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.
ZHAO Guozhen1, JIA Yulin2,*, YAN Zongbu3, Christopher W DEREN3, Melissa H JIA2, DAI Luyuan1,*
. An Efficient, Economic, and Rapid Rice DNA Extraction Method and Its Application[J]. Chinese Journal OF Rice Science, 2012
, 26(4)
: 495
-499
.
DOI: 10.3969/j.issn.10017216.2012.04.016
\[1\]Stewart C, Via L. A rapid CTAB DNA isolation technique useful for RAPD fingerprinting and other PCR applications. Biotechniques, 1993, 14(5): 748750.
\[2\]孙川, 陈刚, 饶玉春, 等. 水稻基因组DNA简易制备方法.中国水稻科学, 2010, 24(6): 677680.
\[3\]邱福林， 王和和， 陈洁， 等．用于水稻突变体大量筛选的ＤＮＡ微量快速提取法．中国水稻科学， 2006, 20(3): 329332.
\[4\]陈文岳, 包劲松, 周祥胜, 等．一种可用于PCR分析的水稻ＤＮＡ简易提取法．中国水稻科学, 2005, 19(6): 561563.
\[5\]汪秀峰, 杨剑波, 向太和, 等．一种叶片直接用作PCR扩增的新方法及其应用．中国水稻科学， 2002, 16(1): 6770.
\[6\]卢扬江, 郑康乐. 提取水稻的一种简易方法.中国水稻科学，1992, 6(1)：4748.
\[7\]赵红霞, 谢攀, 黄志坚, 等. 一种改良的水稻总DNA提取方法. 湖北大学学报: 自然科版, 2006, 28(4): 389392.
\[8\]杨杰, 仲维功, 王才林, 等. 一种简便快速制备水稻PCR模板的方法及其应用.江苏农业学报, 2005, 17(4): 7578.
\[9\]朱世杨, 罗天宽, 张小玲, 等. 8种水稻基因组DNA提取方法的比较. 安徽农业科学， 2009, 37(5): 19291931.
\[10\]Xin Z, Velten J, Oliver M, et al. HighThroughput DNA extraction method suitable for PCR. BioTechniq, 2003, 34(4): 820826.
\[11\]Jia Y, Wang Z, Singh P. Development of dominant rice blast Pita resistance gene markers. Crop Sci, 2002, 42 (6): 2145214.
\[12\]Jia Y, Wang Z, Fjellstrom R G, et al. Rice Pita gene confers resistance to the major pathotypes of the rice blast fungus in the United States. Phytopathology, 2004, 94(3): 296301.
\[13\]王忠华, 贾育林, 吴殿星, 等. 水稻抗稻瘟病基因Pita的分子标记辅助选择. 作物学报, 2004, 30(12): 12591265.
\[14\]王忠华, 贾育林. 粳稻品种Katy和Kaybonnet对稻瘟病抗性的遗传分析. 江苏农业科学, 2005(1): 4549.
\[15\]王忠华, 贾育林, Fjellstrom G. 水稻抗稻瘟病基因Pita 与Pita2之间的关系.浙江万里学院学报, 2004, 17(2): 8992.
\[16\]王军, 杨杰, 陈志德, 等. 利用分子标记辅助选择聚合水稻抗病基因Pita、Pib 和Stvbi. 作物学报, 2011, 37(6): 975981.
\[17\]Jia Y, Valent B, Lee F N. Determination of host responses to Magnaporthe grisea on detached rice leaves using a spot inoculation method. Plant Dis, 2003, 87(2): 129133.
