(Note that many of these activities are integrated into the Comparative Genomics activities)
Sitka Spruce cDNA Libraries Related to Sitka Spruce Adaptation
New cDNA libraries to support gene discovery with an emphasis on cold hardiness and local adaptation have been developed from Sitka spruce FB3-425 needle tissue, and are currently being sequenced at the GSC (November 2007).
Candidate Genes Related to Sitka Spruce Adaptation
For Sitka spruce adaptation, candidate gene selection was undertaken de-novo. RNA was extracted on five dates from foliage collected at different stages of cold acclimation between Aug. 28th and Dec. 13th 2005, from a BC population. 1257 elements on the 21.8K cDNA microarray were up-regulated, and 967 down-regulated. From these expression data and the cold hardiness literature, 311 candidates were selected for SNP discovery. PCR single-products were observed for 198 genes (a success rate comparable to white spruce and also comparable to pine with our co-funders at UC Davis). Interestingly, among the candidate genes strongly up-regulated are many pathenogenesis-related proteins. As antifreeze proteins in other systems are homologous to pathogenesis-related genes; these genes may also be antifreeze genes. Antifreeze genes can only be conclusively identified through testing protein function: collaboration is being established with Dr. Patrick von Aderkas at the University of Victoria to characterize a subset of these putative antifreeze genes. This involves heterologous expression of genes, purification of candidate proteins, and testing of antifreeze activity.
SNP Panel for Genes Related to Sitka Spruce Adaptation
In Sitka spruce, a panel of 24 individuals from across the species range of Sitka spruce serves as the discovery panel; these individuals originate from six widely distributed geographical populations. A sample size of 24 takes into the account the larger geographical differentiation among Sitka spruce, whose latitude spans from California to Alaska. These same genes will also be sequenced in the 12-member white spruce panel, discussed above.
Sitka Spruce Association Populations
Branch cuttings have been rooted from 500 individuals collected from across the species range. The rooted cuttings will also provide genetic material for a growth chamber experiment designed to separate candidate gene expression and cold acclimation phenotype responses to low temperature and critical night length. We have also sown the offspring of 215 open-pollinated mother trees from southern Vancouver Island to test associations between candidate SNPs and cold acclimation and phenology phenotypes within a single large population.