Graduate Thesis Or Dissertation


Candidate Genes Associated with Blister Rust Resistance in Whitebark Pine (Pinus albicaulis) Revealed through de novo Transcriptome Assembly and Differential Gene Expression Analysis Public Deposited

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  • Whitebark pine (Pinus albicaulis) is an iconic North American high-elevation tree species currently threatened by climate change, mountain pine beetle, and white pine blister rust (WPBR), a lethal disease caused by the non-native fungal pathogen Cronartium ribicola. In collaboration with the USDA Forest Service Dorena Genetic Resource Center, germplasm was collected from whitebark pine trees throughout the Cascade Range in Oregon and Washington to evaluate the frequency and variation in genetic resistance among naturally occurring populations. Based on operational resistance screening trials, progeny of ‘Shadow Lake 39’ from Mt. Rainier National Park exhibited significant within-family variation in resistance and were the primary focus of bioinformatic investigation. In an effort to identify candidate genes associated with WPBR resistance, we designed an inoculation experiment to investigate differential gene expression in needle tissues that distinguished resistant from susceptible seedlings in this whitebark pine half-sibling family. Seedlings were distributed in a randomized complete block design and exposed to C. ribicola inoculum (~3000 spores cm²) for 24 hours in a fog chamber to initiate WPBR infection. Three days after inoculation, needle tissue was sampled and flash-frozen to preserve each individual's RNA expression profile. While half of the seedlings were inoculated with C. ribicola prior to needle sampling, the other half were inoculated after sampling, allowing us to capture the expression profiles of uninoculated seedlings. This sampling strategy permitted comparisons between both experimental treatments (inoculated vs. uninoculated), while still revealing the resistance phenotype of each individual tree seedling. Needle tissues from four representative seedlings were sequenced with RNA-Seq and used to assemble a reference transcriptome de novo. These assembled sequences were annotated with inferred transcript function, gene ontology terms, and predicted proteins to create an annotated reference transcriptome for P. albicaulis ‘Shadow Lake 39’ needle tissue. To identify candidate genes that are differentially expressed in association with WPBR resistance, individual libraries were generated for 24 representative seedlings and sequenced using RNA-Seq. This design allowed for group comparisons between uninoculated susceptible, uninoculated resistant, inoculated susceptible, and inoculated resistant trees. Comparative transcriptomic analyses produced a list of differentially expressed genes, which were curated to reveal the top candidate genes associated with blister rust resistance in whitebark pine. We identified a variety of differentially expressed genes with annotations related to the plant defense response to fungal diseases, including several encoding pathogenesis-related proteins, enzymes involved in the biosynthesis of secondary metabolic compounds, and a suite of other gene products putatively related to WPBR resistance. We also searched the P. albicaulis ‘Shadow Lake 39’ needle transcriptome for SSR and SNP variants and designed corresponding primer sequences for future investigations. Following validation, these markers may help to expedite and economize resistance breeding programs by enabling forest geneticists to screen individual whitebark pines for blister rust resistance. In summary, this exploratory survey of the P. albicaulis ‘Shadow Lake 39’ seed family has produced an annotated reference needle transcriptome, lists of differentially expressed candidate genes putatively associated with blister rust resistance, and primer sequences designed to validate potential markers, representing a significant contribution toward WPBR resistance breeding in whitebark pine.
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