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久性和尺寸稳定性，使其在户外增值应用中广受欢迎，供不应求。矛盾的是，高达40%的采伐量由于广泛的心材腐烂而被淘汰。这种差异归因于心材中tropolones水平的广泛变化，tropolones可以防止生长树木中引起腐烂的真菌的传播，而木脂素则可以抵抗木材的腐烂。这些化学物质在树木生长的前20年是检测不到的，这使得它们的水平无法作为选择改善心材抗腐性的标志。因此，需要在WRC树异常短的两年代和选择周期中使用DNA标记。为了确定可用于标记开发的基因，我们利用下一代测序技术（NGS）鉴定了在边材通过大量有毒次生代谢物转化为心材的区域中具有高水平活性的bbb700个基因。除了tropolones和木脂素生物合成的候选基因外，我们还鉴定了倍半萜类和二萜类、单宁类和蜡类生物合成的基因，将目标代谢物类别从2个扩大到5个。迄今为止的体外分析表明，表达的酶可以产生tropolone生物合成的前体以及WRC新型倍半萜和二萜。我们还发现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的基因的高水平表达，为激活和调节次生代谢物的产生提供了途径。我们还确定了对真菌感染具有抗性的WRC家族，这为鉴定抵抗真菌通过树皮和边材组织渗透的基因提供了机会。在这里，我们提出了一个双管齐下的方法，以确定一组核心基因和基因变异参与抗腐。首先，我们将通过细胞培养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tropolone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异来识别。总而言之，这些方法可能提供一组核心基因和变异，用于选择改善WRC心材抗腐性，并提供可应用于其他物种的心材形成遗传基础的新知识。

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.