Silene Latifolia Research Articles

Cell Death and Cell Cycle Arrest of Silene latifolia Stamens and Pistils After Microbotryum lychnidis-dioicae Infection.

Mechanisms of suppression of pistil primordia in male flowers and of stamen primordia in female flowers differ in diclinous plants. In this study, we investigated how cell death and cell cycle arrest are related to flower organ formation in Silene latifolia. Using in situ hybridization and a TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) assay, we detected both cell cycle arrest and cell death in suppressed stamens of female flowers and suppressed pistils of male flowers in S. latifolia. In female flowers infected with Microbotryum lychnidis-dioicae, developmental suppression of stamens is released, and cell cycle arrest and cell death do not occur. Smut spores are formed in S. latifolia anthers infected with M. lychnidis-dioicae, followed by cell death in the endothelium, middle layer, tapetal cells and pollen mother cells. Cell death is difficult to detect using a fluorescein isothiocyanate-labeled TUNEL assay due to strong autofluorescence in the anther. We therefore combined a TUNEL assay in an infrared region with transmission electron microscopy to detect cell death in anthers. We show that following infection by M. lychnidis-dioicae, a TUNEL signal was not detected in the endothelium, middle layer or pollen mother cells, and cell death with outflow of cell contents, including the nucleoplast, was observed in tapetal cells.

Adaptive and non‐adaptive evolution of trait means and genetic trait correlations for herbivory resistance and performance in an invasive plant

The EICA‐hypothesis predicts that invading plants adapt to their novel environment by evolving increased performance and reduced resistance in response to the release from natural enemies, and assumes a resource allocation tradeoff among both trait groups as mechanistic basis of this evolutionary change. Using the plant Silene latifolia as a study system, we tested these predictions by investigating whether 1) invasive populations evolved lower resistance and higher performance, 2) this evolutionary change is indeed adaptive, and 3) there is a negative genetic correlation between performance and resistance (i.e. a tradeoff) in native and introduced individuals. Moreover, we sampled eight native and eight invasive populations and determined their population co‐ancestry based on neutral SSR‐markers. We performed controlled crossings to produce five sib‐groups per population and exposed them to increased and reduced levels of enemy attack in a full‐factorial experiment to estimate performance and resistance. With these data, we performed trait‐by‐trait comparisons between ranges with ‘animal models’ that account for population co‐ancestry to quantify the amount of variance in traits explained by non‐adaptive versus adaptive evolution. Moreover, we tested for genetic correlations among performance and resistance traits within sib‐groups. We found significant reductions in resistance and increases in performance in invasive versus native populations, which could largely be attributed to adaptive evolution. While we detected a non‐significant trend towards negative genetic performance × resistance correlations in native populations, invasive populations exhibited both significant and non‐significant positive correlations. In summary, these results do not support a shift of performance and resistance trait values along a tradeoff line in response to enemy release, as predicted by EICA. They rather suggest that the independent evolution of both traits is not constrained by a tradeoff, and that various selective agents (including resource availability) interact in shaping both traits and in weakening negative genetic correlations in the invaded habitat.

Polymorphic Microsatellite Markers for the Tetrapolar Anther-Smut Fungus Microbotryum saponariae Based on Genome Sequencing.

BackgroundAnther-smut fungi belonging to the genus Microbotryum sterilize their host plants by aborting ovaries and replacing pollen by fungal spores. Sibling Microbotryum species are highly specialized on their host plants and they have been widely used as models for studies of ecology and evolution of plant pathogenic fungi. However, most studies have focused, so far, on M. lychnidis-dioicae that parasitizes the white campion Silene latifolia. Microbotryum saponariae, parasitizing mainly Saponaria officinalis, is an interesting anther-smut fungus, since it belongs to a tetrapolar lineage (i.e., with two independently segregating mating-type loci), while most of the anther-smut Microbotryum fungi are bipolar (i.e., with a single mating-type locus). Saponaria officinalis is a widespread long-lived perennial plant species with multiple flowering stems, which makes its anther-smut pathogen a good model for studying phylogeography and within-host multiple infections.Principal FindingsHere, based on a generated genome sequence of M. saponariae we developed 6 multiplexes with a total of 22 polymorphic microsatellite markers using an inexpensive and efficient method. We scored these markers in fungal individuals collected from 97 populations across Europe, and found that the number of their alleles ranged from 2 to 11, and their expected heterozygosity from 0.01 to 0.58. Cross-species amplification was examined using nine other Microbotryum species parasitizing hosts belonging to Silene, Dianthus and Knautia genera. All loci were successfully amplified in at least two other Microbotryum species.SignificanceThese newly developed markers will provide insights into the population genetic structure and the occurrence of within-host multiple infections of M. saponariae. In addition, the draft genome of M. saponariae, as well as one of the described markers will be useful resources for studying the evolution of the breeding systems in the genus Microbotryum and the evolution of specialization onto different plant species.

Recombination changes at the boundaries of fully and partially sex-linked regions between closely related Silene species pairs.

The establishment of a region of suppressed recombination is a critical change during sex chromosome evolution, leading to such properties as Y (and W) chromosome genetic degeneration, accumulation of repetitive sequences and heteromorphism. Although chromosome inversions can cause large regions to have suppressed recombination, and inversions are sometimes involved in sex chromosome evolution, gradual expansion of the non-recombining region could potentially sometimes occur. We here test whether closer linkage has recently evolved between the sex-determining region and several genes that are partially sex-linked in Silene latifolia, using Silene dioica, a closely related dioecious plants whose XY sex chromosome system is inherited from a common ancestor. The S. latifolia pseudoautosomal region (PAR) includes several genes extremely closely linked to the fully Y-linked region. These genes were added to an ancestral PAR of the sex chromosome pair in two distinct events probably involving translocations of autosomal genome regions causing multiple genes to become partially sex-linked. Close linkage with the PAR boundary must have evolved since these additions, because some genes added in both events now show almost complete sex linkage in S. latifolia. We compared diversity patterns of five such S. latifolia PAR boundary genes with their orthologues in S. dioica, including all three regions of the PAR (one gene that was in the ancestral PAR and two from each of the added regions). The results suggest recent recombination suppression in S. latifolia, since its split from S. dioica.

Analysis of transposable elements and organellar DNA in male and female genomes of a species with a huge Y chromosome reveals distinct Y centromeres.

Few angiosperms have distinct Y chromosomes. Among those that do are Silene latifolia (Caryophyllaceae), Rumex acetosa (Polygonaceae) and Coccinia grandis (Cucurbitaceae), the latter having a male/female difference of 10% of the total genome (female individuals have a 0.85pg genome, male individuals 0.94pg), due to a Y chromosome that arose about 3 million years ago. We compared the sequence composition of male and female C.grandis plants and determined the chromosomal distribution of repetitive and organellar DNA with probes developed from 21 types of repetitive DNA, including 16 mobile elements. The size of the Y chromosome is largely due to the accumulation of certain repeats, such as members of the Ty1/copia and Ty3/gypsy superfamilies, an unclassified element and a satellite, but also plastome- and chondriome-derived sequences. An abundant tandem repeat with a unit size of 144bp stains the centromeres of the X chromosome and the autosomes, but is absent from the Y centromere. Immunostaining with pericentromere-specific markers for anti-histone H3Ser10ph and H2AThr120ph revealed a Y-specific extension of these histone marks. That the Y centromere has a different make-up from all the remaining centromeres raises questions about its spindle attachment, and suggests that centromeric or pericentromeric chromatin might be involved in the suppression of recombination.

Genetic variation in natural Melandrium album populations exposed to chronic ionizing radiation.

The effect of radiation pollution on genetic variation in natural populations of Melandrium album was investigated at the head part of the East-Ural Radioactive Trace (EURT) and background areas. The highest genetic differentiation estimated using F ST was revealed between compared pairs of the background and impact samples in populations of M. album. The highest rate of polymorphism was observed at the closest to nuclear accident, Impact-1 site. The unique alleles (Mdh-3104, Pgi-2106, Lap 105, Mdh-296, and Dia 94) were discovered at the EURT. Individuals from chronically low-level irradiated sites were genetically closer than to plants from background sites using Nadhdh locus. The increase of the frequency of unique homozygous and heterozygous genotypes was identified in populations of M. album growing under chronic radiation exposure conditions. The largest contribution to the group of unique heterozygous genotypes at the EURT was made by three loci-Lap, Pgi-2, and Nadhdh; the main role in interpopulation differentiation of samples was made by the alleles Sod-2115, Skdh 100, and Nadhdh 100. Our results provide evidence for the correlation between the increase of genetic variation other than the «genetic erosion» and chronic radiation exposure factor in natural plant populations.

Strong phylogeographic co-structure between the anther-smut fungus and its white campion host.

Although congruence between host and pathogen phylogenies has been extensively investigated, the congruence between host and pathogen genetic structures at the within-species level has received little attention. Using an unprecedented and comprehensive collection of associated plant-pathogen samples, we investigated the degree of congruence between the genetic structures across Europe of two evolutionary and ecological model organisms, the anther-smut pathogen Microbotryum lychnidis-dioicae and its host plant Silene latifolia. We demonstrated a significant and particularly strong level of host-pathogen co-structure, with three main genetic clusters displaying highly similar spatial ranges in Western Europe, Eastern Europe and Italy, respectively. Correcting for the geographical component of genetic variation, significant correlations were still found between the genetic distances of anther-smut and host populations. Inoculation experiments suggested plant local adaptation, at the cluster level, for resistance to pathogens. These findings indicate that the pathogen remained isolated in the same fragmented southern refugia as its host plant during the last glaciation, and that little long-distance dispersal has occurred since the recolonization of Europe for either the plant or the pathogen, despite their known ability to travel across continents. This, together with the inoculation results, suggests that coevolutionary and competitive processes may be drivers of host-pathogen co-structure.

Identification of herbaceous hosts of the Grapevine Pinot gris virus (GPGV)

Grapevine Pinot gris Virus (GPGV) is a single stranded RNA of the genus Trichovirus infecting grapevine (Vitis vinifera) and associated with stunting, chlorotic mottling and leaf deformation symptoms. During a monitoring of GPGV infection in vineyards of the Trentino region in Italy, we have detected the virus in the herbaceous plants Silene latifolia subsp. Alba (Mill.) (bladder campion) and Chenopodium album L. (white goosefoot), which showed symptoms of viral infection. The full-length GPGV RNA genome, amplified from these infected hosts, was sequenced and a phylogenetic analysis revealed that its closest relative is the strain SK13, recently isolated in Slovakia. Our results indicate that herbaceous plants can be considered as a reservoir for the GPGV virus. This finding is important for studying the epidemiological aspects of GPGV disease and to formulate appropriate control measures.

Lower prevalence but similar fitness in a parasitic fungus at higher radiation levels near Chernobyl.

Nuclear disasters at Chernobyl and Fukushima provide examples of effects of acute ionizing radiation on mutations that can affect the fitness and distribution of species. Here, we investigated the prevalence of Microbotryum lychnidis-dioicae, a pollinator-transmitted fungal pathogen of plants causing anther-smut disease in Chernobyl, its viability, fertility and karyotype variation, and the accumulation of nonsynonymous mutations in its genome. We collected diseased flowers of Silene latifolia from locations ranging by more than two orders of magnitude in background radiation, from 0.05 to 21.03μGy/h. Disease prevalence decreased significantly with increasing radiation level, possibly due to lower pollinator abundance and altered pollinator behaviour. Viability and fertility, measured as the budding rate of haploid sporidia following meiosis from the diploid teliospores, did not vary with increasing radiation levels and neither did karyotype overall structure and level of chromosomal size heterozygosity. We sequenced the genomes of twelve samples from Chernobyl and of four samples collected from uncontaminated areas and analysed alignments of 6068 predicted genes, corresponding to 1.04×10(7) base pairs. We found no dose-dependent differences in substitution rates (neither dN, dS, nor dN/dS). Thus, we found no significant evidence of increased deleterious mutation rates at higher levels of background radiation in this plant pathogen. We even found lower levels of nonsynonymous substitution rates in contaminated areas compared to control regions, suggesting that purifying selection was stronger in contaminated than uncontaminated areas. We briefly discuss the possibilities for a mechanistic basis of radio resistance in this nonmelanized fungus.

Robinia pseudoacacia and Melandrium album in trace elements biomonitoring and air pollution tolerance index study

The accumulation efficiency of selected trace elements in the leaves of Melandrium album and Robinia pseudoacacia grown on heavy metal contaminated sites in comparison with a non-contaminated one was evaluated. The study was undertaken to calculate air pollution tolerance index and to determine the contents of selected metabolites: glutathione, non-protein thiols, ascorbic acid, chlorophyll and the activity of antioxidant enzymes: guaiacol peroxidase and superoxide dismutase. Such estimations can be useful in better understanding of plants defense strategies and potential to grow in contaminated environments. The results in the most contaminated site revealed higher contents of metals in M. album leaves, especially Zn, Cd and Pb (3.4, 6 and 2.3 times higher, respectively) in comparison with the R. pseudoacacia. Better accumulation capacity found in M. album was shown by metal accumulation index values. The plants could be used as indicators of Zn, Cd (both species) and Pb (M. album) in the soil. Glutathione content (in both species) and peroxidase activity (in M. album), general markers of heavy metals contamination, were increased in contaminated sites. In most cases in contaminated areas R. pseudoacacia had decreased ascorbic acid and chlorophyll levels. Opposite tendency was recorded in M. album leaves, where similar or higher contents of the above-mentioned metabolites were found. In our study, M. album and R. pseudoacacia proved to be sensitive species with the air pollution tolerance index lower than 11 and can be recommended as bioindicators.

Sex and heavy metals: Study of sexual dimorphism in response to soil pollution

In dioecious plants, males and females often show distinct morphological, physiological and life history traits as result of their different demands for reproduction. Such sexual dimorphism is likely to be accentuated under stressful conditions, such as that imposed by exposure to heavy metals. However little is known about the response of dioecious plants to stress by heavy metals. Here we use the dioecious herb Silene latifolia to investigate the growth and reproduction of males and females growing in soil polluted with either Cu or Cd. We also examined whether the sexes differed in the patterns of metal accumulation in their tissues. Patterns of biomass allocation to reproduction, roots, leaves and shoots (stem and leaves) were compared in male and female S. latifolia plants that were harvested after growing for 14 weeks in different soil conditions (non-polluted soil, Cu-polluted soil and Cd-polluted soil). In addition, patterns of metal accumulation between the sexes were also compared post-harvest by analysing the metal content in their tissues. Overall, metals decreased plants’ total dry mass allocated to leaves and to roots- particularly in males. Females accumulated more Cu in their tissues. However, this did not seem to correspond with females performing worse (in terms of growth and reproduction) than males when growing in soil polluted with this metal. Despite males and females having similar levels of Cd in their tissues, males seemed to have lower tolerance to this metal, as indicated by a lower total and leaf dry mass than females, and also by a lower number of flowers when growing in Cd-polluted soil. We also found contrasting differences in female seed production due to heavy metals, with number of seeds (but not mass) decreasing with Cu and increasing with Cd. Our results indicate the presence of sexual dimorphism in response to heavy metals, with the sexes differing in both patterns of accumulation and tolerance.

The large-X effect in plants: increased species divergence and reduced gene flow on the Silene X-chromosome.

The disproportionately large involvement of the X-chromosome in the isolation of closely related species (the large-X effect) has been reported for many animals, where X-linked genes are mostly hemizygous in the heterogametic sex. The expression of deleterious recessive mutations is thought to drive the frequent involvement of the X-chromosome in hybrid sterility, as well as to reduce interspecific gene flow for X-linked genes. Here, we evaluate the role of the X-chromosome in the speciation of two closely related plant species - the white and red campions (Silene latifolia and S.dioica) - that hybridize widely across Europe. The two species evolved separate sexes and sex chromosomes relatively recently (~10(7) years), and unlike most animal species, most X-linked genes have intact Y-linked homologs. We demonstrate that the X-linked genes show a very small and insignificant amount of interspecific gene flow, while gene flow involving autosomal loci is significant and sufficient to homogenize the gene pools of the two species. These findings are consistent with the hypothesis of the large-X effect in Silene and comprise the first report of this effect in plants. Nonhemizygosity of many X-linked genes in Silene males indicates that exposure of recessive mutations to selection may not be essential for the occurrence of the large-X effect. Several possible causes of the large-X effect in Silene are discussed.

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.

Two complete chloroplast genomes of white campion (Silene latifolia) from male and female individuals

In this study, we assembled two individuals’ complete chloroplast genomes with one male and one female from the dioecious plant species white campion (Silene latifolia). The two chloroplast genomes have an identical composition with each other as a circular molecule of 150 931 bp in length, with an overall GC content of 36.4%. The genomes consist of a pair of inverted repeats (IRs) of 25 503 bp, separated by a large single-copy (LSC) region and a small single-copy (SSC) region of 82 708 and 17 217 bp, respectively. The genomes contain 111 single copy genes, including 77 protein-coding genes, 4 ribosomal RNA genes and 30 transfer RNA genes. In these protein-coding genes, eight genes (rpl16, rpoC1, rps16, petD, petB, ndhB, ndhA and atpF) contain a single intron and three genes (rps12, clpP and ycf3) contain two introns. The two newly sequenced chloroplast genomes provide valuable information for detecting polymorphisms within species and between sexes.

Deciphering evolutionary strata on plant sex chromosomes and fungal mating-type chromosomes through compositional segmentation

Sex chromosomes have evolved from a pair of homologous autosomes which differentiated into sex determination systems, such as XY or ZW system, as a consequence of successive recombination suppression between the gametologous chromosomes. Identifying the regions of recombination suppression, namely, the "evolutionary strata", is central to understanding the history and dynamics of sex chromosome evolution. Evolution of sex chromosomes as a consequence of serial recombination suppressions is well-studied for mammals and birds, but not for plants, although 48 dioecious plants have already been reported. Only two plants Silene latifolia and papaya have been studied until now for the presence of evolutionary strata on their X chromosomes, made possible by the sequencing of sex-linked genes on both the X and Y chromosomes, which is a requirement of all current methods that determine stratum structure based on the comparison of gametologous sex chromosomes. To circumvent this limitation and detect strata even if only the sequence of sex chromosome in the homogametic sex (i.e. X or Z chromosome) is available, we have developed an integrated segmentation and clustering method. In application to gene sequences on the papaya X chromosome and protein-coding sequences on the S. latifolia X chromosome, our method could decipher all known evolutionary strata, as reported by previous studies. Our method, after validating on known strata on the papaya and S. latifolia X chromosome, was applied to the chromosome 19 of Populus trichocarpa, an incipient sex chromosome, deciphering two, yet unknown, evolutionary strata. In addition, we applied this approach to the recently sequenced sex chromosome V of the brown alga Ectocarpus sp. that has a haploid sex determination system (UV system) recovering the sex determining and pseudoautosomal regions, and then to the mating-type chromosomes of an anther-smut fungus Microbotryum lychnidis-dioicae predicting five strata in the non-recombining region of both the chromosomes.

Effects of pollination timing and distance on seed production in a dioecious weed Silene latifolia

Silene latifolia Poir. (white cockle or white campion) is an important invasive weed in North American agriculture. It exhibits dioecy, therefore, both male and female plants are required in order for seed production to occur. However, dioecious species being invasive is not common because of their limitations in pollination and subsequent seed production. The objective of this study is to determine the effect of pollination timing and distance on seed production of Silene latifolia. A series of experiments including pollination exclusion, timing and pollination distance were conducted in 2009 and 2010 at or around Saskatoon, Saskatchewan. For pollination exclusion, exclosures were built around the natural female plants for exclosure, sham-exclosure, and male and female combined treatments. Pollination timing was studied by applying exclosure, non-exclosure, night-exclosure, and day-exclosure treatments to individual female plants. Female plants were transplanted along a linear interval at six different distances from the pollen source to study the effect of pollination distance. S. latifolia was exclusively insect-pollinated and pollination occurred both day and night; however, in one year, pollination occurred mainly at night. Female plants that were in the range of 0–4 m from a compatible pollen source experienced no limitation to pollination. However, when the distance was increased further up to 128 m, pollination levels and subsequent seed production were declined. Moreover, there were differences in seed production between years suggesting that pollination was affected by the environmental conditions during pollination and the crop that white cockle was grown in. These experiments indicate that seed production in S. latifolia is limited by insect-pollination. Although there was pollination limitation for seed production at greater distances from a pollen source, the high fecundity rate (3000–18000 seeds per plant) resulted in a large seed output. Thus, we believe that a dioecious species may require characteristics that compensate the pollination limitation for a successful invasion.

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.

Rapid Y degeneration and dosage compensation in plant sex chromosomes

The nonrecombining regions of animal Y chromosomes are known to undergo genetic degeneration, but previous work has failed to reveal large-scale gene degeneration on plant Y chromosomes. Here, we uncover rapid and extensive degeneration of Y-linked genes in a plant species, Silene latifolia, that evolved sex chromosomes de novo in the last 10 million years. Previous transcriptome-based studies of this species missed unexpressed, degenerate Y-linked genes. To identify sex-linked genes, regardless of their expression, we sequenced male and female genomes of S. latifolia and integrated the genomic contigs with a high-density genetic map. This revealed that 45% of Y-linked genes are not expressed, and 23% are interrupted by premature stop codons. This contrasts with X-linked genes, in which only 1.3% of genes contained stop codons and 4.3% of genes were not expressed in males. Loss of functional Y-linked genes is partly compensated for by gene-specific up-regulation of X-linked genes. Our results demonstrate that the rate of genetic degeneration of Y-linked genes in S. latifolia is as fast as in animals, and that the evolutionary trajectories of sex chromosomes are similar in the two kingdoms.

RAD mapping reveals an evolving, polymorphic and fuzzy boundary of a plant pseudoautosomal region.

How loss of genetic exchanges (recombination) evolves between sex chromosomes is a long-standing question. Suppressed recombination may evolve when a sexually antagonistic (SA) polymorphism occurs in a partially sex-linked 'pseudoautosomal' region (or 'PAR'), maintaining allele frequency differences between the two sexes, and creating selection for closer linkage with the fully sex-linked region of the Y chromosome in XY systems, or the W in ZW sex chromosome systems. Most evidence consistent with the SA polymorphism hypothesis is currently indirect, and more studies of the genetics and population genetics of PAR genes are clearly needed. The sex chromosomes of the plant Silene latifolia are suitable for such studies, as they evolved recently and the loss of recombination could still be ongoing. Here, we used RAD sequencing to genetically map sequences in this plant, which has a large genome (c. 3 gigabases) and no available whole-genome sequence. We mapped 83 genes on the sex chromosomes, and comparative mapping in the related species S. vulgaris supports previous evidence for additions to an ancestral PAR and identified at least 12 PAR genes. We describe evidence that recombination rates have been reduced in meiosis of both sexes, and differences in recombination between S. latifolia families suggest ongoing recombination suppression. Large allele frequency differences between the sexes were found at several loci closely linked to the PAR boundary, and genes in different regions of the PAR showed striking sequence diversity patterns that help illuminate the evolution of the PAR.

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.