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

对西部红杉（Thuja plicata）心材形成的功能基因组分析，以进行抗腐烂标记辅助选择

• 批准号: RGPIN-2020-06918
• 负责人: Mattsson, Jim
• 金额: $ 2.04万
• 依托单位: Simon Fraser University
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2020
• 资助国家: 加拿大
• 起止时间: 2020-01-01 至 2021-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=718560
• 关键词: Functional; genomic; analysis; heartwood; formation

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.

根据木材价格，Western Redcedar（WRC； Thuja Plicata）是加拿大最珍贵的森林树种之一，年度销售约为10亿加元。 WRC木材的高价值基于其耐用性和尺寸稳定性，使其在室外增值应用程序中很受欢迎，并且不足以满足需求。矛盾的是，由于心脏广泛的腐烂，高达40％的收获体积被淘汰。这种差异归因于心脏托架水平的广泛差异，这些差异可防止腐烂的真菌在生长的树木中以及提供对木材的腐烂耐药性中的木叶散布。在树木生长的头20年中，未检测到这些化学物质，使其水平不切实际，因为标志物可以选择以提高心脏腐烂的耐药性。因此，需要在WRC树的异常短两年和选择周期中使用的DNA标记。为了识别可以针对标记发育的基因，我们使用了下一代测序（NGS）来识别> 700个基因，这些基因通过大量积累有毒的二级代谢物的大量积累而在Sapwood转化为心脏的区域中具有活性的高水平。除了候选基因的基因，我们还鉴定了塞斯奎 - 和二萜，单宁和蜡的生物合成基因，从而将靶代代谢物类别从两个扩大到五。迄今为止的体外测定表明，表达的酶为Tropolone生物合成以及WRC小说sesqui-and Diterpenes产生前体。我们还发现，基因的高水平表达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作用，从而为次级代谢产物的激活和调节提供了途径。我们还确定了对受控真菌感染有抵抗力的WRC家族，提供了一个机会，可以鉴定出抗菌通过树皮和Sapwood组织抗真菌渗透的基因。在这里，我们提出了一种两管齐的方法，以识别涉及腐烂抗性的核心基因和基因变体。首先，我们将通过过表达和RNA干扰在细胞培养物和木芯中操纵鉴定基因的活性，以鉴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，我们将使用一个普通的花园，其中已知的树木在Tropolones和Lignans的化学含量上有所不同，以通过NGS基因和与化学物质水平相关的变体鉴定。综上所述，这些方法可能会提供一组核心基因和变体，用于选择可改善WRC心脏腐烂的耐药性以及可以应用于其他物种的心脏形成遗传基础的新知识。