Rapid advances in short-read DNA sequencing technologies have revolutionized population genomic studies, but there are genomic regions where this technology reaches its limits. Limitations mostly arise due to the difficulties in assembly or alignment to genomic regions of high sequence divergence and high repeat content, which are typical characteristics for loci under strong long-term balancing selection. Studying genetic diversity at such loci therefore remains challenging. Here, we investigate the feasibility and error rates associated with targeted long-read sequencing of a locus under balancing selection. For this purpose, we generated bacterial artificial chromosomes (BACs) containing the Brassicaceae S-locus, a region under strong negative frequency-dependent selection which has previously proven difficult to assemble in its entirety using short reads. We sequence S-locus BACs with single-molecule long-read sequencing technology and conduct de novo assembly of these S-locus haplotypes. By comparing repeated assemblies resulting from independent long-read sequencing runs on the same BAC clone we do not detect any structural errors, suggesting that reliable assemblies are generated, but we estimate an indel error rate of 5.7x10(-5). A similar error rate was estimated based on comparison of Illumina short-read sequences and BAC assemblies. Our results show that, until de novo assembly of multiple individuals using long-read sequencing becomes feasible, targeted long-read sequencing of loci under balancing selection is a viable option with low error rates for single nucleotide polymorphisms or structural variation. We further find that short-read sequencing is a valuable complement, allowing correction of the relatively high rate of indel errors that result from this approach.

A crucial step in the transition from outcrossing to self-fertilization is the loss of genetic self-incompatibility (SI). In the Brassicaceae, SI involves the interaction of female and male specificity components, encoded by the genes SRK and SCR at the self-incompatibility locus (S-locus). Theory predicts that S-linked mutations, and especially dominant mutations in SCR, are likely to contribute to loss of SI. However, few studies have investigated the contribution of dominant mutations to loss of SI in wild plant species. Here, we investigate the genetic basis of loss of SI in the self-fertilizing crucifer species Capsella orientalis, by combining genetic mapping, long-read sequencing of complete S-haplotypes, gene expression analyses and controlled crosses. We show that loss of SI in C. orientalis occurred S-locus. We identify a fixed frameshift deletion in the male specificity gene SCR and confirm loss of male SI specificity. We further identify an S-linked small RNA that is predicted to cause dominance of self-compatibility. Our results agree with predictions on the contribution of dominant S-linked mutations to loss of SI, and thus provide new insights into the molecular basis of mating system transitions.

The question of whether dominance-recessivity relationships between associated alleles in a diploid genotype can evolve independently from the activity of the gene products encoded has been a hot topic in evolutionary genetics throughout the 20th century. In hermaphroditic plants of the Brassicaceae family, the self-incompatibility locus (S-locus) confers the ability to recognize and reject self-pollen. Dominance relationships between self-incompatibility alleles (S-alleles) in pollen are governed by small RNA (sRNA) transcriptional regulators produced by dominant S-alleles and their target sites on recessive S-alleles. These regulators and their target sites segregate together with but are distinct from the genes encoding self-recognition specificities themselves, providing the opportunity for dominance to evolve independently from the recognition specificities. Dominance interactions between the many segregating S-alleles have been described in the distantly related Arabidopsis and Brassica, but little is known about the evolutionary stability of the dominance networks given that divergent sets of S-alleles are segregating in these two genera. In this study, we take advantage of the extensive trans-specific sharing of S-haplotypes between the self-incompatible species Capsella grandiflora and Arabidopsis halleri to investigate the conservation of S-locus dominance relationships across their approximately 8 million years of divergence. For this purpose, we use a combination of controlled crosses and full-length long-read sequencing of S-haplotypes. We find that the dominance network among six C. grandiflora S-alleles has a largely parallel structure to that among their orthologous S-alleles in A. halleri. We test the theoretical prediction that dominant S-alleles should be found at lower population frequencies using a large sample of a natural C. grandiflora population. Finally, we test whether dominant C. grandiflora S-alleles show increased accumulation of repeats (TEs) than recessive S-alleles, as expected due to their lower chance of recombination and lower effective population sizes. Our results contribute to an improved understanding of the maintenance of dominance relationships at loci under balancing selection.

Polyploidy, or whole-genome duplication, is a common speciation mechanism in plants. An important barrier to polyploid establishment is a lack of compatible mates. Because self-compatibility alleviates this problem, it has long been hypothesized that there should be an association between polyploidy and self-compatibility (SC), but empirical support for this prediction is mixed. Here, we investigate whether the molecular makeup of the Brassicaceae self-incompatibility (SI) system, and specifically dominance relationships among S-haplotypes mediated by small RNAs, could facilitate loss of SI in allopolyploid crucifers. We focus on the allotetraploid species Capsella bursa-pastoris, which formed similar to 300 kya by hybridization and whole-genome duplication involving progenitors from the lineages of Capsella orientalis and Capsella grandiflora. We conduct targeted long-read sequencing to assemble and analyze eight full-length S-locus haplotypes, representing both homeologous subgenomes of C. bursa-pastoris. We further analyze small RNA (sRNA) sequencing data from flower buds to identify candidate dominance modifiers. We find that C. orientalis-derived S-haplotypes of C. bursa-pastoris harbor truncated versions of the male SI specificity gene SCR and express a conserved sRNA-based candidate dominance modifier with a target in the C. grandiflora-derived S-haplotype. These results suggest that pollen-level dominance may have facilitated loss of SI in C. bursa-pastoris. Finally, we demonstrate that spontaneous somatic tetraploidization after a wide cross between C. orientalis and C. grandiflora can result in production of self-compatible tetraploid offspring. We discuss the implications of this finding on the mode of formation of this widespread weed.