• Mycobiology
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• v.46 no.4
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• pp.407-415
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• 2018

Pheromone (PHB)-receptor (RCB) interaction in the mating pheromone response pathway of Lentinula edodes was investigated using synthetic PHBs. Functionality of the C-terminally carboxymethylated synthetic PHBs was demonstrated by concentration-dependent induction of a mating-related gene (znf2) expression and by pseudoclamp formation in a monokaryotic strain S1-11 of L. edodes. Treatment with synthetic PHBs activated the expression of homeodomain genes (HDs) residing in the A mating type locus, and of A-regulated genes, including znf2, clp1, and priA, as well as genes in the B mating type locus, including pheromone (phb) and receptor (rcb) genes. The synthetic PHBs failed to discriminate self from non-self RCBs. PHBs of the B4 mating type (B4 PHBs) were able to activate the mating pheromone response pathway in both monokaryotic S1-11 and S1-13 strains, whose B mating types were B4 (self) and B12 (non-self), respectively. The same was true for B12 PHBs in the B4 (non-self) and B12 (self) mating types. The synthetic PHBs also promoted the mating of two monokaryotic strains carrying B4-common incompatible mating types ($A5B4{\times}A1B4$). However, the dikaryon generated by this process exhibited abnormally high content of hyphal branching and frequent clamp connections and, more importantly, was found to be genetically unstable due to overexpression of mating-related genes such as clp1. Although synthetic PHBs were unable to discriminate self from non-self RCBs, they showed a higher affinity for non-self RCBs, through which the mating pheromone response pathway in non-self cells may be preferentially activated.

• The Korean Journal of Mycology
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• v.47 no.2
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• pp.173-179
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• 2019

Sawdust cultivation of Lentinula edodes has been increasing in Korea. Fourteen strains were used to develop the best varieties of L. edodes, and hybridization was carried out by monohybrid cross. The number of hybridized strains was 1,638 among 3,100 combinations. They were cultivated on sawdust medium, and fruiting bodies were formed in 364 strains. Among them, 65 strains were selected as superior candidate strains based on the shape and size of the fruiting bodies. Forty strains formed fruiting bodies without lamellae structure. The shape of the stipe was cylindrical (255 strains), thick to lower part (15 strains), and thick to upper part (94 strains). By the combinations of 2462 n1-10 and 3420 n1-10, 2462 n1, 2462 n2, 2462 n10, and 3420 n3 were selected as excellent monokaryotic strains. These strains were considered to be superior monokaryotic strains that could be used for hybrid breeding.

• The Korean Journal of Mycology
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• v.14 no.1
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• pp.17-23
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• 1986

The mating system of monokaryotic isolates in Pleurotus sajor-caju was controlled by two incompatibility factors A and B of tetrapola mating system. The mycelia of dikaryotic isolates grew faster than those of their component monokaryons, but no correlation between dikaryotic and their component monokaryotic isolates was found. The primodia formed well on the potato dextrose agar (PDA) media not only in the dikaryotic isolates but also in the monokaryons under irradiated conditions. The dikaryotic isolates produced normal sporophores; however, the monokaryotic isolates produced abnormal sporophores when they were cultivated with sawdust substrates. Some dikaryotic isolates derived by mating between monokaryotic isolates showed high yields of sporophores more than those of parental strains. Both the dikaryotic mycelial growth rate and primodia formation number on the PDA plate showed significant correlation with its sporophore products on sawdust substrates.

• Mycobiology
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• v.30 no.2
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• pp.61-64
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• 2002

A Monokaryotic strain G8M without clamp connections was isolated from germinated basidiospore that was obtained from cultivated fruit body. Strain G8M was used as a tester isolate for 'dikaryon-monokaryon mating'(di-mon mating) with the strains of Ganoderma lucidum, G6 and G35(Korean wild strains), G3(Taiwan), G4(Canada), G15(America), G. oregonense G24, G. resinaceum G28, G. oerstedii G23, and G. subamboinense G29. Isolate G8M was compatible to Korean strains G6 and G35, but was incompatible to foreign strains G3, G4, or G15. Compatible reactions between strains were readily observed macroscopically. Clear barrage lines formed between incompatible strains. These clear lines were not apparent in compatible di-mon matings. The Korean strains were morphologically distinct; they did not form any chlamydospores, and stopped growth at $35^{\circ}C$. The strains of G. lucidum from Korea may be considered as different species from Taiwan, Canadian and American cultures.

• 한국균학회소식:학술대회논문집
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• 2014.10a
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• pp.42-42
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• 2014

Mating of tetrapolar mushrooms is regulated by to chromosomal loci, A and B. A locus contains A gene that expresses a homeodomain protein whereas B locus contains multiple pheromones and receptor genes. In order to characterize the mating loci in Korean cultivated strains of Lentinula edodes, one hundred monokaryotic myclelia were isolated from the basidiospores of cultivated strains, including Cham-A-Ram, Sanjo701, and Sanjo707. Both mating loci were amplified using primer sets targeting conserved sequence regions for homeodomain (HD), pheromone, and receptor genes. Subsequent sequence analysis revealed that the Korean strains contained significant variations in the homeodomain of A locus, even within the same A1 or A2 mating type. Similarly, B locus was also highly diversified in the sequences of pheromones and receptors as well as gene organization. These results enabled us to design mating type-specific probes which can distinguish mating type of each strain. The specificity was confirmed by between intra- and inter-strain mating experiment.

• 한국균학회소식:학술대회논문집
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• 2015.11a
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• pp.37-37
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• 2015

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 an identical sequence to known RdRp 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, although variations in the growth rate existed among progeny and virus infection was observed in highly actively growing progeny, there appeared to be a tendency toward a lower frequency of virus incidence in actively growing progeny. This study attempted to cure the edible mushroom L. 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. We were also explored effects of LeV on fruiting body formation and mushroom yield. The fruiting body formation yield of virus-free L. edodes was larger than virus-infected L. edodes. These results indicate that LeV infection has a deleterious effect on mycelial growth and fruiting body formation. In addition, we have been investigated host-parasite interaction between L. edodes and its mycovirus interaction to study viral mechanism by establishment of proteomics.

• Mycobiology
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• v.39 no.4
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• pp.272-277
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• 2011

Chemical mutagenesis of basidiospores of Hypsizygus marmoreus generated new mushroom strains. The basidospores were treated with methanesulfonate methylester, an alkylating agent, to yield 400 mutant monokaryotic mycelia. Twenty fast-growing mycelia were selected and mated each other by hyphal fusion. Fifty out of the 190 matings were successful (mating rate of 26.3%), judged by the formation of clamp connections. The mutant dikaryons were cultivated to investigate their morphological and cultivation characteristics. Mutant strains No. 3 and No. 5 showed 10% and 6% increase in fruiting body production, respectively. Eight mutant strains showed delayed and reduced primordia formation, resulting in the reduced production yield with prolonged cultivation period. The number of the fruiting bodies of mutant No. 31, which displayed reduced primordial formation, was only 15, compared to the parental number of 65. Another interesting phenotype was a fruiting body with a flattened stipe and pileus. Dikaryons generated by mating with the mutant spore No. 14 produced flat fruiting bodies. Further molecular biological studies will provide details of the mechanism. This work shows that the chemical mutagenesis approach is highly utilizable in the development of mushroom strains as well as in the generation of resources for molecular genetic studies.

• Journal of Mushroom
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• v.13 no.1
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• pp.41-49
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• 2015

In order to breed new strains of oak mushroom, Lentinula edodes, on the sawdust cultivation, we collected 10 from Korea, Taiwan and China respectively and examined somatic incompatibility, morphological features of fruiting body and productivity. Four strains(SANJO701HO, FMRI2534, FMRI2337 and FMRI2613) shown remarkable results, were confirmed with parental strains. 80 monokaryotic strains derived from the selected 4 parental strains, were selected and 117 hybrid strains were made by mono-mono mating. Aslo, Physiological characteristics were investigated. Average of the mycelial growth of hybrid strains of mating combination SANJO701HO-FMRI2337 was approximately 10% lower than other mating combinations. Overall, This study was founded to develop new varieties of L. edodes. The productivity of new 117 strains on sawdust cultivation needs continued research.

• 한국균학회소식:학술대회논문집
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• 2015.05a
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• pp.53-53
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• 2015

Agrobacterium tumefaciens-mediated transformation(ATMT) of Flammulina velutipes was used to produce a diverse number of transformants to discover the functions of gene that is vital for its variation color, spore pattern and cellulolytic activity. Futhermore, the transformant pool will be used as a good genetic resource for studying gene functions. Agrobacterium-mediated transformation was conducted in order to generate intentional mutants of F. velutipes strain KACC42777. Then Agrobacterium tumefaciens AGL-1 harboring pBGgHg was transformed into F. velutipes. This method is use to determine the functional gene of F. velutipes. Inverse PCR was used to insert T-DNA into the tagged chromosomal DNA segments and conducting sequence analysis of the F. velutipes. But this experiment had trouble in diverse morphological mutants because of dikaryotic nature of mushroom. It needed to make monokaryotic fruiting varients which introduced genes of compatible mating types. In this study, next generation sequencing data was generated from 28 strains of Flammulina velutipes with different phenotypes using Illumina Hiseq platform. Filtered short reads were initially aligned to the reference genome (KACC42780) to construct a SNP matrix. And then we built a phylogenetic tree based on the validated SNPs. The inferred tree represented that white- and brown- fruitbody forming strains were generally separated although three brown strains, 4103, 4028, and 4195, were grouped with white ones. This topological relationship was consistently reappeared even when we used randomly selected SNPs. Group I containing 4062, 4148, and 4195 strains and group II containing 4188, 4190, and 4194 strains formed early-divergent lineages with robust nodal supports, suggesting that they are independent groups from the members in main clades. To elucidate the distinction between white-fruitbody forming strains isolated from Korea and Japan, phylogenetic analysis was performed using their SNP data with group I members as outgroup. However, no significant genetic variation was noticed in this study. A total of 28 strains of Flammulina velutipes were analyzed to identify the genomic regions responsible for producing white-fruiting body. NGS data was yielded by using Illumina Hiseq platform. Short reads were filtered by quality score and read length were mapped on the reference genome (KACC42780). Between the white- and brown fruitbody forming strains. There is a high possibility that SNPs can be detected among the white strains as homozygous because white phenotype is recessive in F. velutipes. Thus, we constructed SNP matrix within 8 white strains. SNPs discovered between mono3 and mono19, the parental monokaryotic strains of 4210 strain (white), were excluded from the candidate. If the genotypes of SNPs detected between white and brown strains were identical with those in mono3 and mono19 strains, they were included in candidate as a priority. As a result, if more than 5 candidates SNPs were localized in single gene, we regarded as they are possibly related to the white color. In F. velutipes genome, chr01, chr04, chr07,chr11 regions were identified to be associated with white fruitbody forming. White and Brown Fruitbody strains can be used as an identification marker for F. veluipes. We can develop some molecular markers to identify colored strains and discriminate national white varieties against Japanese ones.

• Journal of Mushroom
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• v.1 no.1
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• pp.9-14
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• 2003

To breed new superior strains, collected strains were characterized and then several white strains were selected as parents. Monokaryons from the parents were isolated and studied. All tested white strains showed same mating genotype. Growth rate of monokaryons were lower than collected dikaryons. New dikaryotic strains were derived from inbreeding method, which means mating between monokaryotic isolates from different white strains having same mating genotype. Some of them showed higher yields of fruitbody than their parents. Specially Fv 4-1 strain showed the best productivity. Furthermore some mating combination showed cytoplasmic effect, when they mated reciprocally.