To avoid negative effects of transposable element (TE) proliferation, plants epigenetically silence TEs using a number of mechanisms, including RNA-directed DNA methylation. These epigenetic modifications can extend outside the boundaries of TE insertions and lead to silencing of nearby genes, resulting in a trade-off between TE silencing and interference with nearby gene regulation. Therefore, purifying selection is expected to remove silenced TE insertions near genes more efficiently and prevent their accumulation within a population. To explore how effects of TE silencing on gene regulation shapes purifying selection on TEs, we analyzed whole genome sequencing data from 166 individuals of a large population of the outcrossing species Capsella grandiflora. We found that most TEs are rare, and in chromosome arms, silenced TEs are exposed to stronger purifying selection than those that are not silenced by 24-nucleotide small RNAs, especially with increasing proximity to genes. An age-of-allele test of neutrality on a subset of TEs supports our inference of purifying selection on silenced TEs, suggesting that our results are robust to varying transposition rates. Our results provide new insights into the processes affecting the accumulation of TEs in an outcrossing species and support the view that epigenetic silencing of TEs results in a trade-off between preventing TE proliferation and interference with nearby gene regulation. We also suggest that in the centromeric and pericentromeric regions, the negative aspects of epigenetic TE silencing are missing.

Gene-body methylation (gbM) refers to an increased level of methylated cytosines specifically in a CG sequence context within genes. gbM is found in plant genes with intermediate expression level, which evolve slowly, and is often broadly conserved across millions of years of evolution. Intriguingly however, some plants lack gbM, and thus it remains unclear whether gbM has a function. In animals, there is support for a role of gbM in reducing erroneous transcription and transcription noise, but so far most studies in plants have tested for an effect of gbM on expression level, not noise. Here, we therefore tested whether gbM was associated with reduced expression noise in Arabidopsis thaliana, using single-cell transcriptome sequencing data from root quiescent centre cells. We find that gbM genes have lower expression noise levels than unmethylated genes. However, an analysis of covariance revealed that, if other genomic features are taken into account, this association disappears. Nonetheless, gbM genes were more consistently expressed across single-cell samples, supporting previous inference that gbM genes are constitutively expressed. Finally, we observed that fewer RNAseq reads map to introns of gbM genes than to introns of unmethylated genes, which indicates that gbM might be involved in reducing erroneous transcription by reducing intron retention.

Genetic diversity is shaped by mutation, genetic drift, gene flow, recombination, and selection. The dynamics and interactions of these forces shape genetic diversity across different parts of the genome, between populations and species. Here, we have studied the effects of linked selection on nucleotide diversity in outcrossing populations of two Brassicaceae species, Arabidopsis lyrata and Capsella grandiflora, with contrasting demographic history. In agreement with previous estimates, we found evidence for a modest population size expansion thousands of generations ago, as well as efficient purifying selection in C. grandiflora. In contrast, the A. lyrata population exhibited evidence for very recent strong population size decline and weaker efficacy of purifying selection. Using multiple regression analyses with recombination rate and other genomic covariates as explanatory variables, we can explain 47% of the variance in neutral diversity in the C. grandiflora population, while in the A. lyrata population, only 11% of the variance was explained by the model. Recombination rate had a significant positive effect on neutral diversity in both species, suggesting that selection at linked sites has an effect on patterns of neutral variation. In line with this finding, we also found reduced neutral diversity in the vicinity of genes in the C. grandiflora population. However, in A. lyrata no such reduction in diversity was evident, a finding that is consistent with expectations of the impact of a recent bottleneck on patterns of neutral diversity near genes. This study thus empirically demonstrates how differences in demographic history modulate the impact of selection at linked sites in natural populations.