Kim A. Steige

The Capsella rubella genome and the genomic consequences of rapid mating system evolution

The shift from outcrossing to selfing is common in flowering plants, but the genomic consequences and the speed at which they emerge remain poorly understood. An excellent model for understanding the evolution of self fertilization is provided by Capsella rubella, which became self compatible <200,000 years ago. We report a C. rubella reference genome sequence and compare RNA expression and polymorphism patterns between C. rubella and its outcrossing progenitor Capsella grandiflora. We found a clear shift in the expression of genes associated with flowering phenotypes, similar to that seen in Arabidopsis, in which self fertilization evolved about 1 million years ago. Comparisons of the two Capsella species showed evidence of rapid genome-wide relaxation of purifying selection in C. rubella without a concomitant change in transposable element abundance. Overall we document that the transition to selfing may be typified by parallel shifts in gene expression, along with a measurable reduction of purifying selection.

Hybrid origins and the earliest stages of diploidization in the highly successful recent polyploid Capsella bursa-pastoris

Significance Plants have undergone repeated rounds of whole-genome duplication, followed by gene degeneration and loss. Using whole-genome resequencing, we examined the origins of the recent tetraploid Capsella bursa-pastoris and the earliest stages of genome evolution after polyploidization. We conclude the species had a hybrid origin from two distinct Capsella lineages within the past 100,000–300,000 y. Our analyses suggest the absence of rapid gene loss but provide evidence that the species has large numbers of inactivating mutations, many of which were inherited from the parental species. Our results suggest that genome evolution following polyploidy is determined not only by genome redundancy but also by demography, the mating system, and the evolutionary history of the parental species. Whole-genome duplication (WGD) events have occurred repeatedly during flowering plant evolution, and there is growing evidence for predictable patterns of gene retention and loss following polyploidization. Despite these important insights, the rate and processes governing the earliest stages of diploidization remain poorly understood, and the relative importance of genetic drift, positive selection, and relaxed purifying selection in the process of gene degeneration and loss is unclear. Here, we conduct whole-genome resequencing in Capsella bursa-pastoris, a recently formed tetraploid with one of the most widespread species distributions of any angiosperm. Whole-genome data provide strong support for recent hybrid origins of the tetraploid species within the past 100,000–300,000 y from two diploid progenitors in the Capsella genus. Major-effect inactivating mutations are frequent, but many were inherited from the parental species and show no evidence of being fixed by positive selection. Despite a lack of large-scale gene loss, we observe a decrease in the efficacy of natural selection genome-wide due to the combined effects of demography, selfing, and genome redundancy from WGD. Our results suggest that the earliest stages of diploidization are associated with quantitative genome-wide decreases in the strength and efficacy of selection rather than rapid gene loss, and that nonfunctionalization can receive a “head start” through a legacy of deleterious variants and differential expression originating in parental diploid populations.

Rapid Evolution of Genomic Imprinting in Two Species of the Brassicaceae

Genomic imprinting is a rapidly evolving phenomenon in plants; however, for those genes where imprinting conveys an advantage, imprinted expression is maintained over long evolutionary timescales. Genomic imprinting is an epigenetic phenomenon occurring in mammals and flowering plants that causes genes to adopt a parent-of-origin-specific mode of expression. While the imprinting status of genes is well conserved in mammals, clear estimates for the degree of conservation were lacking in plants. We therefore analyzed the genome-wide imprinting status of Capsella rubella, which shared a common recent ancestor with Arabidopsis thaliana ∼10 to 14 million years ago. However, only ∼14% of maternally expressed genes (MEGs) and ∼29% of paternally expressed genes (PEGs) in C. rubella were commonly imprinted in both species, revealing that genomic imprinting is a rapidly evolving phenomenon in plants. Nevertheless, conserved PEGs exhibited signs of selection, suggesting that a subset of imprinted genes play an important functional role and are therefore maintained in plants. Like in Arabidopsis, PEGs in C. rubella are frequently associated with the presence of transposable elements that preferentially belong to helitron and MuDR families. Our data further reveal that MEGs and PEGs differ in their targeting by 24-nucleotide small RNAs and asymmetric DNA methylation, suggesting different mechanisms establishing DNA methylation at MEGs and PEGs.

Cis-Regulatory Changes Associated with a Recent Mating System Shift and Floral Adaptation in Capsella

The selfing syndrome constitutes a suite of floral and reproductive trait changes that have evolved repeatedly across many evolutionary lineages in response to the shift to selfing. Convergent evolution of the selfing syndrome suggests that these changes are adaptive, yet our understanding of the detailed molecular genetic basis of the selfing syndrome remains limited. Here, we investigate the role of cis-regulatory changes during the recent evolution of the selfing syndrome in Capsella rubella, which split from the outcrosser Capsella grandiflora less than 200 ka. We assess allele-specific expression (ASE) in leaves and flower buds at a total of 18,452 genes in three interspecific F1 C. grandiflora x C. rubella hybrids. Using a hierarchical Bayesian approach that accounts for technical variation using genomic reads, we find evidence for extensive cis-regulatory changes. On average, 44% of the assayed genes show evidence of ASE; however, only 6% show strong allelic expression biases. Flower buds, but not leaves, show an enrichment of cis-regulatory changes in genomic regions responsible for floral and reproductive trait divergence between C. rubella and C. grandiflora. We further detected an excess of heterozygous transposable element (TE) insertions near genes with ASE, and TE insertions targeted by uniquely mapping 24-nt small RNAs were associated with reduced expression of nearby genes. Our results suggest that cis-regulatory changes have been important during the recent adaptive floral evolution in Capsella and that differences in TE dynamics between selfing and outcrossing species could be important for rapid regulatory divergence in association with mating system shifts.

Genomic analysis reveals major determinants of cis-regulatory variation in Capsella grandiflora

Significance Despite long-standing interest in the contribution of cis-regulatory changes to adaptation, we still have a limited understanding of the selective importance and genomic determinants of cis-regulatory variation in natural populations. We use a combination of analyses of allele-specific expression and population genomic analyses to investigate the selective forces and genomic determinants of cis-regulatory variation in the outcrossing plant species Capsella grandiflora. We conclude that gene-specific functional constraints shape cis-regulatory variation and that genes with cis-regulatory variation are under relaxed purifying selection compared with other genes. Finally, we identify a link between gene body methylation and the extent of cis-regulatory constraint in natural populations. Understanding the causes of cis-regulatory variation is a long-standing aim in evolutionary biology. Although cis-regulatory variation has long been considered important for adaptation, we still have a limited understanding of the selective importance and genomic determinants of standing cis-regulatory variation. To address these questions, we studied the prevalence, genomic determinants, and selective forces shaping cis-regulatory variation in the outcrossing plant Capsella grandiflora. We first identified a set of 1,010 genes with common cis-regulatory variation using analyses of allele-specific expression (ASE). Population genomic analyses of whole-genome sequences from 32 individuals showed that genes with common cis-regulatory variation (i) are under weaker purifying selection and (ii) undergo less frequent positive selection than other genes. We further identified genomic determinants of cis-regulatory variation. Gene body methylation (gbM) was a major factor constraining cis-regulatory variation, whereas presence of nearby transposable elements (TEs) and tissue specificity of expression increased the odds of ASE. Our results suggest that most common cis-regulatory variation in C. grandiflora is under weak purifying selection, and that gene-specific functional constraints are more important for the maintenance of cis-regulatory variation than genome-scale variation in the intensity of selection. Our results agree with previous findings that suggest TE silencing affects nearby gene expression, and provide evidence for a link between gbM and cis-regulatory constraint, possibly reflecting greater dosage sensitivity of body-methylated genes. Given the extensive conservation of gbM in flowering plants, this suggests that gbM could be an important predictor of cis-regulatory variation in a wide range of plant species.

Genomic legacies of the progenitors and the evolutionary consequences of allopolyploidy

The formation of an allopolyploid species involves the merger of genomes with separate evolutionary histories and thereby different genomic legacies. Contrary to expectations from theory, genes from one are often lost preferentially in allopolyploids - there is biased fractionation. Here, we provide an overview of two ways in which the genomic legacies of the progenitors may impact the fate of duplicated genes in allopolyploids. Specifically, we discuss the role of homeolog expression biases in setting the stage for biased fractionation, and the evidence for transposable element silencing as a possible mechanism for homeolog expression biases. Finally, we highlight how differences between the progenitors with respect to accumulation of deleterious variation may affect trajectories of duplicate gene evolution in allopolyploids.

Targeted Long-Read Sequencing of a Locus Under Long-Term Balancing Selection in Capsella

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 5.7×10−5. 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.

Demography and mating system shape the genome-wide impact of purifying selection in Arabis alpina

Significance Intermediate outcrossing rates are theoretically predicted to maintain effective selection against harmful alleles, but few studies have empirically tested this prediction with the use of genomic data. We used whole-genome resequencing data from alpine rock-cress to study how genetic variation and purifying selection vary with mating system. We find that populations with intermediate outcrossing rates have similar levels of genetic diversity as outcrossing populations, and that purifying selection against harmful alleles is efficient in mixed-mating populations. In contrast, self-fertilizing populations from Scandinavia have strongly reduced genetic diversity and accumulate harmful mutations, likely as a result of demographic effects of postglacial colonization. Our results suggest that mixed-mating populations can avoid some of the negative evolutionary consequences of high self-fertilization rates. Plant mating systems have profound effects on levels and structuring of genetic variation and can affect the impact of natural selection. Although theory predicts that intermediate outcrossing rates may allow plants to prevent accumulation of deleterious alleles, few studies have empirically tested this prediction using genomic data. Here, we study the effect of mating system on purifying selection by conducting population-genomic analyses on whole-genome resequencing data from 38 European individuals of the arctic-alpine crucifer Arabis alpina. We find that outcrossing and mixed-mating populations maintain genetic diversity at similar levels, whereas highly self-fertilizing Scandinavian A. alpina show a strong reduction in genetic diversity, most likely as a result of a postglacial colonization bottleneck. We further find evidence for accumulation of genetic load in highly self-fertilizing populations, whereas the genome-wide impact of purifying selection does not differ greatly between mixed-mating and outcrossing populations. Our results demonstrate that intermediate levels of outcrossing may allow efficient selection against harmful alleles, whereas demographic effects can be important for relaxed purifying selection in highly selfing populations. Thus, mating system and demography shape the impact of purifying selection on genomic variation in A. alpina. These results are important for an improved understanding of the evolutionary consequences of mating system variation and the maintenance of mixed-mating strategies.

Analysis of allele-specific expression reveals cis-regulatory changes associated with a recent mating system shift and floral adaptation in Capsella

The selfing syndrome constitutes a suite of floral and reproductive trait changes that have evolved repeatedly across many evolutionary lineages in response to the shift to selfing. Convergent evolution of the selfing syndrome suggests that these changes are adaptive, yet our understanding of the detailed molecular genetic basis of the selfing syndrome remains limited. Here, we investigate the role of cis-regulatory changes during the recent evolution of the selfing syndrome in Capsella rubella, which split from the outcrosser Capsella grandiflora less than 200 kya. We assess allele-specific expression (ASE) in leaves and flower buds at a total of 18,452 genes in three interspecific F1 C. grandiflora x C. rubella hybrids. Using a hierarchical Bayesian approach that accounts for technical variation using genomic reads, we find evidence for extensive cis-regulatory changes. On average, 44% of the assayed genes show evidence of ASE, however only 6% show strong allelic expression biases. Flower buds, but not leaves, show an enrichment of cis-regulatory changes in genomic regions responsible for floral and reproductive trait divergence between C. rubella and C. grandiflora. We further detected an excess of heterozygous transposable element (TE) insertions near genes with ASE, and TE insertions targeted by uniquely mapping 24-nt small RNAs were associated with reduced expression of nearby genes. Our results suggest that cis-regulatory changes have been important during the recent adaptive floral evolution in Capsella and that differences in TE dynamics between selfing and outcrossing species could be important for rapid regulatory divergence in association with mating system shifts.

The impact of natural selection on the distribution of cis-regulatory variation across the genome of an outcrossing plant

Cis-regulatory changes in e.g. promoters or enhancers that affect the expression of a linked focal gene have long been thought to be important for adaptation. In this thesis, I investigate the selective importance and genomic correlates of cis-regulatory variation and divergence in the genus Capsella, using massively parallel sequencing data. This genus provides an opportunity to investigate cis-regulatory changes in response to polyploidization and mating system shifts, as it harbors three diploid species, the outcrosser Capsella grandiflora and the selfers Capsella orientalis and Capsella rubella, as well as the tetraploid Capsella bursa-pastoris. We first identify cis-regulatory changes associated with adaptive floral evolution in connection with the recent switch to self-fertilization in C. rubella and show that cis-regulatory changes between C. rubella and its outcrossing close relative C. grandiflora are associated with differences in transposable element content. Second, we show that variation in positive and purifying selection is important for the distribution of cis-regulatory variation across the genome of C. grandiflora. Interestingly, the presence of polymorphic transposable elements is strongly associated with cis-regulatory variation in C. grandiflora. Third, we show that the tetraploid C. bursa-pastoris is of hybrid origin and investigate the contribution of both parental species to gene expression. We show that gene expression in the tetraploid is partly explained by cis-regulatory divergence between the parental species. Nonetheless, within C. bursa-pastoris there is a great deal of variation in homeolog expression. In summary, this thesis explores the role of cis-regulatory changes for adaptive morphological changes in connection to a shift in mating system, the role of cis-regulatory divergence between progenitor species for an allopolyploid as well as the impact of positive and purifying selection on cis-regulatory variation within a species.Understanding the causes of cis-regulatory variation is a long-standing aim in evolutionary biology. Although cis-regulatory variation has long been considered important for adaptation, we still have a limited understanding of the selective importance and genomic determinants of standing cis-regulatory variation. To address these questions, we studied the prevalence, genomic determinants and selective forces shaping cis-regulatory variation in the outcrossing plant Capsella grandiflora. We first identified a set of 1,010 genes with common cis-regulatory variation using analyses of allele-specific expression (ASE). Population genomic analyses of whole-genome sequences from 32 individuals showed that genes with common cis-regulatory variation are 1) under weaker purifying selection and 2) undergo less frequent positive selection than other genes. We further identified genomic determinants of cis-regulatory variation. Gene-body methylation (gbM) was a major factor constraining cis-regulatory variation, whereas presence of nearby TEs and tissue specificity of expression increased the odds of ASE. Our results suggest that most common cis-regulatory variation in C. grandiflora is under weak purifying selection, and that gene-specific functional constraints are more important for the maintenance of cis-regulatory variation than genome-scale variation in the intensity of selection. Our results agree with previous findings that suggest TE silencing affects nearby gene expression, and provide novel evidence for a link between gbM and cis-regulatory constraint, possibly reflecting greater dosage-sensitivity of body-methylated genes. Given the extensive conservation of gene-body methylation in flowering plants, this suggests that gene-body methylation could be an important predictor of cis-regulatory variation in a wide range of plant species.