| Nuclear phase and genetic attribute of the basidiospores were investigated for 3 strains of Flammutina velutipes. Fluorescence staining observation displayed that the majority of basidiospores were dikaryotic and the ratio of dikaryotic spores, monokaryotic ones and nuclear-free ones was 80.2%, 7.5% and 12.3% respectively. The mycelia derived from single spore isolates were confirmed as polykaryotic septate ones without clamp connection.The suitable dyeing conditions of 3 kinds of different fluorescent dyes for the basidiospore and mycelia were investigated. The results indicated that for basidiospore, the suitable dyeing density of Hoechest33258 was 60μg/mL～80μg/mL when the dyeing time was 3min-5min. Similarly, the suitable dyeing density of DAPI was 6μg/mL when the dyeing time was 3min～5min. The suitable dual dyeing condition of mycelia was that the mycelia was dyed for 4 min by 70μg/mL Hoechst33258, before dyed for 4 min by 3μg/mL Fluorescent Brightener28.The suitable protoplasting conditions were preliminary studied. The results indicated that the suitable enzyme dissolve temperature and time was 30℃and 3h respectively, when the Lywallzyme concentration was 2% and the applied amound was 2mL/1g mucelium.In the mating test, mycelial protoplast pairs between single spore isolates of different strains formed colonies with clamp connection, while mycelial protoplast pairs between the same single spore isolates formed colonies without clamp connection, suggesting that the two nuclei of the spores possessed identical mating type factors.RAPD analysis indicated that the profiles of mycelial protoplasts obtained from same single spore isolates amplified by 10 random primers were entirely identical, corroborating that the two nuclei of a basidiospore were homogeneous.In addition, it is observed that there are 4 basidiospores growing on a basidium.Consequently,F. velutipes is a tetrapolar fungus possessing 4 basidiospores where there are two homogeneous nuclei. |
