Horticultural Science & Technology (원예과학기술지)

Korean Society of Horticultural Science (한국원예학회)
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Molecular Identification of Zoysia japonica and Zoysia sinica (Zoysia Species) Based on ITS Sequence Analyses and CAPS

ITS 염기서열 분석 및 CAPS를 이용한 조이시아 속(Zoysia) 들잔디와 갯잔디의 구별

  • Hong, Min-Ji (Faculty of Biotechnology, Jeju National University) ;
  • Yang, Dae-Hwa (Subtropical Horticulture Research Institute, Jeju National University) ;
  • Jeong, Ok-Cheol (Subtropical Horticulture Research Institute, Jeju National University) ;
  • Kim, Yang-Ji (Faculty of Biotechnology, Jeju National University) ;
  • Park, Mi-Young (Subtropical Horticulture Research Institute, Jeju National University) ;
  • Kang, Hong-Gyu (Subtropical Horticulture Research Institute, Jeju National University) ;
  • Sun, Hyeon-Jin (Subtropical Horticulture Research Institute, Jeju National University) ;
  • Kwon, Yong-Ik (Subtropical Horticulture Research Institute, Jeju National University) ;
  • Park, Shin-Young (Department of Clinical Pathology, Cheju Halla College) ;
  • Yang, Paul (Department of Agronomy, Chungnam National University) ;
  • Song, Pill-Soon (Subtropical Horticulture Research Institute, Jeju National University) ;
  • Ko, Suk-Min (Omicsis. Inc. BVC, Korea Research Institute of Bioscience & Biotechnology) ;
  • Lee, Hyo-Yeon (Faculty of Biotechnology, Jeju National University)
  • 홍민지 (제주대학교 생명공학부) ;
  • 양대화 (제주대학교 아열대원예산업연구소) ;
  • 정옥철 (제주대학교 아열대원예산업연구소) ;
  • 김양지 (제주대학교 생명공학부) ;
  • 박미영 (제주대학교 아열대원예산업연구소) ;
  • 강홍규 (제주대학교 아열대원예산업연구소) ;
  • 선현진 (제주대학교 아열대원예산업연구소) ;
  • 권용익 (제주대학교 아열대원예산업연구소) ;
  • 박신영 (제주한라대학교 임상병리학과) ;
  • 양바오로 (충남대학교 농학과) ;
  • 송필순 (제주대학교 아열대원예산업연구소) ;
  • 고석민 (오믹시스) ;
  • 이효연 (제주대학교 생명공학부)
  • Received : 2015.12.03
  • Accepted : 2016.11.03
  • Published : 2017.06.28
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Abstract

Zoysiagrasses are important turf plants used for school playgrounds, parks, golf courses, and sports fields. The two most popular zoysiagrass species are Zoysia japonica and Zoysia sinica. These are widely distributed across different growing zones and are morphologically distinguishable from each other; however, it is phenotypically difficult to differentiate those that grow along the coastal line from those in beach area habitats. A combination of morphological and molecular approaches is desirable to efficiently identify these two plant cultivars. In this study, we used a rapid identification system based on DNA barcoding of the nrDNA-internal transcribed spacer (ITS) regions. The nrDNA-ITS regions of ITS1, 5.8S nrDNA, and ITS2 from Z. japonica, Z. sinica, Agrostis stolonifera, and Poa pratensis were DNA barcoded to classify these grasses according to their molecular identities. The nrDNA-ITS sequences of these species were found at 686 bp, 687 bp, 683 bp, and 681 bp, respectively. The size of ITS1 ranged from 248 to 249 bp, while ITS2 ranged from 270 to 274 bp. The 5.8S coding region ranged from 163 - 164bp. Between Z. japonica and Z. sinica, nineteen (2.8%) nucleotide sites were variable, and the G+C content of the ITS region ranged from 55.4 to 63.3%. Substitutions and insert/deletion (indel) sites in the nrDNA-ITS sequence of Z. japonica and Z. sinica were converted to cleaved amplified polymorphic sequence (CAPS) markers, and applied to the Zoysia grasses sampled to verify the presence of these markers. Among the 62 control and collected grass samples, we classified three groups: 36 Z. japonica, 22 Z. sinica, and 4 Z. japonica/Z. sinica hybrids. Morphological classification revealed only two groups; Z. japonica and Z. sinica. Our results suggest that used of the nrDNA-ITS barcode region and CAPS markers can be used to distinguish between Z. japonica and Z. sinica at the species level.

Zoysia 속 잔디는 학교운동장 및 공원, 골프장, 스포츠경기장과 같이 다양한 장소에 식재되고 있는 중요한 잔디이다. 해안가에서 자생하는 Zoysia 속 들잔디와 갯잔디는 외부 형태적 특성이 유사하여 외부 형태적 분류 뿐 만 아니라 분자생물학적 분류도 필요하다. 본 연구에서는 nrDNA-ITS(Internal Transcribed Spacer)의 DNA 바코드 분석을 통해서 자생하는 들잔디와 갯잔디의 분자생물학적 신속한 분류체계를 확립하고자 하였다. 이를 위해 난지형 잔디인 Zoysia 속 들잔디(Z. japonica) 및 갯잔디(Z. sinica)와 한지형 대표 잔디인 크리핑 벤트그라스(A. stolonifera) 및 켄터키 블루그라스(P. pratensis)의 nrDNA-ITS 염기서열을 확보하였다. 확보된 들잔디및 갯잔디, 크리핑 벤트그라스, 켄터키 블루그라스의 ITS 염기서열 전체 구간은 각 686bp와 687bp, 683bp, 681bp으로 확인되었으며, nrDNA-ITS 내부 염기서열구간 분석 결과, ITS1의 크기는 248-249bp, ITS2는 270̵-274bp, 5.8S rDNA는 163-164bp의 차이로, 각 4종의 잔디가 ITS 염기서열을 이용하여 식별되었다. 특히, 들잔디와 갯잔디 nrDNA-ITS 염기서열은 19 염기(2.8%) 차이를 나타냈으며, ITS1과 ITS2의 G + C 함량은 55.4-63.3% 임을 확인하였다. 이러한 들잔디와 갯잔디의 ITS 염기서열 차이를 바탕으로 CAPS 마커로 전환하여 대조구 및 수집된 자생 Zoysia 속 잔디 영양체 62개체를 분석한 결과, 외부형태학적 분류법으로 들잔디 개체, 갯잔디 개체로 동정되었지만, ITSCAPS 마커를 이용한 분자생물학적 분류법으로 들잔디 36개체와 갯잔디 22개체 뿐만 아니라 들잔디와 갯잔디간의 자연교배종 4개체도 식별하였다. 이상의 결과에서 들잔디와 갯잔디는 ITS 염기서열 및 ITS 기반 CAPS를 통하여 식별할 수 있을 것으로 판단된다.

Keywords

References

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