Submitted to: Crop Science
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/18/2001
Publication Date: 5/6/2002
Citation: HILL AMBROZ,K.L., BROWN GUEDIRA,G.L., FELLERS,J.P., MODIFIED RAPID DNA EXTRACTION PROTOCOL FOR HIGH THROUGHPUT MICROSATELLITE ANALYSIS IN WHEAT, CROP SCIENCE, 2002. Interpretive Summary: Plant breeders would significantly benefit from molecular tools that allow specific traits, like disease resistance, to easily be followed in a breeding population. The tools would significantly reduce the work load of the breeder and allow the development of new varieties to be more efficient. However, a significant hindrance is cost effective isolation of fDNA from large numbers of plants. This manuscript describes a new procedure to isolate DNA. It allows a single person to isolate DNA from nearly 1000 plant samples, while reducing labor and materials costs. The technique will allow researchers to better use the molecular tools now available for breeding programs. This technique is currently being used to advance breeding lines that are resistant to Fusarium head scab, a significant disease in wheat and barley.
Technical Abstract: New technology is allowing marker-assisted selection to fulfill the promise of increasing efficiency of cultivar development. However, these techniques depend upon the ability to extract DNA from large populations of plants. The objective of this project was to develop a high-throughput DNA extraction procedure without the need for greenhouse space or growing wheat t(Triticum aestivum) plants. A sodium hydroxide rapid DNA extraction was modified into a 96-well format to reduce cost. Seeds were germinated in 8- well tissue culture plates, and 4-day-old seedling tissue was used to extract DNA using sodium hydroxide methodology. Approximately 1 ug of genomic DNA per 10mg of tissue was isolated at a cost of about$0.10. The DNA quality was verified by amplification of microsatellite markers. Results were consistent with either fresh or stored tissue extracts. This technique allows one person to extract nearly 1,000 storage stable DNA samples daily, while keeping costs at a minimum.