Structure and evolution of Apetala3, a sex-linked gene in Silene latifolia
BMC Plant Biology
Structure and evolution of Apetala3, a sex-linked gene in Silene latifolia
Radim Cegan 0
Gabriel AB Marais
Hana Kubekova 0
Boris Vyskot 0
Roman Hobza 0
0 Laboratory of Plant Developmental Genetics, Institute of Biophysics, Academy of Sciences of the Czech Republic , v.v.i.Kralovopolska 135, CZ-612 65 Brno , Czech Republic
Background: The evolution of sex chromosomes is often accompanied by gene or chromosome rearrangements. Recently, the gene AP3 was characterized in the dioecious plant species Silene latifolia. It was suggested that this gene had been transferred from an autosome to the Y chromosome. Results: In the present study we provide evidence for the existence of an X linked copy of the AP3 gene. We further show that the Y copy is probably located in a chromosomal region where recombination restriction occurred during the first steps of sex chromosome evolution. A comparison of X and Y copies did not reveal any clear signs of degenerative processes in exon regions. Instead, both X and Y copies show evidence for relaxed selection compared to the autosomal orthologues in S. vulgaris and S. conica. We further found that promoter sequences differ significantly. Comparison of the genic region of AP3 between the X and Y alleles and the corresponding autosomal copies in the gynodioecious species S. vulgaris revealed a massive accumulation of retrotransposons within one intron of the Y copy of AP3. Analysis of the genomic distribution of these repetitive elements does not indicate that these elements played an important role in the size increase characteristic of the Y chromosome. However, in silico expression analysis shows biased expression of individual domains of the identified retroelements in male plants. Conclusions: We characterized the structure and evolution of AP3, a sex linked gene with copies on the X and Y chromosomes in the dioecious plant S. latifolia. These copies showed complementary expression patterns and relaxed evolution at protein level compared to autosomal orthologues, which suggests subfunctionalization. One intron of the Y-linked allele was invaded by retrotransposons that display sex-specific expression patterns that are similar to the expression pattern of the corresponding allele, which suggests that these transposable elements may have influenced evolution of expression patterns of the Y copy. These data could help researchers decipher the role of transposable elements in degenerative processes during sex chromosome evolution.
Sex chromosomes evolved independently many times in
both animals and plants . The initial steps of their
evolution, including the genetic degeneration of the
non-recombining Y or W chromosomes (which are
analogous to Y chromosomes), have received great interest
from geneticists. To date, most of our knowledge about
sex chromosome evolution stems from a few animal
systems with evolutionary old sex chromosomes .
However, evolutionarily young sex chromosomes are
needed to investigate the early steps in sex chromosome
evolution. Such sex chromosomes can be found in
plants [3,4]. Although the majority of plants are
cosexuals, forming either bisexual flowers (hermaphrodites)
or unisexual flowers of both sexes on one individual
(monoecy), dioecious plant species (with separate sexes)
have evolved multiple times in different plant lineages
. The majority of dioecious plant species lack
morphologically distinguishable sex chromosomes. However,
well differentiated heteromorphic sex chromosomes
were described in Rumex acetosa, Cannabis sativa and
Silene latifolia. The latter has become a model species
for investigations into the evolution of sex chromosomes
Silene latifolia Poiret (syn. Melandrium album Garcke,
syn. Melandrium pratense Roehl.) is a strictly dioecious,
perennial herb of the Caryophyllaceae family. The sex of
individual plants is genetically determined by sex
chromosomes that were first described independently by
Blackburn  and Winge . Females are homogametic
with a pair of X chromosomes, while the males are
heterogametic, XY . The X and Y chromosomes are
about 1.4-fold and 2-fold larger than the largest
autosome, respectively . Therefore, they contribute
substantially to the large genome size of the species and to
the slightly larger genome size in males than in females
. The Y chromosome in S. latifolia seems to lack
some essential genes present on the X, since plants are
not viable unless they have at least one X chromosome
. By analyzing hermaphroditic mutants and their
progeny, Westergaard  showed that all
independently derived hermaphrodites had deletions in one arm
of the Y chromosome. From the studies on deletion
mutants, Westergaard  concluded that one arm of
the Y chromosome contains gene(s) for anther
maturation, while the other arm has gene(s) suppressing carpel
development, and additional genes located close to the
centromere stimulate early stages of stamen
development . More recently, molecular markers in
combination with a panel of deletion mutants were used to
create a detailed map of the Y chromosome [14-16].
Gene and genome duplications have been recognized
as major forces driving the evolution of animal and
plant genomes. Two basic processes can cause
duplication of genes. The first process, segmental duplication,
keeps the structure of a gene (exon-intron order, cis
regulatory sequences) in its original constitution. The
duplicated copy of the gene maintains expression
patterns similar to the original copy. The second process,
retrotransposition, often generates non-functional gene
copies that lack regulatory elements and introns [17,18].
The evolution of sex chromosomes is a complex
genetic and epigenetic process , which is often
accompanied by structural rearrangements and
accumulation of repetitive DNA in non-recombining regions.
Moreover, intensive gene turnover within sex
chromosomes is reflected by a high number of retroposed genes
both on X and Y chromosomes [19,20]. It is known that
over the course of S. latifolia sex chromosome
evolution, many repetitive elements have accumulated on the
Y chromosome . However, we still lack information
about which elements are linked to degenerative
processes in Y chromosome evolution by either genetic or
epigenetic mechanisms , and little is known about
the structural and functional role of repetitive DNA in
Y linked genic regions of this plant.
Here we unravel the structure and evolution of a sex
linked gene, SlAP3, first reported as having originated
by duplication from autosomes to the Y chromosome in
S. latifolia . Since SlAP3Y is located close to the
oldest stratum (4.5-7 MY) in the Y chromosome, this gene
is a candidate to be affected by various degenerative
processes [24,25]. In our study, we did not find evidence
for a duplication event in the case of this gene. Instead,
we identified a new pair of sex linked alleles with no
evidence for autosomal paralogues. We demonstrated
the accumulation of retrotransposon sequences in an
intron region of the Y linked allele. We further analyzed
expression patterns of individual elements identified in
the Y copy of SlAP3 gene to reveal their role in Y
Identification of genomic clones for APETALA3 (AP3) gene
S. latifolia and S. vulgaris BAC libraries were screened
with SlAP3A and SlAP3Y gene derived probes prepared
using the sequences and primers from Matsunaga et al.
. Positively hybridizing clones were selected, and the
presence of the target gene was verified by PCR including
sequencing of PCR products. In total, we identified four
clones containing the AP3 gene in the S. latifolia BAC
library. Two clones contained the presumed autosomal
SlAP3A copy (246/K15, 251/L13) and the other two
contained SlAP3Y (30/L22 and 253/J6). Four copies of an
orthologue, which we called SvAP3, were identified in the
S. vulgaris BAC library. Both BACs containing the
SlAP3Y copy were selected for further complete BAC
sequencing. For the SlAP3A and SvAP3 BAC clones, we
isolated DNA, digested it with HindIII and conducted a
Southern blot hybridization. The original probes from
the BAC library screening were used for the
hybridization. Subsequent experiments revealed no difference in
hybridization patterns within BAC groups (SlAP3A,
SvAP3) (Additional file 1, Figure S1). Differences in signal
intensity of individual hybridizing BACs are due mainly
to the different sizes of BAC inserts, which prevented us
from using equimolar amounts during gel loading. Based
on complete sequence similarity (after sequencing of
PCR products with the same primers as used for probe
preparation) and an identical hybridization profile, we
randomly selected BAC clones 91/M20 (containing
SvAP3), 251/L13 (SlAP3A) and 30/L22, 253/J6 (SlAP3Y)
for a more detailed analysis. The data obtained strongly
suggest that there is just one allelic pair of the SlAP3
gene in both the S. latifolia and S. vulgaris genomes.
Linkage mapping by PCR on microdissected
To confirm the linkage of individual SlAP3 alleles to
specific chromosomes, we microdissected and separated
X chromosomes and autosomes in S. latifolia
(Additional file 2, Figure S2) and ran PCR using these
chromosomes as templates. We used primers for POL
sequence of the Retand retroelement  as a positive
control. Surprisingly, PCR with primers for the SlAP3A
K-domain resulted in a product only with genomic
DNA of both sexes and using microdissected X
chromosomes as a template (figure 1). We did not obtain any
PCR product from microdissected autosomes, an
observation which did not confirm the presumed presence of
a copy of the SlAP3A gene on an autosome. These
observations demonstrate that SlAP3A, originally
described as an autosomal copy in Matsunaga et al.
, is in fact an X linked allele of the SlAP3Y gene,
which we call from here on SlAP3X.
BAC clones 251/L13, 30/L22, 253/J6 and 91/M20 were
purified and sequenced. We identified a copy of the AP3
gene in all the BACs sequenced [GenBank: HQ113124,
HQ113125, HQ113126]. Comparison with mRNA for S.
latifolia SlAP3A [GenBank: AB090863] and SlAP3Y
[GenBank: AB090864] revealed seven exons for SlAP3Y
and SvAP3, while SlAP3X contains only six exons (figure
The size of individual introns differed only slightly
among the different copies of AP3 (figure 2). The only
exception was a very large intron 2 (23,855 bp) in the
SlAP3Y allele. We found that this intron contains two
different retroelements that are shuffled into each other.
The first retroelement contains a pair of LTRs, reverse
transcriptase, RNaseH, integrase and gag gene, while the
sequence of the second retroelement is incomplete and
composed of LTRs and ORF1 product from Athila
ORF-1 family (figure 3).
Genomic distribution of repetitive DNA in SlAP3Y intron
All coding domains (LINE, reverse transcriptase,
integrase) and LTRs were used as probes for FISH on
mitotic metaphase chromosomes (Additional file 3,
Figure S3). Analysis of the distribution of individual signals
did not indicate a specific role of these particular
retroelements in Y chromosome evolution (figure S3). FISH
analysis was also conducted with the tandemly arrayed
repetitive Y promoter motive. There was no signal both
after standard and low stringency FISH experiments,
indicating a low abundance of this repetitive satellite
motif in the S. latifolia genome.
Expression analysis of Y linked retroelements
To reveal activity (expression) of individual repetitive
elements linked to SlAP3Y and their domains, we
conducted RT-PCR experiments (figure 4). Retroelement A
(its LTR part) and LINE were expressed in both males
and females in different tissues (leaves and buds).
Surprisingly, retroelement B had a different pattern of
expression between the LTR part and the rest of its
genes. Although integrase and RT domain showed
similarly to retroelement A, and LINE widespread
expression in both sexes and all tissues, the LTR B region was
expressed only in the floral buds of both sexes.
We quantified the expression of individual domains of
the retroelements localized in the SlAP3Y copy by
identifying the number of hits with the male and female
specific EST database of S. latifolia. This database is
composed of ca 100,000 reads generated by 454
sequencing (Roche FLX 454 pyrosequencing) using cDNA
from male and female buds as a sequencing template.
The accuracy of the selected approach was tested by
comparing the occurrence of part of the actin gene in
both male and female EST databases (Table 1).
Internalpart of retroelement B (integrase, reverse transcriptase)
revealed a similar number of hits (expression level) in
both males and females. LTR A, LTR B and LINE
element expression was stronger in males based on the
EST database data. LTR B, which has 28 higher
expression in males than in females, was the most
Figure 1 PCR on microdissected chromosomes. POL Retand primers were used as a positive control (present in all chromosomes) and
primers for SlAP3A/X K-domain were used for sex chromosomes localization. The template DNA is indicated in the figure (size marker (L) 100 bp,
male genomic DNA () female genomic DNA () microdissected X chromosomes (X), microdissected autosomes (A) and negative control (no
template). PCR products were subjected to electrophoresis on 1% agarose gel and stained with ethidium bromide.
Figure 2 Alignment of a promoter and coding region of SlAP3X, SlAP3Y and SvAP3 genes. Rectangles represent exon regions of the
genes. Corresponding coding sequences are indicated
Promoter structure analysis
By comparing promoter sequence structure, we
discovered significant differences in SlAP3Y in comparison
with SlAP3X and SvAP3. A part of the SlAP3Y promoter
is formed by a 6 bp long direct repeat. Both ends of the
repeat are bordered by inverted tandem structures that
resemble the organization of a MITE element
(Additional file 4, Figure S4). GenBank database searches
revealed no similarity of this part of the promoter to
any known sequence, except for the MROS1 gene 
promoter region [GenBank: AB013446.1]. Although
MROS1 is not a sex-linked gene, like SlAP3Y it is
expressed only in males.
Sequence divergence analysis
In S. latifolia, recombination between the sex
chromosomes has ceased in three steps, and three groups of
genes with different levels of divergence (also called
strata) have been identified [25,28]. The level of
divergence between SlAP3 X and Y copies is about 13% (see
Table 2), between the 20% X-Y divergence typical of the
stratum 1 genes and the 10% X-Y divergence typical of
the stratum 2 genes.
We conducted a dN/dS analysis on the SlAP3
sequence to study the possibility of differences in
intensity and form of selection in the X and Y copies of this
gene (and also with autosomal orthologs). We included
all available orthologous sequences from Silene species
to have as many sequences as possible for the
phylogenetic dN/dS analysis, which tends to give more accurate
results with more sequences, and to have outgroups (the
non-dioecious species S. vulgaris and S. conica). The
results are reported in table 2 and in additional file 5,
figure S5. The dN/dS ratios in X and Y sequences
among dioecious Silene species were not found to be
significantly different, which does not provide clear
evidence of the Y copy degeneration. However, dN/dS
ratios were found to be significantly different (p-value =
0.0149) between dioecious and non-dioecious lineages.
The higher ratio in the dioecious lineage suggests that
selection has been relaxed in both the X and Y
sequences compared to the autosomal copy in S.
vulgaris and S. conica where dN/dS is much lower (see
By screening more than five haploid complements of the
S. latifolia genome, we identified two copies each of
SlAP3Y and SlAP3A genes. The number of identified
BAC clones containing variants of the Y and the
presumed autosomal copy suggests that SlAP3 is a single
copy gene. According to Matsunaga et al. , the Y
copy of SlAP3 is a paralog of an autosomal allele that
was transferred onto the Y chromosome after the
beginning of sex chromosome evolution in Silene. Although
the authors mentioned the existence of introns in both
paralogs, genomic sequence data were not presented. To
decipher the mechanism of the translocation of SlAP3
gene within the S. latifolia genome, we isolated and
sequenced BAC clones containing SlAP3 paralogs.
Moreover, we isolated and sequenced SlAP3 gene from
the S. vulgaris genome (a closely related gynodioecious
plant without sex chromosomes) and named this gene
SvAP3 (Silene vulgaris APETALA3 gene). Comparison of
Figure 3 The structure of SlAP3Y gene. Red rectangles represent coding domains of retrotransposons. Blue rectangles are individual exons of
SlAP3Y. Ovals represent long terminal repeats.
Figure 4 RT-PCR analysis. Actin, LTR A, LTR B, integrase, reverse transcriptase (RT) and LINE primers were used. Genomic DNA with actin
primers and BAC DNA with element specific primers were used as a positive control. Actin reveals a different sized PCR product when genomic
DNA is used as a template than when cDNA is used as a template due to intron excision, and so can be used as an internal control for purity of
RNA used for reverse transcription. Template DNA is indicated in the figure (size marker (M) 100 bp ladder), male genomic DNA (), female
genomic DNA (), BAC DNA (30/L22 and 253/J6) S. latifolia male and female cDNA and RNA from leaves (L) and buds (B) and negative control
(-, no template). PCR products were subjected to electrophoresis on 1% agarose gel and stained with ethidium bromide.
the orthologs between S. latifolia and S. vulgaris
revealed that the Y copy of S. latifolia and the
autosomal copy of S. vulgaris have seven introns, whereas the
X copy of S. latifolia contains only six introns.
Surprisingly, the promoter region of SlAP3Y copy is completely
different from both SvAP3 and SlAP3A.
To study the extent of the translocated autosomal
region on the Y chromosome we intended to select low
copy markers in the Y BAC clone and to map them on
dissected sex chromosomes and autosomes by PCR.
However, PCR with the SlAP3 specific primers revealed
Table 1 Estimation of intensity of expression of different
parts of retroelements in intron 2 based on the number
of reads in S. latifolia cDNA libraries (actin was used as
an internal control)
RT (Retroelement B
Integrase (Retroelement B)
different localization of SlAP3 paralogues in the genome
than expected. While we were able to amplify SlAP3
gene copies using X chromosomes as templates, there
was no PCR product when only autosomes served as a
template. These observations show that SlAP3 is a
regular sex-linked gene with X and Y alleles, and that no
transfer to the Y chromosome has occurred. Our data
suggest different results as shown in Matsunaga et al.
. The simplest explanation in this case could be use
of different techniques for separation of sex
chromosomes. Chromosome sorting used by Matsunaga et al.
 is a powerful method which generates large amount
of DNA for further experiments. The main disadvantage
of this method is that it is subject to impurities even
when the chromosomes being sorted are significantly
morphologically different, as is the case for S. latifolia
sex chromosomes and autosomes . Unlike
chromosome sorting, laser microdissection is more laborious
but produces a pure fraction of selected material [30,31].
Based on comparisons of the X and Y alleles we
showed that SlAP3X and SlAP3Y started to diverge
quite early: their divergence at synonymous sites (13%)
is close to the maximum X-Y divergence recorded so far
for S. latifolia sex linked genes, although the mapping
Table 2 dN and dS analysis in X/Y, dioecious and non-dioecious species
of SlAP3 on the X chromosome is needed to confirm
this. No clear evidence of degeneration of the Y copy
has been found in this case. Rather, the dN/dS values
for dioecious vs. non-dioecious lineages suggest a
subfunctionalization in which X and Y copies have
differentially retained their ancestral functions (still coded by
one gene in the non-dioecious species), as has been
hypothesized for some sex-linked genes in humans .
Moreover, a branch-site analysis (Additional file 6,
Table S1) revealed no significant evidence for positive
selection on the Y sequences (or on the X sequences),
which further supports subfunctionalization.
Furthermore, the expression patterns of the Y copies and the X
copies also suggest subfunctionalization . The
observed differences in expression patterns between
SlAP3X and SlAP3Y are due to regulatory differences of
particular alleles in males, where both SlAP3X and
SlAP3Y are present. Comparisons of promoter
sequences of all three copies of AP3 revealed that the
autosomal promoter in S. vulgaris is identical to the
SlAP3X promoter. Surprisingly, there is a unique 80 bp
promoter region in the SlAP3Y copy. The only
regulatory sequence that shows similarity with this Y
promoter specific structure is a part of the MROS1 gene
promoter in S. latifolia . This gene (localized on
autosomes) is also expressed exclusively in males.
Although the role of the identified promoter with the
same expression pattern of both SlAP3Y and MROS1
should be verified by e.g. transformation of a plant with
promoter-reporter gene construct, an efficient
transformation protocol is not yet available for Silene species.
The most prominent evidence suggesting a role of the
promoter in sex-specific gene expression regulation is
its uniqueness in genome of S. latifolia; it appears only
in a genic context and mainly co-occurrs with sex
specifically expressed genes. Although it is a tandem repeat,
it shows no accumulation in S. latifolia genome. Even a
GenBank search of Silene species derived sequences
shows no hits with these repetitive DNA fractions.
Our results have implications for intron size evolution
in evolving sex chromosomes. We identified a very large
intron containing two retrotransposons between the
exons 2 and 3 of SlAP3Y. A similar phenomenon
showing accumulation of different sequences in the Y linked
introns was previously reported for some other S.
latifolia genes [33,34]. Although it is known that different
repetitive elements such as microsatellites , tandem
repeats , organellar DNA  and different
retrotransposons [38,39] have played a prominent role in the
formation of sex chromosomes in S. latifolia, there is no
direct link between the structural role of such elements
and their impact on degeneration of the Y chromosome.
To assess the role of these repetitive elements in the Y
chromosome evolution we ran FISH experiments and
expression (RT-PCR and in silico) analysis. Although we
did not find specific accumulation of the studied
elements in the Y chromosome, we observed different
expression patterns among different retroelements and
in different tissues. RT-PCR data showed that the LTR
B domain, unlike other parts of retroelement B, is
specifically expressed in the floral buds of both sexes. These
data show an increased activation of a specific type of
retroelement during early developmental stages. This
phenomenon could be linked to meiotic stages of cells
in developing buds when the epigenetic status of genetic
information is reassembled and the regulatory role of
epigenetic mechanisms is suppressed. A higher
expression level was found in LTR A, LINE and especially
LTR B in male bud tissue based on in silico analysis
using EST databases. Although it is known that in
males, germ line activity of some retroelements is
elevated compared to females , we suggest several
explanations that concern the evolution of sex
chromosomes. It is known from recent papers [33,34], that Y
linked genes contain more repetitive elements in their
introns compared to X alleles. Higher expression of
such Y linked retroelements could be a consequence of
co-expression of the element with Y linked genes. Biased
occurrence of both LTRs identified in the SlAP3Y gene
in male EST database could also be explained by
another aspect of sex chromosomes evolution. Once a
retroelement appears in a Y linked gene it follows the
same rules for degenerative processes as Y linked genes.
Entire retrotransposonal genes could loose their
functions by mutation accumulation and the introduction of
stop codons. Retrotranscription begins in the LTR
region, and thus this part of the retrotransposon is kept
in the expressome even after termination of
transcription in other parts of the retroelement (gag, pol). It is
also known that unpaired DNA caused by
retrotransposon insertion into a chromosome is recognized by RNAi
machinery and consequently homologous RNA is
degraded . Since there is no pairing of chromosomes
in non-recombining regions of sex chromosomes, this
mechanism is not triggered in this case and expression
of retrotransposons is not regulated in males.
Our data further suggest that previously published
retroelement types that are accumulated on the Y
chromosome  are different from those that participate in
intron targeting of Y linked genes. Analysis of the Y
promoter linked repetitive microsatellite motif within a
MITE element reveals a low abundance in S. latifolia
genome. Paradoxically, the sequences responsible for the
large size of sex chromosomes may thus be different
from those that have potentially caused degeneration of
Y linked genes.
This study unravels the structure and evolution of AP3,
a sex linked gene with copies on the X and Y
chromosomes in the dioecious plant S. latifolia. This gene was
previously reported to be located on the autosomes,
with one copy having been transferred by duplication to
the Y chromosome. Our results provide evidence for the
location of copies on X and Y chromosomes, and the
absence of this gene on autosomes. Divergence between
the X and Y-linked copies both at sequence (promoter
and coding regions) and gene expression levels suggests
subfunctionalization has been an important process in
the evolutionary dynamics of this gene. One intron of
the Y-linked allele was invaded by retrotransposons that
display sex-specific expression patterns, similar to the
expression pattern of the corresponding allele, which
suggests transposable elements may have contributed to
the evolution of gene expression of this gene
Plant material and isolation of metaphase chromosomes
Silene latifolia Garcke and Silene vulgaris plant material
was obtained from a seed collection of the Institute of
Biophysics, Brno. Sterilized seeds were cultured for 2
days in distilled water and then synchronized with
aphidicoline (30 mmol/l for 12 h) and oryzalin (15 mol/l
for 4 h). Root tips from germinating seedlings were cut
off and enzymatically protoplasted. The protoplasts were
briefly fixed in the mixture of ethanol:acetic acid (3:1) to
avoid further DNA damage. The mitotic protoplast
suspension was dropped on a membrane (for laser
microdissection, stained with Giemsa) or on microscope slides
(for FISH experiments), where naked chromosomes
were released .
BAC library construction and screening
The BAC libraries were constructed from S. latifolia
male and S. vulgaris high molecular weight genomic
DNA. Briefly, DNA was digested with HindIII enzyme
and inserted into a pECBAC1 and pIndigoBAC-5
(Epicentre) vectors, respectively. Clones were then grid in
duplicate on Hybond N+ (Amersham, Biosciences)
nitrocellulose membrane filters in a 4 X 4 pattern that
allowed us to identify well positions and plate numbers
of each clone. The filters were incubated and processed
as described in Bouzidi et al. . The S. latifolia BAC
library (total of 119,808 colonies) was arrayed on six
nylon filters with 18,432 colonies each, and one nylon
filter containing 9,216 clones. The average insert-size of
the library is 128 kb. The S. vulgaris BAC library (total
of 55,296 clones) was arrayed on three nylon filters with
18,432 colonies each. The average insert-size of the
library was 110 kb. Based on nuclear size data by Vagera
et al  and irok et al. , we have estimated that
coverage of the S. latifolia BAC library is 5.327
complements of the male haploid genome and the S. vulgaris
BAC library is 6.8 complements of the haploid genome.
Screnning was performed by radioactive hybridization
with a32P and with Prime-It II Random Primer
Labelling Kit (Stratagene) according to the manufacturers
protocol. Probes were prepared by PCR amplification of
K-box region of SlAP3A and SlAP3Y (see PCR).BAC
DNA was isolated by Large Construct Kit (Qiagen).
DNA for PCR reactions was isolated using NaOH/SDS
precipitation according to Sambrook and Russel .
For K-domain of SlAP3A and SlAP3Y and SvAP3
amplification we used primers according to Matsunaga
et al.,  and for MEF2A domain primers SVMEF2_F
(5-TGCAATTTGTGGGTGCTAGA-3) with male and
female genomic DNA and with DNA of positively
hybridized BAC clones. For amplification of
conservative domains of retrolelements in the intron 2 of
SlAP3Y we used the following primers: GAG_IN2_F
F (5CCTCTTCACCTTGCAACTCC-3), Integ_IN2_R
(5-GTTAATCCTCCCGTCCCAAT-3). Primers for
the POL part of the RETAND element were designed
according to Kejnovsky et al. . PCR conditions
were the same for all primer pairs used. The reaction
profile included 35 cycles of 45 s at 94C, 1 min at
61C and 2 min at 72C preceded by initial
denaturation (4 min at 94C) and followed by final extension
step (10 min at 72C). PTC-200 (MJ Research) and
T3000 (Biometra, Goettingen, Germany) thermal
cyclers were used.
Total RNA was extracted from young flower buds
(0.10.4 cm), and leaf tissues using RNA blue (Top-Bio). One
microgram of total RNA was treated with RNase-free
DNase (Ambion) and then used for cDNA synthesis
using High Capacity RNA-to-cDNA Kit (Applied
Biosystems) according to manufacturers protocol. The
synthesized cDNAs were used as templates for RT-PCR. For
amplification of conservative domains of retrolelements
we used the following primers: LINE_ap3y_F
(5-AAAGCAGGTGGGAGAAACCT-3), LINE_ap3y_R (5-GCAA
(5-TATGCACCGTGTTAGGACCA-3). PCR conditions
were the same as described in DNA amplification. Actin
was used as a positive control with primers ActinS-F
(5AGGGCGTAACCCTCGTAAAT-3). The reaction
profile for actin included 35 cycles of 30 s at 94C, 30 sec
at 55C and 30 sec at 72C preceded by initial
denaturation (4 min at 94C) and followed by final extension
step (10 min at 72C).
Southern blot hybridization
BAC DNA was restricted by HindIII and than
ferred by reverse Southern blotting on Hybond N+
(Amersham, Biosciences) membrane filters. Radioactive
hybridization was performed as described in the BAC
Mitotic slides were prepared according to Lengerova
et al. . The CellCut Plus system (Olympus) was
applied to isolate sex chromosomes and autosomes.
Briefly, protoplasts were dropped on a microdissection
membrane. Chromosomes of interest were selected and
transferred into Eppendorf tube according to
manufacturers protocol (MMI).
Fluorescence in situ hybridization on metaphase
Slides were treated as described in Lengerova et al. 
with slight modifications. Slide denaturation was
performed in 7:3 (v/v) formamide: 2 X SSC for 2 min at
72C. Slides were immediately dehydrated through 50%,
70%, and 100% ethanol (-20C), and air dried. The
probe was denatured at 70C for 10 min, and 100 ng of
the denatured probe was added at room temperature
and hybridized for 18 h at 37C. Slides were analyzed
using Olympus Provis microscope, and image analysis
was performed using ISIS software (Metasystems). DNA
was labeled with Fluorolink Cy3-dUTP (Amersham
Pharmacia Biotech) (red labeling) in combination with
the nick translation mix (Roche).
The probe for the SlAP3Y promoter-repeat was
synthesized by VBC-Genomics (Vienna) with Cy3
modification on 5end. The sequence of this probe is:
BAC DNA was isolated and commercially sequenced
from selected BACs using 454 sequencing with Roche
GS FLX (GATC Biotech, Konstanz). Basic sequence
analysis, sequence assembling and alignment were done
with Geneious software. Multiple sequence comparisons
were performed with MAFFT http://align.bmr.
kyushu-u.ac.jp/mafft/online/server/ and BLAST online
applications. A homology search was performed with
BLAST . Gene structure identification was done
with GENSCAN software
http://genes.mit.edu/GENSCAN.html and FGENESH http://linux1.softberry.
ORFs were found with ORF Finder http://www.
ncbi.nlm.nih.gov/projects/gorf/ and a homology search
for conserved domains was performed with NCBI
Conserved Domain Search http://www.ncbi.nlm.nih.gov/
Structure/cdd/wrpsb.cgi. All primers were designed by
Primer3 Input (v.0.4.0)
http://frodo.wi.mit.edu/primer3/. Other simple sequence analyses were done by
The Sequence Manipulation Suite - version 2 (SMS2),
To compute the dN and dS between X and Y copies of
SlAP3 in different dioecious species both GenBank
sequences and data from BAC sequencing were used.
We then ran codeml with the pairwise option on the X
and Y sequences . We also performed a phylogenetic
analysis of dN/dS using SlAP3 sequences presented in
(S. latifolia and S. vulgaris) and from GenBank (S.
latifolia, S. dioica, S. diclinis and S. conica from Matsunaga
et al. ). We aligned the sequences using Seaview
version 4 . We used the species tree known for the
Silene species in the alignment  instead of the tree
built from the alignment because of tree building
problems with less than 10 sequences (as in , see
Additional file 5, figure S5). We then ran codeml (branch
model and branch-site options) on the alignment and a
tree. Statistical significance was tested using likelihood
ratio tests . For the expression analysis, we used
EST libraries extracted from S. latifolia male and female
individuals (Blavet et al., in preparation). These libraries
are non-normalized, so we estimated the relative gene
expression based on the quantity of reads. To estimate
the expression of the retrotransposon located in the
large intron of SlAP3Y, we performed a BLAST search
with an E-value cut-off 10-40 versus both male and
Additional file 1: Figure S1 Southern hybridization. Southern
hybridization with BAC DNA restricted using HindIII. Individual BAC
identifiers are indicated. Different signal intensity is due to differences in
the amount of DNA loaded for electrophoresis. Hybridization was carried
out with a part of the SlAP3 gene covering exons 3-7 as a probe.
Additional file 2: Figure S2 Laser microdissection of the Silene
latifolia X chromosome and autosomes. Metaphase protoplasts were
dropped on a polyethylene naphthalate membrane and stained with
Giemsa. A suitable X chromosome was localized under the inverted
microscope (A). The membrane was cut around the selected region
using a laser microbeam (B) and the X chromosome was picked up (C)
by the adhesive cap of a PCR tube (D). Microdissection of autosomes (E).
Before collecting dissected chromosomes (autosomes), sex chromosomes
were removed (burned) by the laser microbeam (F). Sex chromosomes
Additional file 3: Figure S3 Chromosomal distribution of reverse
transcriptase (A) and integrase (B) gene derived probes revealed by
FISH experiment. Metaphase chromosomes of S. latifolia male were
counterstained with DAPI (blue); the probe was labeled with
Cy3conjugated nucleotides (red). The X and Y chromosomes are indicated,
bars indicate 10 m.
Additional file 4: Figure S4 Specific structure of SlAP3Y promoter.
Pink shading shows tandemly arrayed DNA within the promoter. Blue
represents a border sequence (inverted repeat) of a hypothetical MITE
element. Yellow represents the start of exon1.
Additional file 5: Figure S5 Tree used for the dN/dS analysis. Tree
topology was that of the species phylogeny and branch length has been
estimated by PAML. BAC sequences derived data are indicated by
Additional file 6: Table S1 Branch-site analysis of SlAP3 sequences.
This research was supported by Czech Science Foundation (grant nos. 522/
09/0083 and 204/09/H002), Grant Agency of AS CR (grant nos. M200040905
and KJB600040901), the Academy of Sciences of the Czech Republic (grants
no. AV0Z50040507 and AV0Z50040702), ETH Zurich (grant no. TH-7 06-3),
and by Agence National de la Recherche for support (grant number
ANR-08JCJC-0109). We thank Dr. Julia M. Svoboda for critical reading of the
RC performed S. vulgaris BAC library screening, PCRs, fluorescence in situ
hybridization and bioinformatic analysis of sequences. GABM performed
sequence divergence analysis and made substantial contributions to data
interpretation. HK performed S. latifolia BAC library screening. NB made in
silico expression analysis of Y linked retroelements. AW made substantial
contributions to data interpretation. BV prepared metaphase chromosomes
for microdissection and fluorescence in situ hybridization. JS and JD
constructed S. vulgaris BAC library. RH designed the experiments, sorted
chromosomes by microdissection and conducted PCR on microdissected
chromosomes, helped to interpret the data and drafted the manuscript. All
authors read and approved the final manuscript
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