Study On Behavior Of Nuclei In Different Stages Of Life Cycle Of Flammulina Velutipes

Flammulina velutipes which is an edible and medicinal basidiomycete, belongs to Flammulina, family Physalacriaceae. Currently, Flammulina velutipes is very popular in the world. So far, the biological characteristic and medicinal value of Flammulina velutipes have been studied in the world; But, there were few studies on its life cycle. This study is about the whole behavior of nuclei in different stages of life cycle of Flammulina velutipes, the main contents and conclusions are as follows:1. Artificial cultivation of Flammulina velutipes in different developmental stages.In this study, we modify the cultivation conditions of Flammulina velutipes and then the different stages of development and drops of the spores were observed, then, we collect the spores of different strains to make the germination experiments. The experimental results were concluded that:the mycelia of Flammulina velutipes at the beginning of the formation were white, and later with the growing of the mycelia, the surface of the mycelium produces a small amount of brown pigment; When the mycelia of heterocaryon and part of homocaryon were matured, they were kinked, and then gradually formed the fruiting body. Basidiospores were produced on the heterocaryon fruiting bodies. The spore could germinate and developed into mycelium. A few primary mycelium could also develop into fruiting body, but the fruiting body is deformed, and do not produce spores.2. Anatomic features in different developmental stages of Flammulina velutipesThe temporary slide in different developmental of Flammulina velutipes staining with Congo red, the results showed that:Mycelia of the homokaryon strains had no clamp connection. And the basidium do not have sterigma. However, mycelia of the heterokaryon strain had clamp connection. Basidium of the heterokaryon strain had four sterigmata, and finally the spores falled off from the sterigmata. Both primary mycelium and secondary mycelium could produce homokaryotic oidia.Mycelia, fruiting bodies, and basidiospores in diffferent stages of life cycle of Flammulina velutipes were observed using fluorescence microscopy. The results showed that isolate of a single basidiospore was monokaryotic and had no clamp connections. In addition, parts of single-basidiospore isolates formed homokaryotic fruiting bodies. Probasidia of homokaryotic fruiting bodies started with one nucleus that underwent one round of mitosis, producing two nuclei upon complete development of the basidium. Thus, the homokaryotic fruiting bodies were sterile. Interestingly, a pair of sex-matched single-basidiospore isolates formed heterokaryotic mycelium via plasmogamy. Heterokaryotic mycelia had two nuclei, clamp connections, and formed heterokaryotic fruiting bodies. Probasidia of heterokaryotic fruiting bodies had two haploid nuclei. These two nuclei fused and formed a diploid nuclei. Subsequently, the diploid nuclei underwent meiosis and produced four haploid nuclei. Each of which migrated into four basidiospores respectively through sterigmata. Each basidiosporic nuclei underwent mitosis that produced two haploid nuclei. Thus, mature basidiospores of the heterokaryon were dikaryotic but homogeneous. Furthermore, both homokaryotic mycelia and heterokaryotic mycelia can produce uninuclear iodia.3. Study on life cycle of Flammulina velutipes using SNPs molecular markersAfter obtaining the sequences from the genomic library,17primers pairs were designed. A total of171SNPs were found in Flammulina velutipes. Flammulina velutipes samples in different habitats and different growth periods were amplification by PCR and sequenced using these primers. We analysis the gene sequence and gene map. At the same time we use SNPs technology to confirm the behavior of nuclei in Flammulina velutipes. The SNPs markers also confirm the heterokaryotize in the cultivation of homokaryon of Flammulina velutipes due to the pollution of spores or oidia from other stains.Based on our observation and data, we proposed the following life cycle of Flammulina velutipes. A spore geminated and developed into primary mycelium. A part of primary mycelium could develop into homokaryon fruiting body, the basidium had two nuclei, and do not produce sterigma. A pair of monokaryotic mycelia with compatible mating types formed heterokaryotic mycelium via plasmogamy. Heterokaryotic mycelia had two nuclei, clamp connection, and formed heterokaryotic fruiting bodies. The basidium of the heterokaryotic fruiting body had four sterigmata, each of which produced a basidiospore. Furthermore, both homokaryotic mycelia and heterokaryotic mycelia can produce uninuclear oidia.