Cellular level bottlenecks: genetic diversity, population dynamics, and technology development

This thesis was motivated by the recognition of the importance of the underlying role that genetic variation plays in disease and how this genetic variation is modulated by evolution; specifically, genetic drift and selection. I have sought to understand how genetic diversity is modulated by population bottleneck events at the cellular level and the effects of evolutionary forces on genetic diversity. I chose two very distinct systems, mitochondria and viruses, to address these topics and I also developed a new inexpensive and high-throughput sequencing sample preparation procedure for these research systems. ❧ Mitochondria are central for cellular energy, and for signaling pathways involved in stem cell proliferation, migration, and apoptosis. Mitochondrial heteroplasmy, (intra‐ and inter‐ cellular genetic diversity), is known to cause stem cell dysfunction in self‐renewal and long-term proliferation. Thus, metazoans develop from a single cell bottleneck event that acts to homogenize the mitochondrial population, which significantly minimizes the risk of heteroplasmy. The freshwater asexual planarian Schmidtea mediterranea does not undergo a single cell bottleneck event, but rather reproduces by transverse fission or multicellular inheritance. We sought to understand the effects of lacking a single cell bottleneck event and found that high levels of genetic diversity and haplotypes exist within individual planaria, and that mitochondrial populations varied spatially across the planarian body. As a result we have proposed a cellular model incorporating regeneration events to explain these patterns of intra‐individual genetic diversity. ❧ Unlike the common homogeneous genomic state of mitochondria, the population of zucchini yellow mosaic virus contains high levels of genetic diversity associated with the lack of proofreading activity of RNA polymerase, high replication rates, and large population size. It is commonly assumed that the viral population will be subjected to substantial genetic bottlenecks as it moves systemically through the plant. Thus we sequenced to deep coverage 23 leaves growing sequentially along a vine, and one side branch leaf. It was determined that bottleneck events associated with systemic movement are not severe enough to reduce the viral population and that viral diversity is circulated in the phloem sap during infection. In addition we found variants clustered within the CI protein, which has implications for viral movement and infection, which is populating new niches during systemic movement or host immune evasion. ❧ While pursuing the mitochondrial and viral studies, I optimized and developed a new sample preparation procedure for next generation sequencing. This sample preparation can be applied to difficult samples that would normally yield very little DNA. In addition the procedure is high-throughput, and very cost effective. ❧ These studies, although very different in nature, determined that the extent of evolutionary pressures affecting genetic diversity as a result of cellular level bottleneck events is not straightforward. These studies described the patterns and amount of intra‐individual genetic diversity and elucidated how this diversity is affected by the absence of a population bottleneck and regeneration in planaria or the presence of population bottlenecks imposed by the host plant during systemic movement. Furthermore, this data offers a deeper understanding of the dynamics of population movement on genetic diversity, either across the planarian body or viral invasion between tissues and cells. Thus, in both systems intra‐individual genetic diversity is one of the contributing factors to disease and dysfunction, therefore understanding the patterns and extent of genetic diversity through sequencing is important to aid in the development of disease management and system specific coping mechanisms.