Nucleotide Sequencing and Identification of Some Wild Mushrooms

The rDNA-ITS (Ribosomal DNA Internal Transcribed Spacers) fragment of the genomic DNA of 8 wild edible mushrooms (collected from Eastern Chota Nagpur Plateau of West Bengal, India) was amplified using ITS1 (Internal Transcribed Spacers 1) and ITS2 primers and subjected to nucleotide sequence determination for identification of mushrooms as mentioned. The sequences were aligned using ClustalW software program. The aligned sequences revealed identity (homology percentage from GenBank data base) of Amanita hemibapha [CN (Chota Nagpur) 1, % identity 99 (JX844716.1)], Amanita sp. [CN 2, % identity 98 (JX844763.1)], Astraeus hygrometricus [CN 3, % identity 87 (FJ536664.1)], Termitomyces sp. [CN 4, % identity 90 (JF746992.1)], Termitomyces sp. [CN 5, % identity 99 (GU001667.1)], T. microcarpus [CN 6, % identity 82 (EF421077.1)], Termitomyces sp. [CN 7, % identity 76 (JF746993.1)], and Volvariella volvacea [CN 8, % identity 100 (JN086680.1)]. Although out of 8 mushrooms 4 could be identified up to species level, the nucleotide sequences of the rest may be relevant to further characterization. A phylogenetic tree is constructed using Neighbor-Joining method showing interrelationship between/among the mushrooms. The determined nucleotide sequences of the mushrooms may provide additional information enriching GenBank database aiding to molecular taxonomy and facilitating its domestication and characterization for human benefits.


Introduction
Mushrooms are defined as "a macro fungi with a distinctive fruiting body" [1] and are traditionally used worldwide as nutritious food and as medicinal sources [2][3][4][5][6][7] including antioxidant activity [8,9]. Further, mushrooms are great recyclers and decomposers [10] and therefore play a significant role in the ecosystem. To date, about 3000 species are regarded as prime edible mushrooms [11]. Proper identification knowledge of edible mushrooms is essential for effective exploration in human benefits.
Molecular markers, PCR (Polymerase Chain Reaction) [12] and non-PCR based [13], are widely used for mushroom identification and characterization. However, direct sequencing of PCR product of repetitive nuclear DNA [14][15][16] of mushrooms is powerful tool for identification and phylogenetic studies [17]. The present study describes the nucleotide sequencing of 8 wild edible mushrooms collected from Eastern Chota Nagpur Plateau of West Bengal, India, using genomic DNA from fruit bodies. The rDNA-ITS (Ribosomal DNA Internal Transcribed Spacers) fragments of the genomic DNA were amplified using ITS1 (Internal Transcribed Spacers 1) and ITS2 primers. The nucleotide sequences of 8 mushrooms were matched from the available known sequences of GenBank database. The ITS region is rather useful for molecular characterization in fungi at the species level and within the species [11]. The objective of the work is to gain proper identification knowledge of the mushrooms, 2 The Scientific World Journal which may provide direction towards domestication and commercialization of the wild species for economic benefits apart from aiding molecular taxonomy.  [20]. Isolated DNA samples were checked in agarose gel (1% agarose gel prepared in TAE (Tris-acetate-ethylenediaminetetraacetic acid) buffer to which 2 L ethidium bromide was added) to confirm the yield of DNA, purity, and concentration. A ladder (1 kb plus) was loaded in the gel in order to compare the size of the isolated DNA samples.

Primer Designing.
Based on morphological identification of the samples, available nucleotide sequences ranging from 2 to 5 kb were downloaded from GenBank data base for primer designing. The FASTA format of all those sequences was arranged in a file and then the sequences were aligned according to their homology of nucleotide using ClustalW software program (http://www.ebi.ac.uk/Tools/msa/clustalw2/). The stretches of conserved sequences (ITS1 and ITS2) were considered for primer designing. The length of the primers was kept within 18 to 25 bp and the GC% was within 40 to 55% so that the melting temperature of the primers will not be high during PCR. It was also considered that the amplicon size should not cross more than 500 bp. Forward and reverse primers ( Table 1) for 8 mushroom samples were synthesized commercially (IDT, USA) for amplification of fungal DNA.

PCR Reactions.
Polymerase chain reaction was performed using the following: 10x PCR buffer 2.5 L, dNTPs (25 mM of each) 2.0 L, MgCl 2 (25 mM) 2.0 L, forward primer (10 pm/ L) 1.0 L, reverse primer (10 pm/ L) 1.0 L, DNA template 10 ng for each sample, Taq. polymerase (2 U/ L) 0.5 L, and DNase-and RNase-free water where volume was adjusted to 25 L. The PCR conditions were determined according to Taq. polymerase, primer pair, and amplicon size. The amplification reactions were performed in a DNA Thermal Cycler (Eppendorf AG, Hamburg, Germany) programed as follows: 1st cycle of 5 min at 95 ∘ C (initial denaturation) followed by 30 cycles of 45 sec at 95 ∘ C (denaturation), 30 sec at 50 ∘ C (annealing), 1 min at 72 ∘ C (extension), and 1 cycle of 10 min at 72 ∘ C (final extension). The final step was held at 4 ∘ C. The PCR products were stored at −20 ∘ C and subsequently run in agarose (Hi-media, USA; in TAE buffer) gel (1%). PCR product (10 L) was loaded in the gel by adding 6x DNA loading dye along with 2-log DNA ladder. The gel was run at 50 volts for 1 hour and the amplified products were visualized in the UV trans-illuminator and photographed in Gel Doc system (Bio-Rad, USA).

2.5.
Sequencing. The PCR products were loaded in the coupled lane of low melting point agarose gel (Sigma, USA). The run was at 40 volts for 1 hour and the bands were cut using sterile blade. The DNA was extracted from the agarose using columns (QIAGEN, Germany). This DNA was quantified The Scientific World Journal 3

Evolutionary Relationships Analysis.
Following alignment of the nucleotide sequences, a phylogenetic tree was constructed using the Neighbor-Joining method [21]. The optimal tree is drawn with the sum of branch length equal to 1.4246. The tree is drawn to scale, with branch lengths in the same unit as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Maximum Composite Likelihood method [22] and are in the unit of the number of base substitutions per site. The analysis involved 8 nucleotide 4 The Scientific World Journal

Results
The size of the DNA of 8 mushroom samples was around 15 kb. The PCR amplification products (Figure 1) showed that CN 1 and 2 gave around 200 to 250 bp amplified band, while CN 3 and 6 had 300 bp amplified band. CN 4, 5, and 7 documented a band around 500 bp, while CN 8 showed 400 bp band. These PCR products were gel purified, run in 1% agarose gel, and processed for nucleotide sequencing.
The partial nucleotide sequences ( Table 2) of 8 samples were obtained and analyzed for Basic Local Alignment Search Tool (BLAST) search program (National Center for Biotechnology Information (NCBI) site) against the whole GenBank data base of nucleotide sequences (using ClustalW software) for identification. Each PCR product was sequenced using forward and reverse primers and both were used for BLAST. The BLAST result is presented in Table 3. Based on % identity, mushrooms may be identified as follows:  (Figures 2(a)-2(h)).
Phylogenetic tree (Figure 3) constructed revealed a close relationship between CN 1 and 2 (A. hemibapha and Amanita

Discussion
The nucleotide sequences of 8 mushroom samples were blasted against available sequences from GenBank data base for identification.    view CN 4 and 5 were identified as Termitomyces spp., and these samples matched morphologically T. clypeatus and T. eurhizus [25], respectively. Tang et al. [26] also characterized Termitomyces species (T. longiradicatus, T. quilonensis, and T. poonensis) different morphologically from India. From phylogenetic tree, V. volvacea showed distinct relationship to A. hemibapha and Amanita sp. although the fungi belong to different families and therefore the finding is rather interesting from taxonomic point of view and may further be explored. The present study possibly suggests that GenBank data base for mushroom is not sufficiently rich in India. However, molecular characterization of 4 mushroom samples up to species level is performed and is essential and the provided nucleotide sequences of the rest of the samples may be relevant to GenBank data base for further exploration in the field.
Rajaratnam and Thiagarajan [11] extracted genomic DNA from the fruit body of wild mushroom and subjected it to nucleotide sequencing using ITS1 and ITS4 conserved primer stretches. The sequence was aligned using Jukes-Cantor Correlated Distance model and the aligned sequence (559 bp) revealed 88% matched score with Perenniporia sp. (GQ982890.1). Dung et al. [27] characterized 6 Oyster mushroom samples based on both morphological and molecular data and found that both were corroborating to each other. Lee et al. [16] performed identification of 3 medicinal mushroom (Ganoderma lucidum, Coriolus versicolor, and Fomes fomentarius) species from Korea based on nuclear large subunit rDNA sequences. Nucleotide sequencing of Termitomyces albuminosus [28], Ganoderma lucidum [29], and Agaricus bisporus [30], among others was also performed.

Conclusion
Molecular identification of the fungal samples may enrich and provide additional information to mushroom biodiversity and GenBank data base resource aiding to molecular phylogenetic analysis. Further, identification knowledge may also be significant for human benefits.