Edodes Strains Research Articles

ITS1/5.8S/ITS2, a Good Marker for Initial Classification of Shiitake Culinary-Medicinal Lentinus edodes (Agaricomycetes) Strains in China.

China is home to rich wild and cultivated strains of Lentinus edodes, an important edible and medicinal mushroom. Artificial selection of L. edodes has a long history, and the widely cultivated strains belong to populations different from those of most wild strains. Internal transcribed spacer (ITS) regions have been used as good markers to identify L. edodes populations. But because ITS regions exhibit incomplete concerted evolution, the use of an ITS to identify L. edodes populations has been questioned. The objective of this study was to determine whether the ITS region is suitable for identifying L. edodes populations and which populations the widely cultivated strains and the most wild strains belong to by investigating intraindividual and differential ITS polymorphisms between 44 cultivars and 44 wild strains of L. edodes in China. Intraindividual ITS polymorphism is common in L. edodes strains, and most strains possessed 2 different ITS sequences, which came from their heterokaryons. The genetic polymorphisms of ITS1, 5.8S, and ITS2 in L. edodes strains are distinct. All strains were divided into one 5.8S type (5.8S-A), 2 ITS1 types (ITS1-A and ITS1-B), and 2 ITS2 types (ITS2-A and ITS2-B), which were subdivided into 2 branches (ITS2-A1 and ITS2-A2; ITS2-B1 and ITS2-B2). ITS1/5.8S/ITS2 could be used as a good marker in preliminary classification of L. edodes strains in China. It not only exhibited classified information of ITS1, 5.8S, and ITS2 for each strain at the same time, it also indicated whether the strain was heterozygous. The 44 cultivated strains were mainly the A/A/A1 type, and the 44 wild strains were mainly the A/A/A2 and other mixed types.

Lp16-PSP, a Member of YjgF/YER057c/UK114 Protein Family Induces Apoptosis and p21WAF1/CIP1 Mediated G1 Cell Cycle Arrest in Human Acute Promyelocytic Leukemia (APL) HL-60 Cells

Lp16-PSP (Latcripin 16-Perchloric acid Soluble Protein) from Lentinula edodes strain C91-3 has been reported previously in our laboratory to have selective cytotoxic activity against a panel of human cell lines. Herein, we have used several parameters in order to characterize the Lp16-PSP-induced cell death using human acute promyeloid leukemia (HL-60) as a model cancer. The results of phase contrast microscopy, nuclear examination, DNA fragmentation detection and flow cytometry revealed that high doses of Lp16-PSP resulted in the induction of apoptosis in HL-60 cells. The colorimetric assay showed the activation of caspase-8, -9, and -3 cascade highlighting the involvement of Fas/FasL-related pathway. Whereas, Western blot revealed the cleavage of caspase-3, increased expression of Bax, the release of cytochrome c and decreased expression of Bcl-2 in a dose-dependent manner, suggesting the intrinsic pathway might be involved in Lp16-PSP-induced apoptosis as well. Low doses of Lp16-PSP resulted in the anchorage-independent growth inhibition, induction of G1 phase arrest, accompanied by the increased expression of p21WAF1/CIP1, along with the decreased expression of cyclin D, E, and cdk6. In addition, Lp16-PSP resulted in constitutive translocation inhibition of transcription factor nuclear factor kappa B (NF-κB) into the nucleus by decreasing the phosphorylation of IκBα. All these findings suggested Lp16-PSP as a potential agent against acute promyeloid leukemia; however, further investigations are ultimately needed.

Lentinula edodes and Pleurotus ostreatus: functional food with antioxidant - antimicrobial activity and an important source of Vitamin D and medicinal compounds

Background: Mushrooms produce a large amount of medicinal compounds, and are also an optimal source of fibres, proteins, vitamins (like groups B and D), and other micronutrients including potassium, magnesium, etc. Consequently, mushrooms are commonly considered to be functional foods. Many works report the high biological potentials of medicinal mushrooms involving their antibacterial, hypoglycaemic, anticholesterolemic, radical scavenging, and anti-inflammatory effects.Context and purpose of this study: First off, this work aimed to find strains of Lentinula edodes and Pleurotus ostreatus from a bank of edible mushrooms bought from international strain banks (Table I) that could possess health benefit related properties, such as a radical scavenging activity (antioxidant effect), antibacterial effects against common pathogenic bacteria, and being able to produce interesting nutrients and secondary metabolites. As the fungal bank comprises of 20 strains of L. edodes and 20 strains of P. ostreatus, a first screening was made by the selection of 13 strains for each mushroom able to grow in multiple wood types or that were particularly productive and had proved good growth reproducibility over the last 5 years. This work also studied the correlation between culture conditions and mushroom quality in terms of the previously reported properties. Comparison among the selected strains was operated by the assessment of antioxidant and antimicrobial activities after different sample treatments. Furthermore, an initial optimization of the analytic techniques was produced for the direct estimation of important secondary metabolites and nutrients by means of HPLC-MS/MS technique. Further research will encompass an evaluation of transformation processes (drying, freezing, rehydration, cooking, etc.) impact on radical scavenging, antibacterial activity, and possible degradation/loss of nutraceutically important substances such as vitamin D2, ergothioneine, eritadenine, lovastatin, lentinan, and lenthionine. Results: 13 strains of each mushroom species have been cultivated on different wood logs. Seven strains of shiitake and six strains of oyster mushroom were able to produce sporocarps. Antioxidant levels in water extracts from dried mushrooms produced significatively different results on the basis of strains and of wood. Both mushrooms demonstrated higher radical scavenging activity in log cultivation than substrates cultivation, which was subsequently used as reference. Furthermore, all strains of P. ostreatus demonstrated the lowest level of antioxidant activity at 4°C, a significant increase towards 50°C and a limited decrease towards 80°C. The same trend was observed for shiitake extracts. Concerning the shiitake mushroom only, crude water extracts showed an interesting antibacterial activity against the model microorganisms Pseudomonas aeruginosa and Staphylococcus aureus. A comparison was also performed between the best performing strain extract and the commercial antibiotic Ceftriaxone against P. aeruginosa, assessing that 20 mg of crude extract corresponds to 0.2 mg of the pure antibiotic when studied by means of disk diffusion assay.Conclusions: The results suggested that the cultivation of both shiitake and oyster mushrooms on logs could enhance the content of antioxidant and antibacterial activities, compared to the cultivation of mushrooms on sawdust substrates. Radical scavenging and antibacterial activity depends both on L. edodes strain and the log type. The bacteriostatic/bactericidal activity of the best performer strain may depend on a pH and solvent treatment sensitive substance. Secondary metabolites such as ergothioneine and vitamin D2 from both shiitake and oyster were released just after water extraction: this suggests that the transformation/cooking processes may produce a loss of characteristic mushroom biological properties in water. Further evaluation of biologically relevant compounds content and loss during different food transformation and cooking processes will be assessed.Key words: Shiitake, Oyster, Pleurotus ostreatus, Lentinula edodes, Pseudomonas aeruginosa, Staphylococcus aureus, Log grown mushroom, antioxidant, radical scavenging, total phenolic content, DPPH, ABTS, Folin-Ciocalteu, antibacterial, fruitbodies, sporocarps.

Valorization of spent oyster mushroom substrate and laccase recovery through successive solid state cultivation of Pleurotus, Ganoderma, and Lentinula strains.

Spent mushroom substrate (SMS) of Pleurotus ostreatus was supplemented with wheat bran and soybean flour in various proportions to obtain C/N ratios of 10, 20, and 30, and their effect was evaluated in successive cultivation of Pleurotus ostreatus, Pleurotus pulmonarius, Ganoderma adspersum, Ganoderma resinaceum, and Lentinula edodes strains with respect to mycelium growth rate, biomass concentration, recovery of the enzyme laccase and crude exopolysaccharides, and also with additional fruiting body production. All fungi showed the highest growth rate on unamended SMS (C/N 30), with G. resinaceum being the fastest colonizer (Kr=9.84mmday-1), while biomass concentration maximized at C/N 10. Moreover, supplementation affected positively laccase activity, with P. pulmonarius furnishing the highest value (44,363.22Ug-1) at C/N 20. On the contrary, L. edodes growth, fruiting, and laccase secretion were not favored by SMS supplementation. Fruiting body formation was promoted at C/N 30 for Ganoderma and at C/N 20 for Pleurotus species. Exopolysaccharide production of further studied Pleurotus strains was favored at a C/N 20 ratio, at the initial stage of SMS colonization. The obtained results support the potential effective utilization of supplemented SMS for laccase production from Ganoderma spp. and for new fruiting body production of Pleurotus spp.

Biological and Molecular Characteristics of a Novel Partitivirus Infecting the Edible Fungus Lentinula edodes.

A new partitivirus named Lentinula edodes partitivirus 1 (LePV1) was isolated from a diseased L. edodes strain with severe degeneration of the mycelium and imperfect browning in bag cultures. The nucleotide sequences of LePV1 dsRNA-1 and dsRNA-2 were determined; they were 2,382 bp and 2,245 bp in length, and each contained a single ORF encoding RNA-dependent RNA polymerase (RdRp) and coat protein (CP), respectively. The purified virus preparation contained isometric particles 34 nm in diameter encapsidating these dsRNAs. Phylogenetic analyses showed LePV1 to be a new member of Betapartitivirus, with the RdRp sequence most closely related to Grapevine partitivirus. RT-PCR analysis showed that 27 of the 56 Chinese L. edodes core collection strains carry LePV1, with the virus being more common in wild strains than cultivated strains. In addition, qPCR analysis suggested that coinfection with L. edodes mycovirus HKB (LeV-HKB) could increase replication of the RdRp gene of LePV1. This study may be essential for the development of more accurate disease diagnostics and the formulation of control strategies for viral diseases in L. edodes.

Mycelial Extracts as Sensitive Element for Acoustoelectronic Gas Sensor

The main problem in development of the acoustoelectronic gas sensors is the search of the elements which are sensitive to the presence of different gases. In this paper we suggest to use for this purpose the piezoelectric quartz resonators with the mycelium films of mushroom Lentinula edodes strain F-249, which was cultivated in synthetic medium. The extract of mycelium was deposited on the surface of the resonator. The measurements of the frequency dependencies of the real and imaginary parts of electrical impedance of the resonator allowed finding the density, elastic constants and viscosity of the films under study. The influence of various gases (ammonia, formaldehyde, ethylacetate) and volatile liquids (acetone, acetic acid, chloroform) on physical properties of the extracts of mushroom Lentinula edodes mycelium has been investigated. It has been found that it is possible to use the mycelium films of mushroom Lentinula edodes prepared by different ways as sensitive film to ammonia, formaldehyde and ethyl acetate.

Acousto-electronic gas sensor based on mushroom mycelial extracts

The main problem in the development of acousto-electronic gas sensors is the search for materials which are sensitive to the presence of different gases. For this purpose in this paper we suggest the use of piezoelectric quartz resonators coated by the mycelium-origin films of the mushroom Lentinula edodes strain F-249, which were cultivated in a synthetic medium. The extract of mycelium was deposited on the surface of the resonator. Measurement of the frequency dependence of the complex electrical impedance of the resonator allowed us to evaluate the density, elastic constants, and mass of the films under test. The influence of different gases, such as ammonia, formaldehyde, ethylacetate, and volatile liquids: acetone, acetic acid, chloroform on the physical properties of the extracts of Lentinula edodes mushroom mycelium was investigated. The results have shown that this material prepared according to different technological procedures is suitable as a sensitive layer for development of gas sensors for detection of ammonia, formaldehyde, and ethylacetate.

Selection of strains for shiitake production in axenic substrate.

Shiitake mushroom consumption is increasing in Brazil. In addition to the implementation of new production methods, it is also important to increase productivity, quality and reduce production costs. In this study, six commercial Lentinula edodes strains were characterized for genetic diversity (rep-PCR analysis) and mushroom production (yield, number and weight of individual mushrooms) using different substrates and cultural conditions. All strains showed genetic differences by repetitive element palindromic based-polymerase chain reaction (rep-PCR). The richest substrate resulted in the greatest production under both environmental conditions. Strains LE4 and LE6 produced the majority of their mushrooms earlier than the other strains. The highest number of mushrooms was observed in the LE6 strain while the highest weights of individual mushrooms were observed in the LE4 strain. Controlled environmental conditions resulted in superior production for all strains, except for LE4, which had empirically greater yield in the semi-controlled environmental condition.

표고 수집종들의 유리당, 아미노산 및 β-glucan 함량

표고 수집종들의 유리당, 아미노산 및 β-glucan 함량

Genetic diversity and population structure of Chinese Lentinula edodes revealed by InDel and SSR markers

Genetic diversity and population structure of 88 Chinese Lentinula edodes strains belonging to four geographic populations were inferred from 68 Insertion-Deletion (InDel) and two simple sequence repeat (SSR) markers. The overall values of Shannon’s information index and gene diversity were 0.836 and 0.435, respectively, demonstrating a high genetic diversity in Chinese L. edodes strains. Among the four geographic populations, the Central China population displayed a lower genetic diversity. Multiple analyses resolved two unambiguous genetic groups that corresponded to two regions from which the samples were collected—one was a high-altitude region (region 1) and the other was a low-altitude region (region 2). Results from analysis of molecular variance suggested that the majority of genetic variation was contained within populations (74.8 %). Although there was a strong genetic differentiation between populations (F ST = 0.252), the variability of ITS sequences from representative strains of the two regions (<3 %) could not support the existence of cryptic species. Pairwise F ST values and Nei’s genetic distances showed that there were relatively lower genetic differentiations and genetic distances between populations from the same region. Geographic distribution could play a vital role in the formation of the observed population structure. Mycelium growth rate and precocity of L. edodes strains displayed significant differences between the two regions. Strains from region 2 grew faster and fructified earlier, which could be a result of adaptation to local environmental factors. To the best of our knowledge, this was the first study on the genetic structure and differentiation between populations, as well as the relationship between genetic structure and phenotypic traits in L. edodes.

English

Lentinula edodes is among the five globally cultivated edible mushrooms, which are wood decaying spore bearing Basidiomycetes possessing separate hyphae. Specific identification of this fungus from others in the division Basidiomycota using specific primers enables a fast and accurate detection through polymerase chain reaction (PCR). As a prelude to additional nutritional and sequence characterization research, we have developed a species-specific PCR assay for this fungus after screening four primer-pairs and two universal primer pairs. The primer-pair LE1F/R was specific in amplifications of ATCC-defined L. edodes strains and did not amplify DNA from six medicinally and nutritionally important fungal reference strains, Oyster (Pleurotus ostreatus), Maitake (Grifola frondosa), Enoki (Flammulina velutipes), Baby bella (Agaricus bisporus), Porcini (Boletus edulis), and Chanterelle (Cantharellus cibarius). However, amplifications using the universal primers were positive for all six strains. This assay will therefore serve to validate morphology-based-identifications of L. edodes strains. Key words: Lentinula edodes, LE1F/R, species-specific primers.

Identification and characterization of genes related to cellulolytic activity in basidiomycetes.

Enzymes produced by basidiomycetes that are involved in the cellulose degradation process, and their respective codifying genes, must be identified to facilitate the development of novel biotechnological strategies and applications in the agro-industry. The objective of this study was to identify prospective cellulase-producing genes and characterize their cellulolytic activity, in order to elucidate the potential biotechnological applications (with respect to vegetal residues) of basidiomycetes. The basidiomycete strains Lentinula edodes U8-1, Lentinus crinitus U9-1, and Schizophyllum commune U6-7 were analyzed in this study. The cellulolytic activities of these fungi were evaluated based on the halo formation in carboxymethyl cellulose culture medium after dyeing with Congo red. The presence of cellulase-codifying genes (cel7A, cel6B, cel3A, and egl) in these fungal strains was also evaluated. L. edodes and S. commune presented the highest cellulolytic halo to mycelial growth radius ratio, followed by L. crinitus. Four genes were amplified in the L. edodes strain, whereas three and one genes were isolated from L. crinitus and S. commune, respectively. The cel6B gene (L. edodes) presented the conserved domain glyco_hydro_6 and characterized as cellobiohydrolase gene. The results of this study contribute to the existing knowledge on cellulases in basidiomycetes, and serve as a basis for future studies on the expression of these genes and the characterization of the catalytic activity of these enzymes. This allows for better utilization of these fungi in degrading vegetal fibers from agro-industrial residues and in other biotechnological applications.

Optimised isolation of polysaccharides from Lentinula edodes strain NCBI JX915793 using response surface methodology and their antibacterial activities

Mycelial growth in a defined medium by submerged fermentation is a rapid and alternative method for obtaining fungal biomass of consistent quality. Biomass, exopolysaccharides (EPS) and intracellular polysaccharides (IPS) production were optimised by response surface methodology in Lentinula edodes strain LeS (NCBI JX915793). The optimised conditions were pH 5.0, temperature 26°C, incubation period of 25 days and agitation rate of 52 r/min for L. edodes strain LeS. Under the calculated optimal culture conditions, biomass production (5.88 mg mL− 1), EPS production (0.40 mg mL− 1) and IPS production (12.45 mg g− 1) were in agreement with the predicted values for biomass (5.93 mg mL− 1), EPS (0.55 mg mL− 1) and IPS production (12.64 mg g− 1). Crude lentinan exhibited highest antibacterial effects followed by alcoholic, crude and aqueous extracts. The results obtained may be useful for highly effective yield of biomass and bioactive metabolites.

Taxonomy, Phylogeny, Cultivation and Biological Activities of a Lentinus species from Andman and Nicobar islands (India)

Fruit bodies of Lentinus were collected from subtropical climate of Andman & Nicobar Islands (India). Pure culture was prepared by tissue culture on Malt-extract-agar (MEA) medium at 25C±2C. The specimen was studied for its identification, taxonomy and phylogeny and identified as Lentinus sajor-caju by morphological and microscopical studies. The identification was confirmed through ITS 5.8S rDNA sequencing and sequence analysis. Its cultivation was done on saw dust and wheat bran. The fruit bodies were obtained at a temperature of 28±2°C and 80-85% RH. The cultivated Lentinus sajor-caju fruit bodies were analysed for nutritional and biological properties and observed that the cultivated mushroom has good nutritional properties while antioxidant, reducing and DPPH free radical scavenging properties are comparable to commercially cultivated Lentinula edodes strains.

Changes in the mycovirus (LeV) titer and viral effect on the vegetative growth of the edible mushroom Lentinula edodes

This study attempted to cure the edible mushroom Lentinula edodes strain FMRI0339 of the L. edodes mycovirus (LeV) in order to obtain an isogenic virus-free fungal strain as well as a virus-infected strain for comparison. Mycelial fragmentation, followed by being spread on a plate with serial dilutions resulted in a virus-free colony. Viral absence was confirmed with gel electrophoresis after dsRNA-specific virus purification, Northern blot analysis, and PCR using reverse transcriptase (RT-PCR). Once cured, all of fungal cultures remained virus-free over the next two years. Interestingly, the viral titer of LeV varied depending on the culture condition. The titer from the plate culture showed at least a 20-fold higher concentration than that grown in the liquid culture. However, the reduced virus titer in the liquid culture was recovered by transferring the mycelia to a plate containing the same medium. In addition, oxygen-depleted culture conditions resulted in a significant decrease of viral concentration, but not to the extent seen in the submerged liquid culture. Although no discernable phenotypic changes in colony morphology were observed, virus-cured strains showed significantly higher growth rates and mycelial mass than virus-infected strains. These results indicate that LeV infection has a deleterious effect on mycelial growth.

First Report of Trichoderma oblongisporum Causing Green Mold Disease on Lentinula edodes (shiitake) in China.

Lentinula edodes (shiitake) is well known for its delicious taste and valuable medical functions, and ranked as the second most important mushroom in terms of total world production. In March 2012, a serious green mold epidemic occurred on many mushroom farms in Suizhou County of China. The infected mycelia of L. edodes in cultivated bags became rotten, yellow, wilted, and finally died, with the surfaces of the cultivated bags covered with dark green fungal colonies. At a temperature above 20°C, disease incidence was nearly 100% on some mushroom farms. Three diseased cultivated bags were collected from three different mushroom farms, and two portions at the junction of the diseased and healthy portions of the bag were plated individually on potato dextrose agar (PDA) and incubated at 25°C for 4 days. Following incubation, agar discs cut from the growing front of colonies were inoculated onto fresh PDA and subcultured to obtain putative pathogens. Three purified isolates were all whitish initially, followed by the emergence of greenish conidial clusters at the outer margin of the colony. The underside of the colony appeared pale yellow. The growth rate of the isolates was about 0.95 to 1.02 cm/day in PDA at 25°C. Aerial mycelia were floccose, white, and septate. Chlamydospores were sub-globose to broadly ellipsoidal. Conidiophore branches arose at right angles, and primary branches arose singly or in pairs. Phialides were ampulliform, 3.1 to 6.7 × 2.7 to 4.0 μm, slightly constricted at the base, swollen in the middle, and narrowed abruptly at the apex. Conidia were produced on the top of the phialides with the shape varying from ellipsoidal to oblong, 3.3 to 4.7 × 2.4 to 3.2 μm. These observations were consistent with the description of Trichoderma oblongisporum by Bissett (1). The ITS and partial tef1 were amplified from the three isolates as previously reported (2) and sequenced (KM110064 to KM110069). Nucleotide alignment showed 99% sequence identity (ITS) with two T. oblongisporum isolates (FJ623268 and DQ083020), and 88% similarity (tef1) with T. oblongisporum (AY750884). Neighbor joining tree of ITS and tef1 nucleotide sequences also showed that our three isolates had the closest relationship with the aforementioned three T. oblongisporum strains. To determine pathogenicity, a sawdust substrate was sterilized for 2 h in polyethylene bags and subsequently inoculated separately with three isolates and L. edodes strain Qiu7, which was cultivated widely in Suizhou. When the mycelia of Qiu7 colonized the bags, 10 cm3 of substrate was withdrawn from each bag. The substrate was then exposed to 10 cm3 of mycelium from each pathogen in 10 bags. A parallel inoculation with 10 cm3 of sterilized sawdust substrate without pathogen mycelia was performed as a control. The inoculated cultivated bags were kept at 25°C. After 2 months, all of the mycelia in the bags became wilted and dead, and the cultivated bags became soft, rotten, and covered with dark green fungal colonies. The controls remained uninfected. The symptoms were similar to those observed on mushroom farms. Pathogens re-isolated from all the inoculated culture bags were confirmed to be T. oblongisporum through morphological characteristics, molecular identification, and phylogenetic analysis. To our knowledge, this is the first report of T. oblongisporum causing green mold disease on L. edodes in the world.

Molecular characterization of shiitake medicinal mushroom, Lentinus edodes strains (higher Basidiomycetes) using RAPD and ITS sequencing.

The molecular phylogeny in seven strains of Lentinus edodes was studied based on RAPD and their internal transcribed spacers (ITS) regions. The strains were analyzed by RAPD with 20 arbitrary primers. Fifteen primers were found efficient for the amplification of the genomic DNA. The size of the polymorphic bands were in the range of 100-1000 bp. However, the size of ITS1-2 and ITS1-4 regions varied among the strains from 278 to 575 bp and from 410 to 616 bp, respectively. The higher alignment score of the ITS 1-2 region indicated more variability in the ITS 1-4 region. Thus, on the basis of RAPD-PCR and ITS sequencing it was found that strains LeC and LeI showed a high degree of divergence from all other strains.

Occurrence of dsRNA Mycovirus (LeV-FMRI0339) in the Edible Mushroom Lentinula edodes and Meiotic Stability of LeV-FMRI0339 among Monokaryotic Progeny.

dsRNA was found in malformed cultures of Lentinula edodes strain FMRI0339, one of the three most popular sawdust cultivated commercial strains of shiitake, and was also found in healthy-looking fruiting bodies and actively growing mycelia. Cloning of the partial genome of the dsRNA revealed the presence of the RdRp sequence of a novel L. edodes mycovirus (LeV), and sequence comparison of the cloned amplicon showed identical sequences sequence to known RNA-dependent RNA polymerase genes of LeV found in strain HKA. The meiotic stability of dsRNA was examined by measuring the ratio of the presence of dsRNA among sexual monokaryotic progeny. More than 40% of the monokaryotic progeny still contained the dsRNA, indicating the persistence of dsRNA during sexual reproduction. Comparing the mycelia growth of monokaryotic progeny suggested that there appeared to be a tendency toward a lower frequency of virus incidence in actively growing progeny.

Cloning of the Lentinula edodes B mating-type locus and identification of the genetic structure controlling B mating

During the life cycle of heterothallic tetrapolar Agaricomycetes such as Lentinula edodes (Berk.) Pegler, the mating type system, composed of unlinked A and B loci, plays a vital role in controlling sexual development and resulting formation of the fruit body. L. edodes is produced worldwide for consumption and medicinal purposes, and understanding its sexual development is therefore of great importance. A considerable amount of mating type factors has been indicated over the past decades but few genes have actually been identified, and no complete genetic structures of L. edodes B mating-type loci are available. In this study, we cloned the matB regions from two mating compatible L. edodes strains, 939P26 and 939P42. Four pheromone receptors were identified on each new matB region, together with three and four pheromone precursor genes in the respective strains. Gene polymorphism, phylogenetic analysis and distribution of pheromone receptors and pheromone precursors clearly indicate a bipartite matB locus, each sublocus containing a pheromone receptor and one or two pheromone precursors. Detailed sequence comparisons of genetic structures between the matB regions of strains 939P42, 939P26 and a previously reported strain SUP2 further supported this model and allowed identification of the B mating type subloci borders. Mating studies confirmed the control of B mating by the identified pheromone receptors and pheromones in L. edodes.

Rapid genotyping by low-coverage resequencing to construct genetic linkage maps of fungi: a case study in Lentinula edodes

BackgroundGenetic linkage maps are important tools in breeding programmes and quantitative trait analyses. Traditional molecular markers used for genotyping are limited in throughput and efficiency. The advent of next-generation sequencing technologies has facilitated progeny genotyping and genetic linkage map construction in the major grains. However, the applicability of the approach remains untested in the fungal system.FindingsShiitake mushroom, Lentinula edodes, is a basidiomycetous fungus that represents one of the most popular cultivated edible mushrooms. Here, we developed a rapid genotyping method based on low-coverage (~0.5 to 1.5-fold) whole-genome resequencing. We used the approach to genotype 20 single-spore isolates derived from L. edodes strain L54 and constructed the first high-density sequence-based genetic linkage map of L. edodes. The accuracy of the proposed genotyping method was verified experimentally with results from mating compatibility tests and PCR-single-strand conformation polymorphism on a few known genes. The linkage map spanned a total genetic distance of 637.1 cM and contained 13 linkage groups. Two hundred sequence-based markers were placed on the map, with an average marker spacing of 3.4 cM. The accuracy of the map was confirmed by comparing with previous maps the locations of known genes such as matA and matB.ConclusionsWe used the shiitake mushroom as an example to provide a proof-of-principle that low-coverage resequencing could allow rapid genotyping of basidiospore-derived progenies, which could in turn facilitate the construction of high-density genetic linkage maps of basidiomycetous fungi for quantitative trait analyses and improvement of genome assembly.