Fluorogenic Ribonuclease Protection (FRIP) Analysis of Single Nucleotide Polymorphisms (SNPs) in Japanese Rice (<i>Oryza sativa</i>L.) DNA for Cultivar Discrimination

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  • KITAOKA Momoko
    Department of Applied Chemistry, Graduate School of Engineering, Kyushu University
  • WADA Takuya
    Department of Crop Science, Fukuoka Agricultural Research Center
  • NISHIO Takeshi
    Graduate School of Agricultural Science, Tohoku University
  • GOTO Masahiro
    Department of Applied Chemistry, Graduate School of Engineering, Kyushu University Center for Future Chemistry, Kyushu University

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  • Fluorogenic Ribonuclease Protection (FRIP) Analysis of Single Nucleotide Polymorphisms (SNPs) in Japanese Rice (Oryza sativa L.) DNA for Cultivar Discrimination

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A rapid and easy method to discriminate plant cultivars is indispensable to confirm food labeling. We established a fluorogenic ribonuclease protection (FRIP) assay to discriminate Japanese rice (Oryza sativa L.) cultivars based on single nucleotide polymorphisms (SNPs). The FRIP assay uses a hybridization technique between fluorescent probes and the target sequence prepared by run-off transcription, but the requirement of two PCR thermocycles is the problem when preparing template DNA for run-off transcription from rice genomic DNA. In this study, we designed new PCR primers with asymmetric melting temperatures. These primers amplified the target SNP marker containing a T7 RNA polymerase promoter sequence upstream of the target sequence in a single PCR. Moreover, 100 cultivars were discriminated with the patterns of 15 SNPs. The assay can be used as a rapid method of analysis to discriminate Japanese rice cultivars.

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