Korean Journal of Environmental Biology (환경생물)

Korean Society of Environmental Biology (한국환경생물학회)
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Development of a Species Identification Method for the Egg and Fry of the Three Korean Bitterling Fishes (Pisces: Acheilognathinae) using RFLP (Restriction Fragment Length Polymorphism) Markers

제한절편 길이 다형성(RFLP) 분자마커를 이용한 납자루아과 담수어류 3종의 난과 치어 종 동정 기법 개발

  • Choi, Hee-kyu (Molecular Ecology and Evolution Laboratory, Department of Biological Science, College of Science & Engineering, Sangji University) ;
  • Lee, Hyuk Je (Molecular Ecology and Evolution Laboratory, Department of Biological Science, College of Science & Engineering, Sangji University)
  • 최희규 (상지대학교 생명과학과 분자생태및진화학실험실) ;
  • 이혁제 (상지대학교 생명과학과 분자생태및진화학실험실)
  • Received : 2018.08.05
  • Accepted : 2018.08.17
  • Published : 2018.09.30
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Abstract

This study aimed to develop a species identification method for the egg and fry of the three Korean bitterling fishes (Pisces: Acheilognathinae), including Acheilognathus signifer, Acheilognathus yamatsutae and Rhodeus uyekii based on the PCR-based Restriction Fragment Length Polymorphism (RFLP) markers. We conducted a field survey on the Deokchicheon River from the North Han River basin, where the three Acheilognathinae species co-occur, and also analyzed the existing sequence dataset available from the GenBank. We found coexistence of the three species at the study site. The egg and fry were obtained from the host mussels (Unio douglasiae sinuolatus) by hand from May to June 2015 and in May 2017. To develop PCR-based RFLP markers for species identification of the three Acheilognathinae fish species, restriction enzymes pinpointing species-specific single nucleotide variation (SNV) sites in mitochondrial DNA COI (cytochrome oxidase I) and cyt b (cytochrome b) genes were determined. Genomic DNA was extracted from the egg and fry and RFLP experiments were carried out using restriction enzymes Apal I, Stu I and EcoR V for A. signifer, A. yamatsutae and R. uyekii, respectively. Consequently, unambiguous discrimination of the three species was possible, as could be seen in DNA band patterns from gel electrophoresis. Our developed PCR-based RFLP markers will be useful for the determination of the three species for the young and would assist in studying the spawning patterns and reproductive ecology of Acheilognathinae fishes. Furthermore, we believe the obtained information will be of importance for future maintenance, management and conservation of these natural and endangered species.

본 연구는 PCR 기반 RFLP (Restriction Fragment Length Polymorphism; 제한절편 길이 다형성) 분자기법을 활용하여 난 및 치어 대상 납자루아과 어류 3종의 동정을 좀 더 빠르고 정확하게 파악하고 납자루아과 어류의 종별 산란양상 및 번식생태 이해에 대한 기여가 목적이다. 본 연구를 위해 기존 선행된 문헌자료를 확인하고 납자루아과 어류가 2종 이상 동서하고 있는 지역을 확인하여 현지조사를 수행하였다. 현지조사 결과 확인된 납자루아과 어류는 묵납자루(Acheilognathus signifer), 줄납자루(A. yamatsutae) 및 각시붕어(Rhodeus uyekii)로 총 3종이 확인되었으며, 확인된 납자루아과 어류와 동서하고 있는 숙주조개(작은말조개; Unio douglasiae sinuolatus)를 채집하여 숙주조개 속 납자루아과 어류의 난 및 치어를 확보하였다. 현지조사 결과 확인된 납자루아과 어류 3종을 대상으로 미토콘드리아 DNA COI과 cyt b 유전자 염기서열을 비교하여 각각 종별로 특이성을 지닌 부위(단일염기변이; Single Nucleotide Variation: SNV)에 맞는 제한효소를 선정하였고, 숙주조개 속 난 및 치어를 대상으로 genomic DNA를 추출하여 PCR-RFLP 실험을 수행한 결과 현지조사 시 확인된 납자루아과 어류 3종의 독특한 제한절편 길이 양상을 전기영동을 통하여 확인하였다. 본 연구를 통해 묵납자루, 줄납자루 및 각시붕어의 종을 판별할 수 있는 RFLP 마커를 개발하였으며, 숙주조개 난 및 치어를 대상으로 정확한 종의 동정을 보다 빠르고 효과적으로 수행하여 각각 납자루아과 종별 산란양상을 보다 정확히 규명하고 향후 이들 자연개체군의 효과적인 유지, 관리 및 보전 방법 개발에 유용하게 활용될 수 있을 것으로 판단된다.

Keywords

References

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