| Russula griseocarnosa is a valuable ectomycorrhizal edible and medicinal mushroom produced in Guangdong,Fujian and Yunnan,etc.,it is rich in nutrients and has remarkable health benefits,so is well received by the market.However,since its mycelium and fruiting body are strictly dependent on the host plant for growth,artificial domestication has not yet been achieved.It was extremely difficult to obtain the mycelial materials required for the research,the relevant basic biological research is stagnant,which seriously restricted the scientific research and industrial application of R.griseocarnosa.In this research,the fruiting bodies,mycorrhizae and soil microorganisms of R.griseocarnosa were studied for the symbiotic characteristics of R.griseocarnosa by means of modern molecular biology and highthroughput omics sequencing.1.Through whole-genome sequencing and gene annotation based on the Illumina and Pac Bio platforms of the fruit bodies of R.griseocarnosa,a more refined R.griseocarnosa genome was obtained,the genome was 50.67 Mbp,including 230 scaffolds and 14229 coding sequences.There were a large number of samples which collected from origin of R.griseocarnosa were found to be inconsistent with the ITS sequence of R.griseocarnosa based on comparative analysis of internal transcribed spacer(ITS)sequences,indicating that there were many similar species of R.griseocarnosa and it was difficult to distinguish them with the naked eye.The research on the genetic diversity of R.griseocarnosa showed that there was obvious genetic differentiation between groups in Guangdong and Fujian.The population haplotype network analysis showed that R.griseocarnosa in Guangdong,Fujian and Yunnan were clearly divided into two clusters,revealing the existence of gene exchange among the geographical groups;the variation significance analysis showed that the genetic variation of R.griseocarnosa in each geographical group mainly came from within the population.2.The ITS amplicon sequencing research was carried out on the fresh fruiting bodies of R.griseocarnosa,and a total of 18 genera of fungi were identified,in addition to the genus Russula,the fungi with high relative abundance were Hypomyces spp,Aspergillus spp,Cladosporium spp and so on.Moreover,the genera Purpureocillium,Neopestalotiopsis,Penicillium and Umbelopsis were found to exist in both fruiting bodies and mycorrhizal tissues by four consecutive years of isolation and identification,these endophytic fungi may play a role in promoting the growth and metabolism R.griseocarnosa.3.The comparative genomics study was carried out to explore their symbiotic-related genes and carbohydrase characteristics,but no small secreted proteins with symbiotic effects and small RNAs with symbiotic regulation and transport functions were found.Therefore,it is speculated that there may have more special symbiotic regulation mechanism in R.griseocarnosa.Compared with saprophytic fungi,hydrolase activity acted on glycosyl bonds,catalytic activity,serine Serine-type peptidase activity,transmembrane transporter activity,transmembrane transport,and extracellular components were significantly down-regulated in R.griseocarnosa,and key carbohydrate metabolism enzymes including glucokinase,phosphoribosyl pyrophosphate kinase,galacturonic acid reductase,galactokinase,α-galactosidase and so on were also lost,revealing the reason that R griseocarnosa is difficult to grow on artificial medium.The phylogenetic tree revealed that there was no clear correlation between the nutritional ecotype of fungi and their kinships and evolution.4.Sequencing and analysis of soil microorganisms showed that there were significant structural differences between soil microorganisms in the habitat of R.griseocarnosa and nonR.griseocarnosa.The relative abundance of Bradyrhizobium japonicum,B.elkanii,Chthoniobacter flavus,Ktedonobacter racemifer and Ktedonobacter sp.in the habitat soil of R.griseocarnosa were significantly higher than that of other Russula mushrooms and the control.Soil microbial function analysis showed that the Carbohydrate-Binding Module 19,which has chitin binding function was significantly increased in the habitat of R.griseocarnosa,and glycoside hydrolase,glycosyl transferase,carbohydrate lipase,glutamine synthase,Nit T/Tau T family transport system substrate-binding protein,DNA-guided RNA polymerase,maltose α-D-glucosyltransferase/α-amylase were significantly increased.Metabolic pathway analysis showed that propionate metabolism,cell cycle,and RNA polymerase-related pathways involved in energy metabolism were significantly increased,which may provide energy support for the rapid growth of R.griseocarnosa.5.The detection results of the nutrition and trace elements of R.griseocarnosa show that R.griseocarnosa was rich in protein,fat,essential amino acids and trace elements,of which the content of the arsenic was higher than that of general edible mushroom,and it is necessary to further clarify the compound form of arsenic and its possible role in the growth and establishment of symbiotic relationship of R.griseocarnosa.A total of 1422 metabolites were detected and annotated in the research on the mycorrhizal metabolomics of R.griseocarnosa,which were mainly carboxylic acids,lipids,steroids,and flavonoids.Functional analysis revealed that the host roots forming mycorrhizal tissue were more enriched in some metabolic pathways involved in energy,hormone signaling and immune defense,which pointed out the direction for revealing the symbiotic mechanism.This research obtained a relatively detailed whole genome sequence of R.griseocarnosa,which laid a data foundation for the subsequent symbiotic gene mining and domestication research of R.griseocarnosa.The endophytic fungi species of R.griseocarnosa were clarified,which provided the basis for the isolation and identification of R.griseocarnosa.Large-scale comparative genomics studies have revealed that key genes related to glucose metabolism were deficient in R.griseocarnosa which provided reference for mycelium separation of R.griseocarnosa.The research on mycorrhizae and habitat microbes of R.griseocarnosa revealed that the host plants and potential growth-promoting bacteria may provide enzymes related to glycometabolism for the growth of R.griseocarnosa,thus playing the role in symbiosis and growth promotion,which further provided a theoretical basis for artificial propagation and wildlike cultivation of R.griseocarnosa. |
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