. Nucleic Acids Res., 18: 7213-7218.
CrossRef | PubMed | Direct Link |
Williams, J.G.K., A.R. Kubelik, K.J. Livak, J.A. Rafalski and S.V. Tingey, 1990. DNA polymorphisms amplified by arbitrary primers are useful as genetic markers. Nucl. Acids Res., 18: 6531-6535.
CrossRef | PubMed | Direct Link |