Towards a molecular approach for the identification of fungal taxa that contain psilocybin
The classical taxonomic methodologies for determining taxa of mushrooms are based primarily on morphological traits. If the mushroom specimens are dried or pulverized, the morphological characteristics are frequently disguised or no longer apparent. Included among these fungi are known to produce psilocybin, which are hard to identify by their macroscopic features. Molecular techniques could help to overcome these problems. In this study, twenty samples of two genera, Psilocybe and Panaeolus, of fungi from genera known to produce psilocybin were analyzed. The profiles of random amplified polymorphic DNA (RAPD) produced by two primer sets were analyzed. Fingerprints of amplified fragment length polymorphism (AFLP) produced by five selective primer pairs were also analyzed. By the analysis of genetic similarity, RAPD and AFLP method were capable of providing genus and species information. AFLP band patterns can provide reliable species test and can be separated in a simple way using PAGE electrophoresis and stained by silver nitrate. The sequencing data of twenty psilocybin-producing mushrooms were established by fluorescent sequencing method on the nuclear small subunit ribosomal DNA (nuc-ssu-rDNA) and the internal transcribed spacer 1 (ITS-1) DNA regions. The sequences of 877 bp DNA fragment of nuc-ssu-rDNA exhibited genus specific DNA sequences. The size (307-344 bp) and sequences variation of ITS-1 showed not only genus-specific but also species-specific DNA motifs. In the results of RNA gene analysis, the internal transcribed spacer DNA has much faster evolving sequences than the small subunit rDNA. A simple method was developed to perform the genus identification using a primer that is specific to either members of the Psilocybe or Panaeolus genus. A second primer was used to amplify a common product. This multiplex PCR was successfully developed for the quick screening of a large number of samples for identification of the genus. The SSCP pattern of the common product is sufficient to reveal the species that was present. If dye-labeled primer was used, the accurate size of common product is also a valuable evidence of species. If an automatic sequencer is available in the lab, the best method of species and genus identification is sequencing the RNA gene. The multiallele DNA fragments of ITS-1 DNA in Panaeolus subalteatus and Psilocybe semilanceata gave strong evidence in species identification. By the phylogenetic analysis of sequence in nuclear small subunit rDNA and the variable internal transcribed spacer 1, even the other species (not included in this study) of the same genus can be easily determined their relationship to the fungal genera known to produce psilocybin. This study developed DNA profiling methods for the identification of members of the genera Psilocybe and Panaeolus, which can provide information not only in taxon determination but also in forensic identification.