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<p>The native distribution range of striped bitterling (<i>Acheilognathus cyanostigma</i>), a freshwater fish endemic to the Japanese archipelago, has become obscured due to the introduction of non-native populations. Although genetic analyses based on captured individuals have been used to identify native lineages, such methods are invasive and labor-intensive. To facilitate rapid and non-invasive identification of native populations, we developed an environmental DNA (eDNA) assay capable of distinguishing between two major intraspecific lineages: the Tokai and Kinki lineages. The assay comprises striped bitterling-specific primers and lineage-specific probes for both the Tokai and Kinki lineages. To validate the assay, water samples were collected from six conservation ponds harboring the Tokai lineage and four ponds for the Kinki lineage. Real-time PCR analyses revealed that only the Tokai-specific probe produced positive signals in Tokai ponds, and only the Kinki-specific probe generated positive signals in Kinki ponds. The limit of detection (LOD) was 3 copies per 2 µL for the Tokai probe and 1 copy per 2 µL for the Kinki probe, indicating high assay sensitivity. This newly developed assay enables rapid and non-invasive identification of striped bitterling lineages, offering a cost- and labor-efficient tool for lineage-specific conservation management.</p>

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Development of a lineage-specific environmental DNA assay to differentiate intraspecific lineages of the endangered freshwater fish, Acheilognathus cyanostigma (Cyprinidae: Acheilognathinae)

  • 期刊论文

  • 发表日期：

    2026年1月19日

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  • Gen Ito,
  • Jyun-ichi Kitamura,
  • Seigo Kawase,
  • Maki Asami,
  • Yuko Goto,
  • Hiroki Yamanaka

摘要

The native distribution range of striped bitterling (Acheilognathus cyanostigma), a freshwater fish endemic to the Japanese archipelago, has become obscured due to the introduction of non-native populations. Although genetic analyses based on captured individuals have been used to identify native lineages, such methods are invasive and labor-intensive. To facilitate rapid and non-invasive identification of native populations, we developed an environmental DNA (eDNA) assay capable of distinguishing between two major intraspecific lineages: the Tokai and Kinki lineages. The assay comprises striped bitterling-specific primers and lineage-specific probes for both the Tokai and Kinki lineages. To validate the assay, water samples were collected from six conservation ponds harboring the Tokai lineage and four ponds for the Kinki lineage. Real-time PCR analyses revealed that only the Tokai-specific probe produced positive signals in Tokai ponds, and only the Kinki-specific probe generated positive signals in Kinki ponds. The limit of detection (LOD) was 3 copies per 2 µL for the Tokai probe and 1 copy per 2 µL for the Kinki probe, indicating high assay sensitivity. This newly developed assay enables rapid and non-invasive identification of striped bitterling lineages, offering a cost- and labor-efficient tool for lineage-specific conservation management.