Plastid genome evolution in the holoparasite Orobanche

The plastid genome shows extensive conservation in size, structure, composition, and rate of nucleotide substitution across a broad spectrum of land plants. The composition of the plastid genome, however, is not totally static, and there is a general trend towards the loss of genetic information over time by gene deletion by, e.g., transfer to the nuclear genome. Although the transfer of functional genes is now rare or has ceased altogether, organelle-to- nucleus transfer resulting in non-coding sequences occurs frequently. Additionally, structural rearrangements can occur in the plastid genome, such as intron loss, loss or expansion of the inverted repeat regions, or inversions and transpositions. A specific group exhibiting major changes in content and structure of the plastid genome are holoparasitic plants. These parasites are non-photosynthetic and thus completely depend on a host plant from which they obtain water, nutrients and macromolecules. Having abandoned photosynthesis altogether, the genes for photosynthesis have lost their function causing their pseudogenization and eventual loss. Significantly, changes are not confined only to genes connected to photosynthesis, but also affect other plastid regions. Although the general pattern of plastid genome reduction is true for all non-photosynthetic species investigated so far, they exhibit considerable differences, e.g., genes involved in photosynthesis and chlororespiration can either be intact, modified as pseudogenes or missing. The aim of this study is to investigate the evolution of the plastid genome in a group of holoparasitic genera in the Orobanchaceae (Orobanche and related genera, among those Epifagus, whose plastid genome has already been sequenced). Specifically, the following aspects will be analyzed: (i) co-linearity and structural rearrangements; (ii) potential functionality of genes involved in photosynthesis; (iii) pseudogenization and gene loss; (iv) accelerated rate of evolution; (v) heteroplasmy and horizontal gene transfer. To this end, the plastid genomes of 10 species will be sequenced. The significance of the proposed research lies in allowing to thoroughly examine mechanisms, patterns, and rates of plastid DNA and genome evolution in a group of closely related holoparasitic plants. Specifically, questions such as the evolution of genes after the loss of selective constraints or the modes of size reduction of the whole plastid genome once deprived of its main function can be addressed in an unprecedented wide comparative context.

Due to the loss of photosynthesis, the prime function of plastid organelles, large parts of the plastid genome in non- photosynthetic parasitic flowering plants are no longer under functional constraints and free to evolve. Consequences of these relaxed constraints include overall elevated substitution rates as well as loss of gene function and eventual loss of entire genes involved in photosynthesis and related processes, leading to a considerable size reduction compared to plastid genomes of photosynthetically active plants. Here, for the first time these evolutionary trends have been investigated in a broad comparative context, allowing testing their generality. The major results are: (1) Reduction of plastid genomes is confirmed as a general phenomenon in non-photosynthetic parasitic plants, but this reduction does not occur progressively, but in a highly idiosyncratic and lineage-specific manner disconnected from phylogenetic relationships. (2) In addition to genome reduction, genome rearrangements are identified as a second major force in structural changes of parasite plastid genomes. Significantly, such rearrangements already occur in photosynthetically active parasitic plants, indicating that these changes are not connected to the loss of photosynthesis, but rather to parasitism itself. (3) Patterns of loss of gene function and of gene loss are more complex than previously anticipated, also in photosynthesis genes. Specifically, loss of gene function and gene loss are not determined by the dispensability of a given function, but is determined by other as yet undetermined factors. Furthermore, genes deemed no longer necessary after loss of photosynthesis appear to be still functional (e.g., genes of the thylakoid ATP-synthase complex), whereas others, which have been considered conserved, actually show various degrees of reduction (e.g., plastid ribosomal genes). Recent advances in sequencing technology (i.e., next generation sequencing, such as 454-pyrosequencing) provide the methods to use a brute-force approach to obtain full plastid genome sequences from species, which lack the canonical plastid genome structure and content and are thus notoriously difficult to sequence. Using simulation studies, we quantify the trade-off between coverage of the plastid genome (the more sequencing data, the higher the coverage of a given plastid genome) and the problems resulting from an increasing amount of data (e.g., computational burden, decreasing assembly quality). We also devise a novel strategy for data handling and processing suitable for single personal computers, allowing high-throughput plastid genome sequencing without the need for high-end computational power.