| Summary: | The arbuscular mycorrhizal (AM) symbiosis is a mutualistic association between plant roots and fungi that belong to the Phylum Glomeromycota. In this symbiosis, the plant provides the fungus with carbohydrate and gets phosphorus in return. Study of these fungi is fettered because of their obligatory symbiotic nature and lack of basic knowledge of their genetics. These fungi produce multinucleate spores and it is not clear whether nuclei within a spore are genetically similar or dissimilar. In this thesis, we discuss techniques that address some fundamental questions about the organization of genetic material in AM fungi. Sequence variants from coding and non-coding genes have been reported from single Glomus isolates but it is not known whether these arise from different nuclei or constitute multiple gene copies within each nucleus. Several alleles (variants) of Binding protein (Bip) gene were observed after cloning this gene from each of the four isolates of G. irregulare studied (Chapter-2). A Sanger sequencing trace of a sample with a mixture of allelic sequences shows two peaks at single nucleotide polymorphism (SNP) positions. A new approach of measuring peak heights was developed that can monitor changes in allelic frequency of selected genes without involving cloning and RFLP-screening, which are both expensive and time consuming. Using this technique addressed two important questions, first that spores are genetically different in a culture Petri plate within and among G. irregulare isolates (Chapter-3) and second, plant hosts have a significant effect in changing the allele frequency of selected fungal genes (Chapter-4). Single nuclei were isolated by Laser Capture Microdissection from spores and hyphae after optimising the experimental conditions to minimise autofluorescence. Sequences of two marker genes BiP and internal transcribed spacer-2 region (ITS2), were amplified from the whole genome amplified DNA of the catapulted single nuclei using multiplex PCR (Chapter-5). This study reports for first time the isolation of single nuclei from spores and hyphae using LCM. Isolation of single nuclei can provide useful information about the genome size, organization of genetic variation and genetic processes that are poorly understood in AM fungi.
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