Optimization of a high-throughput single spore DNA extraction protocol for the model species of arbuscular mycorrhizal fungi, Rhizophagus irregularis.

Single spore DNA extraction methods of arbuscular mycorrhizal fungi (AMF) typically involve manually crushing spores to release the DNA into solution. However, there are no consistent protocols for DNA extraction from single spores, nor methods for extracting DNA from many single spores at once. We tested several single spore DNA extraction methods on four isolates of the species Rhizophagus irregularis in 96-well plates: direct polymerase chain reaction (PCR), freeze-thaw, manually crushing, bead beating, and chemical lysis. The extracted DNA was amplified via PCR and visualized using gel electrophoresis. We evaluated each method based on cost, time efficiency, preparation effort, safety, ease, and number of spores with successful DNA extraction and amplification. Overall, we found that crushing spores using a sewing needle had the greatest success for all four isolates and can be easily conducted in 250µL 96-well plates. The direct PCR and freeze-thaw methods show promise of being practical high-throughput methods for single spore DNA extraction; however, they should be further tested on other isolates and species to account for both intra and interspecies variability.