Page Banner

United States Department of Agriculture

Agricultural Research Service

Title: Rapid DNA Extraction and Routine Use of a Microsatellite Marker Associated with the Waxy Gene

Authors
item Bergman, Christine
item Fjellstrom, Robert
item McClung, Anna
item McClung, Anna

Submitted to: Rice Technical Working Group Meeting Proceedings
Publication Type: Proceedings
Publication Acceptance Date: February 27, 2000
Publication Date: N/A

Technical Abstract: Amylose content is the most important rice constituent that influences its end-use quality. A microsatellite marker has been reported that strongly correlates with the various classes of AA content in rice. This marker has been used to decrease development time for new varieties (e.g. Cadet) by several years. Adopting this technology as a routine method by quality evaluation programs has been hindered due to the DNA extraction time, toxic chemicals required and low scoring accuracy of gels. A method has been developed that largely overcomes those problems and is relatively inexpensive. The DNA extraction protocol requires that brown or milled rice kernels be immersed in a sodium hydroxide solution overnight at room temperature. Samples are vortexed and centrifuged and an aliquot of the supernatant is diluted with a neutralization buffer before its use in PCR amplification. The procedure is effective, although the concentration of DNA obtained using this technique is approximately half that obtained by precipitation techniques. Allelic mixtures can often be identified using a multiple kernel extraction procedure. In some instances, single kernels must be extracted to establish which specific alleles are in the mixture. A modification of a previously reported PCR amplification technique was used to amplify the microsatellite and samples were electrophoresed in polyacrylamide gels. Microsatellite allele scoring accuracy and speed were greatly improved by running standards every five samples. The standards consist of 3 microsatellite classes loaded in a single lane. Approximately 350 samples can be analyzed in four days. Because milled or brown rice is used in the procedure, no additional form or quantity of sample is needed other than what is typically used for rice quality screening.

Last Modified: 8/21/2014