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.     

C-Banding/DAPI and in situ hybridization reflect karyotype structure and sex chromosome differentiation in Humulus japonicus Siebold & Zucc

Japanese hop (Humulus japonicus Siebold & Zucc.) was karyotyped by chromosome measurements, fluorescence in situ hybridization with rDNA and telomeric probes, and C-banding/DAPI. The karyotype of this species consists of sex chromosomes (XX in female and XY1Y2 in male plants) and 14 autosomes difficult to distinguish by morphology. The chromosome complement also shows a rather monotonous terminal distribution of telomeric repeats, with the exception of a pair of autosomes possessing an additional cluster of telomeric sequences located within the shorter arm. Using C-banding/DAPI staining and 5S and 45S rDNA probes we constructed a fluorescent karyotype that can be used to distinguish all autosome pairs of this species except for the 2 largest autosome pairs, lacking rDNA signals and having similar size and DAPI-banding patterns. Sex chromosomes of H. japonicus display a unique banding pattern and different DAPI fluorescence intensity. The X chromosome possesses only one brightly stained AT-rich terminal segment, the Y1 has 2 such segments, and the Y2 is completely devoid of DAPI signal. After C-banding/DAPI, both Y chromosomes can be easily distinguished from the rest of the chromosome complement by the increased fluorescence of their arms. We discuss the utility of these methods for studying karyotype and sex chromosome evolution in hops.     

A genetic method to resolve gender complements investigations on sex ratios in Rumex acetosa

The perennial dioecious weed, Rumex acetosa, possesses sex chromosomes (XX in females, XY1Y2 in males). Yet, the operational sex ratios are female-biased. Until now, sex ratio studies in R. acetosa, as in most plants, have relied on data obtained at sexual maturity. To resolve gender among the seeds and nonflowering plants of R. acetosa, a genetic method involving a DNA marker located on both Y chromosomes has now been developed and applied. The results suggest that the sex ratios are about 1 : 1 in the whole seed pool, but that a significant female bias develops by the time of flowering. Since the age of sexually mature plants is two years or more, the time frame during which the female bias present at sexual maturity develops can be several years. It appears that male seeds germinate at a lower rate and males suffer from a greater mortality during the years-long lifespan of R. acetosa. However, there are no considerable sex-related differences in vegetative vigour.     

Accumulation of Y-specific satellite DNAs during the evolution of <Emphasis Type="Italic">Rumex acetosa</Emphasis> sex chromosomes

The study of the molecular structure of young heteromorphic sex chromosomes of plants has shed light on the evolutionary forces that control the differentiation of the X and Y during the earlier stages of their evolution. We have used the model plant Rumex acetosa, a dioecious species with multiple sex chromosomes, 2n = 12 + XX female and 2n = 12 + XY₁Y₂ male, to analyse the significance of repetitive DNA accumulation during the differentiation of the Y. A bulk segregant analysis (BSA) approach allowed us to identify and isolate random amplified polymorphic DNA (RAPD) markers linked to the sex chromosomes. From a total of 86 RAPD markers in the parents, 6 markers were found to be linked to the Ys and 1 to the X. Two of the Y-linked markers represent two AT-rich satellite DNAs (satDNAs), named RAYSII and RAYSIII, that share about 80% homology, as well as with RAYSI, another satDNA of R. acetosa. Fluorescent in situ hybridisation demonstrated that RAYSII is specific for Y₁, whilst RAYSIII is located in different clusters along Y₁ and Y₂. The two satDNAs were only detected in the genome of the dioecious species with XX/XY₁Y₂ multiple sex chromosome systems in the subgenus Acetosa, but were absent from other dioecious species with an XX/XY system of the subgenera Acetosa or Acetosella, as well as in gynodioecious or hermaphrodite species of the subgenera Acetosa, Rumex and Platypodium. Phylogenetic analysis with different cloned monomers of RAYSII and RAYSIII from both R. acetosa and R. papillaris indicate that these two satDNAs are completely separated from each other, and from RAYSI, in both species. The three Y-specific satDNAs, however, evolved from an ancestral satDNA with repeating units of 120 bp, through intermediate satDNAs of 360 bp. The data therefore support the idea that Y-chromosome differentiation and heterochromatinisation in the Rumex species having a multiple sex chromosome system have occurred by different amplification events from a common ancestral satDNA. Since dioecious species with multiple XX/XY₁Y₂ sex chromosome systems of the section Acetosa appear to have evolved from dioecious species with an XX/XY system, the amplification of tandemly repetitive elements in the Ys of the section Acetosa is a recent evolutionary process that has contributed to an increase in the size and differentiation of the already non-recombining Y chromosomes.     

Evolution of ZZ/ZW and XX/XY sex-determination systems in the closely related medaka species, <Emphasis Type="Italic">Oryzias hubbsi</Emphasis> and <Emphasis Type="Italic">O. dancena</Emphasis>

A DM-domain gene on the Y chromosome was identified as the sex-determining gene in the medaka, Oryzias latipes, and named DMY (also known as dmrt1bY). However, this gene is absent in most Oryzias fishes, suggesting that closely related species have another sex-determining gene. In fact, it has been demonstrated that the Y chromosome in O. dancena is not homologous to that in O. latipes, whereas both species have an XX/XY sex-determination system. Through a progeny test of sex-reversed fish and a linkage analysis of isolated sex-linked DNA markers, we show that O. hubbsi, which is one of the most closely related species to O. dancena, has a ZZ/ZW system. In addition, genetic and fluorescence in situ hybridization mapping of the sex-linked markers revealed that sex chromosomes in O. hubbsi and O. dancena are not homologous, indicating different origins of these ZW and XY sex chromosomes. Furthermore, we found that O. hubbsi has morphologically heteromorphic sex chromosomes, in which the W chromosome has 4,6-diamidino-2-phenylindole (DAPI)-positive heterochromatin blocks and is larger than the Z chromosome, although such differentiated sex chromosomes have not been observed in other Oryzias species. These findings suggest that a variety of sex-determining mechanisms and sex chromosomes have evolved in Oryzias.     

Heterogeneous Histories of Recombination Suppression on Stickleback Sex Chromosomes

How consistent are the evolutionary trajectories of sex chromosomes shortly after they form? Insights into the evolution of recombination, differentiation, and degeneration can be provided by comparing closely related species with homologous sex chromosomes. The sex chromosomes of the threespine stickleback (Gasterosteus aculeatus) and its sister species, the Japan Sea stickleback (G. nipponicus), have been well characterized. Little is known, however, about the sex chromosomes of their congener, the blackspotted stickleback (G. wheatlandi). We used pedigrees to obtain experimentally phased whole genome sequences from blackspotted stickleback X and Y chromosomes. Using multispecies gene trees and analysis of shared duplications, we demonstrate that Chromosome 19 is the ancestral sex chromosome and that its oldest stratum evolved in the common ancestor of the genus. After the blackspotted lineage diverged, its sex chromosomes experienced independent and more extensive recombination suppression, greater X–Y differentiation, and a much higher rate of Y degeneration than the other two species. These patterns may result from a smaller effective population size in the blackspotted stickleback. A recent fusion between the ancestral blackspotted stickleback Y chromosome and Chromosome 12, which produced a neo-X and neo-Y, may have been favored by the very small size of the recombining region on the ancestral sex chromosome. We identify six strata on the ancestral and neo-sex chromosomes where recombination between the X and Y ceased at different times. These results confirm that sex chromosomes can evolve large differences within and between species over short evolutionary timescales.     

Distribution and Evolution of Repeated Sequences in Genomes of Triatominae (Hemiptera-Reduviidae) Inferred from Genomic In Situ Hybridization

The subfamily Triatominae, vectors of Chagas disease, comprises 140 species characterized by a highly homogeneous chromosome number. We analyzed the chromosomal distribution and evolution of repeated sequences in Triatominae genomes by Genomic in situ Hybridization using Triatoma delpontei and Triatoma infestans genomic DNAs as probes. Hybridizations were performed on their own chromosomes and on nine species included in six genera from the two main tribes: Triatomini and Rhodniini. Genomic probes clearly generate two different hybridization patterns, dispersed or accumulated in specific regions or chromosomes. The three used probes generate the same hybridization pattern in each species. However, these patterns are species-specific. In closely related species, the probes strongly hybridized in the autosomal heterochromatic regions, resembling C-banding and DAPI patterns. However, in more distant species these co-localizations are not observed. The heterochromatic Y chromosome is constituted by highly repeated sequences, which is conserved among 10 species of Triatomini tribe suggesting be an ancestral character for this group. However, the Y chromosome in Rhodniini tribe is markedly different, supporting the early evolutionary dichotomy between both tribes. In some species, sex chromosomes and autosomes shared repeated sequences, suggesting meiotic chromatin exchanges among these heterologous chromosomes. Our GISH analyses enabled us to acquire not only reliable information about autosomal repeated sequences distribution but also an insight into sex chromosome evolution in Triatominae. Furthermore, the differentiation obtained by GISH might be a valuable marker to establish phylogenetic relationships and to test the controversial origin of the Triatominae subfamily.     

Preliminary Observatiun of the Sex-Chromosomes of Rumex acetosa in China

The Rumex acetosa L. in China possesses sex-chromosomes 2n=12A+XX in female plant and 2n=12A+XY1Y2 in male plant. The sex-chromosomes of male plant exhibit a chain of trivalent (Y1-X-Y2) in diakinesis of PMC meiosis. At metaphase Ⅰ, they make a V-shaped or a ring configuration. Some of them may form a XY-bivalent and a Y-univalent. Finally two kinds of reale gametes e.g. 6A+X and .6A+YY, are produced. Monoecious plant may be found in R. acetosa and has 22 chromosomes (22=18A+ XX+YY). This is a triplont with sterility. Cells with different chromosome number in the same plant have also seen in R. acetosa, and such plant never blooms in its life.     

Rumex acetosa Y chromosomes: constitutive or facultative heterochromatin?

Condensed Y chromosomes in Rumex acetosa L. root-tip nuclei were studied using 5-azaC treatment and immunohistochemical detection of methylated histones. Although Y chromosomes were decondensed within root meristem in vivo, they became condensed and heteropycnotic in roots cultured in vitro. 5-azacytidine (5-azaC) treatment of cultured roots caused transitional dispersion of their Y chromosome bodies, but 7 days after removal of the drug from the culture medium, Y heterochromatin recondensed and again became visible. The response of Rumex sex chromatin to 5-azaC was compared with that of condensed segments of pericentromeric heterochromatin in Rhoeo spathacea (Sw.) Steam roots. It was shown that Rhoeo chromocentres, composed of AT-rich constitutive heterochromatin, did not undergo decondensation after 5-azaC treatment. The Y-bodies observed within male nuclei of R. acetosa were globally enriched with H3 histone, demethylated at lysine 4 and methylated at lysine 9. This is the first report of histone tail-modification in condensed sex chromatin in plants. Our results suggest that the interphase condensation of Y chromosomes in Rumex is facultative rather than constitutive. Furthermore, the observed response of Y-bodies to 5-azaC may result indirectly from demethylation and the subsequent altered expression of unknown genes controlling tissue-specific Y-inactivation as opposed to the global demethylation of Y-chromosome DNA.     

Sex chromosome differentiation in Belostoma (Insecta: Heteroptera: Belostomatidae)

Belostoma, a genus of the family Belostomatidae, includes species of great ecological importance as biocontrol agents. Few species of these species have been the subject of cytogenetic analyses. Karyotypic evolution in this genus involves agmatoploidy and simploidy; there are also different sex chromosome systems. We examined two Belostoma species (B. dilatatum and B. candidulum) collected from the Paranapanema River Basin (Brazil). Mitotic and meiotic analysis revealed 2n(♂) = 26 + X(1)X(2)X(3)Y for B. dilatatum and 2n(♂) = 14 + XY for B. candidulum; both karyotypes have holokinetic chromosomes. Differences in heterochromatin distribution were also observed between the species, besides variation in the localization of CMA(3)(+)/DAPI(-) blocks. The existence of different types of sex chromosome systems in these species was confirmed based on arrangements of the chromosomes in different meiotic stages. We identified a new sex system in B. dilatatum, and make the first cytogenetic report on B. candidulum.     

Excessive variation in Y chromosomal DNA in Rumex acetosa (Polygonaceae)

Rumex acetosa is one of the few angiosperms that possesses sex chromosomes. The same types of abundant repetitive sequences cover both heterochromatic Y chromosomes present in males. The aim of this study was to investigate genetic variation in paternally inherited Y chromosomal DNA and in maternally inherited cpDNA, and to find out whether the examined genomic regions are suited to a phylogeographic study in R. acetosa. DNA sequence polymorphisms present in the 850-bp heterochromatic segment on the Y chromosomes were compared to variation in the 409-bp long chloroplast section (trnL- trnF spacer) in R. acetosa originating from several European locations and from the Altai mountains in Russia. A great amount of genetic variation was detected within the Y chromosomal region while only four chloroplast genotypes were detected. Although the chloroplast haplotypes possessed some geographic pattern, no clear phylogeographic pattern was detected based on the variable Y chromosomes. The mean Y chromosomal nucleotide diversity among all samples equaled 6.6 %, and the mean proportion of polymorphic sites per individual equaled 8.2 % among SNP sites and 1.7 % among all sites investigated. The high number of substitutions detected in the Y chromosomal DNA shows that this heterochromatic sequence has a high mutation rate. The diversity pattern indicates that gene flow via pollen is extensive and it blurs any geographical pattern in the Y chromosomal variation. The high number of repeats and uncertainty concerning the extent of recombination between the two Y chromosomes impair the usability of the Y chromosomal segment for phylogeographic or population genetic studies.     

Does differential selection on the 5S rDNA explain why the rainbow trout sex chromosome heteromorphism is not linked to the SEX locus?

Many but not all rainbow trout strains have morphologically distinguishable sex chromosomes. In these strains, the short arm of the X has multiple copies of 5S rDNA and a bright DAPI band near the centromere, both of which are missing from the Y chromosome, which has a very small short arm. We examined the presence of these markers using fluorescence in situ hybridization (FISH) in four different YY clonal lines derived from different strains and compared the results with sexed fish of the Donaldson strain with the normal X/Y heteromorphism. The Y chromosome in two of the YY clonal lines (Arlee and Swanson) is indistinguishable from the X chromosome and it is positive for 5S rDNA and the DAPI bright band. On the other hand, both 5S rDNA sequences and the DAPI band were not found on the Y chromosome in Hot Creek and Clearwater which have the normal Y. Thus the presence of these two cytogenetic markers may account for the size difference between the short arm of the X and Y chromosome found in most rainbow trout strains. In fishes the expression of one type of 5S rRNA is restricted to oocytes and previous work suggests that although XX males are fairly common, XY females are rare, implying a selective disadvantage for XY females. A hypothesis is presented to explain why this sex chromosome heteromorphism is not closely linked to the SEX locus, which is found on the long arm of the Y chromosome in rainbow trout.     

Genomics of sex chromosomes

Sex chromosomes in plants and animals are distinctive, not only because of their gender-determining role but also for genomic features that reflect their evolutionary history. The genomic sequences in the ancient sex chromosomes of humans and in the incipient sex chromosomes of medaka, stickleback, papaya, and poplar exhibit unusual features as consequences of their evolution. These include the enormous palindrome structure in human MSY, a duplicated genomic fragment that evolved into a Y chromosome in medaka, and a 700 kb extra telomeric sequence of the W chromosome in poplar. Comparative genomic analysis of ancient and incipient sex chromosomes highlights common features that implicate the selection forces that shaped them, even though evolutionary origin, pace, and fate vary widely among individual sex-determining systems.     

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.     

Chromosomal divergence and evolutionary inferences in Rhodniini based on the chromosomal location of ribosomal genes

In this study, we used fluorescence in situ hybridisation to determine the chromosomal location of 45S rDNA clusters in 10 species of the tribe Rhodniini (Hemiptera: Reduviidae: Triatominae). The results showed striking inter and intraspecific variability, with the location of the rDNA clusters restricted to sex chromosomes with two patterns: either on one (X chromosome) or both sex chromosomes (X and Y chromosomes). This variation occurs within a genus that has an unchanging diploid chromosome number (2n = 22, including 20 autosomes and 2 sex chromosomes) and a similar chromosome size and genomic DNA content, reflecting a genome dynamic not revealed by these chromosome traits. The rDNA variation in closely related species and the intraspecific polymorphism in Rhodnius ecuadoriensis suggested that the chromosomal position of rDNA clusters might be a useful marker to identify recently diverged species or populations. We discuss the ancestral position of ribosomal genes in the tribe Rhodniini and the possible mechanisms involved in the variation of the rDNA clusters, including the loss of rDNA loci on the Y chromosome, transposition and ectopic pairing. The last two processes involve chromosomal exchanges between both sex chromosomes, in contrast to the widely accepted idea that the achiasmatic sex chromosomes of Heteroptera do not interchange sequences.     

Molecular cytogenetic analysis of supernumerary heterochromatic segments in Rumex acetosa.

The dioecious plant Rumex acetosa shows intraspecific karyotype variation, caused by supernumerary heterochromatic segments or DAPI (4',6-diamidino-2 phenylindole)-bands at the ends of the short arms of three pairs of autosomes. A DNA sequence (RAE730) specific to the supernumerary heterochromatic segments was cloned and sequenced. RAE730 was about 730 bp and AT-rich (71% AT-content). Using fluorescence in situ hybridization (FISH), RAE730 was localized in the supernumerary DAPI-positive heterochromatic segments on several mitotic chromosomes and chromocenters in interphase nuclei, but not in the DAPI-bands of Y or B chromosomes. RAE730 was tandemly arranged in the genome, and the copy number varied between plants from 40000 to 304000 copies per 2C, corresponding to the relative amount of supernumerary heterochromatic segments per genome. These results indicate that the karyotype variation caused by the supernumerary heterochromatic segment was generated by amplification or reduction of the tandem repeats of RAE730.     

Sex-specific SCAR markers in the dioecious plant<Emphasis Type="Italic"> Rumex nivalis</Emphasis> (Polygonaceae) and implications for the evolution of sex chromosomes

We developed SCAR primers based on isolated and sequenced male-specific fragments as identified in an AFLP analysis of the dioecious plant Rumex nivalis. PCR amplification using these primers on females and males resulted in fragments exclusively present in males. Co-amplification of the nuclear rDNA internal transcribed spacer 2 together with the male-specific fragment was applied as an internal control for successful PCR reactions to avoid false-negative sex scoring. With a length of about 164 bp, the AFLP fragment was of a similar size as the tandemly arranged, repetitive sequences of 180 bp located on the Y chromosomes of Rumex acetosa. The genetic distances between the Y-chromosomal sequences of R. nivalis and R. acetosa, both members of the section Acetosa, were substantial. We found intra-individual divergence among cloned sequences of the male-specific fragment in R. nivalis. The patterns of interspecific and intra-individual sequence variation found are in accordance with proposed modes of the evolution of sex chromosomes. Y chromosomes possibly arose only once in the genus Rumex and consist mainly of heterochromatic DNA. Due to the almost complete absence of selection on them, Y chromosomes are likely to accumulate large numbers of mutations.Communicated by R. Hagemann     

Fundamentally different repetitive element composition of sex chromosomes in Rumex acetosa

Background and AimsDioecious species with well-established sex chromosomes are rare in the plant kingdom. Most sex chromosomes increase in size but no comprehensive analysis of the kind of sequences that drive this expansion has been presented. Here we analyse sex chromosome structure in common sorrel (Rumex acetosa), a dioecious plant with XY₁Y₂ sex determination, and we provide the first chromosome-specific repeatome analysis for a plant species possessing sex chromosomes.MethodsWe flow-sorted and separately sequenced sex chromosomes and autosomes in R. acetosa using the two-dimensional fluorescence in situ hybridization in suspension (FISHIS) method and Illumina sequencing. We identified and quantified individual repeats using RepeatExplorer, Tandem Repeat Finder and the Tandem Repeats Analysis Program. We employed fluorescence in situ hybridization (FISH) to analyse the chromosomal localization of satellites and transposons.Key ResultsWe identified a number of novel satellites, which have, in a fashion similar to previously known satellites, significantly expanded on the Y chromosome but not as much on the X or on autosomes. Additionally, the size increase of Y chromosomes is caused by non-long terminal repeat (LTR) and LTR retrotransposons, while only the latter contribute to the enlargement of the X chromosome. However, the X chromosome is populated by different LTR retrotransposon lineages than those on Y chromosomes.ConclusionsThe X and Y chromosomes have significantly diverged in terms of repeat composition. The lack of recombination probably contributed to the expansion of diverse satellites and microsatellites and faster fixation of newly inserted transposable elements (TEs) on the Y chromosomes. In addition, the X and Y chromosomes, despite similar total counts of TEs, differ significantly in the representation of individual TE lineages, which indicates that transposons proliferate preferentially in either the paternal or the maternal lineage.     

Further evidence for early sex chromosome differentiation of anuran species

Chromosome banding and meiotic evidence show that XX/XY systems found in two Eupsophus species (Amphibia-Leptodactylidae) represent early stages of sex chromosome differentiation. Pair 14 is heteromorphic in E. migueli males and represents the heterochromosomes. In E. roseus this pair is metacentric and does not show heteromorphism. Paracentromeric constitutive heterochromatin is present in all chromosomes except in the E. migueli and E. roseus metacentric Y chromosomes. Constitutive heterochromatin loss is the structural modification responsible for Y chromosome differentiation. Pericentric inversions may have modified the morphology of the X chromosome of Eupsophus species.