BORIS Theses

BORIS Theses
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Reconstitution of pollinator-mediated speciation in Petunia through single gene mutations

Lüthi, Martina Nadine (2022). Reconstitution of pollinator-mediated speciation in Petunia through single gene mutations. (Thesis). Universität Bern, Bern

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Identifying the major genes involved in speciation processes is a key step towards understanding the molecular basis of evolution. A major component of the speciation processes is reproductive isolation, which can occur in plants through differential visitation of pollinators due to diverging pollination syndromes. In the genus Petunia multiple shifts in pollination syndromes can be observed, allowing the genetic changes underlying evolutionary shifts in pollinator attraction to be studied. Here I aimed to mimic changes in phenotypic traits occurring during the transition from hawkmoth to bee pollination in the long tube clade in Petunia. Mimicking this transition required restoration of the AN2 gene while rendering MYB-FL, ODO1 and CNL1 non-functional. Through multiple transgenic complementation approaches AN2 function was restored, although full visible color as observed in P. secreta was not achieved. Genetic modification of the MYB-FL gene in a P. axillaris background through a CRISPR/Cas9 approach rendered it non-functional, altering the floral UV phenotype from absorbent to reflective. Not only were changes in UV color observed, but visible color changed from white to light pink as well, suggesting a trade-off between the anthocyanin and flavonol branches in the flavonoid biosynthetic pathway. Pollinator assays with primary pollinators (hawkmoths) demonstrated a significant preference towards the P. axillaris wildtype compared to the myb-fl mutant lines while the opposite preference was observed for secondary pollinators (bumblebees, solitary bees). However, when presented with the choice of P. secreta vs. the myb-fl mutant, these two lines were not yet indistinguishable for bee pollinators, demonstrating the need for further phenotypic changes needed to obtain a complete P. secreta mimic. To account for these additional phenotypic changes, I also aimed to combine the altered color genes with mutated scent speciation genes ODO1 and CNL1 in a P. axillaris background. Knocking-out the ODO1 transcription factor eliminated production of major scent compounds methylbenzoate, benzaldehyde and isoeugenol/eugenol, creating a scentless mutant. Mutation of the CNL1 gene eliminated scent volatiles methylbenzoate and benzaldehyde but did not alter production of isoeugenol/eugenol compounds. Ultimately analyzing the P. secreta mimic in pollinator assays should demonstrate whether altering these few speciation genes is enough to obtain a shift in primary and secondary pollinator preference strong enough to cause reproductive isolation. This should help settle the debate about the contributions of few mutations of large effect during the process of speciation.

Item Type: Thesis
Dissertation Type: Single
Date of Defense: 27 January 2022
Subjects: 500 Science > 580 Plants (Botany)
Institute / Center: 08 Faculty of Science > Department of Biology > Institute of Plant Sciences (IPS)
Depositing User: Hammer Igor
Date Deposited: 01 Nov 2022 11:31
Last Modified: 27 Jan 2023 23:25

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