The expansion of heterochromatin blocks in rye reflects the co-amplification of tandem repeats and adjacent transposable elements
- E. V. Evtushenko†1,
- V. G. Levitsky†2, 3,
- E. A. Elisafenko2,
- K. V. Gunbin2, 3,
- A. I. Belousov1,
- J. Šafář4,
- J. Doležel4 and
- A. V. Vershinin1Email author
© Evtushenko et al. 2016
Received: 17 December 2015
Accepted: 25 April 2016
Published: 4 May 2016
A prominent and distinctive feature of the rye (Secale cereale) chromosomes is the presence of massive blocks of subtelomeric heterochromatin, the size of which is correlated with the copy number of tandem arrays. The rapidity with which these regions have formed over the period of speciation remains unexplained.
Using a BAC library created from the short arm telosome of rye chromosome 1R we uncovered numerous arrays of the pSc200 and pSc250 tandem repeat families which are concentrated in subtelomeric heterochromatin and identified the adjacent DNA sequences. The arrays show significant heterogeneity in monomer organization. 454 reads were used to gain a representation of the expansion of these tandem repeats across the whole rye genome. The presence of multiple, relatively short monomer arrays, coupled with the mainly star-like topology of the monomer phylogenetic trees, was taken as indicative of a rapid expansion of the pSc200 and pSc250 arrays. The evolution of subtelomeric heterochromatin appears to have included a significant contribution of illegitimate recombination. The composition of transposable elements (TEs) within the regions flanking the pSc200 and pSc250 arrays differed markedly from that in the genome a whole. Solo-LTRs were strongly enriched, suggestive of a history of active ectopic exchange. Several DNA motifs were over-represented within the LTR sequences.
The large blocks of subtelomeric heterochromatin have arisen from the combined activity of TEs and the expansion of the tandem repeats. The expansion was likely based on a highly complex network of recombination mechanisms.
Cultivated rye (Secale cereale, 2n = 2x = 14) is, after wheat and barley, a major temperate cereal species. Its large nuclear genome of around 8 Gb/1C  exceeds that of the average angiosperm (5.6 Gb) . A distinctive feature of the karyotype is that each chromosome arm harbors one or more large blocks of subtelomeric heterochromatin , which is not the case in either wheat or barley chromosomes [4, 5]. Within the genus Secale, nuclear genome size varies by some 15 % , consistent with the variation in the size of the terminal heterochromatic blocks . It would appear, therefore, that the expansion of subtelomeric heterochromatin is fundamental to the determination of genome size in the genus Secale.
The rye genome as a whole comprises >90 % repetitive DNA . Eukaryotic tandemly arranged repetitive sequences are typically based on monomers longer than 100 nt ; transposable elements (TEs) represent the other major class of multi-copy sequence. In rye, unlike in human and many plant species , tandem repeats are concentrated in the subtelomeric region rather than around the centromere [10–12]. Notably, the size of heterochromatic blocks in different rye species correlates with the copy number of tandem DNA families . Molecular organization of the three most abundant of them, pSc119.2, pSc200, and pSc250, was characterized previously [11–13]. They are composed of monomers 118, 379, and 571 bp long, correspondingly, and each family constitutes several percent of the rye nuclear genome . Fluorescence in situ hybridization (FISH) experiments have suggested that the pSc200 and pSc250 blocks coincide close to the telomere, while some pSc119.2 copies are located at interstitial sites. The pSc119.2 sequence is also represented in a number of other cereal genomes, but pSc200 and pSc250 are largely rye-specific . Another tandem repeat family (TaiI), which is present in many Triticeae species , is present on two of the seven rye chromosomes, including the short arm of chromosome 1R (1RS) .
Despite a wealth of information regarding the monomers’ size and sequence, their higher order structure remains obscure, as do the mechanisms underlying their amplification. Direct sequencing is hampered by their repetitive nature . Hence their long-range organization, mutual arrangement within arrays, and molecular features of flanking regions between tandem arrays and neighboring non-tandem DNA remain poorly explored (except perhaps for humans). These obstacles can be overcome by approaches allowing one to combine long- and short-range sequence information. These include the construction of BAC (Bacterial Artificial Chromosome) libraries with individual BAC clones containing long (~200 kb) stretches of DNA, and chromosome isolation , which enables analysis of DNA organization in individual chromosomes. In a previous work, a BAC library was constructed from the 1RS arm, which was purified by flow sorting from a wheat-rye ditelosomic 1RS addition line . At least 84 % of 1RS arm was found by BAC-end sequencing and Roche 454 sequencing to consist of repetitive DNA and more than 5 % of the 1RS DNA was occupied by 3121 genes [19, 20].
With the exception of 1RS arm, DNA composition, molecular structure of rye genome received little attention, as compared to the genomes of the closest relatives, wheat and barley. It was not until recently that a virtual linear gene order model encompassing over 22,000 out of 31,008 detected rye genes has been established using a combination of high-throughput transcript mapping, 454 sequencing DNA of flow-sorted rye chromosomes, and synteny information of sequenced model grass genomes . Nonetheless, large-scale molecular organization of subtelomeric heterochromatin in rye chromosomes remains completely unknown.
Here we address a number of questions, such as whether major families of tandem repeats are present as a single or multiple arrays in chromosome arms, what is the organization of monomers belonging to each family in different arrays, whether arrays composed by different family members are immediately adjacent to each other or are separated by some DNA sequences, what is the nature of non-tandemly repeated DNA flanking the arrays, and whether it shows any peculiar sequence features. This information may shed light on the molecular organization of heterochromatic regions as well as on the expansion mechanisms of tandem repeat families in the genome of cultivated rye, S.cereale.
The pSc200 and pSc250 repeats are present in rye chromosomes as multiple arrays
The FISH signal intensity varied along the length of the rye arm (Fig. 1a), in line with the idea that each family of tandem arrays is present as multiple interspaced arrays. Furthermore, this is confirmed by the results of pulsed field gel electrophoresis (PFGE) when BstXI-digested high molecular weight CS/1RS DNA was subjected PFGE and hybridized with pSc250. Six hybridizing fragments were revealed, ranging in length from 40 kb to 270 kb (Fig. 1b). The observed variation in hybridization intensity, taken to indicate that at least some of the arrays harbored non-pSc250 sequence, complicated the quantification of copy number of arrays in 1RS. A restriction analysis, followed by sequencing of the two 1RS BAC clones 12I5 and 122 F3, suggested that both harbored uninterrupted pSc250 arrays (of length, respectively, 38 kb and 57 kb) flanked on either side by non-array sequence. The conclusion is that the 1RS arm harbors several tandem arrays of pSc200 and pSc250 monomers.
Heterogeneity of tandem array organization
When analysing BAC clones from the 1RS library we identified five partially overlapping clones with inserts of different size, each containing pSc200, TaiI and pSc119.2 arrays (shown in Additional file 1, central part). Identical non-tandem DNA was sequenced in all five clones indicating that they originated from the same genomic region. Differences in the hybridization patterns produced by these BACs allowed us to accurately position multimeric units within the tandem arrays. For example, unlike the pSc200 monomers and dimers, the tetramers of pSc200 are absent in BAC 130H7 (Additional file 1, right side, line 2). In contrast, other BACs with longer segments of pSc200 array contain tetramers. As inferred from the hybridization patterns obtained with both pSc119.2 and pSc200 (Additional file 1, left and right sides), pSc200 monomers and dimers, and similarly pSc119.2 trimers and tetramers, tended to lie at the ends of the arrays, whereas the higher order multimers were positioned more centrally.
Percent identity of pSc200 monomers present in BAC clone 119C15
Sequence of the pSc200 monomers in contig
Phylogenetic relationships between the pSc200 and pSc250 copies
The pSc250 sequences formed a single clade with a star-like topology (Fig. 3b), which arose from the overall high level of sequence identity (85–98 %) between the monomers. Such a situation probably reflects a relatively constant amplification rate over time. Differences in the topology of phylogenetic networks derived for the pSc200 and pSc250 monomers are consistent with their distinct evolutionary ages: the former originated some 30 My before the latter, allowing ample time for sufficient sequence divergence to have occurred to generate branching in the pSc200 second clade.
The nature of the sequences flanking the arrays of tandemly arranged monomers in BAC clones
Characterization of the rye genome composition
The availability of 454 reads derived from each rye chromosome  has provided an opportunity to characterize the sequence composition of the rye genome more globally, and to extend the analysis of sequences flanking the tandem arrays in single BAC clones to a genome-wide level. After trimming the adapters and applying quality filtering, the retained set of 14.66 million 454 reads covered about 7 Gbp (mean length: 478 nt). After a filtration step based on RepeatMasker and TREP, two subsets were generated – one containing pSc200 and flanking DNA (314 reads) and the other (494 reads) pSc250 and flanking DNA. These were considered to represent the junction regions (sets “junction”) between non-array sequence and the tandem arrays.
Sequence composition of genome-wide 454 reads and of the sequences adjacent to pSc200 and pSc250 arrays
Reads with junctions of pSc200
Reads with junctions of pSc250
Type of sequence
Cumulative length, bp
Proportion to cumulative length, %
Cumulative length of non-tandem DNA, bp
Cumulative length of non-tandem DNA, bp
Class I TE
4 142 315 628
799 492 074
60 362 185
57 651 280
1 201 224
Class II TE
Simple repeats, low complexity
27 883 202
8 782 174
16 953 966
48 627 218
The vicinity of tandem arrays is populated by certain TE families
Gypsy-13_TA-I, Xalax/Xalas and Olivia are all relatively rare in the rye genome (0.5, 0.3 and 0.05 %, respectively), but their abundance was noticeably higher in the flanking DNA of the pSc200 and pSc250 arrays, particularly in the case of Xalax/Xalas. The Xalax/Xalas supergroup cannot be classified as a single family given the level of sequence diversity present, if one follows the rules of the unified classification , rather it should be considered as two groups. Xalas elements are very homogeneous, while homology between Xalax and Xalas is restricted to relatively short sequence blocks (shown in Additional file 4). Thus, here, Xalax and Xalas were treated as independent TEs; enrichment around the pSc250 arrays was predominantly composed of Xalas sequence (Fig. 5).
Several other families of TEs show behavior similar to that of Xalax/Xalas around the pSc200 and pSc250 arrays (Fig. 5). While Gypsy-13_TA-I was more highly enriched around the pSc250 than around the pSc200 arrays, the opposite was the case for Olivia. There was a pronounced difference between the genome-wide and tandem-array associated frequency of some other TEs. For instance, Daniela was enriched 7.5 fold in the vicinity of the pSc200 arrays, and Laura occurred 11.5 fold more frequently in regions adjacent to pSc250 (Fig. 5). At the same time, sequences around tandem arrays are depleted with respect to Sabrina, the most abundant TE in the genome as a whole. The differences between genome-average and local enrichment values for some other TE families, such as CACTA and Sabine, are small. Several TEs were virtually absent from the tandem array flanking sequence, namely Cereba, Derami, Sumana around pSc250 and Fatima around pSc200. Thus, our analysis clearly shows that the local sequence composition around tandem arrays differs dramatically from the genome average, and displays several peculiar features.
Structural features of the TE/tandem repeat junctions
Enrichment estimates (t-test) for top-scoring motifs
А. Top-scoring motifs in TE - pSc200 junctions, Logo
В. Top-scoring motifs in TE - pSc250 junctions, Logo
Next, we explored whether there is any regularity in the localization of TEs in these regions, i.e. whether TEs tend to break at LTRs, or at their central domains, and whether unrelated spacer sequences may be present at the junctions. Additional file 5 illustrates this distribution for the four chosen TE families. The spacer sequences between the TEs and the arrays were either absent, or at best short (1–10 nt). About 90 % of the junctions between pSc250 and Laura/Xalas elements fell into this category, as did most of the junctions between pSc200 and Daniela (70 %) and Olivia (58 %).
At the junction points, most TE copies began with the LTR’s 5′- or 3′-terminal nucleotide, or a nucleotide very close to the terminus (the distribution of distances is shown in Additional file 6). Analysis based on RepeatMasker software showed that half of the Laura elements present in the vicinity of pSc250 began with their first or last 1–10 nucleotides. Similarly, 54 % of Daniela copies and 68 % of Olivia copies in the vicinity of pSc200 began within the first 20 nucleotides of their LTR; as for pSc250, 69 % of adjacent Xalas copies began with nucleotides 1–20 of one or other LTR. Mapping the top-scoring motifs against the sequences of Daniela, Olivia, Laura, and Xalas extracted from TREP database revealed the same trend, namely the motif density was the highest within the first 300 bp of their 5′- or 3′- LTRs (data not shown) particularly with respect to motifs 2 and 9 in the Daniela and Olivia LTRs.
Thus, our analysis of genomic DNA composition and de novo identification of DNA motifs overrepresented in the vicinity of pSc200 and pSc250 sequences uncovered enrichment of these regions with 5′- and 3′-LTRs of Olivia/Daniela and Laura/Xalas TEs, respectively. These results point to the substantial role of nucleotide context in the formation of DNA flanking tandem repeats, which is likely based on its involvement in the molecular mechanisms taking place during amplification of tandem arrays and associated recombination events.
Multiple arrays of tandem repeats with distinct higher-order organization are present in the chromosomes of rye
The organization of tandemly repeated sequences is poorly understood, not just in the cereals, but in eukaryotes generally. A notable exception is the human α-satellite, comprising a large array at each centromere which emerged as a paradigm for understanding the genomic organization of other tandem DNA sequences [22, 23, 31–34]. Although these latter arrays are mostly homogeneous, a few chromosomes harbour two or more distinct arrays each defined by different HORs [31, 32]. Here, the application of FISH clarified that the pSc200 and pSc250 arrays located close to the 1RS telomere are organized into discrete domains, a conclusion supported by the Southern hybridization analysis. We believe that this observation can be extrapolated to heterochromatin regions of other rye chromosomes. The evidence for this extrapolation is supported by the size of the set of junction site sequences (314 involving pSc200 and 494 involving pSc250), which number far more than the number of chromosome arms. It is possible that some of these reads arose from non-array sequence embedded in the monomer array, but these cannot be common since none emerged from the sequencing of several 1RS BAC clones. The frequency of direct junctions between pSc200 and pSc250 arrays is very low; only six reads fell into this category, which further reinforces our conclusion that each of these two families has its own, distinct localization domain on the rye chromosomes. Nonetheless, the fact that pSc200 and pSc250 FISH signals display partial overlap is indicative of the close proximity and short junction regions between both domains.
HORs are composed of monomers with nearly identical nucleotide sequence and are found located in the centers of alpha-satellite DNA arrays . In this work we show that in rye, pSc200 and pSc250 sequences form multimeric units with the number of monomers varying from 2 to 8 and that the multimeric units map to the centres of the arrays. Multiplicity of monomers within these multimeric units is specific for each individual array found in 1RS. This may argue in favor of multiple recombination events involving distinct tandem arrays within one chromosome arm, which led to HOR formation. Supportive evidence of active recombination within tandem arrays is provided by the level of sequence divergence (up to 7 %) observed between pSc200 monomers arranged as dimers within a BAC119C15, which implied that two monomers first formed a single unit, which was later amplified as a unit. It has been suggested that, during the evolution of tandem arrays, early duplication events were more frequent than subsequent amplification steps . The dimeric repeat structure is universal for alpha satellite DNA, as it is present across various Old World monkey species  and is 15–20 MY old based on the estimated evolutionary divergence of these species .
A comparison of rye tandem repeat families and primate α-satellite DNA
Most of the pSc200 and pSc250 arrays ranged in length from 40–300 kb , while the human α-satellite forms much longer arrays of up to 6 Mbp . The two array types are also located in a different part of the chromosome (subtelomere vs centromere). The postulated mechanisms for the generation and maintenance of tandem arrays include unequal sister-chromatid exchange, sequence conversion, translocation exchange and transposition [8, 37, 38]. As most α-satellite subsets are chromosome-specific, the within homologs exchange frequency is thought likely to be significantly higher than that occurring between non-homologs . The pSc200 and pSc250 tandem arrays appear to have distinct evolutionary histories. Several pSc200 copies are present in hexaploid wheat and other Triticeae species [14, 39], but has also been identified in the more distantly related rice and oat. As a result, it must have arisen at least 45 Mya, when the rice and oat lineages diverged , making it more ancient than the human α-satellite, whose presence throughout the primate order dates it to some 35 Mya . The pSc250 sequence is much younger; its appearance as isolated copies in a few Triticeae species  dates it to 15 Mya. Despite their representation across multiple grass species, the expansion of both families has postdated the divergence of Secale from its closest relatives . Thus, both families have been amplified over a much shorter timescale than α-satellite DNA.
The topology of both the pSc200 and pSc250 phylogenies was largely star-like, in contrast to the tree-like form of the human α-satellite phylogeny . With the exception of the chromosome 5R and 7R sub-clades, the chromosomal origin of the pSc200 monomers was heterogeneous. The presence of multiple, relatively short arrays on each rye chromosome, along with a predominantly star-like phylogeny, are consistent with the rapid evolution of these arrays, likely accelerated by illegitimate recombination including interchromosomal recombination events. This model is supported by FISH-identified presence/absence and intensity polymorphisms for both pSc200 and pSc250 between homologs of different cereal rye accessions [41, as well as the readiness with which introgression occurs in S. cereale x S. montanum hybrids . Exchange of satellite sequences between chromosomes is not unprecedented and was demonstrated for allopolyploid Nicotiana species . The presence of the 5R- and 7R-specific pSc200 sub-clades may be connected with the observation that it is only these chromosomes which still harbor fragments of the ancestral Triticeae chromosome a6 , but how such ancient DNA segments may have escaped interchromosomal exchanges is not clear.
The abundance of certain TE families in the vicinity of pSc200 and pSc250 arrays
TEs are responsible for much of the genome enlargement seen in the cereals [29, 44, 45], and their concentration in heterochromatin is well-established. Thus it was expected that TE sequence would be common in the regions flanking the pSc200 and pSc250 arrays. The sequences appeared as a mosaic of incomplete, heterogeneous TEs, likely resulting from nested insertions subjected to subsequent recombination, duplication and indel formation [46, 47]. The analyses of barley and wheat genomic sequence has shown that most TE families are present in relatively low copy numbers and that just 15 families make up at least 50 % of the genome complement . Similarly in rye, the Sabrina family constituted an estimated 15.5 % of the nuclear genome. Why particular TE families have been able to expand in a species-specific manner is quite unknown. Sabrina was first identified in barley  but is widespread in the Triticeae  including wild species of Secale . Although similar to Gypsy, it contains an env-like gene, the product of which includes predicted transmembrane domain which may aid its horizontal transfer. Notably, in S. cereale, Sabrina is only seldom seen in subtelomeric regions , suggesting that it has not been actively involved in the formation of the prominent heterochromatin blocks.
A striking feature of pSc200 and pSc250 array flanking sequence is that although it has been enriched for TE sequence, the TEs involved were not highly abundant across the genome as a whole. The frequency of solo-LTR elements is particularly notable around pSc250. Ectopic exchanges were likely commonplace in the vicinity of the arrays as this is in line with the predictions of the ectopic exchange model . The solo-LTRs present in the flanking sequence were largely a heterogeneous mixture of Xalas and Xalax. The former element was first identified in barley , and despite its relatively large size (~4 kb), it has not been assigned to any of the LTR-retrotransposon superfamilies, as no coding domain-like sequences have yet been identified. Various representatives of Xalas/Xalax share relatively short regions of incomplete homology (rarely >80 %). Thus, these elements cannot be classified as a single family according to the 80-80-80 rule ; a similar level of identity applied between the terminal segment of the Daniela and Olivia LTRs. The major processes likely responsible for the formation of solo-LTRs are unequal crossing over and intrachromosomal ectopic recombination between LTRs of the same or even different elements, when they share the regions of homology. If recombination involves the LTRs of different elements, a range of recombination products may result, potentially leading to chromosome rearrangement .
Multiple recombination mechanisms were likely involved in the expansion of rye tandem repeats and their flanking TEs
Whereas the molecular basis of recombination between tandem repeat has long been an active research topic, little attention has been given to resolving whether the sequences adjacent to the arrays affect the expansion process. The sequences flanking human α-satellite DNA are highly heterogeneous  and do not seem to be enriched for TEs . Any recombination event involves the formation of double-strand breaks and their subsequent repair. The latter process is achieved by non-homologous end joining (NHEJ) and homologous recombination (HR) . The present analysis of the array/TE junctions indicated that most of the TE sequence was integrated either directly into the monomers or attached to it via a very short (1–10 bp long) spacer, consistent with the NHEJ scenario. Most of the junctions between pSc250 and Laura/Xalas and between pSc200 and either Daniela or Olivia followed this pattern.
A degenerate 13 nt motif has been demonstrated to be associated with ~40 % of human crossover hotspots . Currently, no such clear association between recombination and specific DNA sequence motifs has been established in plants . Nonetheless, the junction regions in rye are clearly enriched with respect to several DNA motifs, some of which may be involved in other known DNA repair mechanisms acting independently of NHEJ and HR . The heterogeneity of DNA motifs found in the TEs, combined with the relatively low level of sequence similarity within the homologous regions of Xalas/Xalax and Daniela/Olivia, fit the requirements for microhomology-mediated break-induced replication and gene conversion to function . The length of the motifs identified (8–12 nt) agrees well with a recognition mechanism allowing recombinases to align single-stranded DNA with a homologous duplex (dsDNA). Once a presynaptic complex has engaged a particular 8-nt (or longer) tract of microhomology, it may become exchanged with other region of dsDNA bearing the same microhomology, yet resist exchanges with unrelated sequences . The number of rearrangements induced by microhomology-driven pathways is likely to be higher than is currently thought . The outcomes of these currently under-appreciated repair pathways could include an increased copy number of the sequences being repaired . Consequently, these mechanisms may be a significant contributor to the formation of heterochromatic blocks.
Shaping the rye genome by tandem repeats
Although the barley and wheat-rye lineages diverged approximately 10–13 Mya , and wheat and rye shared a common ancestor only 6–7 Mya , the karyotypes of these three species vary drastically with respect to both their size and structure , although not with respect to their gene content . It is widely accepted that differences in genome size between closely related species are largely attributable to the quantity of intergenic DNA present, which in turn is heavily influenced by TE copy number and composition. In the case of rye, an increased TE content has not been the sole factor contributing to its genome expansion; in addition there has been a massive amplification of tandemly repeated DNA, based on pSc200 and pSc250 (and other) monomers. This conclusion is supported by the positive correlation between larger heterochromatic blocks and higher content of tandem DNA repeat families in the cultivated rye (S. cereale) as compared to wild rye species . The high copy tandem repeats found in barley and wheat, HvRT , pSc119.2 , dpTa1  and TaiI , are significantly less abundant than are pSc200 and pSc250 in rye.
The presence of multiple copies of the repeated DNA sequences in each subtelomeric region might be expected to promote pairing between homologous and non-homologous chromosomes. The termini of rye chromosomes are known to play a key role in the initiation of synapsis , and since they remain associated for a longer period than other parts of the chromosome, it has been suggested that this explains why the frequency of recombination increases along the centromere-telomere axis . The recombination rate gradient along the centromere-telomere axis is steeper in the wheat close relative Aegilops speltoides (the chromosomes of which feature large subtelomeric heterochromatic blocks) than in einkorn wheat (which lacks major blocks) .
Early studies have noted that tandemly repeated DNA can increase in copy number over a relatively short evolutionary time by replication conversion-like events or via some other unexplained mechanisms . This phenomenon has recently received further support via analysis of the evolutionary fate of various satellite repeats in species from Nicotiana section Polydicliae . Significant progress has been made over the past twenty years in understanding the molecular nature of various recombination pathways. Direct involvement of pSc200 DNA in the association of subtelomeric regions of two or more bivalents was demonstrated by FISH . It is highly probable that the heterogeneous composition of pSc200 and pSc250 multimeric units and the localization of multimers to the central part of monomer arrays is a by-product of unequal crossing over and homologous recombination. Gene conversion and ectopic exchanges between homologous and non-homologous chromosomes have promoted the formation of multiple arrays of each repeat family and contributed to a significant enrichment of the flanking sequences with solo LTRs and several TE families. The presence of short microhomology tracts in these elements implies a contribution of other known recombination pathways . Thus, all the above-listed mechanisms may have been involved in creating the bewildering complexity of recombination events that have ultimately resulted in expansion of tandem repeat families pSc200 and pSc250 as well as several TEs in the rye genome.
Plant material and FISH
The plant materials used were the bread wheat cv. Chinese Spring (CS), the cereal rye cv. Imperial and wheat-based ditelosome addition line involving rye chromosome arm 1RS (CS/1RS) . Chinese Spring cultivar is an international standard for wheat research, much as the rye cv Imperial. Spikelets at the appropriate meiotic stage were fixed and prepared for FISH as described elsewhere . FISH was performed according to a protocol optimized for rye meiotic chromosomes .
DNA plug preparation, PFGE and Southern hybridization
High molecular weight DNA was isolated from protoplasts prepared from CS/1RS seedlings . The agarose plugs containing the DNA were loaded into a CHEF-DRIII PFGE system device (Bio-Rad) for PFGE through a 1 % agarose gel. The separated DNA fragments were transferred to a Hybond-N+ membrane, which was then subjected to Southern hybridization at 65 °C following , rinsed once at 65 °C in 0.1 M Na2HPO4, 0.1 % (w/v) SDS for 30 min, and then in 0.04 M Na2HPO4, 0.5 % (w/v) SDS for 30 min.
DNA probes and labeling
For FISH experiments, pSc200 (accession number Z50039.1) and pSc250 (accession number Z50040.1) were labeled with, respectively, digoxigenin-11-dUTP (Roche) and biotin-11-dUTP (Roche) via PCR . For Southern hybridization experiments, pSc119.2, TaiI, pSc200 and pSc250 were labeled with [α-32P]dATP (GE Healthcare, Amersham) either by PCR or by random priming .
Analysis of 1RS-specific BAC library and BAC clone sequencing
Filters with spotted BAC clones from the 1RS-specific BAC library SccImp1RShA  were sequentially hybridized with pSc200 and pSc250 probes. Positive clones were selected for preliminary analysis of insert sizes and patterns of restriction fragments. Clones displaying distinct restriction digestion patterns and positive for pSc200 or pSc250 were chosen for finer analyses. Namely, these clones were first subjected to a stability analysis , then restriction mapped using either partial digestion with one enzyme or a complete digestion with two .
Digested BAC fragments were subjected to pulsed-field gel electrophoresis using CHEF-DR III apparatus (BioRad) in 1 % agarose gel on 0.5хТBЕ at 14 °С. The settings used were as follows: initial switch time - 0.5 s, final switch time – 4 s, voltage - 6 V/cm, running time 10–12 hours, depending on the expected DNA fragment sizes. Following gel electrophoresis, the DNA was transferred onto Hybond-XL membrane (Amersham Biosciences) and subjected to Southern-blotting, as described above.
The primer walking sequencing of the BACs was performed using a ABI PRISM BigDye™ Terminator Cycle Sequencing Ready Reaction kit (Applied BioSystems); the reaction products were separated using an ABI3730xl capillary sequencer. Primers annealing to the ends of the pIndigoBAC-5 vector (used to construct the SccImp1RShA library) were used for the initial walking step. Downstream sequencing reactions used primers designed from de novo acquired sequence.
Subcloning of BAC sequences and sequencing of pSc200 arrays
The portion of the pSc200 array in ВАС clone 119С15 was sequenced by initially digesting it with HindIII, NdeI and XbaI. The products were separated by PFGE and probed with pSc200. A hybridizing fragment was gel-eluted using a Min Elute Gel Extraction kit (Qiagen) and the DNA then ligated to NdeI/SpeI restricted pGem-5Zf(+). The ligation products were transferred into E. coli XL10-GOLD (Stratagene) competent cells . A series of deletion clones was obtained by treatment with exonuclease III and SI nuclease (ThermoScientific) . Protruding 5′- and 3′-ends were generated by SphI/NcoI digestion (Promega). The resulting 300–400 bp inserts were sequenced and assembled into a contig which comprised 11 full length pSc200 monomers.
Processing of 454 reads
Chromosome-based rye genome sequence  was used to characterize the flanking regions of the pSc200 and pSc250 arrays. Adapter sequences were removed from the reads using tagcleaner 0.12 , quality sorting was performed as described in , and makeblastdb software  was run to create a relevant reads database. Phred quality scores were set as follows: Av(Q) –Z * σ(Q), where Av(Q) and σ(Q) denoted the quality score mean and standard deviation. The Z values were set as 2.6 and 1.6 for the phylogenetic analysis and the analysis of tandem array/non-array junctions, respectively.
Subsampling of 454 reads for phylogeny construction
Consensus pSc200 and pSc250 sequences, established from archival and BAC clone sequences, were used as blastn and blastcmdsearch queries  to extract homologous 454 reads (length thresholds were, respectively, 95 and 80 %). In order to select only distinct monomers with the homology level at most 98 % we run the nucmer and show-coords routines implemented in MUMmer v3.23 . A multiple alignment of the chosen sequences was performed using sate v2.2.7 software .
Phylogenetic analysis of the pSc200 and pSc250 families
Jack-knife analysis  was performed to assess the statistical robustness of the predicted phylogeny; this involved the removal of 25 % random aligned regions. This threshold was chosen based on the observation that substitutions/deletions were infrequent and were uniformly distributed. For each jackknifed alignment, the maximum likelihood algorithm implemented in raxml v7.4.2 software  and the GTRGAMMA model were used to construct the phylogeny. Based on the set of 500 trees, dendroscope v3.2.8 software  was used to build a Galled phylogenetic network of the original set of trees. Pairwise distances were calculated based on the original (all sites intact) alignment of repeats using the distmat program implemented in the EMBOSS v6.3.1 package .
Sampling of the junctions between TEs and tandem repeats
Quantification of the various DNA families was based on the entire set of high-quality 454 rye whole genome DNA reads. Two subsamples of reads (termed “junction”) were compiled: each member’s sequence included a segment homologous to either the pSc200 or the pSc250 sequence (E < e−06) using WUBLAST (http://blast.wustl.edu). The non-array portions were oriented and aligned to begin at the TE/tandem repeat junction. Both the total set of reads and the two “junction” subsamples were scanned using RepeatMasker (http://www.repeatmasker.org), TREP (http://wheat.pw.usda.gov/ITMI/Repeats/) software, applying default settings. Finer positioning of the repeats within the reads was achieved using FASTA software . The remaining reads were then filtered to retain those harboring at least 200 nt of non-array sequence.
Analysis of nucleotide context at the TE/tandem repeat junctions
The “junction” reads from which array sequence had been were removed were trimmed by 80 nt at their 3′-end. We applied the threshold of sequence identity 90 % to non-array DNA in order to analyse only non-redundant junctions. Homer software (http://homer.salk.edu/homer/motif/) was used for the de novo identification of enriched motifs. The required set of background sequences was generated by the shuffling of sequences of the test sample. The 12 top-scoring motifs from the output of Homer tool for each of the pSc200/pSc250 families were selected, because this number was sufficient to confirm the hypothesis on the relationship between the most overrepresented motifs and the most abundant TEs. This hypothesis is based on the enrichment of tandem-genomic DNA junction with certain types of TEs. For each top-scoring motif a position weight matrix was obtained from the matrix of nucleotide frequencies using log-odds weights . Each of these matrices was based on the threshold values computed as in  applying a P value of 5e−5. The statistical significance of association between the motif hit occurrence and TE mapping in a read (the “DNA motif – TE” association) was estimated by Fischer’s t-test for angular (arcsine square root) transform proportions . The proportions were computed from the ratio between the number of junctions with hits of motif to the total number of junctions. The first proportion A/(A + C) referred to the total set of junctions, and the second B/(B + D) to the subset of junctions which included a TE, where A through D represented the relevant number of reads (the details are described in Additional file 7). According to Bonferroni’s correction only “DNA motif – TE” associations for which P value was <0.00417 (0.05/12) were considered as significant.
The sequence data described are available in GenBank under accession numbers KT724931-48.
We thank Adam Lukaszewski (University of California, Riverside, USA) for the gift of grain of cv. CS and cv. Imperial, and B. Friebe (Kansas State University, Manhattan, USA) for that of CS/1RS. This research was financially supported by the IMCB SB RAS budget project 0310-2014-0002, the Russian Foundation for Basic Research (grant 12-04-00512) and the Czech Ministry of Education, Youth and Sports (grant award LO1204 from the National Program of Sustainability I). The bioinformatics analysis was undertaken with the support of the ICG SB RAS budget project 0324-2015-0003.
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- Bennett MD, Leitch IJ. Angiosperm DNA C-values database (release 8.0, Dec 2012). http://www.kew.org/cvalues/.
- Rabinowicz PD, Bennetzen JL. The maize genome as a model for efficient sequence analysis of large plant genomes. Curr Opin Plant Biol. 2006;9:149–56.PubMedView ArticleGoogle Scholar
- Gill BS, Kimber G. The Giemsa C-banded karyotype of rye. Proc Natl Acad Sci USA. 1974a;71:1247–9.Google Scholar
- Gill BS, Kimber G. Giemsa C-banding and the evolution of wheat. Proc Natl Acad Sci USA. 1974b;71:4086–4090.Google Scholar
- Linde-Laursen I. Giemsa C-banding of barley chromosomes. I. Banding pattern polymorphism. Hereditas. 1978;88:55–64.View ArticleGoogle Scholar
- Bennett MD, Gustafson JP, Smith JB. Variation in nuclear DNA in the genus Secale. Chromosoma. 1977;62:149–76.View ArticleGoogle Scholar
- Flavell RB, Bennett MD, Smith JB, Smith DB. Genome size and the proportion of repeated nucleotide sequence DNA in plants. Biochem Genet. 1974;12:257–69.PubMedView ArticleGoogle Scholar
- Charlesworth B, Sniegowski P, Stephan W. The evolutionary dynamics of repetitive DNA in eukaryotes. Nature. 1994;371:215–20.PubMedView ArticleGoogle Scholar
- Melters DP, Bradnam KR, Young HA, Telis N, May MR, Ruby JG, et al. Comparative analysis of tandem repeats from hundreds of species reveals unique insights into centromere evolution. Genome Biol. 2013;14:R10.PubMedPubMed CentralView ArticleGoogle Scholar
- Jones JDG, Flavell RB. The structure, amount and chromosomal localisation of defined repeated DNA sequences in species of the genus Secale. Chromosoma. 1982;86:613–41.View ArticleGoogle Scholar
- McIntyre CL, Pereira S, Moran LB, Appels R. New Secale cereale (rye) DNA derivatives for the detection of rye chromosome segments in wheat. Genome. 1990;33:317–23.View ArticleGoogle Scholar
- Vershinin AV, Schwarzacher T, Heslop-Harrison JS. The large-scale organization of repetitive DNA families at the telomeres of rye chromosomes. Plant Cell. 1995;7:1823–33.PubMedPubMed CentralGoogle Scholar
- Alkhimova OG, Mazurok NA, Potapova TA, Zakian SM, Heslop-Harrison JS, Vershinin AV. Diverse patterns of the tandem repeats organization in rye chromosomes. Chromosoma. 2004;113:42–52.PubMedView ArticleGoogle Scholar
- Vershinin AV, Alkhimova EG, Heslop-Harrison JS. Molecular diversification of tandemly organized DNA sequences and heterochromatic chromosome regions in some Triticeae species. Chromosome Res. 1996;4:517–25.PubMedView ArticleGoogle Scholar
- Kishii M, Tsujimoto H. Genus-specific localization of the TaiI family of tandem-repetitive sequences in either the centromeric or subtelomeric regions in Triticeae species (Poaceae) and its evolution in wheat. Genome. 2002;45:946–55.PubMedView ArticleGoogle Scholar
- Vershinin AV, Evtushenko EV. What is the specificity of plant subtelomeres? In: Louis EJ, Becker MM, editors. Subtelomeres. Heidelberg-New York-Dordrecht-London: Springer; 2014. p. 195–209.View ArticleGoogle Scholar
- El-Metwally S, Hamza T, Zakaria M, Helmy M. Next-generation sequence assembly: four stages of data processing and computational challenges. PLoS Comput Biol. 2013;9:e1003345.PubMedPubMed CentralView ArticleGoogle Scholar
- Šimková H, Šafář J, Suchánková P, Kovářová P, Bartoš J, Kubaláková M, et al. A novel resource for genomics of Triticeae: BAC library specific for the short arm of rye (Secale cereale L.) chromosome 1R (1RS). BMC Genomics. 2008;9:237.PubMedPubMed CentralView ArticleGoogle Scholar
- Bartoš J, Paux E, Kofler R, Havránková M, Kopecký D, Suchálková P, et al. A first survey of the rye (Secale cereale) genome composition through BAC end sequencing of the short arm of chromosome 1R. BMC Plant Biol. 2008;8:95.PubMedPubMed CentralView ArticleGoogle Scholar
- Fluch S, Kopecky D, Burg K, Šimková H, Taudien S, Petzold A, et al. Sequence composition and gene content of the short arm of rye (Secale cereale) chromosome 1. PLoS One. 2012;7:e30784.PubMedPubMed CentralView ArticleGoogle Scholar
- Martis MM, Zhou R, Haseneyer G, Schmutzer T, Vrána J, Kubaláková M, et al. Reticulate evolution of the rye genome. Plant Cell. 2013;25:3685–98.PubMedPubMed CentralView ArticleGoogle Scholar
- Rudd MK, Willard HF. Analysis of the centromeric regions of the human genome assembly. Trends Genet. 2004;20:529–33.PubMedView ArticleGoogle Scholar
- Warburton PE, Hasson D, Guillem F, Lescale C, Jin X, Abrusan G. Analysis of the largest tandemly repeated DNA families in the human genome. BMC Genomics. 2008;9:533.PubMedPubMed CentralView ArticleGoogle Scholar
- Henikoff S. Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing. Gene. 1984;28:351–9.PubMedView ArticleGoogle Scholar
- Vicient CM, Kalendar R, Schulman AH. Variability, recombination, and mosaic evolution of the barley BARE-1 retrotransposon. J Mol Evol. 2005;61:275–91.PubMedView ArticleGoogle Scholar
- Tek AL, Song J, Macas J, Jiang J. Sobo, a recently amplified satellite repeat of potato, and its implications for the origin of tandemly repeated sequences. Genetics. 2005;170:1231–8.PubMedPubMed CentralView ArticleGoogle Scholar
- Sharma A, Wolfgruber TK, Presting GG. Tandem repeats derived from centromeric retrotransposons. BMC Genomics. 2013;14:142.PubMedPubMed CentralView ArticleGoogle Scholar
- Wicker T, Guyot R, Yahiaoui N, Keller B. CACTA transposons in Triticeae. A diverse family of high-copy repetitive elements. Plant Physiol. 2003;132:52–63.PubMedPubMed CentralView ArticleGoogle Scholar
- Middleton CP, Stein N, Keller B, Kilian B, Wicker T. Comparative analysis of genome composition in Triticeae reveals strong variation in transposable element dynamics and nucleotide diversity. Plant J. 2013;73:347–56.PubMedView ArticleGoogle Scholar
- Wicker T, Sabot F, Hua-Van A, Bennetzen JL, Capy P, Chalhoub B, et al. A unified classification system for eukaryotic transposable elements. Nature Rev Genet. 2007;8:973–82.PubMedView ArticleGoogle Scholar
- Choo KH, Earle E, Vissel B, Filby RG. Identification of two distinct subfamilies of alpha satellite DNA that are highly specific for human chromosome 15. Genomics. 1990;7:143–51.PubMedView ArticleGoogle Scholar
- Wevrick R, Willard HF. Physical map of the centromeric region of human chromosome 7: relationship between two distinct alpha satellite arrays. Nucl Acids Res. 1991;19:2295–301.PubMedPubMed CentralView ArticleGoogle Scholar
- Rudd MK, Wray GA, Willard HF. The evolutionary dynamics of α-satellite. Genome Res. 2006;16:88–96.PubMedPubMed CentralView ArticleGoogle Scholar
- Ames D, Murphy N, Helentjaris T, Sun N, Chandler V. Comparative analyses of human single- and multilocus tandem repeats. Genetics. 2008;7:603–13.Google Scholar
- Alkan C, Ventura M, Archidiacono N, Rocchi M, Sahinalp SC, Eichler EE. Organization and evolution of primate centromeric DNA from whole-genome shotgun sequence data. PLoS Comput Biol. 2007;3:e181.PubMed CentralView ArticleGoogle Scholar
- Goodman M. The genomic record of Humankind’s evolutionary roots. Am J Hum Genet. 1999;64:31–9.PubMedPubMed CentralView ArticleGoogle Scholar
- Smith GP. Evolution of repeated DNA sequences by unequal crossover. Science. 1976;191:528–35.PubMedView ArticleGoogle Scholar
- Willard HF. Evolution of alpha satellite. Curr Opin Genet&Dev. 1991;1:509–14.View ArticleGoogle Scholar
- Xin Z-Y, Appels R. Occurrence of rye (Secale cereale) 350-family DNA sequences in Agropyron and other Triticeae. Pl Syst Evol. 1987;160:65–76.View ArticleGoogle Scholar
- Gaut BS. Evolutionary dynamics of grass genomes. New Phytol. 2002;154:15–28.View ArticleGoogle Scholar
- Cuadrado A, Jouve N. Distribution of highly repeated DNA sequences in species of the genus Secale. Genome. 1997;40:309–17.PubMedView ArticleGoogle Scholar
- Heemert CV, Sybenga J. Identification of the three chromosomes involved in the translocation which structurally differentiates the genome of Secalecereale L., from those of Secale montanum and Secale vavilovii Grossh. Genetica. 1972;43:387–93.View ArticleGoogle Scholar
- Koukalova B, Moraes AP, Renny-Byfield S, Matyasek R, Leitch AR, Kovarik A. Fall and rise of satellite repeats in allopolyploids of Nicotiana over c.5 million years. New Phytol. 2010;186:148–60.PubMedView ArticleGoogle Scholar
- Charles M, Belcram H, Just J, Huneau C, Viollet A, Couloux A, et al. Dynamics and differential proliferation of transposable elements during the evolution of the B and A genomes of wheat. Genetics. 2008;180:1071–86.PubMedPubMed CentralView ArticleGoogle Scholar
- Daron J, Glover N, Pingault L, Theil S, Jamilloux V, Paux E, et al. Organization and evolution of transposable elements along the bread wheat chromosome 3B. Genome Biol. 2014;15:546.PubMedPubMed CentralView ArticleGoogle Scholar
- Shirasu K, Schulman A, Lahaye T, Schulze-Lefert P. A contiguous 66-kb barley DNA sequence provides evidence for reversible genome expansion. Genome Res. 2000;10:908–15.PubMedPubMed CentralView ArticleGoogle Scholar
- Wicker T, Zimmermann W, Perovic D, Paterson AH, Ganal M, Graner A, et al. A detailed look at 7 million years of genome evolution in a 439 kb contiguous sequence at the barley Hv-elF4E locus: recombination, rearrangements and repeats. Plant J. 2005;41:184–94.Google Scholar
- Klemme S, Banaei-Moghaddam AM, Macas J, Wicker T, Novak P, Houben A. High-copy sequences reveal distinct evolution of the rye B chromosome. New Phytol. 2013;199:550–8.PubMedView ArticleGoogle Scholar
- Peterson-Burch BD, Nettleton D, Voytas DF. Genomic neighbourhoods for Arabidopsis retrotransposons: a role for targeted integration in the distribution of the Metaviridae. Genome Biol. 2004;5:R78.PubMedPubMed CentralView ArticleGoogle Scholar
- Horvath JE, Viggiano L, Loftus BJ, Adams MD, Archidiacono N, Rocchi M, et al. Molecular structure and evolution of an alpha satellite/non-alpha satellite junction at 16p11. Hum Mol Genet. 2000;9:113–23.Google Scholar
- Symington LS, Gautier J. Double-strand break end resection and repair pathway choice. Ann Rev Genet. 2011;45:247–71.PubMedView ArticleGoogle Scholar
- Myers S, Freeman C, Auton A, Donnelly P, McVean G. A common sequence motif associated with recombination hot spots and genome instability in humans. Nat Genet. 2008;40:1124–9.PubMedView ArticleGoogle Scholar
- Choi K, Henderson IR. Meiotic recombination hotspots – a comparative view. Plant J. 2015;83:52–61.PubMedView ArticleGoogle Scholar
- Ottaviani D, LeCain M, Sheer D. The role of microhomology in genomic structural variation. Trends Genet. 2014;30:85–94.PubMedView ArticleGoogle Scholar
- Qi Z, Redding S, Lee JY, Gibb B, Kwon YH, Niu H, et al. DNA sequence alignment by microhomology sampling during homologous recombination. Cell. 2015;160:856–69.PubMedPubMed CentralView ArticleGoogle Scholar
- Hastings PJ, Ira G, Lupski JR. A microhomology-mediated break-induced replication model for the origin of human copy number variation. PLoS Genet. 2009;5:e1000327.PubMedPubMed CentralView ArticleGoogle Scholar
- Kellogg EA. Evolutionary history of the grasses. Plant Physiol. 2001;125:1198–205.PubMedPubMed CentralView ArticleGoogle Scholar
- Gill BS, Friebe B. Cytogenetic analysis of wheat and rye genomes. In: Feuillet C, Muehlbauer GJ, editors. Genetics and genomics of the Triticeae, plant genetics and genomics: crops and models 7. LLC.: Springer Science + Business Media; 2009. p. 121–35.View ArticleGoogle Scholar
- Belostotsky DA, Ananiev EV. Characterization of relic DNA from barley genome. Theor Appl Genet. 1990;80:374–80.PubMedView ArticleGoogle Scholar
- Vershinin AV, Svitashev S, Gummesson P-O, Salomon B, von Bothmer R, Bryngelsson T. Characterization of a family of tandemly repeated DNA sequences in Triticeae. Theor Appl Genet. 1994;89:217–25.PubMedView ArticleGoogle Scholar
- Lukaszewski AJ, Rybka K, Korzun V, Malyshev SV, Lapinski B, Whitkus R. Genetic and physical mapping of homoeologous recombination points involving wheat chromosome 2B and rye chromosome 2R. Genome. 2004;47:36–45.PubMedView ArticleGoogle Scholar
- Dvorak J. Triticeae genome structure and evolution. In: Feuillet C, Muchlbauer GJ, editors. Genetics and genomics of the triticeae. Plant genetics and genomics: crops and models 7. LLC.: Springer Science + Business Media; 2009. p. 685–711.View ArticleGoogle Scholar
- Luo MC, Deal KR, Young ZL, Dvorak J. Comparative genetic maps reveal extreme crossover localization in the Aegilops speltoides chromosomes. Theor Appl Genet. 2005;111:1098–106.PubMedView ArticleGoogle Scholar
- Gonzalez-Garcia M, Gonzalez-Sanchez M, Puertas MJ. The high variability of subtelomeric heterochromatin and connections between nonhomologous chromosomes, suggest frequent ectopic recombination in rye meiocytes. Cytogenet Genome Res. 2006;115:179–85.PubMedView ArticleGoogle Scholar
- Driscoll CS, Sears ER. Individual addition of the chromosomes of Imperial rye to wheat. Agron Abstr. 1971: 6.Google Scholar
- Schwarzacher T, Heslop-Harrison JS. Practical in situ Hybridization. Oxford: BIOS; 2000.Google Scholar
- Cheung WY, Gale MD. The isolation of high molecular weight DNA from wheat, barley and rye for analysis by pulse-field gel electrophoresis. Plant Mol Biol. 1990;14:881–8.PubMedView ArticleGoogle Scholar
- Church GM, Gilbert W. Genomic sequencing. Proc Natl Acad Sci U S A. 1984;81:1991–5.PubMedPubMed CentralView ArticleGoogle Scholar
- Song J, Dong F, Lilly JW, Stupar RM, Jiang J. Instability of bacterial artificial chromosome (BAC) clones containing tandemly repeated DNA sequences. Genome. 2001;44:463–9.PubMedView ArticleGoogle Scholar
- Sambrook J, Fritsch EF, Maniatis T. Molecular cloning: a laboratory manual. 2nd ed. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 1989.Google Scholar
- Schmieder R, Lim YW, Rohwer F, Edwards R. TagCleaner: identification and removal of tag sequences from genomic and metagenomic datasets. BMC Bioinformatics. 2010;11:341.PubMedPubMed CentralView ArticleGoogle Scholar
- Ewing B, Green P. Base-calling of automated sequencer traces using phred. II. Error probabilities. Genome Res. 1998;8:186–94.PubMedView ArticleGoogle Scholar
- Camacho C, Coulouris G, Avagyan V, Ma N, Papadopoulos J, Bealer K, et al. BLAST+: architecture and applications. BMC Bioinformatics. 2009;10:421.Google Scholar
- Kurtz S, Phillippy A, Delcher AL, Smoot M, Shumway M, Antonescu C, et al. Versatile and open software for comparing large genomes. Genome Biol. 2004;5:R12.Google Scholar
- Liu K, Warnow TJ, Holder MT, Nelesen SM, Yu J, Stamatakis AP, et al. SATe-II: very fast and accurate simultaneous estimation of multiple sequence alignments and phylogenetic trees. Syst Biol. 2012;61:90–106.Google Scholar
- Freudenstein JV, Davis JI. Branch support via resampling: an empirical study. Cladistics. 2010;26:643–56.View ArticleGoogle Scholar
- Stamatakis A. RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models. Bioinformatics. 2006;22:2688–90.PubMedView ArticleGoogle Scholar
- Huson DH, Scornavacca C. Dendroscope 3: an interactive tool for rooted phylogenetic trees and networks. Syst Biol. 2012;61:1061–7.PubMedView ArticleGoogle Scholar
- Rice P, Longden I, Bleasby A. EMBOSS: the European molecular biology open software suite. Trends Genet. 2000;16:276–7.PubMedView ArticleGoogle Scholar
- Pearson WR, Lipman DJ. Improved tools for biological sequence comparison. Proc Natl Acad Sci U S A. 1988;85:2444–8.PubMedPubMed CentralView ArticleGoogle Scholar
- Levitsky VG, Ignatieva EV, Ananko EA, Turnaev II, Merkulova TI, Kolchanov NA, et al. Effective transcription factor binding site prediction using a combination of optimization, a genetic algorithm and discriminant analysis to capture distant interactions. BMC Bioinformatics. 2007;8:481.Google Scholar
- Touzet H, Varre J-S. Efficient and accurate P-value computation for Position Weight Matrices. Algorithms Mol Biol. 2007;2:15.PubMedPubMed CentralView ArticleGoogle Scholar
- Sokal RR, Rohlf FJ. Biometry: the principles and practice of statistics in biological research. New York: W.H.Freeman and Co.; 2012.Google Scholar