Kerstin Diekmann 2010 Submission for Ph.D. University of Dublin, Trinity College Declaration I, the undersigned, hereby declare that I am the sole author of this dissertation and that the work presented in it, unless otherwise referenced, is my own. I also declare that the work has not been submitted, in whole or in part, to any other university or college for a degree or other qualification. I authorize the library of Trinity College Dublin to lend or copy this dissertation on request. _________________________________ Kerstin Diekmann Dedicated to my parents and my brother. Thank you, for always being there for me! ACKNOWLEDGEMENTS I am very grateful to: My supervisors Dr. Susanne Barth and Dr. Trevor R. Hodkinson for their immense support, guidance and especially motivation throughout the time of this Ph.D. project. Professor Kenneth Wolfe for his advice and patience with the assembly and annotation of the organelle genomes. Dr. Dan Milbourne, Dr. Alfonso Blanco, Colum Kennedy and Dr. Paul McDermott for their help with the DNA isolation from perennial ryegrass organelles in general and Dr. Sophie Kiang and Professor Matthew Harmey for their advice on the isolation of mitochondrial DNA in particular. The Teagasc Oak Park grass breeding group especially Olivia Aylesbury for supplying me with the large amount of seeds necessary to succeed in this project. The Teagasc Oak Park Administration in particular Cheryl Austin, Eleanor Butler, Connie Conway, Sharon Cosgrove, Sadie Fleming, Philomena Kelly and Eugene Brennan who helped me on many occasions. The friends that I met in Ireland particularly Ulrike Anhalt, Bicheng Yang, Celine Tomaszewski, Marta Cristilli, Stephen Byrne, Jeanne Mehenni-Ciz and Sinead Phelan. Thank you for your friendship and support that made my life in Ireland in general and the Ph.D. time in particular so much easier and nicer. My friends back at home in Germany especially Kristin Elvers, Lena and Jörn Uwe Starcke with Julius, Meike Wildung and Luise Lange. Thank you for your friendship in general and for having been so supportive, understanding and patient with me during this time. Teagasc for funding this Ph.D. project within the Teagasc Walsh Fellow Programme. Summary 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. Chloroplast DNA was extracted with an in this study optimised protocol and sequenced using a shotgun sequencing approach from the L. perenne cv. Cashel. After sequence assembly the L. perenne chloroplast genome was found to comprise of 135,282 bp, divided into a large (79,972 bp) and a small (12,428 bp) single copy region separated from each other by two copies of an inverted repeat (each 21,441 bp). The chloroplast genome encodes 76 protein-coding, 30 transfer and four ribosomal RNA genes and therefore was, regarding its size and gene content/order, in the range expected for Poaceae chloroplast genomes. Thirty-three protein-coding genes of the L. perenne chloroplast genome were searched for RNA editing sites via reverse transcriptase PCR. RNA editing is a posttranscriptional process that can increase the gene conservation, create start and stop codons and restore the functionality of genes. Thirty-one editing sites (all C-U) were detected in 18 L. perenne chloroplast protein-coding genes. Chloroplast genomes are generally highly conserved, however, an unexpected and considerable high amount of variation in forms of insertion deletion events and single nucleotide polymorphisms was found throughout the genome of L. perenne cv. Cashel. Nine new Poaceae-universal chloroplast markers were designed from some of those highly variable regions. The markers were tested on a subset of the Teagasc Oak Park grass collection. All markers revealed diversity across the subset although to different extent. Three markers were especially interesting due to their ability to detect high amounts of variation, to distinguish haplotypes via inexpensive agarose gel-electrophoresis, or to distinguish annual from perennial ryegrass. The mitochondrial genome was sequenced from the L. perenne cv. Shandon using a hybrid sequencing approach based on Sanger and GS FLX sequencing. Due to timely restrictions of the project and the very challenging nature of plant mitochondrial genomes, the L. perenne mitochondrial genome could not be completely assembled. The draft assembly consists to date of 43 contigs (ca. 560 kb in total), containing all 33 expected protein- coding genes that are commonly found in Poaceae mitochondrial genomes as well as three ribosomal and twenty transfer RNA genes. Three major regions of intracellular gene transfer of approximately 2, 10 and 12 kb, respectively were detected. The fragments had their origin in the large single copy and inverted repeat region of the L. perenne chloroplast genome. Analyses for horizontal gene transfer using the mitochondrial genome sequence of the abuscular mycorrhizal fungus Glomus interradices revealed a fragment of approximately 300 bp that showed 76% identity to one region in the L. perenne genome. Further investigation showed that this fragment is part of a large ribosomal subunit in Glomus as well as Lolium. Therefore further studies need to be carried out to reveal if this fragment expresses similarity due to horizontal gene transfer or a homologous function in both genomes. Despite the high level of variation across mitochondrial genomes of different Poaceae subfamilies a 9-kb region consisting of five genes and exons (ccmFN. rps1, matK, nad1 exon 5, nad5 exon3), respectively, could be detected and might prove suitable for phylogenetic and population genetic studies in the future. The protein-coding genes of both organelles proved useful in a phylogenetic study using sequence information of all publicly available chloroplast and mitochondrial Poaceae genomes. Analyses revealed that both genomes contain enough sequence information to distinguish between all Poaceae species, the different Poaceae subfamilies and were also able to reveal the relationship between the Poaceae subfamilies. Neither of the whole genome analyses supported the BEP clade hypothesis for the relationships between grass subfamilies. This result showed how promising future analyses based on a combination of sequence information from both organelles could be. TABLE OF CONTENT TABLE OF CONTENT.................................................................................................... I LIST OF FIGURES ...................................................................................................... VII LIST OF TABLES...........................................................................................................X 1. General introduction to ‘Ryegrass organelle genomes: phylogenomics and sequence evaluation’..............................................................................................1 1.1 The endosymbiotic theory...............................................................................1 1.2 The hydrogen hypothesis.................................................................................4 1.3 Metabolic functions of chloroplasts and mitochondria................................6 1.4 Why have organelle genomes been retained in plants?................................8 1.5 Mitochondrial and chloroplast mutations in plants .....................................9 1.6 Lolium perenne L. ..........................................................................................11 1.7 Aims and objectives .......................................................................................12 2. The chloroplast genome of Lolium perenne L. .......................................................15 2.1 Introduction....................................................................................................15 2.1.1 Discovery of the chloroplast genome .....................................................15