Rapid de novo evolution of X chromosome dosage compensation in Silene latifolia, a plant with young sex chromosomes.

Abstract

Silene latifolia is a dioecious plant with heteromorphic sex chromosomes that have originated only ∼10 million years ago and is a promising model organism to study sex chromosome evolution in plants. Previous work suggests that S. latifolia XY chromosomes have gradually stopped recombining and the Y chromosome is undergoing degeneration as in animal sex chromosomes. However, this work has been limited by the paucity of sex-linked genes available. Here, we used 35 Gb of RNA-seq data from multiple males (XY) and females (XX) of an S. latifolia inbred line to detect sex-linked SNPs and identified more than 1,700 sex-linked contigs (with X-linked and Y-linked alleles). Analyses using known sex-linked and autosomal genes, together with simulations indicate that these newly identified sex-linked contigs are reliable. Using read numbers, we then estimated expression levels of X-linked and Y-linked alleles in males and found an overall trend of reduced expression of Y-linked alleles, consistent with a widespread ongoing degeneration of the S. latifolia Y chromosome. By comparing expression intensities of X-linked alleles in males and females, we found that X-linked allele expression increases as Y-linked allele expression decreases in males, which makes expression of sex-linked contigs similar in both sexes. This phenomenon is known as dosage compensation and has so far only been observed in evolutionary old animal sex chromosome systems. Our results suggest that dosage compensation has evolved in plants and that it can quickly evolve de novo after the origin of sex chromosomes.

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Rapid De Novo Evolution of X Chromosome Dosage
Compensation in Silene latifolia, a Plant with Young Sex
Chromosomes
Aline Muyle1., Niklaus Zemp2., Clothilde Deschamps3, Sylvain Mousset1, Alex Widmer2"*,
Gabriel A. B. Marais1"*
1Laboratoire de Biome ´trie et Biologie Evolutive (UMR 5558), CNRS/Universite ´ Lyon 1, Villeurbanne, France, 2Institute of Integrative Biology (IBZ), ETH Zurich, Zu ¨rich,
Switzerland, 3Po ˆle Rho ˆne-Alpes de Bioinformatique (PRABI), Villeurbanne, France
Abstract
Silene latifolia is a dioecious plant with heteromorphic sex chromosomes that have originated only ,10 million years ago
and is a promising model organism to study sex chromosome evolution in plants. Previous work suggests that S. latifolia XY
chromosomes have gradually stopped recombining and the Y chromosome is undergoing degeneration as in animal sex
chromosomes. However, this work has been limited by the paucity of sex-linked genes available. Here, we used 35 Gb of
RNA-seq data from multiple males (XY) and females (XX) of an S. latifolia inbred line to detect sex-linked SNPs and identified
more than 1,700 sex-linked contigs (with X-linked and Y-linked alleles). Analyses using known sex-linked and autosomal
genes, together with simulations indicate that these newly identified sex-linked contigs are reliable. Using read numbers, we
then estimated expression levels of X-linked and Y-linked alleles in males and found an overall trend of reduced expression
of Y-linked alleles, consistent with a widespread ongoing degeneration of the S. latifolia Y chromosome. By comparing
expression intensities of X-linked alleles in males and females, we found that X-linked allele expression increases as Y-linked
allele expression decreases in males, which makes expression of sex-linked contigs similar in both sexes. This phenomenon
is known as dosage compensation and has so far only been observed in evolutionary old animal sex chromosome systems.
Our results suggest that dosage compensation has evolved in plants and that it can quickly evolve de novo after the origin
of sex chromosomes.
Citation: Muyle A, Zemp N, Deschamps C, Mousset S, Widmer A, et al. (2012) Rapid De Novo Evolution of X Chromosome Dosage Compensation in Silene latifolia,
a Plant with Young Sex Chromosomes. PLoS Biol 10(4): e1001308. doi:10.1371/journal.pbio.1001308
Academic Editor: Detlef Weigel, Max Planck Institute for Developmental Biology, Germany
Received September 22, 2011; Accepted March 1, 2012; Published April 17, 2012
Copyright: ? 2012 Muyle et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This study was supported by ETH Zurich and SNF grants (TH-07 06-3 and 31003A-116455) to A.W., and the work done in Lyon by Agence Nationale de
la Recherche (ANR) to G.A.B.M. (grant number ANR-08-JCJC-0109). The funders had no role in study design, data collection and analysis, decision to publish, or
preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: gabriel.marais@univ-lyon1.fr (GABM); alex.widmer@env.ethz.ch (AW)
. These authors contributed equally to this work as first authors
" These authors contributed equally to this work as senior authors.
Introduction
In humans, where the evolution of sex chromosomes is probably
best known, the XY chromosome pair was originally a
recombining pair of autosomes that progressively stopped
recombining, most likely because of a series of inversions on the
Y chromosome [1–4]. This started ,150 million years ago [5,6]
and the non-recombining human Y chromosome subsequently
suffered from degenerating processes known as Hill-Robertson
effects (inefficient selection and reduced polymorphism, see [7–9]),
which explain the massive loss of Y genes (,97%) and the
concomitant accumulation of DNA repeats on the non-recombin-
ing Y compared to the X chromosome and the still recombining
pseudoautosomal regions (PARs) [2,3]. Even the few genes that
persisted on the Y show signs of degeneration [10,11]. The
classical view is that the massive loss of Y-linked genes has been
balanced by the evolution of dosage compensation (equal dosage
of X and autosomal transcripts in both males and females [12–
14]), which is achieved by the inactivation of one X chromosome
in females [15]. The question whether this three-step scenario (X–
Y recombination suppression, Y degeneration, X dosage compen-
sation) is similar for all species with sex chromosomes, in particular
those with much younger sex chromosomes, has received much
attention from evolutionary biologists, and several alternative
model organisms to study the evolution of sex chromosomes have
emerged, some of them very recently [9,16–18].
S. latifolia (white campion) is one such model organism. It is a
dioecious plant from the Caryophyllaceae family with heteromor-
phic sex chromosomes that have originated only ,10 million years
ago [19–22] and is a promising model organism to study sex
chromosome evolution in plants [23,24]. Previous work suggests
that S. latifolia XY chromosomes have stopped recombining
gradually [21,22,25] and that the Y is undergoing degeneration
(gene loss, reduced polymorphism, accumulation of repeats,
maladapted proteins, reduced gene expression) as in animal sex
chromosomes [26–34]. Despite these highly interesting results,
work on sex chromosome evolution in S. latifolia has been limited
by the slow pace of sex-linked gene identification (one to two new
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genes/year) [21,25,35–40]. This situation is now changing rapidly,
thanks to next-generation sequencing (NGS) approaches, which
are helping reveal the strong potential of the S. latifolia model
[23,24,41–43].
Here we report a study using such an NGS approach, RNA-seq,
applied to several males and females of an S. latifolia inbred line.
Using a de novo assembly strategy followed by SNP analysis, we
identified .1,700 sex-linked contigs, increasing by almost 100-fold
the number of sex-linked sequences available until recently in S.
latifolia. Studying these 1,700 sex-linked contigs, we found that
expression of alleles on the Y is significantly reduced compared to
those on the X chromosome, providing evidence for large-scale
ongoing degeneration of the S. latifolia Y chromosome. By
comparing the expression of X-linked alleles in males and females,
which differ in the number of X chromosomes, we further found
evidence of equal dosage of X transcripts among sexes for sex-
linked genes showing Y degeneration, a phenomenon known as
dosage compensation. To our knowledge, this is the first evidence
for dosage compensation in plants and reveals that dosage
compensation is not an animal-specific phenomenon. Moreover,
the finding of dosage compensation in evolutionary young sex
chromosomes has novel implications for the evolution of sex
chromosomes because it shows that 10 million years are sufficient
to evolve dosage compensation de novo. By contrast, dosage
compensation in animals has to date been documented only in
.100-million-year-old sex chromosome systems.
Results
Identification and Validation of New Sex-Linked Genes
We used RNA-seq—a next-generation transcriptome-sequenc-
ing approach—to identify new sex-linked genes and to study gene
expression (find more details in Text S1). We obtained ,35 Gb of
sequence data from three males and three females from a ten-
generation inbred population of S. latifolia using Illumina
technology (Table S1). Male and female reads were pooled and
assembled de novo (see Material and Methods) (Figure S1), and we
obtained 141,855 contigs (Table S2). From these, we identified
sex-linked contigs using a segregation analysis similarly to [42,43]
and found 1,736 contigs with at least one sex-linked SNP (Table
S2). We tested the reliability of our inference of sex-linkage by first
using known autosomal genes [44] to see whether sex-linked SNPs
have been wrongly inferred for these, but could not find any for
the ten autosomal genes tested (Table S3). This very low rate of
false positives was confirmed when running our scripts to detect
sex-linked SNPs on a set of simulated autosomal SNPs (Text S2).
We thus concluded that our inferences of sex-linkage are highly
reliable. To estimate how many sex-linked contigs we missed with
our method, we checked how many of the previously identified
sex-linked genes were among our sex-linked contigs (Table S3).
42% of these were not found, which means that our rate of false
negatives is quite high, and we identified a subset (probably about
half; see Figure S2; Text S1) of the sex-linked genes in S. latifolia.
Many of our sex-linked contigs should be full-length transcripts as
suggested by the size distribution plot (Figure S3).
Expression Analysis of X-Linked and Y-Linked Alleles
We used read numbers to estimate expression levels of the sex-
linked contigs (see Material and Methods). We first compared
expression levels of X-linked and Y-linked alleles in males. The
read numbers were normalized to be able to combine data from
different male individuals. As shown in Figure 1, we found that the
Y/X expression ratio is significantly less than 1 (median 0.77,
mean 0.89, significant Wilcoxon paired test p,10216). This is in
agreement with previous work on six experimentally identified sex-
linked genes [33] and also with recent work using RNA-seq data
[42,43]. Why Y expression is reduced over evolutionary time is not
fully understood. It could be because of the accumulation of
Author Summary
The mammalian sex chromosomes originated from an
ancestral pair of autosomes about 150 million years ago
and the Y chromosome subsequently degenerated, losing
most of its genes. During this process, a phenomenon
called dosage compensation evolved to compensate for
the gene loss on the Y chromosome and to equalize
expression of X-linked genes in the two sexes. In humans,
this is achieved by inactivating one of the two X
chromosomes in females. Dosage compensation has also
been reported in other animal XY systems such as fruit flies
and worms, each 100 million years old or more. Here we
studied dosage compensation in plants. We used high-
throughput RNA sequencing in male and female Silene
latifolia (white campion)—a dioecious plant whose XY
chromosomes originated only about 10 million years
ago—to identify hundreds of sex-linked genes. Analysis
of their expression patterns in males and females revealed
equal doses of sex-linked transcripts in both sexes,
regardless of the degree of reduction of Y expression
due to degeneration. Our results thus show that dosage
compensation occurs in plants and is thus not an animal-
specific phenomenon. They also reveal that proportionate
dosage compensation can evolve rapidly de novo after the
origin of sex chromosomes.
Figure 1. Distribution of Y/X expression ratios in S. latifolia
males for the 1,736 sex-linked contigs. Total Y and X read
numbers were summed at sex-linked SNP locations for each contig and
normalized for each male separately, then averaged across males to
obtain the Y/X ratio. The median is shown in red.
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slightly deleterious mutations in promoters and cis-regulatory
elements, and/or the insertion of transposable elements when the
methylation of these elements spreads to nearby genes. However,
this trend is considered a hallmark of Y chromosome degeneration
and has been observed in several animal systems [45,46]. Y
degeneration is thus clearly visible in S. latifolia but may not be as
pronounced as expected because of haploid selection on pollen
preventing the degeneration of many pollen-expressed Y genes
[42] (but see [43,47]).
The observation that many X/Y pairs show reduced Y
expression (Figure 1) raises the question whether dosage
compensation has evolved in S. latifolia. To test this, we compared
expression levels of sex-linked genes between males and females
following a normalization procedure that allows comparing
different individuals (see Material and Methods). First, we
computed the ratio of the expression intensities of X-linked
contigs in males and females and called this the Xmale/2Xfemale
expression ratio (to stress the difference in gene copy number
between male and female). In the absence of dosage compensa-
tion, the Xmale/2Xfemale expression ratio is expected to be 0.5,
simply because males (XY) have one X-linked copy and females
(XX) have two. This is what we observe for contigs that do not
show reduced expression of the Y-linked allele relative to the X-
linked allele, i.e., that have a Y/X expression ratio close to 1
(median of Xmale/2Xfemale ratio is 0.51 for contigs with 1#Y/
X,1.5; see Figure 2). However, for contigs with reduced Y
expression and therefore low Y/X ratios, we observe an Xmale/
2Xfemale expression ratio very close to 1 (median of contigs with
Y/X,0.5 is 0.93; see Figure 2). This suggests that for contigs with
reduced Y expression, for which expression of sex-linked genes
would thus be unbalanced between males and females, a
mechanism has evolved that compensates for the reduced Y
expression by increasing X expression in males.
To study this phenomenon further, we compared expression of
X-linked and Y-linked alleles in males and females for different Y/
X expression ratio categories (Figure 3). We excluded sex-linked
Figure 2. Distribution of the ratio between the expression of the single X in males and the two X copies in females (Xmale/
2Xfemale) for all sex-linked contigs. Different categories of sex-linked contigs are shown: Y/X ratio below 0.5 (379 contigs), Y/X ratio between 0.5
and 1 (656 contigs), Y/X ratio between 1 and 1.5 (315 contigs), Y/X ratio above 1.5 (195 contigs). Medians are indicated in the colour corresponding to
each Y/X ratio category. When the contigs with high Xmale/2Xfemale ratios are removed as in Figure 3 (see text for explanations) the medians remain
unaltered except for the category Y/X,0.5 where it changes to 0.76 but is still significantly different from 0.5 (Wilcoxon test, p,10216). Total X read
numbers were summed at sex-linked SNP locations in each contig and normalized for each individual separately, then averaged among males and
females to get the Xmale/2Xfemale ratio.
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contigs that showed either an elevated Y expression (high Y/X
ratios) or male-biased X expression (high Xmale/2Xfemale ratios).
Such male-biased expression patterns suggest that these genes may
be sexually antagonistic genes. The evolutionary dynamics of such
genes is known to be distinct from other sex-linked genes and no
dosage compensation is expected [48,49]. Figure 3 shows the
results for the remaining 75% of sex-linked genes. We found that
X expression in males increases with decreasing Y expression,
which results in similar expression levels of sex-linked contigs in
both sexes and provides further evidence of dosage compensation
in S. latifolia. Importantly, this result is consistent even when we
include only sex-linked contigs with at least two sex-linked SNPs,
for which we estimated the rate and number of erroneous sex-
linked contigs to be extremely low (0.001 and 1.38, respectively;
see Figure S4). We also looked at expression patterns of the contigs
corresponding to known sex-linked genes. Although this analysis
can only be qualitative due to the small number of such genes, we
found that Y/X ratios for most genes are consistent with previous
work [33] and some known sex-linked genes show evidence for
dosage compensation (Table S4).
Discussion
Evidence for X Chromosome Dosage Compensation in S.
latifolia
There was a recent claim of absence of dosage compensation in
S. latifolia [42], which seems to contradict our findings. However,
the test for dosage compensation performed in this recent work is
very different from ours. As Chibalina and Filatov (2011) analyzed
crosses (parents and progeny), they were able to identify X-linked
genes without detectable homologous Y-linked copies (called
hemizygous genes). They compared the expression levels of these
hemizygous genes between sexes, found a significantly reduced
expression in males compared to females, and concluded that this
was evidence for the absence of dosage compensation in S. latifolia
[42]. Their test however may be overly conservative, as it requires
Figure 3. Expression levels of sex-linked contigs in both sexes for different Y/X expression ratio categories. Total read numbers were
summed at sex-linked SNP locations and normalized for each individual and contig separately; medians for all contigs and individuals of the same sex
were then obtained. Contigs with Y/X expression ratios above 1.5 were excluded, as well as contigs with Xmale/2Xfemale ratios above 2 (see text for
explanations), which reduces the dataset to 1,346 sex-linked contigs. XX females, median expression level of both X-linked alleles in females; X males,
median expression level of the single X-linked allele in males; Y males, median expression level of the Y-linked allele in males; XY males, median
expression level of the X-linked plus Y-linked alleles in males. To compare different Y/X expression ratio categories, medians were normalized using
the XX expression levels in females. Sample sizes are: 0–0.25, 110; 0.25–0.5, 269; 0.5–0.75, 315; 0.75–1, 341; 1–1.5, 315. Note that we do not have any
contig with Y/X=0 as our method did not allow us to detect such contigs (see Material and Methods). Error bars indicate 95% confidence intervals.
doi:10.1371/journal.pbio.1001308.g003
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a strict Xmale/2Xfemale ratio of 1 to infer for dosage
compensation. Their figure 4 suggests the Xmale/2Xfemale ratio
is not 0.5, as expected under a complete absence of dosage
compensation, but instead is close to 0.7, which is consistent with
many hemizygous genes being dosage compensated. Importantly,
the hemizygous genes were interpreted as sex-linked genes with
fully degenerated Y copies, which may not always be the case as
genes that have recently moved from the autosomes to the X
chromosome will also be detected as hemizygous genes but dosage
compensation is clearly not expected for those genes [43]. Such
gene movement has been documented in S. latifolia [39] and may
account for the intermediate Xmale/2Xfemale value (between 0.5
and 1) found in [42]. By contrast, we looked for departure from a
Xmale/2Xfemale of 0.5 and did not restrict the test to sex-linked
genes with no Y expression but included the many sex-linked
genes with reduced but still detectable Y expression. We thus
performed a more permissive test for dosage compensation, which
may be more suitable in the case of young sex chromosomes with
incipient X chromosome dosage compensation.
Sex Bias in Gene Expression and Dosage Compensation
Dosage compensation is not the only sex-specific gene
expression regulation that is expected on the X chromosome.
Indeed, X-linked genes involved in sexual conflicts—for instance
those underlying sexual dimorphism and having sexually antag-
onistic effects—can show sex-biased expression and this can
substantially affect the global X expression pattern in both sexes if
these genes are numerous [50]. A way to distinguish dosage
compensation from such sex-specific expression regulation is to
look at the X over autosome (X/A) expression ratio as only dosage
compensation predicts a X/A expression of 1 [50]. However, this
test is difficult to perform here for several reasons. First, our set of
sex-linked genes is expected to exclude those with very low
expression levels because the detection of sex-linked SNPs requires
reasonably high read coverage. This should bias upward the
average expression level of sex-linked genes compared to the
‘‘autosonal’’ set, which is what we actually found (the mean
number of reads per base is 466.7 for sex-linked contigs and 101.4
for non–sex-linked contigs). Second, we do not have a reliable
‘‘autosomal’’ set as this includes a mixture of autosomal contigs
and sex-linked contigs not detected by our method (,40% of all
sex-linked genes, see above). Although we excluded possible
candidates for sexually antagonistic genes (some of the contigs with
high Xmale/2Xfemale may be ‘‘male-beneficial and female-
detrimental’’ genes), we cannot completely rule out the possibility
that others remained in the set of contigs used to assess dosage
compensation (especially some contigs with low Xmale/2Xfemale
may be ‘‘female-beneficial and male-detrimental’’ genes). Howev-
er, Figure 3 shows that the increase of X expression in males
follows the level of degeneration of Y expression, which is not
expected in case of sexually antagonistic selection. Moreover,
increased expression of the X-linked allele in males always
compensates for the reduced Y expression, such that the total
expression of these sex-linked genes is similar in both sexes (i.e.,
X+Y expression in males=X+X expression in females), which is
not in agreement with sexually antagonistic selection. On the
contrary, sexually antagonistic selection predicts between-sex
differences in expression of sex-linked genes. The results presented
in Figure 3 are thus better explained by dosage compensation than
by sexually antagonistic selection.
Dosage Compensation in XY and ZW Systems
Global dosage compensation has previously been documented in
male heterogametic systems (XY) such as Drosophila, Caenorhabditis
elegans, and mammals [14,51], whereas only partial (or no) dosage
compensation has been found in female heterogametic systems (ZW)
[52]. Indeed, in zebra finch, chicken, and crow, no global
mechanism to balance avian Z chromosome gene dosage (such as
X chromosomeinactivation) hasbeenfound [53–56]and inchicken,
dosage compensation seems to be local, with only few Z-linked genes
being dosage compensated [57]. Similar observations have been
made in silkworm [58,59], indicating that the lepidopteran Z is not
fully dosage compensated, and also in the parasite Schistosoma mansoni
[60]. Moreover, studies on the platypus [61,62] and on sticklebacks
[63] suggest that partial dosage compensation can also exist in male
heterogametic systems(XY). Overall, these new data suggestthat full
dosage compensation is not a necessary outcome of sex chromosome
evolution [50]. An important point of whether dosage compensation
will evolve or not is the presence of dosage-sensitive genes on the
proto-sex chromosomes, as these genes are the only ones for which
dosage compensation is vital [50,64]. Although we do not have any
data about the fraction of dosage-sensitive genes in the different sex
chromosome systems, it has been suggested that resistance to
aneuploidy and polyploidization may indicate whether the genome
as a whole includes many such genes or not [50]. Polyploidization is
known to be common in plants [65]. However, plant polyploids do
have dosage problems that cause endosperm development failure
and reduced fertility [64,66]. Following polyploidization events, the
retention of plant duplicate genes seems to be driven by dosage
constraints as in animals [64]. All this suggests that the success of
polyploids in plants may not be related to lack of dosage constraints
but to other reasons (e.g., vegetative propagation). It is also known
that aneuploidy has more severe phenotypic consequences than
polyploidyinplants, whichfurther supports the ideaof strongdosage
constraints in plant genomes [64]. As far as we know, there is no
documented caseof fertile polyploids indioecious Silene speciesand it
is possible that the S. latifolia genome includes enough dosage-
sensitive genes for dosage compensation to evolve.
Mechanisms of Dosage Compensation in Plants
Our results reveal that dosage compensation is not restricted to
animals but also occurs in plants and raise questions about the
mechanisms underlying dosage compensation. In animals, three
different dosage compensation mechanisms have been uncovered
(reviewed in [67]): hyper-expression of X-linked alleles in male
Drosophila, down-regulation of the two X-linked alleles in hermaph-
rodites of C. elegans, and inactivation of one of the two female X
chromosomesin mammals. Wetestedwhether such a chromosome-
wide inactivation exists in S. latifolia by checking whether both X-
linked alleles are expressed in females. Although heterozygosity is
low in our X-linked alleles because our individuals are inbred, we
foundthatthe levelofheterozygosityoftheX-linkedallelesissimilar
for sex-linked contigs with dosage compensation and those without
dosage compensation (Table S5). This suggests that both X-linked
alleles are expressed, whatever the level of dosage compensation is,
anddoesnotsupportanX-inactivation-likemechanisminS.latifolia.
Further work will be needed to identify the molecular mechanism
underlying dosage compensation in S. latifolia.
De Novo Evolution of Dosage Compensation in a Young
XY System
Previous work in animals has reported dosage compensation in
old X chromosomes (see above) and also in young neoX
chromosomes such as the D. miranda neoX. The fusion between X
and the autosome that formed the D. miranda neoX is very recent
(1.5 million years old), but dosage compensation is achieved by a
protein complex (the MSL complex) that pre-dates neoX formation
and has been shown to be very old [68]. Evidence for de novo
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evolution of dosage compensation in evolutionary young animal sex
chromosomes is therefore lacking [50]. In the Silene genus, most
species are hermaphroditic or gynodioecious and do not have sex
chromosomes. Sex chromosomes have evolved recently in two
independent lineages, one including S. latifolia and one containing S.
colpophylla [20,44,69]. Our results therefore reveal that dosage
compensation has evolved de novo in evolutionarily young sex
chromosomes in probably less than 10 million years. Furthermore,
Figure 2 shows that many dosage-compensated contigs have an
Xmale/2Xfemale ratio that is not exactly 1 (although the median is
close to 1, there is no peak at 1 for Y/X,0.5 contigs). This is
consistent with the mechanism being evolutionarily young and not
optimized yet. Our results also reveal that dosage compensation can
evolve as soon as Y expression starts declining. This way, dosage
compensation already exists when the Y copy is ultimately lost (and
can even facilitate such loss, see [70]). Instead of being a later step of
sex chromosome evolution following Y degeneration, our results
suggest that the evolution of dosage compensation and Y
degeneration probably occur at the same time.
Material and Methods
Plant Material, RNA Extraction, Sequencing, and
Assembly of Illumina Data
Plants used in this study belong to a population of S. latifolia that
hasbeen inbredforten generations with brother-sistermating: three
males (U10_11, U10_49, and U10_09) and three females (U10_34,
U10_37, and U10_39) that were grown in a temperature-controlled
greenhouse. The QiagenRNeasy Mini Plant extraction kit was used
to extract total RNA two times separately from four flower buds at
developmental stages B1–B2 after removing the calyx. Samples
were treated additionally with QiagenDNase. RNA quality was
assessed with an Aligent Bioanalyzer (RIN.9) and quantity with an
Invitrogen Qubit. An intron-spanning PCR product was checked
on an agarose gel to exclude the possibility of genomic DNA
contamination. Then, the two extractions of the same individual
were pooled. Samples were sequenced by FASTERIS SA on an
Illumina HiSeq2000 following an Illumina paired-end protocol
(fragment lengths 150–250 bp, 100 bp sequenced from each end).
Individuals were tagged and pooled for sequencing in two different
runs (U10_49 male and U10_37 female in the first run and the
others in the second). See Table S1 for sizes of the different libraries.
Our Illumina reads are available in the GEO database (through the
GEO Series GSE35563).
De novo assembly was conducted on a computer cluster (Figure
S1). Illumina reads from all individuals were pooled together for
assembly with AbySS 1.2.5 (E=10, n=5) [71] with the paired-end
option and with all k-mers ranging from 51 to 96 in order to address
variable transcript expression [72]. A k-mer length equal to 51 was
the minimum possible to avoid contigs shorter than the reads, and
96 is the maximum allowed by AbySS. Only contigs were kept at
this stage, singlets were discarded. Contigs that exactly matched
anotherlongercontigwerethen removed bypairwisecomparison of
AbySS outputs using Trans-ABySS 1.2.0 [72]. A non-redundant set
of contigs was thus obtained and further assembled through two
runs of CAP3 version 12/21/07 [73]. Singlets and contigs were
conserved after each CAP3 run. CAP3 runs increased the chance
for X and Y copies to be assembled into the same contig, which is
crucial for further sex-linked SNP detection. Contigs shorter than
200 bp were not included in the final set of contigs.
Mapping, SNPs Analysis, and Sex-Linkage Detection
Illumina reads weremappedonto referencesequences(finalsetof
contigs and also CDS from known sex-linked genes retrieved from
GenBank for adjusting SNP detection, see below) for each
individual separately using BWA 0.5.9 [74] (using default
parameters for paired-end reads, and gap and mismatch maximum
number of 5 as suggested for 100 bp reads in [74]), which was
shown to be efficient and to use much less RAM than other
programs for Illumina read mapping [75]. Alignments of all
individuals were then merged together using Samtoolsmerge
version 0.1.12 [76]. The percentage of mapped reads was assessed
using Samtoolsflagstat version 0.1.12 [76] and the average coverage
was determined using the Genome Analysis Toolkit (GATK
1.0.5315) Depth of Coverage [77].
SNPs were detected with the GATK Unified Genotyper (using
the following parameters: -stand_call_conf 4 -stand_emit_conf 0 -
mbq 17 -mmq 0 -mm40 40 -bad_mates -dcov 2000) [77], which is
considered the best currently available tool for SNP detection [78].
Thresholds for the different SNP detection parameters were set to
be very low (except for the base quality parameter) in order not to
disfavour Y SNPs that are expected to be found in low numbers
and low mapping quality if a contig contains mainly X reads,
which can happen when X-linked alleles are more strongly
expressed than Y-linked alleles [33].
The detected SNPs were then filtered using Perl scripts to
retrieve SNPs for which all males are heterozygous (XY) and all
females homozygous (XX). All contigs with at least one SNP
showing this pattern were considered sex-linked. For females, the
genotypes inferred by GATK were directly used for analysis. For
males, this information is not reliable since the Y-linked allele is
expected to be less expressed than the X-linked allele [33] while
GATK genotyper makes the assumption that both alleles are
expressed at a similar level. The read numbers of each SNP were
thus used to infer male genotypes (see Text S3 for details).
Polymorphism on the X chromosome (at least one male or
female heterozygous or all individuals homozygous but not for the
same polymorphism) was detected on sex-linked contigs with a
similar filter as the one described above.
Estimates of Expression Levels of the Sex-Linked Contigs
Expression levels of the X-linked and Y-linked alleles in males
and both X copies in females were computed by counting reads at
sex-linked SNP locations only, and not for the entire contigs, in
order to clearly distinguish between X and Y reads. Total read
numbers of all X or Y SNPs provided by the GATK Unified
Genotyper [77] were summed for each X-linked or Y-linked alleles
and each individual separately and then normalized using the total
number of mapped reads per individuals (library size) and the
number of sex-linked SNPs in the contigs:
E~
r
n|l
With E=normalized expression level, r=sum of total read counts,
n=n sex-linked SNPs, l=normalized library size.
The library size of the six individuals was normalized to take
into account the difference in mitochondrial, chloroplast, and
transposable element (TE) transcript quantity between sexes and
the difference in rRNA quantity between the first and the second
Illumina run. The Arabidopsis thaliana rRNA genes, complete S.
latifolia mtDNA genome [79], S. latifolia chloroplast genes rpoB,
rpoC1, rpoC2, rps2, atpI, atpH, atpF, atpA, psbI, psbK, rps16, matK,
psbA, rpl2, ycf2, ndhB, rps7, and the TEs known in Silene [80] were
retrieved from GenBank. The read numbers of rRNA, TEs and
mtRNA, and cpRNA were determined by mapping the Illumina
reads onto the known CDS sequences of these elements using the
default parameters in BWA (results presented in Table S1).
Dosage Compensation in Young Plant Sex Chromosomes
PLoS Biology | www.plosbiology.org6 April 2012 | Volume 10 | Issue 4 | e1001308

Page 7

The expression levels were normalized for each contig and for
each individual in number of reads per kilobase per million mapped
reads (RPKM) [81], and then the mean for each sex was computed.
Supporting Information
Figure S1
of the de novo assembly. From left to right: during first assembly
with ABySS, k-mers ranging from 51 from 96, only contigs were
kept. Pairwise comparisons of contigs were then done by Trans-
ABySS in order to remove small contigs that exactly matched
longer contigs. Contigs were then further assembled by two runs of
CAP3 (mismatches and partial overlaps allowed); singlets and
contigs were kept after each run. Illumina reads were mapped onto
the contigs with BWA and SNPs were detected with GATK. SNPs
were then analyzed in order to detect sex-linked SNPs (all males
heterozygous XY, and all females homozygous XX).
(TIFF)
Assembly, mapping, and SNP analysis. Steps
Figure S2
coverage for known sex-linked genes. cDNA sequences of
previously identifiedsex-linkedgeneswereretrievedfromGenBank.
Illumina reads were mapped on the cDNA sequences using BWA
and SNP detection was done as in Material and Methods. We then
computed the number of sex-linked SNPs detectedover the number
of known sex-linked SNPs for these genes and compared this with
the number of reads (=coverage) for each X/Y gene pairs. Sex-
linked genes were grouped by strata as in [82].
(TIFF)
Number of sex-linked SNPs detected and
Figure S3
(TIFF)
Size (bp) distribution of sex-linked contigs.
Figure S4
genders for different Y/X expression ratio categories
for contigs with $ $2 sex-linked SNPs (1,009 contigs). The
legend is the same as for Figure 3 except for contig numbers: 0–
0.25, 66; 0.25–0.5, 165; 0.5–0.75, 248; 0.75–1, 279; 1–1.5, 251.
(TIFF)
Expression levels of sex-linked contigs in both
Table S1
bly.
(DOC)
Raw Illumina data and results of the assem-
Table S2
(DOC)
Contig statistics.
Table S3
and sex-linked genes.
(RTF)
Results of SNP analysis for known autosomal
Table S4
linked genes.
(RTF)
Analysis of expression patterns in known sex-
Table S5
with and without dosage compensation.
(DOC)
Levels of heterozygosity of the X-linked alleles
Text S1
genes.
(RTF)
Identification and validation of new sex-linked
Text S2
positive sex-linked genes.
(DOC)
Simulations to estimate the rate of false
Text S3
(DOC)
SNP detection and filtering.
Acknowledgments
We thank the Genetic Diversity Centre (GDC) for support.
Author Contributions
The author(s) have made the following declarations about their
contributions: Conceived and designed the experiments: AW GABM
SM. Performed the experiments: NZ. Analyzed the data: AM NZ CD
GABM. Contributed reagents/materials/analysis tools: AW. Wrote the
paper: GABM AW.
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