Genome structure and diversity in the perennial ryegrass (Lolium perenne L.) fungal endophyte Neotyphodium lolii

Submission note: A thesis submitted in total fulfilment of the requirements for the degree of Doctor of Philosophy to the Department of Botany, School of Life Sciences, Faculty of Science, Technology and Engineering, La Trobe University, Bundoora.

The prevalent fungal endophyte of Lolium perenne L., Neotyphodium lolii (Clavicipitaceae), protects the host grass against biotic and abiotic stresses. Enhanced resistance to herbivory is due to the production of fungal alkaloids, which are toxic to invertebrate and vertebrate animals. While peramine, ergot alkaloids and lolitrem alkaloids are the major classes of toxins produced by N. lolii, the alkaloid profile has been shown to differ between strains. The most common N. lolii strain found in association with L. perenne, Standard Toxic (ST), was subjected to genome sequencing. A reference N. lolii nuclear genome sequence was generated (strain ST), which consisted of 7,875 contigs (> 500 bp; N50, 7.2 kb) with a total size of 31.8 Mb. In addition, another five genetically distinct N. lolii strains with differing alkaloid profiles were selected and subjected to Genome Survey Sequencing to examine intraspecific genome sequence variation. Comparisons with genomic regions containing gene clusters involved in toxin production, demonstrated tagging of all the genes present within the regions. This analysis also demonstrated that gene loss could be a cause of some of the differences between toxin profiles among the different endophyte strains. Availability of the 31 Mb genome sequence of the sexual relative Epichloë festucae permitted analysis of genome wide differences in DNA content between N. lolii (ST) and E. festucae (E2368). In addition to the nuclear genome, the complete mitochondrial DNA (mtDNA) sequence (88 kb) was determined for each N. lolii strain and analysed by comparison to the highly compact mtDNA (25 kb) of the clavicipitaceous fungus Metarhizium anisopliae. This analysis revealed considerable conservation of mitochondrial gene order and content between both related organellar genomes, but a remarkable difference in intron content.