• An Optimized Chloroplast DNA Extraction Protocol for Grasses (Poaceae) Proves Suitable for Whole Plastid Genome Sequencing and SNP Detection

      Diekmann, Kerstin; Hodkinson, Trevor R; Fricke, Evelyn; Barth, Susanne; Teagasc Walsh Fellowship Programme (PLoS, 30/07/2008)
      Background Obtaining chloroplast genome sequences is important to increase the knowledge about the fundamental biology of plastids, to understand evolutionary and ecological processes in the evolution of plants, to develop biotechnological applications (e.g. plastid engineering) and to improve the efficiency of breeding schemes. Extraction of pure chloroplast DNA is required for efficient sequencing of chloroplast genomes. Unfortunately, most protocols for extracting chloroplast DNA were developed for eudicots and do not produce sufficiently pure yields for a shotgun sequencing approach of whole plastid genomes from the monocot grasses. Methodology/Principal Findings We have developed a simple and inexpensive method to obtain chloroplast DNA from grass species by modifying and extending protocols optimized for the use in eudicots. Many protocols for extracting chloroplast DNA require an ultracentrifugation step to efficiently separate chloroplast DNA from nuclear DNA. The developed method uses two more centrifugation steps than previously reported protocols and does not require an ultracentrifuge. Conclusions/Significance The described method delivered chloroplast DNA of very high quality from two grass species belonging to highly different taxonomic subfamilies within the grass family (Lolium perenne, Pooideae; Miscanthus×giganteus, Panicoideae). The DNA from Lolium perenne was used for whole chloroplast genome sequencing and detection of SNPs. The sequence is publicly available on EMBL/GenBank.
    • Ryegrass organelle genomes: phylogenomics and sequence evaluation

      Diekmann, Kerstin; Teagasc Walsh Fellowship Programme (2010)
      Perennial ryegrass (Lolium perenne L.) is the most important forage grass of temperate regions of the world. The main objective in breeding perennial ryegrass cultivars is to increase its biomass. Chloroplasts and mitochondria are two organelles of the plant cell that are actively involved in biomass production. Chloroplasts derive from cyanobacteria and are the location of photosynthesis in plant cells. Mitochondria derive from α-proteobacteria and are involved in cell respiration. Due to their evolutionary history both organelles still contain their own genome which is in general maternally inherited. The interest in chloroplast genome sequences increased in recent years because they offer a useful option for plant genetic engineering. The risk of transgene escape via pollen flow is reduced while the expression of the transgene due to the high number of chloroplast genome copies is increased (in comparison to nuclear genome transformation). Mitochondrial genomes are of special interest because they are involved in cytoplasmic male sterility. Cytoplasmic male sterility is a very important trait in plant breeding programmes because it enables the cost efficient production of hybrid seed. Additionally, both organelle genomes can be used for molecular evolution or phylogenetic studies, as well as for population genetic approaches. Therefore the major aim of this thesis was to sequence the entire chloroplast and mitochondrial genomes of L. perenne to provide sequence information for chloroplast genetic engineering approaches, insights into the mitochondrial genome of a male fertile L. perenne cultivar and to gather knowledge about sequence variation in both genomes that can be used to design new markers for phylogenetic and population genetic studies.