BACKGROUND
plant genome evolution is closely associated with polyploidy and gene amplification, the most highly amplified genes being long terminal repeat retrotransposons   <cit> .
ltr-rt proliferation is regulated by the genome at both the transcriptional and post-transcriptional level  <cit> .
ltr-rts, like other transposable elements , can not only affect a genome by expansion by proliferation, but also by, for example, providing a template for recombination, inserting into coding regions and disrupting gene expression, or affecting the transcription of neighboring genes .
based on the massive amounts of genomic sequence data released in the last  <dig> years, phylogenetic analysis of plant ltr-rts has identified distinct evolutionary lineages within gypsy and copia, the two main plant ltr-rt superfamilies  <cit> .
these lineages are widespread in both monocot and eudicot genomes.
uncovering of this fine structuring within gypsy and copia plant ltr-rts has lead to a better understanding of the diversification of ltr-rts and shed light on their structure and genomic distribution  <cit> .
sugarcane is an important crop worldwide, being a major source of sugar, and is also increasingly being used for the production of renewable energy sources such as ethanol.
despite its economic importance, the sequencing of the sugarcane genome is at the pilot stage  <cit> .
modern cultivars are highly polyploid and have one of the largest  and most complex crop genomes, and chromosome numbers varying between 100- <dig>  <cit> .
there are a few reports on transposable elements in sugarcane sequences  <cit>  but there is no detailed study on sugarcane ltr-rts, their phylogenetic classification, and potential impact on genomic distribution and transcriptional activity.
in addition, the importance of accurate and complete te annotation is increasingly recognized as a priority in plant genome sequencing projects to minimize the inaccuracy of gene annotation and facilitate functional gene studies  <cit> .
to set the basis for future genome interpretation, ltr-rts in the sugarcane genome were characterized using available public resources and bac sequences from the bioen project  <cit> .
we examined the structure, genomic distribution, phylogenetic diversity, transcriptional activity and regulation of sugarcane ltr-rts.
this study reveals that within well-defined phylogenetic lineages, that while tes within ltr-rts families are structurally similar, they have distinct transcriptional and regulatory signatures.
taken together, these results support the growing evidence that ltr-rts contribute to genomic diversity, but with a wide range of potential outcomes.
RESULTS
characterization of ltr retrotransposons in sugarcane: phylogeny and structural features
plant ltr-rt evolutionary lineage names are not used consistently within the literature  <cit> , we therefore chose to include sequences from more than one source, so that we could directly compare our results with published data.
our inferred evolutionary histories suggest that, at least for the sequences we analyzed within gypsy superfamily, the del lineage is equivalent to tekay, and within the copia superfamily, the maximus lineage is equivalent to sire, ale to retrofit and ivana to oryco .
the relationship between the tar, angela, tork and bianca lineages is more problematic.
while the bianca lineage is not included in the gydb  <cit> tork appears to be the angela and tar lineages combined .
of the sixty full-length sequences extracted from sugarcane bac sequences, thirty-two sequences were assigned to the copia superfamily and twenty-eight to the gypsy superfamily .
we identified four of the six major plant evolutionary copia lineages described by wicker and keller  <cit>  in the sugarcane genome , and three of the six gypsy lineages described by du et al.
<cit>  and the gydb  <cit>  .
in terms of sequence numbers, the maximus lineage was the most highly represented within the copia superfamily, and the del and tat lineages were equally represented within the gypsy superfamily.
based on sequence identity within the ltrs, the elements were classified into  <dig> families   <cit> .
in order to simplify reading of the text we have referred to these families as simply 'ale1' for example, rather than the by the full name 'rlc_scale1'.
we were able to identify all the internal coding domains  in all the sugarcane sequences, apart from the gag domain in rlg_sctat_ <dig>  .
in addition, a putative envelope domain was identified in elements from the maximus <dig> family  and a chromodomain in all del and reina elements   <cit> .
there was a general pattern in overall size of elements from lineages in each of the two superfamilies.
in both superfamilies, there was one lineage that is very large , and at least one lineage that is comparatively smaller  .
within the copia superfamily, there is a lineage that is an in-between size , while within the gypsy superfamily, the sequences of the tat lineage  are more comparable in size to those of the del lineage  .
differences in total length were chiefly due to differences in ltr size, and the presence and size of spacer regions between the internal coding domain and the ltrs, rather than insertions within the gag/pol coding regions .
the exception is the tat elements, which contain insertions within the pol region, but in most cases between actual coding domains .
distribution of ltr retrotransposons in metaphases
given that transposable elements can be distributed throughout a genome, it is difficult to distinguish between random and real signals from a probe derived from a transposable element.
the probe for the del lineage was therefore prepared twice, one probe labeled with dig and the other with biotin, and hybridized separately to the same slide before and after stripping.
the distribution of the del probe was compared on the same  <dig> metaphases visualized with anti-digoxigenin-rhodamine  or with neutravidin-oregon green- <dig>  .
the distribution of the probe signal was similar in all  <dig> metaphases, and therefore the signal was considered to be valid.
similar in situ hybridization conditions were used for all subsequent experiments.
a bac clone , identified by our group  <cit> , contains known sugarcane centromeric repeats  <cit> .
this bac clone hybridized to the middle region of the chromosome, which consists mainly of centromeric specific sequences .
seven ltr-rt probes were used, three of which are from gypsy superfamily members and four from copia superfamily members, representing all the major lineages already described.
no convincing signal was obtained for the reina <dig>  ivana <dig> and angela <dig> probes, using the same conditions that were used for the other ltr-rt probes.
the probe from one of the two gypsy lineages, del <dig>  localized almost exclusively in a broad band around the centromeres.
this observation suggests that it is a heterochromatic or pericentromeric specific element .
the second gypsy element examined, tat <dig>  displays a generally strong broad dispersed pattern, but with some concentration along some chromosome arms .
two copia probes were found dispersed along the chromosome arms and none localized exclusively at or near the centre of the chromosomes.
the ale <dig> lineage probe was found in high concentrations along particular chromosome arms .
signals from the maximus lineage probe were generally dispersed .
transcriptional activity of sugarcane ltr retrotransposons
we associated  <dig> ests from the sugarcane cultivar sp80- <dig> related to full-length ltr retrotransposons.
the largest number of transcripts was identified from the root libraries , followed by the internode, lateral bud and calli libraries.
the copia superfamily was more highly represented in the est database than the gypsy superfamily, with  <dig> and  <dig> sequences, respectively .
previously described full length cdna sequences  <cit>  were assigned to  <dig> of the  <dig> families we identified from bac sequences .
in this study, no ests were identified for  <dig> families .
we assigned ests to another  <dig> families not previously described as transcriptionally active  <cit>  , however for one family, ale <dig>  for which a previously described cdna sequence was assigned, no ests were identified.
rt-pcr analysis confirmed the transcriptional activity of sugarcane ltr-rts in leaves and lateral buds.
we also experimentally confirmed transcriptional activity for five new families: ale <dig>  ivana <dig>  del <dig>  reina <dig> and reina <dig>
the most represented family in the est data, ale <dig>  had an intense band, confirming its transcriptional activity, and had the same intensity of signal in both leaves and lateral buds.
transcriptional patterns differed between tissues and ltr-rt families .
the ivana and reina lineages illustrate that individual families have distinct transcriptional patterns: for instance, the ivana <dig> signal is more intense than that of ivana1; moreover, ivana <dig> has higher transcriptional activity in lateral buds, while ivana <dig> is more active in leaves.
reina <dig> and reina <dig> are both transcriptionally active in lateral buds, but clear differences in band intensity can be observed .
distribution of small rnas among ltr retrotransposon lineages
we divided the srna population into two major classes: 20-22nt and 23-25nt srna sequences.
we refer hereafter to these classes as 21-nt class  and 24-nt class .
in this analysis we allowed a 0- <dig> nt mismatch between the srna and ltr-rt reference sequences, in which 25% and 22% of the  <dig> and  <dig> ntrnas class, respectively, showed a perfect match with the ltr-rts sequences.
all identified lineages had at least one family with srna from both classes mapped to ltr-rts.
in the copia superfamily, all families within the maximus lineage had more than  <dig> srnas mapped, mostly belonging to the 24-nt class .
these elements had the highest srna counts, about  <dig>  to  <dig>  fold higher than other lineages and these counts mapped chiefly to the ltrs .
del <dig> is the only representative of the gypsy superfamily that has comparable srna counts.
the ivana and reina lineages were the lineages with the least number of srna counts .
within the ivana lineage, the element rlc_scivana <dig>  had the highest number of srna matches, most of which belong to the 21-nt class .
the same pattern was observed in rlg_reina <dig> .
the remaining ivana and reina families had lower srna counts.
to evaluate whether srnas preferentially matched specific regions of each ltr-rt, we compared the srna distribution within ltrs and coding domains .
various distribution patterns, both within the ltrs and the coding domains were observed.
families of the maximus lineage and del <dig> had the high 24-nt srna counts within the ltrs.
the 5' region of both ltrs from del <dig> and del <dig> families had a peak of 21-nt srnas.
most ltr-rt families did not have high srna counts within the coding domains, but some cases of high counts of srnas were noted.
the ale <dig> family had >  <dig> counts of the 24-nt class srnas within the integrase and protease domains and the tat <dig> family had >  <dig> counts of the 24-nt class srnas within the rnaseh domain .
all other cases of high counts in coding domains are related to 21-nt class srnas, as seen in the ivana <dig> and reina <dig> families, both with around  <dig> counts.
interestingly, angela <dig> had peaks of >  <dig> 24-nt srna counts in a  <dig>  kb non-coding region between the 5'ltr and the gag domain .
discussion
we characterized ltr-rts in the sugarcane genome using bac sequences available from the bioen project and publicly available genomic and est resources, which allowed us to provide the most complete overview of the structural and phylogenetic diversity of these elements to date.
the  <dig> ltr-rt families identified belong to four lineages from the copia superfamily and to three from the gypsy superfamily.
among all these families we observed distinct patterns of differences in structural features, chromosomal distribution, transcriptional activity, and srnas targeting.
sugarcane ltr-rts are structurally diverse and belong to evolutionary lineages shared by monocots and eudicots
previous surveys of ltr-rts in plants defined six major common evolutionary copia lineages  <cit>  and six major common evolutionary gypsy lineages  <cit> .
bianca was the only copia superfamily lineage in which sugarcane ltr-rts were not found.
absence of bianca in the sugarcane genome dataset analyzed may be explained by its relatively low representation in other plants.
indeed, rice has few bianca families  <cit>  and soybean has none  <cit> .
we also did not find sugarcane ltr-rts elements from the galadriel and crm lineages in gypsy superfamily.
the galadriel lineage seems to be an under-represented lineage among monocots, since there are only reports of galadriel elements in banana  <cit> .
crm elements are also minor components of gypsy retrotransposons in the model grass rice  <cit> , which may explain its low representation in sugarcane.
although very little sequence of the sugarcane genome is available, the number of ltr-rt families we identified was comparable to previous large scale ltr-rts analyses in bac clones from medicago truncatula   <cit>  and capsicum annuum   <cit> .
this finding suggests that we have a good overview of the diversity of sugarcane ltr-rts.
sugarcane ltr-rt families have distinct chromosomal distributions
transposable elements are not distributed randomly along eukaryotic chromosomes  <cit> .
in particular, gypsy elements tend to be found in heterochromatic regions, copia elements are more dispersed throughout the genome  <cit> .
in addition, heterochromatic and pericentromeric regions are enriched in tes, such as those of the cr/crm and tat lineages, compared with euchromatic regions  <cit> .
using fluorescence in situ hybridization , we examined the distribution of representatives from seven lineages identified within the gypsy and copia superfamilies in sugarcane chromosomes.
no detectable fish signal was obtained for the angela, ivana and reina lineages; in the case of ivana preliminary estimates from the r <dig> bac library suggests that there is approximately  <dig> copies of ivana <dig> per haploid genome in sugarcane , which suggests that the lack of signal is due to low copy number.
we suspect that this observation may be also valid for angela and reina.
the two copia probes, ale <dig> and maximus <dig>  had the expected euchromatic patterns, but they were distinctly different.
the signals from the ale probe had localized clusters along particular chromosome arms, while those of the maximus <dig> probe were widespread and dispersed, without any signs of concentration in particularly regions .
it has been suggested that as tes accumulate in a genome, even if they insert randomly, they are more likely to insert within other tes, forming clusters of tes.
because these clusters of tes are most likely selectively neutral, they will be free to expand  <cit> .
the ale distribution pattern observed in sugarcane is reminiscent of such clusters of tes.
the sugarcane bac schrba 239_n <dig> used as a centromeric-specific probe in this study contains the scen repeat interspersed with ltr-rts from the tat lineage  <cit> .
previous studies in sugarcane  <cit>  identified a centromere-specific repeat  and centromere associated ltr-rts, classified as cr/crm-like.
we did not identify any full length ltr-rts from the cr/crm lineage in this study, but sequence from a related gypsy lineage, del <dig>  did hybridize to a broad region around and within the centromeric region .
del lineage sequences have been identified at other plant centromeres  <cit> , however our results suggest that the del sequence we used as a probe is not strictly centromeric specific, but is rather preferentially found in and around heterochromatic regions of the centromeric repeats.
the tat sequences identified in the bac schrba 239_n <dig> clustered with the tat <dig>  tat <dig>  tat <dig> and tat <dig> families,  which is quite distinct from the tat <dig> family .
the tat fish probe used is most closely related to the tat <dig> sequences  and had a clusters of localization along some chromosomes, like those seen with the ale <dig> probe, but with much more intense signals.
modern sugarcane cultivars are interspecific hybrids between saccharum officinarum and s. spontaneum and backcrossed with s. officinarum  <cit> .
the resulting genomes are highly polyploid, 70-80% of which is from s. officinarum, 10-23% from s. spontaneum, the rest being recombinants  <cit> .
the derivation of the modern sugarcane genome suggests that, rather than being the result of tes inserting into clusters of tes, the distribution of tat <dig> and ale <dig> may be the result of differential transmission of tes from the parental genomes.
future work using genomic in situ hybridization  <cit>  combined with fish should enable us to distinguish between these two hypotheses.
transcriptional activity of ltr-rts and their associated srnas
transposable elements make up a substantial proportion of plant genomes, and are often transcriptionally active.
a very stringent search for sugarcane ests associated with tes confirmed that transcriptional activity appears to be a general feature of some sugarcane ltr-rts, as reported for other monocots  <cit> .
since we do not have detailed information about cdna library preparation of est data, we cannot identify if transcribed sequences were in sense or antisense orientation.
interestingly, within each lineage, the number of transcripts mapped to each family was variable .
the most striking example is the ale lineage, which has the highest number of transcripts, all of which mapped to a single family, ale <dig>  there is a similar, but not as definitive pattern in all other lineages, except for maximus, where all families have similar numbers of transcripts.
in terms of tissue specificity , the highest number of ltr-rt associated transcripts was identified from the root libraries, followed by the internode, lateral bud and calli libraries.
a previous macroarray analysis identified calli as the tissue with the highest number of tes being expressed in sugarcane  <cit> , however in this study only calli, apical meristem, leaf roll and flower were analyzed.
these differences in expression according to tissue, in particular that leaf is the tissue with least number of ests identified, may help guide future studies of transposable elements in sugarcane.
as they are potentially highly mutagenic, the activity of ltr-rts is usually controlled by the host genome through the sirna machinery.
the specificity of this response is achieved by a surveillance system that detects aberrant rna.
the proliferative nature of tes makes them prone to insert in the genome in such way that both sense and anti-sense transcripts are produced, generating dsrna, and activating the sirna system.
two main classes of sirnas are generated, the 21-nt class regulates post-transcriptionally related mrnas while the 24-nt class is involved in rna-dependent dna methylation  and heterochromatin maintenance and therefore suppresses gene expression at transcriptional level  <cit> .
previous studies mapping srnas to ltr-rts in wheat and maize genomes  <cit> , presented an overall study of te superfamilies and described a pattern of concentration of 24nt srna in the ltrs.
our work, which focused on mapping to individual ltr-rt elements, identified distinct patterns of srna targeting within sugarcane ltr-rt lineages and families.
the previously described '24nt ltr' pattern was observed for all references sequences in the maximus families, and for del <dig> and tat <dig> .
for all other reference sequences a different type of pattern was observed.
for almost half of the families  very few srnas were mapped to the reference copy .
two other patterns were observed, one in which high numbers of 21nt srnas mapped along the coding region, represented by ivana <dig> and reina <dig> , and one in which a very large number of 24nt srnas mapped within the coding region, seen only in ale <dig>
very few mapped srnas indicates that elements from those families either are not transcriptionally active, or they are very recently activate and have not yet triggered the host small rna-dependent silencing machinery.
if they are not transcriptionally active, silencing may be being maintained by ancient methylation.
a previous study has shown that ~63% of methylated regions were maintained without persistent targeting by srnas  <cit> , explaining the absence of both  <dig> and 24nt srnas.
the high numbers of 21nt srnas mapped to the coding region of some families suggests that they are being regulated primarily post-transcriptionally.
this has been previously reported for mites  <cit>  and may indicate recent activation of transcription, pior to triggering of the rddm machinery.
the unique pattern observed in the ale <dig> family suggests that the rddm machinery is guiding methylation to the coding region of the element, not the promoter region.
the "body-methylated gene" concept was first described in plants in  <dig>  when zhang and collaborators showed that over one third of arabidopsis expressed genes were methylated in the coding region, but not in the promoter region  <cit> .
the ale <dig> family has a distinct profile, it has a 'body-gene'-like srna pattern, it is the most transcriptionally active ltr-rt in sugarcane and is concentrated in euchromatic regions and gene-rich bac sequences .
at this point it is difficult to make conclusions from these observations, however these results are intriguing and warrant further investigation.
CONCLUSIONS
this is the first study to perform a concomitant survey of phylogenetic diversity, chromosomal distribution, structure, transcriptional activity and interaction with srnas of ltr-rts in a plant genome.
we assigned  <dig> ltr-rt elements to  <dig> families within four copia and three gypsy lineages.
two lineages, one copia and one gypsy lineage, showed distinct patterns of signal clustering along some chromosomes in the fish analysis.
given that the modern sugarcane cultivars are hybrids with highly unequal contributions from the ancestral genomes, the fish patterns suggest that for these lineages there has been higher numbers of elements from one ancestral genome than the other.
for the transcriptional and srna mapping analyses we chose to analyze at the family level.
individual families had distinct transcript and srna mapping profiles, suggesting that they are differentially expressed and regulated.
the ale <dig> family was particularly unusual in that it had 'body-gene'-like srna pattern, it is the most transcriptonally active ltr-rt in sugarcane and is concentrated in euchromatic regions.
overall, our results indicate that ltr-rts could impact the genome in different ways at the family levels.

 plant genome evolution is closely associated with polyploidy and gene amplification, the most highly amplified genes being long terminal repeat retrotransposons. ltr-rt proliferation is regulated by the genome at both the transcriptional and post-transcriptional level. ltr-rts, like other transposable elements, can not only affect a genome by expansion by proliferation, but also by, for example, providing a template for recombination, inserting into coding regions and disrupting gene expression, or affecting the transcription of neighboring genes. based on the massive amounts of genomic sequence data released in the last years, phylogenetic analysis of plant ltr-rts has identified distinct evolutionary lineages within gypsy and copia, the two main plant ltr-rt superfamilies. these lineages are widespread in both monocot and eudicot genomes. uncovering of this fine structuring within gypsy and copia plant ltr-rts has lead to a better understanding of the diversification of ltr-rts and shed light on their structure and genomic distribution. sugarcane is an important crop worldwide, being a major source of sugar, and is also increasingly being used for the production of renewable energy sources such as ethanol. despite its economic importance, the sequencing of the sugarcane genome is at the pilot stage. modern cultivars are highly polyploid and have one of the largest and most complex crop genomes, and chromosome numbers varying between 100-. there are a few reports on transposable elements in sugarcane sequences but there is no detailed study on sugarcane ltr-rts, their phylogenetic classification, and potential impact on genomic distribution and transcriptional activity. in addition, the importance of accurate and complete te annotation is increasingly recognized as a priority in plant genome sequencing projects to minimize the inaccuracy of gene annotation and facilitate functional gene studies. to set the basis for future genome interpretation, ltr-rts in the sugarcane genome were characterized using available public resources and bac sequences from the bioen project. we examined the structure, genomic distribution, phylogenetic diversity, transcriptional activity and regulation of sugarcane ltr-rts. this study reveals that within well-defined phylogenetic lineages, that while tes within ltr-rts families are structurally similar, they have distinct transcriptional and regulatory signatures. taken together, these results support the growing evidence that ltr-rts contribute to genomic diversity, but with a wide range of potential outcomes.  characterization of ltr retrotransposons in sugarcane: phylogeny and structural features plant ltr-rt evolutionary lineage names are not used consistently within the literature, we therefore chose to include sequences from more than one source, so that we could directly compare our results with published data. our inferred evolutionary histories suggest that, at least for the sequences we analyzed within gypsy superfamily, the del lineage is equivalent to tekay, and within the copia superfamily, the maximus lineage is equivalent to sire, ale to retrofit and ivana to oryco. the relationship between the tar, angela, tork and bianca lineages is more problematic. while the bianca lineage is not included in the gydb tork appears to be the angela and tar lineages combined. of the sixty full-length sequences extracted from sugarcane bac sequences, thirty-two sequences were assigned to the copia superfamily and twenty-eight to the gypsy superfamily. we identified four of the six major plant evolutionary copia lineages described by wicker and keller in the sugarcane genome, and three of the six gypsy lineages described by du et al. and the gydb. in terms of sequence numbers, the maximus lineage was the most highly represented within the copia superfamily, and the del and tat lineages were equally represented within the gypsy superfamily. based on sequence identity within the ltrs, the elements were classified into families. in order to simplify reading of the text we have referred to these families as simply'ale1' for example, rather than the by the full name'rlc_scale1'. we were able to identify all the internal coding domains in all the sugarcane sequences, apart from the gag domain in rlg_sctat_. in addition, a putative envelope domain was identified in elements from the maximus family and a chromodomain in all del and reina elements. there was a general pattern in overall size of elements from lineages in each of the two superfamilies. in both superfamilies, there was one lineage that is very large, and at least one lineage that is comparatively smaller. within the copia superfamily, there is a lineage that is an in-between size, while within the gypsy superfamily, the sequences of the tat lineage are more comparable in size to those of the del lineage. differences in total length were chiefly due to differences in ltr size, and the presence and size of spacer regions between the internal coding domain and the ltrs, rather than insertions within the gag/pol coding regions. the exception is the tat elements, which contain insertions within the pol region, but in most cases between actual coding domains. distribution of ltr retrotransposons in metaphases given that transposable elements can be distributed throughout a genome, it is difficult to distinguish between random and real signals from a probe derived from a transposable element. the probe for the del lineage was therefore prepared twice, one probe labeled with dig and the other with biotin, and hybridized separately to the same slide before and after stripping. the distribution of the del probe was compared on the same metaphases visualized with anti-digoxigenin-rhodamine or with neutravidin-oregon green-. the distribution of the probe signal was similar in all metaphases, and therefore the signal was considered to be valid. similar in situ hybridization conditions were used for all subsequent experiments. a bac clone, identified by our group, contains known sugarcane centromeric repeats. this bac clone hybridized to the middle region of the chromosome, which consists mainly of centromeric specific sequences. seven ltr-rt probes were used, three of which are from gypsy superfamily members and four from copia superfamily members, representing all the major lineages already described. no convincing signal was obtained for the reina ivana and angela probes, using the same conditions that were used for the other ltr-rt probes. the probe from one of the two gypsy lineages, del localized almost exclusively in a broad band around the centromeres. this observation suggests that it is a heterochromatic or pericentromeric specific element. the second gypsy element examined, tat displays a generally strong broad dispersed pattern, but with some concentration along some chromosome arms. two copia probes were found dispersed along the chromosome arms and none localized exclusively at or near the centre of the chromosomes. the ale lineage probe was found in high concentrations along particular chromosome arms. signals from the maximus lineage probe were generally dispersed. transcriptional activity of sugarcane ltr retrotransposons we associated ests from the sugarcane cultivar sp80- related to full-length ltr retrotransposons. the largest number of transcripts was identified from the root libraries, followed by the internode, lateral bud and calli libraries. the copia superfamily was more highly represented in the est database than the gypsy superfamily, with and sequences, respectively. previously described full length cdna sequences were assigned to of the families we identified from bac sequences. in this study, no ests were identified for families. we assigned ests to another families not previously described as transcriptionally active, however for one family, ale for which a previously described cdna sequence was assigned, no ests were identified. rt-pcr analysis confirmed the transcriptional activity of sugarcane ltr-rts in leaves and lateral buds. we also experimentally confirmed transcriptional activity for five new families: ale ivana del reina and reina the most represented family in the est data, ale had an intense band, confirming its transcriptional activity, and had the same intensity of signal in both leaves and lateral buds. transcriptional patterns differed between tissues and ltr-rt families. the ivana and reina lineages illustrate that individual families have distinct transcriptional patterns: for instance, the ivana signal is more intense than that of ivana1; moreover, ivana has higher transcriptional activity in lateral buds, while ivana is more active in leaves. reina and reina are both transcriptionally active in lateral buds, but clear differences in band intensity can be observed. distribution of small rnas among ltr retrotransposon lineages we divided the srna population into two major classes: 20-22nt and 23-25nt srna sequences. we refer hereafter to these classes as 21-nt class and 24-nt class. in this analysis we allowed a 0- nt mismatch between the srna and ltr-rt reference sequences, in which 25% and 22% of the and ntrnas class, respectively, showed a perfect match with the ltr-rts sequences. all identified lineages had at least one family with srna from both classes mapped to ltr-rts. in the copia superfamily, all families within the maximus lineage had more than srnas mapped, mostly belonging to the 24-nt class. these elements had the highest srna counts, about to fold higher than other lineages and these counts mapped chiefly to the ltrs. del is the only representative of the gypsy superfamily that has comparable srna counts. the ivana and reina lineages were the lineages with the least number of srna counts. within the ivana lineage, the element rlc_scivana had the highest number of srna matches, most of which belong to the 21-nt class. the same pattern was observed in rlg_reina. the remaining ivana and reina families had lower srna counts. to evaluate whether srnas preferentially matched specific regions of each ltr-rt, we compared the srna distribution within ltrs and coding domains. various distribution patterns, both within the ltrs and the coding domains were observed. families of the maximus lineage and del had the high 24-nt srna counts within the ltrs. the 5' region of both ltrs from del and del families had a peak of 21-nt srnas. most ltr-rt families did not have high srna counts within the coding domains, but some cases of high counts of srnas were noted. the ale family had > counts of the 24-nt class srnas within the integrase and protease domains and the tat family had > counts of the 24-nt class srnas within the rnaseh domain. all other cases of high counts in coding domains are related to 21-nt class srnas, as seen in the ivana and reina families, both with around counts. interestingly, angela had peaks of > 24-nt srna counts in a kb non-coding region between the 5'ltr and the gag domain. discussion we characterized ltr-rts in the sugarcane genome using bac sequences available from the bioen project and publicly available genomic and est resources, which allowed us to provide the most complete overview of the structural and phylogenetic diversity of these elements to date. the ltr-rt families identified belong to four lineages from the copia superfamily and to three from the gypsy superfamily. among all these families we observed distinct patterns of differences in structural features, chromosomal distribution, transcriptional activity, and srnas targeting. sugarcane ltr-rts are structurally diverse and belong to evolutionary lineages shared by monocots and eudicots previous surveys of ltr-rts in plants defined six major common evolutionary copia lineages and six major common evolutionary gypsy lineages. bianca was the only copia superfamily lineage in which sugarcane ltr-rts were not found. absence of bianca in the sugarcane genome dataset analyzed may be explained by its relatively low representation in other plants. indeed, rice has few bianca families and soybean has none. we also did not find sugarcane ltr-rts elements from the galadriel and crm lineages in gypsy superfamily. the galadriel lineage seems to be an under-represented lineage among monocots, since there are only reports of galadriel elements in banana. crm elements are also minor components of gypsy retrotransposons in the model grass rice, which may explain its low representation in sugarcane. although very little sequence of the sugarcane genome is available, the number of ltr-rt families we identified was comparable to previous large scale ltr-rts analyses in bac clones from medicago truncatula and capsicum annuum. this finding suggests that we have a good overview of the diversity of sugarcane ltr-rts. sugarcane ltr-rt families have distinct chromosomal distributions transposable elements are not distributed randomly along eukaryotic chromosomes. in particular, gypsy elements tend to be found in heterochromatic regions, copia elements are more dispersed throughout the genome. in addition, heterochromatic and pericentromeric regions are enriched in tes, such as those of the cr/crm and tat lineages, compared with euchromatic regions. using fluorescence in situ hybridization, we examined the distribution of representatives from seven lineages identified within the gypsy and copia superfamilies in sugarcane chromosomes. no detectable fish signal was obtained for the angela, ivana and reina lineages; in the case of ivana preliminary estimates from the r bac library suggests that there is approximately copies of ivana per haploid genome in sugarcane, which suggests that the lack of signal is due to low copy number. we suspect that this observation may be also valid for angela and reina. the two copia probes, ale and maximus had the expected euchromatic patterns, but they were distinctly different. the signals from the ale probe had localized clusters along particular chromosome arms, while those of the maximus probe were widespread and dispersed, without any signs of concentration in particularly regions. it has been suggested that as tes accumulate in a genome, even if they insert randomly, they are more likely to insert within other tes, forming clusters of tes. because these clusters of tes are most likely selectively neutral, they will be free to expand. the ale distribution pattern observed in sugarcane is reminiscent of such clusters of tes. the sugarcane bac schrba 239_n used as a centromeric-specific probe in this study contains the scen repeat interspersed with ltr-rts from the tat lineage. previous studies in sugarcane identified a centromere-specific repeat and centromere associated ltr-rts, classified as cr/crm-like. we did not identify any full length ltr-rts from the cr/crm lineage in this study, but sequence from a related gypsy lineage, del did hybridize to a broad region around and within the centromeric region. del lineage sequences have been identified at other plant centromeres, however our results suggest that the del sequence we used as a probe is not strictly centromeric specific, but is rather preferentially found in and around heterochromatic regions of the centromeric repeats. the tat sequences identified in the bac schrba 239_n clustered with the tat tat tat and tat families, which is quite distinct from the tat family. the tat fish probe used is most closely related to the tat sequences and had a clusters of localization along some chromosomes, like those seen with the ale probe, but with much more intense signals. modern sugarcane cultivars are interspecific hybrids between saccharum officinarum and s. spontaneum and backcrossed with s. officinarum. the resulting genomes are highly polyploid, 70-80% of which is from s. officinarum, 10-23% from s. spontaneum, the rest being recombinants. the derivation of the modern sugarcane genome suggests that, rather than being the result of tes inserting into clusters of tes, the distribution of tat and ale may be the result of differential transmission of tes from the parental genomes. future work using genomic in situ hybridization combined with fish should enable us to distinguish between these two hypotheses. transcriptional activity of ltr-rts and their associated srnas transposable elements make up a substantial proportion of plant genomes, and are often transcriptionally active. a very stringent search for sugarcane ests associated with tes confirmed that transcriptional activity appears to be a general feature of some sugarcane ltr-rts, as reported for other monocots. since we do not have detailed information about cdna library preparation of est data, we cannot identify if transcribed sequences were in sense or antisense orientation. interestingly, within each lineage, the number of transcripts mapped to each family was variable. the most striking example is the ale lineage, which has the highest number of transcripts, all of which mapped to a single family, ale there is a similar, but not as definitive pattern in all other lineages, except for maximus, where all families have similar numbers of transcripts. in terms of tissue specificity, the highest number of ltr-rt associated transcripts was identified from the root libraries, followed by the internode, lateral bud and calli libraries. a previous macroarray analysis identified calli as the tissue with the highest number of tes being expressed in sugarcane, however in this study only calli, apical meristem, leaf roll and flower were analyzed. these differences in expression according to tissue, in particular that leaf is the tissue with least number of ests identified, may help guide future studies of transposable elements in sugarcane. as they are potentially highly mutagenic, the activity of ltr-rts is usually controlled by the host genome through the sirna machinery. the specificity of this response is achieved by a surveillance system that detects aberrant rna. the proliferative nature of tes makes them prone to insert in the genome in such way that both sense and anti-sense transcripts are produced, generating dsrna, and activating the sirna system. two main classes of sirnas are generated, the 21-nt class regulates post-transcriptionally related mrnas while the 24-nt class is involved in rna-dependent dna methylation and heterochromatin maintenance and therefore suppresses gene expression at transcriptional level. previous studies mapping srnas to ltr-rts in wheat and maize genomes, presented an overall study of te superfamilies and described a pattern of concentration of 24nt srna in the ltrs. our work, which focused on mapping to individual ltr-rt elements, identified distinct patterns of srna targeting within sugarcane ltr-rt lineages and families. the previously described'24nt ltr' pattern was observed for all references sequences in the maximus families, and for del and tat. for all other reference sequences a different type of pattern was observed. for almost half of the families very few srnas were mapped to the reference copy. two other patterns were observed, one in which high numbers of 21nt srnas mapped along the coding region, represented by ivana and reina, and one in which a very large number of 24nt srnas mapped within the coding region, seen only in ale very few mapped srnas indicates that elements from those families either are not transcriptionally active, or they are very recently activate and have not yet triggered the host small rna-dependent silencing machinery. if they are not transcriptionally active, silencing may be being maintained by ancient methylation. a previous study has shown that ~63% of methylated regions were maintained without persistent targeting by srnas, explaining the absence of both and 24nt srnas. the high numbers of 21nt srnas mapped to the coding region of some families suggests that they are being regulated primarily post-transcriptionally. this has been previously reported for mites and may indicate recent activation of transcription, pior to triggering of the rddm machinery. the unique pattern observed in the ale family suggests that the rddm machinery is guiding methylation to the coding region of the element, not the promoter region. the "body-methylated gene" concept was first described in plants in when zhang and collaborators showed that over one third of arabidopsis expressed genes were methylated in the coding region, but not in the promoter region. the ale family has a distinct profile, it has a'body-gene'-like srna pattern, it is the most transcriptionally active ltr-rt in sugarcane and is concentrated in euchromatic regions and gene-rich bac sequences. at this point it is difficult to make conclusions from these observations, however these results are intriguing and warrant further investigation.  this is the first study to perform a concomitant survey of phylogenetic diversity, chromosomal distribution, structure, transcriptional activity and interaction with srnas of ltr-rts in a plant genome. we assigned ltr-rt elements to families within four copia and three gypsy lineages. two lineages, one copia and one gypsy lineage, showed distinct patterns of signal clustering along some chromosomes in the fish analysis. given that the modern sugarcane cultivars are hybrids with highly unequal contributions from the ancestral genomes, the fish patterns suggest that for these lineages there has been higher numbers of elements from one ancestral genome than the other. for the transcriptional and srna mapping analyses we chose to analyze at the family level. individual families had distinct transcript and srna mapping profiles, suggesting that they are differentially expressed and regulated. the ale family was particularly unusual in that it had'body-gene'-like srna pattern, it is the most transcriptonally active ltr-rt in sugarcane and is concentrated in euchromatic regions. overall, our results indicate that ltr-rts could impact the genome in different ways at the family levels. 
four distinct patterns were observed in srna mapping, the most unusual of which was that of ale with very large numbers of 24nt srnas in the coding region. sixty complete ltr-rt elements were classified into families within four copia and three gypsy lineages. for instance, these transposable elements may affect nearby genes by generating a diverse set of small rna's that trigger gene silencing mechanisms. modern cultivars have polyploid, large complex genomes, with highly unequal contributions from ancestral genomes. there is also some evidence that ancestral genomes contribute significantly different element numbers from particular ltr-rt lineages to the modern sugarcane cultivar genome. the results presented support the conclusion that distinct small rna-regulated pathways in sugarcane target the lineages of ltr-rt elements. our results indicate that in sugarcane individual ltr-rt families have distinct behaviors and can potentially impact the genome in diverse ways. sugarcane is an important crop worldwide for sugar production and increasingly, as a renewable energy source. fish analysis resulted in the expected pattern of gypsy/heterochromatin, copia/euchromatin, but in two lineages there was localized clustering on some chromosomes. long terminal repeat retrotransposons are the single largest components of most plant genomes and can substantially impact the genome in many ways. individual ltr-rt sugarcane families have distinct structures, and transcriptional and regulatory signatures.
 sugarcane is an important crop worldwide for sugar production and increasingly, as a renewable energy source. modern cultivars have polyploid, large complex genomes, with highly unequal contributions from ancestral genomes. long terminal repeat retrotransposons are the single largest components of most plant genomes and can substantially impact the genome in many ways. it is therefore crucial to understand their contribution to the genome and transcriptome, however a detailed study of ltr-rts in sugarcane has not been previously carried out.  sixty complete ltr-rt elements were classified into families within four copia and three gypsy lineages. structurally, within lineages elements were similar, between lineages there were large size differences. fish analysis resulted in the expected pattern of gypsy/heterochromatin, copia/euchromatin, but in two lineages there was localized clustering on some chromosomes. analysis of related ests and rt-pcr showed transcriptional variation between tissues and families. four distinct patterns were observed in srna mapping, the most unusual of which was that of ale with very large numbers of 24nt srnas in the coding region. the results presented support the conclusion that distinct small rna-regulated pathways in sugarcane target the lineages of ltr-rt elements.  individual ltr-rt sugarcane families have distinct structures, and transcriptional and regulatory signatures. our results indicate that in sugarcane individual ltr-rt families have distinct behaviors and can potentially impact the genome in diverse ways. for instance, these transposable elements may affect nearby genes by generating a diverse set of small rna's that trigger gene silencing mechanisms. there is also some evidence that ancestral genomes contribute significantly different element numbers from particular ltr-rt lineages to the modern sugarcane cultivar genome. ltr retrotransposonssugarcanegenomefishepigeneticsmall rna 