Isolation of Y Chromosome-Specific Sequences from Silene Latifolia and Mapping of Male Sex-Determining Genes Using Representational Difference Analysis

The genomic subtraction method representational difference analysis (RDA) was used to identify male-specific restriction fragments in the dioecious plant Silene latifolia. Male-specific restriction fragments are linked to the male sex chromosome (the Y chromosome). Four RDA-derived male-specific restriction fragments were used to identify polymorphisms in a collection of X-ray-generated mutant plants with either hermaphroditic or asexual flowers. Some of the mutants have cytologically detectable deletions in the Y chromosome that were correlated with loss of male-specific restriction fragments. One RDA-derived probe detected a restriction fragment present in all mutants, indicating that each has retained Y chromosomal DNA. The other three probes detected genomic fragments that were linked in a region deleted in some hermaphroditic and some asexual mutants. Based on the mutant phenotypes and the correlation of cytologically visible deletions with loss of male-specific restriction fragments, these markers were assigned to positions on the Y chromosome close to the carpel suppression locus. This RDA mapping also revealed a Y-linked locus, not previously described, which is responsible for early stamen development.     

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.     

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.     

The inter-specific hybrid Silene latifolia x S. viscosa reveals early events of sex chromosome evolution

The dioecious plant species Silene latifolia has a sex determination mechanism based on an active Y chromosome. Here, we used inter-specific hybrids in the genus Silene to study the effects of gene complexes on the Y chromosome. If the function of Y-linked genes has been maintained in the same state as in the hermaphrodite progenitor species, it should be possible to substitute such genes by genes coming from a related hermaphrodite species. In the inter-specific hybrid, S. latifolia x S. viscosa, anthers indeed develop far beyond the early bilobal stage characteristic of XX S. latifolia female plants. The S. viscosa genome can thus replace the key sex determination gene whose absence abolishes early stamen development in females (loss of the stamen-promoting function, SPF), so that hybrid plants are morphologically hermaphrodite. However, the hybrids have two anther development defects, loss of adhesion of the tapetum to the endothecium, and precocious endothecium maturation. Both these defects were also found in independent Y-chromosome deletion mutants of S. latifolia. The data support the hypothesis that the evolution of complete gender dimorphism from hermaphroditism involved a major largely recessive male-sterility factor that created females, and the appearance of new, dominant genes on the Y chromosome, including both the well-documented gynoecium-suppressing factor, and two other Y specific genes promoting anther development.     

Floral development of an asexual and female-like mutant carrying two deletions in gynoecium-suppressing and stamen-promoting functional regions on the Y chromosome of the dioecious plant Silene latifolia

Sexual dimorphism is controlled by genes on the Y chromosome in the dioecious plant Silene latifolia. K034 is the first mutant with female flowers and asexual flowers in one individual. Its stamens are suppressed completely, and its gynoecium exhibits two suppression patterns. One gynoecium resembles a thin rod, as in wild-type males (asexual flower); the other is imperfectly suppressed, having 1-3 carpels (female-like flower). The ratio of these patterns was 9 : 1. To exclude the possibility of chimerism in K034, we crossed a female-like flower of K034 with a wild-type male. Progeny obtained from this crossing had asexual and female-like flowers in one individual. This two-flower-type phenotype was inherited without separating. To examine the identity of flower organs in K034, we analyzed the development of asexual and female-like flowers using scanning electron microscopy and in situ hybridization with SLM1 and SLM2 (orthologs of AGAMOUS and PISTILLATA, respectively) as probes. Mitotic spreads of root tip chromosomes from hairy root cultures showed that K034 had 25 chromosomes. Fluorescent in situ hybridization analysis, using a subtelomeric repetitive sequence (KpnI subfamily) as a probe, indicated that K034 possessed two X chromosomes and one Y chromosome (Y(d)), of which Y(d) had been rearranged to lose the pseudoautosomal region (PAR). PCR analysis using Y-specific sequence-tagged site (STS) markers clarified that Y(d) of K034 had two other deletions in gynoecium-suppressing and stamen-promoting regions. It is reasonable to suggest that these sex chromosomal abnormalities resulted in two abnormal sexual phenotypes: the asexual and imperfect female (female-like) flowers in K034.     

Defining regions and rearrangements of the Silene latifolia Y chromosome

We combine data from published marker genotyping of three sets of S. latifolia Y chromosome deletion mutants with changed sex phenotypes and add genotypes for several new genic markers to refine the deletion map of the Y chromosome and compare it with the X chromosome genetic map. We conclude that the Y chromosome of this species has been derived through multiple rearrangements of the ancestral gene arrangement and that none of the rearrangements so far detected was involved in stopping X-Y recombination. Different Y genotypes may also differ in their gene content and possibly arrangements, suggesting that mapping the Y-linked sex-determining genes will be difficult, even if many further genic markers are obtained. Even in determining the map of Y chromosome markers to discover all the rearrangements, physical mapping by FISH or other experiments will be essential. Future deletion mapping work should ensure that markers are studied in the parents of deletion mutants and should probably include additional deletions that were not ascertained by causing mutant sex phenotypes.     

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.     

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.     

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.     

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.     

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.     

Genetic and functional analysis of DD44, a sex-linked gene from the dioecious plant Silene latifolia,provides clues to early events in sex chromosome evolution

Silene latifolia is a dioecious plant with heteromorphic sex chromosomes. The sex chromosomes of S. latifolia provide an opportunity to study the early events in sex chromosome evolution because of their relatively recent emergence. In this article, we present the genetic and physical mapping, expression analysis, and molecular evolutionary analysis of a sex-linked gene from S. latifolia, DD44 (Differential Display 44). DD44 is homologous to the oligomycin sensitivity-conferring protein, an essential component of the mitochondrial ATP synthase, and is ubiquitously expressed in both sexes. We have been able to genetically map DD44 to a region of the Y chromosome that is genetically linked to the carpel-suppressing locus. Although we have physically mapped DD44 to the distal end of the long arm of the X chromosome using fluorescence in situ hybridization (FISH), DD44 maps to the opposite arm of the Y chromosome as determined by our genetic map. These data suggest that chromosomal rearrangements have occurred on the Y chromosome, which may have contributed to the genetic isolation of the Y chromosome. We discuss the implications of these results with respect to the structural and functional evolution of the S. latifolia Y chromosome.     

Sex determination in papaya

Sex determination is an intriguing system in trioecious papaya. Over the past seven decades various hypotheses, based on the knowledge and information available at the time, have been proposed to explain the genetics of the papaya's sex determination. These include a single gene with three alleles, a group of closely linked genes, a genic balance of sex chromosome over autosomes, classical XY chromosomes, and regulatory elements of the flower development pathway. Recent advancements in genomic technology make it possible to characterize the genomic region involved in sex determination at the molecular level. High density linkage mapping validated the hypothesis that predicted recombination suppression at the sex determination locus. Physical mapping and sample sequencing of the non-recombination region led to the conclusion that sex determination is controlled by a pair of primitive sex chromosomes with a small male-specific region (MSY) of the Y chromosome. We now postulate that two sex determination genes control the sex determination pathway. One, a feminizing or stamen suppressor gene, causes stamen abortion before or at flower inception while the other, a masculinizing or carpel suppressor gene, causes carpel abortion at a later flower developmental stage. Detailed physical mapping is beginning to reveal structural details about the sex determination region and sequencing is expected to uncover candidate sex determining genes. Cloning of the sex determination genes and understanding the sex determination process could have profound application in papaya production.     

A rearrangement of the Z chromosome topology influences the sex-linked gene display in the European corn borer, <Emphasis Type="Italic">Ostrinia nubilalis</Emphasis>

Males are homogametic (ZZ) and females are heterogametic (WZ) with respect to the sex chromosomes in many species of butterflies and moths (insect order Lepidoptera). Genes on the Z chromosome influence traits involved in larval development, environmental adaptation, and reproductive isolation. To facilitate the investigation of these traits across Lepidoptera, we developed 43 degenerate primer pairs to PCR amplify orthologs of 43 Bombyx mori Z chromosome-linked genes. Of the 34 orthologs that amplified by PCR in Ostrinia nubilalis, 6 co-segregated with the Z chromosome anchor markers kettin (ket) and lactate dehydrogenase (ldh), and produced a consensus genetic linkage map of ~89 cM in combination with 5 AFLP markers. The O. nubilalis and B. mori Z chromosomes are comparatively co-linear, although potential gene inversions alter terminal gene orders and a translocation event disrupted synteny at one chromosome end. Compared to B. mori orthologs, O. nubilalis Z chromosome-linked genes showed conservation of tissue-specific and growth-stage-specific expression, although some genes exhibited species-specific expression across developmental stages or tissues. The O. nubilalis Z chromosome linkage map provides new tools for isolating quantitative trait loci (QTL) involved in sex-linked traits that drive speciation and it exposes genome rearrangements as a possible mechanism for differential gene regulation in Lepidoptera.     

Sexual dimorphism in white campion: deletion on the Y chromosome results in a floral asexual phenotype

White campion is a dioecious plant with heteromorphic X and Y sex chromosomes. In male plants, a filamentous structure replaces the pistil, while in female plants the stamens degenerate early in flower development. Asexual (asx) mutants, cumulating the two developmental defects that characterize the sexual dimorphism in this species, were produced by gamma ray irradiation of pollen and screening in the M1 generation. The mutants harbor a novel type of mutation affecting an early function in sporogenous/parietal cell differentiation within the anther. The function is called stamen-promoting function (SPF). The mutants are shown to result from interstitial deletions on the Y chromosome. We present evidence that such deletions tentatively cover the central domain on the (p)-arm of the Y chromosome (Y2 region). By comparing stamen development in wild-type female and asx mutant flowers we show that they share the same block in anther development, which results in the production of vestigial anthers. The data suggest that the SPF, a key function(s) controlling the sporogenous/parietal specialization in premeiotic anthers, is genuinely missing in females (XX constitution). We argue that this is the earliest function in the male program that is Y-linked and is likely responsible for "male dimorphism" (sexual dimorphism in the third floral whorl) in white campion. More generally, the reported results improve our knowledge of the structural and functional organization of the Y chromosome and favor the view that sex determination in this species results primarily from a trigger signal on the Y chromosome (Y1 region) that suppresses female development. The default state is therefore the ancestral hermaphroditic state.     

The Y chromosome-specific STS marker MS2 and its peripheral regions on the Y chromosome of the dioecious plant Silene latifolia.

Sex determination in Silene latifolia uses the XX/XY system. The recent evolution of dioecy in S. latifolia provides a unique opportunity to study the early stages of Y chromosome evolution. However, the current Y chromosome map still contains many large gaps with no available markers. In this study, a sequence tagged site (STS) marker, MS2, was isolated and mapped to the same locus as L8 on the Y chromosome. To investigate the peripheral regions of MS2, a bacterial artificial chromosome (BAC) library was constructed from a male plant, and the BAC clone containing MS2 (MS2-9d12F) was isolated from 32 640 clones with an average insert size of 115 kb. A 109-kb insert of the BAC clone was analyzed. BLASTX analysis showed 11 sequences similar to some known proteins, most of which are retrotransposon-like elements. The ORF Finder predicted 9 ORFs within MS2-9d12F. RT-PCR analyses revealed that only 4 of the 9 predicted ORFs are expressed in both male and female plants. These 4 ORFs are candidates for genes having counterparts on both the X and Y chromosomes. Dot-matrix plot analysis and a BLASTN search revealed LTR-like sequences close to the retrotransposon-like elements and high similarity to 3 known genomic sequences of S. latifolia. These results suggest an accumulation of retrotransposons and segmental duplications in peripheral regions of MS2 during the early stage of sex chromosome evolution.