Sexy ways: the methodical approaches to study plant sex chromosomes.

Sex chromosomes have evolved in many plant species with separate sexes. Current plant research is shifting from examining the structure of sex chromosomes to exploring their functional aspects. New studies are progressively unveiling the specific genetic and epigenetic mechanisms responsible for shaping distinct sexes in plants. While the fundamental methods of molecular biology and genomics are generally employed for the analysis of sex chromosomes, it is often necessary to modify classical procedures not only to simplify and expedite analyses but sometimes to make them possible at all. In this review, we demonstrate how, at the level of structural and functional genetics, cytogenetics, and bioinformatics, it is essential to adapt established procedures for sex chromosome analysis.

The Outline of Seed Silhouettes: A Morphological Approach to Silene (Caryophyllaceae).

Seed morphology is an important source of information for plant taxonomy. Nevertheless, the characters under study are diverse, and a simple, unified method is lacking in the literature. A new method for the classification of seeds of the genus Silene based on optical images and image analysis has recently been described on the basis of morphological measurements of the lateral seed views. According to the outline of their silhouettes, seeds from 52 species (49 of Silene and three related species) were classified in three groups: smooth, rugose and echinate, revealing remarkable differences between these groups. This methodology has been applied here to 51 new species, making a total of 100 species of Silene analyzed so far. According to our data, a new group was described, termed papillose. The results showed morphological differences between the four mentioned seed groups, with reduced values of circularity for dorsal and lateral seed views in the papillose and echinate groups and reduced values of solidity in the papillose seeds. The method was applied to the analysis of individual as well as to average seed silhouettes and some of the differences between groups were maintained in both cases.

New Geometric Models for Shape Quantification of the Dorsal View in Seeds of Silene Species.

The description of shape in Silene seeds is based on adjectives coined by naturalists in the 19th century. The expressions reniform, dorso plana, and dorso canaliculata were applied in reference to lateral or dorsal views of seeds, but the characters described can be submitted now to an analytical description by quantitative methods, allowing shape quantification and the comparison between species or populations. A quantitative morphological analysis is based on the comparison with geometric models that adjust to the shape of seeds. Morphological analysis of the dorsal view of Silene seeds based on geometric models is applied here to 26 seed populations belonging to 12 species. According to their dorsal views, the seeds are classified as convex and non-convex. New geometric models are presented for both types, including figures such as super-ellipses and modified ellipses. The values of J index (percent of similarity of a seed image with the model) are obtained in representative seed samples from diverse populations and species. The quantitative description of seed shape based on the comparison with geometric models allows the study of variation in shape between species and in populations, as well as the identification of seeds in Silene species. The method is of application to other plant species.

Adaptive changes of the autosomal part of the genome in a dioecious clade of Silene.

The genus Silene brings many opportunities for the study of various processes involved in the evolution of dioecy and young sex chromosomes. Here we focus on a dioecious clade in Silene subgenus Silene and closely related species. This study provides improved support for monophyly of this clade (based on inclusion of further dioecious species) and a new estimate of its age (ca 2.3 million years). We observed a rise in adaptive evolution in the autosomal and pseudoautosomal parts of the genome on the branch where dioecy originated. This increase is not a result of the accumulation of sexually antagonistic genes in the pseudoautosomal region. It is also not caused by the coevolution of genes acting in mitochondria (despite the possibility that dioecy along this branch could have evolved from a nucleo-cytoplasmic male sterility-based system). After considering other possibilities, the most parsimonious explanation for the increase seen in the number of positively selected codons is the adaptive evolution of genes involved in the adaptation of the autosomal part of the genome to dioecy, as described in Charnov's sex-allocation theory. As the observed coincidence cannot prove causality, studies in other dioecious clades are necessary to allow the formation of general conclusions. This article is part of the theme issue 'Sex determination and sex chromosome evolution in land plants'.

The evolution of huge Y chromosomes in Coccinia grandis and its sister, Coccinia schimperi.

Microscopically dimorphic sex chromosomes in plants are rare, reducing our ability to study them. One difficulty has been the paucity of cultivatable species pairs for cytogenetic, genomic and experimental work. Here, we study the newly recognized sisters Coccinia grandis and Coccinia schimperi, both with large Y chromosomes as we here show for Co. schimperi. We built genetic maps for male and female Co. grandis using a full-sibling family, inferred gene sex-linkage, and, with Co. schimperi transcriptome data, tested whether X- and Y-alleles group by species or by sex. Most sex-linked genes for which we could include outgroups grouped the X- and Y-alleles by species, but some 10% instead grouped the two species' X-alleles. There was no relationship between XY synonymous-site divergences in these genes and gene position on the non-recombining part of the X, suggesting recombination arrest shortly before or after species divergence, here dated to about 3.6 Ma. Coccinia grandis and Co. schimperi are the species pair with the most heteromorphic sex chromosomes in vascular plants (the condition in their sister remains unknown), and future work could use them to study mechanisms of Y chromosome enlargement and parallel degeneration, or to test Haldane's rule about lower hybrid fitness in the heterogametic sex. This article is part of the theme issue 'Sex determination and sex chromosome evolution in land plants'.

Seed Morphology in Silene Based on Geometric Models.

Seed description in morphology is often based on adjectives such as "spherical", "globular", or "reniform", but this does not provide a quantitative method. A new morphological approach based on the comparison of seed images with geometric models provides a seed description in Silene species on a quantitative basis. The novelty of the proposed method is based in the comparison of the seed images with geometric models according to a cardioid shape. The J index is a measurement that indicates the seed percentage of similarity with a cardioid or cardioid-derived figures used as models. The seeds of Silene species have high values of similarity with the cardioid and cardioid-derived models (J index superior to 90). The comparison with different figures allows species description and differentiation. The method is applied here to seeds of 21 species and models are proposed for some of them including S. diclinis, an endangered species. The method is discussed in the context of previous comparison with the measures used in traditional morphometric analysis. The similarity of seed images with geometric figures opens a new perspective for the automatized taxonomical evaluation of samples linking seed morphology to functional traits in endangered Silene species.

Evolution of sex determination and heterogamety changes in section Otites of the genus Silene.

Switches in heterogamety are known to occur in both animals and plants. Although plant sex determination systems probably often evolved more recently than those in several well-studied animals, including mammals, and have had less time for switches to occur, we previously detected a switch in heterogamety in the plant genus Silene: section Otites has both female and male heterogamety, whereas S. latifolia and its close relatives, in a different section of the genus, Melandrium (subgenus Behenantha), all have male heterogamety. Here we analyse the evolution of sex chromosomes in section Otites, which is estimated to have evolved only about 0.55 MYA. Our study confirms female heterogamety in S. otites and newly reveals female heterogamety in S. borysthenica. Sequence analyses and genetic mapping show that the sex-linked regions of these two species are the same, but the region in S. colpophylla, a close relative with male heterogamety, is different. The sex chromosome pairs of S. colpophylla and S. otites each correspond to an autosome of the other species, and both differ from the XY pair in S. latifolia. Silene section Otites species are suitable for detailed studies of the events involved in such changes, and our phylogenetic analysis suggests a possible change from female to male heterogamety within this section. Our analyses suggest a possibility that has so far not been considered, change in heterogamety through hybridization, in which a male-determining chromosome from one species is introgressed into another one, and over-rides its previous sex-determining system.

Sex and the flower - developmental aspects of sex chromosome evolution.

Background: The evolution of dioecious plants is occasionally accompanied by the establishment of sex chromosomes: both XY and ZW systems have been found in plants. Structural studies of sex chromosomes are now being followed up by functional studies that are gradually shedding light on the specific genetic and epigenetic processes that shape the development of separate sexes in plants. Scope: This review describes sex determination diversity in plants and the genetic background of dioecy, summarizes recent progress in the investigation of both classical and emerging model dioecious plants and discusses novel findings. The advantages of interspecies hybrids in studies focused on sex determination and the role of epigenetic processes in sexual development are also overviewed. Conclusions: We integrate the genic, genomic and epigenetic levels of sex determination and stress the impact of sex chromosome evolution on structural and functional aspects of plant sexual development. We also discuss the impact of dioecy and sex chromosomes on genome structure and expression.

The evolutionary fate of the horizontally transferred agrobacterial mikimopine synthase gene in the genera Nicotiana and Linaria.

Few cases of spontaneously horizontally transferred bacterial genes into plant genomes have been described to date. The occurrence of horizontally transferred genes from the T-DNA of Agrobacterium rhizogenes into the plant genome has been reported in the genus Nicotiana and in the species Linaria vulgaris. Here we compare patterns of evolution in one of these genes (a gene encoding mikimopine synthase, mis) following three different events of horizontal gene transfer (HGT). As this gene plays an important role in Agrobacterium, and there are known cases showing that genes from pathogens can acquire plant protection function, we hypothesised that in at least some of the studied species we will find signs of selective pressures influencing mis sequence. The mikimopine synthase (mis) gene evolved in a different manner in the branch leading to Nicotiana tabacum and N. tomentosiformis, in the branch leading to N. glauca and in the genus Linaria. Our analyses of the genus Linaria suggest that the mis gene began to degenerate soon after the HGT. In contrast, in the case of N. glauca, the mis gene evolved under significant selective pressures. This suggests a possible role of mikimopine synthase in current N. glauca and its ancestor(s). In N. tabacum and N. tomentosiformis, the mis gene has a common frameshift mutation that disrupted its open reading frame. Interestingly, our results suggest that in spite of the frameshift, the mis gene could evolve under selective pressures. This sequence may still have some regulatory role at the RNA level as suggested by coverage of this sequence by small RNAs in N. tabacum.

Evolution of sex determination systems with heterogametic males and females in silene.

The plant genus Silene has become a model for evolutionary studies of sex chromosomes and sex-determining mechanisms. A recent study performed in Silene colpophylla showed that dioecy and the sex chromosomes in this species evolved independently from those in Silene latifolia, the most widely studied dioecious Silene species. The results of this study show that the sex-determining system in Silene otites, a species related to S. colpophylla, is based on female heterogamety, a sex determination system that is unique among the Silene species studied to date. Our phylogenetic data support the placing of S. otites and S. colpophylla in the subsection Otites and the analysis of ancestral states suggests that the most recent common ancestor of S. otites and S. colpophylla was most probably dioecious. These observations imply that a switch from XX/XY sex determination to a ZZ/ZW system (or vice versa) occurred in the subsection Otites. This is the first report of two different types of heterogamety within one plant genus of this mostly nondioecious plant family.

Comparative analysis of a plant pseudoautosomal region (PAR) in Silene latifolia with the corresponding S. vulgaris autosome.

BACKGROUND: The sex chromosomes of Silene latifolia are heteromorphic as in mammals, with females being homogametic (XX) and males heterogametic (XY). While recombination occurs along the entire X chromosome in females, recombination between the X and Y chromosomes in males is restricted to the pseudoautosomal region (PAR). In the few mammals so far studied, PARs are often characterized by elevated recombination and mutation rates and high GC content compared with the rest of the genome. However, PARs have not been studied in plants until now. In this paper we report the construction of a BAC library for S. latifolia and the first analysis of a > 100 kb fragment of a S. latifolia PAR that we compare to the homologous autosomal region in the closely related gynodioecious species S. vulgaris. RESULTS: Six new sex-linked genes were identified in the S. latifolia PAR, together with numerous transposable elements. The same genes were found on the S. vulgaris autosomal segment, with no enlargement of the predicted coding sequences in S. latifolia. Intergenic regions were on average 1.6 times longer in S. latifolia than in S. vulgaris, mainly as a consequence of the insertion of transposable elements. The GC content did not differ significantly between the PAR region in S. latifolia and the corresponding autosomal region in S. vulgaris. CONCLUSIONS: Our results demonstrate the usefulness of the BAC library developed here for the analysis of plant sex chromosomes and indicate that the PAR in the evolutionarily young S. latifolia sex chromosomes has diverged from the corresponding autosomal region in the gynodioecious S. vulgaris mainly with respect to the insertion of transposable elements. Gene order between the PAR and autosomal region investigated is conserved, and the PAR does not have the high GC content observed in evolutionarily much older mammalian sex chromosomes.

Bisprimer--a program for the design of primers for bisulfite-based genomic sequencing of both plant and Mammalian DNA samples.

Plants and animals differ in the sequence context of the methylated sites in DNA. Plants exhibit cytosine methylation in CG, CHG, and CHH sites, whereas CG methylation is the only form present in mammals (with an exception of the early embryonic development). This fact must be taken into account in the design of primers for bisulfite-based genomic sequencing because CHG and CHH sites can remain unmodified. Surprisingly, no user-friendly primer design program is publicly available that could be used to design primers in plants and to simultaneously check the properties of primers such as the potential for primer-dimer formation. For studies concentrating on particular DNA loci, the correct design of primers is crucial. The program, called BisPrimer, includes 2 different subprograms for the primer design, the first one for mammals and the second one for angiosperm plants. Each subprogram is divided into 2 variants. The first variant serves to design primers that preferentially bind to the bisulfite-modified primer-binding sites (C to U conversion). This type of primer preferentially amplifies the bisulfite-converted DNA strands. This feature can help to avoid problems connected with an incomplete bisulfite modification that can sometimes occur for technical reasons. The second variant is intended for the analysis of samples that are supposed to consist of a mixture of DNA molecules that have different levels of cytosine methylation (e.g., pollen DNA). In this case, the aim is to minimize the selection in favor of either less methylated or more methylated molecules.

What can we learn from tobacco and other Solanaceae about horizontal DNA transfer?

In eukaryotic organisms, horizontal gene transfer (HGT) is regarded as an important though infrequent source of reticulate evolution. Many confirmed instances of natural HGT involving multicellular eukaryotes come from flowering plants. This review intends to provide a synthesis of present knowledge regarding HGT in higher plants, with an emphasis on tobacco and other species in the Solanaceae family because there are numerous detailed reports concerning natural HGT events, involving various donors, in this family. Moreover, in-depth experimental studies using transgenic tobacco are of great importance for understanding this process. Valuable insights are offered concerning the mechanisms of HGT, the adaptive role and regulation of natural transgenes, and new routes for gene trafficking. With an increasing amount of data on HGT, a synthetic view is beginning to emerge.

Dioecious Silene latifolia plants show sexual dimorphism in the vegetative stage.

BACKGROUND: Prior to this study, no differences in gene expression between male and female dioecious plants in the vegetative state had been detected. Among dioecious plants displaying sexual dimorphism, Silene latifolia is one of the most studied species. Although many sexually dimorphic traits have been described in S. latifolia, all of them are quantitative, and they usually become apparent only after the initiation of flowering. RESULTS: We present RT-PCR-based evidence that in S. latifolia, sexual dimorphism in gene expression is present long before the initiation of flowering. We describe three ESTs that show sex-specific (two male specific and one female specific) transcription at the rosette stage before the first flowering season. CONCLUSIONS: To our knowledge, this study provides the first molecular evidence of early pre-flowering sexual dimorphism in angiosperms.

Independent origin of sex chromosomes in two species of the genus Silene.

Here we introduce a new model species, Silene colpophylla, that could facilitate research of sex chromosome evolution and sex-determining systems. This species is related to the well-established dioecious plant model Silene latifolia. Our results show that S. colpophylla is, similarly to S. latifolia, a male heterogametic species, but its sex chromosomes have evolved from a different pair of autosomes than in S. latifolia. The results of our phylogenetic study and mapping of homologs of S. latifolia X-linked genes indicate that the sex determination system in S. colpophylla evolved independently from that in S. latifolia. We assert that this model species pair will make it possible to study two independent patterns of sex chromosome evolution in related species.

An interspecific hybrid as a tool to study phylogenetic relationships in plants using the GISH technique.

We established a new auxiliary phylogenetic approach based on genomic in situ hybridization technique (GISH). We used an interspecific hybrid Silene latifolia x Silene viscosa to compare two different genomes simultaneously on one slide. By using GISH with genomic DNA from another closely related species as a probe, we directly compared the level of relatedness between the genomes of the studied species and parental species. This experimental design enabled us to approximately estimate evolutionary relationships between the genome of tested plant species and genomes of both parental species of the hybrid by using the ratio of intensities of fluorescence signals. We tested this technique in various Silene species and the results were in accordance with the topology of the phylogenetic tree we constructed based on rDNA sequences. The results were also well correlated with phylogenetic distances between species that we estimated from an rDNA-based phylogenetic tree. Our experimental approach could help to improve tree topology and serve as a useful complementary tool in molecular phylogenetic studies in related species.

Early events in the evolution of the Silene latifolia Y chromosome: male specialization and recombination arrest.

Understanding the origin and evolution of sex chromosomes requires studying recently evolved X-Y chromosome systems such as those in some flowering plants. We describe Y chromosome deletion mutants of Silene latifolia, a dioecious plant with heteromorphic sex chromosomes. The combination of results from new and previously described deletions with histological descriptions of their stamen development defects indicates the presence of two distinct Y regions containing loci with indispensable roles in male reproduction. We determined their positions relative to the two main sex determination functions (female suppressing and the other male promoting). A region proximal to the centromere on the Y p arm containing the putative stamen promoting sex determination locus includes additional early stamen developmental factors. A medial region of the Y q arm carries late pollen fertility factors. Cytological analysis of meiotic X-Y pairing in one of the male-sterile mutants indicates that the Y carries sequences or functions specifically affecting sex chromosome pairing.

MK17, a specific marker closely linked to the gynoecium suppression region on the Y chromosome in Silene latifolia.

The aim of this work was to isolate new DNA markers linked to the Silene latifolia Y chromosome. To do this we created a chromosome-specific plasmid library after DOP-PCR amplification of laser-microdissected Y-chromosomes. The library screening led to the isolation of several clones yielding mostly to exclusive male specific hybridization signals. Subsequent PCR confirmed the Y-unique linkage for one of the sequences. This DNA sequence called MK17 has no homology to any known DNA sequence and it is not expressed. Based on PCR and Southern analyses, MK17 is present only in dioecious species of the Elisanthe section of the genus Silene (S. latifolia, S. dioica, and S. diclinis) and it is absent in related gynodioecious and hermaphroditic species. The mapping analysis using a panel of deletion mutants showed that MK17 is closely linked to the region controlling suppression of gynoecium development. Hence MK17 represents a valuable marker to isolate genes controlling the gynoecium development suppression on the Y chromosome of S. latifolia.