PJB-2025-360
Technical development for the isolation of single nuclei from multinucleate spores and hyphae using Glomus irregulare isolate DAOM 181602
Muhammad Shakeel
Abstract
The isolation of single nuclei from multinucleate spores and the hyphae of arbuscular mycorrhizal fungi (Glomeromycota) are technically challenging, but they provide important information about genetic diversity and genome organization. Thinner hyphae grown across Laser Capture Microdissection (LCM) membrane slides in bicompartmental Petri plates exhibited reduced autofluorescence, enabling excellent visualization and successful catapulting of nuclei. Single nuclei were 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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