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.

Development of a Molecular Marker Linked to the A4 Locus and the Structure of HD Genes in Pleurotus eryngii

Allelic differences in A and B mating-type loci are a prerequisite for the progression of mating in the genus Pleurotus eryngii; thus, the crossing is hampered by this biological barrier in inbreeding. Molecular markers linked to mating types of P. eryngii KNR2312 were investigated with randomly amplified polymorphic DNA to enhance crossing efficiency. An A4-linked sequence was identified and used to find the adjacent genomic region with the entire motif of the A locus from a contig sequenced by PacBio. The sequence-characterized amplified region marker 7-2299 distinguished A4 mating-type monokaryons from KNR2312 and other strains. A BLAST search of flanked sequences revealed that the A4 locus had a general feature consisting of the putative HD1 and HD2 genes. Both putative HD transcription factors contain a homeodomain sequence and a nuclear localization sequence; however, valid dimerization motifs were found only in the HD1 protein. The ACAAT motif, which was reported to have relevance to sex determination, was found in the intergenic region. The SCAR marker could be applicable in the classification of mating types in the P. eryngii breeding program, and the A4 locus could be the basis for a multi-allele detection marker.

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 (M(AF)) and the average number of alleles (N(A)) were 0.50 and 5.47, respectively. The average number of genotypes (N(G)), observed heterozygosity (H(O)), expected heterozygosity (H(E)), and polymorphic information content (PIC) were 6.177, 0.227, 0.619, and 0.569, respectively. Pearson's correlation coefficient showed that M(AF) was negatively correlated with N(G) (−0.683), N(A) (−0.600), H(O) (−0.584), and PIC (−0.941). N(G), N(A), H(O), and PIC were positively correlated with other polymorphic parameters except for M(AF). 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.

Genome-Wide Comparison of Carbohydrate-Active Enzymes (CAZymes) Repertoire of Flammulina ononidis

Whole-genome sequencing of Flammulina ononidis, a wood-rotting basidiomycete, was performed to identify genes associated with carbohydrate-active enzymes (CAZymes). A total of 12,586 gene structures with an average length of 2009 bp were predicted by the AUGUSTUS tool from a total 35,524,258 bp length of de novo genome assembly (49.76% GC). Orthologous analysis with other fungal species revealed that 7051 groups contained at least one F. ononidis gene. In addition, 11,252 (89.5%) of 12,586 genes for F. ononidis proteins had orthologs among the Dikarya, and F. ononidis contained 8 species-specific genes, of which 5 genes were paralogous. CAZyme prediction revealed 524 CAZyme genes, including 228 for glycoside hydrolases, 21 for polysaccharide lyases, 87 for glycosyltransferases, 61 for carbohydrate esterases, 87 with auxiliary activities, and 40 for carbohydrate-binding modules in the F. ononidis genome. This genome information including CAZyme repertoire will be useful to understand lignocellulolytic machinery of this white rot fungus F. ononidis.

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.

Ribosomal Intergenic Spacer 1 Based Characterization of Button Mushroom (Agaricus bisporus) Strains

Breeding the button mushroom requires genetic information about its strains. This study was undertaken to genetically characterize four domestically bred button mushroom strains (Saea, Saejung, Saedo, Saeyeon cultivars) and to assess the possibility of using the intergenic spacer 1 (IGS1) region of rDNA as a genetically variable region in the genetic characterization. For the experiment, 34 strains of Agaricus bisporus, two strains of A. bitorquis, and one strain of A. silvaticus, from 17 countries were used. Nucleotide sequence analysis of IGS1 rDNA in these 37 Agaricus strains confirmed that genetic variations exist, not only among the four domestic strains, but also between the four domestic strains and foreign strains. Crossing two different haploid strains of A. bisporus seems to generate genetic variation in the IGS1 region in their off-spring haploid strains. Phylogenetic analysis based on the IGS1 sequence revealed all A. bisporus strains could be differentiated from A. silvaticus and A. bitorquis strains. Five genetic groups were resolved among A. bisporus strains. Saejung and Saeyeon cultivars formed a separate genetic group. Our results suggest that IGS1 could be complementarily applied in the polymorphism analysis of button mushroom.

Taxonomic Position and Species Identity of the Cultivated Yeongji 'Ganoderma lucidum' in Korea

Ganoderma lucidum has a long history of use as a traditional medicine in Asian countries. However, the taxonomy of Ganoderma species remains controversial, since they were initially classified on the basis of their morphological characteristics. Recently, it was proposed that G. lucidum from China be renamed as G. sichuanense or G. lingzhi. In the present study, phylogenetic analysis using the internal transcribed spacer region rDNA sequences of the Ganoderma species indicated that all strains of the Korean 'G. lucidum' clustered into one group together with G. sichuanense and G. lingzhi from China. However, strains from Europe and North American, which were regarded as true G. lucidum, were positioned in a clearly different group. In addition, the average size of the basidiospores from the Korean cultivated Yeongji strains was similar to that of G. lingzhi. Based on these results, we propose that the Korean cultivated Yeongji strains of 'G. lucidum' should be renamed as G. lingzhi.

Genetic and Biochemical Characterization of Monokaryotic Progeny Strains of Button Mushroom (Agaricus bisporus)

To promote the selection of promising monokaryotic strains of button mushroom (Agaricus bisporus) during breeding, 61 progeny strains derived from basidiospores of two different lines of dikaryotic parental strains, ASI1038 and ASI1346, were analyzed by nucleotide sequencing of the intergenic spacer I (IGS I) region in their rDNA and by extracellular enzyme assays. Nineteen different sizes of IGS I, which ranged from 1,301 to 1,348 bp, were present among twenty ASI1346-derived progeny strains, while 15 different sizes of IGS I, which ranged from 700 to 1,347 bp, were present among twenty ASI1038-derived progeny strains. Phylogenetic analysis of the IGS sequences revealed that different clades were present in both the ASI10388- and ASI1346-derived progeny strains. Plating assays of seven kinds of extracellular enzymes (beta-glucosidase, avicelase, CM-cellulase, amylase, pectinase, xylanase, and protease) also revealed apparent variation in the ability to produce extracellular enzymes among the 40 tested progeny strains from both parental A. bisporus strains. Overall, this study demonstrates that characterization of IGS I regions and extracellular enzymes is useful for the assessment of the substrate-degrading ability and heterogenicity of A. bisporus monokaryotic strains.

Cloning and Expression Analysis of Phenylalanine Ammonia-Lyase Gene in the Mycelium and Fruit Body of the Edible Mushroom Flammulina velutipes

Phenylalanine ammonia-lyase (PAL) gene is known to be expressed in plants, and is involved in the differentiation, growth and synthesis of secondary metabolites. However, its expression in fungi remains to be explored. To understand its expression in mushroom fungi, the PAL gene of the edible mushroom Flammulina velutipes (Fvpal) was cloned and characterized. The cloned Fvpal consists of 2,175 bp, coding for a polypeptide containing 724 amino acids and having 11 introns. The translated amino acid sequence of Fvpal shares a high identity (66%) with that of ectomycorrhizal fungus Tricholoma matsutake. Distinctively, the Fvpal expression in the mycelium was higher in minimal medium supplemented with L-tyrosine than with other aromatic amino acids. During cultivation of the mushroom on sawdust medium, Fvpal expression in the fruit body correspondingly increased as the mushroom grew. In the fruiting body, Fvpal was expressed more in the stipe than in the pileus. These results suggest that F. velutipes PAL activity differs in the different organs of the mushroom. Overall, this is first report to show that the PAL gene expression is associated with mushroom growth in fungi.

Genome-Wide Identification and Characterization of Novel Laccase Genes in the White-Rot Fungus Flammulina velutipes

The aim of this study was to identify and characterize new Flammulina velutipes laccases from its whole-genome sequence. Of the 15 putative laccase genes detected in the F. velutipes genome, four new laccase genes (fvLac-1, fvLac-2, fvLac3, and fvLac-4) were found to contain four complete copper-binding regions (ten histidine residues and one cysteine residue) and four cysteine residues involved in forming disulfide bridges, fvLac-1, fvLac-2, fvLac3, and fvLac-4, encoding proteins consisting of 516, 518, 515, and 533 amino acid residues, respectively. Potential N-glycosylation sites (Asn-Xaa-Ser/Thr) were identified in the cDNA sequence of fvLac-1 (Asn-454), fvLac-2 (Asn-437 and Asn-455), fvLac-3 (Asn-111 and Asn-237), and fvLac4 (Asn-402 and Asn-457). In addition, the first 19~20 amino acid residues of these proteins were predicted to comprise signal peptides. Laccase activity assays and reverse transcription polymerase chain reaction analyses clearly reveal that CuSO4 affects the induction and the transcription level of these laccase genes.

Overproduction of Laccase by the White-Rot Fungus Pleurotus ostreatus Using Apple Pomace as Inducer

Laccase activity of Pleurotus ostreatus is significantly increased by the addition of apple pomace. Among various conditions, the best concentration of apple pomace and cultivation time for the production of laccase by P. ostreatus was 2.5% and 9 days, respectively. Reverse transcription polymerase chain reaction analyses of laccase isoenzyme genes, including pox1, pox3, pox4, poxc, poxa3, and poxa1b, revealed a clear effect of apple pomace on transcription induction. Our findings reveal that the use of apple pomace can be a model for the valuable addition of similar wastes and for the development of a solid-state fermenter and commercial production of oyster mushroom P. ostreatus.

Highly Efficient Electroporation-mediated Transformation into Edible Mushroom Flammulina velutipes

In this study, we developed an efficient electroporation-mediated transformation system featuring Flammulina velutipes. The flammutoxin (ftx) gene of F. velutipes was isolated by reverse transcription-PCR. pFTXHg plasmid was constructed using the partial ftx gene (410 bp) along with the hygromycin B phosphotransferase gene (hygB) downstream of the glyceraldehydes-3-phosphate dehydrogenase (gpd) promoter. The plasmid was transformed into protoplasts of monokaryotic strain 4019-20 of F. velutipes by electroporation. High transformation efficiency was obtained with an electric-pulse of 1.25 kV/cm by using 177 transformants/microg of DNA in 1 x 107 protoplasts. PCR and Southern blot hybridization indicated that a single copy of the plasmid DNA was inserted at different locations in the F. velutipes genome by non-homologous recombination. Therefore, this transformation system could be used as a useful tool for gene function analysis of F. velutipes.

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).

Isolation and Characterization of Dikaryotic Mutants from Pleurotus ostreatus by UV Irradiation

Protoplasts of the wild type strain of Pleurotus osteatus were mutagenized with UV light, and 3,000 colonies were examined for abnormal mycelial and fruiting phenotypes. Forty one strains displayed variant phenotypes in mycelia and fruiting processes. The variant phenotypes were classified into 6 groups: (1) auxotrophic strains, which are incapable of growing on minimal media and can only grow when provided with their specific requirements; (2) abnormal vegetative strains, which grow very slowly on minimal and complete media; (3) primordiumless strains, which fail to develop to the formation of primordia; (4) maturationless strains, which form primordia, but do not form mature fruiting bodies; (5) specifically colored strains, which have Specific bluish grey or bluish white pileus; (6) poorly spored strains, which fail to produce basidiospore or which produce few spores. These variant strains may be useful in genetic breeding programs and for the studies of fungal development and genetics.

Molecular Marker Related to Fruitbody Color of Flammulina velutipes

White and brown strains of Flammulina velutipes were inter-crossed. All F1 showed light-brown fruitbody, suggesting that a gene for the brown fruitbody was incompletely dominant against the white one. And backcross experiment showed that more than two genes were involved in color determination. To isolate a molecular marker linked to fruitbody color, a set of primers was designed from a sequence of clones derived by a bulked segregant analysis. These markers showed a specific band which co-segregated with brown fruitbody forming strains.

Breeding of Flammulina velutipes Strains Adaptable to Elevated-temperature

Winter mushroom, Flammulina velutipes, needs low temperature during its cultivation. To save on farm costs, especially during summer, a strain adaptable to a higher or elevated-temperature must be developed. At the start of breeding program, parental strains which could endure high temperature were obtained. Seuenty four dikaryotic strains were collected and divided into four groups according to the nature of temperature. They also had different fruiting temperature. Finally we selected three brown strains ASI 4048, 4057 and 4072, and collected their spores. These selected strains can germinate even at a high temperature of 32degrees C, which were dramatically higher than the other strains. Based on these results, the new white strain adapted to mid-temperature by backcross mating was developed. Molecular markers were applied to select white fruit-body producing strains without cultivation. They showed a specific band which co-segregated with brown fruitbody forming strains in BC1F1 progenies. Selected white strains were tested under several elevated temperature conditions.

The Efficient Transformation of Pleurotus ostreatus using REMI Method

Restriction enzyme-mediated integration (REMI) was used to transform uracil auxotrophs of Pleurotus ostreatus to prototrophy. When protoplasts of Pleurotus ostreatus were treated by the reaction mixture containing 10 units of BamHI, the frequency of REMI was about 64 transformants per 1 microg of DNA. This efficiency was increased by 14.2 times compared with that of the conventional PEG transformation. The optimal condition for REMI of P. ostreatus was achieved when 1 microg of linearized pTRura3-2 DNA was added into 1x10(7) protoplasts along with 10 units BamHI. Southern blot analysis revealed that about 50% of transformants examined were caused by REMI event and 30% carried single copy insertion at the genome. This suggested that the REMI method might be a useful tool for efficient transformation and tagging mutagenesis of P. ostreatus.

Analysis of Cultural Characteristics and Phylogenic Relationships of Collected Strains of Pholiota species

Cultural characteristics and phylogenic relationships were investigated and classified among collected strains in Pholiota spp. which contain P. adiposa, P. squarrosa, P. nameko etc. They were tested on the four different media (PDA, MCM, YM, MEA) and sawdust (Alder, Oak, Pine, Popular) substrates. There was a little variation according to the media and sawdust substrates, although PDA and popular sawdust substrate seemed to be better. Most strains showed white colonies, but some strains were brown. Mycelial growth length differed according to the strains. To classify species, the internal transcribed spacer regions (ITS) of the ribosomal DNA (rDNA) repeats from Pholiota spp. were amplified using polymerase chain reaction (PCR) and then sequenced. According to the analysis of ITS sequences, they were classified into five clusters. Their spacer regions were 644~700 nucleotides in length. The reciprocal homologies of each ITS region among these strains were ranged from 49.6~99.9%. The phylogenic analysis might give a criterion to classify species in the collected strains.