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

根据木材价格，西部红柏(WRC；Thuja plicata)是加拿大最珍贵的森林树种之一，年销售额约10亿美元。WRC木材的高价值基于其耐用性和尺寸稳定性，使其在户外附加值应用中广受欢迎，但供应不足，无法满足需求。矛盾的是，由于大量的心材腐烂，高达40%的采伐量被扑杀。这种差异归因于可防止引起树木腐烂的真菌在生长的树木中扩散的托洛酮的心材水平的广泛差异，以及在使用过程中提供木材防腐性能的木脂素。在树木生长的前20年里，这些化学物质没有被检测到，这使得它们的水平不适用于选择提高心材抗腐性的标记。因此，需要能够在WRC树异常短的两年世代和选择周期中使用的DNA标记。为了确定可以作为标记开发目标的基因，我们使用下一代测序(NGS)来识别&gt；700基因，这些基因在边材通过大量积累有毒的次生代谢物而转化为心材的区域高水平活跃。除了孕酮和木脂素的生物合成候选基因外，我们还确定了倍半萜、二萜、单宁和蜡类的生物合成基因，将目标代谢物类别从2个扩大到5个。到目前为止的体外分析表明，表达的酶产生特罗波隆生物合成的前体，以及WRC新的倍半萜和二萜的前体。我们还发现了可能参与激素乙烯和许多转录因子的生物合成和信号传递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一条途径。我们还鉴定了对可控真菌感染具有抵抗力的WRC家族，这为鉴定抵抗真菌穿透树皮和边材组织的基因提供了机会。在这里，我们提出了一种双管齐下的方法来识别与抗腐性有关的一组核心基因和基因变体。首先，我们将通过细胞培养和木芯中的过度表达和RNA干扰来操纵已识别基因的活性，以确定下游对基因表达和次生代谢物积累的影响，并提供基因功能的证据。其次，我们将使用一个常见的花园，这些树木的已知化学成分不同于三叶皂甙和木脂素，以通过NGS基因和与化学物质水平相关的变异来识别。综上所述，这些方法很可能提供一组核心基因和变种，用于选择提高WRC心材腐烂抗性的基因，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.