BACKGROUND
retrotransposons correspond to class i transposable elements, inserting into a host genome through an rna intermediate  <cit> .
based on structural features and phylogenetic relationships, five orders of retrotransposons were defined  <cit> .
ltr retrotransposons , an order related to retroviruses, usually encode two open reading frames : one called gag, which encodes a structural protein for virus-like particles, and another called pol, which encodes enzymatic domains involved in the transposition cycle, such as an aspartic protease, a reverse transcriptase, an rnaseh and an integrase  <cit> .
the two major superfamilies of plant ltr retrotransposons are copia and gypsy, in which pol genes differ in their domain order  <cit> .
in most angiosperm genomes, the ltr-rtes are the most significant contributor to genome size, contributing over 70 % of the nuclear dna in grasses like maize  <cit> .
most ltr retrotransposons families exist in low copy numbers, but the amplification of few individual families contribute with large differences in genome size among closely related species  <cit> .
despite their stringent regulation, ltr-rtes are transcriptionally active in plants .
although epigenetic regulation is an important feature of most plant transposable elements [reviewed in  <cit> , cis-regulation has a crucial role regulating ltr-rte transcription, since ltrs represent promoter sequences.
the modulation of ltr-rtes transcriptional levels has been observed in different tissues, organs and development stages i.e.
<cit> .
the eucalypts are the most widely planted hardwoods in the world due to their ability to adapt, grow and provide quality wood for multiple applications  <cit> .
species of subgenus symphyomyrtus account for > 95 % of the world's planted eucalypts  <cit> .
these include three members of section latoangulatae: e. grandis, e. urophylla and e. saligna, broadly planted in tropical areas due to their fast growth and disease resistance; and e. tereticornis , known for their drought tolerance and rapid growth  <cit> .
other species are better known for their potential for introgressing new traits in breeding, i.e., e. brassiana  <cit> .
the eucalyptus grandis genome assembly into  <dig> pseudochromosomes ), is composed by  <dig>  % of retrotransposons, and ltr-rtes are the most representative, constituting  <dig>  % of the e. grandis genome  <cit> .
up to now, te dynamics were scarcely studied in the eucalyptus genus and most analysis were based on private est data [i.e.
<cit> .
in the present study, we identify and comprehensively characterize a selected group of eucalyptus ltr-rtes, emphasizing the characterization of elements with putative transcriptional activity.
we analyzed the phylogenetic pattern of nine e. grandis ltr-rtes families and we extended this analysis, understanding dna interaction properties of selected encoding domains.
comparative classifications of ltr-rtes from closely related species were performed on monocots in which transposable elements  had previously been well characterized  <cit> , however this is the first time this approach was employed in forest trees.
our study is the first to exhaustively sample and classify transcriptionally active tes in eucalyptus species, identifying their structure, genomic distribution, insertion time estimation, genomic polymorphism and transcriptional activity in five eucalyptus species and one intrageneric hybrid.
methods
eucalyptus grandis transcriptionally active ltr-rtes: selection and annotation
ltr-rtes of copia and gypsy superfamilies described in wicker and keller  <cit>  and lloréns et al.
<cit>  were used as queries in a blastx against eucalyptus spp ests from the dbest database at the national center for biotechnology information , website on 10/10/ <dig>  we selected ests that aligned over 200 bp or more  for further analyses, similar to rossi et al.
<cit> .
in order to confirm whether the est codes for a ltr-rte, we analyzed sequences using censor implemented in repbase  <cit>  and the ones where the ltr-rte sequence matched more than 80 % of an est were selected for other analyses.
these selected ests were used as queries in a blastn search to identify full-length ltr-rtes in e. grandis genome v.  <dig>  .
the first  <dig> regions  with over 85 % identity in a region over 250 bp were selected for full-length ltr-rte screening.
regions 20 kb up- and downstream from these hits were analyzed using the ltr-finder  <cit>  and ltr_struc  <cit> .
only full-length ltr-rtes that aligned with ests in blastn were retrieved for further analyses, and redundant sequences were discarded.
these reference sequences were deposited at genbank under accessions km <dig> to km <dig>  target site duplications  were identified by submitting the full-length sequences as a query and subject to a blast2seq on ncbi website.
putative orfs were retrieved using fgenesh + tool  <cit>  on softberry platform  and manually inspected.
conserved domains were annotated using pfam .
in the case of the two rlc_egangela families, 5’ non coding leader sequences were compared using lalign .
rna minimum free energy secondary structure of aligned regions  was performed using rnafold .
phylogenetic analysis and rte family name assignment
all sequences from two previous large-scale analyses of plant ltr-rtes  <cit>  were used to develop phylogenetic trees based on a reverse transcriptase fragment.
we used a total of  <dig> copia  <cit>  and  <dig> gypsy  <cit>  families to classify the nine eucalyptus ltr-rte families.
dna sequences were aligned using muscle  <cit>  with default parameters and the phylogenetic trees were made using mega  <dig>   <cit> , applying the maximum likelihood method, with  <dig>  bootstrap replicates.
we used the kimura-2-parameter substitution model and gap positions were excluded when present in more than 5 % of the sequences.
eucalyptus ltr-rtes were assigned to families within lineages on the basis of 80 % sequence identity in at least 80 % of their ltrs, based on the universal classification of tes  <cit> .
we standardized the name of eucalyptus ltr-rte sequences similar to domingues et al.
<cit>  rationale: they were named 'rlc'  or 'rlg' , 'eg' for 'eucalyptus grandis', the lineage name  and the family number .
theoretical protein modeling and molecular dynamics simulations
we selected amino acid sequence of rlg_egtekay_ <dig> chromodomain, integrase and reverse transcriptase, as well as the integrase of rlc_egale_ <dig> and reverse transcriptase of rlc_egbianca_ <dig> by manual selected translation of nucleotide sequences.
the sequences were submitted to hhpred server  <cit> , and further to muscle  <cit>  and muster   <cit> .
the best alignments were used to generate models with modeller v. <dig>   <cit> .
the models were calculated with rampage  <cit>  and prosa-web  <cit>  based on structural comparison with template.
dna/rna molecules were added to the integrases and reverse transcriptases theoretical models by superimposition of dna binding region.
the best  <dig> models were submitted to molecular dynamics  simulations using groningen machine for chemical simulation  v. <dig> .
<dig>   <cit> .
the charmm force field  <cit>  was chosen with explicit solvent  <cit>  and a minimum of 50 ns unrestrained simulation.
theoretical models stability was evaluated by average root mean square deviation /time graph and by overall stereochemical and energy quality.
figure and surface illustrations were generated in chimera  <cit> , with the electrostatic surface generated by apbs  <cit> , pdb2pqr server  and propka  <cit> .
copy number determination in e. grandis genome
we used megablast to extract the full-length copies of the nine families from the e. grandis genome v  <dig>   <cit> .
all matches that were at least 80 % of the length of the reference full-length sequences and had a similarity level higher than 80 % were considered for copy number analysis.
copies that did not harbor the canonical 5'tg..ca3' were manually removed.
complete copies were plotted in e. grandis genome using circos  <cit> .
estimation ltr-rte insertion time and average ltr divergence sequence
the insertion time of intact ltr-rte families with two complete ltr sequences and tsds was calculated based on the assumption that they are identical at the time of integration  <cit> .
for each element, we aligned 5’ and 3’ ltrs using the muscle program implemented in mega  <dig>   <cit> , with default parameters.
divergence between ltrs  was calculated using mega  <dig> , using kimura-2-parameter distance  <cit> .
the insertion time  for each intact element was calculated with the formula: t = k/2r.
the value of  <dig>  x 10- <dig> substitution per site per year   <cit>  was recently used for the calculation of ltr-rtes age in grape  <cit> .
average divergence  of ltrs for each ltr-rte family was also calculated, using dnasp program  <cit> .
in silico transcriptional analysis: eucalyptus spp est screening
ltr-rtes full-length sequences were used as blastn queries against eucalyptus ests from eucanext database  <cit> , http://bioinfo <dig> ibi.unicamp.br/eucalyptusdb/).
ests similar to ltr-rtes were assigned to a family according to the criteria adapted from wicker et al.
<cit> : 80 % coverage with 80 % nucleotide identity, in a region over 200 bp.
plant material and nucleic acid extraction
for irap  and remap  analyses, leaves were collected from  <dig> unrelated individuals of five eucalyptus species: eucalyptus brassiana s.t.
blake, eucalyptus grandis w. hill ex maid., eucalyptus saligna sm., eucalyptus tereticornis sm.
and eucalyptus urophylla s.t.
blake.
these individuals were maintained in field by suzano papel and cellulose breeding program.
total dna was extracted from fresh young leaves using the protocol described in ferreira and grattapaglia  <cit> .
for all other analyses, eucalyptus seedlings were grown under naturally fluctuating conditions of temperature and air relative humidity, and were fertilized and irrigated as necessary in a greenhouse from the suzano papel and cellulose breeding program, in itapetininga, são paulo, brazil.
throughout the experiment, the plants were randomized periodically to minimize any variation within light environment.
all plants were harvested 90 days after seed planting.
in the relative quantification of ltr-rte families, we used the total dna of e. grandis  and e. urophylla .
in this case, genomic dna was obtained from young leaves using the dneasy plant kit , as recommended by the manufacturer.
for rna extraction we used freshly harvested leaves, stalk and secondary roots from the five eucalyptus species mentioned above and one hybrid e. grandis x e. urophylla .
for each tissue, total rna was extracted from two groups composed by five plants each according to modificated ctab protocol proposed by korimbocus et al.
<cit> .
rna integrity was checked by electrophoresis, in denaturing agarose gel.
rna quality and quantification was analyzed by spectrophotometry at 260 nm and 280 nm  and stored at -80 °c until used.
irap and remap profile and data analysis
sixteen single irap primers were designed based on nine ltr-rte families of e. grandis genome v.  <dig>  .
the procedures of pcr amplification were adapted from the protocol of smýkal  <cit> .
reactions were done in a total volume of 10 μl, containing 25 ng of genomic dna,  <dig>  x pcr buffer 2so4), 25 mm mgcl <dig>  40 ng of primer,  <dig>  mm of each dntp and  <dig>  u of taq dna polymerase .
the amplification profile consisted of an initial denaturation at 94 °c for 4 min, followed by 35 cycles at 94 °c for 40 s, 50 °c for 2 min, 72 °c for 3 min and 50 s and a final extension of 5 min at 72 °c.
for remap, we used the combination of sixteen irap primers with  <dig> ssr primers described in kalendar et al.
<cit>  .
pcr reactions contained 40 ng of genomic dna,  <dig>  x pcr buffer 2so4), 25 mm mgcl <dig>  10 ng of each primer,  <dig>  mm of each dntp, and  <dig>  u of taq dna polymerase .
the amplification profile composed of an initial denaturation at 94 °c for 4 min, followed by 35 cycles at 94 °c for 1 min, 56 °c for 1 min, 72 °c for 1 min and 50 s, and a final extension of 5 min at 72 °c.
all irap and remap reactions were carried out in a ptc- <dig> thermocycler  and pcr products were resolved in  <dig>  % agarose gels stained with ethidium bromide.
each irap and remap band was treated as a single locus.
the presence or absence of a given fragment length in each sample was recorded manually in binary code.
dna marker data was processed by ntsys-pc version  <dig>   <cit>  and using the simqual module with the jaccard genetic similarity coefficient , and the similarity data was used to perform an unweighted pair group method with arithmetic mean  cluster analysis using the shan module, following fan et al.
<cit> .
ltr-rte quantification in eucalyptus urophylla: quantitative real time pcr
eight ltr-rte families from e. grandis were quantified in e. urophylla using quantitative real time pcr  using the method of kraitshtein et al.
<cit>  and baruch and kashkush  <cit> , which is based on a comparative 2−ΔΔct method, using a single-copy gene as a reference.
our reference gene was dur <dig>  a urea transporter, which is a single-copy gene in several eukaryotes  <cit> , and in e. grandis genome we found only a single copy in chromosome  <dig> .
primers were designed using perlprimer v <dig> .
<dig> software  in ltr and internal regions .
in order to confirm target specificity we cloned and sequenced amplified regions .
quantitative pcr melt curves also revealed single and unique peaks for each primer pair, confirming high specificity to the target sequence fragments.
pcr efficiencies of the target and reference genes were determined by generating standard curves, based on serial dilutions prepared from cloned dna templates.
we made serial dilutions of the control dna from 5x10- <dig> to 5x10-6 ng/μl, with  <dig>  ng of each primer.
fold amplification in each cycle was calculated according to pcr efficiency, which was deduced by the software from the slope of the regression line  according to the equation e =  ×  <dig>  for primers with 100 % efficiency, the fold equals  <dig>
qpcr reactions were conducted in a step one plus real time pcr system  and analyzed in step one  <dig>  software .
each qpcr reaction was performed in 5 μl of gotaq® qpcr master mix , with  <dig>  ng of each primer and  <dig>  μl of ultra-pure water.
the cycling conditions were as follows: 5 min at 95 °c, followed by 45 cycles each of 15 s at 95 °c, 60 s at 60 °c.
in order to confirm the reproducibility of our results, reactions were done in technical triplicates in three independent experiments using  <dig> ,  <dig>  and  <dig>  ng of genomic dna.
the relative quantities of ltrs and internal regions of copia and gypsy-like ltr-rtes families were calculated according to kraitshtein et al.
<cit>  and baruch and kashkush  <cit> .
ltr-rte transcriptional analysis by rt-qpcr
in addition to plant material mentioned above, in rt-qpcr analysis, we also evaluated the transcriptional impact of osmotic shock in secondary roots of e. grandis submerged in hydroponic solution with peg.
plants were harvested  <dig>   <dig> and 24 h after osmotic stress.
additional details of this experiment are detailed in rodrigues et al.
<cit> .
all rna samples were treated with dnase i  and reverse transcribed using goscript™ reverse transcription system kit .
rt-qpcr reactions were performed in technical triplicates from at least two biological replicates in a steponeplus real time pcr system .
primers used for rt-qpcr are described in additional file 1: table s <dig>  and fragments were cloned and sequenced confirming target amplification.
each biological replicate was represented by bulks of leaves, stalk and secondary roots from five plants.
reactions contained 5 μl of  <dig> x mix gotaq® master mix ,  <dig>  ng of each primer,  <dig> to 280 ng of cdna and ultra-pure water up to a final volume of 10 μl.
the cycling conditions were as follows: 5 min at 95 °c, followed by 45 cycles each of 15 s at 95 °c, 60 s at 60 °c.
relative expression levels were normalized using the gapdh gene  <cit> .
the gapdh stability was evaluated using genorm and normfinder.
expression stability was < <dig>  in genorm and < <dig>  in normfinder for all species and tissues .
these values were compatible with the most stable normalizers from a previous publication in eucalyptus  <cit> .
in genorm, stability values below  <dig>  do not require an additional gene as a reference  <cit> .
the tissue/organ with lowest expression  was used as calibrator .
rt-qpcr efficiency was calculated using linreg v.  <dig>   <cit> , and reactions with efficiency >90 % were used for analysis.
relative expression was calculated using ΔΔct method  <cit>  with the formula ΔΔct, where e represents the efficiency.
the statistical analysis was performed using assistat  <dig>  beta  <cit> .
we used one-way analysis of variance  and in cases where significant differences were found, the least square deviation  method for multiple comparisons was performed.
results were considered significant at p <  <dig> .
RESULTS
ltr-retrotransposons in eucalyptus: overall view and phylogenetic structure
by using a homology-based approach, we mined a total of nine transcriptionally active ltr-rte families in the e. grandis genome.
seven families belong to the copia superfamily and two correspond to ltr-rte elements from the gypsy superfamily.
copia superfamily ltr-rtes were classified into five major plant evolutionary lineages  <cit>   and two gypsy lineages  <cit>  .
we did not found any copia element from gmr and tar lineages.
three gypsy lineages - crm, reina and athila - did not harboured any transcriptionally active ltr-rte in our analysis.fig.
<dig> domains in eucalyptus ltr-rtes.
a
copia superfamily; b
gypsy superfamily.
abbreviations and domain color coding: ltr = long terminal repeat ; pbs = protein binding site and ppt = poly-purine tract ; gag ; rnaseh = ribonuclease h ; integrase = integrase
the reference full-length sequences of these families  have between  <dig> and 99 % identity among 5’and 3’ltrs, and for all reference sequences we annotated at least two internal coding domains among gag, reverse transcriptase, integrase and rnaseh .
rlc_egangela_ <dig> and rlc_egangela_ <dig> families have > 95 % identity between internal coding domains; however, ltrs have < 80 % similarity; consequently, we preferred to classify these elements as separate families.
we also compared 5’ non coding region in order to confirm if they correspond to distinct families, similar to tanskanen et al.
<cit> .
the folding pattern of 5’ leader region was completely distinct between the two families , confirming that they represent two different families.table  <dig> overall features of ltr-rtes identified in e. grandis genome
copia
ale
ale
maximus
bianca
angela
angela
ivana
gypsy
tat
tekay
aid: identity among 5’ and 3’ltrs
fig.
<dig> classification of eucalyptus grandis ltr-rtes copia and gypsy superfamily sequences into nine new families.
phylogenetic analyses used  <dig> copia sequences from wicker et al.
<cit>  and  <dig> gypsy sequences from du et al.
<cit>  a evolutionary lineages of seven elements from copia superfamily in eucalyptus
b evolutionary lineages of two eucalyptus gypsy ltr-rtes
overall size followed previous large-scale reports for ltr-rtes, as well for pbs and ppt sequences .
both gypsy families have >  <dig> kbp and the average size of copia is around  <dig> kpb .
ltrs from gypsy elements are also larger: only rlc_egmax_ <dig> has ltrs longer than 500 bp .
five families contained a spacer region between internal coding domain and ltrs .
structural characterization and molecular dynamics simulations of ltr retrotransposon domains
by threading modeling, we generated the first theoretical models of ltr-rte proteins in plants.
we used three domains of rlg_egtekay_1: chromodomain  , integrase   and reverse transcriptase  .
we also generated domains theoretical models on the domains integrase of rlc_egale_ <dig>   and reverse transcriptase of rlc_egbianca_ <dig>  .
the template used to generate cd <dig> was the nmr structure of a chromodomain  from schizosaccharomyces pombe  <cit> .
the template used to generate int <dig> was the crystallographic structure of an inhibited retroviral integrase  from human spumaretrovirus  <cit>  and m-phase phosphoprotein  <dig>  from homo sapiens  <cit> .
the template used to generate rt <dig> was the crystallographic structure of a reverse transcriptase/ribonuclease h p <dig> complexed with rna/dna hybrid  from schizosaccharomyces pombe  <cit> .
the template used to generate int <dig> was the crystallographic structure of pfv integrase  from human spumaretrovirus  <cit> .
the template used to generate rt <dig> was the crystallographic structure of ty <dig> reverse transcriptase complexed to rna/dna hybrid  from saccharomyces cerevisiae  <cit> .
the cd <dig>  int <dig>  rt <dig>  int <dig> and rt <dig> had original models with  <dig>  %,  <dig>  %,  <dig>  %,  <dig>  % and  <dig>  % of residues in favored and allowed region which were improved to 100 %,  <dig>  %,  <dig>  %  <dig>  % and  <dig>  %, respectively, after md dynamics.
these models started with a z-score of - <dig> , - <dig> , - <dig> , - <dig>  and - <dig>  which was either improved or maintained to - <dig> , - <dig> , - <dig> , - <dig>  and - <dig> .
all of the models were submitted to 50 ns and had its stabilization within the first 30 ns with a rmsd of  <dig>  to  <dig>  nm.
thus, the generated models were stabilized by long md simulations and validated by different approaches.fig.
<dig> three dimension theoretical models of rlg_egtekay_1: integrase  , reverse transcriptase   domains complexed with dna and chromodomain .
in  <dig>  protein cartoon representation.
in  <dig> and  <dig>  electrostatic surface in red  acid regions and in blue  basic regions rotated in 180°
fig.
<dig> three dimension theoretical models of integrase  of rlc_egale_ <dig>  and reverse transcriptase   of rlc_egbianca_ <dig> proteins complexed with dna.
in  <dig>  protein cartoon representation.
in  <dig> and  <dig>  electrostatic surface in red  acid regions and in blue  blue regions rotated in 180°
fig.
<dig> estimative insertion time of ltr-rte families in eucalyptus grandis genome
quantification of ltr-rtes
a total of  <dig> full-length elements from copia superfamily and  <dig> from gypsy superfamily were identified, and copy number per family ranged from  <dig> to  <dig> .
the distribution of each family is distinct among e. grandis chromosomes when compared to gene and repeats density .
rlc_egmax_ <dig> is the most ubiquitous, in accordance with the prevalence of maximus ltr-rte families in plants.
this family is dispersed along the chromosome arms in both gene-rich and repetitive-rich regions  without any specific preference.
full-length rlc_egmax_ <dig> copies correspond to ~1 % of e. grandis genome.
rlc_egbianca_ <dig> and rlc_egangela_ <dig> families presented a similar distribution, preferentially inserted in repeat-rich regions .
rlc_egangela_ <dig> have only one copy in chromosome  <dig>  also located in a repeat-rich region .
both ltr-rtes from gypsy superfamily also have an insertional preference over repeat-rich regions .
on the other hand, rlc_egivana_ <dig> was preferentially inserted in gene-rich regions of chromosomes  <dig>   <dig>   <dig> and  <dig> .
additionally, we also quantified ltrs and internal regions of families by qpcr in e. urophylla using e. grandis as a calibrator.
e. urophylla is among the most commonly used species in the paper industry in brazil and belongs to the same subgenus of e. grandis  <cit> .
in silico quantification and qpcr had similar results, except for rlc_egale_ <dig> which qpcr analyses showed more copies than in silico analyses.
the proportion of ltrs for four retrotransposons families, rlc_egale_ <dig>  rlc_egmax_ <dig>  rlc_egivana_ <dig> and rlg_tat_ <dig>  is higher in e. urophylla than e. grandis .
this pattern is different in internal regions, where only two, rlc_egangela_ <dig> and rlc_egtekay_ <dig>  have a higher copy number in e. urophylla, suggesting a diversification in ltr regions.
rlc_egangela_ <dig> and rlc_egangela_ <dig> families display the most prominent difference in coding regions, increasing significantly in e. urophylla compared to e. grandis .fig.
<dig> copy number of copia and gypsy ltr-rte families e. grandis and e. urophylla using qpcr.
a rq of ltrs; b rq of internal regions
insertion time of intact elements
we estimated the insertion time of all ltr-rte families that harbor at least five intact copies.
the  <dig> rlc_egale_ <dig> copies were inserted into the genome < <dig>  mya, whereas most rlc_egmax_ <dig> copies  were inserted into the genome between  <dig> –  <dig>  mya .
rlc_egbianca_ <dig> and rlc_egangela_ <dig> copies have a similar pattern of insertion time where most of the copies were inserted into the genome  <dig>  –  <dig>  mya.
approximately one third of the copies from rlc_egbianca_ <dig>  and rlc_egangela_ <dig>  insertion has > <dig>  mya, including two copies dated to > <dig>  mya in both families .
the majority of rlc_egivana_ <dig> copies were inserted in the genome between  <dig>  -  <dig>  mya .
the two ltr-rtes from gypsy superfamily have distinct patterns: while rlg_egtat_ <dig> have most of these copies inserted  <dig>  –  <dig>  mya , rlg_egtekay_ <dig> has a uniform distribution.
we found one copy with two identical ltrs, in rlc_egangela_ <dig>  five identical ltrs in rlc_egmax_ <dig>  the diversity  of ltrs ranged from  <dig>    to  <dig>     and both gypsy families have an average diversity of  <dig>  .
irap and remap polymorphisms within eucalyptus genus
fifty eucalyptus spp unrelated individuals were scored by irap and remap, yielding in total  <dig> fragments, among which  <dig> were polymorphic.
irap bands ranged from 250 bp to  <dig>  kb  and remap fragments ranged from 50 bp to 1 kb, except for primers from rlc_egale_ <dig> family .
fifteen single irap primers and  <dig> remap primer combinations yielded reliable results.
the number of scorable bands per primer in irap ranged from  <dig>  to  <dig>  .
for remap bands ranged from  <dig>   to  <dig>   .
the genetic relationships of these genotypes were unrevealed using the upgma method based on jaccard  computed with irap and remap markers .
e. brassiana remained an outgroup, and the most related species were e. tereticornis and e. urophylla.
e. grandis was the second most external species, after e. brassiana.fig.
<dig> upgma dendrogram of five eucalyptus species using irap and remap data based on jaccard distance
transcriptional activity of ltr retrotransposons families
in order to further characterize the transcriptional profile of ltr-rte families, we performed blast searches against est of six eucalyptus species available in the eucanext database  <cit> .
more than  <dig> ests matched the selected ltr-rte families, and results are shown in additional file 1: table s <dig>  rlc_egbianca_ <dig> and rlg_egtat_ <dig> were the most represented families .
rlc_egale_ <dig>  rlc_egale_ <dig>  and rlg_egtekay_ <dig> showed similarity with ests from three of six analyzed eucalyptus species , and one est of e. globulus displayed high similarity rlc_egivana_ <dig> .
rlc_egmax_ <dig> only had similarity with expressed sequences from e. globulus, .
a second approach, rt-qpcr of coding regions, was employed to detect transcriptional levels of ltr-rte families in three tissues  from five eucalyptus species  and one hybrid .
e. grandis secondary roots were also evaluated in osmotic stress imposed by peg treatment  <cit>  .fig.
<dig> transcriptional profile of eucalyptus ltr-rtes in three organs  from five eucalyptus species and one interspecific hybrid using rt-qpcr.
asterisk indicating standard error  in bars
this analysis expanded and detailed est analysis, confirming that families have differential transcriptional activity eucalyptus spp tissues.
rlc_egale_ <dig> highest transcriptional levels were in leaves from “e.
urograndis” and e. brassiana, with a remarkable level in stalks of e. tereticornis and e. grandis .
regarding the rlc_egale_ <dig> family, leaves from e. urophylla presented the highest transcriptional activity .
rlc_egmax_ <dig> family showed a high level of transcriptional activity in secondary roots from e. brassiana and e. grandis, and in “e.
urograndis” we observed a notable difference compared to parentals e. grandis and e. urophylla .
rlc_egbianca_ <dig> family also displayed a higher transcriptional level in secondary roots of “e.
urograndis”, and it was the most expressed family in stalks and roots of e. brassiana .
ltr-rtes of the angela lineage  have a remarkable level in stalks in three eucalyptus species and “e.
urograndis”, and a low transcriptional level in leaves of all eucalyptus species studied .
the rlc_egivana_ <dig> family had a ubiquitous expression level .
gypsy elements showed a distinct transcriptional level according to the family, tissue and eucalyptus species evaluated.
rlg_egtat_ <dig> showed a remarkable expression in leaves of two eucalyptus species , and in stalks from e. saligna and secondary roots from “e.
urograndis” .
on the other hand, rlg_egtekay _ <dig> family showed the highest transcriptional activity in stalks from e. tereticornis .
the transcriptional level of ltr-rtes in roots submitted to osmotic stress by peg treatment increased after 6 h in five families  and decreased after 24 h .
only in the rlc_egale_ <dig> family did we observe a higher expression after 24 h .
rlc_egale_ <dig> and rlc_egbianca_ <dig> families showed a reduction in transcription level following peg application .
discussion
novel ltr-rte families in eucalyptus genus have individual molecular patterns
this study represents a fine-scale analysis of transcriptionally active ltr-rtes in eucalyptus species, taking advantage of the recently released e. grandis genome and expanding in silico analyses to a comparative study in terms of copy number, polymorphism insertion and tissue-specific transcriptional pattern.
the majority of the identified ltr-rte families were from the copia superfamily, consistent with previous est analysis of ltr-rtes in sugarcane  <cit>  and coffee trees  <cit>  but in contrast to wheat  <cit>  and maize  <cit> .
these findings confirm that the superfamily transcriptional preference is variable among plant genomes.
most families from the same evolutionary lineage have similar size, ltr length and high similarity between ppt and pbs regions with previous multi-scale reports  <cit> .
three copia families displayed a spacer region between orf and ltrs: rlc_egmax_ <dig>  with a 1 kb spacer region at 3-utr.
the rlc_egangela_ <dig> and rlc_egangela_ <dig> has a spacer region of  <dig> pb and  <dig> pb at 5-utr, respectively.
despite the high identity between internal coding domains between angela families, their ltrs display differences in size and similarity; moreover, folding analyses of the 5’utr also support that they represent distinct families .
rlc_egangela_ <dig> has a larger spacer region between ltrs and this region has 99 % identity with an eucalyptus spp est , suggesting that spacer regions are also transcriptionally active.
rlc_egangela_ <dig> and rlc_egangela_ <dig> have strikingly high similarity in coding regions, but they habour dissimilar ltr and untranslated regions.
this feature is particularly common in copia superfamily,   <cit> .
we also discovered that one transcriptionally active ltr-rte family initially classified as a member of the gypsy superfamily are in fact caulimoviruses, with  <dig> complete copies in the e. grandis genome with est support .
previous studies in plants provided similar results  <cit> , and the identification of ests matching plant caulimoviruses suggest that these elements may have a significant impact in transcriptome of angiosperms.
gypsy families can be roughly divided according to the presence of a chromodomain in the c-terminal of the integrase domain.
rlg_egtekay_ <dig> family is a typical chromoviridae member, closely related to the sugarbeet element bongo <dig>  <cit> .
but, differing from bongo <dig>  a high-copy element spread throughout all sugarbeet chromosomes, only  <dig> e. grandis chromosomes have full-length rlg_egtekay_ <dig> copies.
eucalyptus ltr-rte proteins interact with dna and rna
up to now, few studies address tes molecular structure  and here we deliver the first study to theoretically model and stabilize with long md simulations domains of ltr-rte proteins in plants.
the generated models posses a wide distribution of unordered regions, above all the copia models   <cit> .
comparison of integrase models  clearly show variability between members , although superimposition of dna interacting region yields lower value .
the same is observed in reverse transcriptases  with lower values, which shows higher structural conservancy.
the high rmsd values are due to the flexibility between cores given by connecting loops and unordered regions, although the cores are similar explaining the conservancy of function.
the variance between models corroborates the differences observed in previous sequence analysis, since rlc_egale_ <dig> and rlc_egbianca_ <dig> are from different families and the rlg_egtekay_ <dig> from a different superfamily.
the electrostatic surface of integrase and reverse transcriptase show the complementarities region with rna/dna .
the chromodomain was not modeled with rna since its interaction region is not fully understood.
this model consists in its general structure, an n-terminal three-stranded anti-parallel β-sheet  packed against c-terminal α-helix  and it has mostly acid regions.
further studies should be performed to aid transposons domains structural comprehension, such as modeling the whole orfs from the ltr-rte.
most ltr-rte families are recently inserted in eucalyptusgrandis genome
our analysis highlights the fact that even in transcriptionally active ltr-rtes we could track contrasting insertion peaks.
we dated the most part of full-length ltr-rtes insertions dated < <dig> mya .
this characteristic is similar to other plants, such as rice  <cit> , vitis  <cit>  and tomato  <cit> .
the identification of insertions >3mya old is difficult in plant genomes  <cit> , because old ltr-rtes insertions could be removed by recombination processes, as well as mutational events on nucleotide composition.
six out of  <dig> analyzed families showed recently inserted copies , with rlc_egangela_ <dig> and rlc_egmax_ <dig> families having copies with estimated insertion of < <dig>  mya.
rlc_egmax_ <dig>  which represents ~1 % of e. grandis genome, has a peak of amplification between  <dig>  and  <dig>  mya.
most insertions over  <dig>  mya correspond to elements from  <dig> families: rlc_egbianca_ <dig> and rlc_egangela_ <dig>  suggesting that forces driving rlc_egangela_ <dig> amplification are evolutionarily old and still in action.
this is consistent with analyses in other plants genomes that insertion > <dig> mya, such as populus tricocarpa  <cit> , onion and asparagus  <cit> .
ltr-rtes in plants can be either localized in gene-rich regions  <cit>  or in repeat-rich regions  <cit> , which also represents the scenario of eucalyptus retrotransposons here characterized.
although chromosome  <dig>   <dig> and  <dig> concentrated most of annotated full-length copies, eucalyptus ltr-rte families had distinct copy distributions, both in genic and repetitive regions.
the most part of younger  ltr-rtes inserted in genic regions, similar to the gene-rich euchromatin distribution from salire family in beta vulgaris  <cit>  and ltr-rtes in tomato genome  <cit> .
few full length copies of rlc_egivana_ <dig> , rlc_egbianca_ <dig> , rlc_egangela_ <dig>  and rlc_egmax_ <dig>  are inserted near to telomeric regions, suggesting that these families are not directly related to telomeric repeats.
the quantification of ltr-rte families by qpcr has been performed in several species  <cit>  and we successfully used this strategy to compare amplification of copia and gypsy elements in e. grandis and e. urophylla.
qpcr quantification did not follow in silico quantification just for rlc_egale_ <dig> which qpcr analyses showed more copies than in silico analyses.
this fact may be due to the e. grandis genotype used in qpcr analyses  that is not the same from genome sequencing , and defective ltr-rtes may be overrepresented in e. grandis genotype here sampled.
this approach also helped us to track out the proportion of non-autonomous ltrs and internal regions.
e. grandis  and e. urophylla  genomes have similar size and diverged < <dig> mya  <cit>  enabling the comparison of ltr-rte families shared by both genomes.
rlc_egale_ <dig> have similar ltr and internal domain proportions for both genomes, suggesting that this family have not gone through an expansion burst since e. grandis and e. urophylla divergence.
rlc_egale_ <dig>  rlc_egmax_ <dig>  rlc_egivana_ <dig> and rlg_egtat_ <dig> families had an increase of ltrs compared to internal domains in e. urophylla, which may indicate the propagation of ltrs, including non-autonomous elements, in this genome and may also reflect a fast substitution processes among internal domains of these families.
rlc_egangela_ <dig> and rlg_egtekay_ <dig> had the opposite profile, with a higher proportion of internal domains.
this observation may indicate a higher divergence of ltrs that were not recognized by the primer combination used in our approach and conservation in coding domains, similar to the pattern of the angela family within the e. grandis genome.
three ltr-rtes families, rlc_egale_ <dig>  rlc_egmax_ <dig> and rlg_egtat_ <dig>  showed an approximate proportion of two ltrs to each element.
these families have most of their copies have young insertions, indicating that they probably did not harboured recombination processes.
the qpcr analyses from rlc_egale_ <dig>  rlc_egangela_ <dig> and rlc_egbianca_ <dig> families showed more copies of internal regions than ltrs, which suggest a loss of ltr and internal region conservation in e. grandis genomes.
irap and remap markers suggest distinct activities of ltr-rtes families in eucalyptus species
irap and remap markers may contribute to understanding the insertion activity of ltr-rte families in eucalyptus species.
most primer combinations were successfully applied in eucalyptus species, showing the ubiquity character of families among the eucalyptus genus.
e. grandis showed more fragments and polymorphic bands, indicating that ltr-rtes families had distinct activity after speciation events in this specie.
copia ltr-rtes studied in four species from vitis genus had a similar pattern, with polymorphic bands suggesting an amplification burst after speciation events  <cit> .
the average size of remap fragments was probably the result of proximity between ltr and ssr regions than ltr-rtes in tandem insertions.
the pattern of remap fragments per eucalyptus species reflect the preferential insertion events of ltr-rte families in ssr regions with repetition motifs  and , probably sampling ltr-rtes located in pericentromeric regions, which are gene-poor and enriched in repetitive sequences, especially retrotransposons  <cit> .
the high level of polymorphisms suggests that ltr-rtes are extensively heterogeneous among eucalyptus species, as observed in diospyros sp.
and medicago sativa  <cit> .
this is the first report using irap and remap markers for genetic diversity in eucalyptus, and genomic polymorphism suggests differential activity among rtes within subgenus symphyomyrtus.
those species occupy the same clade within subgenus symphyomyrtus  <cit>  but it is important to notice that the separation between those eucalyptus species was not completely supported by bootstrap analyses.
distribution of eucalyptus species in dendrogram has some differences comparing to molecular analyses based on dart markers  <cit> .
e. tereticornis and e. urophylla were the most close in rte-based tree differently from the close relation usually present between e. grandis and e. urophylla, also observed for e. brassiana and e. tereticornis using other nuclear markers.
on the other hand, our analysis shows a small distance between e. saligna and e. grandis, in agreement with a previous report using a nuclear gene  <cit> .
transcriptional activity of eucalyptus ltr-rte families is variable among organs and species
the annotation of ests related to ltr-rtes was an initial assessment of transcriptional activity of these elements in eucalyptus genomes.
rlc_egale_ <dig> was the family with the largest number of ests.
rlc_egbianca_ <dig> was the most ubiquitous element, with est in the six mined eucalyptus species.
detailed information of ests matching ltr-rtes is available in supporting information additional file 1: table s <dig>
rt-qpcr clearly demonstrates that families have differences of transcriptional activity among eucalyptus spp.
tissues and species .
ltr-rtes copy number have been suggested a cause to transcriptional increase, because more copies inserted in gene-rich euchromatin region  <cit>  and probably near genes.
nevertheless, our data showed that no relation between ltr-rtes copy number and expression level increase.
the transcriptional activity of families with the highest copy number, rlc_egmax_ <dig>  rlc_egbianca_ <dig> and rlc_egivana_ <dig>  was higher in secondary roots when compared to leaves.
on the other hand, families with lower genomic copies are highly expressed in stalks and lower expressed in roots, i.e, rlc_egale_ <dig>  rlc_egale_ <dig> and rlc_egangela_ <dig>
rlc_egale_ <dig> and rlc_egale_ <dig> families, despite their similar structure, have remarkable differences in their transcriptional patterns.
the most striking differences can be observed in e. brassiana, e. urophylla and “e.
urograndis” leaves, where each element displayed a species-specific transcriptional pattern.
ltr-rtes activation in hybrids was also described in other eudicots, like sunflower  <cit>  and tobacco  <cit> .the activation of ltr-rtes in hybrids may reflect two issues: an organ-specific deregulation of transcription factors that target ltrs in “urograndis” and/or a specific deregulation of silencing mechanisms regulating te transcription.
the expression levels from rlc_egale_ <dig> in roots were lower to other tissues in all eucalyptus species and “e.
urograndis”, rlc_egale_ <dig> has similar expression characteristic in stalks from eucalyptus species and “e.
urograndis”.
families from the same lineage in sugarcane had distinct transcriptional pattern in leaves and buds  <cit> .
high transcriptional levels in roots were described for several copia ltr-rtes families in different plant species, such as citrus  <cit>  and quercus suber  <cit> .
this is the first work that ltr-rtes were evaluated in roots submitted to peg osmotic stress.
e. grandis roots submitted to osmotic stress showed variable transcriptional activity.
three families with young insertion  and with more genomic copies, rlc_egivana_ <dig>  rlc_egale_ <dig> and rlc_egale_ <dig>  had transcriptional activity modification, except rlc_egmax_ <dig>
the transcriptional activity from rlc_egale_ <dig>  rlc_egangela_ <dig>  rlc_egivana_ <dig> e rlg_egtekay_ <dig> families had a peak after 6 h of osmotic stress by peg followed by a decrease in expression level in rlc_egivana_ <dig> and rlg_egtat_ <dig> families.
this observation suggests that both families have their transcription triggered by similar stress conditions, a common feature among tes  <cit> .
future functional studies validating the promoter specificity of these ltrs may shed a light on the stress activation of tes in eucalyptus.
CONCLUSIONS
this study demonstrated that each copia and gypsy families in eucalyptus  have a different amplification pattern.
particularly in e. grandis and e. urophylla, that have diverged from a common ancestor ~  <dig> mya ago, we observed lower copy number in most ltr-rtes at e. urophylla compared to e. grandis.
these differences warrant further investigation to determine if recombination, nucleotide divergence or a specific burst of amplification can explain this pattern.
despite conservation to ltr-rtes between species, irap and remap markers analyses based on transcriptionally active ltr-rtes suggest different level of transpositional activity within eucalyptus genus.
this hypothesis is reinforced taking account that transcriptional activity is not the same among eucalyptus species.
future studies can address if ltr-rtes are specifically modulated by other stresses beside osmotic shock.
another important issue is to address if eucalyptus ltr-rtes families characterized here are in expansion in eucalyptus genus, or even if they are conserved across other families rather than myrtaceae, which may indicate horizontal transfer and/or purifying selection.
availability of supporting data
the data sets supporting the results of this article are available in the dryad repository, doi: <dig> /dryad.h2t <dig>
additional files
additional file 1: tables s1-s <dig>
table s <dig>  primers sequences using different techniques from ltr-rtes families.
table s <dig>  pbs and ppt sequences from the ltr-rte families characterized in e. grandis genome.
table s <dig>  diversity  between ltrs of retrotransposon families in eucalyptus grandis genome.
we used data from intact complete copies.
table s <dig>  total fragments and polymorphic bands in irap primers of five eucalyptus species.
table s <dig>  total fragments and polymorphic bands in remap combinations of five eucalyptus species.
table s <dig>  expressed sequence tags  matched to complete ltr-rtes elements.
additional file 2: figures s1-s <dig>
figure s <dig>  rna folding of rlc_egangela_ <dig> and rlc_egangela_ <dig> 5’spacer region using rnafold  with minimum free energy .
the  rlc_egangela_ <dig> region 185-561;  rlc_egangela_ <dig> region 404- <dig>  figure s <dig>  distribution of full-length copia and gypsy elements across  <dig> chromosomes from e. grandis.
data was plotted using circus.
tracks:  gene density  ;  repetitive coverage  ;  rlc_egmax_ <dig> density ;  rlc_egbianca_ <dig> density ;  rlc_egangela_ <dig> density ;  rlc_egangela_ <dig> density ;  rlc_egivana_ <dig> density ;  rlc_egale_ <dig> density ;  rlc_egale_ <dig> density ;  rlg_egtat_ <dig> density ;  rlg_egtekay_ <dig> density .
figure s <dig>  irap analysis for a set of four eucalyptus species using primers egbiancaltr- <dig>  and egtatltr- <dig> .
l:  <dig> kb molecular weight marker: gene ruler dna ladder mix .
figure s <dig>  remap analysis for a set of three eucalyptus species with primer micro <dig> and egale2ltr- <dig>  or egtatltr- <dig> .
l: 1kb molecular weight marker: gene ruler dna ladder mix .
figure s <dig>  upgma dendrogram of individuals from five eucalyptus species using irap and remap data based on jaccard distance.
abbreviations
testransposable elements
ltr-rtesltr retrotransposons
orfsopen reading frames
mbmegabases
ncbinational center for biotechnology information
irapinter-retrotransposon amplified polymorphism
remapretrotransposon-microsatellite ampliflied polymorphism
tsdstarget site duplications
mustermulti-sources threader
mdmolecular dynamics
gromacsgroningen machine for chemical simulation
rmsdroot mean square deviation
upgmaunweighted pair group method with arithmetic mean
qpcrquantitative real time pcr
int1rlg_egtekay_ <dig> integrase
cd1rlg_egtekay_ <dig> chromodomain
rt1rlg_egtekay_ <dig> reverse transcriptase
int2rlc_egale_ <dig> integrase
rt2reverse transcriptase of rlc_egbianca_1
helena sanches marcon and douglas silva domingues contributed equally to this work.
competing interests
the authors declare that they have no competing interests.
authors’ contributions
the study was conceived by hsm and dsd, and co-ordinated by clm, who participated in its design and helped to draft the manuscript.
hsm and dsd carried out the molecular genetic and phylogenetic studies.
jcs, helped by hsm and dsd, did the bioinformatic analyses.
rjb, ffm and mrmf carried out modelling studies.
hsm, dsd and rjb wrote the manuscript.
all authors read and approved the final manuscript.
authors thank drs.
ivan de godoy maia and juliana pereira bravo by their assistance in transcriptional analyses and cession of plant material; to alan p. lorenzetti, by his help in ltr-rte distribution analysis, and to izabel gava and shinitiro oda, who kindly provided access to plant material at suzano papel e celulose.
this work was supported by a cnpq grant .
hsm was also supported by fellowships from capes and cnpq.
clm is a research fellow from cnpq.

 retrotransposons correspond to class i transposable elements, inserting into a host genome through an rna intermediate. based on structural features and phylogenetic relationships, five orders of retrotransposons were defined. ltr retrotransposons, an order related to retroviruses, usually encode two open reading frames: one called gag, which encodes a structural protein for virus-like particles, and another called pol, which encodes enzymatic domains involved in the transposition cycle, such as an aspartic protease, a reverse transcriptase, an rnaseh and an integrase. the two major superfamilies of plant ltr retrotransposons are copia and gypsy, in which pol genes differ in their domain order. in most angiosperm genomes, the ltr-rtes are the most significant contributor to genome size, contributing over 70 % of the nuclear dna in grasses like maize. most ltr retrotransposons families exist in low copy numbers, but the amplification of few individual families contribute with large differences in genome size among closely related species. despite their stringent regulation, ltr-rtes are transcriptionally active in plants. although epigenetic regulation is an important feature of most plant transposable elements [reviewed in, cis-regulation has a crucial role regulating ltr-rte transcription, since ltrs represent promoter sequences. the modulation of ltr-rtes transcriptional levels has been observed in different tissues, organs and development stages i.e.. the eucalypts are the most widely planted hardwoods in the world due to their ability to adapt, grow and provide quality wood for multiple applications. species of subgenus symphyomyrtus account for > 95 % of the world's planted eucalypts. these include three members of section latoangulatae: e. grandis, e. urophylla and e. saligna, broadly planted in tropical areas due to their fast growth and disease resistance; and e. tereticornis, known for their drought tolerance and rapid growth. other species are better known for their potential for introgressing new traits in breeding, i.e., e. brassiana. the eucalyptus grandis genome assembly into pseudochromosomes ), is composed by  % of retrotransposons, and ltr-rtes are the most representative, constituting  % of the e. grandis genome. up to now, te dynamics were scarcely studied in the eucalyptus genus and most analysis were based on private est data [i.e.. in the present study, we identify and comprehensively characterize a selected group of eucalyptus ltr-rtes, emphasizing the characterization of elements with putative transcriptional activity. we analyzed the phylogenetic pattern of nine e. grandis ltr-rtes families and we extended this analysis, understanding dna interaction properties of selected encoding domains. comparative classifications of ltr-rtes from closely related species were performed on monocots in which transposable elements had previously been well characterized, however this is the first time this approach was employed in forest trees. our study is the first to exhaustively sample and classify transcriptionally active tes in eucalyptus species, identifying their structure, genomic distribution, insertion time estimation, genomic polymorphism and transcriptional activity in five eucalyptus species and one intrageneric hybrid. methods eucalyptus grandis transcriptionally active ltr-rtes: selection and annotation ltr-rtes of copia and gypsy superfamilies described in wicker and keller and lloréns et al. were used as queries in a blastx against eucalyptus spp ests from the dbest database at the national center for biotechnology information, website on 10/10/ we selected ests that aligned over 200 bp or more for further analyses, similar to rossi et al.. in order to confirm whether the est codes for a ltr-rte, we analyzed sequences using censor implemented in repbase and the ones where the ltr-rte sequence matched more than 80 % of an est were selected for other analyses. these selected ests were used as queries in a blastn search to identify full-length ltr-rtes in e. grandis genome v.. the first regions with over 85 % identity in a region over 250 bp were selected for full-length ltr-rte screening. regions 20 kb up- and downstream from these hits were analyzed using the ltr-finder and ltr_struc. only full-length ltr-rtes that aligned with ests in blastn were retrieved for further analyses, and redundant sequences were discarded. these reference sequences were deposited at genbank under accessions km to km target site duplications were identified by submitting the full-length sequences as a query and subject to a blast2seq on ncbi website. putative orfs were retrieved using fgenesh + tool on softberry platform and manually inspected. conserved domains were annotated using pfam. in the case of the two rlc_egangela families, 5’ non coding leader sequences were compared using lalign. rna minimum free energy secondary structure of aligned regions was performed using rnafold. phylogenetic analysis and rte family name assignment all sequences from two previous large-scale analyses of plant ltr-rtes were used to develop phylogenetic trees based on a reverse transcriptase fragment. we used a total of copia and gypsy families to classify the nine eucalyptus ltr-rte families. dna sequences were aligned using muscle with default parameters and the phylogenetic trees were made using mega, applying the maximum likelihood method, with bootstrap replicates. we used the kimura-2-parameter substitution model and gap positions were excluded when present in more than 5 % of the sequences. eucalyptus ltr-rtes were assigned to families within lineages on the basis of 80 % sequence identity in at least 80 % of their ltrs, based on the universal classification of tes. we standardized the name of eucalyptus ltr-rte sequences similar to domingues et al. rationale: they were named'rlc' or'rlg','eg' for'eucalyptus grandis', the lineage name and the family number. theoretical protein modeling and molecular dynamics simulations we selected amino acid sequence of rlg_egtekay_ chromodomain, integrase and reverse transcriptase, as well as the integrase of rlc_egale_ and reverse transcriptase of rlc_egbianca_ by manual selected translation of nucleotide sequences. the sequences were submitted to hhpred server, and further to muscle and muster. the best alignments were used to generate models with modeller v.. the models were calculated with rampage and prosa-web based on structural comparison with template. dna/rna molecules were added to the integrases and reverse transcriptases theoretical models by superimposition of dna binding region. the best models were submitted to molecular dynamics simulations using groningen machine for chemical simulation v... the charmm force field was chosen with explicit solvent and a minimum of 50 ns unrestrained simulation. theoretical models stability was evaluated by average root mean square deviation /time graph and by overall stereochemical and energy quality. figure and surface illustrations were generated in chimera, with the electrostatic surface generated by apbs, pdb2pqr server and propka. copy number determination in e. grandis genome we used megablast to extract the full-length copies of the nine families from the e. grandis genome v. all matches that were at least 80 % of the length of the reference full-length sequences and had a similarity level higher than 80 % were considered for copy number analysis. copies that did not harbor the canonical 5'tg..ca3' were manually removed. complete copies were plotted in e. grandis genome using circos. estimation ltr-rte insertion time and average ltr divergence sequence the insertion time of intact ltr-rte families with two complete ltr sequences and tsds was calculated based on the assumption that they are identical at the time of integration. for each element, we aligned 5’ and 3’ ltrs using the muscle program implemented in mega, with default parameters. divergence between ltrs was calculated using mega, using kimura-2-parameter distance. the insertion time for each intact element was calculated with the formula: t = k/2r. the value of x 10- substitution per site per year was recently used for the calculation of ltr-rtes age in grape. average divergence of ltrs for each ltr-rte family was also calculated, using dnasp program. in silico transcriptional analysis: eucalyptus spp est screening ltr-rtes full-length sequences were used as blastn queries against eucalyptus ests from eucanext database, http://bioinfo ibi.unicamp.br/eucalyptusdb/). ests similar to ltr-rtes were assigned to a family according to the criteria adapted from wicker et al.: 80 % coverage with 80 % nucleotide identity, in a region over 200 bp. plant material and nucleic acid extraction for irap and remap analyses, leaves were collected from unrelated individuals of five eucalyptus species: eucalyptus brassiana s.t. blake, eucalyptus grandis w. hill ex maid., eucalyptus saligna sm., eucalyptus tereticornis sm. and eucalyptus urophylla s.t. blake. these individuals were maintained in field by suzano papel and cellulose breeding program. total dna was extracted from fresh young leaves using the protocol described in ferreira and grattapaglia. for all other analyses, eucalyptus seedlings were grown under naturally fluctuating conditions of temperature and air relative humidity, and were fertilized and irrigated as necessary in a greenhouse from the suzano papel and cellulose breeding program, in itapetininga, são paulo, brazil. throughout the experiment, the plants were randomized periodically to minimize any variation within light environment. all plants were harvested 90 days after seed planting. in the relative quantification of ltr-rte families, we used the total dna of e. grandis and e. urophylla. in this case, genomic dna was obtained from young leaves using the dneasy plant kit, as recommended by the manufacturer. for rna extraction we used freshly harvested leaves, stalk and secondary roots from the five eucalyptus species mentioned above and one hybrid e. grandis x e. urophylla. for each tissue, total rna was extracted from two groups composed by five plants each according to modificated ctab protocol proposed by korimbocus et al.. rna integrity was checked by electrophoresis, in denaturing agarose gel. rna quality and quantification was analyzed by spectrophotometry at 260 nm and 280 nm and stored at -80 °c until used. irap and remap profile and data analysis sixteen single irap primers were designed based on nine ltr-rte families of e. grandis genome v.. the procedures of pcr amplification were adapted from the protocol of smýkal. reactions were done in a total volume of 10 μl, containing 25 ng of genomic dna, x pcr buffer 2so4), 25 mm mgcl 40 ng of primer,  mm of each dntp and u of taq dna polymerase. the amplification profile consisted of an initial denaturation at 94 °c for 4 min, followed by 35 cycles at 94 °c for 40 s, 50 °c for 2 min, 72 °c for 3 min and 50 s and a final extension of 5 min at 72 °c. for remap, we used the combination of sixteen irap primers with ssr primers described in kalendar et al.. pcr reactions contained 40 ng of genomic dna, x pcr buffer 2so4), 25 mm mgcl 10 ng of each primer,  mm of each dntp, and u of taq dna polymerase. the amplification profile composed of an initial denaturation at 94 °c for 4 min, followed by 35 cycles at 94 °c for 1 min, 56 °c for 1 min, 72 °c for 1 min and 50 s, and a final extension of 5 min at 72 °c. all irap and remap reactions were carried out in a ptc- thermocycler and pcr products were resolved in  % agarose gels stained with ethidium bromide. each irap and remap band was treated as a single locus. the presence or absence of a given fragment length in each sample was recorded manually in binary code. dna marker data was processed by ntsys-pc version and using the simqual module with the jaccard genetic similarity coefficient, and the similarity data was used to perform an unweighted pair group method with arithmetic mean cluster analysis using the shan module, following fan et al.. ltr-rte quantification in eucalyptus urophylla: quantitative real time pcr eight ltr-rte families from e. grandis were quantified in e. urophylla using quantitative real time pcr using the method of kraitshtein et al. and baruch and kashkush, which is based on a comparative 2−ΔΔct method, using a single-copy gene as a reference. our reference gene was dur a urea transporter, which is a single-copy gene in several eukaryotes, and in e. grandis genome we found only a single copy in chromosome. primers were designed using perlprimer v. software in ltr and internal regions. in order to confirm target specificity we cloned and sequenced amplified regions. quantitative pcr melt curves also revealed single and unique peaks for each primer pair, confirming high specificity to the target sequence fragments. pcr efficiencies of the target and reference genes were determined by generating standard curves, based on serial dilutions prepared from cloned dna templates. we made serial dilutions of the control dna from 5x10- to 5x10-6 ng/μl, with  ng of each primer. fold amplification in each cycle was calculated according to pcr efficiency, which was deduced by the software from the slope of the regression line according to the equation e =  ×  for primers with 100 % efficiency, the fold equals qpcr reactions were conducted in a step one plus real time pcr system and analyzed in step one software. each qpcr reaction was performed in 5 μl of gotaq® qpcr master mix, with  ng of each primer and  μl of ultra-pure water. the cycling conditions were as follows: 5 min at 95 °c, followed by 45 cycles each of 15 s at 95 °c, 60 s at 60 °c. in order to confirm the reproducibility of our results, reactions were done in technical triplicates in three independent experiments using, and  ng of genomic dna. the relative quantities of ltrs and internal regions of copia and gypsy-like ltr-rtes families were calculated according to kraitshtein et al. and baruch and kashkush. ltr-rte transcriptional analysis by rt-qpcr in addition to plant material mentioned above, in rt-qpcr analysis, we also evaluated the transcriptional impact of osmotic shock in secondary roots of e. grandis submerged in hydroponic solution with peg. plants were harvested and 24 h after osmotic stress. additional details of this experiment are detailed in rodrigues et al.. all rna samples were treated with dnase i and reverse transcribed using goscript™ reverse transcription system kit. rt-qpcr reactions were performed in technical triplicates from at least two biological replicates in a steponeplus real time pcr system. primers used for rt-qpcr are described in additional file 1: table s and fragments were cloned and sequenced confirming target amplification. each biological replicate was represented by bulks of leaves, stalk and secondary roots from five plants. reactions contained 5 μl of x mix gotaq® master mix,  ng of each primer, to 280 ng of cdna and ultra-pure water up to a final volume of 10 μl. the cycling conditions were as follows: 5 min at 95 °c, followed by 45 cycles each of 15 s at 95 °c, 60 s at 60 °c. relative expression levels were normalized using the gapdh gene. the gapdh stability was evaluated using genorm and normfinder. expression stability was < in genorm and < in normfinder for all species and tissues. these values were compatible with the most stable normalizers from a previous publication in eucalyptus. in genorm, stability values below do not require an additional gene as a reference. the tissue/organ with lowest expression was used as calibrator. rt-qpcr efficiency was calculated using linreg v., and reactions with efficiency >90 % were used for analysis. relative expression was calculated using ΔΔct method with the formula ΔΔct, where e represents the efficiency. the statistical analysis was performed using assistat beta. we used one-way analysis of variance and in cases where significant differences were found, the least square deviation method for multiple comparisons was performed. results were considered significant at p < .  ltr-retrotransposons in eucalyptus: overall view and phylogenetic structure by using a homology-based approach, we mined a total of nine transcriptionally active ltr-rte families in the e. grandis genome. seven families belong to the copia superfamily and two correspond to ltr-rte elements from the gypsy superfamily. copia superfamily ltr-rtes were classified into five major plant evolutionary lineages and two gypsy lineages. we did not found any copia element from gmr and tar lineages. three gypsy lineages - crm, reina and athila - did not harboured any transcriptionally active ltr-rte in our analysis.fig. domains in eucalyptus ltr-rtes. a copia superfamily; b gypsy superfamily. abbreviations and domain color coding: ltr = long terminal repeat; pbs = protein binding site and ppt = poly-purine tract; gag; rnaseh = ribonuclease h; integrase = integrase the reference full-length sequences of these families have between and 99 % identity among 5’and 3’ltrs, and for all reference sequences we annotated at least two internal coding domains among gag, reverse transcriptase, integrase and rnaseh. rlc_egangela_ and rlc_egangela_ families have > 95 % identity between internal coding domains; however, ltrs have < 80 % similarity; consequently, we preferred to classify these elements as separate families. we also compared 5’ non coding region in order to confirm if they correspond to distinct families, similar to tanskanen et al.. the folding pattern of 5’ leader region was completely distinct between the two families, confirming that they represent two different families.table overall features of ltr-rtes identified in e. grandis genome copia ale ale maximus bianca angela angela ivana gypsy tat tekay aid: identity among 5’ and 3’ltrs fig. classification of eucalyptus grandis ltr-rtes copia and gypsy superfamily sequences into nine new families. phylogenetic analyses used copia sequences from wicker et al. and gypsy sequences from du et al. a evolutionary lineages of seven elements from copia superfamily in eucalyptus b evolutionary lineages of two eucalyptus gypsy ltr-rtes overall size followed previous large-scale reports for ltr-rtes, as well for pbs and ppt sequences. both gypsy families have >  kbp and the average size of copia is around kpb. ltrs from gypsy elements are also larger: only rlc_egmax_ has ltrs longer than 500 bp. five families contained a spacer region between internal coding domain and ltrs. structural characterization and molecular dynamics simulations of ltr retrotransposon domains by threading modeling, we generated the first theoretical models of ltr-rte proteins in plants. we used three domains of rlg_egtekay_1: chromodomain, integrase and reverse transcriptase. we also generated domains theoretical models on the domains integrase of rlc_egale_ and reverse transcriptase of rlc_egbianca_. the template used to generate cd was the nmr structure of a chromodomain from schizosaccharomyces pombe. the template used to generate int was the crystallographic structure of an inhibited retroviral integrase from human spumaretrovirus and m-phase phosphoprotein from homo sapiens. the template used to generate rt was the crystallographic structure of a reverse transcriptase/ribonuclease h p complexed with rna/dna hybrid from schizosaccharomyces pombe. the template used to generate int was the crystallographic structure of pfv integrase from human spumaretrovirus. the template used to generate rt was the crystallographic structure of ty reverse transcriptase complexed to rna/dna hybrid from saccharomyces cerevisiae. the cd int rt int and rt had original models with  %,  %,  %,  % and  % of residues in favored and allowed region which were improved to 100 %,  %,  %  % and  %, respectively, after md dynamics. these models started with a z-score of -, -, -, - and - which was either improved or maintained to -, -, -, - and -. all of the models were submitted to 50 ns and had its stabilization within the first 30 ns with a rmsd of to  nm. thus, the generated models were stabilized by long md simulations and validated by different approaches.fig. three dimension theoretical models of rlg_egtekay_1: integrase, reverse transcriptase domains complexed with dna and chromodomain. in protein cartoon representation. in and electrostatic surface in red acid regions and in blue basic regions rotated in 180° fig. three dimension theoretical models of integrase of rlc_egale_ and reverse transcriptase of rlc_egbianca_ proteins complexed with dna. in protein cartoon representation. in and electrostatic surface in red acid regions and in blue blue regions rotated in 180° fig. estimative insertion time of ltr-rte families in eucalyptus grandis genome quantification of ltr-rtes a total of full-length elements from copia superfamily and from gypsy superfamily were identified, and copy number per family ranged from to. the distribution of each family is distinct among e. grandis chromosomes when compared to gene and repeats density. rlc_egmax_ is the most ubiquitous, in accordance with the prevalence of maximus ltr-rte families in plants. this family is dispersed along the chromosome arms in both gene-rich and repetitive-rich regions without any specific preference. full-length rlc_egmax_ copies correspond to ~1 % of e. grandis genome. rlc_egbianca_ and rlc_egangela_ families presented a similar distribution, preferentially inserted in repeat-rich regions. rlc_egangela_ have only one copy in chromosome also located in a repeat-rich region. both ltr-rtes from gypsy superfamily also have an insertional preference over repeat-rich regions. on the other hand, rlc_egivana_ was preferentially inserted in gene-rich regions of chromosomes and. additionally, we also quantified ltrs and internal regions of families by qpcr in e. urophylla using e. grandis as a calibrator. e. urophylla is among the most commonly used species in the paper industry in brazil and belongs to the same subgenus of e. grandis. in silico quantification and qpcr had similar results, except for rlc_egale_ which qpcr analyses showed more copies than in silico analyses. the proportion of ltrs for four retrotransposons families, rlc_egale_ rlc_egmax_ rlc_egivana_ and rlg_tat_ is higher in e. urophylla than e. grandis. this pattern is different in internal regions, where only two, rlc_egangela_ and rlc_egtekay_ have a higher copy number in e. urophylla, suggesting a diversification in ltr regions. rlc_egangela_ and rlc_egangela_ families display the most prominent difference in coding regions, increasing significantly in e. urophylla compared to e. grandis.fig. copy number of copia and gypsy ltr-rte families e. grandis and e. urophylla using qpcr. a rq of ltrs; b rq of internal regions insertion time of intact elements we estimated the insertion time of all ltr-rte families that harbor at least five intact copies. the rlc_egale_ copies were inserted into the genome < mya, whereas most rlc_egmax_ copies were inserted into the genome between – mya. rlc_egbianca_ and rlc_egangela_ copies have a similar pattern of insertion time where most of the copies were inserted into the genome – mya. approximately one third of the copies from rlc_egbianca_ and rlc_egangela_ insertion has > mya, including two copies dated to > mya in both families. the majority of rlc_egivana_ copies were inserted in the genome between - mya. the two ltr-rtes from gypsy superfamily have distinct patterns: while rlg_egtat_ have most of these copies inserted – mya, rlg_egtekay_ has a uniform distribution. we found one copy with two identical ltrs, in rlc_egangela_ five identical ltrs in rlc_egmax_ the diversity of ltrs ranged from to and both gypsy families have an average diversity of. irap and remap polymorphisms within eucalyptus genus fifty eucalyptus spp unrelated individuals were scored by irap and remap, yielding in total fragments, among which were polymorphic. irap bands ranged from 250 bp to  kb and remap fragments ranged from 50 bp to 1 kb, except for primers from rlc_egale_ family. fifteen single irap primers and remap primer combinations yielded reliable results. the number of scorable bands per primer in irap ranged from to. for remap bands ranged from to. the genetic relationships of these genotypes were unrevealed using the upgma method based on jaccard computed with irap and remap markers. e. brassiana remained an outgroup, and the most related species were e. tereticornis and e. urophylla. e. grandis was the second most external species, after e. brassiana.fig. upgma dendrogram of five eucalyptus species using irap and remap data based on jaccard distance transcriptional activity of ltr retrotransposons families in order to further characterize the transcriptional profile of ltr-rte families, we performed blast searches against est of six eucalyptus species available in the eucanext database. more than ests matched the selected ltr-rte families, and results are shown in additional file 1: table s rlc_egbianca_ and rlg_egtat_ were the most represented families. rlc_egale_ rlc_egale_ and rlg_egtekay_ showed similarity with ests from three of six analyzed eucalyptus species, and one est of e. globulus displayed high similarity rlc_egivana_. rlc_egmax_ only had similarity with expressed sequences from e. globulus,. a second approach, rt-qpcr of coding regions, was employed to detect transcriptional levels of ltr-rte families in three tissues from five eucalyptus species and one hybrid. e. grandis secondary roots were also evaluated in osmotic stress imposed by peg treatment.fig. transcriptional profile of eucalyptus ltr-rtes in three organs from five eucalyptus species and one interspecific hybrid using rt-qpcr. asterisk indicating standard error in bars this analysis expanded and detailed est analysis, confirming that families have differential transcriptional activity eucalyptus spp tissues. rlc_egale_ highest transcriptional levels were in leaves from “e. urograndis” and e. brassiana, with a remarkable level in stalks of e. tereticornis and e. grandis. regarding the rlc_egale_ family, leaves from e. urophylla presented the highest transcriptional activity. rlc_egmax_ family showed a high level of transcriptional activity in secondary roots from e. brassiana and e. grandis, and in “e. urograndis” we observed a notable difference compared to parentals e. grandis and e. urophylla. rlc_egbianca_ family also displayed a higher transcriptional level in secondary roots of “e. urograndis”, and it was the most expressed family in stalks and roots of e. brassiana. ltr-rtes of the angela lineage have a remarkable level in stalks in three eucalyptus species and “e. urograndis”, and a low transcriptional level in leaves of all eucalyptus species studied. the rlc_egivana_ family had a ubiquitous expression level. gypsy elements showed a distinct transcriptional level according to the family, tissue and eucalyptus species evaluated. rlg_egtat_ showed a remarkable expression in leaves of two eucalyptus species, and in stalks from e. saligna and secondary roots from “e. urograndis”. on the other hand, rlg_egtekay _ family showed the highest transcriptional activity in stalks from e. tereticornis. the transcriptional level of ltr-rtes in roots submitted to osmotic stress by peg treatment increased after 6 h in five families and decreased after 24 h. only in the rlc_egale_ family did we observe a higher expression after 24 h. rlc_egale_ and rlc_egbianca_ families showed a reduction in transcription level following peg application. discussion novel ltr-rte families in eucalyptus genus have individual molecular patterns this study represents a fine-scale analysis of transcriptionally active ltr-rtes in eucalyptus species, taking advantage of the recently released e. grandis genome and expanding in silico analyses to a comparative study in terms of copy number, polymorphism insertion and tissue-specific transcriptional pattern. the majority of the identified ltr-rte families were from the copia superfamily, consistent with previous est analysis of ltr-rtes in sugarcane and coffee trees but in contrast to wheat and maize. these findings confirm that the superfamily transcriptional preference is variable among plant genomes. most families from the same evolutionary lineage have similar size, ltr length and high similarity between ppt and pbs regions with previous multi-scale reports. three copia families displayed a spacer region between orf and ltrs: rlc_egmax_ with a 1 kb spacer region at 3-utr. the rlc_egangela_ and rlc_egangela_ has a spacer region of pb and pb at 5-utr, respectively. despite the high identity between internal coding domains between angela families, their ltrs display differences in size and similarity; moreover, folding analyses of the 5’utr also support that they represent distinct families. rlc_egangela_ has a larger spacer region between ltrs and this region has 99 % identity with an eucalyptus spp est, suggesting that spacer regions are also transcriptionally active. rlc_egangela_ and rlc_egangela_ have strikingly high similarity in coding regions, but they habour dissimilar ltr and untranslated regions. this feature is particularly common in copia superfamily,. we also discovered that one transcriptionally active ltr-rte family initially classified as a member of the gypsy superfamily are in fact caulimoviruses, with complete copies in the e. grandis genome with est support. previous studies in plants provided similar results, and the identification of ests matching plant caulimoviruses suggest that these elements may have a significant impact in transcriptome of angiosperms. gypsy families can be roughly divided according to the presence of a chromodomain in the c-terminal of the integrase domain. rlg_egtekay_ family is a typical chromoviridae member, closely related to the sugarbeet element bongo. but, differing from bongo a high-copy element spread throughout all sugarbeet chromosomes, only e. grandis chromosomes have full-length rlg_egtekay_ copies. eucalyptus ltr-rte proteins interact with dna and rna up to now, few studies address tes molecular structure and here we deliver the first study to theoretically model and stabilize with long md simulations domains of ltr-rte proteins in plants. the generated models posses a wide distribution of unordered regions, above all the copia models. comparison of integrase models clearly show variability between members, although superimposition of dna interacting region yields lower value. the same is observed in reverse transcriptases with lower values, which shows higher structural conservancy. the high rmsd values are due to the flexibility between cores given by connecting loops and unordered regions, although the cores are similar explaining the conservancy of function. the variance between models corroborates the differences observed in previous sequence analysis, since rlc_egale_ and rlc_egbianca_ are from different families and the rlg_egtekay_ from a different superfamily. the electrostatic surface of integrase and reverse transcriptase show the complementarities region with rna/dna. the chromodomain was not modeled with rna since its interaction region is not fully understood. this model consists in its general structure, an n-terminal three-stranded anti-parallel β-sheet packed against c-terminal α-helix and it has mostly acid regions. further studies should be performed to aid transposons domains structural comprehension, such as modeling the whole orfs from the ltr-rte. most ltr-rte families are recently inserted in eucalyptusgrandis genome our analysis highlights the fact that even in transcriptionally active ltr-rtes we could track contrasting insertion peaks. we dated the most part of full-length ltr-rtes insertions dated < mya. this characteristic is similar to other plants, such as rice, vitis and tomato. the identification of insertions >3mya old is difficult in plant genomes, because old ltr-rtes insertions could be removed by recombination processes, as well as mutational events on nucleotide composition. six out of analyzed families showed recently inserted copies, with rlc_egangela_ and rlc_egmax_ families having copies with estimated insertion of < mya. rlc_egmax_ which represents ~1 % of e. grandis genome, has a peak of amplification between and mya. most insertions over mya correspond to elements from families: rlc_egbianca_ and rlc_egangela_ suggesting that forces driving rlc_egangela_ amplification are evolutionarily old and still in action. this is consistent with analyses in other plants genomes that insertion > mya, such as populus tricocarpa, onion and asparagus. ltr-rtes in plants can be either localized in gene-rich regions or in repeat-rich regions, which also represents the scenario of eucalyptus retrotransposons here characterized. although chromosome and concentrated most of annotated full-length copies, eucalyptus ltr-rte families had distinct copy distributions, both in genic and repetitive regions. the most part of younger ltr-rtes inserted in genic regions, similar to the gene-rich euchromatin distribution from salire family in beta vulgaris and ltr-rtes in tomato genome. few full length copies of rlc_egivana_, rlc_egbianca_, rlc_egangela_ and rlc_egmax_ are inserted near to telomeric regions, suggesting that these families are not directly related to telomeric repeats. the quantification of ltr-rte families by qpcr has been performed in several species and we successfully used this strategy to compare amplification of copia and gypsy elements in e. grandis and e. urophylla. qpcr quantification did not follow in silico quantification just for rlc_egale_ which qpcr analyses showed more copies than in silico analyses. this fact may be due to the e. grandis genotype used in qpcr analyses that is not the same from genome sequencing, and defective ltr-rtes may be overrepresented in e. grandis genotype here sampled. this approach also helped us to track out the proportion of non-autonomous ltrs and internal regions. e. grandis and e. urophylla genomes have similar size and diverged < mya enabling the comparison of ltr-rte families shared by both genomes. rlc_egale_ have similar ltr and internal domain proportions for both genomes, suggesting that this family have not gone through an expansion burst since e. grandis and e. urophylla divergence. rlc_egale_ rlc_egmax_ rlc_egivana_ and rlg_egtat_ families had an increase of ltrs compared to internal domains in e. urophylla, which may indicate the propagation of ltrs, including non-autonomous elements, in this genome and may also reflect a fast substitution processes among internal domains of these families. rlc_egangela_ and rlg_egtekay_ had the opposite profile, with a higher proportion of internal domains. this observation may indicate a higher divergence of ltrs that were not recognized by the primer combination used in our approach and conservation in coding domains, similar to the pattern of the angela family within the e. grandis genome. three ltr-rtes families, rlc_egale_ rlc_egmax_ and rlg_egtat_ showed an approximate proportion of two ltrs to each element. these families have most of their copies have young insertions, indicating that they probably did not harboured recombination processes. the qpcr analyses from rlc_egale_ rlc_egangela_ and rlc_egbianca_ families showed more copies of internal regions than ltrs, which suggest a loss of ltr and internal region conservation in e. grandis genomes. irap and remap markers suggest distinct activities of ltr-rtes families in eucalyptus species irap and remap markers may contribute to understanding the insertion activity of ltr-rte families in eucalyptus species. most primer combinations were successfully applied in eucalyptus species, showing the ubiquity character of families among the eucalyptus genus. e. grandis showed more fragments and polymorphic bands, indicating that ltr-rtes families had distinct activity after speciation events in this specie. copia ltr-rtes studied in four species from vitis genus had a similar pattern, with polymorphic bands suggesting an amplification burst after speciation events. the average size of remap fragments was probably the result of proximity between ltr and ssr regions than ltr-rtes in tandem insertions. the pattern of remap fragments per eucalyptus species reflect the preferential insertion events of ltr-rte families in ssr regions with repetition motifs and, probably sampling ltr-rtes located in pericentromeric regions, which are gene-poor and enriched in repetitive sequences, especially retrotransposons. the high level of polymorphisms suggests that ltr-rtes are extensively heterogeneous among eucalyptus species, as observed in diospyros sp. and medicago sativa. this is the first report using irap and remap markers for genetic diversity in eucalyptus, and genomic polymorphism suggests differential activity among rtes within subgenus symphyomyrtus. those species occupy the same clade within subgenus symphyomyrtus but it is important to notice that the separation between those eucalyptus species was not completely supported by bootstrap analyses. distribution of eucalyptus species in dendrogram has some differences comparing to molecular analyses based on dart markers. e. tereticornis and e. urophylla were the most close in rte-based tree differently from the close relation usually present between e. grandis and e. urophylla, also observed for e. brassiana and e. tereticornis using other nuclear markers. on the other hand, our analysis shows a small distance between e. saligna and e. grandis, in agreement with a previous report using a nuclear gene. transcriptional activity of eucalyptus ltr-rte families is variable among organs and species the annotation of ests related to ltr-rtes was an initial assessment of transcriptional activity of these elements in eucalyptus genomes. rlc_egale_ was the family with the largest number of ests. rlc_egbianca_ was the most ubiquitous element, with est in the six mined eucalyptus species. detailed information of ests matching ltr-rtes is available in supporting information additional file 1: table s rt-qpcr clearly demonstrates that families have differences of transcriptional activity among eucalyptus spp. tissues and species. ltr-rtes copy number have been suggested a cause to transcriptional increase, because more copies inserted in gene-rich euchromatin region and probably near genes. nevertheless, our data showed that no relation between ltr-rtes copy number and expression level increase. the transcriptional activity of families with the highest copy number, rlc_egmax_ rlc_egbianca_ and rlc_egivana_ was higher in secondary roots when compared to leaves. on the other hand, families with lower genomic copies are highly expressed in stalks and lower expressed in roots, i.e, rlc_egale_ rlc_egale_ and rlc_egangela_ rlc_egale_ and rlc_egale_ families, despite their similar structure, have remarkable differences in their transcriptional patterns. the most striking differences can be observed in e. brassiana, e. urophylla and “e. urograndis” leaves, where each element displayed a species-specific transcriptional pattern. ltr-rtes activation in hybrids was also described in other eudicots, like sunflower and tobacco.the activation of ltr-rtes in hybrids may reflect two issues: an organ-specific deregulation of transcription factors that target ltrs in “urograndis” and/or a specific deregulation of silencing mechanisms regulating te transcription. the expression levels from rlc_egale_ in roots were lower to other tissues in all eucalyptus species and “e. urograndis”, rlc_egale_ has similar expression characteristic in stalks from eucalyptus species and “e. urograndis”. families from the same lineage in sugarcane had distinct transcriptional pattern in leaves and buds. high transcriptional levels in roots were described for several copia ltr-rtes families in different plant species, such as citrus and quercus suber. this is the first work that ltr-rtes were evaluated in roots submitted to peg osmotic stress. e. grandis roots submitted to osmotic stress showed variable transcriptional activity. three families with young insertion and with more genomic copies, rlc_egivana_ rlc_egale_ and rlc_egale_ had transcriptional activity modification, except rlc_egmax_ the transcriptional activity from rlc_egale_ rlc_egangela_ rlc_egivana_ e rlg_egtekay_ families had a peak after 6 h of osmotic stress by peg followed by a decrease in expression level in rlc_egivana_ and rlg_egtat_ families. this observation suggests that both families have their transcription triggered by similar stress conditions, a common feature among tes. future functional studies validating the promoter specificity of these ltrs may shed a light on the stress activation of tes in eucalyptus.  this study demonstrated that each copia and gypsy families in eucalyptus have a different amplification pattern. particularly in e. grandis and e. urophylla, that have diverged from a common ancestor ~  mya ago, we observed lower copy number in most ltr-rtes at e. urophylla compared to e. grandis. these differences warrant further investigation to determine if recombination, nucleotide divergence or a specific burst of amplification can explain this pattern. despite conservation to ltr-rtes between species, irap and remap markers analyses based on transcriptionally active ltr-rtes suggest different level of transpositional activity within eucalyptus genus. this hypothesis is reinforced taking account that transcriptional activity is not the same among eucalyptus species. future studies can address if ltr-rtes are specifically modulated by other stresses beside osmotic shock. another important issue is to address if eucalyptus ltr-rtes families characterized here are in expansion in eucalyptus genus, or even if they are conserved across other families rather than myrtaceae, which may indicate horizontal transfer and/or purifying selection. availability of supporting data the data sets supporting the results of this article are available in the dryad repository, doi: /dryad.h2t additional files additional file 1: tables s1-s table s primers sequences using different techniques from ltr-rtes families. table s pbs and ppt sequences from the ltr-rte families characterized in e. grandis genome. table s diversity between ltrs of retrotransposon families in eucalyptus grandis genome. we used data from intact complete copies. table s total fragments and polymorphic bands in irap primers of five eucalyptus species. table s total fragments and polymorphic bands in remap combinations of five eucalyptus species. table s expressed sequence tags matched to complete ltr-rtes elements. additional file 2: figures s1-s figure s rna folding of rlc_egangela_ and rlc_egangela_ 5’spacer region using rnafold with minimum free energy. the rlc_egangela_ region 185-561; rlc_egangela_ region 404- figure s distribution of full-length copia and gypsy elements across chromosomes from e. grandis. data was plotted using circus. tracks: gene density; repetitive coverage; rlc_egmax_ density; rlc_egbianca_ density; rlc_egangela_ density; rlc_egangela_ density; rlc_egivana_ density; rlc_egale_ density; rlc_egale_ density; rlg_egtat_ density; rlg_egtekay_ density. figure s irap analysis for a set of four eucalyptus species using primers egbiancaltr- and egtatltr-. l: kb molecular weight marker: gene ruler dna ladder mix. figure s remap analysis for a set of three eucalyptus species with primer micro and egale2ltr- or egtatltr-. l: 1kb molecular weight marker: gene ruler dna ladder mix. figure s upgma dendrogram of individuals from five eucalyptus species using irap and remap data based on jaccard distance. abbreviations testransposable elements ltr-rtesltr retrotransposons orfsopen reading frames mbmegabases ncbinational center for biotechnology information irapinter-retrotransposon amplified polymorphism remapretrotransposon-microsatellite ampliflied polymorphism tsdstarget site duplications mustermulti-sources threader mdmolecular dynamics gromacsgroningen machine for chemical simulation rmsdroot mean square deviation upgmaunweighted pair group method with arithmetic mean qpcrquantitative real time pcr int1rlg_egtekay_ integrase cd1rlg_egtekay_ chromodomain rt1rlg_egtekay_ reverse transcriptase int2rlc_egale_ integrase rt2reverse transcriptase of rlc_egbianca_1 helena sanches marcon and douglas silva domingues contributed equally to this work. competing interests the authors declare that they have no competing interests. authors’ contributions the study was conceived by hsm and dsd, and co-ordinated by clm, who participated in its design and helped to draft the manuscript. hsm and dsd carried out the molecular genetic and phylogenetic studies. jcs, helped by hsm and dsd, did the bioinformatic analyses. rjb, ffm and mrmf carried out modelling studies. hsm, dsd and rjb wrote the manuscript. all authors read and approved the final manuscript. authors thank drs. ivan de godoy maia and juliana pereira bravo by their assistance in transcriptional analyses and cession of plant material; to alan p. lorenzetti, by his help in ltr-rte distribution analysis, and to izabel gava and shinitiro oda, who kindly provided access to plant material at suzano papel e celulose. this work was supported by a cnpq grant. hsm was also supported by fellowships from capes and cnpq. clm is a research fellow from cnpq.
we also evaluated genomic polymorphism and transcriptional profile in three organs of five eucalyptus species. irap and remap markers suggested genomic polymorphisms among eucalyptus species. in eucalyptus genus, studies on genome composition and transposable elements are particularly scarce. using est analysis and qrt-pcrs, we observed transcriptional activity in several tissues and in all evaluated species. we characterized nine families of transcriptionally active ltr retrotransposons from copia and gypsy superfamilies in eucalyptus grandis genome and we depicted genomic distribution and copy number in two eucalyptus species. overall, our results show that retrotransposon activity have differentially affected genome and transcriptome among eucalyptus species. rlc_egmax_ was the most prevalent family and rlc_egangela_ was the family with the lowest copy number. nearly half of the recently released eucalyptus grandis genome is composed by retrotransposons and this data provides an important opportunity to understand te dynamics in eucalyptus genome and transcriptome. ltr retrotransposonsdynamicsgenomic distributioncomparative analysiseucalyptus genomesissue-copyright-statement© the author 2015
 in eucalyptus genus, studies on genome composition and transposable elements are particularly scarce. nearly half of the recently released eucalyptus grandis genome is composed by retrotransposons and this data provides an important opportunity to understand te dynamics in eucalyptus genome and transcriptome.  we characterized nine families of transcriptionally active ltr retrotransposons from copia and gypsy superfamilies in eucalyptus grandis genome and we depicted genomic distribution and copy number in two eucalyptus species. we also evaluated genomic polymorphism and transcriptional profile in three organs of five eucalyptus species. we observed contrasting genomic and transcriptional behavior in the same family among different species. rlc_egmax_ was the most prevalent family and rlc_egangela_ was the family with the lowest copy number. most families of both superfamilies have their insertions occurring < million years, except one copia family, rlc_egbianca_ protein theoretical models suggest different properties between copia and gypsy domains. irap and remap markers suggested genomic polymorphisms among eucalyptus species. using est analysis and qrt-pcrs, we observed transcriptional activity in several tissues and in all evaluated species. in some families, osmotic stress increases transcript values.  our strategy was successful in isolating transcriptionally active retrotransposons in eucalyptus, and each family has a particular genomic and transcriptional pattern. overall, our results show that retrotransposon activity have differentially affected genome and transcriptome among eucalyptus species. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords ltr retrotransposonsdynamicsgenomic distributioncomparative analysiseucalyptus genomesissue-copyright-statement© the author 2015 