Establishment Of Core Collections And Analysis Of Substrate Degradation Ability Of Stropharia Rugosoannulata

Cultivation scale of Stropharia rugosoannulata is expanding year by year in China,because:1)it is crispy texture and rich in nutrition;2)it is able to use crop straw as a growth substrate and can be cultivated in winter idle fields,thus generating good ecological and economic benefits.In this study,Inter-Simple Sequence Repeat（ISSR）markers were used to analyze genetic diversity of 42 S.rugosoannulata strains.Bsed on it,a core collection of S.rugosoannulata was constructed.The substrate degradation abilities of different strains in the core collection were evaluated by combining extracellular enzyme activity,the content of degraded lignin and cellulose in rice straw etc.Furthermore,two strains with significantly different degradation ability were selected for transcriptome sequencing to elucidate the molecular mechanism underlying the difference in substrate degradation ability of S.rugosoannulata.The main results are as follows:1.Ten ISSR markers were used to analyze the genetic diversity of 42 S.rugosoannulata strains,and were to construct a core collection of S.rugosoannulata.The number of polymorphic loci（A）,observed number of alleles（Na）,effective number of alleles（Ne）,Nei’s gene diversity index（H）,and Shannon information index（I）were 102,1.93,1.52,0.27,and 0.44 respectively,indicating that the tested strains was relatively rich in genetic diversity.Cluster analysis divided the 42 strains into two groups.Core3 and Core4 were constructed by gradually clustering priority sampling sites and Power Core software,respectively.Core5 was finally determined by combining Core3 and Core4 based on different sampling weights and proportions,consisting of 14 strains.2.Mycelial growth rate of S.rugosoannulata in rice straw plates,activities of five extracellular enzymes（laccase,carboxymethyl cellulase,filter paper enzyme,xylanase,and peroxidase）,and lignin and cellulose contents of rice straw after degradation,were comprehensively measured in the core collection.Based on these eight indexs,the substrate degradation ability of S.rugosoannulata core collection was evaluated.Strong degradation ability strains included SA05,SA09,SA22,SA24,SA26 and SA44,and weak ones were SA07,SA17,SA19 and SA48.3.Two S.rugosoannulata strains,SA44 with strong degradation ability and SA07 with weak degradation ability,were selected and their transcriptomes were sequenced at three time points（7d,14d and 21d）during growth on rice straw media.Transcriptomic analysis identified 2200,1672 and 2037 differentially expressed genes between the two strains at each of the three stages.Combined with the process of lignocellulose degradation,the molecular mechanism underlying the difference in substrate degradation ability between S.rugosoannulata strains was elucidated.Laccase and cytochrome P450 initiate redox reactions that generate free radicals to degrade lignin.Genes encoding these two enzymes were differentially expressed between the two strains,with SA44 showing a higher number of significantly upregulated genes than SA07.Therefore,SA44 is capable of producing more free radicals to degrade lignin.In the course of lignin depolymerization,some intermediate products provide substrates for alcohol oxidase to produce H₂O₂.Meanwhile,glucose oxidase and glyoxal oxidase oxidize corresponding small-molecule substrates to generate H₂O₂.Among the genes encoding these three enzymes,SA44 showed a higher number of upregulated genes.Therefore,SA44 can produce more H₂O₂,which provides substrates for manganese peroxidase and versatile peroxidase to generate free radicals with specific substrates and further degrade lignin.Meanwhile,genes encoding manganese peroxidase and versatile peroxidase were found to be differentially expressed between the two strains,with SA44 showing a higher number of upregulated genes.This allows SA44to continuously produce more free radicals to degrade lignin than SA07,and to increase the contact chances of carbohydrate enzymes with cellulose and hemicellulose.Thus glycolysis reaction in SA44 is stronger than that in SA07.While these oxidation reactions generate reactive oxygen species to degrade lignin,they also cause damage to S.rugosoannulata themselves.Here,27 toxin metabolism-related genes that are differentially expressed genes between strains were identified,with SA44 showing a higher number of significantly upregulated genes than SA07.In addition,a large number of genes related to DNA fragmentation,replication,and repair were enriched in SA07,indicating that SA44 is better able to cope with the damage caused by reactive oxygen species compared to SA07.DNA damage will affect the normal life activity of SA07,thereby affecting its degradation ability.In this study,utilization efficiency of S.rugosoannulata germplasm resources was improved by constructing a core collection.The degradation abilities of strains within the core collection were evaluated,and a batch of excellent strains were screened for practical production,which could improve the production efficiency of S.rugosoannulata and promote the recycling of agricultural and forestry waste such as rice straw and stalks.Analyzing the molecular mechanisms underlying differences of substrate degradation ability of S.rugosoannulata would lay a foundation for a comprehensive understanding of substrate degradation mechanisms in edilbe mushrooms.