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Mol Ecol Resour. 2017 Mar;17(2):324-333. doi: 10.1111/1755-0998.12586. Epub 2016 Aug 31.

Nuclear internal transcribed spacer-1 as a sensitive genetic marker for environmental DNA studies in common carp Cyprinus carpio.

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Graduate School of Human Development and Environment, Kobe University, 3-11 Tsurukabuto, Nada-ku, Kobe City, Hyogo, 657-8501, Japan.
Institute for Sustainable Sciences and Development, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima, Hiroshima, 739-8530, Japan.
Faculty of Pharmacy, Osaka Ohtani University, 3-11-1 Nishikiori-kita, Tondabayashi, Osaka, 584-8540, Japan.
Faculty of Life and Environmental Science, Shimane University, 1060 Nishikawatsu-cho, Matsue, Shimane, 690-8504, Japan.
Faculty of Science and Technology/Graduate School of Science and Technology, Ryukoku University, 1-5 Yokotani, Seta Oe-cho, Otsu, Shiga, 520-2194, Japan.
Graduate School of Simulation Studies, University of Hyogo, 7-1-28 Minatojima-minamimachi, Chuo-ku, Kobe, 650-0047, Japan.


The recently developed environmental DNA (eDNA) analysis has been used to estimate the distribution of aquatic vertebrates by using mitochondrial DNA (mtDNA) as a genetic marker. However, mtDNA markers have certain drawbacks such as variable copy number and maternal inheritance. In this study, we investigated the potential of using nuclear DNA (ncDNA) as a more reliable genetic marker for eDNA analysis by using common carp (Cyprinus carpio). We measured the copy numbers of cytochrome b (CytB) gene region of mtDNA and internal transcribed spacer 1 (ITS1) region of ribosomal DNA of ncDNA in various carp tissues and then compared the detectability of these markers in eDNA samples. In the DNA extracted from the brain and gill tissues and intestinal contents, CytB was detected at 95.1 ± 10.7 (mean ± 1 standard error), 29.7 ± 1.59 and 24.0 ± 4.33 copies per cell, respectively, and ITS1 was detected at 1760 ± 343, 2880 ± 503 and 1910 ± 352 copies per cell, respectively. In the eDNA samples from mesocosm, pond and lake water, the copy numbers of ITS1 were about 160, 300 and 150 times higher than those of CytB, respectively. The minimum volume of pond water required for quantification was 33 and 100 mL for ITS1 and CytB, respectively. These results suggested that ITS1 is a more sensitive genetic marker for eDNA studies of C. carpio.


cytochrome b (CytB); environmental DNA (eDNA); internal transcribed spacer 1 (ITS1); mitochondrial DNA (mtDNA); nuclear DNA (ncDNA); real-time PCR

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