Leaf-punch method to prepare a large number of PCR templates from plants for SNP analysis |
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Authors: | Sachiko Shiokai Hiroyasu Kitashiba Kenta Shirasawa Kuniaki Nagano Takeshi Nishio |
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Affiliation: | (1) Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai 981-8555, Japan;(2) Miyagi Prefecture Furukawa Agricultural Experiment Station, Osaki, Furukawa Miyagi, 989-6227, Japan;(3) Present address: Kazusa DNA Research Institute, 2-6-7 Kazusa-kamatari, Kisarazu Chiba, 292-0818, Japan |
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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. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. |
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Keywords: | DNA preparation PCR template SNP analysis Large-scale genotyping Marker-assisted selection |
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