Assessment of stress-induced and developmentally-induced DNA methylation changes in barley (Hordeum vulgare L.)

DNA methylation is involved in both plant development and adaptation to environmental stress. Changes in DNA methylation can affect the expression of genes that are important for both plant tissue differentiation and stress response. Characterisation of tissue and stress specific methylation markers generates an invaluable tool for epiallele discovery that can be used for future functional and crop improvement studies. We used barley as a plant model, and salinity as a stress model, to study methylation markers that discriminate the plant tissues and that are specific to salinity stress. This choice presented the advantage of using a crop plant with a reference genome sequence, which allows for genomic analyses; and an abiotic stress factor that is relatively easy to control. Nine barley varieties subjected to mild salt stress (75 mM NaCl) were studied for their response to the stress by measuring phenotypic traits, such as biomass, yield and ion accumulation in the leaves. Then, Methylation Sensitive Amplified Polymorphisms (MSAP) were used to analyse changes induced by salt stress in their DNA methylation profiles, which were tested for correlation with the phenotypic data from the same plants. This study revealed that, although the MSAP approach can detect differentially methylated markers induced by a mild salt stress in barley, it presented a limitation in the number of differentially methylated markers (DMMs) detected. This study also revealed that the detection of DMMs by MSAPs was significantly influenced by genotypic differences among varieties. Finally, analysis of the epigenetic variability detected by MSAP indicated that microclimatic differences experienced by different plants in the study contributed to what was previously considered to be stochastic variability. The results from the MSAP suggested an alternative approach was required to identify DMMs that are conserved across barley varieties. Using the high throughput DNA sequencing approach methylation-sensitive genotyping by sequencing (ms-GBS), we detected thousands of saltinduced DMMs and similar numbers of tissue-specific DMMs. Ms-GBS-generated DMMs were potentially universal, since they were conserved in five barley varieties used in the study. Sequence analysis of the ms-GBS generated DMMs indicate that both tissue-specific and salt induced changes in DNA methylation happen preferentially in repeat regions, but also target other gene types, such as protein-coding and Transfer RNA genes. Ontology analysis of differentially methylated protein-coding genes revealed that many are likely to play a role in stress response and organ-specific functions. However, further studies, including expression analyses, are needed to link gene methylation to gene expression.Thesis (Ph.D.) (Research by Publication) -- University of Adelaide, School of Agriculture, Food and Wine, 2017