Phenylalanine Ammonia-Lyase: A Key Gene for Color Discrimination of Edible Mushroom Flammulina velutipes.

In nature; Flammulina velutipes, also known as winter mushrooms, vary in the color of their fruiting bodies, from black, yellow, pale yellow, or beige to white. The purpose of this study was to compare the genome sequences of different colored strains of F. velutipes and to identify variations in the genes associated with fruiting body color. Comparative genomics of six F. velutipes strains revealed 70 white-strain-specific variations, including single nucleotide polymorphisms (SNPs) and insertions/deletions (indels), in the genome sequences. Among them, 36 variations were located in the open reading frames, and only one variation was identified as a mutation with a disruptive in-frame deletion (ΔGCGCAC) within the annotated gene phenylalanine ammonia-lyase 1 (Fvpal1). This mutation was found to cause a deletion, without a frameshift, of two amino acids at positions 112 and 113 (arginine and threonine, respectively) in the Fvpal1 gene of the white strain. Specific primers to detect this mutation were designed, and amplification refractory mutation system (ARMS) polymerase chain reaction (PCR) was performed to evaluate whether the mutation is color specific for the F. velutipes fruiting body. PCR analysis of a total of 95 F. velutipes strains revealed that this mutation was present only in white strains. In addition, monospores of the heterozygous mutant were isolated, and whether this mutation was related to the color of the fruiting body was evaluated by a mating assay. In the mating analysis of monospores with mutations in Fvpal1, it was found that this mutation plays an important role in determining the color of the fruiting body. Furthermore, the deletion (Δ112RT113) in Fvpal1 is located between motifs that play a key role in the catalytic function of FvPAL1. These results suggest that this mutation can be used as an effective marker for the color-specific breeding of F. velutipes, a representative edible mushroom.

Evaluation of Genetic Diversity and Population Structure Analysis among Germplasm of Agaricus bisporus by SSR Markers.

Agaricus bisporus is a popular edible mushroom that is cultivated worldwide. Due to its secondary homothallic nature, cultivated A. bisporus strains have low genetic diversity, and breeding novel strains is challenging. The aim of this study was to investigate the genetic diversity and population structure of globally collected A. bisporus strains using simple sequence repeat (SSR) markers. Agaricus bisporus strains were divided based on genetic distance-based groups and model-based subpopulations. The major allele frequency (MAF), number of genotypes (NG), number of alleles (NA), observed heterozygosity (HO), expected heterozygosity (HE), and polymorphic information content (PIC) were calculated, and genetic distance, population structure, genetic differentiation, and Hardy-Weinberg equilibrium (HWE) were assessed. Strains were divided into two groups by distance-based analysis and into three subpopulations by model-based analysis. Strains in subpopulations POP A and POP B were included in Group I, and strains in subpopulation POP C were included in Group II. Genetic differentiation between strains was 99%. Marker AB-gSSR-1057 in Group II and subpopulation POP C was confirmed to be in HWE. These results will enhance A. bisporus breeding programs and support the protection of genetic resources.

Interspecific hybridization between Ganoderma lingzhi and G. applanatum through protoplast fusion.

Interspecific hybridization between Ganoderma lingzhi and G. applanatum was attempted through polyethylene glycol (PEG) induced fusion technique. The protoplast isolation procedure was simplified, and we obtained a significant number of protoplasts from both Ganoderma species. The number of protoplasts obtained was 5.27 ± 0.31 × 107/mL in G. lingzhi and 5.57 ± 0.49 × 106/mL in G. applanatum. Osmotic stabilizer NaCl (0.4 M) at pH 5.8 and enzymolysis time 3.5 h have supported high frequency of protoplast regeneration. G. lingzhi and G. applanatum regeneration frequency was 1.73 ± 0.04% and 0.23 ± 0.02%, respectively. 40% of PEG induced high number of protoplast fusion the regeneration frequency was 0.09% on a minimal medium. Two hundred fifty-two fusant colonies were isolated from the following four individual experiments. Among them, ten fusants showed the mycelial morphological difference compared to their parents and other fusant isolates. The fruiting body could be generated on oak sawdust and wheat bran substrate, and a few of them showed recombined morphology of the parental strains. The highest yield and biological efficacy (BE) were recorded in GF248, while least in GF244. The hybridity of the fusant was established based on mycelia, fruiting morphology, and PCR fingerprinting. ISSR and RAPD profile analysis of ten fusants and parents depicted that fusants contained polymorphic bands, which specified the rearrangement and deletion of DNA in the fusants. A Dendrogram was constructed based on the RAPD profile, and the clustering data exhibited two major clusters: cluster I included the G. lingzhi and Cluster II, including the G. applanatum and fusant lines. Total polysaccharide (α, ß and total glucan) content was compared with fusants and parental strains. The present study highlighted the efficient methods for protoplast isolation from Ganoderma species. PEG-induced fusants showed high polymorphic frequency index, while the phenotypic characters showed high similarity to G. applanatum. A significant difference was observed in the mushroom yield and its total polysaccharide between the fusants and parental strains.

SNP-Based Genetic Linkage Map and Quantitative Trait Locus Mapping Associated with the Agronomically Important Traits of Hypsizygus marmoreus.

White strains of Hypsizygus marmoreus are more difficult to cultivate than are brown strains; therefore, new white strain breeding strategies are required. Accordingly, we constructed the genetic map of H. marmoreus with 1996 SNP markers on 11 linkage groups (LGs) spanning 1380.49 cM. Prior to analysis, 82 backcrossed strains (HM8 lines) were generated by mating between KMCC03106-31 and the progenies of the F1 hybrid (Hami-18 × KMCC03106-93). Using HM8, the first 23 quantitative trait loci (QTLs) of yield-related traits were detected with high limit of detection (LOD) scores (1.98-9.86). The length, thickness, and hardness of the stipe were colocated on LG 1. Especially, length of stipe and thickness of stipe were highly correlated given that the correlation coefficients were negative (-0.39, p value ≤ .01). And a typical biomodal distribution was observed for lightness of the pileus and the lightness of the pileus trait belonged to the LG 8, as did traits of earliness and mycelial growth in potato dextrose agar (PDA) medium. Therefore, results for color traits can be suggested that color is controlled by a multi-gene of one locus. The yield trait was highly negatively correlated with the traits for thickness of the stipe (-0.45, p value ≤ .01). Based on additive effects, the white strain was confirmed as recessive; however, traits of mycelial growth, lightness, and quality were inherited by backcrossed HM8 lines. This new genetic map, finely mapped QTLs, and the strong selection markers could be used in molecular breeding of H. marmoreus.

Bioactive Sterol Derivatives Isolated from the Pleurotus djamor var. Roseus Induced Apoptosis in Cancer Cell Lines.

OBJECTIVE: The aim of the present study is to isolate and characterize the bioactive compounds from Pleurotus djamor against human breast cancer (MDA-MD-231) and mouse T cell lymphoma (EL4) cell lines. MATERIALS AND METHODS: Sequential fractionization and column chromatography methods were involved in compound isolation. The structures of the isolated compound were determined by NMR, GC/MS, and X-ray crystallography studies. RESULTS: The isolated compounds 1- 4 [D-mannitol (C1), ergosta-5,7,22-trien-3ß-ol (C2), 5,8- epidioxy-ergosta-6-22-dien-3ß-ol (C3), and palmitic acid (C4)] are white crystal and amorphous powder in nature. All these compounds were isolated from this mushroom for the first time. In vitro lipid peroxidation activities of isolated compounds were determined by ferric thiocyanate (FTC) and thiobarbituric acid (TBA) method. The sterol derivatives C2 and C3 compounds displayed strong antioxidant activity and were not significantly different (p<0.05) to α-tocopherol. This finding elaborates on the isolation of a cytotoxic compound C2 and C3 from P. djamor via a rapid elution method. CONCLUSION: The compound C3 has exhibited better cytotoxic activity against MDA-MD-231 and EL4 cells. The present finding and data might provide new insights into the possible therapeutic and pharmaceutical use for the design of anti-cancer drugs from this edible mushroom.

Cultivation and Nutritional Value of Prominent Pleurotus spp.: An Overview.

Pleurotus species are commercially essential mushrooms and widely cultivated throughout the world. The production of Pleurotus mushrooms alone accounts for around 25% of that total cultivated mushrooms globally. In America and Europe, Pleurotus species are considered specialty mushrooms, whereas, in Korea, their cultivation is economically profitable, and it is one of the highly consumed species. Pleurotus species are predominantly found in tropical forests and often grow on fallen branches, dead and decaying tree stumps, and wet logs. Biographical studies have shown that the Pleurotus genus is among the more conspicuous fungi that induce wood decay in terrestrial ecosystems worldwide due to its formidable lignin-modifying enzymes, including laccase and versatile peroxidases. Pleurotus species can be grown easily due to their fast colonization nature on diversified agro-substrates and their biological efficiency 100%. Pleurotus mushrooms are rich in proteins, dietary fiber, essential amino acids, carbohydrates, water-soluble vitamins, and minerals. These mushrooms are abundant in functional bioactive molecules, though to influence health. Pleurotus mushrooms are finding unique applications as flavoring, aroma, and excellent preservation quality. Apart from its unique applications, Pleurotus mushrooms have a unique status delicacy with high nutritional and medicinal values. The present review provides an insight into the cultivation of Pleurotus spp. using different agro-waste as growth substances paying attention to their effects on the growth and chemical composition.

Evaluating Genetic Diversity of Agaricus bisporus Accessions through Phylogenetic Analysis Using Single-Nucleotide Polymorphism (SNP) Markers.

Agaricus bisporus, commonly known as the button mushroom, is widely cultivated throughout the world. To breed new strains with more desirable traits and improved adaptability, diverse germplasm, including wild accessions, is a valuable genetic resource. To better understand the genetic diversity available in A. bisporus and identify previously unknown diversity within accessions, a phylogenetic analysis of 360 Agaricus spp. accessions using single-nucleotide polymorphism genotyping was performed. Genetic relationships were compared using principal coordinate analysis (PCoA) among accessions with known origins and accessions with limited collection data. The accessions clustered into four groups based on the PCoA with regard to genetic relationships. A subset of 67 strains, which comprised a core collection where repetitive and uninformative accessions were not included, clustered into 7 groups following analysis. Two of the 170 accessions with limited collection data were identified as wild germplasm. The core collection allowed for the accurate analysis of A. bisporus genetic relationships, and accessions with an unknown pedigree were effectively grouped, allowing for origin identification, by PCoA analysis in this study.

Molecular Characterization of 170 New gDNA-SSR Markers for Genetic Diversity in Button Mushroom (Agaricus bisporus).

We designed 170 new simple sequence repeat (SSR) markers based on the whole-genome sequence data of button mushroom (Agaricus bisporus), and selected 121 polymorphic markers. A total of 121 polymorphic markers, the average major allele frequency (MAF) and the average number of alleles (NA) were 0.50 and 5.47, respectively. The average number of genotypes (NG), observed heterozygosity (HO), expected heterozygosity (HE), and polymorphic information content (PIC) were 6.177, 0.227, 0.619, and 0.569, respectively. Pearson's correlation coefficient showed that MAF was negatively correlated with NG (-0.683), NA (-0.600), HO (-0.584), and PIC (-0.941). NG, NA, HO, and PIC were positively correlated with other polymorphic parameters except for MAF. UPGMA clustering showed that 26 A. bisporus accessions were classified into 3 groups, and each accession was differentiated. The 121 SSR markers should facilitate the use of molecular markers in button mushroom breeding and genetic studies.

Genomic discovery of the hypsin gene and biosynthetic pathways for terpenoids in Hypsizygus marmoreus.

BACKGROUND: Hypsizygus marmoreus (Beech mushroom) is a popular ingredient in Asian cuisine. The medicinal effects of its bioactive compounds such as hypsin and hypsiziprenol have been reported, but the genetic basis or biosynthesis of these components is unknown. RESULTS: In this study, we sequenced a reference strain of H. marmoreus (Haemi 51,987-8). We evaluated various assembly strategies, and as a result the Allpaths and PBJelly produced the best assembly. The resulting genome was 42.7 Mbp in length and annotated with 16,627 gene models. A putative gene (Hypma_04324) encoding the antifungal and antiproliferative hypsin protein with 75% sequence identity with the previously known N-terminal sequence was identified. Carbohydrate active enzyme analysis displayed the typical feature of white-rot fungi where auxiliary activity and carbohydrate-binding modules were enriched. The genome annotation revealed four terpene synthase genes responsible for terpenoid biosynthesis. From the gene tree analysis, we identified that terpene synthase genes can be classified into six clades. Four terpene synthase genes of H. marmoreus belonged to four different groups that implies they may be involved in the synthesis of different structures of terpenes. A terpene synthase gene cluster was well-conserved in Agaricomycetes genomes, which contained known biosynthesis and regulatory genes. CONCLUSIONS: Genome sequence analysis of this mushroom led to the discovery of the hypsin gene. Comparative genome analysis revealed the conserved gene cluster for terpenoid biosynthesis in the genome. These discoveries will further our understanding of the biosynthesis of medicinal bioactive molecules in this edible mushroom.

Development of Polymorphic Simple Sequence Repeat Markers using High-Throughput Sequencing in Button Mushroom (Agaricus bisporus).

The white button mushroom (Agaricus bisporus) is one of the most widely cultivated species of edible mushroom. Despite its economic importance, relatively little is known about the genetic diversity of this species. Illumina paired-end sequencing produced 43,871,558 clean reads and 69,174 contigs were generated from five offspring. These contigs were subsequently assembled into 57,594 unigenes. The unigenes were annotated with reference genome in which 6,559 unigenes were associated with clusters, indicating orthologous genes. Gene ontology classification assigned many unigenes. Based on genome data of the five offspring, 44 polymorphic simple sequence repeat (SSR) markers were developed. The major allele frequency ranged from 0.42 to 0.92. The number of genotypes and the number of alleles ranged from 1 to 4, and from 2 to 4, respectively. The observed heterozygosity and the expected heterozygosity ranged from 0.00 to 1.00, and from 0.15 to 0.64, respectively. The polymorphic information content value ranged from 0.14 to 0.57. The genetic distances and UPGMA clustering discriminated offspring strains. The SSR markers developed in this study can be applied in polymorphism analyses of button mushroom and for cultivar discrimination.

Comparative genomics of the mating-type loci of the mushroom Flammulina velutipes reveals widespread synteny and recent inversions.

BACKGROUND: Mating-type loci of mushroom fungi contain master regulatory genes that control recognition between compatible nuclei, maintenance of compatible nuclei as heterokaryons, and fruiting body development. Regions near mating-type loci in fungi often show adapted recombination, facilitating the generation of novel mating types and reducing the production of self-compatible mating types. Compared to other fungi, mushroom fungi have complex mating-type systems, showing both loci with redundant function (subloci) and subloci with many alleles. The genomic organization of mating-type loci has been solved in very few mushroom species, which complicates proper interpretation of mating-type evolution and use of those genes in breeding programs. METHODOLOGY/PRINCIPAL FINDINGS: We report a complete genetic structure of the mating-type loci from the tetrapolar, edible mushroom Flammulina velutipes mating type A3B3. Two matB3 subloci, matB3a that contains a unique pheromone and matB3b, were mapped 177 Kb apart on scaffold 1. The matA locus of F. velutipes contains three homeodomain genes distributed over 73 Kb distant matA3a and matA3b subloci. The conserved matA region in Agaricales approaches 350 Kb and contains conserved recombination hotspots showing major rearrangements in F. velutipes and Schizophyllum commune. Important evolutionary differences were indicated; separation of the matA subloci in F. velutipes was diverged from the Coprinopsis cinerea arrangement via two large inversions whereas separation in S. commune emerged through transposition of gene clusters. CONCLUSIONS/SIGNIFICANCE: In our study we determined that the Agaricales have very large scale synteny at matA (∼350 Kb) and that this synteny is maintained even when parts of this region are separated through chromosomal rearrangements. Four conserved recombination hotspots allow reshuffling of large fragments of this region. Next to this, it was revealed that large distance subloci can exist in matB as well. Finally, the genes that were linked to specific mating types will serve as molecular markers in breeding.

Screening of Biodegradable Function of Indigenous Ligno-degrading Mushroom Using Dyes.

The process of biodegradation in lingo-cellulosic materials is critically relevant to biospheric carbon. The study of this natural process has largely involved laboratory investigations, focused primarily on the biodegradation and recycling of agricultural by-products, generally using basidiomycetes species. In order to collect super white rot fungi and evaluate its ability to degrade lingo-cellulosic material, 35 fungal strains, collected from forests, humus soil, livestock manure, and dead trees, were screened for enzyme activities and their potential to decolorize the commercially used Poly-R 478 dye. In the laccase enzymatic analysis chemical test, 33 white rot fungi and 2 brown rot fungi were identified. The degradation ability of polycyclic aromatic hydrocarbons (PAHs) according to the utilized environmental conditions was higher in the mushrooms grown in dead trees and fallen leaves than in the mushrooms grown in humus soil and livestock manure. Using Poly-R 478 dye to assess the PAH-degradation activity of the identified strains, four strains, including Agrocybe pediades, were selected. The activities of laccase, MnP, and Lip of the four strains with PAH-degrading ability were highest in Pleurotus incarnates. 87 fungal strains, collected from forests, humus soil, livestock manure, and dead trees, were screened for enzyme activities and their potential to decolorize the commercially used Poly-R 478 dye on solid media. Using Poly-R 478 dye to assess the PAHdegrading activity of the identified strains, it was determined that MKACC 51632 and 52492 strains evidenced superior activity in static and shaken liquid cultures. Subsequent screening on plates containing the polymeric dye poly R-478, the decolorization of which is correlated with lignin degradation, resulted in the selection of a strain of Coriolus versicolor, MKACC52492, for further study, primarily due to its rapid growth rate and profound ability to decolorize poly R-478 on solid media. Considering our findings using Poly-R 478 dye to evaluate the PAH-degrading activity of the identified strains, Coriolus versicolor, MKACC 52492 was selected as a favorable strain. Coriolus versicolor, which was collected from Mt. Yeogi in Suwon, was studied for the production of the lignin-modifying enzymes laccase, manganese-dependent peroxidase (MnP), and lignin peroxidase (LiP).

Analysis of the Chemical Constituents of Agaricus brasiliensis.

This study examined the chemical composition of A. blasiliensis and the chemical structural properties of an immuno-stimulating polysaccharide. The amino acids, free sugars, and organic acids by HPLC and fatty acids by GC were analyzed. The immuno-stimulating substance from A. blasiliensis was extracted with hot water and purified by ethanol precipitation. It underwent ion exchange chromatography on DEAE-cellulose and gel filtration on Toyopearl HW 65F. Through GP-HPLC, the substance was found to be homogeneous. Its chemical structure was determined by (13)C-NMR. Fatty acids, organic acids, and sugar alcohol composition consisted exclusively of linoleic acid, fumaric acid and mannitol, respectively. The amino acids were mainly glutamic acid, glycine, and arginine. By (13)C-NMR analysis, the immuno-stimulating substance was identified as ß-(1→3) (1→6)-glucan, composed of a backbone with (1→3)-linked D-glucopyranosyl residues branching a (1→6)-linked D-glucopyranosyl residue. The ß-glucan from A. blasiliensis showed pronounced immuno-stimulating activity on the antibody-production ability of B-lymphocytes by the hemolytic suspension assay. In these results, A. blasiliensis was estimated to have potent pharmacological properties and potential nutritional values.

Pedobacter suwonensis sp. nov., isolated from the rhizosphere of Chinese cabbage (Brassica campestris).

A mesophilic bacterium, strain 15-52(T), was isolated from the rhizosphere of Chinese cabbage (Brassica campestris). On the basis of phenotypic and genotypic characteristics, the bacterium was identified as representing a novel species belonging to the genus Pedobacter. The strain is non-flagellated, non-spore-forming and grows at temperatures in the range 1-37 degrees C. Physiological tests of the strain showed the presence of oxidase, catalase, protease (gelatin and casein hydrolysis), beta-glucosidase and beta-galactosidase activities. The highest levels of 16S rRNA gene sequence similarity were found with respect to Pedobacter roseus CL-GP80(T) (97.3 %) and Pedobacter sandarakinus DS-27(T) (97.2 %). A phylogenetic analysis based on 16S rRNA gene sequence data indicated that strain 15-52(T) is a member of the genus Pedobacter. DNA-DNA hybridization analysis revealed low levels of relatedness (<42.3 %) between the isolate and two phylogenetically related type strains, P. roseus KCCM 42272(T) and P. sandarakinus KCTC 12559(T). The DNA G+C content is 44.2 mol% and the predominant fatty acids are iso-C(15 : 0) (35.4 %), iso-C(15 : 0) 2-OH and/or C(16 : 1)omega7c (27.8 %) and iso-C(17 : 0) 3-OH (15.8 %). On the basis of these data, strain 15-52(T) represents a novel species of the genus Pedobacter, for which the name Pedobacter suwonensis sp. nov. is proposed. The type strain is 15-52(T) (=KACC 11317(T)=DSM 18130(T)).

Changes of Enzyme Activities and Compositions of Abnormal Fruiting Bodies Grown under Artificial Environmental Conditions in Pleurotus ostreatus.

This study investigated the biochemical changes of abnormal fruiting bodies grown under artificial environmental conditions in P. ostreatus. Abnormal mushroom growth during cultivation damages the production of good quality mushroom. This study showed that different environmental conditions produced morphological changes in the fruiting bodies of P. ostreatus. The fruiting bodies with morphological changes were collected and examined for differences in biochemical properties, enzyme activities, and carbohydrates composition. The enzyme activities assay showed that glucanase and chitinase activities decreased when the temperature was below or above the optimum cultivation temperature for P. ostreatus. The biochemical compositions of the abnormal mushroom were significantly different from the normal fruiting bodies. It was suggested that the changes in the biochemical composition of abnormal mushroom were caused by the unfavorable environmental conditions during mushroom cultivation.