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Leaf-punch method to prepare a large number of PCR templates from plants for SNP analysis

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Abstract

DNA preparation is indispensable for genotyping by DNA polymorphism analysis, and that for a large number of plants is laborious. In the present study, a small leaf disk of rice, 1–2 mm in diameter, punched by a mini cork borer was found to be directly usable as a PCR template. DNA fragments <300 bp were amplified efficiently. Leaf disks of 1–1.5 mm in diameter were better than those of 2 mm for a small volume of reaction mixture. Multiplex PCR was possible with four or eight primer pairs using the small leaf disk as a template. Leaf disks of Arabidopsis, Lotus, wheat, soybean, tomato, Chinese cabbage, and melon were also good PCR templates. This method for preparation of PCR templates, named the leaf-punch method, was applicable to SNP analysis of a large number of plants by dot-blot-SNP analysis.

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Acknowledgments

This work was supported in part by a Grant-in-Aid for Scientific Research (A) (no. 19208001) from the Japan Society for the promotion of science (JSPS). CSSL228 was provided by Rice Genome Resource Center, Japan. S. Shiokai is a recipient of a research fellowship from the Japan Society for the promotion of science for young Scientists.

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Author notes

  1. Kenta Shirasawa

    Present address: Kazusa DNA Research Institute, 2-6-7 Kazusa-kamatari, Kisarazu, Chiba, 292-0818, Japan

Authors and Affiliations

  1. Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai, 981-8555, Japan

    Sachiko Shiokai, Hiroyasu Kitashiba, Kenta Shirasawa & Takeshi Nishio

  2. Miyagi Prefecture Furukawa Agricultural Experiment Station, Osaki, Furukawa, Miyagi, 989-6227, Japan

    Kuniaki Nagano

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  1. Sachiko Shiokai
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  2. Hiroyasu Kitashiba
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  3. Kenta Shirasawa
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  4. Kuniaki Nagano
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  5. Takeshi Nishio
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Correspondence to Takeshi Nishio.

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Shiokai, S., Kitashiba, H., Shirasawa, K. et al. Leaf-punch method to prepare a large number of PCR templates from plants for SNP analysis. Mol Breeding 23, 329–336 (2009). https://doi.org/10.1007/s11032-008-9244-9

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  • DOI: https://doi.org/10.1007/s11032-008-9244-9

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