A target cultivar-specific identification system based on the chromatographic printed array strip method for eight prominent Japanese citrus cultivars

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  • Okamoto Mitsutoshi
    Ehime Research Institute of Citrus Fruits Present address: Department of Agricultural Research, Ehime Research Institute of Agriculture, Forestry and Fisheries
  • Monden Yuki
    Graduate School of Environmental and Life Science, Okayama University
  • Shindo Akiko
    Graduate School of Environmental and Life Science, Okayama University
  • Takeuchi Tomoyuki
    FASMAC Co., Ltd.
  • Endo Tomoko
    National Agriculture and Food Research Organization Institute of Fruit and Tea Tree Science
  • Shigematsu Yukinori
    Ehime Research Institute of Citrus Fruits
  • Takasaki Kazuto
    FASMAC Co., Ltd.
  • Fujii Hiroshi
    National Agriculture and Food Research Organization Institute of Fruit and Tea Tree Science
  • Shimada Takehiko
    National Agriculture and Food Research Organization Institute of Fruit and Tea Tree Science

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<p>Citrus is a major cultivated crop in Japan, and new cultivars are of great interest in the Japanese and global market. Recently, the infringement of breeders’ rights to citrus cultivars bred in Japan has become a problem related to the agricultural product export strategy promoted by the Japanese government. Cultivar identification systems using DNA markers are an effective tool for protecting breeders’ rights. Here, a novel target cultivar-specific identification system using the chromatographic printed array strip method was developed for eight prominent Japanese citrus cultivars. A polymorphic InDel fragment specific to each cultivar was explored through the screening of published citrus InDel markers and next-generation sequencing of retrotransposon libraries. The cultivar-specific DNA marker set for each cultivar comprised 1–3 polymorphic InDel fragments in combination with a PCR-positive DNA marker for the ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit gene. The DNA markers were detected within 3 hours from DNA extraction to the detection by the C-PAS4 membrane stick following multiplex PCR. The developed system is superior as a convenient, rapid, and cost-effective DNA diagnostic method during inspection. The proposed target cultivar-specific identification system is expected to serve as an efficient tool for the injunction of suspicious registered cultivars, contributing to the protection of breeders’ rights.</p>

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