Graduation date: 2007In forest trees from temperate and boreal regions, cold acclimation is an important adaptive trait that involves changes in gene expression and physiology. Genecological, quantitative genetic, and QTL studies have been used to study the genetics of cold acclimation in Douglas-fir (Pseudotsuga menziesii (Mirb.) Franco), but the genes responsible for variation among individuals and populations remain unknown. Douglas-fir seedlings from a low elevation, coastal Oregon population and a high elevation, inland Washington population were grown in a common garden in Corvallis, Oregon, and artificial freeze tests were used to compare their cold hardiness from September, 2004 to May, 2005. Cold hardiness was associated with the timing of bud set and bud flush, and there were large population differences in cold acclimation, deacclimation, and bud phenology. In late October, for example, population differences in percent cold damage (predicted at -7°C) were as large as 58% for needles (OR=62% and WA=3%), 48% for stems (OR=53% and WA=5%), and 43% for buds (OR=43% and WA=0%). A custom oligonucleotide microarray was used to analyze gene expression differences between cold susceptible and cold hardy seedlings from the two populations during acclimation. The microarray was developed from >18,000 ESTs from four Douglas-fir cDNA libraries, including three new libraries specifically targeting the stages of cold acclimation, maximum hardiness, and cold deacclimation. Oligonucleotides designed from loblolly pine and white spruce ESTs were included on the microarray to test whether heterologous hybridizations could be used to study Douglas-fir cold acclimation and other processes. Three hundred forty-three differentially expressed Douglas-fir genes were identified (FDR adjusted p-value ≤ 0.01). Many of these genes had similarities to genes encoding dehydrins, heat shock proteins, pathogenesis-related proteins, and genes associated with ABA, GA, and dormancy. Preliminary results suggest that loblolly pine and white spruce ESTs are useful for identifying genes that are differentially expressed during cold acclimation. These differentially expressed genes are considered cold hardiness candidate genes and will be used in genetic association studies of adaptive traits in Douglas-fir from Oregon and Washington.