miRNA-like secondary structures in maize (Zea mays) genes and transposable elements correlate with small RNAs, methylation, and expression.

RNA molecules carry information in their primary sequence and also their secondary structure. Secondary structure can confer important functional information, but it is also a signal for an RNAi-like host epigenetic response mediated by small RNAs (smRNAs). In this study, we used two bioinformatic methods to predict local secondary structures across features of the maize genome, focusing on small regions that had similar folding properties to pre-miRNA loci. We found miRNA-like secondary structures to be common in genes and most, but not all, superfamilies of RNA and DNA transposable elements (TEs). The miRNA-like regions map to a higher diversity of smRNAs than regions without miRNA-like structure, explaining up to 27% of variation in smRNA mapping for some TE superfamilies. This mapping bias is more pronounced among putatively autonomous TEs relative to nonautonomous TEs. Genome-wide, miRNA-like regions are also associated with elevated methylation levels, particularly in the CHH context. Among genes, those with miRNA-like secondary structure are 1.5-fold more highly expressed, on average, than other genes. However, these genes are also more variably expressed across the 26 nested association mapping founder lines, and this variability positively correlates with the number of mapping smRNAs. We conclude that local miRNA-like structures are a nearly ubiquitous feature of expressed regions of the maize genome, that they correlate with higher smRNA mapping and methylation, and that they may represent a trade-off between functional requirements and the potentially negative consequences of smRNA production.

Dioecy Is Associated with High Genetic Diversity and Adaptation Rates in the Plant Genus Silene.

About 15,000 angiosperm species (∼6%) have separate sexes, a phenomenon known as dioecy. Why dioecious taxa are so rare is still an open question. Early work reported lower species richness in dioecious compared with nondioecious sister clades, raising the hypothesis that dioecy may be an evolutionary dead-end. This hypothesis has been recently challenged by macroevolutionary analyses that detected no or even positive effect of dioecy on diversification. However, the possible genetic consequences of dioecy at the population level, which could drive the long-term fate of dioecious lineages, have not been tested so far. Here, we used a population genomics approach in the Silene genus to look for possible effects of dioecy, especially for potential evidence of evolutionary handicaps of dioecy underlying the dead-end hypothesis. We collected individual-based RNA-seq data from several populations in 13 closely related species with different sexual systems: seven dioecious, three hermaphroditic, and three gynodioecious species. We show that dioecy is associated with increased genetic diversity, as well as higher selection efficacy both against deleterious mutations and for beneficial mutations. The results hold after controlling for phylogenetic inertia, differences in species census population sizes and geographic ranges. We conclude that dioecious Silene species neither show signs of increased mutational load nor genetic evidence for extinction risk. We discuss these observations in the light of the possible demographic differences between dioecious and self-compatible hermaphroditic species and how this could be related to alternatives to the dead-end hypothesis to explain the rarity of dioecy.

Plant epigenetics: phenotypic and functional diversity beyond the DNA sequence.

Phenotypic variation determines the capacity of plants to adapt to changing environments and to colonize new habitats. Deciphering the mechanisms contributing to plant phenotypic variation and their effects on plant ecological interactions and evolutionary dynamics is thus central to all biological disciplines. In the past few decades, research on plant epigenetics is showing that (1) epigenetic variation is related to phenotypic variation and that some epigenetic marks drive major phenotypic changes in plants; (2) plant epigenomes are highly diverse, dynamic, and can respond rapidly to a variety of biotic and abiotic stimuli; (3) epigenetic variation can respond to selection and therefore play a role in adaptive evolution. Yet, current information in terms of species, geographic ranges, and ecological contexts analyzed so far is too limited to allow for generalizations about the relevance of epigenetic regulation in phenotypic innovation and plant adaptation across taxa. In this report, we contextualize the potential role of the epigenome in plant adaptation to the environment and describe the latest research in this field presented during the symposium "Plant epigenetics: phenotypic and functional diversity beyond the DNA sequence" held within the Botany 2020 conference framework in summer 2020.

Loss of Gene Body Methylation in Eutrema salsugineum Is Associated with Reduced Gene Expression.

Gene body methylation (gbM) is typically characterized by DNA methylation in the CG context within coding regions and is associated with constitutive genes that have moderate to high expression levels. A recent study discovered the loss of gbM in two plant species (Eutrema salsugineum and Conringia planisiliqua), illustrating that gbM is not necessary for survival and reproduction. The same paper stated there was no detectable effect of gbM loss on gene expression (GE). Here, we reanalyzed the GE data and accounted for experimental variability in expression level estimates. We show that the loss of gbM in E. salsugineum is associated with a small but highly significant decrease in GE relative to the closely related species Arabidospis thaliana. Our results are consistent with various evolutionary analyses that suggest gbM has a function, perhaps as a homeostatic effect on GE.

Rapid de novo evolution of X chromosome dosage compensation in Silene latifolia, a plant with young sex chromosomes.

Silene latifolia is a dioecious plant with heteromorphic sex chromosomes that have originated only ∼10 million years ago and is a promising model organism to study sex chromosome evolution in plants. Previous work suggests that S. latifolia XY chromosomes have gradually stopped recombining and the Y chromosome is undergoing degeneration as in animal sex chromosomes. However, this work has been limited by the paucity of sex-linked genes available. Here, we used 35 Gb of RNA-seq data from multiple males (XY) and females (XX) of an S. latifolia inbred line to detect sex-linked SNPs and identified more than 1,700 sex-linked contigs (with X-linked and Y-linked alleles). Analyses using known sex-linked and autosomal genes, together with simulations indicate that these newly identified sex-linked contigs are reliable. Using read numbers, we then estimated expression levels of X-linked and Y-linked alleles in males and found an overall trend of reduced expression of Y-linked alleles, consistent with a widespread ongoing degeneration of the S. latifolia Y chromosome. By comparing expression intensities of X-linked alleles in males and females, we found that X-linked allele expression increases as Y-linked allele expression decreases in males, which makes expression of sex-linked contigs similar in both sexes. This phenomenon is known as dosage compensation and has so far only been observed in evolutionary old animal sex chromosome systems. Our results suggest that dosage compensation has evolved in plants and that it can quickly evolve de novo after the origin of sex chromosomes.

Phased gap-free genome assembly of octoploid cultivated strawberry illustrates the genetic and epigenetic divergence among subgenomes.

The genetic and epigenetic mechanisms underlying the coexistence and coordination of the four diverged subgenomes (ABCD) in octoploid strawberries (Fragaria × ananassa) remains poorly understood. In this study, we have assembled a haplotype-phased gap-free octoploid genome for the strawberry, which allowed us to uncover the sequence, structure, and epigenetic divergences among the subgenomes. The diploid progenitors of the octoploid strawberry, apart from subgenome A (Fragaria vesca), have been a subject of public controversy. Phylogenomic analyses revealed a close relationship between diploid species Fragaria iinumae and subgenomes B, C, and D. Subgenome A, closely related to F. vesca, retains the highest number of genes, exhibits the lowest content of transposable elements (TEs), experiences the strongest purifying selection, shows the lowest DNA methylation levels, and displays the highest expression level compared to the other three subgenomes. Transcriptome and DNA methylome analyses revealed that subgenome A-biased genes were enriched in fruit development biological processes. In contrast, although subgenomes B, C, and D contain equivalent amounts of repetitive sequences, they exhibit diverged methylation levels, particularly for TEs located near genes. Taken together, our findings provide valuable insights into the evolutionary patterns of subgenome structure, divergence and epigenetic dynamics in octoploid strawberries, which could be utilized in strawberry genetics and breeding research.

GC-biased gene conversion and selection affect GC content in the Oryza genus (rice).

Base composition varies among and within eukaryote genomes. Although mutational bias and selection have initially been invoked, more recently GC-biased gene conversion (gBGC) has been proposed to play a central role in shaping nucleotide landscapes, especially in yeast, mammals, and birds. gBGC is a kind of meiotic drive in favor of G and C alleles, associated with recombination. Previous studies have also suggested that gBGC could be at work in grass genomes. However, these studies were carried on third codon positions that can undergo selection on codon usage. As most preferred codons end in G or C in grasses, gBGC and selection can be confounded. Here we investigated further the forces that might drive GC content evolution in the rice genus using both coding and noncoding sequences. We found that recombination rates correlate positively with equilibrium GC content and that selfing species (Oryza sativa and O. glaberrima) have significantly lower equilibrium GC content compared with more outcrossing species. As recombination is less efficient in selfing species, these results suggest that recombination drives GC content. We also detected a positive relationship between expression levels and GC content in third codon positions, suggesting that selection favors codons ending with G or C bases. However, the correlation between GC content and recombination cannot be explained by selection on codon usage alone as it was also observed in noncoding positions. Finally, analyses of polymorphism data ruled out the hypothesis that genomic variation in GC content is due to mutational processes. Our results suggest that both gBGC and selection on codon usage affect GC content in the Oryza genus and likely in other grass species.

Gene Body Methylation in Plants: Mechanisms, Functions, and Important Implications for Understanding Evolutionary Processes.

Gene body methylation (gbM) is an epigenetic mark where gene exons are methylated in the CG context only, as opposed to CHG and CHH contexts (where H stands for A, C, or T). CG methylation is transmitted transgenerationally in plants, opening the possibility that gbM may be shaped by adaptation. This presupposes, however, that gbM has a function that affects phenotype, which has been a topic of debate in the literature. Here, we review our current knowledge of gbM in plants. We start by presenting the well-elucidated mechanisms of plant gbM establishment and maintenance. We then review more controversial topics: the evolution of gbM and the potential selective pressures that act on it. Finally, we discuss the potential functions of gbM that may affect organismal phenotypes: gene expression stabilization and upregulation, inhibition of aberrant transcription (reverse and internal), prevention of aberrant intron retention, and protection against TE insertions. To bolster the review of these topics, we include novel analyses to assess the effect of gbM on transcripts. Overall, a growing body of literature finds that gbM correlates with levels and patterns of gene expression. It is not clear, however, if this is a causal relationship. Altogether, functional work suggests that the effects of gbM, if any, must be relatively small, but there is nonetheless evidence that it is shaped by natural selection. We conclude by discussing the potential adaptive character of gbM and its implications for an updated view of the mechanisms of adaptation in plants.

Dosage compensation evolution in plants: theories, controversies and mechanisms.

In a minority of flowering plants, separate sexes are genetically determined by sex chromosomes. The Y chromosome has a non-recombining region that degenerates, causing a reduced expression of Y genes. In some species, the lower Y expression is accompanied by dosage compensation (DC), a mechanism that re-equalizes male and female expression and/or brings XY male expression back to its ancestral level. Here, we review work on DC in plants, which started as early as the late 1960s with cytological approaches. The use of transcriptomics fired a controversy as to whether DC existed in plants. Further work revealed that various plants exhibit partial DC, including a few species with young and homomorphic sex chromosomes. We are starting to understand the mechanisms responsible for DC in some plants, but in most species, we lack the data to differentiate between global and gene-by-gene DC. Also, it is unknown why some species evolve many dosage compensated genes while others do not. Finally, the forces that drive DC evolution remain mysterious, both in plants and animals. We review the multiple evolutionary theories that have been proposed to explain DC patterns in eukaryotes with XY or ZW sex chromosomes. This article is part of the theme issue 'Sex determination and sex chromosome evolution in land plants'.

Gene body methylation is under selection in Arabidopsis thaliana.

In plants, mammals and insects, some genes are methylated in the CG dinucleotide context, a phenomenon called gene body methylation (gbM). It has been controversial whether this phenomenon has any functional role. Here, we took advantage of the availability of 876 leaf methylomes in Arabidopsis thaliana to characterize the population frequency of methylation at the gene level and to estimate the site-frequency spectrum of allelic states. Using a population genetics model specifically designed for epigenetic data, we found that genes with ancestral gbM are under significant selection to remain methylated. Conversely, ancestrally unmethylated genes were under selection to remain unmethylated. Repeating the analyses at the level of individual cytosines confirmed these results. Estimated selection coefficients were small, on the order of 4 Nes = 1.4, which is similar to the magnitude of selection acting on codon usage. We also estimated that A. thaliana is losing gbM threefold more rapidly than gaining it, which could be due to a recent reduction in the efficacy of selection after a switch to selfing. Finally, we investigated the potential function of gbM through its link with gene expression. Across genes with polymorphic methylation states, the expression of gene body methylated alleles was consistently and significantly higher than unmethylated alleles. Although it is difficult to disentangle genetic from epigenetic effects, our work suggests that gbM has a small but measurable effect on fitness, perhaps due to its association to a phenotype-like gene expression.

Epigenetics drive the evolution of sex chromosomes in animals and plants.

We review how epigenetics affect sex chromosome evolution in animals and plants. In a few species, sex is determined epigenetically through the action of Y-encoded small RNAs. Epigenetics is also responsible for changing the sex of individuals through time, even in species that carry sex chromosomes, and could favour species adaptation through breeding system plasticity. The Y chromosome accumulates repeats that become epigenetically silenced which leads to an epigenetic conflict with the expression of Y genes and could accelerate Y degeneration. Y heterochromatin can be lost through ageing, which activates transposable elements and lowers male longevity. Y chromosome degeneration has led to the evolution of meiotic sex chromosome inactivation in eutherians (placentals) and marsupials, and dosage compensation mechanisms in animals and plants. X-inactivation convergently evolved in eutherians and marsupials via two independently evolved non-coding RNAs. In Drosophila, male X upregulation by the male specific lethal (MSL) complex can spread to neo-X chromosomes through the transposition of transposable elements that carry an MSL-binding motif. We discuss similarities and possible differences between plants and animals and suggest future directions for this dynamic field of research. This article is part of the theme issue 'How does epigenetics influence the course of evolution?'

Gene capture by transposable elements leads to epigenetic conflict in maize.

Transposable elements (TEs) regularly capture fragments of genes. When the host silences these TEs, siRNAs homologous to the captured regions may also target the genes. This epigenetic crosstalk establishes an intragenomic conflict: silencing the TEs has the cost of silencing the genes. If genes are important, however, natural selection may maintain function by moderating the silencing response, which may also advantage the TEs. In this study, we examined this model by focusing on Helitrons, Pack-MULEs, and Sirevirus LTR retrotransposons in the maize genome. We documented 1263 TEs containing exon fragments from 1629 donor genes. Consistent with epigenetic conflict, donor genes mapped more siRNAs and were more methylated than genes with no evidence of capture. However, these patterns differed between syntelog versus translocated donor genes. Syntelogs appeared to maintain function, as measured by gene expression, consistent with moderation of silencing for functionally important genes. Epigenetic marks did not spread beyond their captured regions and 24nt crosstalk siRNAs were linked with CHH methylation. Translocated genes, in contrast, bore the signature of silencing. They were highly methylated and less expressed, but also overrepresented among donor genes and located away from chromosomal arms, which suggests a link between capture and gene movement. Splitting genes into potential functional categories based on evolutionary constraint supported the synteny-based findings. TE families captured genes in different ways, but the evidence for their advantage was generally less obvious; nevertheless, TEs with captured fragments were older, mapped fewer siRNAs, and were slightly less methylated than TEs without captured fragments. Collectively, our results argue that TE capture triggers an intragenomic conflict that may not affect the function of important genes but may lead to the pseudogenization of less-constrained genes.

Chromosome-scale assembly of the genome of Salix dunnii reveals a male-heterogametic sex determination system on chromosome 7.

Sex determination systems in plants can involve either female or male heterogamety (ZW or XY, respectively). Here we used Illumina short reads, Oxford Nanopore Technologies (ONT) long reads and Hi-C reads to assemble the first chromosome-scale genome of a female willow tree (Salix dunnii), and to predict genes using transcriptome sequences and available databases. The final genome sequence of 328 Mb in total was assembled in 29 scaffolds, and includes 31,501 predicted genes. Analyses of short-read sequence data that included female and male plants suggested a male heterogametic sex-determining factor on chromosome 7, implying that, unlike the female heterogamety of most species in the genus Salix, male heterogamety evolved in the subgenus Salix. The S. dunnii sex-linked region occupies about 3.21 Mb of chromosome 7 in females (representing its position in the X chromosome), probably within a pericentromeric region. Our data suggest that this region is enriched for transposable element insertions, and about one-third of its 124 protein-coding genes were gained via duplications from other genome regions. We detect purifying selection on the genes that were ancestrally present in the region, though some have been lost. Transcriptome data from female and male individuals show more male- than female-biased genes in catkin and leaf tissues, and indicate enrichment for male-biased genes in the pseudo-autosomal regions. Our study provides valuable genomic resources for further studies of sex-determining regions in the family Salicaceae, and sex chromosome evolution.

Evidence for Dosage Compensation in Coccinia grandis, a Plant with a Highly Heteromorphic XY System.

About 15,000 angiosperms are dioecious, but the mechanisms of sex determination in plants remain poorly understood. In particular, how Y chromosomes evolve and degenerate, and whether dosage compensation evolves as a response, are matters of debate. Here, we focus on Coccinia grandis, a dioecious cucurbit with the highest level of X/Y heteromorphy recorded so far. We identified sex-linked genes using RNA sequences from a cross and a model-based method termed SEX-DETector. Parents and F1 individuals were genotyped, and the transmission patterns of SNPs were then analyzed. In the >1300 sex-linked genes studied, maximum X-Y divergence was 0.13-0.17, and substantial Y degeneration is implied by an average Y/X expression ratio of 0.63 and an inferred gene loss on the Y of ~40%. We also found reduced Y gene expression being compensated by elevated expression of corresponding genes on the X and an excess of sex-biased genes on the sex chromosomes. Molecular evolution of sex-linked genes in C. grandis is thus comparable to that in Silene latifolia, another dioecious plant with a strongly heteromorphic XY system, and cucurbits are the fourth plant family in which dosage compensation is described, suggesting it might be common in plants.

The genetic basis of sex determination in grapes.

It remains a major challenge to identify the genes and mutations that lead to plant sexual differentiation. Here, we study the structure and evolution of the sex-determining region (SDR) in Vitis species. We report an improved, chromosome-scale Cabernet Sauvignon genome sequence and the phased assembly of nine wild and cultivated grape genomes. By resolving twenty Vitis SDR haplotypes, we compare male, female, and hermaphrodite haplotype structures and identify sex-linked regions. Coupled with gene expression data, we identify a candidate male-sterility mutation in the VviINP1 gene and potential female-sterility function associated with the transcription factor VviYABBY3. Our data suggest that dioecy has been lost during domestication through a rare recombination event between male and female haplotypes. This work significantly advances the understanding of the genetic basis of sex determination in Vitis and provides the information necessary to rapidly identify sex types in grape breeding programs.

Evolution of Young Sex Chromosomes in Two Dioecious Sister Plant Species with Distinct Sex Determination Systems.

In the last decade, progress has been made in methods to identify the sex determination system in plants. This gives the opportunity to study sex chromosomes that arose independently at different phylogenetic scales, and thus allows the discovery and the understanding of early stages of sex chromosome evolution. In the genus Silene, sex chromosomes have evolved independently in at least two clades from a nondioecious ancestor, the Melandrium and Otites sections. In the latter, sex chromosomes could be younger than in the section Melandrium, based on phylogenetic studies and as no heteromorphic sex chromosomes have been detected. This section might also exhibit lability in sex determination, because male heterogamy and female heterogamy have been suggested to occur.In this study, we investigated the sex determination system of two dioecious species in the section Otites (Silene otites and its close relative Silene pseudotites). Applying the new probabilistic method SEX-DETector on RNA-seq data from cross-controlled progenies, we inferred their most likely sex determination system and a list of putative autosomal and sex-linked contigs. We showed that the two phylogenetically close species differed in their sex determination system (XY versus ZW) with sex chromosomes that derived from two different pairs of autosomes. We built a genetic map of the sex chromosomes and showed that both pairs exhibited a large region with lack of recombination. However, the sex-limited chromosomes exhibited no strong degeneration. Finally, using the "ancestral" autosomal expression of sex-linked orthologs of nondioecious S. nutans, we found a slight signature of dosage compensation in the heterogametic females of S. otites.

The genome-wide dynamics of purging during selfing in maize.

Self-fertilization (also known as selfing) is an important reproductive strategy in plants and a widely applied tool for plant genetics and plant breeding. Selfing can lead to inbreeding depression by uncovering recessive deleterious variants, unless these variants are purged by selection. Here we investigated the dynamics of purging in a set of eleven maize lines that were selfed for six generations. We show that heterozygous, putatively deleterious single nucleotide polymorphisms are preferentially lost from the genome during selfing. Deleterious single nucleotide polymorphisms were lost more rapidly in regions of high recombination, presumably because recombination increases the efficacy of selection by uncoupling linked variants. Overall, heterozygosity decreased more slowly than expected, by an estimated 35% to 40% per generation instead of the expected 50%, perhaps reflecting pervasive associative overdominance. Finally, three lines exhibited marked decreases in genome size due to the purging of transposable elements. Genome loss was more likely to occur for lineages that began with larger genomes with more transposable elements and chromosomal knobs. These three lines purged an average of 398 Mb from their genomes, an amount equivalent to three Arabidopsis thaliana genomes per lineage, in only a few generations.

Early Sex-Chromosome Evolution in the Diploid Dioecious Plant Mercurialis annua.

Suppressed recombination allows divergence between homologous sex chromosomes and the functionality of their genes. Here, we reveal patterns of the earliest stages of sex-chromosome evolution in the diploid dioecious herb Mercurialis annua on the basis of cytological analysis, de novo genome assembly and annotation, genetic mapping, exome resequencing of natural populations, and transcriptome analysis. The genome assembly contained 34,105 expressed genes, of which 10,076 were assigned to linkage groups. Genetic mapping and exome resequencing of individuals across the species range both identified the largest linkage group, LG1, as the sex chromosome. Although the sex chromosomes of M. annua are karyotypically homomorphic, we estimate that about one-third of the Y chromosome, containing 568 transcripts and spanning 22.3 cM in the corresponding female map, has ceased recombining. Nevertheless, we found limited evidence for Y-chromosome degeneration in terms of gene loss and pseudogenization, and most X- and Y-linked genes appear to have diverged in the period subsequent to speciation between M. annua and its sister species M. huetii, which shares the same sex-determining region. Taken together, our results suggest that the M. annua Y chromosome has at least two evolutionary strata: a small old stratum shared with M. huetii, and a more recent larger stratum that is probably unique to M. annua and that stopped recombining ∼1 MYA. Patterns of gene expression within the nonrecombining region are consistent with the idea that sexually antagonistic selection may have played a role in favoring suppressed recombination.

Genomic imprinting mediates dosage compensation in a young plant XY system.

Sex chromosomes have repeatedly evolved from a pair of autosomes. Consequently, X and Y chromosomes initially have similar gene content, but ongoing Y degeneration leads to reduced expression and eventual loss of Y genes1. The resulting imbalance in gene expression between Y genes and the rest of the genome is expected to reduce male fitness, especially when protein networks have components from both autosomes and sex chromosomes. A diverse set of dosage compensating mechanisms that alleviates these negative effects has been described in animals2-4. However, the early steps in the evolution of dosage compensation remain unknown, and dosage compensation is poorly understood in plants5. Here, we describe a dosage compensation mechanism in the evolutionarily young XY sex determination system of the plant Silene latifolia. Genomic imprinting results in higher expression from the maternal X chromosome in both males and females. This compensates for reduced Y expression in males, but results in X overexpression in females and may be detrimental. It could represent a transient early stage in the evolution of dosage compensation. Our finding has striking resemblance to the first stage proposed by Ohno6 for the evolution of X inactivation in mammals.