Study On The Identification Of Color Control Loci And The Mechanism Of Pigment Synthesis In The Fruit Bodies Of Auricularia Cornea Based On Multiomics

Auricularia cornea Ehrenb.is rich in colloid and has high nutritional value for health and medicinal purposes.The color of fruiting bodies is not only an important indicator for germplasm evaluation and an indispensable genetic marker in genetic research of A.cornea,but also one of the main characteristics that must be considered when breeding and improving varieties.Owing to its great market potential for food and health care,white A.cornea,a rare edible fungus,has received increased attention in recent years.Academician Li Yu has cultivated a white fungus variety,"Auricularia cornea.var.Li",which has achieved significant results in promoting and demonstrating poverty alleviation and rural revitalization.With the improvement of consumption capacity,consumer demand has also shifted from "being full" to "eating well".Currently,white variety of A.cornea is scarce in the market,making it difficult to meet the growing consumption demand.Therefore,the issue of efficient breeding of white variety of A.cornea needs to be urgently addressed.To study breeding issues,it is necessary to clarify the gene function of the color control site of A.cornea,the pigment synthesis pathway,and the key metabolites and regulatory genes in the pigment synthesis pathway.Therefore,In this study,two sequencing models combined with Hi-C-assisted assembly were used to perform high-throughput sequencing on the white A.cornea genome to assemble the genome at the chromosome level,using recombinant inbred populations for color typing→Constructing of genetic linkage map→Gene targeting by color control locus→Gene function verification.On the other hand,collect mycelium,primordium,and fruiting bodies of white and purple strains→Transcriptomic sequencing and metabolomic detection→ Identify the pigment synthesis pathway and pigment type of A.cornea → Multigroup association analysis → Verify key metabolites and genes in the pigment synthesis pathway.This study sheds light on the genetic blueprint and evolutionary history of the white A.cornea genome,revealing the mechanism of pigment synthesis in A.cornea.It has important theoretical and practical implications for understanding the evolution of basidiomycetes,molecular breeding of white A.cornea,and deciphering the genetic regulations of edible fungi.Additionally,it provides valuable insights for the study of phenotypic traits in other edible fungi.,The main results are as follows:1.Genomic Assembly and Analysis of A.cornea(1)16 Gb raw data were generated from Pac Bio Sequel and Illumina Nova Seq PE150.After selection,assembly,and optimization,79.01 Mb genome sequences comprising 28 contigs were obtained.The length of N50 is 5.66 Mb,the maximum contig length is 8.18 Mb,and the GC content is 59.06%.BUSCO evaluation showed that only 26 of 1764 single-copy genes were missing,with a 98.53% assembly integrity.Hi-C captured 24,808,063 pairs of reads that could pair with the genome,17,376,358 valid read pairs were obtained after Hi CUP quality control,and the reads were aligned to 23 contigs.Because the intra-chromosome interaction probability markedly exceeded that of inter-chromosome,different contigs were divided into different chromosomes,and 23 contigs were clustered into clusters resembling chromosomes.In addition,99.63% of assembled sequences were mounted to 13 clusters;the N50 value was 6.03 Mb and N90 value was 4.56 Mb.(2)A total of 18,574 coding genes were predicted based on ab initio Augustus prediction of the genome,with a total length of 24.33 Mb and an average length of 1310 bp.The total length of the coding region accounted for 29.41% of the entire genome.78.69%(14,616)of the coding genes were supported in transcripts(coverage>80%).18,574 prediction genes were annotated with KEGG,KOG,GO,NR,Pfam,Swiss Prot,CAZY,and P450 databases,respectively.86.20%(15,933)of predicted genes have assumed functions in these databases.A total of28,142 repetitive sequences were predicted.(3)Gene family analysis of 19 edible fungi found that a total of 1,997 homologous gene families were conserved in all compared genomes,including 641 single copy homologous genes.In the four Auricularia species genomes,there were approximately 8,270 conserved homologous gene families.Functional analysis indicated that these specific genes are associated with carbon source degradation and secondary metabolite metabolism.We then performed phylogenetic evolutionary analysis of 641 conserved single-copy homologous genes in all edible mushrooms,and the phylogenetic tree of 19 species showed that A.cornea was closer to Auricularia subglabra than to Auricularia heimuer in terms of genetics.In the evolutionary process of A.cornea,contraction of gene families is more common than expansion,and a total of997 gene families have been expanded in A.cornea.The number of contracted gene families in A.cornea(1855)is much smaller than in the same genus A.heimuer(4164)and A.subglabra(2837).Functional analysis shows that these contracted gene families are mainly related to degradation metabolism,immune system,sorting,and degradation.These genes play a critical role in adapting to harsh environments and substrate degradation,leading to the widespread distribution of A.cornea in Auricularia.In the comparison between white and purple A.cornea,we found 15 and 17 specific homologous gene families for white/purple A.cornea,functional analysis indicated that these specific genes are associated with carbon source degradation and secondary metabolite metabolism.Based on the divergence time of white/purple A.cornea,it can be inferred that about 40,000 years ago.We conducted a whole-genome collinearity analysis between A.cornea and purple A.cornea and found numerous inversions and translocations between homologous regions of the two genomes,These findings suggest that a series of chromosome fusions or breakages may have occurred during the long evolutionary history of A.cornea.2.Location and Functional Verification of Color Control Sites in A.cornea.(1)It is known that the color control locus of A.cornea is controlled by two alleles(locus A and locus B).Monokaryotic strains 23 and 51 with color locus recombination(color type a B)were selected from 87 mapping populations to mate with 85 other mapping populations to construct a recombinant inbred population.The color loci of strain nos.23 and 51 in the mapping population were the Pr type(a B)and locus B of all 61 recombinant inbred strains was dominant.Therefore,locus A determined the color of the Fruiting bodys of the recombinant inbred strains.In the mapping population,the color locus of the monokaryotic strains that produced purple Fruiting bodys was the A＿type and that of the monokaryotic strains that produced white Fruiting bodys was the a＿type.By associating locus A with phenotypic traits,54 SSR molecular markers were screened to locate locus A and the Join Map 4.0 software was used to perform linkage analysis and generate a genetic linkage map of locus A.The findings revealed that locus A was linked to the markers SSR2507 and SSR2514 and was located between 0 and 197,550 bp of genome Contig9.(2)Fine mapping of candidate genes for color control at locus A.According to the genome contig9 sequence,Base on this sequence,seven pairs of PTG-specific primers were generated for fine mapping,which revealed that the color control locus was located between the PTG-20 and PTG-21 loci.Located in contig9＿29,619-53,463 bp.(3)Prediction of candidate functional genes for color control.The protein-encoding gene A18078 was mapped by gene prediction using the Soft Berry software.Further analysis revealed that the candidate protein-coding gene included a Velvet conserved domain associated with the Ve A protein in the Velvet factor family(pfam11754),named Ac Ve A,which can form dimers with Vel B proteins in filamentous fungi to inhibit pigment synthesis.The interaction between Ac Ve A and Vel B(Acvel B)in A.cornea was confirmed by yeast two hybrid method.Detection of the expression levels of purple and white strains showed that the Acve A sequence had a SNP mutation in purple strain ACP004;Different light tests have found that Ac Ve A-Ac Vel B polymers can inhibit pigment synthesis in A.cornea and the expression of Velvet polymers is affected by light intensity.Under dark conditions,Ac Ve A can carry Ac Vel B from the cytoplasm into the nucleus,forming a Velvet polymer to inhibit pigment synthesis.Under light conditions,polymers mainly exist in the cytoplasm,and the pathway into the nucleus is blocked,unable to inhibit pigment synthesis.The Velvet polymer,commonly found in filamentous fungi,was first discovered in A.cornea.It has the function of regulating secondary metabolism and can inhibit pigment synthesis.(4)Creation of White Germplasm Resources.31 white strains were obtained by recombinant inbred population,and 54 white strains were obtained by hybridization of parent mononuclear strains ACW001-9 and ACW001-33 with white strain AP64,respectively.These white strains can be used as valuable germplasm resources for genetic and breeding research.3.Analysis of Pigment Synthesis Pathway in A.cornea Based on DEGs(1)The white strain ACW001 and purple strain ACP004 were subjected to mycelial culture and fruiting experiment.RNA from three stages of mycelium,primordium,and fruiting body was collected for RNA-seq,and a total of 21,878 differential genes were obtained.KEGG annotated 95 candidate genes for pigment synthesis,and found that ubiquinone and other terpene quinone biosynthesis,glycolysis pathway,pentose phosphate pathway,and TCA cycle pathway were significantly enriched.(2)GSEA enrichment analysis revealed that the glycolysis,phenylalanine,tyrosine,and tryptophan pathways of the purple strain were significantly upregulated at the primordial stage,and a total of 11 core genes were enriched.The pigment synthesis pathway related to the enrichment results of KEGG and GSEA is the shikimate pathway,which is synthesized through glycolysis and pentose phosphate pathways.The phenylalanine,tyrosine,and tryptophan pathways are branches of the shikimate pathway,and the enriched core genes are also related to these pathways.Therefore,it is preliminarily inferred that the pigment of A.cornea is synthesized through the shikimate pathway.4.Analysis of A.cornea Pigment Based on Metabolomic Differential Metabolites(1)Non target LC-MS detected 150 different metabolites in white and purple strains,including 81 positive ion modes and 69 negative ion modes.According to the VIP value in the OPLS-DA analysis and the univariate statistical analysis T-test(VIP ≥ 1 and T-test P<0.05),the differential metabolites were selected.The VIP value and correlation analysis showed that L-glutamine,L-glutamate,malate,citrate,2-oxoglutarate,tyrosine,alanine,and phenylalanine were highly correlated,and the abundance was significantly upregulated in purple strains.These metabolites are important metabolites in the TCA cycle and shikimate pathway.KEGG topology analysis identified 7 metabolic pathways that may be related to pigment synthesis,involving 16 enzyme genes.(2)Non target LC-MS did not detect L-dopa,GDHB,and other pigment synthesis related metabolites,but L-glutamine abundance was significantly upregulated in purple strains.L-glutamine is a precursor of GDHB pigment synthesis,and many compounds involved in the GDHB pigment synthesis pathway contain glutamine groups.Therefore,it is inferred that A.cornea pigment is a single type of pigment synthesized by the GDHB pathway.5.Synergistic Analysis of Main Metabolites and Genes in Pigment Synthesis Pathways by Transcriptomics and Metabolomics(1)A functional model of pigment synthesis pathway was constructed based on differential metabolites and genes: glycolysis and pentose phosphate pathway → shikimate pathway→chorismate → para-aminobenzoate → para-aminophenol → GHB → GDHB → GBQ →melanin.The pigment synthesis pathway of A.cornea is consistent with that of Agaricus bisporus.(2)O2PLS model analysis and correlation coefficient model found that D-glucosamine1-phosphate,L-glutamine,citrate and 2-oxoglutarate are important intermediate metabolites in the pigment synthesis pathway,and 20 genes such as polyphenol oxidase(PPO)are key regulatory factors in the pigment synthesis process.