| Sarcomyxa edulis belongs to Basidiomycota,Mycenaceae,Sarcomyxa,which is a low-temperature fungus characteristic of Northeastern China.Artificial cultivation is available now for this fungus.It is an important edible and medicinal fungal resource in China that has a delicioustaste,rich nutrition,a variety of medicinal values and broad prospects for research,development and utilization.However,with the continuous deterioration of the environment,its wild resources are decreasing at an alarming rate.Therefore,strengthening research on the conservation,utilization and genetic diversity of its germplasm resources is in urgent need..Up to now,domestic research on S.edulis has focused mainly on the basic cultivation and determination of the chemical composition.Studies on omics and related traits of S.edulis,such as bitterness characteristics,have not been reported.In this study,wild and cultivated germplasms of S.edulis were collected,identified and evaluated.The genome of S.edulis was obtained based on SMRT technology de novo sequencing.Further studies evaluating the genetic diversity and population structure of 26 representative germplasms which have differences in phenotype,quality,flavor,disease resistance,etc.were analyzed using morphological and genotype methods(SSR markers and SNP markers).The diversity in the germplasm was examined comprehensively in the context of geographical distribution and cultivation features.The study aimed at evaluating the existing germplasms of S.edulis to provide excellent variety resources and references for breeding and germplasm innovation.Based on the cultivation and assessment of the nutritional quality traits of the 26 representative S.edulis in this study,the different germplasms of S.edulis had different degrees of bitterness which directly affected mushroom quality and commodity value.In order to gain insight on the formation of bitterness,based on the genetic diversity analysis,two strains with long genetic distance and distinct bitterness difference were selected.The variation and expression of genes related to the triterpenoid synthesis pathway,S.edulis germplasms with different levels of bitterness were compared at the transcriptional expression and genome-wide level,respectively.The study provided support for future studies on the phylogenetic relationship and protein interaction of bitter genes in S.edulis which is of guiding significance to clone bitter genes of edible and medicinal fungi and to breed non-bitter varieties.The main results of this study are as follows:1.Results of collection and identification of S.edulis germplasms: In this study,a total of 252 wild and cultivated samples were obtained from herbarium and 25 collection sites in Northeast China,and 229 strains were isolated.It was identified that except for the mycelial strains that were not isolated and a strain with unclear taxonomic status,the other 228 strains were all S.edulis.The germplasm resources of S.edulis were primarily established.In the process of germplasm collection,pathogens of S.edulis were also surveyed.A Green Mold disease that affected the host was identified.To our knowledge,it was the first report of the occurrence of Trichoderma pleuroticola on S.edulis in China.2.Evaluation of the diversity in the germplasm resources of S.edulis: The diversity of the S.edulis germplasms was analyzed by recording and evaluating 11 agronomic characters including the number of primordium,yield,flavor,disease resistance and so on.It showed that there were great phenotypic character differences among the germplasms,based on diversity indexes.It was also found that the number of primordia formed by each strain correlated not positively correlated with yield.A high-quality germplasm,T24,with high yield,short cycle and strong disease resistance was obtained.4 germplasm resources of S.edulis with different characters represented by T95,T21,T17 and T109,respectively were obtained.And four strains with bitterness represented by T115 was also identified by screening.3.Genome analysis and molecular evolution of S.edulis: The size of S.edulis SE1(HMJAU2016092521)genome was 35.65 Mb,and 41 contigs were obtained.N50 was 1772559 bp,G+C content was 48.31%,and 9364 protein-coding genes were annotated.S.edulis was enriched with genes encoding enzymes predicted to be involved in secondary metabolite biosynthesis,with 39 obtained by comparison.Four of them belonged to the Terpene gene cluster.The phylogenetic tree was constructed by single-copy homologous protein genes annotated with 33 typical genomes of fungi.The results supported the independent existence of Sarcomyxa,which was later than Pleurotus in evolution.4.Genetic diversity analysis of S.edulis germplasm resources: The genetic diversity of the germplasms was studied based on SSR markers and genome-wide resequencing(SNP makers).Ten pairs of SSR markers with high polymorphism were obtained.It was found that there was high polymorphism in SSR loci among the germplasms.The results of genetic diversity studies using both approaches were similar,and the differentiation degree of the wild type germplasm was higher than that of the cultivated type,and the genetic distance of the cultivated type germplasm was closer.The distribution of wild type T24 was special.After domestication and cultivation,the differences between the germplasms of S.edulis and its wild ancestor germplasms were obvious.5.Results on genes related to triterpene biosynthetic pathway in S.edulis: MVA pathway was found to be the main synthesis of IPP,a precursor of triterpenes in S.edulis.The important enzymatic genes of triterpene in the synthesis pathway of triterpene precursors and skeleton compounds were obtained from S.edulis,and their expression level were all up-regulated.There were structural differences in the gene sequences,which caused large numbers of non-synonymous mutations.A total of 82 P450 genes with significantly different expressions were obtained in the P450 modification after the triterpene synthesis pathway.Among them,21 genes were involved in the further oxygenation modification of the triterpene scaffold(?-amyrin scaffold).Moreover,it was found that there were more genes modifying the first product in T115 strain with bitterness,while the genes modifying the secondary product were more in the T184 strain with no bitterness.However,the amount in T184 strain with no bitterness was more after secondary modification.What's more,two homologous protein coding genes of cucumber for the formation of the first step gene Bi were found in S.edulis.Both genes were significantly up-regulated in strain T115 with bitterness.There was a large number of structural variations in the DNA sequence.The above results showed that the genes related to triterpenoid synthesis pathway in S.edulis might be related to its bitterness.In this study,the germplasm resources of S.edulis in northeast China were preliminarily established.The genome and transcriptome databases of typical strains were established.The genetic diversity of S.edulis germplasm resources were evaluated at the phenotype and genotype level.Also the bitterness characteristics of this mushroom was studied at omics the level for the first time.The results lay a foundation for the breeding,classification and cultivation of edible and medicinal mushroom. |
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