Evolutionary studies of uncultivated Radiolaria and their symbionts using Single Cell Whole Genome Amplification

Abstract

Understanding the evolution of eukaryotes and resolving the Tree of Life is one of the major goals of biology. Protists (unicellular eukaryotes) play a crucial role in this quest being the earliest diverging eukaryotes and as ancestors to all multicellular species. They display the largest diversity of all cellular forms, having photosynthetic-, heterotrophic-, saprotrophic-, parasitic- and symbiotic representatives in almost all of the world's ecosystems. The molecular study of protists, however, is currently burdened by the fact that many protists cannot be cultivated in the laboratory the traditional approach to grow the hundreds of cells needed for molecular studies. Methods needs to be developed to overcome this problem. Radiolaria belong to one such group of unculturable protists, and in this thesis they are used as a test system for developing a method that allows molecular study of protists based on single cell whole genome amplification (SCWGA).

Briefly the method consists of isolation of single cells from natural samples followed by an amplification step that in theory can amplify the entire genome from a single cell using the phi29 polymerase and random primers. The amplified genome is then subjected to 18S and 28S rDNA PCR and to shotgun sequencing using the 454 GS FLX Titanium pyrosequencing platform.

Retained genomic information is used in combined 18S and 28S rDNA phylogenies covering the major groups of Radiolaria in order to resolve their evolutionary relationship. In addition the 18S rDNA gene is obtained from several symbiotic cells from several different radiolarian species and used to infer the identity and diversity of the symbionts. Finally several mitochondrial markers are identified from the high throughput sequences.