Species-specific identification of the major cooked and fresh poisonous mushrooms in Japan was performed using a real-time PCR system. Specific fluorescence signals were detected, and no nonspecific signals were detected. Therefore, we succeeded in developing a species-specific test for the identification of poisonous mushrooms within 1.5 h.

Nucleotide sequence and polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) analysis of the 3 0 -portion of the mitochondrial 16S RNA gene (rDNA) coding sequence were used to differentiate between flying fishes and other fishes that are frequently used in ago-noyaki production. In this study, we successfully distinguished between flying fishes and the other fishes by combining amplified DNA fragments with universally designed primers and digesting the PCR products with AfaI and MfeI restriction endonucleases. PCR-RFLP of 15 ago-noyaki, 2 noyaki, and 8 other processed flying-fish products were analyzed to authenticate flyingfish content among processed seafood products. After digestion of the PCR products with both enzymes, we found that all ago-noyaki and processed flying fish products contained either two or three DNA fragments of *200, 300, and 530 bp, and noyaki samples (which do not contain flying fish) contained only one fragment of *530 bp. Here, we present a new procedure to confirm the content of flying-fish meal in ago-noyaki.

Real-time polymerase chain reaction (PCR) analysis of the 3 0 -portion of the mitochondrial 16S RNA gene (rDNA) coding sequence was used to determine flying fish paste in ago-noyaki. We quantified the amount of flying fish paste in ago-noyaki samples using flying fishspecific primers (Tobi16SF3/Tobi16SR) and universal primers (Univ16SF2/Univ16SR2). Using real-time PCR of standard ago-noyaki, a standard equation was obtained (y = 1.08x -3.20; R 2 = 0.977). This equation was then used to estimate the relative flying fish paste contents of eight commercially available ago-noyaki and two similar products. These results verified that the ago-noyaki products that had already been labeled with the E-mark deserved this status.

In order to develop a novel method of obtaining monokaryons for a mycorrhizal fungus, Lyophyllum shimeji, monokaryotization of dikaryotic stock culture via protoplast formation and regeneration was performed using 12 dikaryotic stocks. From 6 dikaryotic stocks, a total of 120 monokaryons were isolated, and their mating compatibility was tested. Mating-compatible monokaryons were successfully derived from a dikaryotic stock (NBRC 100325), and monokaryons of only 1 mating type relative to the parental dikaryons were isolated from another 3 strains (MH01710, OK2L-1, and HY7L-1). We successfully prepared monokaryotic stocks via protoplast monokaryotization, a technique that can be used to identify biological species of L. shimeji. This technique could be used for breeding various mycorrhizal mushrooms, including Tricholoma matsutake, for which the preparation of monospore cultures is extremely diffi cult.

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