Hypsizygus marmoreus is a mushroom with abundant flavor and medicinal properties. However, its application is limited by problems such as long cultivation period, low biological efficiency, and microbiological contamination; therefore, there is a substantial need for development of new cultivars of this species. In this study, 55 strains of H. marmoreus were subjected to inter simple sequence repeat (ISSR) analysis to identify markers for the selection of mother strains for breeding from the collected germplasm. ISSR 13 and 15 were confirmed as polymorphic markers. The three strains (KMCC03106, KMCC03107, and KMCC03108) with white cap color were found to be genetically closely related upon UPGMA analysis of both ISSR 13 and 15. Based on the PCR analysis results for ISSR 15, the collected germplasm were differentiated into three groups according to the strain collection year. Thus, ISSR 15 could be a marker for determining the phylogeny of cap color and genetic variations according to the strain collection year. These results suggest that ISSR markers can be effective tools for the selection of mother strains for breeding of H. marmoreus.

Flammulina velutipes belonging to white rot fungi is one of the commercially important edible mushrooms and is produced in large quantities due to the introduction of a automated and mechanized cultivation system in Korea. Despite the chief item of export among edible mushrooms, Flammulina velutipes has the lowest distribution rate of domestic cultivar, estimated that about 20 percent. As the result that most white cultivars of Flammulina velutipes produced and exported in Korea were introduced from Japan, farmers pay a large amount of royalties. Therefore, we try to develop a new pure domestic cultivars as a substitute for Japanese cultivars. To breed both white and gold superior strains, we selected the crossing mother groups including 10 white strains of ASI 4198 etc. and 7 brown strains of ASI 4049 etc. and mated each of the 17 strains by mon-mon hybridization. 19 white and 14 brown strains were chosen through two selection experiment over 2014 2016. In the third selection experiment this year, we finally selected one white(Fv 16 c 37) and the other gold(Fv 15 a 31) strain. Two selected strains were cultivated in the same environmental conditions. Spawn running period on the sawdust substrate required 30days at 20°C. The cultivation period and optimum temperature were 12±1 days at 14°C for primordia formation, 5 days at 4°C for inhibition phase, and 14±1 days at 7°C for fruiting body development. The length of pilei and stipes in two selected strains and Megumi as a control Japanese cultivar harvested in optimal stage was as follows: 10.5±0.81mm and 139.7±4.23mm in Fv 16 c 37, 10.8±0.43mm and 128..2±7.31mm in Fv 15 a 31, and 10.9±0.41mm and 141.8±4.64mm in Megumi respectively. The Yield of Fv 16 c 37, Fv 15 a 31 and Megumi was 271.2±11.84g, 237.7±9.05g and 270.7±16.87g per 1100ml in bottle cultivation.

Volvariella volvacea is mainly cultivated in subtropics area like South-East Asia. Because it is cultivated in rice straws, it is called a straw mushroom. That mushroom grows well in high temperature about 30~38°C and high humidity. Straw mushroom is a homothallic mushroom, so it is difficult to identify whether the offspring is different from the parents. This study was carried out to investigate RAPD primers that can be used for identification the DNA polymorphism of Volvariella volvacea genetic resources. 9 strains were collected from various countries like China, Vietnam etc. When ITS regions of their DNA were analyzed, they proved to be Volvariella volvacea. A cultivation test was conducted to measure the morphological characteristics of them 2 strains, KMCC04380 and KMCC04382 were selected for breeding resources because the mycelium of them grew well on medium and fruiting bodies were formed quickly. The Universal PCR Fingerprinting kits(UPF primers) were used to confirm the genetic polymorphisms of the 2 strains. As a result of confirming the DNA bands, 2 of 12 primers could be used to genetically distinguish 2 strains. About 50 spores were isolated from their fruiting bodies respectively and they also will be confirmed DNA polymorphisms by using UPF primers.

H. mamoreus is a mushroom with abundant favor and medicinal use. However, its cultivation has problems such as the long cultivation period, low biological efficiency and microbiological contamination, so new cultivars should be developed. In this study, 55 strains of H.marmoreus were analyzed with ISSR markers to identify precise genetic information in collected germplasm as mother strains in breeding. ISSR 13 and 15 markers were confirmed polymorphism. The three strains (KMC03106, KMC03107, and KMC03108) with white cap color were close genetic relationship in the UPGMA analysis of both the ISSR 13 and 15 markers. Especially in the PCR result of the ISSR 15, the collected germplasm were differentiated to three groups following collecting year. As these results, the ISSR 15 marker would be able to classify the phylogeny of cap color and genetic variation along the collecting year. Therefore ISSR markers will confer effective selection of mother strains for breeding of H. mamoreus.

‘Baekseung’, a new variety of Flammulina velutipes, was bred by mating two monokaryotic strains isolated from KMCC 4210 and KMCC 4216 in Mushroom Research Division, Baekseung ARES in 2016. Baekseung showed fast mycelial growth and high mycelial density on MEA (Malt Extract Agar) media for 7days of incubation. Spawn running period on the sawdust substrate required 30days at 25°C. The cultivation period and optimum temperature were 11±1 days at 14°C for primordia formation and 14±1 days at 7°C for fruiting body development. The length of pilei and stipes in Baekseung harvested in optimal stage exhibited 11.3±0.4㎜ and 89.2±7.1㎜ and Megumi harvested in optimal stage showed 8.2±1.0㎜ and 95.9±5.0㎜ respectively. Yield of Baekseung and Megumi strain grown of sawdust substrate was 153.7±12.5g and 150.5±29.7g per 850ml in bottle cultivation. The inferred tree exhibited the difference of phylogenetic relationship between the Korean white fruiting body strains such as Baekseung, Uri1ho, Fv-14-a-38, and Fv-14-a-51 and the Japanese white fruiting body strain Megumi.

Microsatellite SSR markers were developed and utilized to reveal the genetic diversity of 32 strains of Flammulina velutipes collected in Korea, China, and Japan. From SSR-enriched library, 490 white colonies were randomly selected and sequenced. In the 490 sequenced clones, 85 clones (17.35%) were redundant. Among the remaining 405 unique clones, 201 clones (49.6%) contained microsatellite sequences. As a result, 12 primer pairs produced reproducible polymorphic bands within diverse 4 strains and these selected markers were further characterized in 32 Flammulina velutipes strains. A total of 34 alleles were detected using the 12 markers, with an average of 3.42 alleles and the number of alleles ranged from two to seven per locus. The major allele frequency ranged from 0.42 (GB-FV-127) to 0.98 (GB-FV-166), and values for observed (HO) and expected (HE) heterozygosity ranged from 0.00 to 0.94 (mean = 0.18) and from 0.03 to 0.67 (mean = 0.32), respectively. SSR loci amplified with GB-FV-127 markers gave the highest polymorphism information content (PIC) of 0.61 and mean allele number of five, while for loci amplified with GB-FV-166 markers these values were the lowest, namely 0.03 and two. The mean PIC value (0.29) observed in the present study with average number of alleles (3.42). The genetic relationships among 32 Flammulina velutipes strains based on SSR data were generated by UPGMA cluster analysis. In conclusion, we succeeded in developing 12 polymorphic SSRs markers from SSR-enriched library of Flammulina velutipes. These SSRs are presently being used for phylogenic analysis and evaluation of genetic variations. In future, these SSR markers will be used in clarifying taxonomic relationships among the Flammulina velutipes.

In this study, we made population with high biological efficiency to investigate the complex genetic architecture of yield-related traits in A. bisporus. MB013 that crossed with bisp 15-p2 and bisp 34-p2 had high biological efficiency. 170 homokaryons was isolated with CAPS marker (PIN primer/HaeⅢ) from 1000 ISSs. And 100 BC1F1 hybrids obtained by crossing the homokaryons of MB013 with bisp15-p1. Parental line bisp 15-p2 and bisp 34-p2 and 100 homokaryons of MB013 will analyze genome sequencing. Also 100 BC1F1 hybrids will evaluate yield-related traits.

In the present study, the effect of cysteine and NT or bisphenol A (BP) on in vitro aturation (IVM) of porcine oocytes were examined. COCs was cultured in NCSU-23 medium supplement with 10% FCS which had previously been covered with mineral oil and equilibrated in a humidified atmosphere of 5% CO2 and 95% air at 38℃. The IVM rate of oocytes cultured for 48 hrs in NCSU-23 medium supplement with 0.5～10.0 mM cysteine were 34.0±3.2%, 36.0±3.5%, 48.0±3.8%, 22.0±3.2%, respectively. The IVM rate of oocytes cultured in NCSU-23 medium supplement with 0.5～5.0 mM NT for 48 hrs were 24.0±4.2%, 18.0±4.9%, 8.0±2.2%, respectively. NT affects oocyte in vitro maturation rate in a dose-dependent. This result were significantly lower than the control group. The IVM rate of oocytes cultured for 48 hrs in NCSU-23 medium supplement with 1.0 mM NT+5.0 mM cysteine (38.0±4.3%) were significantly higher than that of NT treatment. The IVM rate of oocytes cultured in NCSU-23 medium supplement with 0.05～5.0 mM BP for 48 hrs were 20.0±4.7%, 10.0±5.3%, 6.0±3.2%, respectively. The IVM rate of oocytes cultured in NCSU-23 medium supplement with BP was significantly lower cultured non supplement of BP (44.0±3.5%). BP affects porcine oocyte maturation rate in a dose-dependent manner. The IVM rate of oocytes cultured for 48 hrs in NCSU-23 medium supplement with 1.0 mM BP+5.0 mM cysteine (32.0±3.2%) were increased than that of BP treatment.

These study was carried out to investigate the effects of the supplementation with sodium nitroprusside (SN) and nitric oxide (NO) of canine oocytes on IVM rates. Oocytes were incubated in TCM-199 supplement with at 0.03～0.10 mM SN and 0.3～1.0 mM NO for 48 hrs. Oocytes were transferred to 50 ul drops of maturation medium covered mineral oil and cultured in a CO2 incubator (5% CO2, 95% air, 38℃). The in vitro maturation rate of oocytes cultured for 48 hrs in TCM-199 medium supplement with 0.03, 0.05, 0.07, 0.10 mM SN were 25.9±3.5%, 36.4±3.2%, 33.3±3.5%, 28.8±3.2%, respectively. The in vitro maturation rate of oocytes cultured for 48 hrs in TCM-199 medium supplement with 0.03～0.07 mM SN were significantly increased compare to the control (26.0±2.2%). The in vitro maturation rates of oocytes cultured for 48 hrs in TCM-199 medium supplement with 0.3, 0.5, 0.7, 1.0 mM NO were 28.0±4.2%, 36.5± 3.6%, 30.0±3.8%, 19.2±3.5%, respectively. The in vitro maturation rate of oocytes in TCM-199 medium supplemented with 0.3 and 0.5 mM NO were significantly increased compare to the control (26.0±2.2%). The in vitro maturation rates of oocytes cultured for 12～48 hrs in TCM-199 medium supplement with 0.05 mM SN were 26.0±3.2%, 28.0±3.4%, 38.0±3.2%, respectively. The in vitro maturation rate of oocytes cultured for 12～48 hrs in TCM-199 medium supplement with 0.5 mM NO were 22.0±3.0%, 30.0±3.8%, 36.0±4.2%, respectively. These result was significantly increased compare to the control.

These study was to investigate the in vitro fertilization and viability of fresh and vitrified oocytes. Also, the developmental capacity of IVF and intracytoplasmic sperm injection (ICSI) oocytes were investigated. Then vitrification was performed with the use of 20% ethylene glycol + 20% DMSO + 0.5 M sucrose + 10% FCS + TCM-199 medium. Vitrification immature oocytes are cultured in vitrification solution for 10 min afterwards transferred to expose at room temperature for 5 min. and transferred to the ice water for 5 min. The oocytes were sealed in a 1.0 mm straw and placed in a LN2 container. Frozen oocytes were rapidly thawed in a water bath at 30～35℃, and then placed in TCM-199 medium containing 0.5 M sucrose for 5 min each, respectively, at 38℃. After being washed for 2～3 times, using fresh medium the oocytes were cultured in TCM-199 medium supplemented with 5% FCS at 38℃ in 5% CO2 and air. The normal morphology of fresh and vitrified-thawed oocytes were 87.1±2.1% and 54.8±2.5%, respectively. The viability rates of fresh and vitrified-thawed oocytes were 70.0±2.2% and 41.9±2.6%, respectively. Viability rates of vitrified-thawed oocytes were lower than that of fresh follicular oocytes (p<0.05). The in vitro maturation rates of fresh and vitrified oocytes were 45.1±3.6% and 28.9±4.4%, respectively. The IVF rates of fresh follicular and vitrified-thawed oocytes were 34.0±2.2% and 20.2±2.6%, respectively. The in vitro maturation and fertilization rates of vitrified-thawed oocytes were lower than those of the fresh follicular oocytes (p<0.05). A total of 350 oocytes were fixed and stained after co-incubation with spermatozoa, of which 88 had identifiable nuclear material. After IVF for 20 hrs, 25.1±3.4% of the oocytes found to have been penetrated by spermatozoas. Oocytes were fixed and stained after ICSI, and 105 oocytes contained identifiable nuclear material. After IVF and ICSI for 20 hrs, 34.3±3.4% and 59.0±2.0% of the oocytes were found to have been penetrated by spermatozoas. The developmental rates upon ICSI were significantly higher than those of the IVF method (p<0.05).

The in vitro maturation rate of vitrified-thawed canine oocytes was 30.8±3.4%. The in vitro maturation rate of vitrified oocytes was lower than that of the control (52.0±2.5%, p<0.05). The in vitro maturation rate of vitrified-thawed oocytes were significantly (p<0.05) lower than those of fresh oocytes. The in vitro maturation and developmental rates of the vitrified-thawed oocytes were 17.5±2.5% and 8.8±3.4%, respectively. This results were lower than the control group (43.6±3.2% vs 20.0±3.0%). SOD1 gene expression of 1～2 mm of follilce size were higher than those of above 6 mm follicle size. SOD2 gene expression of 1～2 mm of follicle size were significantly higher than those of above 6 mm follicle size (p<0.01). The expression pattern of SOD1, 2 was constantly expressed in both groups but strongly expressed in follicles (1～2 mm) group when compared to the above 6 mm follicles. SOD gene expression between groups the fresh and vitrified oocytes groups were significant differences in rates. However, RGS gene expression between groups the fresh and vitrified oocytes groups were no significant differences in rates.