Next Generation Sequencing Reveals Genome Downsizing in Allotetraploid Nicotiana tabacum, Predominantly through the Elimination of Paternally Derived Repetitive DNAs
Mol. Biol. Evol.
Next Generation Sequencing Reveals Genome Downsizing in Allotetraploid Nicotiana tabacum, Predominantly through the Elimination of Paternally Derived Repetitive DNAs
Simon Renny-Byfield 2
Michael Chester 1 2
Ales Kovaˇr´ık 0
Steven C. Le Comber 2
Marie-Ange`le Grandbastien 5
Marc Deloger 5
Richard A. Nichols 2
Jiri Macas 4
Petr Nova´k 4
Mark W. Chase 3
Andrew R. Leitch 2
0 Institute of Biophysics, Academy of Sciences of the Czech Republic , v.v.i, Brno , Czech Republic
1 Laboratory of Molecular Systematics and Evolutionary Genetics, Florida Museum of Natural History, University of Florida
2 School of Biological and Chemical Sciences, Queen Mary University of London , London , United Kingdom
3 Jodrell Laboratory , Royal Botanic Gardens, Kew, Richmond, Surrey , United Kingdom
4 Biology Centre, Institute of Plant Molecular Biology, Academy of Sciences of the Czech Republic , C
5 Institute Jean-Pierre Bourgin, Institut National de la Recherche Agronomique-Versailles , France
We used next generation sequencing to characterize and compare the genomes of the recently derived allotetraploid, Nicotiana tabacum (,200,000 years old), with its diploid progenitors, Nicotiana sylvestris (maternal, S-genome donor), and Nicotiana tomentosiformis (paternal, T-genome donor). Analysis of 14,634 repetitive DNA sequences in the genomes of the progenitor species and N. tabacum reveal all major types of retroelements found in angiosperms (genome proportions range between 17-22.5% and 2.3-3.5% for Ty3-gypsy elements and Ty1-copia elements, respectively). The diploid N. sylvestris genome exhibits evidence of recent bursts of sequence amplification and/or homogenization, whereas the genome of N. tomentosiformis lacks this signature and has considerably fewer homogenous repeats. In the derived allotetraploid N. tabacum, there is evidence of genome downsizing and sequences loss across most repeat types. This is particularly evident amongst the Ty3-gypsy retroelements in which all families identified are underrepresented in N. tabacum, as is 35S ribosomal DNA. Analysis of all repetitive DNA sequences indicates the T-genome of N. tabacum has experienced greater sequence loss than the S-genome, revealing preferential loss of paternally derived repetitive DNAs at a genome-wide level. Thus, the three genomes of N. sylvestris, N. tomentosiformis, and N. tabacum have experienced different evolutionary trajectories, with genomes that are dynamic, stable, and downsized, respectively.
next generation sequencing; allopolyploidy; genome downsizing; transposable elements; retroelements; paternal genome; Nicotiana tabacum; Nicotiana sylvestris; Nicotiana tomentosiformis
Angiosperm evolution has been heavily impacted by
polyploidy, which has occurred in the ancestry of most,
if not all, species (Soltis et al. 2009). Polyploidy itself
may induce revolutionary changes in genome composition
in early generations (Leitch and Leitch 2008), a
phenomenon explored here. Interspecific hybridization combined
with whole-genome multiplication (allopolyploidy)
provides a natural experiment in genome perturbation where
the fate of DNA sequences can be examined by studying
the descendants of the two progenitor species and their
allopolyploid offspring. McClintock (1984) first proposed
that allopolyploidy can induce ‘‘genomic shock’’ and we
now know that changes can occur at the DNA sequence,
epigenetic, karyotypic, and transcription levels (Wendel
2000). Moreover, polyploid-associated genetic change
has been observed to occur rapidly in some species,
occurring after only a few generations, leading many to
envisage a ‘‘genome revolution’’ where perturbation of
the progenitor genomes is induced by their unification
(Wendel 2000; Comai et al. 2003; Liu and Wendel 2003;
Feldman and Levy 2009).
Repetitive DNA sequences, which comprise a large
proportion of the genomes of many plant species, may
be subject to change in sequence, copy number, and/or
epigenetic profile following allopolyploidy (Matyasek
et al. 2002, 2003; Adams and Wendel 2005; Leitch et al.
2008). However, there are only a few examples of
allopolyploid-associated or interspecific hybridization–associated
changes for the transposable elements (TEs; Parisod
et al. 2010). Such evidence includes 1) the activation
and movement of retroelements in natural (Petit et al.
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2007) and synthetic Nicotiana tabacum (Petit et al. 2010) in
addition to the loss of some retroelements which may
occur rapidly (within a few generations). 2) The activation
of retroelements and miniature inverted-repeat
transposable elements in rice following alien DNA introgression
from related wild species (Liu and Wendel 2000), in which
activation was transient, involving amplification of a few
copies (10–20 copies) and methylation of the new
insertions, which were stably inherited in subsequent
generations. 3) Allopolyploid induced activation of Wis2
retroelement transcription in synthetic crosses of Aegilops
sharonensis Triticum monococcum, although this was
not associated with any observed increase in copy number
or element mobility (Kashkush et al. 2003).
There are examples where repeat sequence activation
following allopolyploidy is not apparent, notably in
Gossypium synthetic allopolyploids (Liu et al. 2001; Hu et al.
2010) and in recently formed (within last 150 years) natural
Spartina anglica (Ainouche et al. 2009). Similarly,
sequencespecific amplified polymorphism analysis of Arabidopsis
thaliana Arabidopsis lyrata revealed the CAC family
of transposons was not activated in neotetraploids
(Beaulieu et al. 2009). However, there were substantial
epigenetic changes influencing establishment of nucleolar
dominance and degree of cytosine methylation at 25%
of loci examined as well as a large chromosomal deletion.
Genome size estimates have indicated that many
allopolyploids have undergone genome downsizing (Dolezel
et al. 1998; Leitch and Bennett 2004; Beaulieu et al.
2009). Certainly, the balance between retrotransposition
and DNA deletion will influence genome size and turnover
of DNA sequences (Leitch and Leitch 2008). Indeed,
analysis of rice bacterial artificial chromosome clones revealed
that retroelement insertions may only have a half-life of
a few million years, an indication of the speed with which
these retroelement replacement mechanisms can operate
(Ma et al. 2004). Such turnover of sequences may explain
why genomic in situ hybridization fails, even in some
relatively young Nicotiana allopolyploids where loss of
homology with progenitor species can be detected after only
approximately 5 million years of divergence (Clarkson
et al. 2005; Lim et al. 2007).
It is apparent that the dynamism of plant genomes is not
restricted to changes in DNA sequence. In Spartina and
Dactylorhiza allopolyploid hybrids, epigenetic alterations
have been shown to occur rapidly; such changes are often
associated with TEs and can be specific to the maternally
derived portion of the genome (Parisod et al. 2009; Paun
et al. 2010).
The genus Nicotiana (Solanaceae) provides an excellent
model group for studies on the consequences of
polyploidy, because the genus consists of approximately 70
species, and ;40% of which are documented to be
allotetraploids derived from six independent polyploidy events
(Clarkson et al. 2005, 2010; Leitch et al. 2008). The
allopolyploid species studied here, N. tabacum (tobacco), is
particularly worth studying because it is relatively young species
(less than 200,000 years old; Leitch et al. 2008) and is
derived from known ancestors that are related to Nicotiana
sylvestris (the maternal genome donor, the S-genome
component of N. tabacum) and Nicotiana tomentosiformis (the
paternal genome donor, the T-genome component of
Previous molecular and cytogenetics studies have
suggested that for noncoding tandemly repeated DNA,
N. tabacum is typically additive for its two diploid parents
(Murad et al. 2002; Koukalova et al. 2010), exceptions being
for 35S nuclear ribosomal DNA (rDNA), a satellite called
NTRS and A1/A2 repeats derived from the intergenic
spacer (IGS) of 35S rDNA. The IGS in N. tabacum has
experienced near complete replacement with a novel unit
most closely resembling the N. tomentosiformis type
(Volkov et al. 1999; Lim, Kovarik, et al. 2000). In addition,
A1/A2 repeats that are found within the IGS and scattered
across the N. tomentosiformis genome have fewer than
expected dispersed copies in N. tabacum (Lim et al. 2004).
Similarly, for Tnt2 retroelements, there is evidence for
the gain of new insertion sites as well as element loss (Petit
et al. 2007). Other variation includes translocations
between the S- and T-genomes, some of which appear
ubiquitous, and probably fixed, whereas others are specific to
particular N. tabacum cultivars (Lim, Matyasek, et al.
2004). In generation S3 of synthetic N. tabacum, a similar
translocation to the one fixed in N. tabacum is observed in
some plants, suggesting a fitness advantage for such
a change (Skalicka et al. 2005). Furthermore, in some
synthetic N. tabacum lines, there is already replacement of
several thousand rDNA units with a novel unit type (Skalicka
et al. 2003) and evidence for the loss of N. tomentosiformis–
derived Tnt1 insertion sites (Petit et al. 2010). These events
suggest a rapidly diverging genome, perhaps responding to
the genomic shock of allotetraploidy (McClintock 1984).
The emergence of next generation sequencing
technologies (Margulies et al. 2005) has enabled, for the first time,
the possibility of studying in detail and at modest cost, the
repetitive elements of any genome. Using DNA sequence
data produced with 454 pyrosequencing and a genome
coverage of ;1%, Macas et al. (2007) have been able to
calculate copy number and genome proportions of
wellrepresented repeat sequences in pea (Pisum sativum).
In addition, Swaminathan et al. (2007) have used a similar
approach to classify the repeats present in soybean,
whereas others have investigated the genome of barley
(Wicker et al. 2006, 2009). More recently, Hribova et al.
(2010) have used 454 read-depth analysis to characterize
the repeat component of the banana genome. However,
these studies did not focus on addressing the question
of how repeat sequences respond to allopolyploidy, the
principal objective of this paper.
Here, we compare the genomes of N. tabacum and the
extant lineages most closely related to its two diploid
progenitors by using 454 GS FLX Titanium Technology,
sequencing in each case at least 0.5% of the genome. Such
data combined with clustering based repeat identification
and abundance estimates using established approaches
(Novak et al. 2010) enabled us to analyze patterns of
evolution subsequent to polyploidy for abundant repetitive
sequences. We present here our analysis of the nuclear
ecology (sensu Brookfield 2005) and population dynamics
of repeat sequences associated with the divergence of
allotetraploid N. tabacum.
Materials and Methods
Nicotiana sylvestris Speg. & Comes (ac. ITB626) was
obtained from the Tobacco Institute, Imperial Tobacco
Group, Bergerac, France. Nicotiana tomentosiformis
Goodsp. (ac. NIC 479/84) was from the Institute of Plant
Genetics and Crop Plant Research, Gatersleben, Germany.
Nicotiana tabacum cv. SR1, Petit Havana, was obtained
from the Tobacco Institute, Imperial Tobacco Group,
Bergerac, France. The N. tomentosiformis accession was
selected because it is the most similar of the accessions
to the T-genome of N. tabacum, with which it shares
several cytological markers (Murad et al. 2002) and
amplified fragment length polymorphisms (MA Grandbastien,
unpublished data). Of the N. sylvestris accessions available,
none is particularly more suitable than any other as they
are all closely related (Petit et al. 2007).
DNA Extraction and 454 Sequencing
To reduce organellar contamination of reads, genomic
DNA was isolated from purified nuclei prepared from
fresh leaf tissue as described in Fojtova et al. (2003).
Extracted DNA was checked for integrity by gel
electrophoresis. Approximately 5 lg of genomic DNA was
submitted for sequencing at the NERC Biomolecular
Analysis Facility—Liverpool, United Kingdom. DNA
was randomly sheared by nebulization and sequenced
using a 454 GS FLX Instrument with Titanium reagents
(Roche Diagnostics). For each species, we used
oneeighth of a 70 75 picotiter plate. Sequence reads were
submitted to the NCBI sequence read archive (SRA)
under the study accession number SRA023759.
Preparation and Analysis of 454 Reads
Using custom Perl scripts sequence reads and associated
quality files, the first ten bases were clipped to remove
any associated adapter sequences. The stand-alone Blast
program (http://www.ncbi.nlm.nih.gov/) was used to screen
454 reads for similarity to the appropriate plastid genome (N.
sylvestris NCBI#: NC_007500.1, N. tomentosiformis NCBI#:
NC_007602.1, and N. tabacum NCBI#: NC_001879.2). Reads
with significant hits (e-value , e 6) to plastid DNA were
excluded from further analysis, whereas the remaining
454 reads were considered nuclear in origin.
Comparative Genome Analysis Using Blast
The stand-alone Blast program was used to assess sequence
similarity at the genome-wide level. Complete pairwise
analysis was performed on the N. tabacum data set and
the proportion of reads with significant hits (E value ,
e 8) was recorded for each sequence. All other Blast
parameters were set to default throughout the analysis.
The same analysis was repeated using N. tabacum sequences
to probe the N. sylvestris and N. tomentosiformis data sets,
and for each N. tabacum read, the number of sequences
(from the progenitor data set) with significant sequence
similarity hits to the N. tabacum reads was recorded. Due to the
number of reads in each data set being unequal, the number
of hits recorded in all cases was standardized to the N.
tabacum data set, where hit numbers were scaled up or down
depending on the difference in the number of reads between
data sets. For example, the N. tabacum data set consists of
70,616 reads, whereas the N. tomentosiformis data set has
65,858 reads and to standardize these data, the number
of hits recorded for N. tomentosiformis was multiplied by
1.072 (number of reads in the N. tabacum data set/number
of reads in the N. tomentosiformis data set). We constructed
an in silico N. tabacum consisting of a random set of 35,000
reads from each of the parental data sets. An equal
contribution from the parents was used to reflect the
equivalence of genome size in the progenitor species. A control
analysis consisting of the in silico N. tabacum in place of the
454 N. tabacum reads was performed (supplementary fig. 1,
Supplementary Material online).
Individual N. tabacum 454 reads, for annotation
purposes, were subjected to sequence similarity searches to
known repeat elements, including those submitted to
RepBase and a custom database consisting of known
satellite and rDNA repeats from the three Nicotiana species.
We also annotated the reads with known protein domains
by sequence similarity to the pfam database. The
resulting data were plotted in the R statistical package
(R Development Core Team 2010; fig. 1 and supplementary
fig. 1, Supplementary Material online).
Genome-Wide Analysis of Mean Similarity of
We analyzed the genomes of parental and progenitor species
by comparisons of the mean sequence similarity of related
sequences. Blast analysis with an e-value cutoff of e 6 was
used to identify related sequences. Custom Perl scripts and
the R statistical package were used to calculate the mean
sequence similarity for each sequence with a Blast hit under
the condition that the HSP was above 80 bp in length.
Clustering, Contig Assembly, Graph Visualization,
and Species-Specific Reference Assembly
Repeat sequence assembly was performed with a combined
data set of 454 reads from all three species using a
graphbased clustering approach as described in Novak et al.
(2010). Briefly, the reads were subjected to a complete
pairwise sequence comparison, and their mutual
similarities were represented as a graph in which the vertices
corresponded to sequence reads; overlapping reads were
connected with edges and their similarity scores were
expressed as edge weights. Distances between a given node
(a single sequence) and other related nodes are
determined, in part, by the bit score (edge weight) of a Blast
analysis between sequences and a Fruchterman–Reingold
FIG. 1. Genome comparisons using pairwise similarity analysis of
individual 454 reads. Nicotiana tabacum reads compared with the
N. tabacum data set (x axis) and Nicotiana sylvestris (A) or
Nicotiana tomentosiformis (B) data sets (y axis) using the Blast
program with e value cutoff of 10 8. The number of similarity hits
was normalized to take into account the varying size of each data
set. Reads highlighted red and green are rDNA and NTRS
sequences; respectively, 1:1 and 2:1 lines are labeled and indicated
in blue. In (A), reads on the 2:1 line are likely from N. sylvestris
with reduced frequency in N. tabacum caused by the unification
(as a result of allopolyploidy) with the N. tomentosiformis genome.
Reads on the 1:1 line in (A) and (B) are sequences inherited from
both parents where they occur in similar copy numbers.
algorithm is used to position the nodes. This results in more
similar sequences being placed closer together, whereas
more distantly related reads are placed further apart.
The graph structure was analyzed using custom-made
programs in order to detect clusters of frequently
connected nodes representing groups of similar sequences.
These clusters, corresponding to families of genomic
repeats, were separated and analyzed with respect to
the number of reads they contained (which is proportional
to their genomic abundance) and similarity to known
repeats. Graphs of selected clusters were also visually
examined using the SeqGrapheR program (Novak et al. 2010) in
order to assess structure and variability of the repeats.
We then used the CLC Genomics Workbench v. 3 to
independently map reads derived from each species to
reference sequences derived from the clustering and
assembly algorithm described above. Default parameters
of at least 80% sequence identity along 50% of the
sequence read were used. This approach allowed us to
estimate the average read depth along the length of
the contig (RD), genome representation (GR, the average
RD the length of the contig), and genome proportion
(GP, calculated as (GR/database size in bp) 100) for all
reference sequences in each species. Sequence similarity
searches and custom Perl scripts were used to sort
resulting clusters and contigs according to sequence type, RD,
and GR. Clusters were annotated using sequence
similarity (BlastN and BlastX) searches to the entire RepBase
(edition 14.10, accessed 9/1/2009), using an e value cutoff
score of e 6. Additional annotation using the Blast
function on the GyDB was required in order to establish the
clade to which Ty3-gypsy-like elements belonged (Llorens
et al. 2008). The total GR and GP of a given repeat was
calculated by summing all GR and GP estimates for
clusters associated with that repeat type.
All scripts are available on request.
454 High-Throughput DNA Sequencing
454 GS FLX Titanium sequencing of genomic DNA of
N. sylvestris, N. tomentosiformis, and N. tabacum returned
between 68,000 and 75,000 reads per species, with an
average read length of 360–370 bp. This totals 22–29
Mb of DNA sequence per species. Filtering for plastid
contaminants and trimming of primer sequences
resulted in 19–25 Mb of DNA sequence for each
accession. This amounts to ;0.9% coverage of the N. sylvestris
(1C genome size of 2,650 Mb) genome, ;0.8% coverage
of the N. tomentosiformis (1C genome size of 2,650 Mb)
genome, and ;0.5% coverage for N. tabacum (1C
genome size of ;5,100 Mb) (Leitch et al. 2008). Sequence
reads were submitted to NCBI SRA under the study
accession number SRA023759.
Genome-Wide Comparisons via 454 Read Similarity
To estimate abundance of sequences within and between
species, we conducted pairwise sequence similarity
searches. The data are shown as 2D plots where the
number of sequence similarity hits in N. tabacum is plotted
against the number of hits in each parent (fig. 1A and B).
The output reflects the abundance of sequences in the
Nicotiana genomes. We would expect those sequences that
were faithfully inherited in N. tabacum exclusively from one
parent to fall on a 2:1 line. This is because these sequences
will be twice as abundant in the parent as in N. tabacum,
given the normalized data sets and the effective dilution by
the other parental genome. Those sequences falling above
the 2:1 line are likely underrepresented in N. tabacum and
derived from the parent in the analysis. Similarly, sequences
falling on a 1:1 line are expected to be in similar abundance
in both parents.
Figure 1A and B show complete pairwise sequence
similarity analysis of individual 454 reads from N. tabacum
against all reads in the N. tabacum, N. sylvestris, and
N. tomentosiformis data sets. In figure 1A, which shows
the analysis of N. tabacum against N. sylvestris, there is a
distinct clustering of reads on or close to a 2:1 line, suggesting
these are N. tabacum reads that have been inherited solely
or predominantly from N. sylvestris. Few reads in this
category were identifiable using similarity searches to
RepBase or pfam domains. In comparison, when the same
analysis is conducted using the N. tomentosiformis data set,
sequences on the 2:1 line are less abundant (fig. 1B).
The analysis in figure 1A also shows a spike of sequences
that reach substantially higher copy number (i.e., higher
frequency of sequence similarity hits) in N. sylvestris than
in N. tabacum. We found that these sequences are
predominantly rDNA (highlighted red in fig. 1A). The
corresponding sequences are also highlighted red in figure
1B. This spike was absent in a control genome, generated in
silico from an equal mixture (35,000 reads) from each
parental data set (totaling 70,000 reads) (supplementary
fig. 1A and B, Supplementary Material online).
In figure 1B, 3,078 sequence reads have a higher
frequency of sequence similarity hits in N. tomentosiformis
than would be expected from their observed frequency in
N. tabacum (i.e., sequences that fall above the 2:1 line in
fig. 1B). This pattern is absent in the in silico N. tabacum
(supplementary fig 1B, Supplementary Material online).
For the reads above the 2:1 line (i.e., they are
underrepresented in N. tabacum relative to expectation), the
mean and sum of the residuals (i.e., deviation from the
line) was 22.2 and 68,332, respectively. In N. sylvestris,
there are 5,919 sequences above the 2:1 line, but the mean
of residual of these sequences is only 9.6 and the sum
totals 56,822. Amongst the reads above the 2:1 line in
figure 1B (plotting N. tabacum against N. tomentosiformis),
there are NTRS-like repeat sequences (highlighted in
green), previously shown to occur in N. tomentosiformis,
other species of section Tomentosae and N. tabacum
but not in N. sylvestris (Matyasek et al. 1997). The
remainder of the reads had few significant hits to RepBase, but
several were related to retrotransposon gag
(retrotransgag) and reverse transcriptase (RVT) pfam domains (data
Comparison of Genome-Wide Sequence Similarity
For all reads in each of the three data sets, we calculated the
mean sequence similarity between that read and all related
sequences within the same data set. Figure 2 shows
histograms of mean sequence similarity in N. tabacum, its
diploid progenitors and an in silico N. tabacum. A major peak
with a mean of ;0.86 is seen in all three species, and in the
in silico N. tabacum. Nicotiana sylvestris has a secondary
peak where the mean sequence similarity is close to one
(fig. 2A). These latter sequences are likely either highly
constrained by selection or have experienced recent
expansion/homogenization. The sequences from N. sylvestris
with a mean sequence similarity (to other related reads
in the N. sylvestris data set) above 0.98 and a coefficient
of variation less than one (2,248 reads in total) were
identified (supplementary table 1, Supplementary Material
online) and shown to include 5S and 35S rDNA sequences
(totaling 353 of the reads). In addition, there were a number
of gypsy-like repeat sequences, although they were in
considerably lower abundance than rDNA repeats. Nicotiana
tomentosiformis (fig. 2B) lacks such an abundance of
sequences with a high mean sequence similarity. The in silico
N. tabacum (fig. 2C) exhibits a secondary peak of high mean
sequence similarity as seen in N. sylvestris (fig. 2A), but the
peak is absent in natural N. tabacum (fig. 2D).
Clustering and Contig Assembly
We combined all 454 high-throughput DNA sequencing
reads from the three Nicotiana species and subjected this
combined data set (.70 Mb of DNA sequence) to a
clustering based repeat identification procedure, leading to
partitioning of sequencing data into groups of overlapping
reads representing individual repeat families as described in
Novak et al. (2010). As the average read depth in each
cluster reflects the genomic proportions of the corresponding
repeat, read-depth analysis was used to estimate the repeat
composition in the genomes of the species studied. Details
of the repeat identification and assembly output are given
in table 1. The normalized (by total number of reads in the
data set) contribution of each species to the 30 largest
clusters is shown in supplementary fig. 2 (Supplementary
Material online). Clusters were then assembled to provide
reference sequences that were used as a scaffold for the
independent mapping of reads for each of the three
Nicotiana species (table 1 and fig. 3). This allowed
characterization of the average read depth along the length of the
contig (RD), genome representation (GR, calculated as
RD contig length), and genome proportion (GP,
calculated as (GR/total size of the data set in base pairs) 100)
for each of the three species. GP is therefore the percentage
of the data set (and therefore the genome) that can be
attributed to a given repeat. This allowed characterization of
the most abundant repeats in the three genomes. Others
have used similar approaches to measure repeat sequence
abundance (Macas et al. 2007; Swaminathan et al. 2007;
Hribova et al. 2010).
An example of the output of the clustering and assembly
procedure is provided for cluster CL2, which contains reads
from all three species (fig. 4) and has sequence similarity to
Ogre-like LTR retroelements (Macas and Neumann 2007).
Each node within the network corresponds to a single 454
read and similar reads are placed more closely together
than more distantly related sequences. We observe in this
network that most reads fall along a contiguous line, similar
to an assembly into a single contig. However, it is clear that
some related reads deviate from this main axis and become
FIG. 2. Histogram showing frequency of mean sequence similarity between each read in a 454 data set and all related sequences in the same
data set in (A) Nicotiana sylvestris, (B) Nicotiana tomentosiformis, (C) an in silico Nicotiana tabacum, and (D) natural Nicotiana. tabacum. In (A),
many reads have high mean sequence similarity values generating a secondary peak. This peak is much reduced in N. tomentosiformis (B) and is
absent in Nicotiana tabacum (D).
a linked but separate string of sequences (boxed in fig. 4).
These are likely to be alternative variants of this repeat, one
of which is found in the genome of N. sylvestris and another
in N. tomentosiformis (red and blue in fig. 4, respectively).
Both repeat variants are present in N. tabacum.
To check the validity of the contigs developed in silico
(as described above), we cloned and sequenced a region of
cluster 3 contig 8 and found clones sharing between 92%
and 96% identity with the consensus (data not shown).
Characterizing Nicotiana Genomes
Table 2 shows GR and GP estimates for the major repeat
sequence fraction of the N. tabacum, N. sylvestris, and
N. tomentosiformis genomes. The sequence type with
largest GPs in all three species was the retroelements,
comprising at least 20.52%, 27.17%, and 22.90% of the
genomes of N. tabacum, N. sylvestris, and N. tomentosiformis,
respectively. In N. tabacum, comparison of observed GPs with
expected percentages (average of the parents) reveals the GP
and the Species-Specific Read Mapping Analysesa.
NOTE.—N/A, not available.
a The repeat identification algorithm is described in detail in Novak et al. (2010).
FIG. 3. Venn diagram where the area of each circle (and the
intersections) is proportional to number of 16,229 clusters that have
significant similarity to sequence reads from the species as indicated.
Absolute numbers are given in each section.
of retroelements to be reduced by over 18% from expectation.
The majority of retroelements are Ty3-gypsy-like (estimates
ranging from 17% to 23% in the three species), and in
N. tabacum, there is a reduction in their GP by 19.8% from
expectation. Figure 5A shows the contribution of the major
groups of the Ty3-gypsy-like elements present in all three
species. The group with the highest GP in N. sylvestris is
Tat, which includes the large Ogre and Atlantys elements. This
group is also well represented in N. tabacum but less so in
N. tomentosiformis, where the largest group are the
Del (Chromovirus) elements. All families of Ty3-gypsy have
a GP lower in N. tabacum than would be expected based
on the proportions observed in the diploid progenitors
(fig. 5B), indicating that sequence loss may have occurred
subsequent to allotetraploidy.
Estimates of 35S rDNA abundance in the three Nicotiana
species have shown that these repeats make up a substantial
fraction of the N. sylvestris genome (1.70%). The observed
abundance of 35S rDNA in N. tomentosiformis is lower
(0.48%), whereas in N. tabacum it is lower still (0.17%), which
is more than an 80% reduction in GP compared with that
expected. We also observed that one cluster (CL3) is
particularly abundant in N. tomentosiformis (GP 5 1.91%),
whereas in N. tabacum, the abundance of this repeat was
considerably lower (GP 5 0.1%). In addition, pararetrovirus-like
sequences are more abundant in the N. tomentosiformis
genome (0.54%) than they are in both N. tabacum (0.25%) and
N. sylvestris (0.22%), revealing a 34% reduction in GP from
expectation (table 2).
Comparing Observed with Expected Genome
Proportions in N. tabacum
We used linear regression to compare GP estimates of 14,634
repeat clusters in N. tabacum against those in the two
progenitor species (fig. 6). If the N. tabacum genome was
an equal mixture of the two progenitors, the slope of the
regression would have been 0.5 for each (fig. 6). The actual
estimate of the slope (fig. 6) was close to, although significantly
different from, the expected slope of 0.5 for N. tabacum versus
N. sylvestris (0.472, standard error [SE] 0.004) whereas the GP
contribution from N. tomentosiformis in N. tabacum was
considerably lower and significantly different from expectation
(0.300, SE 0.003). In fig. 6, notice that 1) the fitted surface
(red) through the observed data falls below the expected
surface (green), which assumes N. tabacum has inherited
sequences faithfully from the progenitors; 2) repetitive DNAs
inherited from N. tomentosiformis appear underrepresented
along the length of the data range, and 3) this discrepancy
is greatest for the most common repeat elements in N.
454 Titanium Sequencing to Estimate Repeat
The major repeat composition of the genomes of three
Nicotiana species has been characterized using 454 GS FLX
pyrosequencing, providing between ;0.5% and 1% coverage of
NOTE.—N/A, not available.
a The genic but not intergenic regions were estimated here.
b These estimates were based on Blast read depth to cloned sequences.
the genome per species. Such coverage theoretically allows
the reconstruction of repeat units that have copy number in
excess of ;1,000 copies per 1C genome. This approach
provides estimates of repeat abundance (copy number and
genome proportion estimates) that are in broad agreement
with other experimental approaches and have been used
to characterize repeats in pea (Macas et al. 2007), soybean
(Swaminathan et al. 2007), barley (Wicker et al. 2009),
and banana (Hribova et al. 2010).
We observed a relatively low abundance of TEs in the
Nicotiana genomes analyzed (table 2). Hordeum vulgare
with a genome size similar to N. tabacum (1C 5 5,439
Mb) has higher proportions of Ty1-copia (;16%) and
Ty3-gypsy (;30%) elements than N. tabacum (Wicker
et al. 2009). Likewise, Zea mays, with a genome size similar
to the progenitor diploids of N. tabacum, has a total TE
abundance of around 84% of the genome (;46% gypsy
elements) (Schnable et al. 2009). However, N. tabacum has
similar abundance of TEs to P. sativum (1C 5 4,778 Mb),
with 5% and 24% of the genome consisting of Ty1-copia
and Ty3-gypsy elements, respectively (Macas et al. 2007),
whereas for the much smaller genome (1C 5 490 Mb)
of Oryza sativa ;4% of the DNA consists of Ty1-copia
elements and ;11% of Ty3-gypsy (International Rice
Genome Sequencing Project 2005).
It is possible that our GP estimates of repeat sequences
could be low because of an abundance of a diverse range of
low copy repeats in the genome. Furthermore, we may be
unable to identify repeats because RepBase has only 67
Solanaceae entries (October 2010), including complete
and incomplete Ty1-copia and Ty3-gypsy elements.
However, these interpretations seem unlikely as conserved
domains are normally detectable across wide phylogenetic
distances. Importantly, on lowering the Blast threshold
to e 10 2, we observed only a small (3–5%) increase in
the total proportion of the genome that was annotated
(data not shown). Further support for our estimates of
TE abundance in N. tabacum (;22% of genome) is
provided by reassociation kinetics of long DNA fragments,
where only 25% of the genome is shown to consist of
repetitive DNA in excess of 1,500 bp in length (Zimmerman
and Goldberg 1977).
The satellite repeat content of Nicotiana genomes seems
to be underestimated in 454 data sets (6-fold for NTRS and
.10-fold for HRS60) as compared with Southern blot
estimates (Koukalova et al. 1989; Matyasek et al. 1997). There
is also a 2- and 10-fold variation in read depths along the
NTRS and HRS60 monomers, respectively (supplementary
fig. 3, Supplementary Material online). Nevertheless,
although the absolute numbers of tandem repeats may be
underestimated in our 454 data sets, the relative
abundance of these repeats in the three species of Nicotiana
is fully concordant (data not shown).
Angiosperm Genome Dynamism
The genomes of angiosperms are thought to be highly
dynamic in relation to other eukaryotes, in particular
mammals (Kejnovsky et al. 2009), and many studies have
FIG. 5. (A) Histogram showing genome proportions of the major
Ty3-gypsy families detected in the genomes of three Nicotiana
species. The Ty3-gypsy clades are indicated along the x axis, with the
proportion in each species given as a percentage of the genome on
the y axis. (B) Genome proportion values of Ty3-gypsy families for
Nicotiana tabacum compared with the expected value (parental
average, blue line), plotted on a log scale. Genome proportion
estimates are below the expected value for all Ty3-gypsy families
suggested that hybridization can trigger extensive genetic
change in some young hybrids and allopolyploids (Comai
et al. 2003; Adams and Wendel 2005; Leitch and Leitch
2008; Feldman and Levy 2009; Parisod et al. 2009; Paun
et al. 2010; Petit et al. 2010), although the extent of change
is variable between taxa (Baumel et al. 2002).
Occurrence of repeats with a high degree of sequence
similarity can be indicative of either repeat expansion or
homogenization, and previous research has used this
signature to identify recent episodes of repeat turnover (Kim
et al. 1998; Jordan and McDonald 1999). There is an
indication that expansion of repeats and/or homogenization is
occurring in the diploid genome of N. sylvestris because
there are many repeats with high mean sequence similarity
to related sequences in the same genome (fig. 2A). Some
FIG. 6. We used linear regression analysis to generate a smoothed
contour plot of genome proportion (GP) values for clusters in each
of the three Nicotiana species (red surface, for a description of this
analysis, see Materials and Methods). The expected plane assuming
faithful inheritance of sequences in Nicotiana tabacum is shown
(green surface). The observed trend is for repeat clusters in N.
tabacum to be underrepresented compared with expected,
particularly from clusters that are abundant in Nicotiana
tomentosiformis. The data range is for clusters with a GP between 0% and
0.5% on each axis.
sequences in N. sylvestris showing evidence of recent
expansion belong to the Ty3-gypsy superfamily, other TE
groups, and rDNA sequences (supplementary table 1,
Supplementary Material online). We know that rDNA units can
be homogenized astonishingly rapidly in Nicotiana; in
synthetic N. tabacum, thousands of units were converted to
a novel type in just a few generations (Skalicka et al. 2003).
Importantly, only a few sequences have the signature of
high mean sequence similarity in the genomes of N.
tomentosiformis (fig. 2B) and N. tabacum (fig. 2D). The low levels
of these sequences in N. tabacum perhaps indicates
homogenization/expansion of repeats in N. sylvestris after
the formation of N. tabacum or that these sequences have
been eliminated from the N. tabacum genome. There
appears not to be large-scale repeat expansion following
polyploidy in N. tabacum, as also observed for
retroelements in the polyploid Gossypium hirsutum (cotton) which
formed 1–2 million years ago (Hu et al. 2010). Some reads
that do have high mean sequence similarity in N. tabacum
are rDNA sequences. Such an observation is expected
considering that 35S rDNA has been recently homogenized in
this species (Volkov et al. 1999; Lim, Kovarik, et al. 2000;
Kovarik et al. 2008). However, the number of rDNA repeats
is much lower in N. tabacum compared with its progenitors
(table 2), explaining why we find only a few copies.
Genome Downsizing in Polyploids
The 1C genome size (GS) of N. tabacum (5,100 Mb) is ;
3.7% less than would be expected by summing the sizes of
the two parental genomes (N. sylvestris 5 2,650 Mb, N.
tomentosiformis 5 2,650 Mb), which has been proposed as
evidence of genome downsizing in this species (Bennett
and Leitch 2005; Leitch et al. 2008). Analysis of more than
3,000 diploid and polyploid angiosperm species indicated
that genome downsizing following polyploidy may be
a common, although not ubiquitous occurrence (Leitch
and Bennett 2004). Indeed one allotetraploid of Nicotiana
has been shown to have a larger that predicted based on
the sum of their parent’s genomes (Leitch et al. 2008).
The following sequence types have materially lower GP
in N. tabacum than expected compared with the diploid
progenitors. 1) Retroelements overall are reduced in N.
tabacum from expected GP by 18.0% (expectation is the
mean GP of the two diploid parents, reflecting the
equivalence of GS in the progenitors) of which Ty3-gypsy
elements are lower by 19.8% (table 2). All major
Ty3-gypsylike families detected exhibit a reduction in GP from
expectation (fig. 5B), indicating a general reduction in their
abundance in N. tabacum (or increase in one or both
progenitors). 2) 35S rDNA exhibits a reduction of GP in N.
tabacum by 84% from expectation (table 2). All observations
are in line with the genome downsizing hypothesis and the
disparity between the expected genome size of N. tabacum
(1C 5 5,300 Mb) and that estimated by flow cytometry (1C
5 5,100 Mb) (Leitch et al. 2008). 3) In addition, using
Southern hybridization data, Skalicka et al. (2005)
determined the proportion of NTRS satellite repeats to be 3%
of the genome in N. tomentosiformis, 0% in N. sylvestris,
and 0.5% in N. tabacum (cultivar SR1), a 85% negative
deviation from expectation assuming N. tabacum inherited
units in the abundance found in the diploids.
Nicotiana tabacum may have lost ;200 Mb of DNA,
much of which could be accounted for by the sequences
described here. Regression analysis of GPs in N. tabacum
and the progenitor species reveals the general trend is
for repeats to be underrepresented in N. tabacum
compared with expected (fig. 6) These data indicate that
sequence loss seems to be particularly prevalent among
the most common repeat types. The large
underrepresentation of the Ty3-gypsy-like sequences may simply be a
consequence of their high abundance.
Loss of Repeats from Paternally Derived T-Genome
of N. tabacum
From cytogenetic analysis of chromosomes, Gill (1991)
proposed in the nuclear cytoplasmic interaction hypothesis that
the paternally inherited genome of an allopolyploid is more
prone to genetic change than the maternally derived
genome. In support of this hypothesis, Southern blot (Skalicka
et al. 2005) and cytogenetic (Koukalova et al. 1989; Lim,
Matyasek, et al. 2000) data have revealed that in N. tabacum, the
subtelomeric satellite repeat HRS60, inherited from N.
sylvestris, does not deviate from expectation. In contrast, Skalicka
et al. (2005) reported that four families of repeats inherited
from N. tomentosiformis were in lower copy number in N.
tabacum. In addition, there is preferential loss of Tnt1
insertions from the N. tomentosiformis–derived genome in
synthetic N. tabacum (Petit et al. 2010) and of Tnt2
elements in natural N. tabacum (Petit et al. 2007). We report
here that the most well-represented cluster in N.
tomentosiformis (GP 5 1.91%) is CL3, and this cluster is in much
lower abundance in N. tabacum (GP5 0.1%), perhaps
indicative of sequence loss in the T-genome of N. tabacum.
On a genome-wide scale, we provide evidence that the
T-genome of N. tabacum has experienced preferential
sequence loss compared with the S-genome. Cross-species
sequence similarity analyses (fig. 1B) showed many
sequences in N. tomentosiformis that deviate substantially from
the 2:1 line and are less well represented in N. tabacum than
expected. Although N. sylvestris has a greater number of
sequences that are underrepresented in N. tabacum (fig.
1A), they do not deviate substantially from expectation
and the sum of residuals is less than that observed for
N. tomentosiformis, indicating that repeats derived from
N. sylvestris may not to be affected to the same degree
as those of the T-genome.
We compared a smoothed contour plot that best
describes the observed GPs in N. tabacum with a theoretical
surface that assumes an average GP of that found in the
parents GPs (fig. 6). Linear regression analysis revealed unequal
contribution from the progenitor species to the N. tabacum
genome, with particular underrepresentation of repeats from
N. tomentosiformis (fig. 6), regardless of their abundance in
N. sylvestris. The degree to which a repetitive DNA sequence
is underrepresented in N. tabacum increases with the
corresponding abundance in N. tomentosiformis. In contrast for
clusters that were absent or had a low abundance in N.
tomentosiformis, GP values were close to expectation (fig. 6)
indicating faithful inheritance from N. sylvestris.
The lack of evidence for repeat expansion in N.
tomentosiformis (fig. 2) indicates that the underrepresentation of
N. tomentosiformis–derived repeat sequences in N.
tabacum is likely to be the result of sequence erosion in the
latter rather than sequence gains in the former
postallotetraploidy. Collectively, our data are congruent with the loss
of repeats derived from the T-genome of N. tabacum and
the maintenance of repeats from the S-genome.
Instability of a particular genomic component of an
allopolyploid has also been observed in crosses between
Brassica nigra and Brassica rapa, where directional loss of
restriction fragments was apparent in some lines (Song
et al. 1995). Mechanisms that induce the preferential
elimination of paternally derived sequences are unknown.
Potentially, they could involve the 24 nt class of siRNAs that
are known to be highly abundant and uniparentally
expressed in the endosperm (Baulcombe 2009). Perhaps
these small RNAs provide an opportunity for the epigenetic
modification of N. sylvestris–derived repeats, which were
maternally inherited. The small RNAs may have acted to
enhance the stability and reduce the frequency of DNA loss
from the S-genome of N. tabacum in early generations after
What is apparent from these data is that not all repeats
have responded in the same way subsequent to
allopolyploidy. The processes giving rise to repeat copy number
changes may be stochastic or directed. Evidence for the
latter may be the preferential loss of repeats from the
N. tomentosiformis–derived T-genome of N. tabacum.
Our data suggest that the three species studied have
experienced distinctive patterns of genome evolution. Nicotiana
sylvestris and N. tomentosiformis probably diverged early in
the evolution of genus Nicotiana which split from
Symonanthus around 15 million years ago (Chase et al. 2003;
Clarkson et al. 2005). Nicotiana sylvestris, the maternal
progenitor of N. tabacum, appears to have many features
indicating recent expansion of repeats. In contrast, this
signature is much less apparent in N. tomentosiformis,
suggesting that its genome has not experienced substantial
rounds of homogenization/amplification in its recent
evolutionary history. In N. tabacum, which formed less than 0.2
million years ago (Clarkson et al. 2005) there is evidence for
a third pattern of genome evolution, that of genome
reduction. In a broad sense, the three genomes of Nicotiana
studied here are experiencing different evolutionary
trajectories, and the same families of repeats, in N. sylvestris,
N. tomentosiformis, and N. tabacum are dynamic, stable,
and downsizing, respectively, indicating the same families
of repeats can have varying fates in different, yet closely
Supplementary figures 1–3 and table 1 are available at
Molecular Biology and Evolution online (http://www.mbe.
We thank NERC and the grants OC10037 from the Ministry
of Education, Youth and Sports of the Czech Republic and
AVOZ50510513 from the Academy of Sciences of the
Czech Republic for supporting the project. We thank
two anonymous referees for their valuable comments,
R. Verity for assistance with the plotting of some of the
data using R. In addition, we thank B. Chalhoub, R. Buggs,
and L. Kelly for their insightful comments and useful
Adams KL , Wendel JF . 2005 . Polyploidy and genome evolution in plants . Curr Opin Plant Biol . 8 : 135 - 141 .
Ainouche ML , Fortune PM , Salmon A , Parisod C , Grandbastien M-A , Fukunaga K , Ricou M , Misset MT . 2009 . Hybridization, polyploidy and invasions: lessons from Spartina (Poaceae) . Biol Invasions . 11 : 1159 - 1173 .
Baulcombe D. 2009 . The diverse roles of small, non-coding RNA in plants . Mech Dev . 126 : S24 .
Baumel A , Ainouche M , Kalendar R , Schulman AH . 2002 . Retrotransposons and genomic stability in populations of the young allopolyploid species Spartina anglica CE Hubbard (Poaceae) . Mol Biol Evol . 19 : 1218 - 1227 .
Beaulieu J , Jean M , Belzile F. 2009 . The allotetraploid Arabidopsis thaliana-Arabidopsis lyrata subsp petraea as an alternative model system for the study of polyploidy in plants . Mol Genet Genomics . 281 : 421 - 435 .
Bennett MD , Leitch IJ . 2005 . Angiosperm DNA C-values database . Richmond , UK.
Brookfield JFY . 2005 . The ecology of the genome-mobile DNA elements and their hosts . Nat Rev Genet . 6 : 128 - 136 .
Chase MW , Knapp S , Cox AV , Clarkson JJ , Butsko Y , Joseph J , Savolainen V , Parokonny AS . 2003 . Molecular systematics, GISH and the origin of hybrid taxa in Nicotiana (Solanaceae) . Ann Bot . 92 : 107 - 127 .
Clarkson JJ , Kelly LJ , Leitch AR , Knapp S , Chase MW . 2010 . Nuclear glutamine synthetase evolution in Nicotiana: phylogenetics and the origins of allotetraploid and homoploid (diploid) hybrids . Mol Phylogenet Evol . 55 : 99 - 112 .
Clarkson JJ , Lim KY , Kovarik A , Chase MW , Knapp S , Leitch AR . 2005 . Long-term genome diploidization in allopolyploid Nicotiana section Repandae (Solanaceae) . New Phytol . 168 : 241 - 252 .
Comai L , Madlung A , Josefsson C , Tyagi A. 2003 . Do the different parental 'heteromes' cause genomic shock in newly formed allopolyploids ? Philos Trans R Soc Lond B Biol Sci . 358 : 1149 - 1155 .
Dolezel J , Greilhuber J , Lucretti S , Meister A , Lysak MA , Nardi L , Obermayer R . 1998 . Plant genome size estimation by flow cytometry: inter-laboratory comparison . Ann Bot . 82 : 17 - 26 .
Feldman M , Levy AA . 2009 . Genome evolution in allopolyploid wheat-a revolutionary reprogramming followed by gradual changes . J Genet Genomics . 36 : 511 - 518 .
Fojtova M , Van Houdt H , Depicker A , Kovarik A. 2003 . Epigenetic switch from posttranscriptional to transcriptional silencing is correlated with promoter hypermethylation . Plant Physiol . 133 : 1240 - 1250 .
Gill BS . 1991 . Nucleocytoplasmic interaction (NCI) hypothesis of genome evolution and speciation in polyploid plants . In: Sasakuma T, Kinoshita T, editors. Kihara Memorial International Symposium on cytoplasmic engineering in wheat. Yokohama (Japan): Kihara Memorial Foundation . p 48 - 53 .
Hribova E , Neumann P , Matsumoto T , Roux N , Macas J , Dolezel J. 2010 . Repetitive part of the banana (Musa acuminata) genome investigated by low-depth 454 sequencing . BMC Plant Biol . 10 : 204 .
Hu GJ , Hawkins JS , Grover CE , Wendel JF . 2010 . The history and disposition of transposable elements in polyploid Gossypium . Genome 53 : 599 - 607 .
International Rice Genome Sequencing Project . 2005 . The mapbased sequence of the rice genome . Nature 436 : 793 - 800 .
Jordan IK , McDonald JF . 1999 . Tempo and mode of Ty element evolution in Saccharomyces cerevisiae . Genetics 151 : 1341 - 1351 .
Kashkush K , Feldman M , Levy AA . 2003 . Transcriptional activation of retrotransposons alters the expression of adjacent genes in wheat . Nat Genet . 33 : 102 - 106 .
Kejnovsky E , Leitch IJ , Leitch AR . 2009 . Contrasting evolutionary dynamics between angiosperm and mammalian genomes . Trends Ecol Evol . 24 : 572 - 582 .
Kim JM , Vanguri S , Boeke JD , Gabriel A , Voytas DF . 1998 . Transposable elements and genome organization: a comprehensive survey of retrotransposons revealed by the complete Saccharomyces cerevisiae genome sequence . Genome Res . 8 : 464 - 478 .
Koukalova B , Moraes AP , Renny-Byfield S , Matyasek R , Leitch AR , Kovarik A. 2010 . Fall and rise of satellite repeats in allopolyploids of Nicotiana over c. 5 million years . New Phytol . 186 : 148 - 160 .
Koukalova B , Reich J , Matyasek R , Kuhrova V , Bezdek M. 1989 . A BamHI family of highly repeated DNA sequences of Nicotiana tabacum . Theor Appl Genet . 78 : 77 - 80 .
Kovarik A , Dadejova M , Lim YK , Chase MW , Clarkson JJ , Knapp S , Leitch AR . 2008 . Evolution of rDNA in Nicotiana allopolyploids: a potential link between rDNA homogenization and epigenetics . Ann Bot . 101 : 815 - 823 .
Leitch AR , Leitch IJ . 2008 . Perspective genomic plasticity and the diversity of polyploid plants . Science 320 : 481 - 483 .
Leitch IJ , Bennett MD . 2004 . Genome downsizing in polyploid plants . Biol J Linn Soc . 82 : 651 - 663 .
Leitch IJ , Hanson L , Lim KY , Kovarik A , Chase MW , Clarkson JJ , Leitch AR . 2008 . The ups and downs of genome size evolution in polyploid species of Nicotiana (Solanaceae) . Ann Bot . 101 : 805 - 814 .
Lim KY , Kovarik A , Matyasek R , Bezdek M , Lichtenstein CP , Leitch AR . 2000 . Gene conversion of ribosomal DNA in Nicotiana tabacum is associated with undermethylated, decondensed and probably active gene units . Chromosoma 109 : 161 - 172 .
Lim KY , Kovarik A , Matyasek R , Chase MW , Clarkson JJ , Grandbastien MA , Leitch AR . 2007 . Sequence of events leading to near-complete genome turnover in allopolyploid Nicotiana within five million years . New Phytol . 175 : 756 - 763 .
Lim KY , Matyasek R , Kovarik A , Leitch AR . 2004 . Genome evolution in allotetraploid Nicotiana . Biol J Linn Soc . 82 : 599 - 606 .
Lim KY , Matyasek R , Lichtenstein CP , Leitch AR . 2000 . Molecular cytogenetic analyses and phylogenetic studies in the Nicotiana section Tomentosae . Chromosoma 109 : 245 - 258 .
Lim KY , Skalicka K , Koukalova B , Volkov RA , Matyasek R , Hemleben V , Leitch AR , Kovarik A. 2004 . Dynamic changes in the distribution of a satellite homologous to intergenic 26-18S rDNA spacer in the evolution of Nicotiana . Genetics 166 : 1935 - 1946 .
Liu B , Brubaker CL , Mergeai G , Cronn RC , Wendel JF . 2001 . Polyploid formation in cotton is not accompanied by rapid genomic changes . Genome 44 : 321 - 330 .
Liu B , Wendel JF . 2000 . Retrotransposon activation followed by rapid repression in introgressed rice plants . Genome 43 : 874 - 880 .
Liu B , Wendel JF . 2003 . Epigenetic phenomena and the evolution of plant allopolyploids . Mol Phylogenet Evol . 29 : 365 - 379 .
Llorens C , Futami R , Bezemer D , Moya D. 2008 . The Gypsy Database (GyDB) of mobile genetic elements . Nucleic Acids Res . 26 : 38 - 46 .
Ma JX , Devos KM , Bennetzen JL . 2004 . Analyses of LTR-retrotransposon structures reveal recent and rapid genomic DNA loss in rice . Genome Res . 14 : 860 - 869 .
Macas J , Neumann P. 2007 . Ogre elements-a distinct group of plant Ty3/gypsy-like retrotransposons . Gene 390 : 108 - 116 .
Macas J , Neumann P , Navratilova A. 2007 . Repetitive DNA in the pea (Pisum sativum L.) genome: comprehensive characterization using 454 sequencing and comparison to soybean and Medicago truncatula . BMC Genomics . 8 : 427 .
Margulies M , Egholm M , Altman WE , et al. ( 56 co-authors) . 2005 . Genome sequencing in microfabricated high-density picolitre reactors . Nature 437 : 376 - 380 .
Matyasek R , Fulnecek J , Lim KY , Leitch AR , Kovarik A. 2002 . Evolution of 5S rDNA unit arrays in the plant genus Nicotiana (Solanaceae) . Genome 45 : 556 - 562 .
Matyasek R , Gazdova B , Fajkus J , Bezdek M. 1997 . NTRS, a new family of highly repetitive DNAs specific for the T1 chromosome of tobacco . Chromosoma 106 : 369 - 379 .
Matyasek R , Lim KY , Kovarik A , Leitch AR . 2003 . Ribosomal DNA evolution and gene conversion in Nicotiana rustica . Heredity . 91 : 268 - 275 .
McClintock B. 1984 . The significance of responses of the genome to challenge . Science 226 : 792 - 801 .
Murad L , Lim KY , Christopodulou V , Matyasek R , Lichtenstein CP , Kovarik A , Leitch AR . 2002 . The origin of tobacco's T genome is traced to a particular lineage within Nicotiana tomentosiformis (Solanaceae) . Am J Bot . 89 : 921 - 928 .
Novak P , Neumann P , Macas J. 2010 . Graph-based clustering and characterization of repetitive sequences in next-generation sequencing data . BMC Bioinformatics . 11 : 378 .
Parisod C , Alix K , Just J , Petit M , Sarilar V , Mhiri C , Ainouche M , Chalhoub B , Grandbastien MA . 2010 . Impact of transposable elements on the organization and function of allopolyploid genomes . New Phytol . 186 : 37 - 45 .
Parisod C , Salmon A , Zerjal T , Tenaillon M , Grandbastien M-A , Ainouche M. 2009 . Rapid structural and epigenetic reorganization near transposable elements in hybrid and allopolyploid genomes in Spartina . New Phytol. 184 : 1003 - 1015 .
Paun O , Bateman RM , Fay MF , Hedren M , Civeyrel L , Chase MW . 2010 . Stable epigenetic effects impact adaptation in allopolyploid orchids (Dactylorhiza: Orchidaceae) . Mol Biol Evol . 27 : 2465 - 2473 .
Petit M , Guidat C , Daniel J , et al. ( 11 co-authors) . 2010 . Mobilization of retrotransposons in synthetic allotetraploid tobacco . New Phytol . 186 : 135 - 147 .
Petit M , Lim KY , Julio E , Poncet C , de Borne FD , Kovarik A , Leitch AR , Grandbastien MA , Mhiri C. 2007 . Differential impact of retrotransposon populations on the genome of allotetraploid tobacco (Nicotiana tabacum) . Mol Genet Genomics . 278 : 1 - 15 .
R Development Core Team . 2010 . R: a language and environment for statistical computing . Vienna (Austria): R Foundation for Statistical Computing.
Schnable PS , Ware D , Fulton RS , et al. (157 co-authors) 2009 . The B73 maize genome: complexity , diversity, and dynamics. Science 326 : 1112 - 1115 .
Skalicka K , Lim KY , Matyasek R , Koukalova B , Leitch AR , Kovarik A. 2003 . Rapid evolution of parental rDNA in a synthetic tobacco allotetraploid line . Am J Bot . 90 : 988 - 996 .
Skalicka K , Lim KY , Matyasek R , Matzke M , Leitch AR , Kovarik A. 2005 . Preferential elimination of repeated DNA sequences from the paternal, Nicotiana tomentosiformis genome donor of a synthetic, allotetraploid tobacco . New Phytol . 166 : 291 - 303 .
Soltis DE , Albert VA , Leebens-Mack J , Bell CD , Paterson AH , Zheng CF , Sankoff D , dePamphilis CW , Wall PK , Soltis PS . 2009 . Polyploidy and angiosperm diversification . Am J Bot . 96 : 336 - 348 .
Song KM , Lu P , Tang KL , Osborn TC . 1995 . Rapid genome chane in synthetic polyploids of Brassica and its implications for polyploid evolution . Proc Natl Acad Sci U S A . 92 : 7719 - 7723 .
Swaminathan K , Varala K , Hudson ME . 2007 . Global repeat discovery and estimation of genomic copy number in a large, complex genome using a high-throughput 454 sequence survey . BMC Genomics . 8 : 132 - 145 .
Volkov RA , Borisjuk NV , Panchuk II , Schweizer D , Hemleben V. 1999 . Elimination and rearrangement of parental rDNA in the allotetraploid Nicotiana tabacum . Mol Biol Evol . 16 : 311 - 320 .
Wendel JF . 2000 . Genome evolution in polyploids . Plant Mol Biol . 42 : 225 - 249 .
Wicker T , Schlagenhauf E , Graner A , Close TJ , Keller B , Stein N. 2006 . 454 sequencing put to the test using the complex genome of barley . BMC Genomics . 7 : 275 .
Wicker T , Taudien S , Houben A , Keller B , Graner A , Platzer M , Stein N. 2009 . A whole-genome snapshot of 454 sequences exposes the composition of the barley genome and provides evidence for parallel evolution of genome size in wheat and barley . Plant J 59 : 712 - 722 .
Zimmerman JL , Goldberg RB . 1977 . DNA sequence organization in the genome of Nicotiana tabacum . Chromosoma 59 : 227 - 252 .