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Abstract

Isolation of single nuclei from multinucleate spores and hyphae of arbuscular mycorrhizal fungi (Glomeromycota) is technically challenging but provides important information about genetic diversity and genome organization. This study aimed to isolate single nuclei from hyphae and spores of Glomus irregulare isolate DAOM 181602, amplify marker genes and get an idea about presence of homokaryons and heterokaryons in arbuscular mycorrhizal fungi. Arbuscular mycorrhizal fungi grown on Laser Capture Microdissection (LCM) slides in bicompartmental Petri plates produced hyphae which showed reduced autofluorescence, enabling excellent visualization and successful catapulting of nuclei. Single nucleus was also successfully isolated from spores by gentle crushing and staining on LCM slides. Isolated nuclei were subjected to multiple-strand displacement amplification (MDA) followed by multiplex PCR targeting nine Glomus irregulare genes. The Binding protein (BiP) gene amplified successfully from approximately 50% of single nuclei, while most other genes showed inconsistent amplification. Cloning and sequencing of BiP from six single nuclei revealed 1-7 allelic variants per nucleus. Direct sequencing traces from additional nuclei confirmed that some nuclei contain single BiP sequences while others harbor multiple variants, providing insights into the genetic organization of this arbuscular mycorrhizal fungus

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