Identification of the dinoflagellate community during Cochlodinium polykrikoides (Dinophyceae) blooms using amplified rDNA melting curve analysis and real-time PCR probes |
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| Authors: | Tae-Gyu Park Gi-Hong Park Young-Tae Park Yang-Soon Kang Heon-Meen Bae Chang-Hoon Kim Hae-Jin Jeong Yoon Lee |
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| Institution: | 1. Marine Ecology Research Division, National Fisheries Research and Development Institute, Sirang-ri, Gijang-gun, Busan 619-705, Republic of Korea;2. Department of Aquaculture, Pukyong National University, Busan 608-737, Republic of Korea;3. School of Earth and Environmental Sciences, College of Natural Sciences, Seoul National University, Seoul 151-747, Republic of Korea;1. Key Laboratory of Applied Marine Biotechnology (Ningbo University), Ministry of Education, Ningbo, China;2. Institute of Marine and Environmental Technology, University of Maryland Center for Environmental Science, Baltimore, MD, USA;1. Institute of Chemical, Environmental and Bioscience Engineering, Research Area Biochemical Engineering, Technische Universität Wien, 1060 Vienna, Austria;2. CD Laboratory on Mechanistic and Physiological Methods for Improved Bioprocesses, Technische Universität Wien, 1060 Vienna, Austria;3. Institute of Chemical Technologies and Analytics, Technische Universität Wien, 1060 Vienna, Austria;4. Lackner Ventures & Consulting GmbH, Hofherr Schrantz Gasse 2, 1210 Vienna, Austria;5. University of Applied Sciences FH Technikum Wien, 1200 Vienna, Austria;1. Bachok Marine Research Station, Institute of Ocean and Earth Sciences, University of Malaya, 16310 Bachok, Kelantan, Malaysia;2. Institute of Biodiversity and Environmental Conservation, Universiti Malaysia Sarawak, 94300 Kota Samarahan, Sarawak, Malaysia;3. Faculty of Resource Science and Technology, Universiti Malaysia Sarawak, 94300 Kota Samarahan, Sarawak, Malaysia;4. Fisheries Research Institute Batu Maung, Department of Fisheries Malaysia, 11960 Batu Maung, Pulau Pinang, Malaysia;5. Faculty of Science and Technology, National University of Malaysia, 43600 Bangi, Selangor, Malaysia;6. Asian Natural Environmental Science Center, The University of Tokyo, Tokyo 113-8657, Japan;1. Research Unit for Palaeontology, Gent University, Krijgslaan 281 s8, 9000 Gent, Belgium;2. University of South Florida, Fish and Wildlife Research Institute, 100 Eighth Avenue SE, St. Petersburg, FL 33701, USA;3. Department of Botany and Plant Pathology, College of Agricultural Sciences, Oregon State University, Corvallis, OR 97331-2902, USA;4. International Ocean Discovery Program and Department of Geology and Geophysics, Texas A&M University, 1000 Discovery Drive, College Station, TX 77845, USA;5. Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen, Thorvaldsensvej 40, 1st Floor, DK-1871 Frederiksberg C, Denmark;6. GEOTOP, Université du Québec à Montréal, P.O. Box 8888, Montréal, Québec H3C 3P8, Canada;7. Laboratory of Aquatic Science Consultant Co., LTD. (LASC), 1-14-1, Kami-ikedai, Ota-ku, 145-0064 Tokyo, Japan;8. Qatar University, Department of Biological and Environmental Sciences, Doah, Qatar;9. Institute for East China Sea Research (ECSER), 1551-7 Taira-machi, Nagasaki 852-8521, Japan;1. Key Laboratory of Marine Ecology and Environmental Sciences, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China;2. Graduate University of Chinese Academy of Sciences, Beijing 100049, China;1. IRTA, Ctra. Poble Nou km 5.5, 43540, Sant Carles de la Ràpita, Tarragona, Spain;2. Departament d’Enginyeria Química, Universitat Rovira i Virgili, Av. Països Catalans 26, 43007, Tarragona, Spain;3. ICREA, Pg. Lluís Companys 23, 08010, Barcelona, Spain |
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| Abstract: | The dinoflagellate community present during blooms of the fish killing dinoflagellate Cochlodinium polykrikoides was characterized by DNA melting curve analysis and direct sequencing of the SSU rDNA amplified from environmental sample extracts. PCR amplification of genomic DNA from Gaedo water samples using dinoflagellate-specific SSU rDNA primers yielded 280 clones, which were screened by closed tube PCR-melting curve analysis targeting a region of the SSU rDNA, enabling high throughput analysis. Twenty-eight clones producing distinct melting curve patterns were sequenced, and their phylogenetic information revealed that C. polykrikoides co-occurred with morphologically similar species including Gymnodinium impudicum and Gymnodinium catenatum. Temporal variations of C. polykrikoides and G. impudicum abundances in South Sea were also examined by species-specific real-time TaqMan-based PCR probes developed in this study. C. polykrikoides- and G. impudicum-specific real-time PCR probes were designed targeting the internal transcribed spacer 2 ribosomal DNA region. The probe specificity was confirmed by testing against related dinoflagellates and verified by sequencing PCR products from environmental samples. The real-time PCR assays showed that C. polykrikoides cell densities peaked in August at 16,928 cells mL?1, while G. impudicum was present at low abundances (below 25 cells mL?1). Our amplified rDNA melting curve protocol provides a facile method for the characterization of the dinoflagellate community, and the real-time PCR assay could be an alternative method for rapid and sensitive enumeration of harmful dinoflagellates in the marine environment. |
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