Molecular basis of chemodiversity in the genome

Tanacetum vulgare表现出显著的α-和β-化学多样性，特别是在叶单萜。在第一个资助期内，我们的目标是在以P5和P6为特征的单一人群中使用RNA-seq、性状：转录本关联和生物化学的组合来确定化学多样性的分子来源。我们能够表明，萜烯合酶候选转录与特定的化学型。RNA-seq数据表明，表达变异而不是存在不存在变异是观察到的化学型的可能原因。已对T.长读技术产生的vulgare支持这一假设。由P5进行并与P9一起分析的杂交实验表明，萜类化合物生物合成的酶可能是密切相关的，并且性状的表达受反式作用转录因子（TF）控制。基因组提供了一个支持连锁观察的候选专门代谢岛。我们还组装了S. dulcamara用P4进行了食草动物和茉莉酸甲酯RNA-seq实验，在测试条件下得到了控制专门代谢的转录因子，并验证了这些转录因子确实结合专门代谢基因的启动子。在第二个资助期内，我们的目标是检验α和β化学多样性在很大程度上控制在转录因子介导的表达变异水平上的假设。验证这一假设将进一步加深我们对化学多样性是如何被分子编码的理解。于T.对于普通植物，我们将提供额外的基因组组装，对更多的萜烯合酶进行生物化学表征，并使用RNA-seq来鉴定控制其在叶子发育过程中表达的转录因子。我们将使用与转录因子结合的DNA的DNA亲和纯化测序，以实验验证基于RNA-seq的预测，并比较分析不同的化学型，以描绘β-化学多样性水平上的调控差异。将通过杂交实验（与P5和P9）进一步验证结论。于T. vulgare和S. dulcamara，我们将使用干旱和草食动物作为应激源（P4，P5，COR）来测试调节对α-化学多样性的影响。我们假设调控网络，即控制诱导的转录因子是保守的，但在每种化学型和两个物种中的专门代谢靶基因是高度分化的。通过确定顺式和反式作用基因在不同水平上，包括在干旱胁迫下的化学多样性，我们将支持参数化的模型在P9。总之，我们在第二个资助期的实验将探索调控对化学多样性的贡献。

Tanacetum vulgare shows remarkably high α- and β-chemodiversity, particularly in the leaf monoterpenoids. In the first funding period, our goal was to characterise the molecular sources of chemodiversity using a combination of RNA-seq, trait:transcript association and biochemistry within a single population characterised by P5 and P6. We were able to show that terpene synthase candidate transcripts associate with particular chemotypes. RNA-seq data indicated that expression variation and not presence absence variation is the likely cause of the observed chemotypes. A preliminary genome sequence of T. vulgare generated by long read technology supported that hypothesis. Crossing experiments performed by P5 and analysed together with P9 showed that enzymes for terpenoid biosynthesis are likely closely linked and that expression of traits is controlled by trans-acting transcription factors (TFs). The genome provided a candidate specialised metabolism island supporting the linkage observation. We also assembled a genome for S. dulcamara, performed a herbivory and methyl jasmonate RNA-seq experiment with P4, derived transcription factors controlling specialised metabolism under the test conditions and validated that these transcription factors indeed bind the promoters of specialised metabolism genes. In a second funding period, our goal is to test the hypothesis that both α- and β-chemodiversity are controlled largely at the level of expression variation mediated by transcription factors. Testing this hypothesis will further our understanding how chemodiversity is molecularly encoded. In T. vulgare, we will provide additional genome assemblies, biochemically characterise more terpene synthases and use RNA-seq to identify the transcription factors governing their expression during leaf development. We will use DNA affinity purification sequencing of DNA bound to transcription factors to experimentally validate the predictions made based on RNA-seq and comparatively analyse different chemotypes to delineate differences in regulation at the level of β-chemodiversity. Conclusions will be further tested by crossing experiments (with P5 and P9). In T. vulgare and S. dulcamara, we will test the influence of regulation on α-chemodiversity using drought and herbivory as stressors (P4, P5, COR). We hypothesise that the regulatory network, i.e. the transcription factors which control induction, are conserved but that the specialised metabolism target genes in each chemotype and in the two species are highly diverged. By identifying the cis- and trans-acting genes contributing to chemodiversity at different levels, including under drought stress, we will support parametrisation of models in P9. Taken together, our experiments in the second funding period will explore the contribution of regulation to chemodiversity.