Molecular basis of chemodiversity in the genome

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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.