Plant Science 236 (2015) 126–135
Contents lists available at ScienceDirect
Plant Science
journal homepage: www.elsevier.com/locate/plantsci
Review
The genomics of plant sex chromosomes
Boris Vyskot∗
, Roman Hobza
Department of Plant Developmental Genetics, Institute of Biophysics, Academy of Sciences of the Czech Republic, 61265 Brno, Czech Republic
a r t i c l e i n f o
Article history:
Received 27 December 2014
Received in revised form 27 February 2015
Accepted 26 March 2015
Available online 2 April 2015
Keywords:
Sex chromosomes
Nuclear genome
Plastid DNA
Recombination
Transposable elements
Silene latifolia
Rumex acetosa
a b s t r a c t
Around six percent of flowering species are dioecious, with separate female and male individuals. Sex
determination is mostly based on genetics, but morphologically distinct sex chromosomes have only
evolved in a few species. Of these, heteromorphic sex chromosomes have been most clearly described in
the two model species – Silene latifolia and Rumex acetosa. In both species, the sex chromosomes are the
largest chromosomes in the genome. They are hence easily distinguished, can be physically separated
and analyzed. This review discusses some recent experimental data on selected model dioecious species,
with a focus on S. latifolia. Phylogenetic analyses show that dioecy in plants originated independently and
repeatedly even within individual genera. A cogent question is whether there is genetic degeneration of
the non-recombining part of the plant Y chromosome, as in mammals, and, if so, whether reduced levels
of gene expression in the heterogametic sex are equalized by dosage compensation. Current data provide
no clear conclusion. We speculate that although some transcriptome analyses indicate the first signs
of degeneration, especially in S. latifolia, the evolutionary processes forming plant sex chromosomes in
plants may, to some extent, differ from those in animals.
© 2015 Published by Elsevier Ireland Ltd.
Contents
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126
2. Evolution of sex chromosomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127
3. Case studies: recent advances through genomic studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127
4. Silene as a model system. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130
5. Y-chromosome degeneration and dosage compensation? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 132
6. Current research strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134
References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134
1. Introduction
Genetic recombination is a key process in the variation achieved
during meiosis when paternal and maternal chromosomes are
combined and exchanged. Random combination of male and female
gametes completes the variation necessary for evolution. However,
there are exceptions demonstrating other mechanisms for
ensuring genetic variation. For example, bdelloid rotifers reproduce
asexually and are propagated by parthenogenesis without
meiosis. Their genome is totally restructured during anhydrobiosis,
including the integration of foreign DNA sequences from adjacent
∗ Corresponding author. Tel.: +420 541240500.
E-mail address: vyskot@ibp.cz (B. Vyskot).
organisms (horizontal gene transfer). Mechanisms like this appear
to be functionally equivalent to genetic exchange and allow a large
divergence and speciation [1].
The flowers of angiosperms are largely bisexual, i.e., they contain
both pistils and stamens. These develop differently from most animals.
Briefly, they do not possess a true germline, and sexual organs
are formed in flowers after transition from the vegetative to the
generative state from somatic axillary meristems late in development.
This enables them to partially maintain the environmentally
induced epigenetic changes occurring during development. The
products of meiosis are not gametes, as in animals, but haploid
spores which require gene expression for differentiation. Finally,
there are two fertilization events between sperms produced by the
male gametophyte (the pollen tube) and the embryo sac: one fertilization
event leads to the formation of a zygote, and the other
http://dx.doi.org/10.1016/j.plantsci.2015.03.019
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B. Vyskot, R. Hobza / Plant Science 236 (2015) 126–135 127
leads to (usually triploid) endosperm, which is critical for embryo
nutrition and viability. Plant bisexuality may result in inbreeding
depression, and dioecy (the separation of the sexes into different
individuals) may have evolved in many plant species in response
to selection to avoid inbreeding.
The separation of male and female organs in different flowers
occurs either as dioecy (pistillate or staminate flowers in different
individuals) or monoecy (pistillate or staminate flowers on the
same individual). Either way, unisexuality in flowers is achieved in
floral development as an arrest of sex organ formation (either the
pistil or the stamen). In dioecious species, sex is most often determined
by genotype, but environmental, hormonal, and epigenetic
cues are also used in determination [2–4]. Sexual differentiation
includes not only floral differentiation and the formation of male
and female gametes. It is also responsible for gender dimorphism,
and, in some groups of organisms, dosage compensation of X-linked
genes and genomic imprinting. Sex-determining genes are often
clustered in heteromorphic sex chromosomes, which are more
common in animals, and less relevant in plants. Heteromorphic
sex chromosomes are defined by being distinguishable under a
microscope [3–5].
Similar to animal species, sex determination systems in plants
can be classified with respect to whether each sex forms different
gametes: in X/Y systems males (XY) are heterogametic and
females (XX) are homogametic (e.g., Silene latifolia, Rumex acetosa,
and Carica papaya). By contrast, in Z/W systems males (ZZ) are
homogametic and females (ZW) are heterogametic (e.g., Populus trichocarpa,
Fragaria chiloensis, Silene ottites). In X/Y species, there are
two basic systems of sex determination, the mammalian type with
the dominant Y chromosome (e.g., S. latifolia) and the Drosophila
type with the critical X/A ratio (e.g., R. acetosa).
2. Evolution of sex chromosomes
According to evolutionary theories [6,7], the sex chromosomes
originated from an ordinary pair of chromosomes (autosomes),
usually in lineages derived from hermaphrodite plants. For dioecy
to evolve from hermaphroditism, two mutations are needed, a
male-sterility (usually the first to occur) and a female-sterility
mutation. These loci had to be linked at one chromosome
pair (the sex chromosomes) for the stability of the sexes.
Later, selection for alleles advantageous to males and disadvantageous
to females is hypothesized to bring about further
genetic differences between the X and Y chromosomes and
sometimes suppression of recombination between them in further
regions [8]. In discussing sex determination mechanisms
and specific features of sex chromosome evolution (especially
degeneration of non-recombining regions of Y or W), one
should bear in mind the variety of sex-determining mechanisms
(genetic, environmental, hormonal, and epigenetic), sex determination
genes and pathways. Further, ageing and degeneration
of Y (or W) sex chromosomes make generalizations difficult
[9].
The early steps in Y-chromosome evolution have been revealed
through experimental interspecific hybrids created between dioecious
and non-dioecious species [2]. In the genus Silene, hybrids
between S. latifolia and the closely related S. viscosa have given
insights into the dominance of Y-linked alleles [10]. In these
hybrids, there is only one sex chromosome (X) inherited from the S.
latifolia seed parent. Its counterpart (an autosome) originates from
the S. viscosa pollen donor. Despite the absence of the Y chromosome,
the hybrid plants should form bisexual flowers. Indeed, in
these hybrids, anthers developed far beyond the early bilobal stage
characteristic of XX S. latifolia female plants. The S. viscosa genome
can thus replace the Y-linked sex determination gene(s) whose
Fig. 1. Immunostaining of male mitotic metaphase chromosomes of R. acetosa.
Chromosomes were mild denaturated with formaldehyde and stained with antiacetyl-lysine5-H4
histone antibody (green) and counterstained with DAPI (red). The
sex chromosomes are indicated. The bar indicates 10 ␮m.
absence, or lack of function, in females abolishes early stamen
development.
3. Case studies: recent advances through genomic studies
Here we provide some recent genomic data on the most commonly
studied dioecious species (Table 1). The following section is
focused on the classical model of dioecy, S. latifolia.
Various types of reproductive systems occur in Rumex:
hermaphroditism, polygamy, gynodioecy, monoecy and dioecy.
Phylogenetic analysis of ITS rDNA sequences suggest that dioecy
appeared in Rumex between 15–16 million years ago [11]. Two
different sex-chromosomal systems and sex-determining mechanisms
have been described in dioecious Rumex species: XX/XY with
an active Y chromosome (e.g., R. acetosella) and XX/XY1Y2 with
sex determination based on the X/A ratio (e.g., R. acetosa). There
is one exceptional species, R. hastatulus, which has two chromosomal
“races”: the Texas race possesses an XX/XY system, while the
North Carolina race has an XX/X Y1Y2 system. In this species, the X/A
ratio controls sex determination, but the presence of the Y chromosome
is necessary for male fertility [12]. The two Y chromosomes
of R. acetosa are large, full of repetitive satellites, and cytologically
heterochromatic [13,14]. Their epigenetic analysis reveals that they
are depleted in acetylated H4 histones, which is a clear marker of
heterochromatin (Fig. 1). In somatic cells of some male tissues,
these Y chromosomes form heterochromatic interphase bodies
that are typically located in the nuclear periphery [15]. R. acetosa
is an ideal subject for cytogenetic studies: its genome is huge
(C ∼ 7.0 Gb), and it is divided into seven pairs of acrocentric autosomes
and larger metacentric sex chromosomes (XX in females and
XY1Y2 in males). In R. acetosa, similar repetitive sequences in both
Y chromosomes suggest that they might originate from one Y chromosome
that underwent centromere fission and gave rise to a pair
of metacentric chromosomes possessing identical chromosomal
arms (isochromosomes). These isochromosomes have been subsequently
modified by deletions. One view suggests evolution of the
XX–XY1Y2 system through autosomal translocation to the original
X chromosome. A phylogenetic study indicates that all dioecious
Rumex species evolved from a common hermaphroditic ancestor
[11]. The switch from a sex-determining mechanism based on the
active role of the Y chromosome to a mechanism based on the X/A
ratio occurred at least twice [16]. The dynamics of microsatellite
expansion vary between closely related Rumex species [17] and the
abundance of microsatellites within the individual genomes differs
with higher frequency in the neighbourhood of transposable elements.
This fact suggests that microsatellites are probably targets
for transposon insertions and that microsatellite expansion is an
128 B. Vyskot, R. Hobza / Plant Science 236 (2015) 126–135
Table 1
List of angiosperm dioecious species, and their basic characteristics, as described in the text.
Family Species Sex determination Sex chromosomes Notes and references
Caryophyllaceae Silene latifolia, white
campion
Dominant Y, heterogametic males
XY
Heteromorphic First described
dominant Y sex
determination, [2,44]
S. diclinis Dominant Y, heterogametic males
XXneoY1Y2
Heteromorphic [45]
S. colpophylla Heterogametic males XY Homomorphic [46]
S. otites, Spanish
catchfly
Heterogametic females ZW Homomorphic [47]
Polygonaceae Rumex acetosa,
common sorrel
X/A ratio, heterogametic males
XY1Y2
Heteromorphic First described sex
chromo-somes in
plants, [11,48]
R. hastatulus,
heartwing dock
X/A ratio, heterogametic males XY
or XY1Y2
Heteromorphic [19]
Caricaceae Carica papaya, papaya
tree
Dominant Y, heterogametic males
XY
Homomorphic Draft genome
sequence, [4]
Cannabinaceae Cannabis sativa, hemp X/A ratio, heterogametic males XY Heteromorphic [27,28]
Humulus lupulus, hop X/A ratio, heterogametic males XY Heteromorphic [26]
Cucurbitaceae Bryonia dioica, white
bryony
Dominant Y, heterogametic males
XY
Homomorphic First genetic evidence
of dioecy, [29]
Coccinia grandis, ivy
gourd
Dominant Y, heterogametic males
XY
Heteromorphic Large Y chromosome,
[30]
Vitaceae Vitis vinifera, grapevine Dominant Y, heterogametic males
XY
Homomorphic Draft genome
sequence, [31,32]
Salicaceae Populus trichocarpa,
black cottonwood
Heterogametic XY males or ZW
females?
Homomorphic Draft genome
sequence, [34–36]
Euphorbiaceae Mercurialis annua,
annual mercury
Dominant Y, heterogametic males
XY
Homomorphic Association of ploidy
levels and sex
expression, [37,38]
Asparagaceae Asparagus officinalis,
asparagus
Dominant Y, heterogametic males
XY
Homomorphic [39,40]
Ebenaceae Diospyros lotus,
Caucasian persimmon
Dominant Y, heterogametic males
XY
Homomorphic First sex-determining
candi-date gene in
plants, [41]
early event that shapes Y chromosome evolution with the accumulation
of transposons and chromosome shrinkage occurring later.
Comprehensive analysis of male and female genomic DNA datasets
has shown not only the accumulation, but also the depletion of
some repetitive DNA within non-recombining regions of the Y chromosomes
(Fig. 2a, c, and e) [18]. R. hastatulus is a species with two
distinct systems of sex chromosomes—the neo-Y sex chromosome
system (XX/XY1Y2) was derived from an ancestral XX/XY system.
SNPs inheritance from parental to F1 generation (X alleles transmitted
from fathers to daughters and Y alleles transmitted from fathers
to sons) has been recently analyzed. Segregation-based experiments
using RNA-Seq have identified hundreds of sex linked genes
in R. hastatulus. This study describes two types of Y-linked genes.
First, genes shared by both R. hastatulus races are old sex-linked
genes. This set includes hemizygous genes suggesting that a gene
loss has occurred as a consequence of Y chromosome degeneration
[19]. Seventy percent of sex-linked genes from the XY system
have been calculated to be present in the XY1Y2 system, and 40% of
genes in the XY1Y2 system are shared with the XY system. Detailed
RNA-Seq-based analysis reveals that 488 “old” sex-linked genes
are shared between the XY and XY1Y2 races, while 607 “young”
genes are unique to the XY1Y2 system. This further suggests that
the XY1Y2 system has acquired many new sex-linked genes since
the fusion event. The younger Y linked genes have a significantly
reduced pattern of genetic degeneration and this confirms the evolution
of XX/XY1Y2 via fusion of the X chromosome and a former
autosome about 600,000 years ago [20]. How much expression
variation of X–Y1 or X–Y2 allelic pairs correlates with the degeneration
(loss of function) of individual Y1Y2 alleles needs to be
confirmed by further proteomic and reverse genetic (functional)
analyses.
The papaya model has been recently reviewed [4]. Although
it does not belong to the two dozen known species with clearly
heteromorphic sex chromosomes, it is a leading dioecious model
with structurally differentiated sex chromosomes [3]. Papaya is a
favourite model because of its small nuclear genome (C ∼ 0.7 Gb)
and its importance as a crop. Since males of papaya are heterogametic
and females are homogametic, sex determination can be
classified as an XY type, with a dominant Y chromosome. A large
amount of DNA polymorphism near the sex locus initially led to
the discovery of the Y chromosome in papaya. There is a small
8.1 Mb region, called the MSY (male-specific region Y), which harbours
the primary sex-determining genes. It has been proposed
that recombination suppression in the sex-determining region of
the Y chromosome is a result of proximity of the centromere to this
region [21]. As shown in other dioecious species with heteromorphic
sex chromosomes, even in papaya with evolutionarily young
and homomorphic sex chromosomes, organelle DNA accumulates
in the sex chromosomes in the absence of recombination [22]. Sex
chromosomes in papaya contain 21 specific repeats that are absent
on autosomes and with no homology to other plant sequences
suggesting a recent origin [23]. Sequencing of sex chromosomes in
this species reveals two large-scale inversions along with numerous
chromosomal rearrangements. Moreover, sequencing data confirm
an accumulation of transposable elements in the initial stages of
sex-chromosome evolution after recombination arrest, leading to
the physical expansion of the sex-determining regions [24]. Papaya
diverged from its close relative, Vasconcellea parviflora, about 27
million years ago. Both species share a homologous pair of X/Y
chromosomes. Surprisingly, the extent of non-recombining region
is much larger in S. latifolia with younger sex chromosomes [25].
S. latifolia, according to cytogenetic data, also accumulated more
transposons and other satellites than papaya. It is a question,
whether higher repetitive DNA content is merely a consequence
of the large non-recombining region in S. latifolia or whether other
processes play a role in the spread of repeats in evolving sex chromosomes.
On the other hand, heterochromatization (cytological
data) of the Y chromosome has been described only in papaya,
B. Vyskot, R. Hobza / Plant Science 236 (2015) 126–135 129
Fig. 2. FISH mapping of microsatellites and retroelements on R. acetosa (a, c, e) and S. latifolia (b, d, f) male metaphase chromosomes. The hybridization signals are in red, the
chromosomes are counterstained with DAPI (blue). The sex chromosomes are indicated. (a) R. acetosa (TA)n microsatellite, (b) S. latifolia (CAA)n microsatellite, (c) R. acetosa
Maximus/SIRE retroelement, (d) S. latifolia Athila CL3 retroelement, (e) R. acetosa CRM retroelement, (f) S. latifolia Athila CL10 retroelement. The bar indicates 10 ␮m and is
shared by all figures.
not in S. latifolia. This may suggest that epigenetic processes play a
role even in early stages of sex chromosome evolution and repetitive
content leading to large Y chromosomes in some dioecious
species reflects only the extent of the non-recombining region. The
correlation between the non-recombining regions of the Y/W chromosomes
(relative proportion to the rest of the chromosome) and
the content of repetitive elements needs to be confirmed by further
experiments. It has been suggested that the relation between the
degree of heteromorphism and the age of sex chromosomes is just
a myth [9].
In spite of its agronomical importance, relatively little is known
about the sex determination mechanism in Humulus. The genus
Humulus comprises only three species: H. lupulus, H. japonicus, and
H. yunnanensis, the most important crop being the first of these.
Males of H. lupulus are heterogametic (with either a simple XY or
multiple sex chromosome systems), whereas females are homogametic
(2n = 18 + XX). It has been suggested that sex expression is
determined by the X/A ratio. The obvious sex-related differences
are found in the plant at the stage of inflorescence initiation [26].
Cytogenetic studies on Humulus reveal that the sex chromosomes
have evolved from a pair of autosomes via ancient translocation
and/or inversion.
Cannabis sativa is another of the few dioecious species possessing
heteromorphic sex chromosomes, albeit a difference in size
130 B. Vyskot, R. Hobza / Plant Science 236 (2015) 126–135
between the X and Y chromosome being rather small (reviewed
in [27]). Males are heterogametic (XY), and females homogametic
(XX). The sex-determining system is of the Drosophila type with the
ratio of Xs to autosomes being decisive. In contrast to other dioecious
models with heteromorphic sex chromosomes (e.g., S. latifolia
and R. acetosa), the Y-chromosome of C. sativa probably does not
contain the genes necessary for male fertility. Several genes that
are involved in sex determination and/or sexual dimorphism were
identified using cDNA AFLP [28].
Most Cucurbitaceae species have unisexual flowers. Of the 800
species in this family, 460 are monoecious, and 340 are dioecious.
The best studied genus containing dioecious species is Bryonia.
Genetic crosses between the dioecious B. dioica and the monoecious
B. alba provided the first clear evidence for Mendelian inheritance
of sexual phenotypes (dioecy) and made B. dioica the first organism
for which the XY sex-determination was experimentally proved
[29]. The Coccinia grandis Y chromosome appears to be completely
heterochromatinised with twice the size of any of the other chromosomes
[30].
Although Vitis vinifera cultivars form bisexual flowers, almost all
wild Vitis relatives are dioecious. In the genus Vitis, sex determination
is putatively controlled by one major locus with three alleles:
male M, hermaphrodite H, and female F, with an allelic dominance
M>H>F. Hermaphrodite alleles appear to derive from male alleles of
wild grapevines. It has been shown that sex is controlled by a single
genomic region. Combination of a high-resolution genetic map and
markers derived from the BAC library screening points to a physical
map of a 143 kb sex-specific region spanning less than 1% of
the sex chromosome [31]. Although sex chromosomes in V. vinifera
resemble a very early evolutionary stage, dioecy is at least as old
as the separation of the Vitis and Muscadinia subgenera, about 18
million years. It has been proposed that this trait makes this species
an ideal model to address the question why some dioecious plants
rapidly developed specific sex-chromosomes, while others did not
[32].
Members of the genus Populus (poplars, cottonwoods and
aspens), along with Salix species that present sister genera, are
composed exclusively of dioecious species. A single ancient origin
of dioecy around 65 million years ago in this genus has been
proposed [33]. A peritelomeric region on chromosome 19 was identified
as a non-recombining region, with ZZ/ZW sex determination,
and <5% of the maternal W chromosome (706 kb) subjected to
recombination suppression [34]. This observation is contradicted
by a new study indicating that the TOZ19 gene is present in males
and absent in females in aspens [35]. A more extensive study has
now confirmed the XX/XY system in poplar, suggesting that assembly
problems are sufficient to explain the incorrect distribution of
the sex-associated markers found in previous studies [36]. This
study further indicates that divergence of P. trichocarpa (poplar)
and P. tremuloides (aspen) predates divergence times of X and Y
haplotype sequences (6–7 million years). Importantly, this observation
is not consistent with the hypothesis of a single origin of
dioecy in this group. The labile nature of sex-determining regions is
well documented in other species and it now seems likely that there
has been at least one “turnover” in sex-determination mechanism
since the divergence of poplars and aspens.
The genus Mercurialis is mostly dioecious, with monoecy having
evolved from dioecy at least twice. In a clade of annual species,
dioecy has given way to monoecy in loose association with polyploidy:
thus, tetraploid populations of M. annua are monoecious,
and hexaploid populations are often androdioecious, with the frequent
co-occurrence of males and hermaphrodites [37]. A closely
related species, M. canariensis, is tetraploid but fully dioecious, and
some Moroccan populations of hexaploid M. annua have become
almost fully dioecious, so that dioecy was probably lost at a couple
of times and was subsequently regained. The sex determination
of M. annua has been recently re-evaluated: rather than having a
3-locus system, sex is determined at a single locus with XY determination
[38].
In Asparagus officinalis, an M-locus controlling sexual dimorphism
was identified on chromosome 5. Although a partial physical
map of this region was constructed based on BAC library screening
with M-locus specific DNA markers, no candidate gene for sex
determination has yet been identified [39]. Comparison of male and
female genomes revealed that most of the repeat groups had similar
abundance in males and females. This suggests that asparagus
sex chromosomes are in an early stage of evolution, a conclusion
supported by the fact that YY plants are viable [40].
Diospyros lotus (Caucasian persimmon) is a dioecious plant with
dominant male (XY) sex determination. The D. lotus was known to
the ancient Greeks as “the fruit of the gods“(Dios pyros). Dioecy is
very ancient in the genus Diospyros and dates back to the origin of
Ebenacea family (35–65 million years ago). Massive DNA sequencing
(32 females and 25 males) revealed a male-specific region with
a total length of ∼1 Mb. Sixty-two genes differentially expressed
in males and females according to RNA-Seq analysis, seven genes
were MSY-linked. One gene with male-specific expression in developing
buds named OGI (class I homeodomain transcription factor)
displays male-specific conservation among Diospyros species. OGI
encodes a small RNA-regulating transcription of MeGI, an autosomally
linked gene with a female-biased bud and flower-specific
expression [41]. In females, MeGI shows elevated expression, followed
by repression of pollen formation. In males, OGI prevents
the accumulation of MeGI. Detailed transcriptional, functional, and
evolutionary analyses of OGI-MeGI complex makes D. lotus the most
advanced model in terms of our understanding sex determination.
The question remains whether a single locus is sufficient for sexdetermination
resembling SRY in humans or an additional Y-linked
locus has to be identified, according to the two-mutation model for
the evolution of dioecy [7].
There are many other dioecious models, but knowledge of their
structure and evolution remains limited. Actinidia chinensis represents
an XX/XY system with a sex-determining region located in a
recombination-suppressed subtelomeric locus within the linkage
group 17 and showing characteristics of early stages of sex chromosome
evolution [42]. Phoenix dactylifera has a smaller Y than
X chromosome (which appears to be exceptional in plants) and
probably possesses one of the most ancient sex chromosomes in
flowering plants so far studied. Its separate male flowers were identified
in fossil sediments dating from the middle Eocene period
[43]. Other emerging dioecious species include mainly Acnida
tamariscina (XY), Datisca cannabiba (ZW), Dioscorea tokoro (XY), Viscum
fischeri (XY, multiple sex chromosomes), Fragaria virginiana
(ZW), and others (reviewed in [5]).
4. Silene as a model system
The genus Silene includes about 700 species, most of which are
hermaphroditic or gynodioecious. However, there are two groups
of dioecious species that have proved invaluable for the study of
sex-determining mechanisms. These dioecious species belong to
two different Silene groups: section Melandrium (e.g., S. latifolia, S.
dioica, S. diclinis) and subsection Otites (e.g., S. otites, S. colpophylla).
The former possesses large heteromorphic sex chromosomes. Their
X-chromosomes have been mapped with reasonable resolution:
except for a recent rearrangement in S. dioica, the gene order
appears to be the same in all three species. This points to a
monophyletic origin of this sex chromosome and its relative short
evolution (less than 10 million years) [49]. Analysis of amino acid
replacements (vs. synonymous substitutions) has shown that Ylinked
alleles appear to be largely functional in all three species
and indications of degenerative processes are negligible.
B. Vyskot, R. Hobza / Plant Science 236 (2015) 126–135 131
S. latifolia is one of the best studied plants possessing heteromorphic
sex chromosomes. In contrast to the human Y chromosome,
which is relatively small, the Y chromosome in S. latifolia is the
largest chromosome in the entire genome. Chromosomes of S. latifolia
(formerly Melandrium album) were described nearly a century
ago, in 1923. The nuclear genome of S. latifolia is arranged in 11
autosomal pairs and one pair of sex chromosomes. Its C value is
rather high (∼2.8 Gb). The X chromosome is approximately 1.4
times larger than the average size of autosomes, being 1.4 times
smaller than the Y chromosome [50].
The sex chromosomes of the dioecious species from the subsection
Otites are homomorphic. A comparative mapping study
of S. colpophylla, which is also a male heterogametic species like
S. latifolia, indicates that its sex chromosomes have evolved from
a different pair of autosomes that those of S. latifolia [46]. The
results of this study indicate that the sex-determination system
in S. colpophylla evolved independently from that of S. latifolia.
Other analyses have been done on S. otites, a close relative of S.
colpophylla. These have revealed that its sex-determining system is
based on female heterogamety, which is unique among the Silene
species so far studied [47]. An analysis of ancestral states indicates
that the most recent common ancestor of S. otites and S.
colpophylla was also dioecious and a switch from an XX/XY sex
determination to a ZZ/ZW system (or vice versa) is implied in
the subsection Otites. Silene species (together with Populus) therefore
possess two different types of heterogamety within one plant
genus.
In principle, there are two options for identifying sex-linked
sequences. Indirect methods are based on the generation of DNA
sequences used as genetic markers that detect sex-linkage (e.g.,
RAPD, AFLP). This approach is frequently accompanied by the
establishment of genetic crosses that support observed data.
Recently, high-throughput sequencing methods (RNA-Seq) have
been employed to increase number of known ESTs (genes) in S. latifolia.
The combination of segregation analysis of individual SNPs
with massive sequencing of transcripts was independently used
to identify sex-linked genes [51–53]. Hundreds to thousands of
genes (sex-linked contigs) have been described as localized on
S. latifolia sex chromosomes in these studies along with expression
levels of identified genes. The question arises, how many
genes are missing in the list due to, e.g., low levels of expression
in examined tissues. Another important issue concerns quantification
of expression (transcription) of individual genes to study
Y degeneration and dosage compensation. Since RNA-Seq data
provide only a very rough view of what is transcribed without
any direct connection to translation (de facto expression),
new methods as, e.g., Ribo-Seq are needed to confirm RNA-Seq
data.
To decrease the complexity of the studied material (due to
the large genomes) and get directly focused on the chromosome
of interest, direct methods are frequently used. One of the main
advantages of direct methods is rapid identification of sex-specific
sequences, typically starting with separation of individual chromosomes.
Historically, manual microdissection was followed by laser
microdissection and flow sorting [54]. The manual dissection of
sex chromosomes was successfully applied experiments leading to
the isolation of the first active gene from a plant Y chromosome
[55].
Functional studies of Y-linked sequences have largely led
to analyses of Y deletion mutants. Large-scale deletions (disruptions)
of the Y chromosome were introduced by means
of either X-ray or gamma-irradiation [56,57] and by heavyion
beam irradiation in a ring cyclotron [58]. With increasing
knowledge of sex-linked genes, analysis of individual deletion
mutants should assist in identifying candidate genes
in flower development. Since anonymous DNA markers have
been replaced by ESTs (expressed sequence tags), analysis
of deletion mutants of known phenotypes is beginning
to be a feasible technique for mapping regions and subsequently
genes responsible for sex expression (male promoting,
male fertility, female suppression). Moreover, local deletions
are currently generated through targeted genome manipulation
using, e.g., TALE and/or CRISPR/Cas9 technologies and the
number of candidate genes “coding for” individual phenotypes can
be significantly decreased.
The structural features of the sex chromosomes in S. latifolia
have been studied over many years using a combination of various
cytogenetic and genomic approaches [2]. The first cytogenetic
map of the S. latifolia genome was constructed using FISH analysis
of selected low copy, sex-linked BAC clones [50]. Although the S.
latifolia Y chromosome is not heterochromatinised, it has accumulated
a significant number of tandemly arranged DNA repeats. It has
been demonstrated that many microsatellites have accumulated on
the Y chromosome (with a greater abundance on the q arm, which
stopped recombination relatively recently compared to the p arm).
This suggests that microsatellite spread predates other structural
changes that occurred during the Y chromosome evolution (Fig. 2b)
[59].
A global survey of all the major types of transposable elements
in S. latifolia revealed that most of the Copia elements had accumulated
on the Y chromosome. Surprisingly, one type of Gypsy
elements, which is similar to Ogre elements known from legumes,
was almost absent on the Y chromosome (with the exception of
the pseudoautosomal region), but otherwise uniformly distributed
in all chromosomes [60]. It was suggested that the absence of
one Ogre family on the Y chromosome may be caused by 24nucleotide
small RNA-mediated silencing leading to exclusively
female-specific spreading [61]. The spread of Ogre elements has
been confirmed to be relatively recent [62]. Ogre retrotransposons
appeared before sex chromosomes evolved but were mobilized
soon after the formation of the Y chromosome. This result is
supported by identical chromosomal distribution of this element
in S. latifolia close relatives possessing sex chromosomes in the
Melandrium section. These data suggest that the appearance of sex
chromosomes is quickly followed by complex genome response
with transposons playing a dominant role. Identification of another
retroelement underrepresented on the Y chromosome in S. latifolia
provides supporting data for rather complex process shaping
genome structure after sex chromosome establishment (Fig. 2d and
f) [63].
S. latifolia represents an intermediate step between early
stages of sex chromosome establishment and genetic degeneration
accompanied by either strong heterochromatinisation or loss
of non-functional DNA. Analysis of genomic libraries constructed
from individual sex chromosomes showed that there is a preferential
accumulation of chloroplast sequences on the Y chromosome
[64]. A subsequent comprehensive study revealed that the majority
of the chloroplast DNA, corresponding to the single copy regions,
was localized near the centromere of the Y chromosome, while the
inverted repeat region was also present in other loci (Fig. 3) [18]. It
has been demonstrated that repetitive DNA also accumulate in the
vicinity and within the Y alleles of sex-linked genes [51]. Although
accumulation of various transposons within the Y chromosome has
been confirmed in many other plant species (e.g., Rumex, papaya,
and Asparagus) accurate quantification is needed. Due to the size
differences between the X and Y chromosomes in S. latifolia, chromosome
sorting was recently employed to begin sequencing of
individual sex chromosomes. The combination of a draft sequence
of S. latifolia sex chromosomes and detailed analysis of transposon
insertions (dating using LTR divergence) will provide comprehensive
data on the extent and timescale of transposon participation
in sex chromosome formation.
132 B. Vyskot, R. Hobza / Plant Science 236 (2015) 126–135
Fig. 3. Frequency and distribution of various DNA repeats in the sex chromosomes and an average autosome of the model dioecious species S. latifolia. STAR-Y, TRAYC, and
X43.1 are tandem repeats isolated from the S. latifolia nuclear genome, and chloroplast DNA (IR and SC) are sequences from the inverted repeat and the single copy region
of chloroplast genome, respectively. Compiled from [77,78].
5. Y-chromosome degeneration and dosage compensation?
Y-linked S. latifolia genes show indications of genetic degeneration
and are probably not fully functional [51–53,65]. Conversely,
due to the differentiation of the male gametophyte (haploid pollen
tube, either AX or AY) and its required gene expression, many
deleterious Y-linked mutations have probably been removed by
purifying selection [52]. The necessary biological functions of the
Y-chromosome in S. latifolia could be summarized as follows:
[*] The gametophyte (i.e., male pollen tube and female embryo
sac) is a phase of the life-cycle unique to plants. It is a haploid
“checkpoint” in development and the only period where the (male)
AY-individual can survive (as pollen grain and pollen tube) without
the X. The Y chromosome carries genes that are important for male
gametophyte development, and its proper function suggests that a
large part of the Y-linked genes cannot be excessively degenerated.
[*] Many dioecious species show female-biased sex ratios (up to
70% female individuals). Most sex-biased species are probably the
result of differential mortality in adult individuals (e.g., because
of different demands on their resource status by sexual reproduction).
In S. latifolia, and perhaps in other species, an observed female
bias reflects the different viability of AAXX vs. AAXY embryos
and the function of corresponding triploid endosperm tissues. The
endosperm consists of AAXX/AX or AAXX/AY genomes in females
and males, respectively. This may indicate that the Y chromosome
is not fully competent to support the nutritional functions of the
endosperm (and the embryo). In reality, it cannot be excluded that
the female bias is a result of a sex-linked meiotic drive system, or
a lower viability of AY-pollen tubes (Fig. 4) [66].
[*] Genomic imprinting is a well-described epigenetic phenomenon,
in flowering plants documented in triploid endosperm.
Imprinting is responsible for the proper function of this feeding tissue
and its malfunction leads to the abortion of both endosperm and
embryo. As depicted in Fig. 4, the S. latifolia endosperm consists of
maternal (AAXX) and paternal (AX or AY) contributions. Obviously,
both genetic and epigenetic modifications of the Y chromosome can
influence its function in the endosperm and may be responsible for
the lower frequency of male individuals in populations (the female
bias).
[*] In studying the behaviour of the Y chromosome in the female
gametophyte (the embryo sac), we isolated two types of S. latifolia
hermaphrodites: one with an aberrant Y (a genetic deletion leading
to the loss of its ability to pass on to next generations) and the other
with an epigenetically modified Y (experimentally hypomethylated
by 5-azacytidine). In the crossed plants, we checked whether the
epigenetically modified Y could pass through the female germinal
pathway. However, in the progeny, only female plants occurred.
Hence, the Y chromosome was insufficient for the embryo sac
development. We conclude that embryo sacs with the AY constitution
are not viable and that the female gametophyte cannot survive
without the X chromosome [67].
[*] Androgenesis is a powerful tool for enabling the formation
of haploid sporophytes from in vitro cultivated pollen grains. The
cultivation of S. latifolia anthers yielded only female haploid and
dihaploid plants (AX and AAXX, resp.) rather than males (AY and
AAYY). A detailed molecular analysis of in vitro plantlets revealed
no Y-markers. Hence, it is clear that abortion of putative AY embryoids
occurred in very early development. Taken together, complete
sporophyte development strictly requires at least one X chromosome.
The Y chromosome is not sufficient, suggesting that it has
lost some important genes [68,69].
[*] Sex determination in S. latifolia is under genetic control and
realized by the expression of at least three (groups of) Y-linked
genes [2]. However, their expression appears to be regulated by
epigenetic mechanisms. An epigenetic activation of pistil development
by a 5-azacytidine treatment in male plants possessing
the AAXY karyotype led to hermaphrodite flowers. This trait was
inherited only from pollen donor (holandric heritable) [70]. The
data indicate that the arrest of pistil formation in AAXY plants was
overcome by inactivation of a Y-linked gynoecium suppressor. This
demonstrates that the basic (original) floral phenotype of S. latifolia
is bisexual and this can be achieved by an artificially hypomethylated
AAXY genotype. Conversely, the epigenetic activation of AAXX
plants did not lead to any change in female floral phenotype. This
could indicate that the AAXX plants do not contain all the genetic
information necessary for the basic bisexual phenotype, which may
be connected with an evolutionary loss of corresponding proto-sexchromosome
genes now linked to the Y-chromosome.
B. Vyskot, R. Hobza / Plant Science 236 (2015) 126–135 133
Fig. 4. Comparison of animal (a) and dioecious plant (b) male development in species possessing the X/Y sex chromosome system. Meiosis produces haploid cells (AX and
AY), which are the gametes (sperm) in animals, but are only spores (pollen) in plants. Formation of the male gametophyte (pollen tubes AX and AY) requires massive gene
expression, thus preventing a large degeneration of the Y chromosome. Fertilization is simple in animals yielding female and male (diploid) individuals in nearly the same
frequency, while in plants there is a double fertilization yielding diploid embryos with triploid endosperm. In many dioecious plants there is female bias that might sometimes
be a result of a partial degeneration of the Y chromosome, which is not fully functional in the AY pollen tubes or the AAXX/AY endosperm. “A” stands for one set of autosomes.
[*] The Y chromosome in S. latifolia is invaded by accumulation
of many types of DNA sequence repeats (satellites, microsatellites,
plastid sequences, transposons [60]). Chiefly as a result of
these sequences, the Y chromosome in S. latifolia is the largest
in the nuclear genome. However, there is yet no direct evidence
for a causal association between accumulation of DNA repeats and
genetic degeneration. Moreover, the Y chromosome has a global
euchromatic character, like autosomes [71], and several dozen Xlinked
genes have been identified that still have functional Y-alleles
[52,53,65]. Clearly, substantial degeneration of the Y chromosome
with global X-dosage compensation has thus not occurred in S. latifolia.
In any event, the numbers of X-linked transcriptome reads
seem to be equalized in AAXX and AAXY individuals: the transcript
levels in males appear to be increased, which indicates a positive
dosage compensation (as known in Drosophila) [65]. Some cytogenetic
experiments have shown DNA hypermethylation and late
replication in one of the two Xs in female somatic cells, which could
indicate a negative type of dosage compensation (the mammalian
type) [72]. Other data show that Y-linked genes evolve faster at the
protein level than their X-homologues and that their expression is
lower [51]. Dosage compensation could certainly evolve, not only at
a global level of the whole X chromosome, but also as a regulatory
mechanism operating at the level of an X-linked locus lacking its
Y-partner. Nevertheless, there is still no clear evidence for this idea:
the gene SlWUS1 (a homolog of the Arabidopsis gene WUSCHEL) is
X-linked and has no Y-linked allele, and yet it is not dosage compensated
[73]. The question of dosage compensation in S. latifolia will be
resolved when more X-linked genes with functional identification
are available for careful quantitative analysis.
[*] An important question is whether the sex chromosomes
in S. latifolia actively influence somatic development as early as
at pre-flowering stages, with differential expression in males
and females. So far, only a few quantitative markers for sexual
dimorphism in dioecious plants have been found in dioecious
plants in general [74]. In S. latifolia, a large functional screening
of EST sequences led to the unambiguous conclusion that some
genes are sex-specifically activated (either male or female) at early
stages of their vegetative development [75].
[*] Many species of the genus Silene are interfertile, despite the
sex differences [10]. For instance, crosses between the dioecious
species S. latifolia (white campion) and S. dioica (red campion) are
especially common in natural populations. The Y chromosome of S.
latifolia is fully functional in these interspecific hybrid plants, which
suggests that its evolutionary divergence is not very large (these
two Silene species separated about one million years ago [47,49]).
6. Current research strategies
Although the advent of sequencing techniques has shed light
on many aspects of sex chromosome structure and evolution in
plants, there is still limited information about the various biological
consequences for dioecious plants.
First, the role of individual genes localized on sex chromosomes
is not commonly confirmed by reverse genetics. Due to the advent
of targeted genome manipulation, there are a variety of methods
that could be optimized and employed to directly study the role of
candidate genes in plant sex development.
Second, the Y chromosome has a low-complexity genomic
region with large DNA stretches composed of tandemly arranged
sequences. These regions present a complicated template for de
novo assembly. These difficulties can be partially overcome by two
genomics approaches: the construction and physical assembly
134 B. Vyskot, R. Hobza / Plant Science 236 (2015) 126–135
of deep coverage BAC libraries for individual species in parallel
with direct sequencing experiments; and flow sorting of sex
chromosomes, with subsequent sequencing of a unique chromosomal
template by various next generation sequencing platforms.
Similar strategies have been successfully employed in sequencing
projects focused on plants with huge genomes such as wheat [76].
Inaccurate data processing and/or assembly of sequenced genomes
can lead to very erroneous conclusions, as documented in poplar.
Third, although the amount of sequencing data (both genomic
and transcriptomic) is progressively increasing, there is almost no
proteomic confirmation of the results obtained. There is as yet no
evidence that the degeneration revealed by lower expression of
individual Y alleles affects gene functionality at the protein level.
Fourth, more information about sex-linked genes from different
dioecious models is needed along with detailed genetic and physical
maps. Only identification of sex-determining genes in different
species can precisely date the age of sex chromosomes systems.
Fifth, it should be tested whether dosage compensation exists
in different dioecious species at various stages of sex chromosome
evolution. If so, the question arises as to whether individual genes
or loci are affected (compensated), or whether there is large-scale
dosage compensation within X (Z) chromosomes.
Sixth, transposable elements can play both direct (genetic)
and indirect (epigenetic) roles in the Y-chromosome degeneration.
Transposon insertion (gene mutation/disruption) has mostly
negative consequences on gene expression. Accumulation of repetitive
sequences can also trigger local/global rearrangements. On the
other hand, introduction of transposons in the vicinity of gene(s)
can affect gene expression resulting in new patterns of transcription
regulation. Indirect effects show mainly epigenetic changes.
Transposon insertion is frequently regulated by the genome itself
(e.g., using RNAi machinery), resulting in the DNA methylation of
target sequences. Methylation spreads into regions surrounding
transposon and locally affects the expression of linked genes. Moreover,
large-scale responses to repetitive DNA accumulation results
in changes in chromatin status (heterochromatisation). It is still an
open question as to whether the accumulation of transposons is
the cause or consequence of sex chromosome degeneration, and
to what extent it contributes to the evolution of sex chromosomes
generally.
Although our understanding of plant sex chromosomes remains
fragmented, rapid developments in sequencing and reverse genetics
in combination with standard approaches promise to yield
interesting results.
Acknowledgements
This research was supported by the Czech Science Foundation
(grant P501/12/G090 to B.V., and P501/12/2220 to R.H.). We would
like to thank Prof. John Pannell (University of Lausanne) and Dr.
Bohuslav Janousek (Institute of Biophysics, Brno) for helpful criticism,
and Dr. Alexander Oulton (Palacky University, Olomouc) for
English corrections.
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