Functional genomic analysis of heartwood formation in Western redcedar (Thuja plicata) towards marker-assisted selection for rot resistance

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Based on lumber prices, Western redcedar (WRC; Thuja plicata) is one of the most prized forest tree species in Canada with yearly sales around $1 billion. The high value of WRC lumber is based on its durability and dimensional stability, making it popular for outdoor value-added applications and insufficient supply to satisfy demand. Paradoxically, up to 40% of harvested volumes are culled due to extensive heartwood rot. This discrepancy is attributed to extensive variation in heartwood levels of tropolones that prevent spread of rot-causing fungi in growing trees as well as lignans providing rot resistance to wood in service. These chemicals are not detected in the first 20 years of tree growth, making their levels impractical as markers to select for improved heartwood rot resistance. Thus, there is a need for DNA markers that can be used in the unusually short two-year generation and selection cycle of WRC trees. To identify genes that can be targeted for marker development, we have used Next Generation Sequencing (NGS) to identify > 700 genes that are active at high levels in zone where sapwood is converted into heartwood by massive accumulation of toxic secondary metabolites. In addition to candidate genes for biosynthesis of tropolones and lignans, we also identified genes for the biosynthesis of sesqui- and diterpenes, tannins and waxes, expanding the target metabolite classes from two to five. In vitro assays to date show that expressed enzymes produce precursors to tropolone biosynthesis as well as for WRC novel sesqui- and diterpenes. We also found high levels of expression of genes likely to act in the biosynthesis and signaling of the hormone ethylene and many transcription factors, providing a pathway for activation and regulation of secondary metabolite production. We have also identified WRC families that are resistant to controlled fungal infection, providing an opportunity to identify genes that act to resist fungal penetration through bark and sapwood tissues. Here we propose a two-pronged approach to identify a core set of genes and gene variants involved in rot resistance. First, we will manipulate the activity of identified genes via overexpression and RNA interference in cell cultures and wood cores to identify downstream effects on gene expression and secondary metabolite accumulation and provide evidence of gene functions. Secondly, we will use a common garden with trees known to differ in the chemical content of tropolones and lignans to identify by NGS genes and variants that correlate with levels of chemicals. Taken together, these approaches are likely to provide a core set of genes and variants to be used to select for improved WRC heartwood rot resistance and novel knowledge of the genetic basis of heartwood formation that can be applied to other species.