Abstract
The isolation of high quantity and intact DNA is of a great significance for molecular identification of higher fungi. The aim of this study was to compare two DNA extraction methods for isolation of DNA from medicinal mushrooms of Agaricomycetes class. A modified CTAB method and a modified SDS method were compared by the yield and purity of the extracted DNA, its fragmentation state and suitability for amplification. The results demonstrated high efficiency of both methods in regard to DNA yield (14.18 -144.28 ng DNA/mg biomass with CTAB method and 15.03 -108.34 ng DNA/mg biomass with SDS method). The CTAB method provided DNA extracts with higher purity (A260/A280 ranged from 1.83 to 1.99) in comparison with the SDS method (A260/A280 = 1.53 -1.86). The modified CTAB method produced amplifiable DNA from all mushroom isolates, while the SDS method demonstrated suitability for amplification only in 50% of the samples. Therefore, the modified CTAB method could be the method of choice for DNA extraction from medicinal mushrooms. The analyzed isolates were subjected to molecular identification by ITS1-5.8S-ITS2 rRNA gene sequence analysis and were identified as Ganoderma resinaceum, Trametes versicolor, Fomitopsis pinicola and Inonotus hispidus.
Highlights
Since ancient time, mushrooms are of interest as a source of nutrients and medicines [1]
The isolation of high quantity and intact DNA is of a great significance for molecular identification of higher fungi
1.90 ± 0.05 1.54 ± 0.04 1.99 ± 0.01 1.83 ± 0.03 1.99 ± 0.01 1.76 ± 0.08 1.91 ± 0.08 1.58 ± 0.01 1.83 ± 0.05 1.53 ± 0.05 1.99 ± 0.01 1.71 ± 0.03 1.97 ± 0.03 1.84 ± 0.01 1.96 ± 0.01 1.86 ± 0.05 were compared by the yield and purity of the extracted DNA
Summary
Mushrooms are of interest as a source of nutrients and medicines [1]. Medicinal mushrooms most of which belong to the higher basidiomycetes are an exceptionally diverse group part of the fungal kingdom. The secondary metabolites of the basidiomycetes from genera Agaricus, Auricularia, Phellinus, Ganoderma, Pleurotus, Trametes and Lentinus and their biological activities have seen a marked main reason for the increased scientific interest regarding their application [2]. Because of their high sensitivity and specificity, the PCR based molecular techniques has become a preferable method for identification and characterization of medical mushrooms [3]. The research of population genetics and biodiversity of mushroom species rely on DNA sequences obtained by PCR [7]
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