Dioecious Plant Silene Latifolia Research Articles

Non-canonical bases differentially represented in the sex chromosomes of the dioecious plant Silene latifolia.

The oxidation of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC), known as oxi-mCs, garners significant interest in plants as potential epigenetic marks. While research in mammals has established a role in cell reprogramming, carcinogenesis, and gene regulation, their functions in plants remain unclear. In rice, 5hmC has been associated with transposable elements (TEs) and heterochromatin. This study utilizes Silene latifolia, a dioecious plant with heteromorphic sex chromosomes and a genome with a large proportion of TEs, which provides a favourable environment for the study of oxi-mCs in individual sexes. Notably, we detected surprisingly high levels of oxi-mCs in S. latifolia comparable with mammals. Nuclei showed enrichment in heterochromatic regions, except for 5hmC whose signal was homogeneously distributed. Intriguingly, the same X chromosome in females displayed overall enrichment of 5hmC and 5fC compared with its counterpart. This fact is shared with 5mC, resembling dosage compensation. Co-localization showed higher correlation between 5mC and 5fC than with 5hmC, indicating no potential relationship between 5hmC and 5fC. Additionally, the promoter of several sex-linked genes and sex-biased TEs clustered in a clear sex-dependent way. Together, these findings unveil a hypothetical role for oxi-mCs in S. latifolia sex chromosome development, warranting further exploration.

Heterochiasmy and Sex Chromosome Evolution in Silene

The evolution of a non-recombining sex-specific region is a key step in sex chromosome evolution. Suppression of recombination between the (proto-) X- and Y-chromosomes in male meiosis creates a non-recombining Y-linked region (NRY), while the X-chromosome continues to recombine in females. Lack of recombination in the NRY defines its main properties-genetic degeneration and accumulation of repetitive DNA, making X and Y chromosomes very different from each other. How and why recombination suppression on sex chromosomes evolves remains controversial. A strong difference in recombination rates between the sexes (heterochiasmy) can facilitate or even cause recombination suppression. In the extreme case-complete lack of recombination in the heterogametic sex (achiasmy)-the entire sex-specific chromosome is automatically non-recombining. In this study, I analyse sex-specific recombination rates in a dioecious plant Silene latifolia (Caryophyllaceae), which evolved separate sexes and sex chromosomes ~11 million years ago. I reconstruct high-density RNAseq-based genetic maps including over five thousand genic markers for the two sexes separately. The comparison of the male and female maps reveals only modest heterochiasmy across the genome, with the exception of the sex chromosomes, where recombination is suppressed in males. This indicates that heterochiasmy likely played only a minor, if any, role in NRY evolution in S. latifolia, as recombination suppression is specific to NRY rather than to the entire genome in males. Other mechanisms such as structural rearrangements and/or epigenetic modifications were likely involved, and comparative genome analysis and genetic mapping in multiple Silene species will help to shed light on the mechanism(s) of recombination suppression that led to the evolution of sex chromosomes.

Recent expansion of the non-recombining sex-linked region on Silene latifolia sex chromosomes.

Evolution of a non-recombining sex-specific region on the Y (or W) chromosome (NRY) is a key step in sex chromosome evolution, but how recombination suppression evolves is not well understood. Studies in many different organisms indicated that NRY evolution often involves several expansion steps. Why such NRY expansions occur remains unclear, although it is though that they are likely driven by sexually antagonistic selection. This paper describes a recent NRY expansion due to shift of the pseudoautosomal boundary on the sex chromosomes of a dioecious plant Silene latifolia. The shift resulted in inclusion of at least 16 pseudoautosomal genes into the NRY. This region is pseudoautosomal in closely related Silene dioica and Silene diclinis, indicating that the NRY expansion occurred in S. latifolia after it speciated from the other species ~120 thousand years ago. As S. latifolia and S. dioica actively hybridise across Europe, interspecific gene flow could blur the PAR boundary in these species. The pseudoautosomal genes have significantly elevated genetic diversity (π ~ 3% at synonymous sites), which is consistent with balancing selection maintaining diversity in this region. The recent shift of the PAR boundary in S. latifolia offers an opportunity to study the process of on-going NRY expansion.

Water availability drives population divergence and sex-specific responses in a dioecious plant.

Water availability is an important abiotic factor, resulting in differences between plant species growing in xeric and mesic habitats. Species with populations occurring in both habitat types allow examination of whether water availability has acted as a selective force at the intraspecific level. Investigating responses to water availability with a dioecious species allows determination of whether males and females, which often have different physiologies and life histories, respond differently. An experiment varying water availability was performed under an outdoor rain-out shelter using plants from two mesic and two xeric populations of the dioecious plant Silene latifolia. Early growth rate, flowering propensity, flower size, and specific leaf area were measured. At the end of the season, the plants were harvested, aboveground and root biomass were measured, and the total number of flowers and fruit produced were counted. Compared to the two mesic populations, plants from the two xeric populations grew more slowly, were less likely to flower, took longer to flower, had thicker leaves, invested less in aboveground biomass and more in root biomass, produced fewer flowers and fruit, but were more likely to live. Many traits exhibited significant habitat type × treatment interactions. Compared to the xeric populations, males-but not females-from mesic populations had less root biomass and greatly reduced their flower production in response to low water availability. Mesic and xeric populations responded in ways congruent with water availability being a selective force for among-population divergence, especially for males.

Development of the VIGS System in the Dioecious Plant Silene latifolia.

(1) Background: Silene latifolia is a dioecious plant, whose sex is determined by XY-type sex chromosomes. Microbotryum lychnidis-dioicae is a smut fungus that infects S. latifolia plants and causes masculinization in female flowers, as if Microbotryum were acting as a sex-determining gene. Recent large-scale sequencing efforts have promised to provide candidate genes that are involved in the sex determination machinery in plants. These candidate genes are to be analyzed for functional characterization. A virus vector can be a tool for functional gene analyses; (2) Methods: To develop a viral vector system in S. latifolia plants, we selected Apple latent spherical virus (ALSV) as an appropriate virus vector that has a wide host range; (3) Results: Following the optimization of the ALSV inoculation method, S. latifolia plants were infected with ALSV at high rates in the upper leaves. In situ hybridization analysis revealed that ALSV can migrate into the flower meristems in S. latifolia plants. Successful VIGS (virus-induced gene silencing) in S. latifolia plants was demonstrated with knockdown of the phytoene desaturase gene. Finally, the developed method was applied to floral organ genes to evaluate its usability in flowers; (4) Conclusion: The developed system enables functional gene analyses in S. latifolia plants, which can unveil gene functions and networks of S. latifolia plants, such as the mechanisms of sex determination and fungal-induced masculinization.

DNA methylation and genetic degeneration of the Y chromosome in the dioecious plant Silene latifolia

BackgroundS. latifolia is a model organism for the study of sex chromosome evolution in plants. Its sex chromosomes include large regions in which recombination became gradually suppressed. The regions tend to expand over time resulting in the formation of evolutionary strata. Non-recombination and later accumulation of repetitive sequences is a putative cause of the size increase in the Y chromosome. Gene decay and accumulation of repetitive DNA are identified as key evolutionary events. Transposons in the X and Y chromosomes are distributed differently and there is a regulation of transposon insertion by DNA methylation of the target sequences, this points to an important role of DNA methylation during sex chromosome evolution in Silene latifolia. The aim of this study was to elucidate whether the reduced expression of the Y allele in S. latifolia is caused by genetic degeneration or if the cause is methylation triggered by transposons and repetitive sequences.ResultsGene expression analysis in S. latifolia males has shown expression bias in both X and Y alleles. To determine whether these differences are caused by genetic degeneration or methylation spread by transposons and repetitive sequences, we selected several sex-linked genes with varying degrees of degeneration and from different evolutionary strata. Immunoprecipitation of methylated DNA (MeDIP) from promoter, exon and intron regions was used and validated through bisulfite sequencing. We found DNA methylation in males, and only in the promoter of genes of stratum I (older). The Y alleles in genes of stratum I were methylation enriched compared to X alleles. There was also abundant and high percentage methylation in the CHH context in most sequences, indicating de novo methylation through the RdDM pathway.ConclusionsWe speculate that TE accumulation and not gene decay is the cause of DNA methylation in the S. latifolia Y sex chromosome with influence on the process of heterochromatinization.

The slowdown of Y chromosome expansion in dioecious Silene latifolia due to DNA loss and male-specific silencing of retrotransposons

BackgroundThe rise and fall of the Y chromosome was demonstrated in animals but plants often possess the large evolutionarily young Y chromosome that is thought has expanded recently. Break-even points dividing expansion and shrinkage phase of plant Y chromosome evolution are still to be determined. To assess the size dynamics of the Y chromosome, we studied intraspecific genome size variation and genome composition of male and female individuals in a dioecious plant Silene latifolia, a well-established model for sex-chromosomes evolution.ResultsOur genome size data are the first to demonstrate that regardless of intraspecific genome size variation, Y chromosome has retained its size in S. latifolia. Bioinformatics study of genome composition showed that constancy of Y chromosome size was caused by Y chromosome DNA loss and the female-specific proliferation of recently active dominant retrotransposons. We show that several families of retrotransposons have contributed to genome size variation but not to Y chromosome size change.ConclusionsOur results suggest that the large Y chromosome of S. latifolia has slowed down or stopped its expansion. Female-specific proliferation of retrotransposons, enlarging the genome with exception of the Y chromosome, was probably caused by silencing of highly active retrotransposons in males and represents an adaptive mechanism to suppress degenerative processes in the haploid stage. Sex specific silencing of transposons might be widespread in plants but hidden in traditional hermaphroditic model plants.

A new physical mapping approach refines the sex-determining gene positions on the Silene latifolia Y-chromosome.

Sex chromosomes are particularly interesting regions of the genome for both molecular genetics and evolutionary studies; yet, for most species, we lack basic information, such as the gene order along the chromosome. Because they lack recombination, Y-linked genes cannot be mapped genetically, leaving physical mapping as the only option for establishing the extent of synteny and homology with the X chromosome. Here, we developed a novel and general method for deletion mapping of non-recombining regions by solving “the travelling salesman problem”, and evaluate its accuracy using simulated datasets. Unlike the existing radiation hybrid approach, this method allows us to combine deletion mutants from different experiments and sources. We applied our method to a set of newly generated deletion mutants in the dioecious plant Silene latifolia and refined the locations of the sex-determining loci on its Y chromosome map.

Fungal Infection Induces Sex-Specific Transcriptional Changes and Alters Sexual Dimorphism in the Dioecious Plant Silene latifolia.

Sexual dimorphism, including differences in morphology, behavior and physiology between females and males, is widespread in animals and plants and is shaped by gene expression differences between the sexes. Such expression differences may also underlie sex-specific responses of hosts to pathogen infections, most notably when pathogens induce partial sex reversal in infected hosts. The genetic changes associated with sex-specific responses to pathogen infections on the one hand, and sexual dimorphism on the other hand, remain poorly understood. The dioecious White Campion (Silene latifolia) displays sexual dimorphism in floral traits and infection with the smut fungus Micobrotryum lychnidis-dioicae induces a partial sex reversal in females. We find strong sex-specific responses to pathogen infection and reduced sexual dimorphism in infected S. latifolia. This provides a direct link between pathogen-mediated changes in sex-biased gene expression and altered sexual dimorphism in the host. Expression changes following infection affected mainly genes with male-biased expression in healthy plants. In females, these genes were up-regulated, leading to a masculinization of the transcriptome. In contrast, infection in males was associated with down-regulation of these genes, leading to a demasculinization of the transcriptome. To a lesser extent, genes with female-biased expression in healthy plants were also affected in opposite directions in the two sexes. These genes were overall down-regulated in females and up-regulated in males, causing, respectively, a defeminization in infected females and a feminization of the transcriptome in infected males. Our results reveal strong sex-specific responses to pathogen infection in a dioecious plant and provide a link between pathogen-induced changes in sex-biased gene expression and sexual dimorphism.

Identifying new sex-linked genes through BAC sequencing in the dioecious plant Silene latifolia.

BackgroundSilene latifolia represents one of the best-studied plant sex chromosome systems. A new approach using RNA-seq data has recently identified hundreds of new sex-linked genes in this species. However, this approach is expected to miss genes that are either not expressed or are expressed at low levels in the tissue(s) used for RNA-seq. Therefore other independent approaches are needed to discover such sex-linked genes.ResultsHere we used 10 well-characterized S. latifolia sex-linked genes and their homologs in Silene vulgaris, a species without sex chromosomes, to screen BAC libraries of both species. We isolated and sequenced 4 Mb of BAC clones of S. latifolia X and Y and S. vulgaris genomic regions, which yielded 59 new sex-linked genes (with S. vulgaris homologs for some of them). We assembled sequences that we believe represent the tip of the Xq arm. These sequences are clearly not pseudoautosomal, so we infer that the S. latifolia X has a single pseudoautosomal region (PAR) on the Xp arm. The estimated mean gene density in X BACs is 2.2 times lower than that in S. vulgaris BACs, agreeing with the genome size difference between these species. Gene density was estimated to be extremely low in the Y BAC clones. We compared our BAC-located genes with the sex-linked genes identified in previous RNA-seq studies, and found that about half of them (those with low expression in flower buds) were not identified as sex-linked in previous RNA-seq studies. We compiled a set of ~70 validated X/Y genes and X-hemizygous genes (without Y copies) from the literature, and used these genes to show that X-hemizygous genes have a higher probability of being undetected by the RNA-seq approach, compared with X/Y genes; we used this to estimate that about 30 % of our BAC-located genes must be X-hemizygous. The estimate is similar when we use BAC-located genes that have S. vulgaris homologs, which excludes genes that were gained by the X chromosome.ConclusionsOur BAC sequencing identified 59 new sex-linked genes, and our analysis of these BAC-located genes, in combination with RNA-seq data suggests that gene losses from the S. latifolia Y chromosome could be as high as 30 %, higher than previous estimates of 10-20 %.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1698-7) contains supplementary material, which is available to authorized users.

Identification of Internal Reference Genes for Gene Expression Normalization between the Two Sexes in Dioecious White Campion

Quantitative real time (qRT)-PCR is a precise and efficient method for studying gene expression changes between two states of interest, and is frequently used for validating interesting gene expression patterns in candidate genes initially identified in genome-wide expression analyses, such as RNA-seq experiments. For an adequate normalisation of qRT-PCR data, it is essential to have reference genes available whose expression intensities are constant among the different states of interest. In this study we present and validate a catalogue of traditional and newly identified reference genes that were selected from RNA-seq data from multiple individuals from the dioecious plant Silene latifolia with the aim of studying gene expression differences between the two sexes in both reproductive and vegetative tissues. The catalogue contains more than 15 reference genes with both stable expression intensities and a range of expression intensities in flower buds and leaf tissues. These reference genes were used to normalize expression differences between reproductive and vegetative tissues in eight candidate genes with sex-biased expression. Our results suggest a trend towards a reduced sex-bias in sex-linked gene expression in vegetative tissues. In this study, we report on the systematic identification and validation of internal reference genes for adequate normalization of qRT-PCR-based analyses of gene expression differences between the two sexes in S. latifolia. We also show how RNA-seq data can be used efficiently to identify suitable reference genes in a wide diversity of species.

Expansion of the pseudo-autosomal region and ongoing recombination suppression in the Silene latifolia sex chromosomes.

There are two very interesting aspects to the evolution of sex chromosomes: what happens after recombination between these chromosome pairs stops and why suppressed recombination evolves. The former question has been intensively studied in a diversity of organisms, but the latter has been studied largely theoretically. To obtain empirical data, we used codominant genic markers in genetic mapping of the dioecious plant Silene latifolia, together with comparative mapping of S. latifolia sex-linked genes in S. vulgaris (a related hermaphrodite species without sex chromosomes). We mapped 29 S. latifolia fully sex-linked genes (including 21 newly discovered from transcriptome sequencing), plus 6 genes in a recombining pseudo-autosomal region (PAR) whose genetic map length is ∼25 cM in both male and female meiosis, suggesting that the PAR may contain many genes. Our comparative mapping shows that most fully sex-linked genes in S. latifolia are located on a single S. vulgaris linkage group and were probably inherited from a single autosome of an ancestor. However, unexpectedly, our maps suggest that the S. latifolia PAR region expanded through translocation events. Some genes in these regions still recombine in S. latifolia, but some genes from both addition events are now fully sex-linked. Recombination suppression is therefore still ongoing in S. latifolia, and multiple recombination suppression events have occurred in a timescale of few million years, much shorter than the timescale of formation of the most recent evolutionary strata of mammal and bird sex chromosomes.

SlWUS1; An X-linked Gene Having No Homologous Y-Linked Copy inSilene latifolia

The dioecious plant Silene latifolia has heteromorphic sex chromosomes, and comparison of the positions of sex-linked genes indicates that at least three large inversions have occurred during the evolution of the Y chromosome. In this article, we describe the isolation of a new sex-linked gene from S. latifolia, which provides new information on the evolution of this plant’s young sex chromosomes. By using reverse-transcription polymerase chain reaction degenerate primers based on the Arabidopsis thaliana sequence of WUSCHEL, a flower-development gene, we found two copies in S. latifolia, which we named SlWUS1 and SlWUS2. Southern blot and genetic segregation analysis showed that SlWUS1 is located on the X chromosome and SlWUS2 is autosomal. No Y-linked copy of SlWUS1 was found by either Southern blot analysis under low-stringency conditions or polymerase chain reaction with degenerate primers, so we conclude that SlWUS1 probably has no Y-linked homolog. It is unknown whether the Y chromosome lost the SlWUS1 copy by degeneration of this individual gene or whether deletion of a larger genome region was involved. Several tests lead us to conclude that dosage compensation has not evolved for this sex-linked gene. We mapped the ortholog in the nondioecious relative S. vulgaris (SvWUS1), to compare the location in a species that has no history of having sex chromosomes. SvWUS1 maps to the same linkage group as other fully X-linked genes, indicating that it was not added to the X, but was lost from the Y. Its location differs in the maps from the two species, raising the possibility that the X chromosome, as well as the Y, may have been rearranged.

Maturation timing of stamens and pistils in the dioecious plant Silene latifolia

The dioecious plant Silene latifolia depends on nocturnal insects for pollination. To increase the chance of cross-pollination, pollen grains seem to be released and stigmas seem to be receptive simultaneously at night. We divided the floral development of S. latifolia into 1-20 stages, and determined the timetables of male and female function. The corolla of both male and female flowers opens at sunset (1900hours) and closes at sunrise (0900hours). To investigate the period of the reproductive phase of male and female function, we measured the germination rate on a pollen medium and the pollen germination rate on stigma during the period when stamens and stigmas were viable in the timetable. Male flowers had early- and late-maturing stamens that had the highest pollen viability, germination rate and pollen tube growth at midnight (0000hours) at 1day after flowering (DAF) and 0000hours at 2 DAF. In contrast, female flowers maintained a germination rate of nearly 100% from 1800hours at 1 DAF to 1200hours at 3 DAF. These results suggested that S. latifolia transferred the matured pollen grains from male flowers to female flowers only at night.

Frequency and genomic distribution of genes with sex-biased expression in the dioecious plant Silene latifolia

Frequency and genomic distribution of genes with sex-biased expression in the dioecious plant Silene latifolia

The contribution of a pollinating seed predator to selection onSilene latifoliafemales

Interactions, antagonistic or mutualistic, can exert selection on plant traits. We explored the role of Hadena bicruris, a pollinating seed predator, as a selective agent on its host, the dioecious plant Silene latifolia. We exposed females from artificial-selection lines (many, small flowers (SF) vs. few, large flowers (LF)) to this moth. Infestation did not differ significantly between lines, but the odds of attacked fruits aborting were higher in SF females. We partitioned selection between that caused by moth attack and that resulting from all other factors. In both lines, selection via moth attack for fewer, smaller flowers contrasted with selection via other factors for more flowers. In LF females, selection via the two components was strongest and selection via moth attack also favoured increased fruit abortion. This suggests that the moths act as more of a selective force on flower size and number via their predating than their pollinating role.

SEXUAL, FECUNDITY, AND VIABILITY SELECTION ON FLOWER SIZE AND NUMBER IN A SEXUALLY DIMORPHIC PLANT

The evolution of sexual dimorphism will depend on how sexual, fecundity and viability selection act within each sex, with the different forms of selection potentially operating in opposing directions. We examined selection in the dioecious plant Silene latifolia using planted arrays of selection lines that differed in flower size (small vs. large). In this species, a flower size/number trade-off exists within each sex, and males produce smaller and more numerous flowers than females. Moreover, floral traits are genetically correlated with leaf physiology. Sexual selection favoring males in the small-flower line occurred via greater overlap in the timing of flower output between males from this line and females. Fecundity selection favored males with high flower production, as siring success was proportionate to pollen production. Viability selection opposed sexual selection, favoring males from the large-flower line. In females, fecundity and viability selection operated in the same direction, favoring those from the large-flower line via greater seed production and survival. These results concur with the pattern of floral sexual dimorphism. Together with previous results they suggest that the outcome of the different forms of selection will be environmentally dependent, and therefore help to explain variation among populations in sexually dimorphic traits.

ELIMINATION OF A GENETIC CORRELATION BETWEEN THE SEXES VIA ARTIFICIAL CORRELATIONAL SELECTION

Genetic correlations between the sexes can constrain the evolution of sexual dimorphism and be difficult to alter, because traits common to both sexes share the same genetic underpinnings. We tested whether artificial correlational selection favoring specific combinations of male and female traits within families could change the strength of a very high between-sex genetic correlation for flower size in the dioecious plant Silene latifolia. This novel selection dramatically reduced the correlation in two of three selection lines in fewer than five generations. Subsequent selection only on females in a line characterized by a lower between-sex genetic correlation led to a significantly lower correlated response in males, confirming the potential evolutionary impact of the reduced correlation. Although between-sex genetic correlations can potentially constrain the evolution of sexual dimorphism, our findings reveal that these constraints come not from a simple conflict between an inflexible genetic architecture and a pattern of selection working in opposition to it, but rather a complex relationship between a changeable correlation and a form of selection that promotes it. In other words, the form of selection on males and females that leads to sexual dimorphism may also promote the genetic phenomenon that limits sexual dimorphism.

Genetic differences among populations in sexual dimorphism: evidence for selection on males in a dioecious plant

Genetic variation among populations in the degree of sexual dimorphism may be a consequence of selection on one or both sexes. We analysed genetic parameters from crosses involving three populations of the dioecious plant Silene latifolia, which exhibits sexual dimorphism in flower size, to determine whether population differentiation was a result of selection on one or both sexes. We took the novel approach of comparing the ratio of population differentiation of a quantitative trait (Q(ST) ) to that of neutral genetic markers (F(ST) ) for males vs. females. We attributed 72.6% of calyx width variation in males to differences among populations vs. only 6.9% in females. The Q(ST) /F(ST) ratio was 4.2 for males vs. 0.4 for females, suggesting that selection on males is responsible for differentiation among populations in calyx width and its degree of sexual dimorphism. This selection may be indirect via genetic correlations with other morphological and physiological traits.

Identification of differentially expressed genes from male and female flowers of kiwifruit

Kiwifruit is one of the dioecious plant species, whereas most flowering plants are hermaphrodite. Sex chromosome-linked genes have been isolated mainly from the dioecious plant Silene latifolia. As an attempt to understand the molecular mechanisms of sex determination and differentiation in kiwifruit, we performed DEG (differentially expressed gene) screening using annealing control primer-based gene fishing method from male and female floral buds. As a result, 15 DEGs in total were isolated and then sequenced. RT-PCR analysis revealed that some of them were differentially expressed in either male or female floral buds. GenBank database searches revealed that 11 DEGs exhibited significant sequence similarity with known function of genes or ESTs of kiwifruit. Among them, kiwiDEG1 encodes partial open reading frame (ORF) for pectin methylesterase, which plays an important role in pollen development as a cell-wall modifying enzyme. It was abundantly expressed in male floral buds compared with those of female, suggesting its functional role in pollen development of kiwifruit. KiwiDEG9 encodes a full-length ORF for adenosine diphosphate (ADP)-ribosylation factor, which is a small guanosine-5'-triphosphate (GTP)-binding protein and plays an essential role in vesicle trafficking. Its expression level was higher in male floral buds than in female. The isolated kiwiDEGs will be useful resources to better understand the molecular events of sex differentiation in kiwifruit. Key words: Actinidia, differentially expressed genes, dioecious plant, male flowers, female flowers, kiwifruit, pectin methylesterase, ADP-ribosylation factor.