Copyright ? 2008 by the Genetics Society of America
Active Miniature Transposons From a Plant Genome and
Its Nonrecombining Y Chromosome
R. Bergero,1A. Forrest and D. Charlesworth
Institute of Evolutionary Biology, Ashworth Laboratories, University of Edinburgh, Edinburgh, EH9 3JT, United Kingdom
Manuscript received October 18, 2007
Accepted for publication December 16, 2007
Mechanisms involved in eroding fitness of evolving Y chromosomes have been the focus of much the-
oretical and empirical work. Evolving Y chromosomes are expected to accumulate transposable elements
(TEs), but it is not known whether such accumulation contributes to their genetic degeneration. Among
TEs, miniature inverted-repeat transposable elements are nonautonomous DNA transposons, often
inserted in introns and untranslated regions of genes. Thus, if they invade Y-linked genes and selection
against their insertion is ineffective, they could contribute to genetic degeneration of evolving Y chromo-
somes. Here, we examine the population dynamics of active MITEs in the young Y chromosomes of the
plant Silene latifolia and compare their distribution with those in recombining genomic regions. To isolate
active MITEs, we developed a straightforward approach on the basis of the assumption that recent trans-
poson insertions or excisions create singleton or low-frequency size polymorphisms that can be detected
in alleles from natural populations. Transposon display was then used to infer the distribution of MITE
insertion frequencies. The overall frequency spectrum showed an excess of singleton and low-frequency
insertions, which suggests that these elements are readily removed from recombining chromosomes. In
contrast, insertions on the Y chromosomes were present at high frequencies. Their potential contribution
to Y degeneration is discussed.
their ability to self-replicate, they can proliferate and
reach high copy numbers and, if fixed, can be retained
in evolutionary lineages across wide taxonomic groups.
However, abundance of TEs in eukaryotic genomes
varies over several orders of magnitude (Wright and
Finnegan 2001) and the factors that control their
population dynamics are not yet completely resolved.
TE insertions can be highly deleterious and selective
pressures oppose their insertion and thus accumula-
tion. Deleterious effects on fitness derived from TE
insertions are of two main types: insertions within or
near genic regions can disturb gene functions by chang-
ing reading frames or disrupting regulatory motifs
(Finnegan 1992), and chromosomal rearrangements
can be caused by ectopic exchange between TE copies
at nonhomologous genomic locations (Montgomery
et al. 1987; Langley et al. 1988).
In sexually dimorphic organisms whose gender is
controlled by sex chromosomes, recombination is sup-
pressed between the Y chromosome, inherited only by
male individuals, and the homologous X chromosome
RANSPOSABLE elements (TEs) have major roles
in genome diversification and expansion. Due to
(or the Z and W chromosomes in species with female
heterogamety). Because Y chromosomes are recombi-
nationally isolated, TE dynamics can be studied in a
nonrecombining chromosome in an otherwise recom-
bining background. Y chromosomes should be less
affected by deleterious effects of TEs causing chromo-
somal rearrangements due to ectopic exchanges, be-
cause meiotic recombination is suppressed. Thus the
main deleterious effects of TEs on fitness of Y chromo-
somes should be insertions affecting functionally impor-
Among TEs, miniature inverted-repeat transposable
elements (MITEs) are a class of DNA transposons that
move by the trans-activity of a transposase encoded by a
related transposable element (Zhang et al. 2001). Un-
like other major classes of TEs, MITEs are preferentially
located in or near genes (Bureau and Wessler 1994;
Feschotte et al. 2002), and, most likely because of their
small size (?100–500 bp), insertions often do not cause
major disruption of the genes or their regulation
(Naito et al. 2006). However, some insertions could
behighlydeleterious (Yanoetal. 2000) and, iftheyoccur
within Y-linked genes, they could contribute to genetic
degeneration of an evolving Y chromosome.
On Y chromosomes, selection is expected to be in-
effective, since recombination is suppressed. Y chromo-
somesare thus expectedtohavelow effective population
size, Ne, due to the ‘‘hitchhiking’’ effects of selection
Sequence data from this article have been deposited with the EMBL/
1Corresponding author: Institute of Evolutionary Biology, Ashworth
Laboratories, University of Edinburgh, King’s Bldgs., W. Mains Rd.,
Edinburgh, EH9 3JT, United Kingdom.E-mail: firstname.lastname@example.org
Genetics 178: 1085–1092 (February 2008)
½selective sweeps, background selection, and weak Hill–
Robertson interference (reviewed by Charlesworth
and Charlesworth 2000)?. This expectation is sup-
ported byempirical data showing low silent-site diversity
of Y-linked genes, compared with their X-linked alleles
(Zurovcova and Eanes 1999; Montell et al. 2001).
Reduced Neshould lead to lower efficacy of natural
selection, so that mildly deleterious mutations, includ-
ing MITE insertions, should be able to rise to interme-
diate frequencies or to fixation (Brookfield and Badge
1997). Moderately deleterious MITE insertions may
It is well known that Y chromosomes and neo-Y chro-
mosomes undergo genetic degeneration in the long
term (Charlesworth and Charlesworth 2000) and
(Bachtrog 2003). There are so far no empirical data
on the dynamics of MITEs in newly evolving Y chro-
mosomes. We here examine the distribution of MITEs
in a dioecious plant species, Silene latifolia, whose sex-
chromosome system is not older than 5–10 MY. Silene
Y chromosomes are 40% larger than their homologous
X chromosomes, but the two sex chromosomes carry a
number of homologous genes (Bergero et al. 2007), so
that it is unlikely that the larger size of the Y is due to a
major autosomal translocation ½forming a neo-sex chro-
mosome (Steinemann and Steinemann 1998)?; most
likely repetitive DNA has accumulated. For the few sex-
linked gene pairs so far known, S. latifolia Y-linked genes
also show lower expression levels than their X counter-
parts (R. Bergero, unpublished results), suggesting
that genetic degeneration is occurring in this species.
To study MITE dynamics, sequences of actively trans-
posing elements are needed. These can be found by
scanning complete genome sequences (or sequences of
large genome regions) for TE insertions (Duret et al.
2000; Surzycki and Belknap 2000; Rizzon et al. 2002;
Wright et al. 2003), but such extensive genomic se-
quences are difficult to obtain from DNA regions rich
in repetitive sequences, such as the Y chromosomes
(Foote et al. 1992; Holt et al. 2002), or are simply not
scan approaches provide no information about the dis-
tribution of insertion frequencies, which is needed to
test the predictions of the theories outlined above, and
it is not always clear whether any given transposable ele-
ment has recently been active. Indeed, a large fraction
of TEs in genomes of higher eukaryotes are probably
inactivated copies (fossils) that have lost transposition
activity (Smit and Riggs 1996; Feschotte et al. 2002;
Pace and Feschotte 2007).
To search for actively transposing MITEs we devel-
oped an approach on the basis of assuming that recent
transposon insertions or excisions create singleton or
low-frequency size polymorphisms, which can be de-
tected in surveys of alleles from natural populations. We
used this approach to isolate MITE elements from S.
latifolia introns and identified two active subfamilies
from this dioecious plant. Transposon display for MITE
insertionsfrom both subfamilieswascarried out toinfer
their frequency distributions and compare Y chromo-
somes with other genome regions sampled from S.
latifolia natural populations.
MATERIALS AND METHODS
Plant material: One male and one female S. latifolia individ-
ual from each of eight European natural populations (sup-
plemental Table 1 at http:/ /www.genetics.org/supplemental/)
were used to investigate intron-size polymorphisms. A trans-
poson display was carried out on 48 individuals (24 females
and 24 males) derived from a larger set of natural populations
(supplemental Table 1) and a collection of 108 F2plants. The
F2family derived from a single cross between two F1plants
obtained by crossing two parents, one obtained from a French
population and one from the Netherlands (Bergero et al.
2007). As these parent plants were not inbred, the elements
used as genetic markers are sometimes heterozygous in one
parent and sometimes in both (other MITE insertions do not
segregate in this family).
Genomic DNA was obtained from fresh leaves using the
FastDNA kit (Q-Biogen), following the manufacturer’s
Identification of MITEs from intron-size polymorphisms:
Introns from a set of 19 genes (two introns were analyzed for
each locus, see supplemental Table 2 at http:/ /www.genetics.
org/supplemental/) were amplified by PCR and size esti-
mated by standard gel electrophoresis in alleles from natural
populations tosearchforlarge-sizepolymorphisms (.150bp)
that could result from recent MITE insertion/excision. Loci
were chosen to be single copy or low copy to limit amplifica-
the results. The set also included the Silene sex-linked genes
SlSSX/Y, SlX3/Y3, SlX1/Y1, and SlCypX/Y. Primers were de-
signed on the basis of S. latifolia cDNA sequences. Intron posi-
tions were inferred according to gene structures reported
for putative Arabidopsis thaliana and Oryza sativa orthologs
(Bergero et al. 2007).
PCR products from introns showing size polymorphisms
were cloned in a T-tailed pBSKS1 vector (Marchuk et al.
1991) and sequenced on an ABI3730 sequencer (Applied Bio-
systems, Foster City, CA). Alignment of intron-size variants
Arbor, MI). As MITEs lack transposase coding sequences,
other features were used for their identification. These were
the presence of terminal inverted repeats (10–15 bp) at the
for other MITEs (Wicker et al. 2007), size in the range of 150–
500 bp, and extensive secondary structure. The web package
MFOLD (Zuker 2003) was used to infer the DNA folding and
secondary structure of putative MITE elements.
MITE insertion variants in a mapping family and in natural
populations: Transposon display (TD) was used to detect
segregating MITE elements in an F2family and MITE poly-
morphisms and copy numbers in a set of natural populations.
TD is an AFLP-based technique (Van Den Broeck et al. 1998;
Casa et al. 2000) that uses a primer annealing to the adapter
and one annealing to conserved regions of the TE element.
Although MITEs do not have conserved coding sequences,
extensive sequence conservation should occur in members of
recently active MITE subfamilies. MITE-specific primers were
designed to face outward from and anneal to subterminal
sequences of the two MITE elements isolated from S. latifolia
1086R. Bergero, A. Forrest and D. Charlesworth
(SlTo1 and EITRI). The procedures were as outlined in Casa
et al. (2004) with the following modifications. Genomic DNA
(0.8–1.5 mg) was digested with DpnII for 3hr at 37?. After
inactivation of DpnII by incubation at 65? for 10 min, re-
striction fragments were ligated to a DpnII linker. The ligation
reactions containing 200 units T4 DNA ligase (New England
Biolabs, Ipswich, MA), 20 units BamHI, and 12 mm of DpnII
adapter were performed for 12 cycles, each consisting of 30
min at 16? and 10 min at 37?. The DpnII adapter was obtained
by spontaneous annealing at room temperature of the oligo-
nucleotides TDADA1 (59-GACAGTTGTGTACCTCGAATG-39)
and TDADA2 (59-GATCCATTCGAGGTACACAACTG-39).
Adapter dimer formation in the ligation reaction (which
could significantly decrease the availability of adapters for li-
gation with genomic DNA) was avoided by designing a 59 GA
ex novo BamHI site at the other end when adapter dimers
formed. Dimers were destroyed by adding BamHI to the
The DpnII library was amplified by a first-round PCR of 20
cycles using adapter-specific primers (with one specific base)
and the MITE-specific external primer (EITRI-ext, 59-TAAA
TAACGTGTCCCGTGTCC-39 and SlTo1-ext, 59-TCCATTCCA
ATCCATTCCAAGAG-39). Thermocycling conditions were as
follows: 4 min at 94?; 20 cycles at 30 sec at 95?, 40 sec at 50?, 30
was used as template in a nested touchdown PCR (Don et al.
1991), using a set of adapter-specific primers with 2 selective
bases at the 39 end (a total of 10 adapter-specific primers of
Biosystems) MITE-specific internal primer (EITRI-int, 59-TCC
CGTGTCCTAAATTTCATG-39 and SlTo1-int, 59-GAGAGCAAAC
CAAACACCCC -39), with the following thermocycling condi-
tions: 2 min at 95?, followed by 10 annealing cycles at 0.5?
carried out using a hot-start TAQ polymerase (JumpStart;
Sigma, St. Louis), which was found to increase product yields,
especially for bigger-size bands (.300 bp), and to produce
VIC-labeled PCR products were first diluted (1:25) in
the size standard GeneScan 500-LIZ (Applied Biosystems),
and directly separated by capillary electrophoresis on an ABI-
3730 DNA analyzer (Applied Biosystems). Transposon inser-
Biosystems), and their frequencies were analyzed and plotted
using the R statistics package (http:/ /www.r-project.org).
MITE sequences from this study were deposited in the
GenBank databases(accessionnos. EU334132 and EU334133).
Data analyses: The average numbersof MITE insertions per
haploid genome were estimated from electropherograms
obtained from the set of 24 females (thus excluding Y-linked
TE insertions whose properties may be unusual). Because of a
preponderance of rare insertions, an estimate of insertion
frequencies can be obtained from the observed frequencies of
frequency, pi, of insertions at the ith site, we assumed that the
population is in Hardy–Weinberg equilibrium, since the spe-
cies is outcrossing, and used pi¼ 1 ? Oqi
frequency of the recessive genotype at this site (with no band
on the gel). The expected number of TE insertions per hap-
loid genome is the total number of insertions weighted by
their frequencies ÆNtotæ ¼Ppi.
sertion and the restriction site will create a new ‘‘spurious’’
observed polymorphisms due to such events by examining
segregation of 203 MITE insertions in an F2progeny made by
2, where qi
Indels in the intervening sequence between a MITE in-
crossing two F1individuals from a cross between two outbred
natural populations. Pairs of transposon display bands that
segregated as alternatives in repulsion were counted as prob-
able insertions that are descended from a single ancestral
insertion, but that have undergone indel events since the
Intron insertion polymorphisms due to MITE activ-
ity: A total of 38 primer pairs amplifying intronic re-
polymorphisms in European populations of S. latifolia.
Sixteen plants were included in the survey, and 5 of the
38 introns showed size polymorphisms, with size differ-
ences .150 bp (Table 1).
PCR amplification of intron 5 from a S. latifolia gene
(SlAnk) produced an ?600-bp amplicon in all individ-
uals, but a single male plant appeared to be heterozy-
gous, having an additional larger PCR product (?1000
bp, Figure 1). A single 407-bp insertion delimited by
11-bp terminal inverted repeats (with the sequence
59-CTAGGTAGCAC-39) and 8-bp TSDs was confirmed
in the larger amplicon by sequencing. The TSD has the
imperfect palindromic sequence 59-CTCTTGAG-39. Ex-
cluding the insertion, the 1-kb product differed from its
allelic counterpart in the same plant by one base sub-
stitution and a 3-bp indel. These data suggest that the
long and short sequences are allelic. The presence of
terminal inverted repeats (TIRs), TSDs, and extensive
secondary structure strongly suggests that this insertion
is a MITE element. Classification of nonautonomous
2007). The size of the TSD (8 bp) suggests that this ele-
ment is either a hATor a P element, but the TIR motifs
known from these two classes of elements were not
found. We therefore identify it as the first MITE ob-
tained from our study species, and, given its uncertain
classification, we named it EITRI.
In contrast to this singleton polymorphic insertion, a
size polymorphism due to a singleton excision was ob-
served in a sex-linked gene. PCR amplification of intron
2 of the recently described S. latifolia sex-linked gene
(710 and 1000 bp). Segregation analysis of these bands
clearly showed Y linkage of the longer intron variant
Loci showing intron-size polymorphisms
MITE Insertions in the Silene latifolia Genome1087
(1000 bp). Sequence alignment of these two variants
shows an insertion of 290 bp, delimited by a 14-bp in-
verted repeat, and the inserted sequence exhibits the
potential for extensive secondary structure (Figure 2).
Surprisingly, its TIR (59-GGGGGTGTTTGGTT-39)
matches perfectly the TIR region of a Tourist element
(Zm20) isolated from Zea mays (Bureau and Wessler
1994) and TIR consensus sequence from 21 rice PIF
families (Zhang et al. 2004), probably because of high
conservation of the catalytic domain of the transposase.
Furthermore, a 3-bp TTA motif flanked this insertion,
which is typical for TSDs of Tourist (mPIF) elements
(Jurka and Kapitonov 2001). We therefore classified
this as a Tourist-like element and named it SlTo1. In a
sample of eight Y chromosomes from natural popula-
tions, we found a singleton excision with a clearly visible
footprint in the SlCypY sequence alignment (Figure 2).
A search for this insertion in Cyp orthologs of other,
closely related, dioecious Silene species revealed the
same transposable element insertion in the Y chromo-
somes of S. diclinis and S. dioica; the sequence identity of
these MITE insertions was estimated to be 99%. Al-
though there are reported cases of independent in-
sertions in the identical site (Walker et al. 1997), the
most parsimonious explanation for this MITE in the
SlCypY gene is that these three sister species split after
X–Y recombination stopped in this region and that this
Tourist insertion occurred before this time. This is con-
sistent with the fact that the divergence between the
SlCyp X and the Y copies (Ks¼ 6.1%, from Bergero
et al. 2007) is considerably higher than that between
these species (Ks¼ 4.4%).
Figure 1.—The EITRI element. (a) A singleton MITE inser-
tion (named EITRI) was detected in SlAnk intron 5. (b) A pos-
sible secondary structure of this insertion predicted by using
the web-based software MFOLD (Zuker 2003).
Figure 2.—The Tourist-related element SlTo1. (a) Invasion
of a Tourist-like element (SlTo1) in intron 2 of the SlCypY gene.
A singleton excision was observed in male G2005-2, from Italy
(*). (b) A possible secondary structure of this MITE insertion.
(c) Alignment of sequences flanking the SlTo1 insertion site
and homologous intronic sequences from the X-linked coun-
terpart. The footprint of SlTo1 excision (boxed region) was rec-
ognized by sequence comparison of X- and Y-linked variants.
1088R. Bergero, A. Forrest and D. Charlesworth
We detected three further intron-size polymorphisms
due to insertions of unknown origins (Table 1). All the
polymorphic insertions from these three loci appear to
be at low to intermediate frequencies, but none are
have inverted repeats, nor did analysis of their sequences
suggest extensive secondary structure. Thus they are not
recognizable MITEs. The origin of these large insertion
variants is puzzling. Their intermediate frequencies in
natural populations suggest that these are not recent
insertions, and this is consistent with the absence of the
conserved features of MITE sequences. They could rep-
resent relics of MITEs, deleted for part(s) of the se-
quence, including the TIRs ½solo LTRs are known in
et al. 2002; Ma et al. 2004)?, or the TIR sequences may
have become unrecognizable due to mutations.
Transposon display of genomic and Y-linked MITE
insertions: A TD analysis of the two MITE elements,
EITRI and SlTo1, was carried out on a set of 48 individ-
uals collected from 24 European natural populations
(supplemental Table 1). Using the frequencies of null
alleles (see materials and methods), we estimated an
average of 230 copies of EITRI and 130 copies of the
SlTo1 element per haploid genome.
Segregation of MITE insertions in a full-sib F2family
allowed us to recognize MITE insertions in the Y
chromosome. Twenty-three EITRI elements and 16
SlTo1 elements showed a clear segregation pattern of
complete Y linkage. From the estimated average num-
whole, we computed the predicted number of Y-linked
insertions that should be found on a Y chromosome.
Taking into account the physical size of the Y chromo-
some ½the largest S. latifolia chromosome, estimated to
distribution of TE insertions in the S. latifolia genome,
there are significantly fewer MITE insertions in the Y
chromosome than the expected copy numbers for both
the EITRI and the SlTo1 subfamilies (49 and 28, re-
spectively; x2¼ 18.9, P , 0.0001).
An excess of singleton or low-frequency MITE geno-
mic insertions was observed from the TD analysis of the
24 female plants sampled from natural populations
(Figure 3); only a small fraction (5%) of insertion sites
were at medium or high frequencies (pi. 0.3). In
contrast, the frequency spectrum for Y-linked insertions
was markedly shifted toward high frequencies, with a
remarkable paucity of singleton and low-frequency
insertions (Figure 3). Thus, the ratio of fixed to poly-
fixed to polymorphic genomic insertions, due to an
excess of fixed Y-linked insertions (3/23 vs. 1/1923, for
the Y-linked and the other insertions, respectively). A
x2-test for independence, with Yates’ correction for con-
tinuity, showed that the two ratios were highly signifi-
cantly different (x2¼ 113.99, P , 0.000001).
MITEs and their contribution to the genetic de-
generation of evolving Y chromosomes: With their
ability to invade genic regions, MITEs could contribute
if selection against their removal is ineffective. Here we
show that MITE elements from two active subfamilies
are invading the evolving S. latifolia Y chromosome and
are present at intermediate and high frequencies or
fixed on this chromosome. This strongly contrasts with
the preponderance of singleton and rare insertions in
the rest of the genome (insertions with frequencies
,0.1 account for 78% of all observed insertions), which
suggests that these elements are readily removed from
recombining chromosomes. MITE insertions in Y-
linked genes at intermediate to high frequencies could
change gene expression and lower functions of Y-linked
genes. Future empirical work should test this possibility
directly, for example,bytesting whether the presence of
insertions correlates with changed expression of Y-
Figure 3.—Frequency distribution of EITRI and SlTo1 in-
sertions in 24 female genomes (a) and 24 Y chromosomes
from European S. latifolia populations (b). (a) The majority
of genomic MITE insertions were singletons or at low fre-
quency (pi, 0.3). (b) Y-linked MITE insertions (recognized
as such by segregation analysis in a full-sib F2family) were at
intermediate to high frequencies (pi. 0.3).
MITE Insertions in the Silene latifolia Genome1089
A caveat to estimating numbers of polymorphic TE
insertions from transposon display is that a proportion
of the polymorphisms may not be due to insertion/
excision events, but to indel mutations and/or muta-
tions changing restriction sites. Transposon display can-
not distinguish bona fide polymorphic TE insertions
from polymorphisms created by indels that change the
size of TD fragments or SNPs that modify restriction
sites. This will lead to overestimated numbers of poly-
morphic TE insertions, which will probably affect auto-
somes and X chromosomes more than Y chromosomes
because sequencing studies show that Y-linked sequen-
ces have lower diversity than is found in other genome
regions (Filatov et al. 2000, 2001; Filatov 2005;
Laporte et al. 2005). The relative number of low-
frequency polymorphic insertions may thus be some-
what inflated for the genome regions other than the Y.
However, this effect is probably very slight. Although
indels are common in intronic sequences from this
plant species (Bergero et al. 2007), indels postdating
MITE insertion events (see materials and methods
proportion (2%) of 203 MITE insertions segregating in
an F2family. This supports our assumption of a recent
age of most of the MITE insertions observed in our data
set and excludes the possibility that intervening indels
have substantially inflated our estimates of MITE poly-
morphisms. Similarly, destruction of restriction sites, or
creation of new ones in the region between the original
restriction sites and the TE insertion, should be in-
frequent in the short period after a TE insertion. Using
the approach of Nei and Li (1979), the probability of a
changed restriction site for a 4-base cutter (DpnII in our
study) by the time t after a TE insertion, given a rate l of
nucleotide substitutions per unit time, is (1 ? e?4lt), and
the expected probability of a new restriction site ap-
pearing in the intervening region (based on surveyed
fragment sizes of 175 678 bp) is 0.68(1 ? e?4lt). If MITE
insertions are recent, t is small, and these probabilities
become very small.
MITE dynamics in an asexual (nonrecombining)
genetic background: High MITE insertion frequencies
will lead to an increased mean copy number of inser-
tions per Ychromosome.The highfrequencies ofMITE
insertions in the S. latifolia Y chromosome indicate in-
creased chances of finding a TE per site. It is of interest
Y-linked insertions than the expected average number
for a genomic region of similar size (i.e., whether there
are more insertion sites per megabase in the Y than in
other genome regions, which might indicate loss of
functional sequences and therefore genetic degenera-
tion). Computing the expected number of Y-linked
per megabase, Y-linked MITEs appear to be somewhat
underrepresented (23 EITRI and 16 SlTo1 insertions
observed, vs. expected numbers of 49 and 28 insertions,
respectively). The estimated numbers of TE copies were
estimated from a single Y chromosome (the one in our
family), and the numbers are likely to vary among Y
are fixed or at high frequencies (see Figure 3 above),
any such variability should be minor and will not greatly
affect our conclusions.
Our result contrasts with findings from the neo-Y of
Drosophila miranda. In a survey of 12 D. miranda lines,
Bachtrog (2003) estimated that the Y-linked retro-
transposon insertions were fixed and thus inferred that
the number of insertions present in the neo-Y exceeds
that in the homologous regions of the neo-X chromo-
some. However, unlike retrotransposons (which use a
versions of DNA transposons, which use a cut-and-paste
et al. 1990). It is therefore plausible that their accumu-
lation will differ from that of retrotransposons. Another
reason for underrepresentationof Y-linked MITEs is that
they may be preferentially located either in regions with
high-recombination rates ½as reported for DNA trans-
posons in the Caenorhabditis elegans genome (Duret et al.
2003). Mammalian and Drosophila Y chromosomes are
notoriously low in gene content (Lahn et al. 2001), and
Silene Y chromosomes could be in a stage where de-
generation is already eroding gene content.
Nevertheless, the Silene Y chromosome has a DNA
content 40% larger than the homologous X chromo-
some (Siroky et al. 2001), and it seems likely that ac-
cumulation of TEs could explain this size difference.
Different behavior of different elements has been
found on the nonrecombining fourth chromosome of
D. melanogaster; there is a significant accumulation of
non-LTR retrotransposon elements, but not ofthe much
younger class of LTR retrotransposons (Kaminker et al.
2002; Bergman and Bensasson 2007). To test whether
TE classes other than the MITEs studied here have
accumulated, more TE types should be studied in the
future, especially RNA transposons.
The role of MITEs in plant genome diversification
and expansion: Our approach of screening for large
polymorphic insertions within a small set of intronic
sequences revealed active MITE elements in the non-
model plant S. latifolia, producing either singleton in-
sertions or excisions. The application of this approach
in nonmodel species is thus straightforward and will
greatly facilitate discovery of new active MITE families,
to study their accumulation and locations in eukaryotic
The finding of hundreds of MITE copies per haploid
genome suggests that the S. latifolia genome probably
contains many active MITEs. A large fraction of the
genome of many higher plants is repetitive, and MITEs
O. sativa transposable elements and a major fraction of
1090 R. Bergero, A. Forrest and D. Charlesworth
Silene, estimated genome sizes range from 1100 Mb in
S. vulgaris to 2646 Mb in S. latifolia and to 3300 in S.
chalcedonica (Siroky et al. 2001; Meagher et al. 2005).
These species all have the same chromosome number
½2n ¼ 24, like all nonpolyploid species in the genus for
which data exist (Goldblatt 1981)?, which excludes a
genome duplication as the cause of genome size differ-
differential accumulation of repetitive sequences and
MITEs could have contributed to genome size expan-
sion over relatively short evolutionary times (the genus
Silene is not older than a few million years, since the
largest synonymous-site divergence between any pair of
species in the genus is 20.8%, based on 1791 bp of
concatenated coding sequences from five loci).
This research was supported by the National Environmental Re-
search Council (grant no. NE/B504230/1).
Bachtrog, D., 2003
non-LTR retrotransposons, on the neo-Y chromosome of Drosoph-
ila miranda. Mol. Biol. Evol. 20: 173–181.
Bergero, R., A. Forrest, E. Kamau and D. Charlesworth, 2007
lutionary strata on the X chromosomes of the dioecious plant
Silene latifolia: evidence from new sex-linked genes. Genetics
Bergman, C. M., and D. Bensasson, 2007
son insertion contrasts with waves of non-LTR insertion since spe-
ciation in Drosophila melanogaster. Proc. Natl. Acad. Sci. USA 104:
Brookfield, J. F., and R. M. Badge, 1997
els of transposable elements. Genetica 100: 281–294.
Bureau, T. E., and S. R. Wessler, 1994
ments of the Tourist family are associated with the genes of many
cereal grasses. Proc. Natl. Acad. Sci. USA 91: 1411–1415.
Casa, A. M., C. Brouwer, A. Nagel, L. Wang, Q. Zhang et al.,
2000 The MITE family Heartbreaker (Hbr): molecular markers
in maize. Proc. Natl. Acad. Sci. USA 97: 10083–10089.
Casa, A. M., A. Nagel and S. R. Wessler, 2004
ods Mol. Biol. 260: 175–188.
Charlesworth, B., and D. Charlesworth, 2000
tion of Y chromosomes. Philos. Trans. R. Soc. Lond. B Biol.
Sci. 355: 1563–1572.
Devos, K. M., J. K. Brown and J. L. Bennetzen, 2002
reduction through illegitimate recombination counteracts ge-
nome expansion in Arabidopsis. Genome Res. 12: 1075–1079.
Don, R. H., P. T. Cox, B. J. Wainwright, K. Baker and J. S. Mattick,
1991‘Touchdown’ PCR to circumvent spurious priming during
gene amplification. Nucleic Acids Res. 19: 4008.
Duret, L., G. Marais and C. Biemont, 2000
retrotransposons are located preferentially in regions of high re-
combination rate in Caenorhabditis elegans. Genetics 156: 1661–
Engels, W. R., D. M. Johnson-Schlitz, W. B. EgglestonandJ. Sved,
1990 High-frequency P element loss in Drosophila is homolog
dependent. Cell 62: 515–525.
Feschotte, C., N. Jiang and S. R. Wessler, 2002
elements: where genetics meets genomics. Nat. Rev. Genet. 3:
Filatov, D. A., 2005 Substitution rates in a new Silene latifolia sex-
linked gene, SlssX/Y. Mol. Biol. Evol. 22: 402–408.
Filatov, D. A., F. Moneger, I. Negrutiu and D. Charlesworth,
2000Low variability in a Y-linked plant gene and its implica-
tions for Y-chromosome evolution. Nature 404: 388–390.
Filatov, D. A., V. Laporte, C. Vitte and D. Charlesworth,
2001DNA diversity in sex-linked and autosomal genes of the
Accumulation of Spock and Worf, two novel
Recent LTR retrotranspo-
Population genetics mod-
Mobile inverted-repeat ele-
MITE display. Meth-
Transposons but not
plant species Silene latifolia and Silene dioica. Mol. Biol. Evol. 18:
Finnegan, D. J., 1992 Transposable elements. Curr. Opin. Genet.
Dev. 2: 861–867.
Foote, S., D. Vollrath, A. Hilton and D. C. Page, 1992
man Y chromosome: overlapping DNA clones spanning the eu-
chromatic region. Science 258: 60–66.
Goldblatt, P., 1981
Index to Plant Chromosome Numbers: 1975–1978.
Missouri Botanical Garden, St. Louis.
Holt, R. A., G. M. Subramanian, A. Halpern, G. G. Sutton, R.
Charlab et al., 2002The genome sequence of the malaria mos-
quito Anopheles gambiae. Science 298: 129–149.
Jiang, N., C. Feschotte, X. Zhang and S. R. Wessler, 2004
rice to understand the origin and amplification of miniature in-
verted repeat transposable elements (MITEs). Curr. Opin. Plant
Biol. 7: 115–119.
Jurka, J., and V. V. Kapitonov, 2001
bingers: a superfamily reunion. Proc. Natl. Acad. Sci. USA 98:
Kaminker, J. S., C. M. Bergman, B. Kronmiller, J. Carlson,
R. Svirskas et al., 2002 The transposable elements of the Dro-
sophila melanogaster euchromatin: a genomics perspective. Genome
Biol. 3: research0084.0081–0084.0020.
Lahn, B. T., N. M. Pearson and K. Jegalian, 2001
chromosome, in the light of evolution. Nat. Rev. Genet. 2:
Langley, C. H., E. Montgomery, R. Hudson, N. Kaplan and B.
Charlesworth, 1988On the role of unequal exchange in
the containment of transposable element copy number. Genet.
Res. 52: 223–235.
Laporte, V., D. A. Filatov, E. Kamau and D. Charlesworth,
2005Indirect evidence from DNA sequence diversity for ge-
netic degeneration of the Y-chromosome in dioecious species
of the plant Silene: the SlY4/SlX4 and DD44-X/DD44-Y gene pairs.
J. Evol. Biol. 18: 337–347.
Ma, J., K. M. Devos and J. L. Bennetzen, 2004
retrotransposon structures reveal recent and rapid genomic
DNA loss in rice. Genome Res. 14: 860–869.
Marchuk, D., M. Drumm, A. Saulino and F. S. Collins,
1991Construction of T-vectors, a rapid and general system
for direct cloning of unmodified PCR products. Nucleic Acids
Res. 19: 1154.
Meagher, T. R., A. C. Gillies and D. E. Costich, 2005
size, quantitative genetics and the genomic basis for flower size
evolution in Silene latifolia. Ann. Bot. 95: 247–254.
Montell, H., A. K. Fridolfsson and H. Ellegren, 2001
trasting levels of nucleotide diversity on the avian Z and W sex
chromosomes. Mol. Biol. Evol. 18: 2010–2016.
Montgomery, E., B. Charlesworth and C. H. Langley, 1987
test for the role of natural selection in the stabilization of trans-
posable element copy number in a population of Drosophila mel-
anogaster. Genet. Res. 49: 31–41.
Naito, K., E. Cho, G. Yang, M. A. Campbell, K. Yano et al.,
2006Dramatic amplification of a rice transposable element
during recent domestication. Proc. Natl. Acad. Sci. USA 103:
Nei, M., and W. H. Li, 1979Mathematical model for studying ge-
netic variation in terms of restriction endonucleases. Proc. Natl.
Acad. Sci. USA 76: 5269–5273.
Pace, 2nd, J. K., and C. Feschotte, 2007
human DNA transposons: evidence for intense activity in the pri-
mate lineage. Genome Res. 17: 422–432.
Rizzon, C., G. Marais, M. Gouy and C. Biemont, 2002
bination rate and the distribution of transposable elements in
the Drosophila melanogaster genome. Genome Res. 12: 400–407.
Siroky, J., M. A. Lysak, J. Dolezel, E. Kejnovsky and B. Vyskot,
2001Heterogeneity of rDNA distribution and genome size in
Silene spp. Chromosome Res. 9: 387–393.
Smit, A. F., and A. D. Riggs, 1996
sils in the human genome. Proc. Natl. Acad. Sci. USA 93: 1443–
Steinemann, M., and S. Steinemann, 1998
some degeneration: neo-Y and neo-X chromosomes of Drosophila
miranda a model for sex chromosome evolution. Genetica 102–
PIFs meet Tourists and Har-
The human Y
Analyses of LTR-
The evolutionary history of
Tiggers and DNA transposon fos-
Enigma of Y chromo-
MITE Insertions in the Silene latifolia Genome1091
Surzycki, S. A., and W. R. Belknap, 2000
are similarly distributed on Caenorhabditis elegans autosomes.
Proc. Natl. Acad. Sci. USA 97: 245–249.
Van den Broeck, D., T. Maes, M. Sauer, J. Zethof, P. De Keukeleire
et al., 1998Transposon display identifies individual transpos-
able elements in high copy number lines. Plant J. 13: 121–129.
Walker, E. L., W. B. Eggleston, D. Demopulos, J. Kermicle and S.
L. Dellaporta, 1997 Insertions of a novel class of transposable
elements with a strong target site preference at the r locus of
maize. Genetics 146: 681–693.
Wicker, T., F. Sabot, A. Hua-Van, J. L. Bennetzen, P. Capy et al.,
2007 A unified classification system for eukaryotic transposable
elements. Nat. Rev. Genet. 8: 973–982.
Wright, S., and D. Finnegan, 2001
transposable element. Curr. Biol. 11: R296–R299.
Wright, S. I., N. Agrawal and T. E. Bureau, 2003
bination rate and gene density on transposable element distribu-
tions in Arabidopsis thaliana. Genome Res. 13: 1897–1903.
Yano, M.,Y. Katayose, M. Ashikari, U. Yamanouchi, L. Monna et al.,
Hd1, a major photoperiod sensitivity quantitative trait lo-
Genome evolution: sex and the
Effects of recom-
cus in rice,is closely relatedto the Arabidopsis floweringtime gene
CONSTANS. Plant Cell 12: 2473–2484.
Zhang, X., C. Feschotte, Q. Zhang, N. Jiang, W. B. Eggleston
et al., 2001
P instability factor: an active maize transposon system
associated with the amplification of Tourist-like MITEs and a
new superfamily of transposases. Proc. Natl. Acad. Sci. USA 98:
Zhang, X., N. Jiang, C. Feschotte and S. R. Wessler, 2004
and Pong-like transposable elements: distribution, evolution
and relationship with Tourist-like miniature inverted-repeat trans-
posable elements. Genetics 166: 971–986.
Zuker, M., 2003Mfold web server for nucleic acid folding and hy-
bridization prediction. Nucleic Acids Res. 31: 3406–3415.
Zurovcova, M., and W. F. Eanes, 1999
morphism in the Y-linked sperm flagellar dynein gene Dhc-
Yh3 of Drosophila melanogaster and D. simulans. Genetics 153:
Lack of nucleotide poly-
Communicating editor: D. Voytas
1092 R. Bergero, A. Forrest and D. Charlesworth