PCR Amplification of Heat Shock Protein Gene

PCR was performed using the touchdown protocol described by Sargent et al. [35 (link)] in a 20 μl reaction containing HotStart Taq Master Mix (Qiagen, Valencia, CA), 0.4 μM each primer, and 1.0 ng genomic DNA. PCR products were separated by electrophoresis through a 1.5% TAE agarose gel and visualized by ethidium bromide staining. Primers were designed to span the intron in the N-terminal domain of a low molecular weight heat shock protein gene identified as an EST (GenBank accession number CX661743.1) from a "Yellow Wonder" (Y2) heat-treated seedling cDNA library. Template DNA for Y1, Y2, F. iinumae J-17, and F. vesca subsp. americana Pawtuckaway was obtained from 50-100 mg young leaf tissue using a DNeasy Plant Mini kit (Qiagen). Y1 and F. vesca subsp. americana Pawtuckaway plants were obtained from T. Davis (University of New Hampshire) and F. iinumae J-17 plants were obtained from the US National Plant Germplasm collection.