• Mycobiology
• /
• v.50 no.5
• /
• pp.374-381
• /
• 2022

In the mating of filamentous basidiomycetes, dikaryotic mycelia are generated through the reciprocal movement of nuclei to a monokaryotic cytoplasm where a nucleus of compatible mating type resides, resulting in the establishment of two different dikaryotic strains having the same nuclei but different mitochondria. To better understand the role of mitochondria in mushrooms, we created four sets of dikaryotic strains of Lentinula edodes, including B2×E13 (B2 side) and B2×E13 (E13 side), B5×E13 (B5 side) and B5×E13 (E13 side), E8×H3 (E8 side) and E8×H3 (H3 side), and K3×H3 (K3 side) and K3×H3 (H3 side). The karyotypes and mitochondrial types of the dikaryotic strains were successfully identified by the A mating type markers and the mitochondrial variable length tandem repeat markers, respectively. Comparative analyses of the dikaryotic strains on the mycelial growth, substrate browning, fruiting characteristics, and mitochondrial gene expression revealed that certain mitochondria are more effective in the mycelial growth and the production of fruiting body, possibly through the activated energy metabolism. Our findings indicate that mitochondria affect the physiology of dikaryotic strains having the same nuclear information and therefore a selection strategy aimed at mitochondrial function is needed in the development of new mushroom strain.

• Journal of Mushroom
• /
• v.13 no.2
• /
• pp.129-134
• /
• 2015

Hybrid strains was selected by crossing between dikaryotic strain ASI 0628 (Dagul) bred from ASI 2596 (Suhan No.3) and ASI 2782(Black pileus mutant) and monokaryotic strain ASI 2344-84. The strain 84 that shown the best cultural characteristics was selected to be a new cytoplasmic hybrid variety and named as 'Chunhwashim'. The 'Chunhwashim' shown incompatibility among them forming the confrontation growth line against parental strains ASI 0628 (Dagul) and ASI 2344 (chunchu 2ho). Fruiting body produced about $161.4{\pm}4.8g$ per bottle. And also the individual generation of fruit body is multiple than chunchu2ho as 40.9. The 'Chunhwashim' was cytoplasmic hybrid included hybrid DNA bands of parental strains ASI 0628 (Dagul) and ASI 2344 (chunchu 2ho) by Random Amplified Polymorphic DNA (RAPD) primer, and mitochondria DNA band of monokaryotic ASI 2344 (chunchu 2ho) using mitochondria microsatellite DNA marker. This new cytoplasmic hybrid variety 'Chunhwashim' of oyster mushroom is characterized by multiple of individual generation and deeply grey color of pileus.

• Journal of Mushroom
• /
• v.13 no.3
• /
• pp.270-273
• /
• 2015

Hypsizigus marmoreus is commercially the most important edible mushroom in Japan. This mushroom is usually cultivated for a longer period (about 85~120 days) than other mushroom. In order to develop a new cultivar that has a shortened cultivation period, the genome analysis of this strain has been considered. This study aims to obtain parental monokaryotic strains reproducing 'Haemi' cultivar in Hypsizigus marmoreus for reference genome sequencing. The mycelia were cultured in MCM and MYG media for various incubation periods. Homogenized mycelia were treated with commercial cell wall degrading enzymes to maximize protoplasts production yield from Hypsizigus marmoreus. The greatest number of protoplasts was obtained from mycelia cultured in MCM media for 3 days using Novozyme enzyme. The isolated protoplasts were grown in regeneration agar media after two weeks. Regenerated colonies were picked and moved on separated dishes for microscopic observation. Neohaplonts regenerated from dikayotic strains were identified by the absence of clamp connections. We confirmed that one of monokaryotic strains is a parental strain by crossing with an original compatible strain of 'Haemi' cultivar. This parental strain will be used for reference genome sequence analysis.

• The Korean Journal of Mycology
• /
• v.51 no.4
• /
• pp.389-396
• /
• 2023

In this study, dikaryotic strains of Lentinula edodes were generated by mono-mono hybridization using mating type analysis and their fruiting body characteristics were investigated. Approximately 100 monokaryotic strains were isolated from the basidiospores of Sanbaekhyhang, and homokaryotic strains were isolated from Chungheung 1ho using protoplast isolation and regeneration. Their mating types were evaluated and a total of 60 dikaryotic strains were hybridized. Using these strains, fruiting bodies were produced and their characteristics were examined after cultivation on sawdust media. The results indicated that the rate of hybridization was 100% and that 55 of 60 strains formed fruiting bodies. These showed normal pileus and gill structures; however,10 strains also produced fruiting bodies with abnormal pileus and gill structures. The weight and size of the fruiting bodies differed depending on the strains. Overall, further studies are needed for predicting the characteristics of hybridized strains based on their parental strains.

• Journal of Mushroom
• /
• v.16 no.3
• /
• pp.140-146
• /
• 2018

Pleurotus eryngii is one of the most commercially important mushrooms cultivated in Korea. However, the shelf-life of the fruiting body is short, limiting its export. A new hybrid strain H17 of P. eryngii was developed to extend the shelf-life by mono-mono crossing between monokaryotic strains derived from DanBi and KNR2774. Although the cultivation period of H17 was slightly longer than that of the reference cultivar Kenneutari No.2, the quality did not change and remained normal after a period of 65.0 days at $4^{\circ}C$. This result was significantly different from that of the reference cultivar Kenneutari No.2. Analysis of the genetic characteristics of the new hybrid strain H17 revealed a different profile from that of the parental and reference cultivars when random amplification of polymorphic DNA (RAPD) primers was used. These results demonstrate that H17 is a new cultivar with improved storability after harvesting.

• Journal of Mushroom
• /
• v.19 no.3
• /
• pp.246-250
• /
• 2021

The main characteristics of a new cultivar 'Jinhyang', a cross of a collection of monokaryotic strains of the brown button mushroom, were examined. 'Jinhyang' was not significantly different from the control cultivar 'Dahyang' in mycelial culture and cultivation period. The cap of 'Jinhyang' was thicker and darker in color than that of 'Dahyang'. The hardness and individual weight of the fruiting body of 'Jinhyang' were slightly higher than those of 'Dahyang'; however, the differences were not significant. The yield of 'Jinhyang' was 14.1 kg/m², which was 8% higher than that of 'Dahyang'. Therefore, it is expected that it will be possible to supply this cultivar growers in the future.

• Mycobiology
• /
• v.49 no.6
• /
• pp.599-603
• /
• 2021

CRISPR/Cas9 genome editing systems have been established in a broad range of eukaryotic species. Herein, we report the first method for genetic engineering in pyogo (shiitake) mushrooms (Lentinula edodes) using CRISPR/Cas9. For in vivo expression of guide RNAs (gRNAs) targeting the mating-type gene HD1 (LeA1), we identified an endogenous LeU6 promoter in the L. edodes genome. We constructed a plasmid containing the LeU6 and glyceraldehyde-3-phosphate dehydrogenase (LeGPD) promoters to express the Cas9 protein. Among the eight gRNAs we tested, three successfully disrupted the LeA1 locus. Although the CRISPR-Cas9-induced alleles did not affect mating with compatible monokaryotic strains, disruption of the transcription levels of the downstream genes of LeHD1 and LeHD2 was detected. Based on this result, we present the first report of a simple and powerful genetic manipulation tool using the CRISPR/Cas9 toolbox for the scientifically and industrially important edible mushroom, L. edodes.

• Journal of Mushroom
• /
• v.13 no.2
• /
• pp.135-138
• /
• 2015

'Daejang 3ho' is a new cultivar of oyster mushroom for high productivity than that of Chunchu 2ho. This cultivar was bred by Di-mon mating between monokaryotic strains isolated from 'Daejang 1ho' and dikaryotic strain 'Jangan 5ho'. The color of pileus and the shape of pileus of new cultivar were dark gray and deeply funnel shape, respectively. The length and thickness of stipe were longer and thicker than those of Chunchu 2ho. It took 4~5 days to formation of primordia, that was a similar to Chunchu 2ho. The optical temperature of fruit body was $14-17^{\circ}C$. Yield of 'Daejang 3ho' was 13.9% higher than that of Chunchu 2ho. Analysis of RAPD by URP-primer of #03, #08, #10 and #11 showed that different band pattern those of other strains. There were no significant differences between the texture properties of 'Daejang 3ho' and Chunchu 2ho.

• 한국균학회소식:학술대회논문집
• /
• 2015.11a
• /
• pp.35-35
• /
• 2015

Pleurotus eryngii has recently become a major cultivated mushroom; it uses tetrapolar heterothallism as a part of its reproductive process. Sexual development progresses only when the A and B mating types are compatible. Such mating incompatibility occasionally limits the efficiency of breeding programs in which crossing within loci-shared strains or backcrossing strategies are employed. Therefore, understanding the mating system in edible mushroom fungi will help provide a short cut in the development of new strains. We isolated and identified pheromone and receptor genes in the B3 locus of P. eryngii and performed a functional analysis of the genes in the mating process by transformation. A genomic DNA library was constructed to map the entire mating-type locus. The B3 locus was found to contain four pheromone precursor genes and four receptor genes. Remarkably, receptor PESTE3.3.1 has just 34 amino acid residues in its C-terminal cytoplasmic region; therefore, it seems likely to be a receptor-like gene. Real-time quantitative RT-PCR (real-time qRT-PCR) revealed that most pheromone and receptor genes showed significantly higher expression in monokaryotic cells than dikaryotic cells. The pheromone genes PEphb3.1 and PEphb3.3 and the receptor gene PESTE3.3.1 were transformed into P5 (A3B4). The transformants were mated with a tester strain (A4B4), and the progeny showed clamp connections and a normal fruiting body, which indicates the proposed role of these genes in mating and fruiting processes. This result also confirms that PESTE3.3.1 is a receptor gene. In this study, we identified pheromone and receptor genes in the B3 locus of P. eryngii and found that some of those genes appear to play a role in the mating and fruiting processes. These results might help elucidate the mechanism of fruiting differentiation and improve breeding efficiency.

• The Korean Journal of Mycology
• /
• v.47 no.2
• /
• pp.143-152
• /
• 2019

A new cultivar 'Sanjanghyang' was bred from monokaryotic strains of 'Sanbaekhyang' and 'Jangan 1ho'. Pileus was flat, round, and reddish brown. The diameters of the pilei and stipe length of the fruiting bodies were 67.1 mm and 16.9 mm, respectively. The scales were white or slightly brown and distributed evenly. The gill density was sparse and showed a rippled texture. The stipe was cream in color and the fluff was medium. 'Sanjanghyang' had a short cultivation period and fruiting bodies occurred sporadically. Temperature for fruiting body formation was a medium, between 15 to $19^{\circ}C$. 'Sanjanghyang' was different from 'Sanbaekhyang' with regard to its pileus diameter (67.1 mm) and autumn and spring fruiting body production period. 'Sanbaekhyang' had pileus diameter of 74.7 mm, and fruiting body formation occurred in spring and autumn. The rate of fruiting body formation was 89% (first flush), 4% (second flush), and 7% (third flush).