| Observation of the micromorphological characteristics of the basidia and basidiospores of Agaricus bisporus is helpful in understanding the developmental process and characteristics of its individual organs.In this paper,optical microscopy、confocal microscopy and scanning electron microscopy were used to observe the micromorphological characteristics of basidia and basidiospores at different stages of development in Agaricus bisporus.The results showed that the mycelium of Agaricus bisporus had polymorphism,according to the morphology of mycelia,they can be divided into creeping mycelia inside the base,aerial mycelia outside the base,spiny aerial mycelia outside the base,and spiral aerial mycelia.The basidia grows closely together and the proportion of different types of basidia in the gill at different developmental stages is not sustained,In other words,the ratio of sporobasidium of 3 spores at the stage of endomycetosis was higher than that at the stage of maturity.In the early stage of development,wich is the top of the basidium was covered with membrane(the top membrane of basidium).The top membrane of the basidium and its nuclear phase changed as follows:the two nuclei fused in the middle of the basidium,and the nuclear phase of the basidium changed during its development.The formation of mononuclear cells migrated back to the top of the basidium and meiosis took place.Finally,four nuclei were formed at the top of the basidium.Sometimes,the second meiosis was different and the phenomenon of three nuclei appeared in the head of the basidia due to the non-synchrony of the secondary mitosis.After meiosis,an average of four nuclei migrated to the sporozoites.Microscopically-observed basidiospores were found and,for the first time,it was observed that the basidiospore walls have two layers.The basidiospores contain lipid droplets of uneven sizes.Hybrid breeding is the main breeding method at present.Since it is difficult to distinguish the homokaryon mycelium and heterocaryon mycelium,molecular markers are needed to distinguish them.This research according to the observation results of basidium under scanning electron microscope,basidiospores were collected from the fruiting bodies of bispora bispora at the early stage of development(the stage of endophore emergence),so as to improve the rate of obtaining homokaryotes from basidiospores,forty-two strains of monospore isolates with clear karyotype were screened by mycelium morphology,growth rate and fruiting rate.Taking the genome of 42 monocytogenes as the template,Finally,12 pairs of molecular markers that can accurately distinguish the homokaryon mycelium of agaricus bisporus were obtained.Fungal RLM gene,a transcription factor of the MADS family,has been confirmed to be regulated by abiotic stress and involved in the biosynthetic pathway that regulates the entry of carbohydrates into cell walls,while the expression of AbRLM gene in the development process of agaricus bisporus has not been studied.In this study,RT-qPCR technology was used to detect the expressions of 8 reference genes of agaricus bisporus at 5 developmental stages,3 different tissues and organs and 4 different temperatures,Using ΔCt method,GeNorm and NormFiner analysis of single gene,tow gene combination and three genes expression differences of stability analysis,and then use the most stable expression of genes for internal genetic analysis AbRLM genes in different development periods,different fruit body tissue,different temperature cultivation hypha.The results showed that 40s*Actin*Eif5 can be used as a stable expression reference gene at different developmental stages,and Eif5*Ubiquitin*PRL14 is the most suitable reference gene in the samples of different tissues.EF1*Ubiquitin*RPL14 gene combination can be used as the best internal reference gene in different temperature treatment of mycelium samples.AbRLM gene in primordium stage and 15℃ cultivated hypha obviously high expression,suggest AbRLM gene is closely related to primordium formation and differentiation. |
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