Logo: to the web site of Uppsala University

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.

Arbuscular mycorrhizal (AM) fungi form an obligate symbiosis with the roots of the majority of land plants and are found in all terrestrial ecosystems. The source and structure of genetic variation in AM fungi has remained an enigma due to difficulties in the axenic cultivation and generation of high-quality genome assemblies from most species. Furthermore, how AM fungi survive long-term without a known single nucleus per cell stage in their life cycle is puzzling. Purifying selection acting at the nuclear level has been hypothesized as a mechanism to purge deleterious mutations. In this study, we aimed to characterize both intra- and inter-organismal genetic variation in AM fungi by analyzing genomic information from individual nuclei of three strains of two species in the genus Claroideoglomus. We observed overall low levels of genetic variation within the strains, most of which represent rare variants and with average dN/dS ratios within nuclei indicating that these are kept at low frequency by purifying selection. We also observed sites that are maintained as polymorphic across both strains and species, and discuss different models to explain this pattern. The results in this study affirm our conceptual understanding that nuclei in AM fungal strains function as populations of asexually reproducing units with strong signals of purifying selection on nuclei within the strains.

Arbuscular mycorrhizal (AM) fungi are obligate root symbionts that play crucial roles in terrestrial ecosystems by aiding land plants in nutrient and water uptake in exchange for photosynthetically fixed carbon. Their nuclear genomes exhibit considerable variation in size across families, ranging from approximately 40 megabases in Paraglomeraceae to several hundred megabases in Gigasporaceae. Notable variations in nuclear size have also been observed in species within Diversisporales, suggesting that genome size may also vary within species. Three species in this order also stood out in our earlier work with broad taxonomic sampling of AM fungi, by exhibiting a distinct separation of forward-scattered light during nuclei sorting with Fluorescence-Activated Cell Sorting (FACS), indicating that nuclei of two distinct sizes existed within these species. AM fungi are multinucleate with coenocytic hyphae and mating is thought to be controlled by allele variation in the mating-type (mat) locus. Species where all nuclei within the cytoplasm contain only one mat allele are referred to as homokaryotic while strains with nuclei carrying different mat alleles are called heterokaryotic.

Here, we compare genome characteristics of the two nuclear sizes by investigating ploidy level, mat alleles across nuclei, and the SNP density of three species in Diversisporales. Irrespective of nuclei size estimated from FACS sorting, all nuclei were found to be haploid. All three species exhibited low intra-organismal genetic variation, and identical mat allele were found when the mat locus was identified across single nuclei within the three species. We thus conclude that the three strains are homokaryotic for the mating type. Interestingly, we observed substantial structural divergence of the putative mat locus in two of the three species, corroborating a previous report where the HD1-like and HD-2 mat genes were found on different genomic contigs in strains of Gigasporaceae. Together, our analysis suggests that the observed different nuclear sizes in species within Diversisporales are due to asynchronous nuclear division within spores.

Arbuscular mycorrhizal (AM) fungi are essential to terrestrial ecosystems, yet their genomic study is fraught with challenges. Traditional sequencing methods are often limited by difficulties in obtaining pure, high-quality DNA due to the obligate symbiosis of AM fungi with land plants. Here, we explore the previously developed wokflow to sequence AM fungal genomes using single nuclei instead of whole DNA extracts. We present a refined workflow that involves the isolation of spores, sorting of individual nuclei through fluorescence-activated cell sorting (FACS), whole genome amplification (WGA), sequencing, and assembly. We assess variability in nuclear sorting accuracy across different

AM fungal taxa, as well as diversity in genome assembly sizes and completeness across different AM fungal families. This method assessment paves the way for the continuous development towards comprehensive genomic studies of AM fungi without the need for extensive culturing.