Won Sik Kong

Comparative Genomics of the Mating-Type Loci of the Mushroom Flammulina velutipes Reveals Widespread Synteny and Recent Inversions

Background Mating-type loci of mushroom fungi contain master regulatory genes that control recognition between compatible nuclei, maintenance of compatible nuclei as heterokaryons, and fruiting body development. Regions near mating-type loci in fungi often show adapted recombination, facilitating the generation of novel mating types and reducing the production of self-compatible mating types. Compared to other fungi, mushroom fungi have complex mating-type systems, showing both loci with redundant function (subloci) and subloci with many alleles. The genomic organization of mating-type loci has been solved in very few mushroom species, which complicates proper interpretation of mating-type evolution and use of those genes in breeding programs. Methodology/Principal Findings We report a complete genetic structure of the mating-type loci from the tetrapolar, edible mushroom Flammulina velutipes mating type A3B3. Two matB3 subloci, matB3a that contains a unique pheromone and matB3b, were mapped 177 Kb apart on scaffold 1. The matA locus of F. velutipes contains three homeodomain genes distributed over 73 Kb distant matA3a and matA3b subloci. The conserved matA region in Agaricales approaches 350 Kb and contains conserved recombination hotspots showing major rearrangements in F. velutipes and Schizophyllum commune. Important evolutionary differences were indicated; separation of the matA subloci in F. velutipes was diverged from the Coprinopsis cinerea arrangement via two large inversions whereas separation in S. commune emerged through transposition of gene clusters. Conclusions/Significance In our study we determined that the Agaricales have very large scale synteny at matA (∼350 Kb) and that this synteny is maintained even when parts of this region are separated through chromosomal rearrangements. Four conserved recombination hotspots allow reshuffling of large fragments of this region. Next to this, it was revealed that large distance subloci can exist in matB as well. Finally, the genes that were linked to specific mating types will serve as molecular markers in breeding.

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 μg 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 μg of linearized pTRura3-2 DNA was added into 1×107 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.

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/μg of DNA in 1 × 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.

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

Abstract 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.

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

Abstract 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.

Development trend of the mushroom industry

세계의 버섯 생산량은 매년 10-20% 증가해 왔으며 다품목화 되어가고 있다. 최근에는 큰느타리, 백령느타리 등이 새로운 품목으로 재배 면적이 증가하고 있다. 특히 중국은 2002년 870만 톤으로 세계생산량 71.5%를 차지하였다. 한국도 유사한 경향을 나타내고 있다. 우리나라에서는 고려시대 저술한 김부식의 삼국사기(114...

Changes in the amino acid contents of fruiting body with growing temperature of common mushroom, Agaricus bisporus

This study was analyzed by HPLC that the changing sugar contents according to the strains of Agaricus bisporus and cultivation temperature. All strains showed that high total amino acid content in 19oC. 103 strain have been identified as the lowest total amino acids and amino acids content compare to the other strains. The amino acid ‘cysteine’ content is the highest, followed by phenylalanine, glutamic acid, lysine, proline, histidine. This big difference is estimated because in the strains of A. bisporus and cultivated temperature.

Nuclear DNA inheritance of intraspecific somatic hybrids by mono-mono cross in Pleurotus ostreatus based on URP-PCR analysis

The primary objective of the present study is the characterization of the somatic hybrids of dikaryon-monokaryon (dimono) crosses in mushroom breeding. We employed this technique for developing superior strains from Pleurotus ostreatus strains with 48 intraspecific hybrids of 12 combinations between six P. ostreatus strains and one P. florida strain. The results on the experiments of hybridization rate, nuclear DNA patterns, and colors and morphology of fruit-bodies, are presented. In di-mono crosses, somatic hybrids among Pleurotus strains showed 100% of crossability as seen in those between P. ostreatus and P. florida strains indicating that the nuclei of a dikaryon is inferred to be migrated to a recipient. 87.5% of the somatic hybrids among Pleurotus strains were similar to the donor dikaryons, and 12.5% of the somatic hybrids presented DNA patterns of both parents. In 16.6% of di-mono crosses between P. ostreatus and P. florida, the nuclear DNA patterns of all hybrids showed the same or similar patterns compared to the donor dikaryons. 70.9% of the hybrids between P. ostreatus and P. ostreatus were similar to the donor dikaryons and 12.5% of them presented the DNA patterns of both parents. 79.2% of fruiting body morphology of the hybrids among Pleurotus strains were similar to the dikaryons and 20.8% of them were similar to both parents. Interestingly, the morphology of all dikaryons were dissimilar each other. All hybrid strains between dikaryon P. florida and monokaryon P. ostreatus showed the fruiting body of which colors were similar to those of the dikaryon, while the hybrids between dikaryon P. ostreatus and monokaryon P. florida were showed the combined colors of both parents. Therefore, the fruiting body color of P. florida tends to be generally dominant. In conclusion, the present study provides a way to find out and suggest superior hybrid strains using the nuclear DNA patterns of hybrids between Pleurotus strains as well as the characteristics of their fruiting bodies. The advantages of the di-mono crossing are needs to be fully utilized in mushroom breeding because it is an ideal way to develop the superior strains of Pleurotus.