Detection of rare variants among nuclei populating the arbuscular mycorrhizal fungal model species Rhizophagus irregularis DAOM197198.

Identifying genuine polymorphic variants is a significant challenge in sequence data analysis, although detecting low-frequency variants in sequence data is essential for estimating demographic parameters and investigating genetic processes, such as selection, within populations. Arbuscular mycorrhizal (AM) fungi are multinucleate organisms, in which individual nuclei collectively operate as a population, and the extent of genetic variation across nuclei has long been an area of scientific interest. In this study, we investigated the patterns of polymorphism discovery and the alternate allele frequency distribution by comparing polymorphism discovery in 2 distinct genomic sequence datasets of the AM fungus model species, Rhizophagus irregularis strain DAOM197198. The 2 datasets used in this study are publicly available and were generated either from pooled spores and hyphae or amplified single nuclei from a single spore. We also estimated the intraorganismal variation within the DAOM197198 strain. Our results showed that the 2 datasets exhibited different frequency patterns for discovered variants. The whole-organism dataset showed a distribution spanning low-, intermediate-, and high-frequency variants, whereas the single-nucleus dataset predominantly featured low-frequency variants with smaller proportions in intermediate and high frequencies. Furthermore, single nucleotide polymorphism density estimates within both the whole organism and individual nuclei confirmed the low intraorganismal variation of the DAOM197198 strain and that most variants are rare. Our study highlights the methodological challenges associated with detecting low-frequency variants in AM fungal whole-genome sequence data and demonstrates that alternate alleles can be reliably identified in single nuclei of AM fungi.

Whole genome analyses based on single, field collected spores of the arbuscular mycorrhizal fungus Funneliformis geosporum.

Arbuscular mycorrhizal (AM) fungi are ubiquitous mutualistic symbionts of most terrestrial plants and many complete their lifecycles underground. Whole genome analysis of AM fungi has long been restricted to species and strains that can be maintained under controlled conditions that facilitate collection of biological samples. There is some evidence suggesting that AM fungi can adapt to culture resulting in phenotypic and possibly also genotypic changes in the fungi. In this study, we used field isolated spores of AM fungi and identified them as Funneliformis geosporum based on morphology and phylogenetic analyses. We separately assembled the genomes of two representative spores using DNA sequences of 19 and 22 individually amplified nuclei. The genomes were compared with previously published data from other members of Glomeraceae including two strains of F. mosseae. No significant differences were observed among the species in terms of gene content, while the single nucleotide polymorphism density was higher in the strains of F. geosporum than in the strains of F. mosseae. In this study, we demonstrate that it is possible to sequence and assemble genomes from AM fungal spores sampled in the field, which opens up the possibility to include uncultured AM fungi in phylogenomic and comparative genomic analysis and to study genomic variation in natural populations of these important plant symbionts.