Journal of Mushroom (한국버섯학회지)

The Korean Society of Mushroom Science (한국버섯학회)
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Selection of parental monokaryons from Korean Hypsizigus marmoreus by protoplast regeneration

원형질체 재생을 통한 느티만가닥버섯 단핵균주 선발

  • Oh, Youn-Lee (Department of Biotechnology College of Life Sciences and Biotechnology Korea University) ;
  • Kong, Won-Sik (Mushroom Science Division, National Institute of Horticultural and Herbal Science, RDA) ;
  • Jang, Kab-Yeul (Mushroom Science Division, National Institute of Horticultural and Herbal Science, RDA) ;
  • Shin, Pyung-Gyun (Mushroom Science Division, National Institute of Horticultural and Herbal Science, RDA) ;
  • Kim, Eun-Sun (Mushroom Science Division, National Institute of Horticultural and Herbal Science, RDA) ;
  • Oh, Min ji (Mushroom Science Division, National Institute of Horticultural and Herbal Science, RDA) ;
  • Choi, In-Geol (Department of Biotechnology College of Life Sciences and Biotechnology Korea University)
  • 오연이 (고려대학교 생명공학과 대학원 생명공학과&BK21플러스) ;
  • 공원식 (농촌진흥청 국립원예특작과학원 버섯과) ;
  • 장갑열 (농촌진흥청 국립원예특작과학원 버섯과) ;
  • 신평균 (농촌진흥청 국립원예특작과학원 버섯과) ;
  • 김은선 (농촌진흥청 국립원예특작과학원 버섯과) ;
  • 오민지 (농촌진흥청 국립원예특작과학원 버섯과) ;
  • 최인걸 (고려대학교 생명공학과 대학원 생명공학과&BK21플러스)
  • Received : 2015.09.08
  • Accepted : 2015.09.30
  • Published : 2015.09.30
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Abstract

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.

만가닥버섯은 일본에서 가장 중요한 식용버섯이다. 그러나 배양기간이 약 85~120일로 길어서 국내에서 다른 버섯에 비해 배양이 어려운 실정이다. 이 연구에서는 만가닥버섯 표준유전체 염기서열분석을 위하여 '해미'품종으로부터 원형질체를 분리하고 재생하여 모균주의 단핵균주를 얻었다. MCM배지 및 MYG배지에서 배양되고, 균질화된 균사체는 원형질체를 얻기 위하여 Novozyme효소를 처리하였다. 원형질체 분리의 가장 높은 효율은 MCM 배지에서 이차배양이 3일된 균사체에 2.5시간 효소처리 조건이었다. 분리된 원형질체는 재생배지에서 2주 동안 배양되어 재생되었다. 재생체는 클램프로 단핵균주를 확인하고 '해미'의 모균주의 단핵균주와 클램프 유무로 친화성을 확인하였다. 분리된 모균주의 단핵균주는 짧은 배양기간을 가진 품종을 개발하기 위한 유전적 기초자료를 완성하는 표준유전체 염기분석 재료로서 사용될 예정이다.

Keywords

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