Evolutionary history of Silene latifolia sex chromosomes revealed by genetic mapping of four genes

The sex chromosomes of dioecious white campion, Silene latifolia (Caryophyllaceae), are of relatively recent origin (10-20 million years), providing a unique opportunity to trace the origin and evolution of sex chromosomes in this genus by comparing closely related Silene species with and without sex chromosomes. Here I demonstrate that four genes that are X-linked in S. latifolia are also linked in nondioecious S. vulgaris, which is consistent with Ohno's (1967) hypothesis that sex chromosomes evolve from a single pair of autosomes. I also report a genetic map for four S. latifolia X-linked genes, SlX1, DD44X, SlX4, and a new X-linked gene SlssX, which encodes spermidine synthase. The order of the genes on the S. latifolia X chromosome and divergence between the homologous X- and Y-linked copies of these genes supports the "evolutionary strata" model, with at least three consecutive expansions of the nonrecombining region on the Y chromosome (NRY) in this plant species.     

A cytogenetic view of sex chromosome evolution in plants

The recent origin of sex chromosomes in plant species provides an opportunity to study the early stages of sex chromosome evolution. This review focuses on the cytogenetic aspects of the analysis of sex chromosome evolution in plants and in particular, on the best-studied case, the sex chromosomes in Silene latifolia. We discuss the emerging picture of sex chromosome evolution in plants and the further work that is required to gain better understanding of the similarities and differences between the trends in animal and plant sex chromosome evolution. Similar to mammals, suppression of recombination between the X and Y in S. latifolia species has occurred in several steps, however there is little evidence that inversions on the S. latifolia Y chromosome have played a role in cessation of X/Y recombination. Secondly, in S. latifolia there is a lack of evidence for genetic degeneration of the Y chromosome, unlike the events documented in mammalian sex chromosomes. The insufficient number of genes isolated from this and other plant sex chromosomes does not allow us to generalize whether the trends revealed on S. latifolia Y chromosome are general for other dioecious plants. Isolation of more plant sex-linked genes and their cytogenetic mapping with fluorescent in situ hybridisation (FISH) will ultimately lead to a much better understanding of the processes driving sex chromosome evolution in plants.     

Mapping of sex determination loci on the white campion (Silene latifolia) Y chromosome using amplified fragment length polymorphism

S. latifolia is a dioecious plant with morphologically distinct sex chromosomes. To genetically map the sex determination loci on the male-specific Y chromosome, we identified X-ray-induced sex determination mutants that had lost male traits. We used male-specific AFLP markers to characterize the extent of deletions in the Y chromosomes of the mutants. We then compared overlapping deletions to predict the order of the AFLP markers and to locate the mutated sex-determining genes. We found three regions on the Y chromosome where frequent deletions were significantly associated with loss of male traits. One was associated with hermaphroditic mutants. A second was associated with asexual mutants that lack genes needed for early stamen development and a third was associated with asexual mutants that lack genes for late stages of stamen development. Our observations confirmed a classical genetic prediction that S. latifolia has three dispersed male-determining loci on the Y chromosome, one for carpel suppression, one for early stamen development, and another for late stamen development. This AFLP map provides a framework for locating genes on the Y chromosome and for characterizing deletions on the Y chromosomes of potentially interesting mutants.     

Evidence for degeneration of the Y chromosome in the dioecious plant Silene latifolia

The human Y--probably because of its nonrecombining nature--has lost 97% of its genes since X and Y chromosomes started to diverge [1, 2]. There are clear signs of degeneration in the Drosophila miranda neoY chromosome (an autosome fused to the Y chromosome), with neoY genes showing faster protein evolution [3-6], accumulation of unpreferred codons [6], more insertions of transposable elements [5, 7], and lower levels of expression [8] than neoX genes. In the many other taxa with sex chromosomes, Y degeneration has hardly been studied. In plants, many genes are expressed in pollen [9], and strong pollen selection may oppose the degeneration of plant Y chromosomes [10]. Silene latifolia is a dioecious plant with young heteromorphic sex chromosomes [11, 12]. Here we test whether the S. latifolia Y chromosome is undergoing genetic degeneration by analyzing seven sex-linked genes. S. latifolia Y-linked genes tend to evolve faster at the protein level than their X-linked homologs, and they have lower expression levels. Several Y gene introns have increased in length, with evidence for transposable-element accumulation. We detect signs of degeneration in most of the Y-linked gene sequences analyzed, similar to those of animal Y-linked and neo-Y chromosome genes.     

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.     

Polymorphism and differentiation of rainbow trout Y chromosomes.

Most fish species show little morphological differentiation in the sex chromosomes. We have coupled molecular and cytogenetic analyses to characterize the male-determining region of the rainbow trout (Oncorhynchus mykiss) Y chromosome. Four genetically diverse male clonal lines of this species were used for genetic and physical mapping of regions in the vicinity of the sex locus. Five markers were genetically mapped to the Y chromosome in these male lines, indicating that the sex locus was located on the same linkage group in each of the lines. We also confirmed the presence of a Y chromosome morphological polymorphism among these lines, with the Y chromosomes from two of the lines having the more common heteromorphic Y chromosome and two of the lines having Y chromosomes morphologically similar to the X chromosome. The fluorescence in situ hybridization (FISH) pattern of two probes linked to sex suggested that the sex locus is physically located on the long arm of the Y chromosome. Fishes appear to be an excellent group of organisms for studying sex chromosome evolution and differentiation in vertebrates because they show considerable variability in the mechanisms and (or) patterns involved in sex determination.     

Selective trade-offs and sex-chromosome evolution in Silene latifolia

Alleles of sexually antagonistic genes (i.e., genes with alleles affecting fitness in opposite directions in the two sexes) can avoid expression in the sex to which they are detrimental via two processes: they are subsumed into the nonrecombining, sex-determining portion of the sex chromosomes or they evolve sex-limited expression. The former is considered more likely and leads to Y-chromosome degeneration. We mapped quantitative trait loci of major effect for sexually dimorphic traits of Silene latifolia to the recombining portions of the sex chromosomes and found them to exhibit sex-specific expression, with the Y chromosome in males controlling a relatively larger proportion of genetic variance than the X in females and the average autosome. Both reproductive and ecophysiological traits map to the recombining region of the sex chromosomes. We argue that genetic correlations among traits maintain recombination and polymorphism for these genes because of balancing selection in males, whereas sex-limited expression represses detrimental alleles in females. Our data suggest that the Y chromosome of S. latifolia plays a major role in the control of key metabolic activities beyond reproductive functions.     

SlY1, the first active gene cloned from a plant Y chromosome, encodes a WD-repeat protein.

Unlike the majority of flowering plants, which possess hermaphrodite flowers, white campion (Silene latifolia) is dioecious and has flowers of two different sexes. The sex is determined by the combination of heteromorphic sex chromosomes: XX in females and XY in males. The Y chromosome of S.latifolia was microdissected to generate a Y-specific probe which was used to screen a young male flower cDNA library. We identified five genes which represent the first active genes to be cloned from a plant Y chromosome. Here we report a detailed analysis of one of these genes, SlY1 (S.latifolia Y-gene 1). SlY1 is expressed predominantly in male flowers. A closely related gene, SlX1, is predicted to be located on the X chromosome and is strongly expressed in both male and female flowers. SlY1 and SlX1 encode almost identical proteins containing WD repeats. Immunolocalization experiments showed that these proteins are localized in the nucleus, and that they are most abundant in cells that are actively dividing or beginning to differentiate. Interestingly, they do not accumulate in arrested sexual organs and represent potential targets for sex determination genes. These genes will permit investigation of the origin and evolution of sex chromosomes in plants.     

The role of chromosomal rearrangements in the evolution of <Emphasis Type="Italic">Silene latifolia</Emphasis> sex chromosomes

Silene latifolia is a model plant for studies of the early steps of sex chromosome evolution. In comparison to mammalian sex chromosomes that evolved 300 mya, sex chromosomes of S. latifolia appeared approximately 20 mya. Here, we combine results from physical mapping of sex-linked genes using polymerase chain reaction on microdissected arms of the S. latifolia X chromosome, and fluorescence in situ hybridization analysis of a new cytogenetic marker, Silene tandem repeat accumulated on the Y chromosome. The data are interpreted in the light of current genetic linkage maps of the X chromosome and a physical map of the Y chromosome. Our results identify the position of the centromere relative to the mapped genes on the X chromosome. We suggest that the evolution of the S. latifolia Y chromosome has been accompanied by at least one paracentric and one pericentric inversion. These results indicate that large chromosomal rearrangements have played an important role in Y chromosome evolution in S. latifolia and that chromosomal rearrangements are an integral part of sex chromosome evolution.     

Evolutionary strata on the X chromosomes of the dioecious plant Silene latifolia: evidence from new sex-linked genes

Despite its recent evolutionary origin, the sex chromosome system of the plant Silene latifolia shows signs of progressive suppression of recombination having created evolutionary strata of different X-Y divergence on sex chromosomes. However, even after 8 years of effort, this result is based on analyses of five sex-linked gene sequences, and the maximum divergence (and thus the age of this plant's sex chromosome system) has remained uncertain. More genes are therefore needed. Here, by segregation analysis of intron size variants (ISVS) and single nucleotide polymorphisms (SNPs), we identify three new Y-linked genes, one being duplicated on the Y chromosome, and test for evolutionary strata. All the new genes have homologs on the X and Y chromosomes. Synonymous divergence estimated between the X and Y homolog pairs is within the range of those already reported. Genetic mapping of the new X-linked loci shows that the map is the same in all three families that have been studied so far and that X-Y divergence increases with genetic distance from the pseudoautosomal region. We can now conclude that the divergence value is saturated, confirming the cessation of X-Y recombination in the evolution of the sex chromosomes at approximately 10-20 MYA.     

Multicolor FISH mapping of the dioecious model plant,<Emphasis Type="Italic"> Silene latifolia</Emphasis>

Silene latifolia is a key plant model in the study of sex determination and sex chromosome evolution. Current studies have been based on genetic mapping of the sequences linked to sex chromosomes with analysis of their characters and relative positions on the X and Y chromosomes. Until recently, very few DNA sequences have been physically mapped to the sex chromosomes of S. latifolia. We have carried out multicolor fluorescent in situ hybridization (FISH) analysis of S. latifolia chromosomes based on the presence and intensity of FISH signals on individual chromosomes. We have generated new markers by constructing and screening a sample bacterial artificial chromosome (BAC) library for appropriate FISH probes. Five newly isolated BAC clones yielded discrete signals on the chromosomes: two were specific for one autosome pair and three hybridized preferentially to the sex chromosomes. We present the FISH hybridization patterns of these five BAC inserts together with previously described repetitive sequences (X-43.1, 25S rDNA and 5S rDNA) and use them to analyze the S. latifolia karyotype. The autosomes of S. latifolia are difficult to distinguish based on their relative arm lengths. Using one BAC insert and the three repetitive sequences, we have constructed a standard FISH karyotype that can be used to distinguish all autosome pairs. We also analyze the hybridization patterns of these sequences on the sex chromosomes and discuss the utility of the karyotype mapping strategy presented to study sex chromosome evolution and Y chromosome degeneration.Communicated by J.S. Heslop-Harrison     

C-banded karyotypes of two Silene species with heteromorphic sex chromosomes.

Mitotic metaphase chromosomes of Silene latifolia (white campion) and Silene dioica (red campion) were studied and no substantial differences between the conventional karyotypes of these two species were detected. The classification of chromosomes into three distinct groups proposed for S. latifolia by Ciupercescu and colleagues was considered and discussed. Additionally, a new small satellite on the shorter arm of homobrachial chromosome 5 was found. Giemsa C-banded chromosomes of the two analysed species show many fixed and polymorphic heterochromatic bands, mainly distally and centromerically located. Our C-banding studies provided an opportunity to better characterize the sex chromosomes and some autosome types, and to detect differences between the two Silene karyotypes. It was shown that S. latifolia possesses a larger amount of polymorphic heterochromatin, especially of the centromeric type. The two Silene sex chromosomes are easily distinguishable not only by length or DNA amount differences but also by their Giemsa C-banding patterns. All Y chromosomes invariably show only one distally located band, and no other fixed or polymorphic bands on this chromosome were observed in either species. The X chromosomes possess two terminally located fixed bands, and some S. latifolia X chromosomes also have an extra-centric segment of variable length. The heterochromatin amount and distribution revealed by our Giemsa C-banding studies provide a clue to the problem of sex chromosome and karyotype evolution in these two closely related dioecious Silene species.     

Plant sex chromosomes: molecular structure and function

Recent molecular and genomic studies carried out in a number of model dioecious plant species, including Asparagus officinalis, Carica papaya, Silene latifolia, Rumex acetosa and Marchantia polymorpha, have shed light on the molecular structure of both homomorphic and heteromorphic sex chromosomes, and also on the gene functions they have maintained since their evolution from a pair of autosomes. The molecular structure of sex chromosomes in species from different plant families represents the evolutionary pathway followed by sex chromosomes during their evolution. The degree of Y chromosome degeneration that accompanies the suppression of recombination between the Xs and Ys differs among species. The primitive Ys of A. officinalis and C. papaya have only diverged from their homomorphic Xs in a short male-specific and non-recombining region (MSY), while the heteromorphic Ys of S. latifolia, R. acetosa and M. polymorpha have diverged from their respective Xs. As in the Y chromosomes of mammals and Drosophila, the accumulation of repetitive DNA, including both transposable elements and satellite DNA, has played an important role in the divergence and size enlargement of plant Ys, and consequently in reducing gene density. Nevertheless, the degeneration process in plants does not appear to have reached the Y-linked genes. Although a low gene density has been found in the sequenced Y chromosome of M. polymorpha, most of its genes are essential and are expressed in the vegetative and reproductive organs in both male and females. Similarly, most of the Y-linked genes that have been isolated and characterized up to now in S. latifolia are housekeeping genes that have X-linked homologues, and are therefore expressed in both males and females. Only one of them seems to be degenerate with respect to its homologous region in the X. Sequence analysis of larger regions in the homomorphic X and Y chromosomes of papaya and asparagus, and also in the heteromorphic sex chromosomes of S. latifolia and R. acetosa, will reveal the degenerative changes that the Y-linked gene functions have experienced during sex chromosome evolution.     

Recombination is suppressed over a large region of the rainbow trout Y chromosome

The previous genetic mapping data have suggested that most of the rainbow trout sex chromosome pair is pseudoautosomal, with very small X-specific and Y-specific regions. We have prepared an updated genetic and cytogenetic map of the male rainbow trout sex linkage group. Selected sex-linked markers spanning the X chromosome of the female genetic map have been mapped cytogenetically in normal males and genetically in crosses between the OSU female clonal line and four different male clonal lines as well as in outcrosses involving outbred OSU and hybrids between the OSU line and the male clonal lines. The cytogenetic maps of the X and Y chromosomes were very similar to the female genetic map for the X chromosome. Five markers on the male maps are genetically very close to the sex determination locus ( SEX ), but more widely spaced on the female genetic map and on the cytogenetic map, indicating a large region of suppressed recombination on the Y chromosome surrounding the SEX locus. The male map is greatly extended at the telomere. A BAC clone containing the SCAR (sequence characterized amplified region) Omy - 163 marker, which maps close to SEX , was subjected to shotgun sequencing. Two carbonyl reductase genes and a gene homologous to the vertebrate skeletal ryanodine receptor were identified. Carbonyl reductase is a key enzyme involved in production of trout ovarian maturation hormone. This brings the number of type I genes mapped to the sex chromosome to six and has allowed us to identify a region on zebrafish chromosome 10 and medaka chromosome 13 which may be homologous to the distal portion of the long arm of the rainbow trout Y chromosome.     

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.     

DNA diversity in sex-linked and autosomal genes of the plant species Silene latifolia and Silene dioica

The relatively recent origin of sex chromosomes in the plant genus Silene provides an opportunity to study the early stages of sex chromosome evolution and, potentially, to test between the different population genetic processes likely to operate in nonrecombining chromosomes such as Y chromosomes. We previously reported much lower nucleotide polymorphism in a Y-linked gene (SlY1) of the plant Silene latifolia than in the homologous X-linked gene (SlX1). Here, we report a more extensive study of nucleotide diversity in these sex-linked genes, including a larger S. latifolia sample and a sample from the closely related species Silene dioica, and we also study the diversity of an autosomal gene, CCLS37.1. We demonstrate that nucleotide diversity in the Y-linked genes of both S. latifolia and S. dioica is very low compared with that of the X-linked gene. However, the autosomal gene also has low DNA polymorphism, which may be due to a selective sweep. We use a single individual of the related hermaphrodite species Silene conica, as an outgroup to show that the low SlY1 diversity is not due to a lower mutation rate than that for the X-linked gene. We also investigate several other possibilities for the low SlY1 diversity, including differential gene flow between the two species for Y-linked, X-linked, and autosomal genes. The frequency spectrum of nucleotide polymorphism on the Y chromosome deviates significantly from that expected under a selective-sweep model. However, we detect population subdivision in both S. latifolia and S. dioica, so it is not simple to test for selective sweeps. We also discuss the possibility that Y-linked diversity is reduced due to highly variable male reproductive success, and we conclude that this explanation is unlikely.     

Duplicative transfer of a MADS box gene to a plant Y chromosome

Y chromosomes carry genes with functions in male reproduction and often have few other loci. Their evolution and the causes of genetic degeneration are of great interest. In addition to genetic degeneration, the acquisition of autosomal genes may be important in Y chromosome evolution. We here report that the dioecious plant Silene latifolia harbors a complete MADS box gene, SlAP3Y, duplicated onto the Y chromosome. This gene has no X-linked homologs but only an autosomal paralog, SlAP3A, and sequence divergence suggests that the duplication is a quite old event that occurred soon after the evolution of the sex chromosomes. Evolutionary sequence analyses using homologs of closely related species, including hermaphroditic Silene conica and dioecious Silene dioica and Silene diclinis, suggest that both SlAP3A and SlAP3Y genes encode functional proteins. Indeed, quantitative RT-PCR and in situ hybridization analyses showed that SlAP3A is expressed specifically in developing petals, but SlAP3Y is much more strongly expressed in developing stamens. The S. conica homolog, ScAP3A, is expressed in developing petals, suggesting subfunctionalization with evolution of male-specific functions, possibly due to evolutionary change in regulatory elements. Our results suggest that the acquisition of autosomal genes is an important event in the evolution of plant Y chromosomes.     

Sex Determination by Sex Chromosomes in Dioecious Plants

Abstract: Sex chromosomes have been reported in several dioecious plants. The most general system of sex determination with sex chromosomes is the XY system, in which males are the heterogametic sex and females are homogametic. Genetic systems in sex determination are divided into two classes including an X chromosome counting system and an active Y chromosome system. Dioecious plants have unisexual flowers, which have stamens or pistils. The development of unisexual flowers is caused by the suppression of opposite sex primordia. The expression of floral organ identity genes is different between male and female flower primordia. However, these floral organ identity genes show no evidence of sex chromosome linkage. The Y chromosome of Rumex acetosa contains Y chromosome-specific repetitive sequences, whereas the Y chromosome of Silene latifolia has not accumulated chromosome-specific repetitive sequences. The different degree of Y chromosome degeneration may reflect on evolutionary time since the origination of dioecy. The Y chromosome of S. latifolia functions in suppression of female development and initiation and completion of anther development. Analyses of mutants suggested that female suppressor and stamen promoter genes are localized on the Y chromosome. Recently, some sex chromosome-linked genes were isolated from flower buds of S. latifolia.