Whole-genome sequencing reveals the extent of heterozygosity in a preferentially self-fertilizing hermaphroditic vertebrate.

The mangrove rivulus, Kryptolebias marmoratus, is one of only two self-fertilizing hermaphroditic fish species and inhabits mangrove forests. While selfing can be advantageous, it reduces heterozygosity and decreases genetic diversity. Studies using microsatellites found that there are variable levels of selfing among populations of K. marmoratus, but overall, there is a low rate of outcrossing and, therefore, low heterozygosity. In this study, we used whole-genome data to assess the levels of heterozygosity in different lineages of the mangrove rivulus and infer the phylogenetic relationships among those lineages. We sequenced whole genomes from 15 lineages that were completely homozygous at microsatellite loci and used single nucleotide polymorphisms (SNPs) to determine heterozygosity levels. More variation was uncovered than in studies using microsatellite data because of the resolution of full genome sequencing data. Moreover, missense polymorphisms were found most often in genes associated with immune function and reproduction. Inferred phylogenetic relationships suggest that lineages largely group by their geographic distribution. The use of whole-genome data provided further insight into genetic diversity in this unique species. Although this study was limited by the number of lineages that were available, these data suggest that there is previously undescribed variation within lineages of K. marmoratus that could have functional consequences and (or) inform us about the limits to selfing (e.g., genetic load, accumulation of deleterious mutations) and selection that might favor the maintenance of heterozygosity. These results highlight the need to sequence additional individuals within and among lineages.

Use of microsatellite markers for the assessment of bambara groundnut breeding system and varietal purity before genome sequencing.

Maximizing the research output from a limited investment is often the major challenge for minor and underutilized crops. However, such crops may be tolerant to biotic and abiotic stresses and are adapted to local, marginal, and low-input environments. Their development through breeding will provide an important resource for future agricultural system resilience and diversification in the context of changing climates and the need to achieve food security. The African Orphan Crops Consortium recognizes the values of genomic resources in facilitating the improvement of such crops. Prior to beginning genome sequencing there is a need for an assessment of line varietal purity and to estimate any residual heterozygosity. Here we present an example from bambara groundnut (Vigna subterranea (L.) Verdc.), an underutilized drought tolerant African legume. Two released varieties from Zimbabwe, identified as potential genotypes for whole genome sequencing (WGS), were genotyped with 20 species-specific SSR markers. The results indicate that the cultivars are actually a mix of related inbred genotypes, and the analysis allowed a strategy of single plant selection to be used to generate non-heterogeneous DNA for WGS. The markers also confirmed very low levels of heterozygosity within individual plants. The application of a pre-screen using co-dominant microsatellite markers is expected to substantially improve the genome assembly, compared to a cultivar bulking approach that could have been adopted.

Evidence for genetic incompatibilities associated with post-zygotic reproductive isolation in the human fungal pathogen Cryptococcus neoformans.

Hybridization is a potent mechanism for generating unique strains with broad host ranges and increased virulence in fungal pathogens. In the opportunistic basidiomycete pathogen Cryptococcus neoformans, intervarietal hybrids are commonly found infecting patients. The two parental varieties C. neoformans var. grubii and C. neoformans var. neoformans mate readily under laboratory conditions, but the hybrid basidiospores have germination rates about four times lower than those from intravarietal crosses. Here, we used microdissection to collect basidiospores from a hybrid cross and analysed the genotypes of germinated basidiospores to identify potentially antagonistic allelic combinations between loci that impact basidiospore germination. Our analyses showed clear evidence for Bateson-Dobzhansky-Muller (BDM) incompatibility affecting basidiospore viability. Antagonistic combinations of alleles from both two loci and three loci were found. Interestingly, most of the hybrid progeny showed segregation distortion in favour of the alleles from var. neoformans, consistent with large-scale epistatic interactions among loci affecting basidiospore viability. Our study presents the first evidence of BDM incompatibility between nuclear genes affecting post-zygotic reproductive isolation in this model basidiomycete yeast.