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United States Department of Agriculture

Agricultural Research Service

Research Project: RESPONSE OF DIVERSE RICE GERMPLASM TO BIOTIC AND ABIOTIC STRESSES

Location: Dale Bumpers National Rice Research Center

Title: Establishment and application of an efficient, economic, and rapid rice DNA extraction method

Authors
item Zhao, Guo-Zhen -
item JIA, YULIN
item Deren, Christopher -
item Yan, Zong-Bu -
item JIA, MELISSA
item Dai, Lu-Yuan -

Submitted to: Chinese Journal of Rice Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: August 20, 2012
Publication Date: September 27, 2012
Citation: Zhao, G., Jia, Y., Deren, C.W., Yan, Z., Jia, M.H., Dai, L. 2012. Establishment and application of an efficient, economic, and rapid rice DNA extraction method. Chinese Journal of Rice Science. 26(4):495-499.

Interpretive Summary: DNA marker assisted breeding and molecular diagnostics of rice are the new tools in rice genetics and breeding. One of the major constraints for molecular analysis of rice genotypes is the cost of DNA preparation. In the present study, a method of DNA extraction was successfully adapted to prepare DNA from as little as half of one seed or 5 mg of root or leaf tissue. Plant DNA was released into the solution by NaOH at 95C for 10 minutes, and the solution was stabilized by Tween 20, Tris and EDTA. Resulting DNA was used for the identification of the Pi-ta blast resistance gene in 165 rice breeding lines. Results of the DNA identification of Pi-ta in the breeding lines were consistent with the results of pathogenicity assays using differential blast races. The entire procedure requires less than one hour, costs less than two cents per sample, and is highly efficient for assaying a large number of samples.

Technical Abstract: A rapid, economic, and efficient 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 Pi-ta. Profiles of Pi-ta in 165 breeding lines detected by DNA markers were verified using differential blast races. This method involved two steps: 1) plant tissue was placed into a 200 ul 96-well plate. A total of 70 uL of Buffer A containing NaOH and Tween 20 was added to each sample and incubated at 95C for 10 min in a PCR machine; 2) A total of 70 ul buffer containing Tris-HCl and EDTA (Buffer B) 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 uL 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 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 Pi-ta 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.

Last Modified: 9/10/2014
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