Arbuscular mycorrhizal (AM) fungi have been living in association with land plants for at least 400 million years. Because they are obligate symbionts, the study of AM symbiosis has focused primarily on its plant host and progress in the molecular biology of AM fungi has been very slow. Using two different approaches, library screening and direct PCR-based assays, genetic information of AM fungi was compared across isolates and species. This allowed the study of novel DNA regions previously unexplored in AM fungi. The following species were investigated: Glomus intraradices, Glomus mosseae, Gigaspora margarita, Scutellospora calospora, Acaulospora scrobiculata and Entrophosphora colombiana. In the first approach, using in vitro grown G. intraradices, a DNA extraction protocol was developed for the construction of a partial genomic library. This library was screened for the presence of microsatellite-containing loci. PCR primers were designed based on five identified loci. Two of these loci were monomorphic for all isolates and species. The second approach used a combination of degenerate and specific primers for fungal chitin synthase genes to explore the variability of this gene family in AM fungi. A total of 21 AM sequences were isolated and sequenced, covering class I and II chitin synthases. RT-PCR with G. intraradices revealed differential expression of chitin synthases in spores and mycelium, as compared to mycorrhized roots. In addition, using primers designed from a highly conserved sequence for plant resistance genes, classical PCR and RT-PCR allowed the detection of a genomic sequence and its cDNA counterpart encoding a putative serine/lysine rich protein in G. intraradices. We have thus investigated genetic variability in AM fungi in functional genes as well as in repetitive DNA regions. Study of gene expression was also possible using in-vitro grown G. intraradices.

Smith, Donald L. (Supervisor)