wheat hybridization and polyploidization results in deregulation … · 2011. 4. 5. · dictating...
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Wheat Hybridization and Polyploidization Results in Deregulation of small
RNAs
Michal Kenan-Eichler1, Dena Leshkowitz2, Lior Tal1, Elad Noor1, Cathy Melamed-Bessudo1, Moshe Feldman1, Avraham A. Levy1*
1 Department of Plant Sciences,
2 Bioinformatics Unit Weizmann Institute of Science,
Rehovot, 76100 Israel
*Corresponding author
Abstract
Speciation via interspecific or intergeneric hybridization and polyploidization triggers
genomic responses involving genetic and epigenetic alterations. Such modifications
may be induced by small RNAs, which affect key cellular processes, including gene
expression, chromatin structure, cytosine methylation and transposable element (TE)
activity. To date, the role of small RNAs in the context of wide hybridization and
polyploidization has received little attention. In this work, we performed high-
throughput sequencing of small RNAs of parental, intergeneric hybrid, and
allopolyploid plants that mimic the genomic changes occurring during bread wheat
speciation. We found that the percent of small RNAs corresponding to miRNAs
increased with ploidy level, while the percent of siRNAs corresponding to TEs
decreased. The abundance of most miRNA species was similar to mid-parent values
in the hybrid, with some deviations, as seen in overrepresentation of miR168, in the
allopolyploid. In contrast, the number of siRNAs corresponding to TEs strongly
decreased upon allopolyploidization, but not upon hybridization. The reduction in
corresponding siRNAs, together with decreased CpG methylation, as shown here for
the Veju element, represent hallmarks of TE activation. TE-siRNA downregulation in
the allopolyploid may contribute to genome destabilization at the initial stages of
speciation. This phenomenon is reminiscent of hybrid dysgenesis in Drosophila.
Genetics: Published Articles Ahead of Print, published on April 5, 2011 as 10.1534/genetics.111.128348
Copyright 2011.
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Article Summary
Evolution through interspecies hybridization and polyploidization plays an important
role in plant speciation. We have analyzed newly synthesized bread wheat-analagous
hybrids and allopolyploids, in order to characterize the epigenetic events that occur
during early speciation via allopolyploidization. The abundance of small RNAs
corresponding to transposable elements was significantly reduced in the
allopolyploid. This observation correlated with hypomethylation of transposable
elements, a hallmark of active elements. These data suggest that small RNAs
involved in maintaining genome integrity, can be de-regulated in wide crosses. This
alteration might present one of the barriers controling gene flow between related
species.
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Introduction
Interspecific or intergeneric hybridization and whole genome doubling provide a
mechanism for “overnight” speciation (LEITCH and LEITCH 2008; RIESEBERG and
WILLIS 2007; SOLTIS and SOLTIS 2009; WOOD et al. 2009). Wheat, Spartina, Senecio,
and Tragopogon are examples of recent speciation via wide hybridization and
polyploidization. It has been argued that in nature, there are plenty of opportunities
for interspecific hybridization and genome doubling, with an estimated 10%
hybridization among animals and 25% among plants (MALLET 2007). Moreover,
unreduced gametes, which are occasionally produced in most plant species
(BRETAGNOLLE and THOMPSON 1995), were shown to occur at a frequency of up to
50% in some combinations of intergeneric hybrids of wheat (KIHARA and LILIENFELD
1949). Therefore, one might expect new hybrid and polyploid species to be formed
more frequently than documented (VAN DE PEER et al. 2009). This apparent paradox
may be resolved by works demonstrating the genome-wide genetic and epigenetic
impact of genome merging or doubling, consequently challenging maintenance of
genome functionality and stability. While some intergenomic combinations, show
hybrid incompatibility (BOMBLIES and WEIGEL 2007; ISHIKAWA and KINOSHITA
2009; WALIA et al. 2009) or hybrid dysgenesis (MALONE and HANNON 2009), or
feature reduced fitness, others show improved vigor (CHEN 2010). The mechanisms
dictating nascent hybrid and polyploid survival are therefore of particular interest
when attempting to understand the opportunities and bottlenecks of speciation.
A genetic model for such hybrid incompatibility was proposed by Dobzhansky
(DOBZHANSKY 1936) and thoroughly reviewed by LANDRY et al. (LANDRY et al.
2007), whereby divergent loci or alleles demonstrate incompatibility when merged
into the same nucleus. More recently, TIROSH et al. (TIROSH et al. 2009) performed a
genome-wide analysis of gene expression in yeast interspecific hybrids and analyzed
interactions between cis and trans-acting regulatory factors and their relation to gene
expression rewiring in hybrid genomes. Distinct expression patterns in the hybrid
were shown to account for both hybrid failure (LANDRY et al. 2007) and hybrid vigor
(BIRCHLER et al. 2010; NI et al. 2009).
Whole genome doubling of interspecific or intergeneric hybrids gives rise to
allopolyploid species, which present the same type of novel intergenomic interactions
as those found in interspecific hybrids while also providing fertility rescue. Plant
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allopolyploidy also offers an additional level of opportunities for the newly formed
species, such as mutation buffering, sub- and neofunctionalization of duplicated
genes, fixed heterozygosity and a broader range of dosage responses (CHEN 2010;
COMAI 2005; DOYLE et al. 2008). Most angiosperms undergo one or more
polyploidization events in their lineage (VAN DE PEER et al. 2009) and 2-4% of all
speciation events seem to occur via polyploidization (OTTO and WHITTON 2000).
Despite these potential advantages, polyploidization events can lead to “genomic
shock” due to gene redundancy, imbalanced and antagonistic gene expression,
orchestration of DNA replication among the multiple and sometimes different
genomes, and pairing between multiple homologous and homeologous chromosomes.
Studies in several species have shown a broad range of adverse genetic and epigenetic
responses, which occur soon after hybridization and polyploidization, including DNA
deletions, rearrangements or cytosine methylation, gene silencing, activation of
transposons and modification of parental imprinting (CHEN 2010; COMAI 2005;
DOYLE et al. 2008). Small RNAs have been associated with all of these events
(MATZKE and BIRCHLER 2005) and therefore, changes in small RNA species in
hybrids and polyploids might provide mechanistic insight into the control of genetic
and epigenetic changes that occur in response to polyploidization.
Endogenous plant small RNAs can be divided into several classes (GHILDIYAL and
ZAMORE 2009), where two of the most prominent classes include the 21-nucleotide
(nt)-long species, mostly corresponding to microRNAs (miRNA), and the small
interfering RNAs (siRNAs), typically 24 nt in length. Several dsRNA-mediated
pathways operating at the level of the nuclear genome, have been described in plants
and include RNA-directed DNA methylation (MATZKE and BIRCHLER 2005) and
small RNA-mediated DNA demethylation (PENTERMAN et al. 2007). Small RNAs
have been shown to be involved in a broad range of functions including
heterochromatin formation and silencing (LIPPMAN and MARTIENSSEN 2004). siRNAs
seem to function as guardians against transposable elements (TEs) during plant
development (MOSHER et al. 2009). However, their role in transposon regulation in
the context of hybridization or polyploidization, has received far less attention.
In this work, we performed a high-throughput screen of small RNAs in parental,
hybrid, and allopolyploid plants mimicking the events that shaped the genome during
the speciation of bread wheat. We show that miRNAs and repeat-derived siRNAs
respond differently to changes in ploidy level. In addition, the siRNA pools
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corresponding to transposons were significantly reduced upon allopolyploidization.
In parallel, we show that the reduction or total disappearance of siRNAs, correlated
with decreased CpG methylation of target transposons. This deregulation of siRNAs,
and the associated reduction in transposon methylation, may contribute to genome
instability and may hinder speciation via hybridization and polyploidization.
Materials and Methods
Plant material
Seeds were germinated on wet 3MM papers in Petri dishes. All seeds were imbibed
for 24 hours at room temperature under regular light. Seeds of Aegilops tauschii then
underwent 3-6 weeks of vernalization, at 4OC in the dark. After germination, plantlets
were placed in 5-liter pots and grown in a greenhouse. All plants were grown during
the winter, at 20OC and short daylight conditions.
Pollen from the diploid wheat Aegilops tauschii, cultivar TQ113 (genome DD, kindly
provided by J. Dvořak), was used to manually pollinate stigmas of the tetraploid
wheat Triticum turgidum ssp. durum, cultivar Langdon (TTR16, genome BBAA),
which served as the female parent, to generate BAD F1 hybrids. Pollination was
performed early in the morning, using freshly collected pollen, two to three days after
emasculation of the female-parent flowers. The hybrid plants were mostly sterile. F1
spikes were bagged to prevent out-crossing. Occasionally, seeds were obtained from
the spikes of F1 plants. Typically, spikes from F1 plants spontaneously gave zero to
one seed, corresponding to an average frequency of ~ 1%, consistent with reports of
frequent occurrence of unreduced gametes, in similar intergeneric hybrids of wheat
(KIHARA and LILIENFELD 1949). Allopolyploid plants derived from these seeds were
then characterized (see results for details).
Preparation of metaphase chromosomes from wheat root tips
To count chromosomes in F1 hybrids and in newly synthesized amphiploids, root tips
of germinated seeds were cut, subjected to 24h cold treatment in double-distilled
water to arrest mitosis, and then transferred to a 2% acetocarmine staining solution for
2-3 days. Before squashing, root tips were briefly heated in a solution containing 4
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drops of 2% acetocarmine and 1 drop of 1N HCl. Squashing was performed on a
preparative slide, in one drop of 2% acetocarmine.
RNA extraction
Total RNA was extracted from 2 g of whole two-month-old plant tiller tissue, using
25 ml TRI reagent (Molecular Research Center, Inc. Cincinnati, Ohio), according to
the manufacturer's protocol, with the exception of isopropanol precipitations and
ethanol washes which were performed overnight, at -20OC. The plant material used
in this study consisted of six TTR16 plants, six TQ113 plants, three F1 hybrid plants,
and six newly synthesized S1 amphiploid plants, whose polyploid nature had been
previously confirmed by karyotype analysis. Tissues from all genotypes were at the
vegetative phase.
Small RNA library preparation
RNA quality of all samples was verified using a bio-analyzer. Cloning of small RNAs
was performed by Illumina (San Diego, California), using the DGE small-RNA
sample preparation kit protocol v1.0 (Illumina Hayward, California). In brief, small
RNAs (18-32 nt long) were size-selected, purified, and ligated with 3’ and 5’
adaptors. Four cDNA libraries of size-selected RNA from the four pools of plants
were prepared by reverse-transcription PCR, followed by PCR.
Bioinformatic analysis of small RNA high-throughput sequencing data
Eighteen million reads were obtained from high-throughput sequencing of small
RNAs from the four libraries. Within each library, the unique sequences were
reported as tags containing sequence information and frequency. There were 9.8
million tags in total for all four libraries. A script was developed to trim the 3’
adaptor: in the first step, the first 7 bases from the adaptor and any downstream
sequence were removed. Then the process was repeated allowing one mismatch in the
adaptor sequence. Sequences which contained more than 3 N’s were also eliminated.
Tag frequencies were recalculated after these steps, and resulted in 8.9 million tags.
Following quality control, tags with a frequency of at least 30 reads were assembled
into short contigs using the Staden sequence analysis program's (STADEN 1996)
"normal shotgun assembly" module with a minimal initial match of 20 bases,
allowing 5% maximal mismatch, and then allowing additional assembly with 17
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bases with no mismatch. Eventually, 21,787 tags were assembled into 6,892 contigs.
The number of reads and tags in each library is summarized in Table 1 of the
Supplementary data. The Staden program produces a file, which reveals the
relationship between the contigs and the sequences and thus helps determine the
contigs composition and library frequencies. Contigs were annotated using Eland
version 0.3 and BLAST 2.2.17 (ALTSCHUL et al. 1990). Searches were performed
against the following databases: NCBI nr/nt (July 2008), the rice genome
(ftp://ftp.plantbiology.msu.edu/pub/data/Eukaryotic_Projects/o_sativa/annotation_dbs
/pseudomolecules /version_5.0 and version_6.0), wheat repeats
(http://www.tigr.org/tdb/e2k1/tae1/wheat_downloads.shtml – wheat repeat database),
Triticum aestivum release 2 (ftp://ftp.tigr.org/pub/data/plantta/Triticum_aestivum),
Triticum turgidum release 1 (ftp://ftp.tigr.org /pub/data/plantta/Triticum_turgidum),
and mature miRNA (http://microrna.sanger.ac.uk/sequences/). Specifically, for
miRNA detection Eland_21 (seed of 21 bases) was run against plant miRNAs using a
script that slides a window of 21 bases along the contig sequence. Only miRNAs with
no mismatches were reported. Using the output of BLASTn table against the TIGR
wheat repeat database, the contig hits were divided into several subcategories:
retroelements, DNA transposons, repetitive element, and unclassified repeats.
Contig frequency was calculated by summing the frequency of each tag comprising
the contig. Interlibrary normalization was performed by dividing the contig frequency
count per library, by a factor which was calculated by dividing by the total number of
reads from tags with more than 30 reads. Therefore, the calculation represents the
frequency per million reads. A rigorous significance test was performed according to
Audic & Claverie (AUDIC and CLAVERIE 1997). For miRNA counting, all contigs
with the same seed sequence were summed.
Northern blot analyses
Northern blot analyses were performed as previously described (BROWN and MACKEY
2001). Briefly, 15 µg of total RNA were loaded on Formamide-agarose gels and later
transferred to a Hybond N+ nylon membrane (Amersham) via the standard wet
transfer method. The Wis2-1A LTR probe was prepared using the forward primer 5’
TGTTGGAAATATGCCCTAGAG 3’ and the reverse primer 5’
GCACAATCTCATGGTCTAAGG 3’; the Veju LTR probe was prepared using the
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forward primer 5’ TAGAATAAATCCGAGGCATACC 3’ and the reverse primer 5’
TTAGGTTACAGTTGGACTTGG 3’, for amplification from the Triticum aestivum
var. Chinese Spring genome.
Probes were labeled with α[32P] dCTP (Amersham) using Klenow DNA polymerase
(Fermentas). The membranes were blotted overnight with 20 pmol of the labeled
probe and then exposed overnight to a phosphorimager screen, and images were
visualized using the Image Gauge programme. Membranes were stripped for re-use,
in 0.1% SDS and verified to give no signal.
Bisulfite sequencing
Hybrid and polyploid genomic DNA was extracted from 100mg of leaf tissue, using
the GeneElute Plant genomic DNA miniprep kit (Sigma). Two micrograms of
genomic DNA suspended in 0.5ml double-distilled water, were sheared on ice by
ultrasound (Microson sonicator, Misonix) set at 3W output power, four pulses of 10
seconds each with 50 second intervals between pulses. Sheared DNA was
concentrated to a final volume of 20µl, using a speed vacuum set at 60OC for 2 hours.
The genomic DNA was then subjected to bisulfite conversion and purification using
the EpiTect Bisulfite kit (Qiagen), according to the manufacturer's instructions.
Bisulfite ions convert non-methylated cytosine residues to uracil residues. Fifty
nanograms of purified DNA were then subjected to PCR, using degenerate primers
(Metabion), designed to amplify the Veju retrotransposon LTR (Veju degenerate
forward primer: 5’ AGTGAATGTYAAGTTGTTGGTG 3’, Veju degenerate reverse
primer: 5’ TCRAACAACCTARCTCATRATAC 3'). The PCR products were then
separated on a 2% agarose gel and purified using a PCR purification kit (RBC).
Cleaned PCR products were ligated to a pGem-T easy vector (Promega), which were
then used to transform E. coli (top 10 strain); transformed bacteria were then plated
on selective plates. Plasmid DNA was extracted from 25 bacterial colonies for each
plant and then sequenced. Sequences were analyzed using the Sequencher 4.9
programme, and methylation patterns were analyzed using the Kismeth programme
http://katahdin.mssm.edu/kismeth/revpage.pl (GRUNTMAN et al. 2008).
Results
Phenotypic analysis of hybrid and polyploid wheat
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In order to gain insight into the molecular mechanisms leading to the genetic and
epigenetic changes occurring upon hybrid and polyploid formation, the small RNA
profiles of parental wheat lines were compared to those of the derived hybrid and the
first allopolyploid generation. More specifically, the four wheat species analyzed
included the parental tetraploid Triticum turgidum ssp. durum (genome BBAA) and
the diploid Aegilops tauschii (genome DD), their synthetic triploid hybrid (genome
BAD), and their derived hexaploid (genome BBAADD). This synthetic hexaploid is
analogous in genome structure to bread wheat Triticum aestivum ssp. aestivum.
Parental lines were self pollinated for several generations, with spike bagging to
prevent cross-pollination, and were thus considered inbred. Hybrid seeds were
obtained by crossing Triticum turgidum and Aegilops tauschii. The hybrid F1 seeds
were obtained from the female tetraploid plants and were found to be very small and
shriveled compared to their parents (Figure 1). Upon germination, F1 seeds gave rise
to F1 plants bearing necrotic leaves (Figure 1), a typical hybrid incompatibility
phenotype (BOMBLIES and WEIGEL 2007). Some F1 plants died shortly after
germination. Nevertheless, those which survived were vigorous and featured spikes
larger than their parents (Figure 1), but most were sterile. Occasionally, seeds were
spontaneously obtained from the spikes of F1 plants (See Materials and Methods) and
were termed "S1". While these seeds were slightly shriveled, they were larger in size
than the seeds of either parent (Figure 1). Plants germinating from S1 seeds, has a
duplicated genome as confirmed by karyotype analysis (Figure S1). The resulting S1
plants were fertile and bore spikes that yielded S2 seeds, which were less shriveled
than but similar in size to S1 seeds. While S1 plant leaves were highly necrotic, the
plants were vigorous, and featured spikes larger than in the hybrid or in the parents,
and leaves similar in size to the tetraploid parent (Figure 1).
High-throughput sequencing of small RNAs
Total RNA was isolated from tillers of two-month-old wheat plants, which primarily
included somatic tissues, namely, leaves and stems, and some meristematic tissues. A
small RNA library was prepared for each of the analyzed wheat types and was
comprised of a pool of RNA collected from 3-6 plants of each genotype. Eighteen
million reads were obtained from small RNA sequencing of the four libraries
(Supplementary data: Table 1). The unique sequences within each library were
reported as tags (containing sequence information and frequency). These tags
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underwent strict quality checks (Figure S2 and Materials & methods), and only tags
that had 30 reads or more were included in the analysis, due to lack of a fully
sequenced reference genome. As the data from a number of small RNA sequencing
experiments proved highly reproducible (with a correlation of Spearman’s ρ=0.95-
0.98) for tags with ≥30 reads (FAHLGREN et al. 2009), this number was set as the
threshold for data analysis. Of the 18 million reads obtained, 6 million high-quality
reads, corresponding to 21,787 unique sequence tags, were analyzed. The small RNA
sizes ranged between 18 and 35 nt and included the two prominent classes of 21 nt-
and 24 nt-long small RNAs (Figure 2). The 21 nt class corresponds mainly to
miRNA, while the 24 nt class corresponds most likely to siRNAs (GHILDIYAL and
ZAMORE 2009). The 24nt-long small RNAs were most abundant within the two
parental and and the hybrid libraries, whereas, the 21nt-long small RNAs were most
prevalent in the allopolyploid library.
The various small RNAs obtained from tags with ≥30 reads, were categorized
according to sequence similarity (Figure S3). miRNAs were one of the most abundant
small RNA species (21-44%, depending on the genotype). Small RNAs that matched
repeats, which were largely TEs, represented ~12% of the total reads for all libraries,
aside from the allopolyploid library, where a significant decrease (P(χ2)<0.0001) to
only 6% was observed. Surprisingly, tRNAs comprised ~9% of total hits in the
parental and hybrid libraries, and rose to ~20% (P(χ2)<0.0001) in the allopolyploid
library (Figure S3). Small RNA contigs matching tRNA genes ranged in size between
18 and 36 bases. However, 55% of the small RNA tags corresponding to tRNAs
showed distinct peaks at 19, 20 and 21nt, suggesting that they were not degradation
products of larger tRNA molecules. No exceptional RNA degradation was observed
in any of the samples, hence degradation could not explain the higher tRNA
frequency observed in allopolyploid samples.
Abundance of miRNAs in parent, hybrid and allopolyploid libraries
Twenty-one known plant miRNA sequences were identified with high certainty, i.e.
with ≥30 reads each, and were identical to known miRNAs from other plant species
(Supplementary data: Table 2). For example, miR168, the most abundant miRNA in
rice (LU et al. 2008), was the most abundant in the present data. Interestingly, the
amount of miRNA, relative to total small RNA, increased with increasing levels of
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ploidy, being the lowest (21%) in the diploid Aegilops tauschii DD genome, 33% in
the triploid hybrid genome (BAD), 38% in the tetraploid Triticum turgidum BBAA
genome, and 44% in the synthetic hexaploid BBAADD genome (Figure 3).
In order to assess the changes in the abundance and profile of miRNAs expressed as a
result of hybridization and polyploidization, we compared the normalized number of
hits (in reads per million) found in the hybrid and the polyploid libraries to the
average number of hits in the two parental libraries, namely the mid-parent value
(MPV). Under the assumption of additive expression, the number of hits in F1 or S1
should be similar to the mid-parent value: log2(F1/MPV) or log2(S1/MPV)=0.
Positive log2 values indicate that the number of small RNAs for a given tag is higher
in the hybrid or the polyploid than in the parent average; negative values indicate the
opposite. When applying a cutoff value in which |log2|<0.5 is considered similar to
MPV, most miRNAs were expressed to similar degrees as the MPV, (Figure 4).
However, miR390a was under-represented (log2(F1/MPV)= -4.8), and miR160f was
over-represented (log2(F1/MPV)=4.86) when comparing hybrid miRNA expression
profiles to those of the parent libraries. Interestingly, both miR390 and miR160 play
key roles in auxin regulation, via regulation of TAS3, a trans-acting siRNA involved
in auxin signaling (FAHLGREN et al. 2006) by the former and via targeting of ARF10
(LIU et al. 2007) by the latter. In the allopolyploid plants, several miRNA expression
patterns significantly deviated from those observed in the parental lines. miR157a,
miR160f, miR159a, miR396d, and miR168 were significantly over-represented
relative to MPV, while miR156b, miR165a, and miR1135 were significantly under-
represented. miR168, had the highest number of reads in almost all libraries,
amounting to 25% of the polyploid library reads and 119,710 and 79,244 reads in T.
turgidum and Ae. tauschii libraries, respectively (Supplementary data: Table 2). It
was over-represented by 1.27-fold in the hybrid, and by 2.46-fold in the synthetic
polyploid, relative to the mid-parent value (MPV=99,477) and is suggested to have
genome-wide effects on small RNA species (see discussion). miR156 was the
second-highest tag expressed in the model, while miR1135 was only moderately
expressed (Supplementary data: Table 2). miR156a was over-represented by 2.4-
fold in the polyploid relative to the MPV. miR156 and its related sequence is of
particular interest, as its overexpression has been suggested to prolong the vegetative
phase and to delay flowering in Arabidopsis (SCHWARZ et al. 2008) and may explain
the heterotic effects seen here in the allopolyploid plants.
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siRNAs that correspond to repeats
Small 24 nt-long RNAs were abundant in all the libraries, where a large percentage of
these siRNAs corresponded to repeats: 1,113 Staden contigs out of a total of 6,892
correspond to transposons and retrotransposons. An additional 130 contigs
corresponded to ribosomal RNA genes, telomeric and centromeric repeats. siRNAs
matching known genes, often corresponded to repeats as well. Here, we focused on
repeats, using the complete wheat repeat database
(http://wheat.pw.usda.gov/ITMI/Repeats/flatfile.total) as reference.
Transposons, which make up approximately 80% of the wheat genome (CHARLES et
al. 2008), comprise a major class of repeats. Recently, small RNAs have been
mapped to the repeat sequences of the Triticeae repeats database (CANTU et al. 2010).
The present work demonstrates the changes in TE-related small RNAs following
hybridization and polyploidization and confirms that TEs are targets of small RNAs,
particularly at their termini. In contrast to miRNAs, the amount of siRNAs
corresponding to transposons decreased with increased ploidy (Figure 3). In the
hybrid, 42% of siRNAs corresponding to transposons were represented similarly to
the MPV (defined here as |log2|<0.5), while 39% were under-represented and 19%
were over-represented. In the allopolyploid, a massive reduction and significant shift
(P(χ2)<0.0001) in the relative abundance of siRNAs corresponding to transposons
was observed, with 85% of the hits below the MPV (Figure 5). This under-
representation affected both DNA elements and retroelements (Table 1). Small RNA
matching to the sequenced and annotated Triticum aestivum BAC genome region
(accession number CT009735), using IGB (Integrative Genome Browser), confirmed
the massive reduction of small RNAs corresponding to transposons, in the polyploid
compared to the parental lines (Figure 6). Note that in the data used by CANTU et al.
(CANTU et al . 2010), the number of reads corresponding to Veju in natural hexaploid
wheat was very low (1/400,000), similar to the data shown here for the synthetic
hexaploid, but contrasting with the present data collected from natural tetraploid and
diploid wheat.
We then examined whether the reduction in TE-related siRNAs correlated with
hallmarks of transposons activation. Table 1 summarizes several examples of
retrotransposons and DNA transposons for which small RNAs were under-
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represented in the polyploid compared to the parental lines. We then tested whether
siRNA under-representation correlated with transcriptional activation of the copia-
like retrotransposon Wis2-1A and of Veju, a “terminal-repeat retrotransposon in
miniature” (TRIM) element (SANMIGUEL et al. 2002). In both cases, the
allopolyploid expressed higher transcript levels than the hybrid, further substantiating
the observed reduction in corresponding siRNAs. However, the tetraploid parent also
expressed high transcript levels, thus, no straightforward correlation can be drawn
between small RNA and corresponding mRNA levels, detected by Northern
hybridization (Figure S4).
Cytosine methylation represents an additional hallmark of transposon activity, and
has been reported to be reduced in active and hypermethylated in silent transposons
(CHANDLER and WALBOT 1986). As a case study, we performed bisulfite sequencing
of the LTR of a Veju element, using the available sequence from a transposon-rich
region in the Triticum aestivum genome (Genebank accession number CT009735,
coordinates 38,989-39,363). Seven different small RNAs, corresponding to hundreds
of reads, were mapped along the LTR sequence (Figure 7, middle panel) and shown
to derive from both strands. All seven were almost fully suppressed in the
allopolyploid line (Figure 7, top panel). Sequencing of 25 different Veju-LTR clones
per plant type, following bisulfite conversion, enabled us to determine the average
methylation of Veju elements similar in sequence to the Triticum aestivum LTR
(Genebank accession number CT009735, see Methods). A decrease in CG
methylation (Figure 7, bottom panel) but not in CHG or CHH methylation (data not
shown), was observed in the allopolyploid line and correlated with decreased
abundance of Veju LTR small RNAs in the polyploid. These results are consistent
with and further elucidate data reported by KRAITSHTEIN et al. with regard to AFLP-
based Veju methylation (KRAITSHTEIN et al. 2010).
Discussion
Small RNAs are involved in a number of key cellular processes, and perturbations in
their steady-state expression levels can lead to genome-wide changes in gene
expression or in chromatin and genome structure. Modified small RNA expression
levels have been widely reported in the context of developmental regulation of plants,
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but to a lesser extent in the context of speciation via interspecific hybridization and
allopolyploidization (HA et al. 2009). Ha et al. (2009) describe an allotetraploid
hybrid, analogous to A. suecica, formed between a synthetic Arabidopsis
autotetraploid line and the natural Arabidopsis tetraploid, A. arenosa and report
significant deviations in hybrid miRNA expression from MPV. In the present study,
most hybrid miRNA expression profiles did not deviate from their MPVs (Figure 4).
However, a number of deviations were found in the allopolyploid (Table 2-
Supplementary data). In addition, some classes, such as miR156 and miR166,
included several sequence variants, each demonstrating differential expression
patterns. However, in the absence of the wheat genome sequence, it is difficult to
associate changes in miRNA levels to the expression of target genes or to plant
phenotypes.
Another difference between the present model and that described in the Arabidopsis
allopolyploid analysis (HA et al. 2009), lies in the nature of the genetic material
analyzed, where the Arabidopsis work involved plants of the same ploidy level
(parents and hybrid were all tetraploids), while this study encompassed lines of
different ploidy levels (parents (2x and 4x), hybrid (3x) and a derived allohexaploid
(6x) analogous to natural bread wheat). The wheat material offered unique and novel
insight into an unexpected response to changes in ploidy levels. Remarkably, and
unexpectedly, the relative amount of small RNAs corresponding to miRNAs,
increased with ploidy (Figure 3). Inversely, the relative amount of 24 nt small RNAs
corresponding to transposons decreased with increased ploidy (Figure 3). Similarly,
in the work of CANTU et al. (2010), the count/bp of small RNAs corresponding to
transposons was lower in hexaploid versus tetraploid wheat varieties (CANTU et al.
2010). This ploidy-dependence seems to be insensitive to genomic composition, but
sensitive to dosage. Indeed, while the diploid, tetraploid and hexaploid wheat had
different genomic composition, the triploid (hybrid) and allohexaploid, which both
featured the same three wheat genomes (A,B and D) but at varying doses (3x versus
6x), expressed divergent small RNAs profiles.
Mechanistically, a correlation can be drawn between specific miRNA over-
representation and repressed siRNAs formation. More specifically, miR168 over-
representation in polyploids may account for ARGONAUTE 1 (AGO1) suppression
(MALLORY and VAUCHERET 2009), which in turn reduces the production of siRNAs
(VAUCHERET 2008). Although, the regulation of AGO1 is quite complex (MALLORY
15
and VAUCHERET 2009), it is conceivable that affecting regulatory loops responsible
for AGO1 activity, e.g. via alterations in miR168, may bear genome-wide effects on
siRNAs. In fact, the novel intergenomic interactions and novel dosage effects
demonstrated in the allopolyploid lines, may account for the disruption of the activity
of several genes related to small RNA expression and stability. Previously, we have
shown that novel interactions between cis and trans-acting regulatory elements can
lead to overexpression or, alternatively, to suppression of ~10% of the genes of an
interspecific hybrid (TIROSH et al. 2009). Deregulation of central genes involved in
the gene silencing machinery could account for some of the alterations in small RNA
profiles reported here, which in turn can affect TE activities, as discussed below.
siRNAs play a critical role in heterochromatin maintenance and in transposable
element silencing (ZARATIEGUI et al. 2007). The strong decrease in the percentage of
siRNAs corresponding to TEs reported here, could thus lead to transposon activation.
Transposons, notoriously responsive to “genomic shocks” (MCCLINTOCK 1984), may
be responsive to hybridization- and polyploidization-induced “shocks”, linked with
downregulation of small RNAs. Indeed, it has been reported that silent transposons
and retrotransposons may become transcriptionally and sometime transpositionally
active upon hybrid and polyploid formation (CHEN and NI 2006; KASHKUSH et al.
2003; KRAITSHTEIN et al. 2010; MADLUNG et al. 2005). The correlation between
small RNAs cytosine methylation and transposon silencing (MATZKE et al. 2007) may
further explain transposon activation following small RNAs-related demethylation, as
reported here for the Veju TE.
A fascinating aspect of TE regulation involves their capacity to transit between
inactive and active states. Remarkably, a hypomethylated TE can become re-
methylated within one generation, as shown here for a specific element (Figure 7) and
as shown on a genome-wide scale for the Veju element (KRAITSHTEIN et al. 2010).
An attractive model for such kinetics is demonstrated via the disappearance of small
RNAs, as seen here in S1 for several elements, including WIS2-1A (Table 1), which
then leads to transcription of otherwise silent TEs. As a result, high levels of aberrant
and double stranded RNAs may be generated, as many TEs are nested within each
other in opposite orientations (SANMIGUEL et al. 1996). In the subsequent generation,
small RNAs produced by such transcriptional activation, might reverse the element to
its original methylated and silenced state. This proposed mechanism may account for
the reported maintenance-silencing of repeats by small RNAs produced by RNA
16
polymerases IV and V (MATZKE et al. 2009; PIKAARD et al. 2008). In other words,
TE activation may catalyze TE silencing via upregulation of small RNA production.
However, the association between small RNAs and TE transcription still remains
elusive. In an earlier work (KASHKUSH et al. 2003), we analyzed an allopolyploid
resulting from a cross between two diploid parents and measured S1 transcriptional
activation of transposons that were silent in the parents. The present model, using
different parental species, is a bit more complex (Figure S4), where S1 transposon
transcript levels was higher than in F1, substantiating an earlier report of
transcriptional activation in the S1 generation (KASHKUSH et al. 2003). However, high
levels of both small RNAs and TE transcripts were found in the tetraploid parent
(Figure S4), which may be due to transcription of a divergent subfamily of
transposons, not targeted by the small RNAs, or to alteration in small RNA mobility,
processing or other unknown phenomenon.
Another intriguing question remaining relates to the changes measured in
allopolyploid small RNA species, and to a lesser extent in the hybrid. This
observation may be linked to polyploidy per se. Alternatively, this may be a result of
the kinetics of small RNA alterations which begin in the hybrid and peak in the S1
generation. Another interesting possibility is that global demethylation and activation
of TEs may occur through disruption of developmentally regulated processes in the
germline. Such modifications may occur during meiosis, or gametogenesis, or during
the development of an embryo derived from “deregulated” gametes. In such cases,
massive changes would be found in the S1 only, and not in the F1.
In summary, the data reported here, together with previous findings, suggest that
deregulation of small RNAs may stimulate TE activation in interspecific hybrids and
allopolyploids. This phenomenon is reminiscent of hybrid dysgenesis in Drosophila
(MALONE and HANNON 2009), a mechanism of incompatibility that can hinder
speciation via hybridization and polyploidization. The correlation between the
different hallmarks of transposon activation (small RNAs, hypomethylation,
transcription and transposition) is complex, as they undergo rapid changes between
generations. In addition, it is important to note that the present study was performed
on plants which survived hybridization and allopolyploidization. The events of
seedling death and seed abortion, which were observed in the hybrid and
allopolyploid lines, may have occurred as a result of severe transposon activation and
were not considered in our analysis.
17
Acknowledgements
We thank Naomi Avivi-Ragolski and other members of the Levy laboratory for their
help and discussions; Idan Efroni for help in data analysis; Yehudit Posen for editing
the manuscript and members of the bioinformatics unit, especially Jaime Prilusky for
writing the quality-control script and Ester Feldmesser for help with statistical analysis.
This work was funded by a grant from the Israeli Science Foundation, #616/09, to
A.A.L.
18
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Figure legends
21
Figure 1: Parents, hybrids and allopolyploid plant material. Triticum turgidum
durum (female parent, genome BBAA) and Aegilops tauschii (male parent, genome
DD) leaves, spikes and seeds are shown in the top left and top right panels,
respectively. Plant crossing yielded small and shriveled hybrid (F1) seeds (genome
BAD, in the center), from which hybrid F1 plants were grown. F1 plants featured
necrotic leaves and were mostly sterile, occasionally and spontaneously giving rise to
allopolyploid (S1) seeds (middle panel), with a doubled chromosome number. S1
seeds were larger than the seeds of both parents, and were somewhat shriveled. S1
plants (bottom panel), derived from S1 seeds, bore leaves as broad as the maternal
plant, spikes larger than those of both parents, and yielded S2 seeds (bottom left),
which were as large as S1 seeds, and a little less shriveled. Scale bar =0.5cm.
Figure 2: Length distribution of small RNAs. Length distribution of small RNAs in
the four libraries. Small RNAs ranged in size from 18-35 nucleotides, with two
prominent peaks at 21 and 24 nucleotides. Note that the X axis is not linear at the
end.
Figure 3: Changes in small RNA expression levels as a function of plant ploidy.
Percent of siRNAs corresponding to repeats, and of miRNA reads, as a function of
genome ploidy (where X is a haploid genome) and genomic composition. The percent
of reads was determined from the total number of small RNA reads in each library,
thus enabling a comparison between the different ploidy groups.
Figure 4: miRNA abundance in the hybrid and polyploid lines. miRNA
abundance in the hybrid (F1, gray) and the polyploid (S1, black) relative to the mid-
parent value is shown as a logarithmic cumulative distribution function (n=21).
Positive values indicate higher abundance in the hybrid or polyploid compared to the
mid-parent value (MPV), negative values indicate the opposite and values close to
zero indicate similar abundance between the analyzed and parental lines.
Figure 5: Abundance of small RNAs corresponding to transposons. The
abundance in the hybrid (F1, gray) and the polyploid (S1, black) relative to the mid-
parent value is shown as a logarithmic cumulative distribution function (n=1113). The
22
box on the top left summarizes the abundance relative to the mid-parent value. In the
hybrid, 42% of the small RNAs corresponding to transposons had an abundance
similar to the mid-parent (|log2|<0.5), 19% were over-represented (log2>0.5), and 39%
were under-represented (log2<-0.5). In the polyploid, 12% of the small RNAs
corresponding to transposons had an abundance similar to the mid-parent value, 3%
were over-represented and 85% were under-represented, compared to the mid-parent
value. This under-representation was highly significant P(χ2)<0.0001.
Figure 6: Viewing of small RNAs on a BAC sequence. A view of small RNAs
mapped to the Triticum aestivum BAC CT009735, using the IGB tool from
Affymetrix, with coordinates indicating the nucleotide number (nt). Horizontal gray
bars correspond to transposable elements, vertical black lines indicate the abundance
of corresponding small RNAs in each library.
Figure 7: Veju-related small RNAs and CpG methylation. The abundance of small
RNAs that correspond to the Veju retrotransposon LTR found on BAC CT009735, is
shown in the top panel for the mid-parent value, the hybrid (F1) and the polyploid
(S1). The coordinates of small RNAs on the Veju LTR genomic DNA are shown in
the middle panel. The percent of CpG methylation was determined by bisulfite
sequencing for the Veju LTR region. A decrease in CpG methylation in the first
generation of the polyploid (S1), compared to Aegilops tauschii (genome DD) was
observed. In the second polyploid generation (S2), CpG methylation increased. A
natural hexaploid (Genome BBAADD, variety Chinese Spring) is shown for
comparison.
23
Table 1: Number of reads (normalized to reads per million) of siRNAs corresponding to transposable elements (TEs), in parents, hybrid and first generation allopolyploid (S1). TEsa durum
BBAA tauschii DD
F1 BAD
S1 BBAADD
WIS2-1A 756 528 475 0 Latidu 1267 486 747 94 Veju 326 211 400 6 WHAM 83 2646 420 69 Caspar 701 315 472 8 Thalos 532 656 753 5 a Caspar and Thalos are DNA transposons, other TEs are retroelements
24
Supporting figure legends
FIGURE S1: Metaphase chromosomes from a root tip of an S1 allopolyploid plant,
spontaneously derived from a TTR16XTQ113 F1 hybrid. The expected 42
chromosomes are shown.
FIGURE S2: Flow chart of small RNA high-throughput sequencing data analysis.
FIGURE S3: Abundance of small RNA classes in the four analyzed libraries, namely,
the tetraploid parent (genome BBAA), the diploid parent (genome DD), their hybrid
and derived allopolyploid (first generation= S1), and the calculated mid-parent value
(MPV). The total number of reads in each library is shown at the top of each column.
FIGURE S4: Northern blot analyses of Wis2-1A (left) and Veju (right) retrotransposon
LTR regions in the tetraploid parent (genome BBAA), the diploid parent (genome
DD), their hybrid and derived allopolyploid. Methylene blue staining of the
membranes shows equal loading of RNA samples. Probes were derived from a
consensus sequence from wheat Wis2-1A LTRs (a 354bp fragment) and from
Veju1_TM_LTR entry at TREP (a 339 bp fragment).
BBAA
F1 seeds (BAD)
S1 plant
X
Triticum turgidum durum Aegilops tauschii
S1 seeds (BBAADD)
DD
S2 seeds (BBAADD)
F1 plant
Figu
re 1
0
10
20
30
40
50
60
70
35 33 28 27 26 25 24 23 22 21 20 19 18
Perc
ent o
f rea
ds p
er s
ampl
e
Nucleotides
parent BBAA parent DD Hybrid Polyploid
Figu
re 2
0
5
10
15
20
25
30
35
40
45
50 Pe
rcen
t fro
m to
tal s
mal
l RN
As
miRNAs
siRNAs
2X 3X 4X 6X
Figu
re 3
DD BBAA BAD BBAADD Ploidy level and genome composition
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
-12 -10 -8 -6 -4 -2 0 2 4 6 8 10
Perc
ent m
iRN
As
(cum
ulat
ive)
Log2[(reads in F1 or S1)/(reads in MPV)]
Hybrid Polyploid
Figu
re 4
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
-16 -14 -12 -10 -8 -6 -4 -2 0 2 4 6 8 10 12
Perc
ent t
rans
poso
ns s
iRN
As
(c
umul
ativ
e)
Log2[(reads in F1 or S1)/(reads in MPV)]
Hybrid Polyploid
~MPV <MPV >MPV Hybrid Polyploid
39% 85%
42% 12%
19% 3%
Figu
re 5
Parent BBAA
Parent DD
Hybrid
Polyploid
CT009735(+)
CT009735(‐)
nt 20,000 40,000 60,000 80,000 100,000 120,000 140,000 160,000 180,000
Figu
re 6
130-114 27-4 0 374 180 90 270
1-22 38-61
317-301
255-270 67-87
gDNA
Figu
re 7