Improved accuracy of geographical origin identification of shiitake grown in sawdust medium: A compound-specific isotope model-based pilot study.

In countries like South Korea and the USA, origin labeling of shiitake grown using imported Chinese-inoculated medium is an issue. Therefore, we evaluated the use of compound-specific isotope analysis (CSIA) for the accurate identification of the geographical origin of shiitake (Korean, Chinese-inoculated medium, and Chinese); Chinese-inoculated medium shiitake were cultivated in Korea using inoculated sawdust medium from China. The CSIA-discriminant model showed an overall accuracy of 100% in the geographical classification of the original set and 96.4% for the cross-validated set. Glutamate and aspartate δ15N values were the most important variables for differentiating shiitake based on their origins. Compared to that observed upon using the bulk stable isotope analysis, the CSIA model was associated with significantly improved predictability of origin identification. Our findings elucidate the importance of isotope signatures in developing a reliable origin labeling method for shiitake cultured on the sawdust medium for the global market.

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.

An origin identification model for labeling of shiitake (Lentinula edodes).

With the increasing globalization of the food trade across countries and continents, reliable identification of the geographical origin of products is critical. In this study, we describe the limitations of the current origin labeling system for non-soil-based agricultural products and suggest alternative strategies for the identification of the geographical origin of such products. An origin identification model based on stable isotope ratio analysis combined with discriminant analysis is used to evaluate the similarities and dissimilarities between domestic and foreign shiitake mushrooms, including Chinese inoculated sawdust blocks and Chinese origin. The results show a classification sensitivity of 92.0%, classification specificity of 91.5%, and overall accuracy of 93.5%. In particular, δ15N was the most important isotope marker for the identification of the origin of shiitake mushrooms. Hence, the current origin labeling system for mushroom species has to be revised to establish fair trade and avoid improper origin labeling in the global shiitake market.

Comparative Analysis of Carbohydrate Active Enzymes in the Flammulina velutipes var. lupinicola Genome.

The purpose of this study was to determine the genome sequence of Flammulina velutipes var. lupinicola based on next-generation sequencing (NGS) and to identify the genes encoding carbohydrate-active enzymes (CAZymes) in the genome. The optimal assembly (71 kmer) based on ABySS de novo assembly revealed a total length of 33,223,357 bp (49.53% GC content). A total of 15,337 gene structures were identified in the F. velutipes var. lupinicola genome using ab initio gene prediction method with Funannotate pipeline. Analysis of the orthologs revealed that 11,966 (96.6%) out of the 15,337 predicted genes belonged to the orthogroups and 170 genes were specific for F. velutipes var. lupinicola. CAZymes are divided into six classes: auxiliary activities (AAs), glycosyltransferases (GTs), carbohydrate esterases (CEs), polysaccharide lyases (PLs), glycoside hydrolases (GHs), and carbohydrate-binding modules (CBMs). A total of 551 genes encoding CAZymes were identified in the F. velutipes var. lupinicola genome by analyzing the dbCAN meta server database (HMMER, Hotpep, and DIAMOND searches), which consisted of 54-95 AAs, 145-188 GHs, 55-73 GTs, 6-19 PLs, 13-59 CEs, and 7-67 CBMs. CAZymes can be widely used to produce bio-based products (food, paper, textiles, animal feed, and biofuels). Therefore, information about the CAZyme repertoire of the F. velutipes var. lupinicola genome will help in understanding the lignocellulosic machinery and in-depth studies will provide opportunities for using this fungus for biotechnological and industrial applications.

Fatty Acids and Stable Isotope Ratios in Shiitake Mushrooms (Lentinula edodes) Indicate the Origin of the Cultivation Substrate Used: A Preliminary Case Study in Korea.

Shiitake mushroom (Lentinula edodes) is commonly consumed worldwide and is cultivated in many farms in Korea using Chinese substrates owing to a lack of knowledge on how to prepare sawdust-based substrate blocks (bag cultivation). Consequently, issues related to the origin of the Korean or Chinese substrate used in shiitake mushrooms produced using bag cultivation have been reported. Here, we investigated differences in fatty acids (FAs) and stable isotope ratios (SIRs) in shiitake mushrooms cultivated using Korean and Chinese substrates under similar conditions (strain, temperature, humidity, etc.) and depending on the harvesting cycle. The total FA level decreased significantly by 5.49 mg∙g-1 as the harvesting cycle increased (p < 0.0001); however, no differences were found in FAs between shiitake mushrooms cultivated using Korean and Chinese substrates. Linoleic acid was the most abundant FA, accounting for 77-81% of the total FAs during four harvesting cycles. Moreover, the SIRs differed significantly between the Korean and Chinese substrates, and the harvesting cycles resulted in smaller maximum differences in SIR values compared to those of the cultivation substrate origins. Our findings contribute to the identification of the geographical origin of shiitake mushrooms and may have potential applications in international shiitake-mushroom markets.

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.

Unusual genome expansion and transcription suppression in ectomycorrhizal Tricholoma matsutake by insertions of transposable elements.

Genome sequencing of Tricholoma matsutake revealed its unusually large size as 189.0 Mbp, which is a consequence of extraordinarily high transposable element (TE) content. We identified that 702 genes were surrounded by TEs, and 83.2% of these genes were not transcribed at any developmental stage. This observation indicated that the insertion of TEs alters the transcription of the genes neighboring these TEs. Repeat-induced point mutation, such as C to T hypermutation with a bias over "CpG" dinucleotides, was also recognized in this genome, representing a typical defense mechanism against TEs during evolution. Many transcription factor genes were activated in both the primordia and fruiting body stages, which indicates that many regulatory processes are shared during the developmental stages. Small secreted protein genes (<300 aa) were dominantly transcribed in the hyphae, where symbiotic interactions occur with the hosts. Comparative analysis with 37 Agaricomycetes genomes revealed that IstB-like domains (PF01695) were conserved across taxonomically diverse mycorrhizal genomes, where the T. matsutake genome contained four copies of this domain. Three of the IstB-like genes were overexpressed in the hyphae. Similar to other ectomycorrhizal genomes, the CAZyme gene set was reduced in T. matsutake, including losses in the glycoside hydrolase genes. The T. matsutake genome sequence provides insight into the causes and consequences of genome size inflation.

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.

Genomic Insights into the Fungal Lignocellulolytic Machinery of Flammulina rossica.

Next-generation sequencing (NGS) of the Flammulina rossica (wood-rotting basidiomycete) genome was performed to identify its carbohydrate-active enzymes (CAZymes). De novo genome assembly (31 kmer) revealed a total length of 35,646,506 bp (49.79% GC content). In total, 12,588 gene models of F. rossica were predicted using an ab initio gene prediction tool (AUGUSTUS). Orthologous analysis with other fungal species revealed that 7433 groups contained at least one F. rossica gene. Additionally, 12,033 (95.6%) of 12,588 genes for F. rossica proteins had orthologs among the Dikarya, and F. rossica contained 12 species-specific genes. CAZyme annotation in the F. rossica genome revealed 511 genes predicted to encode CAZymes including 102 auxiliary activities, 236 glycoside hydrolases, 94 glycosyltransferases, 19 polysaccharide lyases, 56 carbohydrate esterases, and 21 carbohydrate binding-modules. Among the 511 genes, several genes were predicted to simultaneously encode two different CAZymes such as glycoside hydrolases (GH) as well as carbohydrate-binding module (CBM). The genome information of F. rossica offers opportunities to understand the wood-degrading machinery of this fungus and will be useful for biotechnological and industrial applications.

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.

Potential geo-discriminative tools to trace the origins of the dried slices of shiitake (Lentinula edodes) using stable isotope ratios and OPLS-DA.

The Lentinus edodes species, known as shiitake, has a history of extensive use in many cuisines in several East Asian countries owing to its unique and pleasant flavor and texture. As international food trade increases, reliable discrimination of geographical origin is becoming increasingly crucial in Korea to identify cheaper imported Chinese shiitake. Herein, stable isotope ratios (i.e., δ13C, δ15N, δ18O, and δ34S) were measured with a stable isotope ratio mass spectrometer, and a geographical discrimination method using orthogonal projection to latent structure-discriminant analysis was developed. The externally validated discrimination method showed excellent predictability (Q2cum = 0.881) and illustrated that δ18O and δ15N were important isotope markers for the geographical discrimination of dried shiitake slices. This study extends the knowledge of geographical differences between China and Korea evidenced by the shiitake isotope signatures, thereby contributing to potential geographical authentication with broader applications for international shiitake markets.

Characterization of Volatile Profiles of Six Popular Edible Mushrooms Using Headspace-Solid-Phase Microextraction Coupled with Gas Chromatography Combined with Chemometric Analysis.

The classification of six mushroom species (white beech, brown beech, button, oyster, king oyster, and enoki mushrooms) was successfully achieved using canonical discriminant analysis (CDA) on volatile metabolite data sets obtained by headspace-solid-phase microextraction gas chromatography (HS-SPME-GC). Twenty-seven major volatile compounds in six edible mushrooms were positively identified by HS-SPME-GC mass spectroscopy. The total volatile content was highest in brown beech mushroom (P < 0.05). Significant difference in volatile profile was observed between brown beach and white beech mushrooms. Button mushroom contained significantly higher contents of benzaldehyde and benzyl alcohol than the other mushrooms (P < 0.05). Oyster mushroom contained 1-octen-3-ol as the most prevalent volatile, representing 67% out of total volatiles. Hexanal (35.0%) and 1-octen-3-ol (22.5%) were the most abundant volatiles found in king oyster. Hexanal (29.1%) was the most prevalent volatile in enoki mushroom only. Several volatile pairs with very high positive correlation in their levels were identified, representing the highest correlation coefficient (r = 0.970) for the pair of t-2-octenal and 2,4-octandienal. CDA was much more efficient than principal component analysis for the differentiation of mushroom species. PRACTICAL APPLICATION: The present study provided the important information on the volatile metabolite profiles of popular six commercial mushroom species. The present data will be useful for the quality control of mushrooms cultivated in farms and mushroom products processed in food industry. The strategy of canonical discriminant analysis in combination with HS-SPME-GC could be expanded for the determining the authentication of mushroom species.

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.

Genome Sequencing and Carbohydrate-Active Enzyme (CAZyme) Repertoire of the White Rot Fungus Flammulina elastica.

Next-generation sequencing (NGS) of the Flammulina elastica (wood-rotting basidiomycete) genome was performed to identify carbohydrate-active enzymes (CAZymes). The resulting assembly (31 kmer) revealed a total length of 35,045,521 bp (49.7% GC content). Using the AUGUSTUS tool, 12,536 total gene structures were predicted by ab initio gene prediction. An analysis of orthologs revealed that 6806 groups contained at least one F. elastica protein. Among the 12,536 predicted genes, F. elastica contained 24 species-specific genes, of which 17 genes were paralogous. CAZymes are divided into five classes: glycoside hydrolases (GHs), carbohydrate esterases (CEs), polysaccharide lyases (PLs), glycosyltransferases (GTs), and auxiliary activities (AA). In the present study, annotation of the predicted amino acid sequences from F. elastica genes using the dbCAN CAZyme database revealed 508 CAZymes, including 82 AAs, 218 GHs, 89 GTs, 18 PLs, 59 CEs, and 42 carbohydrate binding modules in the F. elastica genome. Although the CAZyme repertoire of F. elastica was similar to those of other fungal species, the total number of GTs in F. elastica was larger than those of other basidiomycetes. This genome information elucidates newly identified wood-degrading machinery in F. elastica, offers opportunities to better understand this fungus, and presents possibilities for more detailed studies on lignocellulosic biomass degradation that may lead to future biotechnological and industrial applications.

Regional discrimination of Agaricus bisporus mushroom using the natural stable isotope ratios.

This study was aimed to verify the regional traceability of Agaricus bisporus mushroom using unique δ13C, δ15N, δ18O, and δ34S features combined with chemometric approaches. Ten cultivars from 15 mushroom farms in six regions of Korea were measured their isotopic signatures by stable isotope ratio mass spectrometer. The δ13C, δ15N, δ18O, and δ34S values were significantly different among mushrooms collected from six cultivation regions (P < 0.05). Multi-dimensional plots including the δ15N and δ34S values demonstrated clear regional discrimination of the Saedo and Saehan cultivars produced from Buyeo, Nonsan, Eumseong, Boryeong, or Gyeongju. Partial least-squares discriminate analysis models showed good discrimination for Saedo (Rx2 = 0.798 and Q2 = 0.563) and Saehan (Rx2 = 0.819 and Q2 = 0.894). These preliminary results can extend knowledge of regional isotope signatures in A. bisporus mushroom produced in Korea, contributing to accurate geographical authenticity with potential broader applications for the international mushroom market.

Anti-inflammatory effect of lovastatin is mediated via the modulation of NF-κB and inhibition of HDAC1 and the PI3K/Akt/mTOR pathway in RAW264.7 macrophages.

Lovastatin is a 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor that is clinically used for the prevention of cardiovascular diseases. Although it has been reported that lovastatin has anti-inflammatory properties in several studies, how lovastatin regulates the inflammation is still unclear. To evaluate the effect of lovastatin on nitric oxide production (NO) in RAW264.7 macrophages, NO production assay was performed. Also, cell viability was measured to confirm cytotoxicity. Level of tumor necrosis factor-α (TNF-α) transcription was measured by reverse transcription polymerase chain reaction (RT-PCR) from total RNA in RAW264.7 cells. Western blot analysis and immunofluorescence staining were used to investigate the regulation of lovastatin on the expression, phosphorylation, and nuclear translocation of cellular proteins. The results of the present study revealed that lovastatin reduced nitric oxide production via the reduction of inducible nitric oxide synthase (iNOS) expression in lipopolysaccharide (LPS)-stimulated RAW264.7 macrophage cells. The mRNA level of TNF-α was reduced in presence of lovastatin. In addition, lovastatin downregulated histone deacetylase 1 (HDAC1), resulting in the accumulation of acetylated histone H3 and heat shock protein 70. Furthermore, the expression of phosphoinositide 3-kinase catalytic subunits α and ß was reduced under lovastatin treatment, and the phosphorylation of Akt and mammalian target of rapamycin was consequently inhibited. Lovastatin also inhibited the phosphorylation of inhibitor of nuclear factor (NF)-κBα and the translocation of NF-κB into the nucleus. Therefore, the present study demonstrates that lovastatin inhibits the expression of pro-inflammatory mediators, including iNOS and TNF-α, through the suppression of HDAC1 expression, PI3K/Akt phosphorylation and NF-κB translocation in LPS-stimulated RAW264.7 macrophage cells.

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.

A detailed analysis of the recombination landscape of the button mushroom Agaricus bisporus var. bisporus.

The button mushroom (Agaricus bisporus) is one of the world's most cultivated mushroom species, but in spite of its economic importance generation of new cultivars by outbreeding is exceptional. Previous genetic analyses of the white bisporus variety, including all cultivars and most wild isolates revealed that crossing over frequencies are low, which might explain the lack of introducing novel traits into existing cultivars. By generating two high quality whole genome sequence assemblies (one de novo and the other by improving the existing reference genome) of the first commercial white hybrid Horst U1, a detailed study of the crossover (CO) landscape was initiated. Using a set of 626 SNPs in a haploid offspring of 139 single spore isolates and whole genome sequencing on a limited number of homo- and heterokaryotic single spore isolates, we precisely mapped all COs showing that they are almost exclusively restricted to regions of about 100kb at the chromosome ends. Most basidia of A. bisporus var. bisporus produce two spores and pair preferentially via non-sister nuclei. Combined with the COs restricted to the chromosome ends, these spores retain most of the heterozygosity of the parent thus explaining how present-day white cultivars are genetically so close to the first hybrid marketed in 1980. To our knowledge this is the first example of an organism which displays such specific CO landscape.