以水稻叶片、根和种子为材料，采用高通量快速法提取基因组DNA，用稻瘟病Pita基因分子标记进行检测，获得与预期片段大小一致的特异性条带，对165份育种材料的检测结果与稻瘟病接种鉴定一致。操作方法如下：将少量水稻叶片、根或种子放入 200 μL PCR盘中，加入70 μL 缓冲液A (含NaOH和Tween20)，在PCR仪中加热到95℃，保持 10 min，再加入70 μL 缓冲液B (TrisHCl和EDTA)，该提取液可以直接用于PCR扩增。该方法具有几个优点：1）成本低，仅用4种化学试剂，共140 μL 提取液；2）操作简便，仅需3步，每人每天可以提取上千份样品；3）仪器设备简单，只用常规的PCR仪；4）可直接提取干种子的DNA；5）DNA质量好，能检测出水稻中的抗稻瘟病单基因Pita，并且与稻瘟病接种鉴定结果一致；6）用量少，只需要 5~20 mg 叶片、20 mg 根或半粒籽粒。尤其是检测大量样本的基因型时, 此种方法更显得高效便捷。
A method for DNA extraction from rice leaf, root and seed was developed, and the extracted DNA was used as a template to successfully amplify the rice blast resistance gene Pita. Profiles of Pita in 165 breeding lines detected by DNA markers were verified using differential blast races. This method involved three steps: 1) Plant tissue was placed into a 200 μL 96well plate; 2) A total of 70 μL of Buffer A containing NaOH and Tween 20 was added to each sample and incubated at 95℃ for 10 min in a PCR machine; and 3) A total of 70 μL of Buffer B containing TrisHCl and EDTA was added to each well and the resulting solution used for PCR amplification. Results demonstrated that this method of DNA extraction has the following advantages: 1) It is economical and only 4 common chemicals totaling 140 μL were used; 2) It is easy to perform, consists of only three steps, and one technician can extract hundreds of samples per day; 3) It requires a standard PCR machine; and 4) It can efficiently extract DNA from as little as a half dried seed, 5 to 20 mg of leaf tissue, or 20 mg root tissue. The resulting DNA quality was good enough to detect a single copy of the rice blast resistance gene Pita which was verified in breeding lines with the expected disease reactions. This method has proven to be highly efficient for evaluating a large number of samples.
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