Periodic Reporting for period 1 - DEFPOLL (Origins of trait diversity in flowering plants: understanding interactions between plant defence and pollination using molecular, phenotypic and ecological studies of natural selection)
Periodo di rendicontazione: 2018-06-01 al 2020-05-31
Project Summary: The extraordinary diversity of flowering plants is arguably most evident in two seemingly unrelated aspects of life history: reproduction, exemplified by the stunning diversity in flower form and function; and defence, exemplified by the remarkable variation in toxic chemistry found in the leaves of most plant species. Despite over 150 years of research on these topics(1), comparatively few studies have addressed how defence and reproduction interact on ecological and evolutionary time scales(2). This is surprising, first, because variation in sexual reproduction determines key aspects of plant populations that are known to influence the frequency and impact of antagonists such as herbivores and pathogens(3); and second, because herbivory and disease are ubiquitous stresses that influence plant reproductive success(4). Feedbacks between defence and sexual reproduction therefore represent a rich set of unexplored mechanisms explaining important components of plant trait diversity. The objective of this research is to address this knowledge gap by answering two questions: First, what are the consequences of a shift in a plant species’ reproductive strategy for interactions with pollinators and herbivores? Second, what is the impact of these altered species interactions for plant fitness and the evolution of leaf defence traits and floral pollination traits? To answer these questions, I will combine cutting edge genome-editing techniques with evolutionary and chemical ecology. With this interdisciplinary approach, I will assess novel genetic and ecological factors underlying variation in defence and pollination phenotypes, two classes of plant traits that are of critical significance in both wild and managed plant species.
Explanation of the work carried out, by Work Package/Objective
Results:
Obj/WP 1) Use CRISPR technology to develop “neo-selfing” genotypes from multiple genetic backgrounds.
We sequenced a conserved region of the S-locus for replicate genetic families from an outcrossing population of the study species, Arabidopsis lyrata ssp. lyrata, and then attempted to implement CRISPR-Cas9 based silencing of the S-locus by designing targets unique to each allele. During this time the EU/ECJ passed legislation which placed CRISPR-Cas9 gene editing under the same regulatory framework as GMOs, making it difficult to continue with the planned approach. We thus implemented a parallel approach, relying on manual pollinations. This technique is extremely laborious, thus we focused on a single population, rather than 4-5 populations, and generated replicate seed families that could be used in experiments in lieu of the GM lines.
Obj/WP 2) Conduct a semi-natural experiment to test how herbivores and pollinators impose natural selection on plant traits following the shift to selfing.
This objective was to be completed in the second phase of the project (mos. 13-24), and was thus not met.
Obj/WP 3) Phenotype relevant plant traits (leaf defence and floral traits) and identify how transitions to selfing change expression of defence-related genes.
As per the DoA, this objective was originally to be completed in the second phase of the project (mos. 13-24). Nevertheless, we were able to conduct pilot-scale phenotyping of defence and growth rate dynamics using a subset of the manually self-pollinated lines described above.
Results:
Obj/WP 1) Use CRISPR technology to develop “neo-selfing” genotypes from multiple genetic backgrounds.
We sequenced a conserved region of the S-locus for replicate genetic families from an outcrossing population of the study species, Arabidopsis lyrata ssp. lyrata, and then attempted to implement CRISPR-Cas9 based silencing of the S-locus by designing targets unique to each allele. During this time the EU/ECJ passed legislation which placed CRISPR-Cas9 gene editing under the same regulatory framework as GMOs, making it difficult to continue with the planned approach. We thus implemented a parallel approach, relying on manual pollinations. This technique is extremely laborious, thus we focused on a single population, rather than 4-5 populations, and generated replicate seed families that could be used in experiments in lieu of the GM lines.
Obj/WP 2) Conduct a semi-natural experiment to test how herbivores and pollinators impose natural selection on plant traits following the shift to selfing.
This objective was to be completed in the second phase of the project (mos. 13-24), and was thus not met.
Obj/WP 3) Phenotype relevant plant traits (leaf defence and floral traits) and identify how transitions to selfing change expression of defence-related genes.
As per the DoA, this objective was originally to be completed in the second phase of the project (mos. 13-24). Nevertheless, we were able to conduct pilot-scale phenotyping of defence and growth rate dynamics using a subset of the manually self-pollinated lines described above.
Not applicable - Project was terminated early.