Understanding phenotypic and epigenetic drought responses in Trifolium repens (white clover) : a thesis presented in partial fulfilment of the requirements for the degree of Master of Philosophy at Massey University, Palmerston North, New Zealand

Abstract

Trifolium repens (white clover) is an allopolyploid species belonging to the clover genus (Trifolium). White clover is very important in New Zealand agriculture as feed for livestock. Yet this species is facing challenges due to drought, an abiotic stress that has impacted New Zealand’s economy in the past and is predicted to have increased effects in the future due to climate change. Therefore, analysing plant phenotypic and epigenetic responses to drought is an important area of research because it will broaden understanding of drought resistance/tolerance and mechanisms that plants use to survive drought stress. An epigenetic mechanism of interest is DNA methylation, which has a role in gene expression in response to drought. There is also evidence that DNA methylation is involved in plant memory and ‘priming’ which assists in subsequent stress responses. However, this response has not been explored in depth in white clover. In other species there has been evidence of transgenerational inheritance of DNA methylation marks associated with stress. Therefore, research into stress-related DNA methylation in white clover is important as it may be able to be incorporated into the plant breeding process in the future to breed more drought tolerant varieties. In this study, a drought trial with two subsequent drought stress periods was run on white clover varieties and related species. A range of phenotypes were measured throughout the drought trial, including relative water content and leaf area. There was evidence that these phenotypes were significantly impacted by drought stress. The relative water content measurements revealed significant differences in drought response depending on whether plants were experiencing their first or second drought, suggesting that some aspect of plant stress memory was involved. Subsequently, DNA methylation analysis was run on a subset of the white clover plants involved in the drought trial. It was found that DNA methylation played a role in the plant drought response and that there were differences in methylation patterns between plants that were experiencing their first and second drought exposures. Further investigation into genomic regions with DNA methylation profiles retained after drought stress revealed examples of stress-related genes. This supported the hypothesis that areas of differential methylation had a role in the stress response. This study revealed insights into white clover’s drought response and provides a starting point for further research into how these findings can be incorporated into plant breeding/generating more drought tolerant varieties in the future.