Molecular basis of chemodiversity in the genome

普通唐乙酸菌表现出显著的α-和β-化学多样性，特别是在叶片中的单萜。在第一个资助期，我们的目标是在一个以P5和P6为特征的单一种群中，使用RNA-seq、特征：转录本关联和生物化学的组合来表征化学多样性的分子来源。我们能够证明萜烯合成酶候选转录本与特定的化学类型相关。RNA-SEQ数据表明，表达变异和不存在变异可能是观察到的化学类型的原因。通过长读技术产生的普氏锥虫的初步基因组序列支持了这一假设。由P5进行的杂交试验和与P9的分析表明，与萜类生物合成有关的酶可能是紧密相连的，性状的表达受反式作用转录因子(TF)控制。该基因组提供了一个支持连锁观察的候选代谢专化岛。我们还组装了杜卡马拉的基因组，用P4进行了食草性和茉莉酸甲酯的RNA-seq实验，获得了在测试条件下控制专门化代谢的转录因子，并验证了这些转录因子确实与专门化代谢基因的启动子结合。在第二个资助期，我们的目标是测试这一假设，即α和β化学多样性在很大程度上是由转录因子介导的表达差异控制的。验证这一假说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。在普通小麦中，我们将提供更多的基因组组合，对更多的萜类合成酶进行生化表征，并使用RNA-SEQ来鉴定在叶片发育过程中控制其表达的转录因子。我们将使用与转录因子结合的DNA的DNA亲和纯化测序来实验验证基于RNA-SEQ的预测，并比较分析不同的化学类型，以描绘在β-化学多样性水平上的调控差异。结论将通过交叉实验(与P5和P9)进一步检验。我们将以干旱和食草性为应激源(P4，P5，COR)，在普通小麦和杜卡马拉中测试调控对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，但在每种化学类型和两个物种中的特定代谢靶基因是高度分化的。通过识别在不同水平上(包括干旱胁迫下)影响化学多样性的顺式和反式作用基因，我们将支持P9中模型的参数化。综上所述，我们在第二个资助期的实验将探索监管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.