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

根据木材价格，西部红杉（WRC; Thuja plicata）是加拿大最珍贵的森林树种之一，年销售额约为10亿美元。WRC木材的高价值是基于其耐用性和尺寸稳定性，使其成为户外增值应用的热门产品，并且供应不足以满足需求。奇怪的是，由于广泛的心材腐烂，高达40%的收获量被淘汰。这种差异是由于广泛的变化，在心材水平的酚酮，防止传播的腐烂引起的真菌在生长的树木，以及木脂素提供腐烂的阻力，以木材在服务。这些化学物质在树木生长的前20年中没有被检测到，使得它们的水平作为选择改善心材腐烂抗性的标记是不切实际的。因此，需要可用于WRC树的异常短的两年世代和选择周期的DNA标记。为了鉴定可被靶向用于标记开发的基因，我们已经使用下一代测序（NGS）来鉴定> 700个基因，这些基因在边材通过有毒次级代谢物的大量积累转化为心材的区域中以高水平活跃。除了用于托酚酮和木脂素的生物合成的候选基因之外，我们还鉴定了用于倍半萜和二萜、单宁和蜡的生物合成的基因，将目标代谢物类别从两个扩展到五个。迄今为止的体外试验表明，表达的酶产生前体的托酚酮生物合成以及WRC新的倍半和二萜。我们还发现了高水平的基因表达，这些基因可能在激素乙烯和许多转录因子的生物合成和信号传导中起作用，为次级代谢产物的活化和调节提供了途径。我们还确定了WRC家庭是耐控制真菌感染，提供了一个机会，以确定基因的作用，以抵抗真菌渗透通过树皮和边材组织。在这里，我们提出了一个双管齐下的方法来确定一组核心的基因和基因变异参与腐烂抗性。首先，我们将通过在细胞培养物和木芯中的过表达和RNA干扰来操纵已鉴定基因的活性，以确定对基因表达和次生代谢物积累的下游影响，并提供基因功能的证据。其次，我们将使用一个普通的花园，其中的树木已知在酚酮和木脂素的化学含量上存在差异，以通过NGS基因和与化学物质水平相关的变体进行鉴定。总之，这些方法很可能提供一组核心基因和变异体，用于选择改进的WRC心材腐烂抗性和心材形成的遗传基础的新知识，可应用于其他物种。

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.