| Ganoderma tsugae is a precious species of cultivated Ganoderma in the Northeast China and has become an important source of health products.Compared with other Ganoderma species,such as G.sichuanense and G.sinense,wild G.tsugae is mainly distributed in Changbai Mountain and grows on coniferous wood,which has the advantage of using coniferous wood.Previous studies have focused on the isolation and pharmacological effects of secondary metabolites in G.tsugae.However,important biological processes such as phylogenetic evolution,genetic diversity,and genetic differentiation of G.tsugae are still unclear.At present,the NCBI database has published the genome of the G.tsugae strain(GCA＿003057275.1),but due to the use of Illumina sequencing technology,the assembly results are fragmented,with a total of6,742 contigs and the N50 length is only 11.7 kb,which hinders the study of functional genes in important biologically active compounds.Therefore,in this paper,the monokaryotic strains of the wild germplasm resources of G.tsugae were subjected to whole genome sequencing,and high-quality whole genome data of G.tsugae was assembled and compared with those of G.sichuanense,G.sinense and G.boninense to explore its gene structure,genetic information,and phylogeny to perform genomics differential analysis.Based on the whole genome data to develop SSR molecular markers,to provide resources for the genetic diversity research of G.tsugae;then,based on whole-genome resequencing analysis to further analyze the genetic structure and diversity of its population;finally,in the whole G.tsugae genome,the terpene synthase gene family was mined,and the SNP variation detection in the coding and regulatory regions of the terpene synthase gene was carried out to lay the foundation for further development of molecular markers and subsequent gene function verification.The main research contents are as follows:1.Whole genome sequencing and comparative genomic analysis of G.tsugae.The monokaryotic strain of wild G.tsugae in Changbai Mountains was obtained by the protoplast mononucleation technique.The sequencing depth of Pac Bio long reads and Illumina short reads were 220 × and 123 × respectively,followed by assembly and annotation.Genome of G.tsugae,based on genome-wide data,SSR molecular markers were developed for G.tsugae,and comparative genomic analysis was carried out with the published Ganoderma species(G.sichuanense,G.sinense,G.boninense).(1)The de novo assembly size of G.tsugae is 43.26 M,consisting of 18 contigs with 3.16 Mb in N50 value,and with 98.5% BUSCO genes.This paper obtains a high-quality G.tsugae genome.(2)A total of 2,508 SSR loci were identified in the 18 contigs of the G.tsugae genome,11,645 pairs of SSR primers were designed,and 100 pairs of SSR primers were selected for detection.Finally,88 pairs of primers were successfully amplified,of which 59 pairs of primers showed polymorphism.(3)Comparative genomic analysis explored the phylogenetic relationship and divergence time of Ganoderma species,and found that the genus Ganoderma and the genus Dichomitus diverged 38 million years ago,while the divergence between G.tsugae and their common ancestor of G.sichuanense,G.sinense,and G.boninense was about 21 million years ago.It was speculated that the movement of the Himalayas may have caused dramatic changes in the climate in northern and southern China at that time,which led to the divergence of Ganoderma species and the current geographical distribution pattern.A total of 151 genes in 16 gene families were expanded in the genome of G.tsugae.KEGG analysis showed that these genes were enriched in fatty acid metabolism,Fanconi anemia pathway,homologous recombination and other pathways,such as genes salh,phea,cyp53a1 and cyp102a;184 genes were positively selected and significantly enriched in mismatch repair,Fanconi anemia pathway,base excision repair,DNA repair,and plant-pathogen interaction pathways,as well as positivly selected genes such as glpk and amie,these expansion and positivly selected genes may play important roles in G.tsugae physiology and environmental adaptation.2.Population genomics analysis of G.tsugae.Whole-genome resequencing analysis of 22 G.tsugae strains and 18 G.sichuanense strains using the next-generation sequencing Illumina platform.(1)A total of 937.70 ～ 2,605.23 Mb and 1,203.90 ～2,225.63 Mb of sequencing data were obtained for G.tsugae and G.sichuanense,respectively,with an average sequencing depth of 32.57×and 40.12×.(2)A total of214,161 high-quality SNP loci were identified for G.tsugae from the sequencing data,and these SNP loci were approximately evenly distributed in 18 contigs,with more than27.7%,12.2%,13.0%,11.6% and 31.5% of the sites were located in exons,gene upstream,gene downstream,intron and intergenic regions,respectively;the SNP density in exon region was the lowest,followed by gene upstream region.The SNP density in downstream,intronic and intergenic regions of genes was significantly higher than in exonic and upstream regions.(3)Then the top 1000 genes with the highest SNP density were functionally enriched,and the results showed that most of these genes were related to pathways such as transport and membrane proteins.(4)The phylogenetic relationship,PCA and population structure analysis showed that significantly different genetic backgrounds between G.tsugae and G.sichuanense.3.Analysis of terpene synthase-related gene family of G.tsugae.Based on the whole genome data of G.tsugae,the gene families involved in the synthesis of terpene in G.tsugae were identified,the homologous gene tree,gene structure and sequence information were analyzed for these genes.Then,genome-wide SNP variants were detected in population,and terpene synthase genes cloning were verified.The results showed:(1)Ten terpene synthase genes were identified in the whole genome of G.tsugae,6 of which encode three terpene synthases(Cop1-4),which are germacrene A synthases(Cop1 and Cop2);γ-muurolene synthase(Cop3)and γ-cadinene synthase(Cop4);and 4 genes encoding trichodiene synthase(TRI5).Those genes distributed on5 contigs,and are from 7 gene clusters.Six terpene synthases contain Terpene＿syn＿C-2domain,and four trichodiene synthases contain Isoprenoid＿Biosyn＿C1 domain,all of which belong to sesquiterpene synthases.(2)The ten terpene synthase genes were cloned and verified by reverse transcription PCR.The results showed that full-length sequences of the 10 genes ranged from 1,178-1,478 bp and contained 2 to 5 introns.The identification results and sequencing results were highly consistent,proved the accuracy of the whole genome data of G.tsugae.(3)Based on the population-wide gene SNP variation results,among the 10 terpene synthase genes,9 genes have non-synonymous mutations in the coding regions,three different G.tsugae strains were selected for gene cloning verification,and the identification results were basically consistent with the predicted SNP variation information,laying a foundation for further development of molecular markers. |
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