David Manyara: A single nuclei approach to understand genomic organization of arbuscular mycorrhizal fungi

Abstract

Arbuscular mycorrhizal (AM) fungi are obligate plant symbionts that significantly enhance plant nutrient and water uptake in exchange for photosynthetically fixed carbon, playing a crucial role in terrestrial ecosystems. The cellular biology of these fungi is characterized by an aseptate hyphae with thousands of nuclei coexisting within a continuous cytoplasm. Thus, individual nuclei function collectively as a population within AM fungi. The structure and extent of within-organism genetic variation has been a subject of interest in AM fungal research. Additionally, their long-term survival without a single nucleus per cell stage and a cryptic sexual cycle remains puzzling. However, investigating within-organism genetic variation in AM fungi has been challenging due to difficulties in their axenic cultivation as well as obtaining high-quality genome assemblies. With this thesis, I sought to elucidate the genomic organization of AM fungi to deepen our understanding of their important evolutionary drivers. In the first paper, I investigated the capacity to detect intra-organismal genetic variation in published genome assemblies of Rhizophagus irregularis DAOM197198 that were obtained from single nuclei and whole organism sequence datasets. The findings showed that the two datasets exhibited different frequency patterns for discovered variants, and highlighted the methodological challenges associated with detecting low-frequency variants in AM fungal whole genome sequence data. The second paper focused on characterizing the distribution of genetic variation in three strains of two species within the genus Claroideoglomus. Here, the findings revealed low levels of genetic variation within the strains, most of which represent rare variants, with average pN/pS ratios indicating purifying selection. Curiously, some polymorphic sites were shared across both strains and species, and I discuss different models to understand these observations. In the third paper, I explored the relationship between nuclear size, ploidy level, and the genetic organization in three Diversisporales species. During nuclear sorting, we observed nuclear size variations, and I hypothesized that larger nuclei might contain more DNA due to either the merging of different haploid nuclei resulting in diploidy or asynchronized nuclear replication within a spore. Analysis revealed significant assembly size differences in two species. Investigation into the genetic organization based on the putative mating-type (mat) locus showed that most single nuclei contained only one mat allele. However, a substantial structural divergence of the mat locus was noted between species. Finally, in the fourth paper, I evaluated the performance of the workflow used to generate whole genome sequence data from single nuclei in AM fungal species. Through this assessment, I highlight the workflow's effectiveness in generating high-quality genomic data from individual nuclei and underscore the potential of the workflow for advancing AM fungal genomic studies. Overall, this thesis provides insights into the genetic organization of various AM fungal species, enhancing our understanding of within-organism genetic variation in these important plant symbionts.