Development of rapid DNA diagnosis system of poisonous mushrooms

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1. In order to develop a novel method of obtaining monokaryons for a mycorrhizal fiungus, Lyophyllum shimeji, monokaryotization of dikaryotic stodc culture via protoplast formation and regeneration was performed using 12 dikaryotic stocks. In conclusion, we successfully prepared monokaryotic stocks via protoplast monookaryotization, a technique that can be used to identify biological species of L shimeji.2. The cladistic analysis of the V4 domain sequences, performed by UPGMA methods, revealed that the twelve Lyophyllum shimeji strains and two L. decastes strains were tested in this study divided into two clusters. For mating compatibility tests, the preparation of monokaryotic stocks were prepared from dikaryon stocks by protoplast monokaryotization. According to mating compatibility tests between monokaryotic stocks belong to cluster 1 and 2, it is suggested that the biological species of strains belong to cluster 1 are different. from that of strains belong to cluster 2. We suggest that the L. shimeji strains tested in this study might. be contained the strains belonging to different biological species.3. Genomic DNAs isolated from fresh, baked, stir-fried, tempura-style and boiled fruiting bodies of flesh and heat-dry Lentinula edodes and fresh, canned and retorted Agaricus bisporus were used as templates for PCR reactions. About. 350 bp and 250 bp fragments could be amplified from genomic DNA of boiled mushroom for more than 120 minutes, and that of canned and retorted mushroom, respectively. Therefore, it is possible that DNA diagnostics is applicable for species identification of cocked mushroom.4. Species-specific identification of the cooked and fresh poisonous mushrooms that are major in Japan was performed using a real-time PCR system. Specific fluorescence signals were detected, and no non-specific signals were detected. Therefore, we succeeded in developing a species-specific test for poisonous mushrooms within 1.5 hours.