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Title: DNA extraction protocols from dormant buds of twelve woody plant genera

Author
item Gilmore, Barbara
item Bassil, Nahla
item Hummer, Kim

Submitted to: Journal of the American Pomological Society
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/28/2011
Publication Date: 11/15/2011
Citation: Gilmore, B.S., Bassil, N.V., Hummer, K.E. 2011. DNA extraction protocols from dormant buds of twelve woody plant genera. Journal of American Pomological Society. 65(4):201-207.

Interpretive Summary: Standard plant DNA extraction methods call for samples of young leaves and shoots. Yet leaf material sometimes is needed when plants are dormant. We tested three DNA extraction methods using dormant buds from a total of 40 species from 12 different plant groups. Two methods were from ready to use kits and the third was using commercial chemical compounds. The plant groups included: hardy kiwi, red raspberry, gooseberry and currant, quince, mountain ash, butternut, service berry, pear, medlar, hazelnut, peony, and blueberry. The amount of DNA recovered varied greatly under different methods: Qiagen extraction chemicals, Omega E-Z 96 Plant DNA Kit and Fast ID 96-Well Genomic DNA Extraction Kit, with the separate plant groups performing consistently across the different extraction methods. Using an average of 33 mg of dormant bud material, we obtained .038 mg of DNA for the Qiagen extraction chemicals, .014 mg DNA for the Omega kit and .005 mg DNA for the Fast ID kit. Walnut DNA was difficult to obtain with all three methods. The Qiagen comercial chemical method was the least expensive of the three methods and resulted in the highest DNA yields, but the buds had to receive more preparation resulting in a greater time expenditure. All three methods resulted in high enough quality DNA that could be used in further DNA applications, the exception being walnut.

Technical Abstract: Standard plant DNA extraction protocols call for samples of newly expanding leaves and shoots yet analysis is sometimes needed when plants are dormant. We evaluated three DNA extraction protocols using dormant buds from 40 species and four hybrids of 12 genera. Two protocols were from ready-to-use kits (the Omega E-Z 96 Plant DNA Kit and the Fast ID 96-Well Genomic DNA Extraction Kit) and the third included commercial lysis and protein precipitation reagents (Qiagen). The genera included: Actinidia (Hardy Kiwi), Rubus (red raspberry), Ribes (gooseberry and currant), Cydonia (quince), Sorbus (mountain ash), Juglans (butternut), Amelanchier (service berry), Pyrus (pear), Mespilus (medlar), Corylus (hazelnut), Paeonia (peony), and Vaccinium (blueberry). In each of the genera tested, except for Juglans, both the Qiagen and Omega protocols generated large amounts of DNA (averaging 40 and 14.8 µg, respectively, from 30 to 36 mg of tissue) from dormant buds. For Juglans, none of these procedures provided satisfactory amounts of DNA from dormant buds. The positive result for 11 genera expanded the options for the sources of tissue as well as time of tissue collection for DNA extraction. The highest DNA yield was obtained with the Qiagen protocol, which was the least expensive of the three. However, in this protocol the bud scales must be removed to obtain a clear DNA extract. The Omega protocol may be more efficient if DNA is to be extracted from a large number of samples. In each of these 11 genera, DNA produced by at least one of the three protocols was of sufficient quality to apply in downstream molecular techniques, such as sequencing.