Active Miniature Transposons From a Plant Genome and Its Nonrecombining Y Chromosome

Institute of Evolutionary Biology, Ashworth Laboratories, University of Edinburgh, Edinburgh, EH9 3JT, United Kingdom.
Genetics (Impact Factor: 5.96). 03/2008; 178(2):1085-92. DOI: 10.1534/genetics.107.081745
Source: PubMed
Mechanisms involved in eroding fitness of evolving Y chromosomes have been the focus of much theoretical 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 chromosomes. 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 transposon 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.


Available from: Roberta Bergero
Copyright 2008 by the Genetics Society of America
DOI: 10.1534/genetics.107.081745
Active Miniature Transposons From a Plant Genome and
Its Nonrecombining Y Chromosome
R. Bergero,
A. 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.
RANSPOSABLE elements (TEs) have major roles
in genome diversification and expansion. Due to
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
(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 recombina tion is suppressed. Thus the
main deleterious effects of TEs on fitness of Y chromo-
somes should be insertions affecting functionally impor-
tant sequences.
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 (100–5 00 bp), insertions often do not cause
major disruption of the genes or their regulation
(Naito et al. 2006). However, some insertions could
be highly deleterious (Yano et al. 2000) and, if they occur
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-
somes are thus expected to have low effective population
size, N
, due to the ‘hitchhiking’ effects of selection
Sequence data from this article have been deposited with the EMBL/
GenBank Data Libraries under accession nos. EU334132 and EU334133.
Corresponding author: Institute of Evolutionary Biology, Ashworth
Laboratories, University of Edinburgh, King’ s Bldgs., W. Mains Rd.,
Edinburgh, EH9 3JT, United Kingdom. E-mail:
Genetics 178: 1085–1092 (February 2008)
Page 1
½selective sweeps, background selection, and weak Hill–
Robertson interference (reviewed by Charlesworth
and Charlesworth 2000). This expectation is sup-
ported by empirical data showing low silent-site diversity
of Y-linked genes , compared with th eir X-linked alleles
(Zurovcova and Eanes 1999; Montell et al. 2001).
Reduced N
should 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 insert ions may
thus contribute to Y chromosome genetic degeneration.
It is well known that Y chromosomes and neo-Y chro-
mosomes undergo genetic degeneration in the long
term (Charlesworth and Charlesworth 2000) and
that TEs can quickly accu mulate on neo-Y chromosomes
(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 (Dure t et al.
2000; S urzycki and Belknap 2000; Rizzon et al. 2002;
Wright et al. 2003), but such extensive genomic se-
quences are difficult to obtain from D NA regions rich
in repetitive sequences, such as the Y chromosomes
(Foote et al. 1992; Holt et al. 2002), or are simply not
available from nonmodel species. Furthermore, genome-
scan approaches provide no information about the dis-
tribution of insert ion frequencies, which is needed to
test the predictions of the theories outlined above, and
it is not always clear whether any given transposab le 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
insertions from both subfamilies was carried out to infer
their frequency distributions and compare Y chromo-
somes with other genome regions sampled from S.
latifolia natural populations.
Plant material: One male and one female S. latifolia individ-
ual from each of eight European natural populations (sup-
plemental Table 1 at
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 F
plants. The
family derived from a single cross between two F
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 to search for large-size polymorphisms (.150 bp)
that could result from recent MITE insertion/excision. Loci
were chosen to be single copy or low copy to limit amplifica-
tion of paralogous genes, which could hinder interpretation of
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
was done using the package Sequencher 4.7 (GeneCodes, Ann
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
ends of the insertion, target site duplication (TSD) as reported
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 F
family 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
1086 R. Bergero, A. Forrest and D. Charlesworth
Page 2
(SlTo1 and EITRI). The procedures were as outlined in Casa
et al. (2004) with the following modifications. Genomic DNA
(0.8–1.5 m g) 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-
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
overhang at one end of the adapter and by the formation of an
ex novo BamHI site at the other end when adapter dimers
formed. Dimers were destroyed by adding BamHI to the
ligation reaction.
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
ATCCATTCCAAGAG-39). Thermocycling conditions were as
follows: 4 min at 94; 20 cycles at 30 sec at 95, 40 sec at 50,30
sec at 72; and 5 min at 72. One microliter of the PCR reaction
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
16 possible combination were used) and a VIC-labeled (Applied
Biosystems) MITE-specific internal primer (EITRI-int, 59-TCC
CAAACACCCC -39), with the following thermocycling condi-
tions: 2 min at 95, followed by 10 annealing cycles at 0.5
decreasing and 18 cycles at 50. The selective amplification was
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
better electropherograms.
VIC-labeled PCR products were first diluted (1:25) in
distilled water, further diluted (1:10) in formamide containing
the size standard GeneScan 500-LIZ (Applied Biosystems),
and directly separated by capillary electrophoresis on an ABI-
3730 DNA analyzer (Applied Biosystems). Transposon inser-
tions were scored using the software Genemapper v. 3.7 (Applied
Biosystems), and their frequencies were analyzed and plotted
using the R statistics package (
MITE sequences from this study were deposited in the
GenBank databases (accession nos. EU334132 and EU334133).
Data analyses: The average numbers of 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
the recessive genotypes (nulls) of each insertion. To obtain the
frequency, p
, of insertions at the ith site, we assumed that the
population is in Hardy–Weinberg equilibrium, since the spe-
cies is outcrossing, and used p
¼ 1 Oq
, where q
is the
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 ÆN
æ ¼
Indels in the intervening sequence between a MITE in-
sertion and the restriction site will create a new ‘spurious’
polymorphic TE insertion. We estimated the proportion of the
observed polymorphisms due to such events by examining
segregation of 203 MITE insertions in an F
progeny made by
crossing two F
individuals 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-
gions from 19 loci were used to search for large insertion
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 amplico n 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 5 9-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
TEs relies on the TIR motifs and TSD sizes (Wicker et al.
2007). The size of the TSD (8 bp) suggests that this ele-
ment is either a hAT or 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
SlCypX/Y (Bergero et al. 2007) produced two amplicons
(710 and 1000 bp). Segregation analysis of these bands
clearly showed Y linkage of the longer intron variant
Loci showing intron-size polymorphisms
location Intron
size (bp)
SlATTPS6 Autosomal 2 232 57 Not detected
SlAnk Autosomal 5 407 61 EITRI
SlCypY Y-linked 2 290 58 SlTo1
SlPI Autosomal 5 348 65 Not detected
SlX3 X-linked 10 226 63 Not detected
MITE Insertions in the Silene latifolia Genome 1087
Page 3
(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 exhi bits the
potential for extensive secondar y 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 K apitonov 2001). We therefore classified
this as a Tourist-like element and named it SlTo1.Ina
sample of eight Y chromosomes from natural popula-
tions, we found a singleton excision with a clearly visib le
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 eleme nt 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 inde pendent 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 cop ies (K
¼ 6.1%, from Bergero
et al. 2007) is considerably higher than that between
these species (K
¼ 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.
1088 R. Bergero, A. Forrest and D. Charlesworth
Page 4
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
singleton insertions or excisions. These insertions did not
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
other plant genomes and are caused by deletions (Devos
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 e lement per haploid genome.
Segregation of MITE insertions in a full-sib F
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-
ber of MITE insertions per megabase in the genome as a
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
be 570 Mb (Siroky et al. 2001), and assuming a uniform
distribution of TE insertions in the S. latifolia genome,
there are significantly fewer MITE insertions in the Y
chromosome than th e expected copy numbers for both
the EITRI and the SlTo1 subfamilies (49 and 28, re-
spectively; x
¼ 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 (p
. 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-
morphic Y-linked insertions was greater than the ratio of
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
-test for independence, with Yates’ correction for con-
tinuity, showed that the two ratios were highly signifi-
cantly different (x
¼ 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
to the genetic degeneration of evolving Y chromosomes,
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 inter mediate 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, by testing whether the presence of
insertions correlates with changed expression of Y-
linked genes.
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 (p
, 0.3). (b) Y-linked MITE insertions (recognized
as such by segregation analysis in a full-sib F
family) were at
intermediate to high frequencies (p
. 0.3).
MITE Insertions in the Silene latifolia Genome 1089
Page 5
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 cre ated 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 (B ergero et al. 2007), indels postdating
MITE insertion events (see materials and methods
for how these are ascertained) are seen in only in a small
proportion (2%) of 203 MITE insertions se gregating in
an F
family. 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 approa ch 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
), and
the expected probability of a new restriction site ap-
pearing in the intervening region (based on surveyed
fragment sizes of 175 6 78 bp) is 0.68(1 e
). 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 Y ch romosome. The high frequencies of MITE
insertions in the S. latifolia Y chromosome indicate in-
creased chances of finding a TE per site. It is of interest
also to ask whether there is also a higher total number of
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
MITEs from the estimated average number of insertions
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
haplotypes. However, given that most of the Y-linked TEs
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
copy-and-paste mechanism), MITEs are nonautonomous
versions of DNA transposons, which use a cut-and-paste
mechanism for their movement and replication (Engels
et al. 1990). It is therefore plausible that their accumu-
lation will differ from that of retrotransposons. Another
reason for underrepresentation of 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.
2000) or in regions with high gene density (Wright 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 of the 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, pro ducing either singleton in-
sertions or excisions. The application of this approach
in nonmodel spec ies 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
are reported to represent the largest component among
O. sativa transposable elements and a major fract ion of
1090 R. Bergero, A. Forrest and D. Charlesworth
Page 6
the Arabidopsis genome (Jiang et al. 2004). In the genus
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-
ences. This high variability in genome sizes suggests that
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 Accumulation of Spock and Wor f, two novel
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 Evo-
lutionary strata on the X chromosomes of the dioecious plant
Silene latifolia: evidence from new sex-linked genes. Genetics
175: 1945–1954.
Bergman, C. M., and D. Bensasson, 2007 Recent LTR retrotranspo-
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 Population genetics mod-
els of transposable elements. Genetica 100: 281–294.
Bureau, T. E., and S. R. Wessler, 1994 Mobile inverted-repeat ele-
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 MITE display. Meth-
ods Mol. Biol. 260: 175–188.
Charlesworth, B., and D. Charlesworth, 2000 The degenera-
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 Genome size
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 Transposons but not
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.Eggleston and J. 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 Plant transposable
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,
2000 Low 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,
2001 DNA diversity in sex-linked and autosomal genes of the
plant species Silene latifolia and Silene dioica. Mol. Biol. Evol.
Finnegan, D. J., 1992 Transposable elements. Curr. Opin. Genet.
Dev. 2: 861–867.
Foote, S., D. Vollrath,A.Hilton and D. C. Page, 1992 The hu-
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., 2002 The genome sequence of the malaria mos-
quito Anopheles gambiae. Science 298: 129–149.
Jiang, N., C. Feschotte,X.Zhang and S. R. Wessler, 2004 Using
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 PIFs meet Tourists and Har-
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 The human Y
chromosome, in the light of evolution. Nat. Rev. Genet. 2:
angley, C. H., E. Montgomery,R.Hudson,N.Kaplan and B.
Charlesworth, 1988 On 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,
2005 Indirect 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 Analyses of LTR-
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,
1991 Construction 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 Genome
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. E llegren, 2001 Con-
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 A
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.
, K., E. Cho,G.Yang,M.A.Campbell,K.Yano et al.,
2006 Dramatic amplification of a rice transposable element
during recent domestication. Proc. Natl. Acad. Sci. USA 103:
Nei, M., and W. H. Li, 1979 Mathematical 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 The evolutionary history of
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 Recom-
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,
2001 Heterogeneity of rDNA distribution and genome size in
Silene spp. Chromosome Res. 9: 387–393.
Smit, A. F., and A. D. Riggs, 1996 Tiggers and DNA transposon fos-
sils in the human genome. Proc. Natl. Acad. Sci. USA 93: 1443–
Steinemann, M., and S. Steinemann, 1998 Enigma of Y chromo-
some degeneration: neo-Y and neo-X chromosomes of Drosophila
miranda a model for sex chromosome evolution. Genetica 102–
103: 409–420.
MITE Insertions in the Silene latifolia Genome 1091
Page 7
Surzycki, S. A., and W. R. Belknap, 2000 Repetitive-DNA elements
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., 1998 Transposon 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 Genome evolution: sex and the
transposable element. Curr. Biol. 11: R296–R299.
Wright, S. I., N. Agrawal and T. E. Bureau, 2003 Effects of recom-
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.,
2000 Hd1, a major photoperiod sensitivity quantitative trait lo-
cus in rice, is closely related to the Arabidopsis
flowering time 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 PIF-
and Pong-like transposable elements: distribution, evolution
and relationship with Tourist-like miniature inverted-repeat trans-
posable elements. Genetics 166: 971–986.
Zuker, M., 2003 Mfold web server for nucleic acid folding and hy-
bridization prediction. Nucleic Acids Res. 31: 3406–3415.
Zurovcova, M., and W. F. Eanes, 1999 Lack of nucleotide poly-
morphism in the Y-linked sperm flagellar dynein gene Dhc-
Yh3 of Drosophila melanogaster and D. simulans. Genetics 153:
Communicating editor: D. Voytas
1092 R. Bergero, A. Forrest and D. Charlesworth
Page 8
  • Source
    • "The rDNA genes are rich in repetitive DNA sequences and have gained wide utility in chromosome characterization through fluorescence in situ hybridization in many important plant species including cucurbits (Gerlach and Bedbrook 1979; Chen et al. 1998; Koo et al. 2002; Xu et al. 2007; Han et al. 2008; Ren et al. 2009; Vasconcelos et al. 2010; Xiang-Hui 2011; Zhao et al. 2011; Waminal and Kim 2012). Repetitive DNA elements have played significant roles in the evolution of plant sex chromosomes (Lengerova et al. 2004; Jamilena et al. 2008; Bergero et al. 2008; Mariotti et al. 2009; Heslop-Harrison and Schwarzacher 2011). Sex chromosomes have been distinctly identified through FISH with 45S rDNA as probes in animals and few plant species (Nakayama et al. 2001; Lan et al. 2006; Novotná et al. 2011; Takehana et al. 2012; Deng et al. 2012). "
    [Show abstract] [Hide abstract] ABSTRACT: Coccinia grandis is a widely distributed dioecious cucurbit in India, with heteromorphic sex chromosomes and X-Y sex determination mode. The present study aids in the cytogenetic characterization of four native populations of this plant employing distribution patterns of 45S rDNA on chromosomes and guanine-cytosine (GC)-rich heterochromatin in the genome coupled with flow cytometric determination of genome sizes. Existence of four nucleolar chromosomes could be confirmed by the presence of four telomeric 45S rDNA signals in both male and female plants. All four 45S rDNA sites are rich in heterochromatin evident from the co-localization of telomeric chromomycin A (CMA)(+ve) signals. The size of 45S rDNA signal was found to differ between the homologues of one nucleolar chromosome pair. The distribution of heterochromatin is found to differ among the male and female populations. The average GC-rich heterochromatin content of male and female populations is 23.27 and 29.86 %, respectively. Moreover, the male plants have a genome size of 0.92 pg/2C while the female plants have a size of 0.73 pg/2C, reflecting a huge genomic divergence between the genders. The great variation in genome size is owing to the presence of Y chromosome in the male populations, playing a multifaceted role in sexual divergence in C. grandis.
    Full-text · Article · Jan 2016 · Protoplasma
  • Source
    • "The early periods of study have been followed by a long gap until researchers continued thorough cytogenetic analyses since the last decade (Guha et al. 2004; Bhowmick et al. 2012 Bhowmick et al. , 2015 Sousa et al. 2013). The large size of Coccinia Y chromosome indicates the accumulation of tremendous repeats in recently evolved Y chromosomes (Bergero et al. 2008; Jamilena et al. 2008; Mariotti et al. 2009; Heslop-Harrison and Schwarzacher 2011). Again, C-banding experiments reveal heterochromatic nature of Coccinia Y chromosome unlike the euchromatic Y chromosome of Silene (Sousa et al. 2013). "
    [Show abstract] [Hide abstract] ABSTRACT: The family Cucurbitaceae showcases a wide range of sexual phenotypes being variedly regulated by biological and environmental factors. In the present context, we have tried to assemble reports of cytogenetic investigations carried out in cucurbits accompanied by information on sex expression diversities and chromosomal or molecular basis of sex determination in dioecious (or other sexual types, if reported) taxa known so far. Most of the Cucurbitaceae tribes have mixed sexual phenotypes with varying range of chromosome numbers and hence, ancestral conditions become difficult to probe. Occurrence of polyploidy is rare in the family and has no influence on sexual traits. The sex determination mechanisms have been elucidated in some well-studied taxa like Bryonia, Coccinia and Cucumis showing interplay of genic, biochemical, developmental and sometimes chromosomal determinants. Substantial knowledge about genic and molecular sex differentiation has been obtained for genera like Momordica, Cucurbita and Trichosanthes. The detailed information on sex determination schemes, genomic sequences and molecular phylogenetic relationships facilitate further comprehensive investigations in the tribe Bryonieae. The discovery of organ identity genes and sex-specific sequences regulating sexual behaviour in Coccinia, Cucumis and Cucurbita opens up opportunities of relevant investigations to answer yet unaddressed questions pertaining to floral unisexuality, dioecy and chromosome evolution in the family. The present discussion brings the genera in light, previously recognized under subfamily Nhandiroboideae, where the study of chromosome cytology and sex determination mechanisms can simplify our understanding of sex expression pathways and its phylogenetic impacts.
    Full-text · Article · Dec 2015 · Journal of Genetics
  • Source
    • "The early periods of study have been followed by a long gap until researchers continued thorough cytogenetic analyses since the last decade (Guha et al. 2004; Bhowmick et al. 2012 Bhowmick et al. , 2015 Sousa et al. 2013). The large size of Coccinia Y chromosome indicates the accumulation of tremendous repeats in recently evolved Y chromosomes (Bergero et al. 2008; Jamilena et al. 2008; Mariotti et al. 2009; Heslop-Harrison and Schwarzacher 2011). Again, C-banding experiments reveal heterochromatic nature of Coccinia Y chromosome unlike the euchromatic Y chromosome of Silene (Sousa et al. 2013). "
    [Show abstract] [Hide abstract] ABSTRACT: Basic cytogenetic investigation of economically important members of Cucurbitaceae is a prerequisite to understand their genetic architecture. The study reports detailed chromosome morphometric analyses with fluorescence banding technique, differential distribution of heterochromatin, flow cytometric nuclear DNA contents and meiotic behavior of the native populations of Benincasa hispida, Luffa cylindrica and Trichosanthes dioica in India. In spite of frequent vegetative propagation, different populations of these cultivated cucurbits show regular genetic processes evident from stable chromosome counts, nuclear DNA sizes, ploidy levels and regular meiotic behavior. Distal CMA+ve signals have been scored in two pairs of chromosomes in B. hispida and four pairs of chromosomes in L. cylindrica, representing the GC rich heterochromatic portions in genomes. The presence of terminal DAPI+ve bands in the chromosomes of male and female populations of T. dioica indicates the predominance of AT rich heterochromatin. In addition to DAPI bands, terminal CMA+ve bands in the female plants of T. dioica depict the chromosomal signs of sexual differentiation in this species. The existence of rod bivalent in meiosis of male plants facilitates further approach to characterize the genetics of chromosomal evolution since gross similarity in genome size and karyotypic features of male and female plants advocates genic control of sexual dimorphism in T. dioica.
    Full-text · Article · Aug 2015 · Scientia Horticulturae
Show more