Investigating evolution of gene regulation with crossspecies comparative transcriptomics
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An essential ability of all lifeforms is to regulate the activity of its genes. This ability is what allows multicellular organisms to generate hundreds of completely different cells despite having the same genome. It is also this ability that allows organisms to adjust dynamically to external conditions, including how plants in temperate climates adjusts their metabolism in preparation for the coming winter as we study in paper 1 and 2 of this thesis. Quantification of gene expression by RNA sequencing or microarrays is a common method for investigating gene regulation. However, comparing gene expression across species to study evolution of gene regulation presents many challenges. In the first paper, we investigated the evolution of cold adaption in grass by comparing the transcriptomic response to cold in five diverse species of temperate grasses (Pooideae). Two of these species, Barley and Brachypodium distachyon, are well characterized and their cold-response has been studied before. The three other species, which are less studied, belong to early diverging Pooideae tribes. By comparing the cold-response from both early and later diverging species it was possible to investigate to what extent the cold-response has been conserved since their common ancestor, or if it evolved gradually within the Pooideae. Although we observed a significant common response in all species, there were extensive differences. These differences did not follow the phylogeny, as we would expect if there was a gradual evolution. These results indicate that any conserved cold-response either involved very few genes or that the cold-response evolved independently in each lineage. In the second paper, instead of looking at the entire transcriptome, we look specifically at orthologs of a handful of well-known cold-tolerance genes. By inspecting their phylogeny, we see that gene duplication have played an important role both early and later in the evolution of some of the cold-tolerance genes. This includes the CBF gene family, which is known to contain master regulators of cold-response. By investigating the protein sequences we also find that some of the functional protein motifs was missing in the early diverging species, suggesting a gradual and lineage specific evolution of cold-tolerance functions. One of the challenges when comparing gene regulation across species is to acquire directly comparable samples. While we designed the experiment in paper 1 so that the samples should be as similar as possible across species, most experiments found in public databases are not suitable for direct comparison. In the third paper we investigate the use of co-expression to indirectly compare the expression similarities of orthologs. This approach makes it possible to use samples that are not directly comparable. The current methods for co-expression based comparison are not so well studied and we find that we can develop an improved method. Applying our new method to public gene expression data from five plant species, we investigate the link between gene duplication and expression divergence.