BACKGROUND
the c-value paradox, the phenomenon describing the lack of correlation between biological complexity of an organism and its genome size  <cit> , is probably best exemplified in plants were genome sizes span several orders of magnitude ranging from the  <dig> mbp fragaria viridis genome  <cit>  to more than  <dig>  mbp genome of the lily fritillaria assiriaca  <cit> .
significant genome size variations are also not uncommon even within single genera such as rice, cotton and sorghum were  <dig> ,  <dig> and  <dig>  fold genome size variation have been reported, respectively  <cit> .
the primary mechanisms that contribute to this variation in plants are polyploidization  <cit>  and transposable element  proliferation  <cit>  and elimination  <cit> .
tes are classified according to the different molecules used as intermediates in the replicative mechanism of transposition as class  <dig> and class  <dig>   <cit> .
class  <dig> elements transpose via an rna intermediate and include long terminal repeat  retrotransposons , long interspersed nuclear elements  and short interspersed nuclear elements   <cit> .
class  <dig> elements transpose via a dna intermediate and have been classified into superfamilies  according to the similarity of transposases, the element-encoded protein that catalyzes transposition and integration  <cit> .
other classes of dna transposable elements are represented by helitrons  <cit>  and polintons  <cit> .
the genus oryza is an ideal model system to study the role of tes on genome size variation.
the genus is composed of  <dig> species: two cultivated  and  <dig> wild  <cit> .
based on evidence derived from interspecific crossing, cytogenetics and genomic dna hybridization, ten oryza genome types have been recognized including  <dig> diploid  and  <dig> tetraploid .
the relevance of rice as staple food resource for the world, its compact  <dig> mbp genome  <cit>  and its role as a "model" species for genomic studies of cereals  <cit>  have driven massive research efforts that include the production of the first finished genome sequence of any crop plant  <cit> .
additionally, our laboratory has developed a comprehensive set of bac libraries, bac end sequences and integrated physical maps representing the  <dig> oryza genome types  <cit> .
to better understand the role transposable elements have played in genome size variation in oryza we generated and sequenced a set of random sheared genomic libraries from  <dig> species representative of the  <dig> genome types of the oryza.
random sheared libraries represent an unbiased sampling of genome content and enable the characterization of their most relevant features without undertaking a massive sequencing effort.
the sequences obtained were analyzed in order to describe, classify and compare the repetitive fraction of the genus oryza.
particular attention was paid to the role played by the most important classes of tes  in shaping these genomes and in contributing to genome size variation.
in the case of four groups of tes  phylogenetic analyses were carried out.
RESULTS
to better understand the repeat content of the genus oryza,  <dig> random sheared libraries were constructed and sequenced from  <dig> vouchered accessions that represent the  <dig> genome types of oryza .
the total number of clones produced was  <dig> , which roughly corresponded to  <dig> × coverage of each genome.
mean insert size ranged from  <dig>  kbp for o. australiensis  to  <dig>  kbp for o. rufipogon .
cloned inserts were bi-directionally sequenced and after removing low quality data ,  <dig>  high quality sequence reads were obtained for a total of  <dig>  mb of sequence ).
all the genome size estimates are from ammiraju et al   <cit>  except those of o. glaberrima and o. minuta which are from martinez et al   <cit>  and of o. coarctata which is a rough estimate because the real genome size of this species is unknown.
we therefore used the value estimated for o. ridleyi , which is also an allotetraploid species and shares the hh genome type with o. coarctata.
repeats abundance and distribution
repetitive sequences were identified by similarity searches against databases of previously characterized repetitive elements, isolated from o. sativa and other oryza species, including centromeric repeats, helitrons, sines, lines, mites, ltr-rts, dna transposable elements , ribosomal sequences and telomeric repeats .
all the major classes of repeats were represented throughout the genus .
the overall amount of repeats was quite variable in different species and ranged from the  <dig> % of reads in o. coarctata  to  <dig> % of reads in o. officinalis .
occurrences are expressed as percentage of sequence reads having significant similarity out of the total.
refers to ltr-rts that could not be classified as ty3-gypsy or ty1-copia
includes all the autonomous dna elements; non autonomous ones such mites are excluded; helitrons are reported separately
unclassified repeats
in all the genomes studied the largest class of repeats consisted of ltr-rts with amounts ranging from the 55% of reads in o. granulata  to  <dig> % of reads in o. brachyantha .
dna transposons, excluding mites and helitrons, appeared to be more abundant in o. ridleyi   and were depleted in o. coarctata  .
the mite sequences used as queries in similarity searches were isolated in o. sativa and not surprisingly they tag the highest value of hits in the three aa genome species: o. glaberrima , o. nivara  and o. rufipogon .
the quantity of mites identified in the remaining genome types was less than 6% of reads except for the notable exception of the distantly related species o. brachyantha  with  <dig> % of reads.
the lower than expected mite content in the non-aa genomes species is likely due to the fact that mites are highly species specific and the repeat database used to identify mites was a curated data set from the o. sativa species only.
differences in te size, host genome size and degrees of interspecific conservation make it particularly difficult to perform a detailed comparison based solely on the percentage of significantly similar sequences out of the total.
a more rigorous approach was recently proposed by hawkins et al.
<cit>  where an equation was derived  that takes into account te mean size, host genome size, the minimum length needed in order to have a significant blastn hit, and the number of matches out of total reads available to estimate the absolute number of repeats in a given genome.
we used this equation for all classes of repeats for which reliable estimates of the average length were available and then, in order to have comparable results, normalized the data to the single mbp .
here we considered only the most represented and conserved families of elements: ltr-rts, lines, helitrons, and the dna-tes – cacta and mutator like.
it should however be noted how the use of average lengths for entire classes of elements could introduce a certain degree of uncertainty and so the following values should be considered as an approximation.
not surprisingly the ltr-rts remained the major contributor to the repeat pool of each species.
species with the highest occurrence of ltr-rts per mbp were o. granulata   and o. australiensis   whereas the most depleted was o. brachyanta  .
in almost all cases, the ty3-gypsy elements outnumbered ty1-copia elements.
this disproportion was particularly striking in the case of o. nivara , o. rufipogon  and o. granulata  were the ratios ty3-gypsy to ty1-copia elements were  <dig> ,  <dig>  and  <dig>  respectively.
the only exception to the overwhelming presence of ty3-gypsy elements in comparison to ty1-copia was found in o. coarctata  where twice as many ty1-copia elements  were found.
among other repeats, it is worth to note that cacta elements seemed to be significantly more frequent in the bb , cc , bbcc , ccdd , ee  and hhjj  genomes as compared with all  <dig> aa genomes  where the number of hits was less than  <dig> per mbp.
in almost all the genomes the frequency of cacta elements was greater than the hat elements, with the exception of o. brachyantha   and o. granulata  in which hat elements outnumber the cacta elements  <dig> to  <dig> .
the distribution of lines was higher in the aa genomes where it was greater than  <dig>  hits per mbp and very low in o. granulata  with only  <dig>  hits per mbp.
the significant variance of ltr-rt representation presented above prompted us to perform a more in depth analysis of the distribution of the different families in each genome.
to identify the primary ltr-rt families responsible for this variance and possibly link them to the genome size variation in each host species, all sequences previously identified as being similar to ltr-rts were screened a second time against a reduced database containing representatives of twenty-six of the most abundant ltr-rt families isolated in oryza genus  .
in all species the overall majority of sequences already annotated as ltr-rts could be easily classified according to these parameters .
elements related to the most abundant families of ltr-rts isolated in o. sativa  were identified in all the oryza genome types considered with very few exceptions.
this indicated that the complement of ltr-rts previously identified in rice is shared throughout the oryza genus.
however, as shown in table  <dig>  different ltr-rts had different fates in different species.
figures represent the percentage of significant hits out of the total number of reads for each species.
ty3-gyspy family names are in bold; ty1-copia ones are not.
unc refers to elements that in repbase were classified as ltr-rt but without any other detail ; nhf refers to elements that are ltr-rts related but do not have significant similarity with the major families isolated in the genus oryza.
"other families" indicates significant hits with other oryza sativa ltr-rts elements than those belonging to the families analyzed here.
a paradigmatic case was that of two ty3-gypsy families atlantys and rire <dig> .
atlantys was the most abundant element in  <dig> of the  <dig> oyrza genome types .
it was also the second most abundant element in o. granulata ,  <dig> % of total reads.
however in the aa and ff genomes, it did not expand significantly in comparison to the other elements.
rire <dig> related elements  expanded significantly in o. australiensis ,  <dig> % of the total reads and in o. granulata ,  <dig> % of total reads.
it is also worth to note that the rire <dig> ty3-gypsy element is more widespread in the aa genomes  than in the remaining genome types.
the o. minuta  genome represents the only polyploid genome for which a comparison with both the original genome counterparts bb  and cc  is possible.
all ltr-rt families identified in o. minuta  were found in at least one of the diploid counterparts and there were only few cases where an element was significantly more represented in the polyploid genome than in the two diploid counterparts.
the most convincing example was that of the ty1-copia element rire <dig> .
for all the genome types it was possible to identify a fraction of ltr-rt related sequences that did not show any significant similarity with the major families isolated from o. sativa.
the amount of this fraction expressed as percentage out of the total number of sequence reads ranged from  <dig> % in o. brachyantha  to  <dig> % in o. ridleyi  and represented the most species specific sets of these elements .
our strategy to rely on similarity searches in order to identify the repetitive fraction of a genome has the obvious pitfall that, if a repeat is not present in the repeat database it will not be detected.
to check for this possibility we adopted a different approach to screen all sequence reads that did not show any significant hits with already characterized repeats.
sequences were clustered using the program blastclust and the results are shown in table  <dig>  only a few clusters containing more than  <dig> sequences were isolated in all the species with the exception of the aa and bb genomes in which no clusters were identified.
the largest cluster identified was found in o. ridleyi  and contained  <dig> sequences out of  <dig>  .
the sequences present in these clusters very likely represent previously uncharacterized repeats.
however as the figures for o. ridleyi  demonstrate, they do not constitute a significant portion of the genomes in which they were isolated.
results of the clustering analysis carried out on the sequence reads that did not show any significant hits with previously annotated repeats.
b: number of sequence reads.
c: number of clusters containing more than  <dig> sequences.
d: number of sequences included in the largest cluster.
phylogenetic analysis of repeats
to establish the phylogeneic relationships among the most prominent superfamilies of tes in oryza, we used amino acid sequences of the most conserved domain tracts  of ty1-copia and ty3-gypsy ltr-rts, lines and cacta and mutator-like dna-tes to build neighbor-joining trees.
for ty1-copia elements  it was possible to retrieve  <dig> reverse transcriptase  sequences out of the  <dig>  available sequences.
these sequences were resolved into at least eight lineages as representatives of the major ty1-copia retroelements isolated.
the amount of sequences in each lineage reflected the abundance of that family in the genome.
furthermore the most abundant lineages contained representatives of all the different species and there was not a single species-specific lineage with the partial exception of rire <dig> in o. australiensis .
this lineage, also including a few sequences from o. minuta , was separated from the others and it was possible to see how the branch lengths are short suggesting that amplification of this element took place after speciation .
rt-like sequences from ty3-gypsy elements  were isolated and used to build a phylogenetic tree .
the complete set is less heterogeneous than the ty1-copia set, however all the major families are represented.
in the case of rire <dig> and atlantys, the two most abundant ty3-gypsy families throughout the genus, all the species were included in the correspondent lineages indicating that their presence in the genus predates its speciation.
however, considering the different species in which the major amplification events of elements belonging to these two families took place, it was possible to note how the elements isolated in different species usually do not mix together in the internal lineages suggesting that their amplification has happened after speciation.
lines are usually not very abundant in plants as compared to ltr-rts with some exceptions such as the del <dig> element in lilium speciosum  <cit> .
the genus oryza in this sense appears to follow this general rule.
this trend is reflected in the number of line reverse transcriptase like sequences  <cit>  isolated from our set.
twenty-two of them cluster in  <dig> major bootstrap supported lineages and many are characterized by very long branches suggestive of an ancient origin of these retroelements in the genus .
these findings suggest that even in the case of the oryza genus, lines exhibit, as in other plants, high sequence divergence and extreme heterogeneity  <cit> .
the two major groups of dna tes, cacta and mutator-like were phylogenetically investigated using tracts of their transposase coding domains.
in the case of cacta elements, we identified  <dig> transposase-like sequences, whereas  <dig> were isolated for mutator-like elements.
for both classes of te elements, it was not possible to identify species specific lineages in the corresponding neighbor joining trees .
the only exception was represented by a large lineage including  <dig> of the  <dig> cacta transposase domains isolated in o. alta .
however this lineage lacks consistent bootstrap support.
discussion
genome survey sequences from  <dig> random sheared genomic libraries representative of the  <dig> genome types of the genus oryza were generated and analyzed to obtain an unbiased sampling of the repeat content of the genus as a whole.
our results showed that the genomic composition of the oryza species is not exceptional when compared to other plant species in that the repetitive fraction consistently makes up a significant portion of each of the  <dig> genome types.
among tes, the major class of repeats in oryza was represented by ltr-rts whose absolute occurrence was estimated to be in the order of thousands of copies for each species.
detailed analysis of the major o. sativa ltr-rt families enabled us to identify the elements responsible for the major differences in genome size variation across the genus oryza.
for example, ltr-rts related to rire <dig> represent more than  <dig> % and 21% of all the sequences obtained for o. australiensis  and o. granulata  respectively.
this is consistent with previous studies which showed that the proliferation of  <dig> family members  in ee genomes  <cit>  and a single family  in the gg genome  accounts for nearly 50% and 25% increases in their genome sizes respectively.
another family that has played a significantly similar role in genome size variation is the ty3-gypsy element atlantys.
the atlantys family has been surprisingly overlooked by previous studies and with its amplification has impacted not only the o. granulata and o. australiensis genomes but also the bb, cc, bbcc and ccdd genomes.
the events leading to the explosive proliferation of both the rire <dig> and atlantys families appear to have taken place after each speciation event.
the consequences of te proliferation subsequent to speciation on genome size variation is reminiscent of zea mays in which five ltr-rt families represent nearly 25% of the genome  <cit>  and of the species gossypium herabaceum and g. exiguum where a group of ty3-gypsy elements, named gorge- <dig>  has extensively proliferated in comparison to other elements and constitutes a significant portion of each genome  <cit> .
in contrast, no single ltr-rt family was found to constitute significant portions of the aa genome oryza species with the only exception being represented by the ty3-gypsy element rire <dig>  the finding that, apart from the polyploids, the largest genomes in the oryza  are those that have seen the "explosive" proliferation of one or two ltr-rt families, once again identifies the pivotal role of this class of tes in the dynamics of genome size variation.
the differential proliferation of ltr-rts in the oryza genus could be explained by taking into account the effects of varying genomic backgrounds on element life cycles, different rates of ltr-rt removal by unequal recombination and/or illegitimate recombination  <cit>  and inherently different retrotranspositional potentials of various element families  <cit> .
however no single convincing explanation of this phenomenon has so far been advanced for other plant species in which similar events have been described  <cit> .
here we have demonstrated that the pool of ltr-rts in the oryza is substantially conserved throughout the genus because in almost all cases relatives of the ltr-rt families identified in o. sativa  could also be found in the other oryza species.
we therefore conclude that its existence predates the speciation events that originated the genus as we know.
this finding is well supported by our phylogenetic analyses and it is in accordance with the results of previous studies analyzing the ty1-copia elements phylogeny and distribution across different genera of the gramineae family  <cit> .
besides the identification of different amounts of elements from the same ltr-rt families, all the genomes studied, except o. coarctata, showed the same quantitative relationship between ty3-gypsy and ty1-copia elements where the ty3-gypsy elements constantly outnumber ty1-copia elements, even if the ratio between the two elements varied .
this data is completely consistent with previous analysis of the cultivated japonica and indica genomes  <cit>  and can now be extended to the genus oryza, with the notable exception of o. coarctata  where we found a ratio close to  <dig>  between the ty3-gypsy and ty1-copia elements.
an even more striking difference between the o. coarctata  genome and the other oryza spp genomes is that it was found to contain the least amount repetitive elements despite it being polyploid and its predicted large genome size.
it should be noted that we have not been able to obtain live plants or tissues in the u.s. in order to determine an accurate genome size measurement by flow cytometry due to quarantine restrictions.
to rule out the possibility that our similarity searches could not detect highly diverged elements we analyzed all the sequences to try to identify repeats de novo through a clustering approach.
in this case no significant de novo clusters were obtained in o. coarctata.
these findings rule out the existence of a single recently originated family of repeats, highly species specific and constituting a significant portion of the genome.
however, the results do not discount the possibility that repeats missed by previous searches in o. coarctata, if present, belong to several different families, possibly diverged .
all evidence combined supports the old taxonomic view that places o. coarctata in the genus porteresia  <cit>  and not oryza.
however more in depth and accurate analyses are needed in order to ascertain the dynamics acting towards this apparently repeat depletion in o. coarctata.
the evolutionary events leading to the present day oryza genus spanned at least  <dig> million years from o. sativa  to the the most basal species o. granulata   <cit> .
similar to what was observed in o. coarctata , the use of repeat collections mainly based on elements isolated in o. sativa  to screen other species of the genus raises some concerns about how nucleotide divergence can impact estimates of ltr-rts abundance.
with the exceptions of o. coarctata  and o. brachyantha  , our work demonstrated that the amount of ltr-rts isolated does not decrease substantially when our analysis moved from the aa genome types to the most distant ones.
the evidence has been indirectly confirmed by the clustering analysis that did not revealed the existence any significantly repeat family previously missed.
this, in turn, demonstrates how similarity searches of ltr-rt families have a good level of "portability" throughout the genus giving substantially reliable and reasonably exhaustive results, if the proper settings are used.
a completely different scenario could be described for tes lacking conserved domains and having higher species sequence specificity such as mites and sines.
in this case the evolutionary distance from aa genomes consistently results in a steady drop in the amount tes identified  prompting the need of de novo and ad hoc identification tools based on structural features beside the nucleotidic sequence.
CONCLUSIONS
here we report the results of a comprehensive analysis of the abundance and relative distribution of major te classes across twelve species of the genus oryza.
we have demonstrated how the ltr-rt complement in of these species is ancient and conserved throughout the genus, and has attained different retrotranpositional success in different species.
we also identified two ltr-rt families  that are responsible for a significant portion of genome size variation in the genus and we demonstrated how their massive increase in copy number is recent.

 the c-value paradox, the phenomenon describing the lack of correlation between biological complexity of an organism and its genome size, is probably best exemplified in plants were genome sizes span several orders of magnitude ranging from the mbp fragaria viridis genome to more than mbp genome of the lily fritillaria assiriaca. significant genome size variations are also not uncommon even within single genera such as rice, cotton and sorghum were, and fold genome size variation have been reported, respectively. the primary mechanisms that contribute to this variation in plants are polyploidization and transposable element proliferation and elimination. tes are classified according to the different molecules used as intermediates in the replicative mechanism of transposition as class and class. class elements transpose via an rna intermediate and include long terminal repeat retrotransposons, long interspersed nuclear elements and short interspersed nuclear elements. class elements transpose via a dna intermediate and have been classified into superfamilies according to the similarity of transposases, the element-encoded protein that catalyzes transposition and integration. other classes of dna transposable elements are represented by helitrons and polintons. the genus oryza is an ideal model system to study the role of tes on genome size variation. the genus is composed of species: two cultivated and wild. based on evidence derived from interspecific crossing, cytogenetics and genomic dna hybridization, ten oryza genome types have been recognized including diploid and tetraploid. the relevance of rice as staple food resource for the world, its compact mbp genome and its role as a "model" species for genomic studies of cereals have driven massive research efforts that include the production of the first finished genome sequence of any crop plant. additionally, our laboratory has developed a comprehensive set of bac libraries, bac end sequences and integrated physical maps representing the oryza genome types. to better understand the role transposable elements have played in genome size variation in oryza we generated and sequenced a set of random sheared genomic libraries from species representative of the genome types of the oryza. random sheared libraries represent an unbiased sampling of genome content and enable the characterization of their most relevant features without undertaking a massive sequencing effort. the sequences obtained were analyzed in order to describe, classify and compare the repetitive fraction of the genus oryza. particular attention was paid to the role played by the most important classes of tes in shaping these genomes and in contributing to genome size variation. in the case of four groups of tes phylogenetic analyses were carried out.  to better understand the repeat content of the genus oryza, random sheared libraries were constructed and sequenced from vouchered accessions that represent the genome types of oryza. the total number of clones produced was, which roughly corresponded to × coverage of each genome. mean insert size ranged from kbp for o. australiensis to kbp for o. rufipogon. cloned inserts were bi-directionally sequenced and after removing low quality data, high quality sequence reads were obtained for a total of mb of sequence ). all the genome size estimates are from ammiraju et al except those of o. glaberrima and o. minuta which are from martinez et al and of o. coarctata which is a rough estimate because the real genome size of this species is unknown. we therefore used the value estimated for o. ridleyi, which is also an allotetraploid species and shares the hh genome type with o. coarctata. repeats abundance and distribution repetitive sequences were identified by similarity searches against databases of previously characterized repetitive elements, isolated from o. sativa and other oryza species, including centromeric repeats, helitrons, sines, lines, mites, ltr-rts, dna transposable elements, ribosomal sequences and telomeric repeats. all the major classes of repeats were represented throughout the genus. the overall amount of repeats was quite variable in different species and ranged from the % of reads in o. coarctata to % of reads in o. officinalis. occurrences are expressed as percentage of sequence reads having significant similarity out of the total. refers to ltr-rts that could not be classified as ty3-gypsy or ty1-copia includes all the autonomous dna elements; non autonomous ones such mites are excluded; helitrons are reported separately unclassified repeats in all the genomes studied the largest class of repeats consisted of ltr-rts with amounts ranging from the 55% of reads in o. granulata to % of reads in o. brachyantha. dna transposons, excluding mites and helitrons, appeared to be more abundant in o. ridleyi and were depleted in o. coarctata. the mite sequences used as queries in similarity searches were isolated in o. sativa and not surprisingly they tag the highest value of hits in the three aa genome species: o. glaberrima, o. nivara and o. rufipogon. the quantity of mites identified in the remaining genome types was less than 6% of reads except for the notable exception of the distantly related species o. brachyantha with % of reads. the lower than expected mite content in the non-aa genomes species is likely due to the fact that mites are highly species specific and the repeat database used to identify mites was a curated data set from the o. sativa species only. differences in te size, host genome size and degrees of interspecific conservation make it particularly difficult to perform a detailed comparison based solely on the percentage of significantly similar sequences out of the total. a more rigorous approach was recently proposed by hawkins et al. where an equation was derived that takes into account te mean size, host genome size, the minimum length needed in order to have a significant blastn hit, and the number of matches out of total reads available to estimate the absolute number of repeats in a given genome. we used this equation for all classes of repeats for which reliable estimates of the average length were available and then, in order to have comparable results, normalized the data to the single mbp. here we considered only the most represented and conserved families of elements: ltr-rts, lines, helitrons, and the dna-tes – cacta and mutator like. it should however be noted how the use of average lengths for entire classes of elements could introduce a certain degree of uncertainty and so the following values should be considered as an approximation. not surprisingly the ltr-rts remained the major contributor to the repeat pool of each species. species with the highest occurrence of ltr-rts per mbp were o. granulata and o. australiensis whereas the most depleted was o. brachyanta. in almost all cases, the ty3-gypsy elements outnumbered ty1-copia elements. this disproportion was particularly striking in the case of o. nivara, o. rufipogon and o. granulata were the ratios ty3-gypsy to ty1-copia elements were, and respectively. the only exception to the overwhelming presence of ty3-gypsy elements in comparison to ty1-copia was found in o. coarctata where twice as many ty1-copia elements were found. among other repeats, it is worth to note that cacta elements seemed to be significantly more frequent in the bb, cc, bbcc, ccdd, ee and hhjj genomes as compared with all aa genomes where the number of hits was less than per mbp. in almost all the genomes the frequency of cacta elements was greater than the hat elements, with the exception of o. brachyantha and o. granulata in which hat elements outnumber the cacta elements to. the distribution of lines was higher in the aa genomes where it was greater than hits per mbp and very low in o. granulata with only hits per mbp. the significant variance of ltr-rt representation presented above prompted us to perform a more in depth analysis of the distribution of the different families in each genome. to identify the primary ltr-rt families responsible for this variance and possibly link them to the genome size variation in each host species, all sequences previously identified as being similar to ltr-rts were screened a second time against a reduced database containing representatives of twenty-six of the most abundant ltr-rt families isolated in oryza genus. in all species the overall majority of sequences already annotated as ltr-rts could be easily classified according to these parameters. elements related to the most abundant families of ltr-rts isolated in o. sativa were identified in all the oryza genome types considered with very few exceptions. this indicated that the complement of ltr-rts previously identified in rice is shared throughout the oryza genus. however, as shown in table different ltr-rts had different fates in different species. figures represent the percentage of significant hits out of the total number of reads for each species. ty3-gyspy family names are in bold; ty1-copia ones are not. unc refers to elements that in repbase were classified as ltr-rt but without any other detail; nhf refers to elements that are ltr-rts related but do not have significant similarity with the major families isolated in the genus oryza. "other families" indicates significant hits with other oryza sativa ltr-rts elements than those belonging to the families analyzed here. a paradigmatic case was that of two ty3-gypsy families atlantys and rire. atlantys was the most abundant element in of the oyrza genome types. it was also the second most abundant element in o. granulata, % of total reads. however in the aa and ff genomes, it did not expand significantly in comparison to the other elements. rire related elements expanded significantly in o. australiensis, % of the total reads and in o. granulata, % of total reads. it is also worth to note that the rire ty3-gypsy element is more widespread in the aa genomes than in the remaining genome types. the o. minuta genome represents the only polyploid genome for which a comparison with both the original genome counterparts bb and cc is possible. all ltr-rt families identified in o. minuta were found in at least one of the diploid counterparts and there were only few cases where an element was significantly more represented in the polyploid genome than in the two diploid counterparts. the most convincing example was that of the ty1-copia element rire. for all the genome types it was possible to identify a fraction of ltr-rt related sequences that did not show any significant similarity with the major families isolated from o. sativa. the amount of this fraction expressed as percentage out of the total number of sequence reads ranged from % in o. brachyantha to % in o. ridleyi and represented the most species specific sets of these elements. our strategy to rely on similarity searches in order to identify the repetitive fraction of a genome has the obvious pitfall that, if a repeat is not present in the repeat database it will not be detected. to check for this possibility we adopted a different approach to screen all sequence reads that did not show any significant hits with already characterized repeats. sequences were clustered using the program blastclust and the results are shown in table only a few clusters containing more than sequences were isolated in all the species with the exception of the aa and bb genomes in which no clusters were identified. the largest cluster identified was found in o. ridleyi and contained sequences out of. the sequences present in these clusters very likely represent previously uncharacterized repeats. however as the figures for o. ridleyi demonstrate, they do not constitute a significant portion of the genomes in which they were isolated. results of the clustering analysis carried out on the sequence reads that did not show any significant hits with previously annotated repeats. b: number of sequence reads. c: number of clusters containing more than sequences. d: number of sequences included in the largest cluster. phylogenetic analysis of repeats to establish the phylogeneic relationships among the most prominent superfamilies of tes in oryza, we used amino acid sequences of the most conserved domain tracts of ty1-copia and ty3-gypsy ltr-rts, lines and cacta and mutator-like dna-tes to build neighbor-joining trees. for ty1-copia elements it was possible to retrieve reverse transcriptase sequences out of the available sequences. these sequences were resolved into at least eight lineages as representatives of the major ty1-copia retroelements isolated. the amount of sequences in each lineage reflected the abundance of that family in the genome. furthermore the most abundant lineages contained representatives of all the different species and there was not a single species-specific lineage with the partial exception of rire in o. australiensis. this lineage, also including a few sequences from o. minuta, was separated from the others and it was possible to see how the branch lengths are short suggesting that amplification of this element took place after speciation. rt-like sequences from ty3-gypsy elements were isolated and used to build a phylogenetic tree. the complete set is less heterogeneous than the ty1-copia set, however all the major families are represented. in the case of rire and atlantys, the two most abundant ty3-gypsy families throughout the genus, all the species were included in the correspondent lineages indicating that their presence in the genus predates its speciation. however, considering the different species in which the major amplification events of elements belonging to these two families took place, it was possible to note how the elements isolated in different species usually do not mix together in the internal lineages suggesting that their amplification has happened after speciation. lines are usually not very abundant in plants as compared to ltr-rts with some exceptions such as the del element in lilium speciosum. the genus oryza in this sense appears to follow this general rule. this trend is reflected in the number of line reverse transcriptase like sequences isolated from our set. twenty-two of them cluster in major bootstrap supported lineages and many are characterized by very long branches suggestive of an ancient origin of these retroelements in the genus. these findings suggest that even in the case of the oryza genus, lines exhibit, as in other plants, high sequence divergence and extreme heterogeneity. the two major groups of dna tes, cacta and mutator-like were phylogenetically investigated using tracts of their transposase coding domains. in the case of cacta elements, we identified transposase-like sequences, whereas were isolated for mutator-like elements. for both classes of te elements, it was not possible to identify species specific lineages in the corresponding neighbor joining trees. the only exception was represented by a large lineage including of the cacta transposase domains isolated in o. alta. however this lineage lacks consistent bootstrap support. discussion genome survey sequences from random sheared genomic libraries representative of the genome types of the genus oryza were generated and analyzed to obtain an unbiased sampling of the repeat content of the genus as a whole. our results showed that the genomic composition of the oryza species is not exceptional when compared to other plant species in that the repetitive fraction consistently makes up a significant portion of each of the genome types. among tes, the major class of repeats in oryza was represented by ltr-rts whose absolute occurrence was estimated to be in the order of thousands of copies for each species. detailed analysis of the major o. sativa ltr-rt families enabled us to identify the elements responsible for the major differences in genome size variation across the genus oryza. for example, ltr-rts related to rire represent more than % and 21% of all the sequences obtained for o. australiensis and o. granulata respectively. this is consistent with previous studies which showed that the proliferation of family members in ee genomes and a single family in the gg genome accounts for nearly 50% and 25% increases in their genome sizes respectively. another family that has played a significantly similar role in genome size variation is the ty3-gypsy element atlantys. the atlantys family has been surprisingly overlooked by previous studies and with its amplification has impacted not only the o. granulata and o. australiensis genomes but also the bb, cc, bbcc and ccdd genomes. the events leading to the explosive proliferation of both the rire and atlantys families appear to have taken place after each speciation event. the consequences of te proliferation subsequent to speciation on genome size variation is reminiscent of zea mays in which five ltr-rt families represent nearly 25% of the genome and of the species gossypium herabaceum and g. exiguum where a group of ty3-gypsy elements, named gorge- has extensively proliferated in comparison to other elements and constitutes a significant portion of each genome. in contrast, no single ltr-rt family was found to constitute significant portions of the aa genome oryza species with the only exception being represented by the ty3-gypsy element rire the finding that, apart from the polyploids, the largest genomes in the oryza are those that have seen the "explosive" proliferation of one or two ltr-rt families, once again identifies the pivotal role of this class of tes in the dynamics of genome size variation. the differential proliferation of ltr-rts in the oryza genus could be explained by taking into account the effects of varying genomic backgrounds on element life cycles, different rates of ltr-rt removal by unequal recombination and/or illegitimate recombination and inherently different retrotranspositional potentials of various element families. however no single convincing explanation of this phenomenon has so far been advanced for other plant species in which similar events have been described. here we have demonstrated that the pool of ltr-rts in the oryza is substantially conserved throughout the genus because in almost all cases relatives of the ltr-rt families identified in o. sativa could also be found in the other oryza species. we therefore conclude that its existence predates the speciation events that originated the genus as we know. this finding is well supported by our phylogenetic analyses and it is in accordance with the results of previous studies analyzing the ty1-copia elements phylogeny and distribution across different genera of the gramineae family. besides the identification of different amounts of elements from the same ltr-rt families, all the genomes studied, except o. coarctata, showed the same quantitative relationship between ty3-gypsy and ty1-copia elements where the ty3-gypsy elements constantly outnumber ty1-copia elements, even if the ratio between the two elements varied. this data is completely consistent with previous analysis of the cultivated japonica and indica genomes and can now be extended to the genus oryza, with the notable exception of o. coarctata where we found a ratio close to between the ty3-gypsy and ty1-copia elements. an even more striking difference between the o. coarctata genome and the other oryza spp genomes is that it was found to contain the least amount repetitive elements despite it being polyploid and its predicted large genome size. it should be noted that we have not been able to obtain live plants or tissues in the u.s. in order to determine an accurate genome size measurement by flow cytometry due to quarantine restrictions. to rule out the possibility that our similarity searches could not detect highly diverged elements we analyzed all the sequences to try to identify repeats de novo through a clustering approach. in this case no significant de novo clusters were obtained in o. coarctata. these findings rule out the existence of a single recently originated family of repeats, highly species specific and constituting a significant portion of the genome. however, the results do not discount the possibility that repeats missed by previous searches in o. coarctata, if present, belong to several different families, possibly diverged. all evidence combined supports the old taxonomic view that places o. coarctata in the genus porteresia and not oryza. however more in depth and accurate analyses are needed in order to ascertain the dynamics acting towards this apparently repeat depletion in o. coarctata. the evolutionary events leading to the present day oryza genus spanned at least million years from o. sativa to the the most basal species o. granulata. similar to what was observed in o. coarctata, the use of repeat collections mainly based on elements isolated in o. sativa to screen other species of the genus raises some concerns about how nucleotide divergence can impact estimates of ltr-rts abundance. with the exceptions of o. coarctata and o. brachyantha, our work demonstrated that the amount of ltr-rts isolated does not decrease substantially when our analysis moved from the aa genome types to the most distant ones. the evidence has been indirectly confirmed by the clustering analysis that did not revealed the existence any significantly repeat family previously missed. this, in turn, demonstrates how similarity searches of ltr-rt families have a good level of "portability" throughout the genus giving substantially reliable and reasonably exhaustive results, if the proper settings are used. a completely different scenario could be described for tes lacking conserved domains and having higher species sequence specificity such as mites and sines. in this case the evolutionary distance from aa genomes consistently results in a steady drop in the amount tes identified prompting the need of de novo and ad hoc identification tools based on structural features beside the nucleotidic sequence.  here we report the results of a comprehensive analysis of the abundance and relative distribution of major te classes across twelve species of the genus oryza. we have demonstrated how the ltr-rt complement in of these species is ancient and conserved throughout the genus, and has attained different retrotranpositional success in different species. we also identified two ltr-rt families that are responsible for a significant portion of genome size variation in the genus and we demonstrated how their massive increase in copy number is recent. 
two families of ty3-gypsy elements account for a significant portion of the genome size variations present in the oryza genus. the genus oryza is composed of distinct genome types, diploid and polyploid, and includes the world's most important food crop – rice. particular attention was paid to the role played by the most important classes of transposable elements in shaping these genomes and in their contributing to genome size variation. genome size variation in the oryza is more than 3-fold and ranges from mbp in oryza glaberrima to mbp in the polyploid oryza ridleyi. because repetitive elements are known to play a significant role in genome size variation, we constructed random sheared small insert genomic libraries from representative oryza species and conducted a comprehensive study of the repetitive element composition, distribution and phylogeny in this genus. we identified the elements primarily responsible for the most strikingly genome size variation in oryza. long terminal repeat retrotransposons are the major component of the oryza genomes analyzed and, along with polyploidization, are the most important contributors to the genome size variation across the oryza genus. we also showed that the pool of long terminal repeat retrotransposons is substantially conserved and ubiquitous throughout the oryza and so its origin is ancient and its existence predates the speciation events that originated the genus. finally we described the peculiar behavior of repeats in the species oryza coarctata whose placement in the oryza genus is controversial.
 the genus oryza is composed of distinct genome types, diploid and polyploid, and includes the world's most important food crop – rice. genome size variation in the oryza is more than 3-fold and ranges from mbp in oryza glaberrima to mbp in the polyploid oryza ridleyi. because repetitive elements are known to play a significant role in genome size variation, we constructed random sheared small insert genomic libraries from representative oryza species and conducted a comprehensive study of the repetitive element composition, distribution and phylogeny in this genus. particular attention was paid to the role played by the most important classes of transposable elements in shaping these genomes and in their contributing to genome size variation.  we identified the elements primarily responsible for the most strikingly genome size variation in oryza. we demonstrated how long terminal repeat retrotransposons belonging to the same families have proliferated to very different extents in various species. we also showed that the pool of long terminal repeat retrotransposons is substantially conserved and ubiquitous throughout the oryza and so its origin is ancient and its existence predates the speciation events that originated the genus. finally we described the peculiar behavior of repeats in the species oryza coarctata whose placement in the oryza genus is controversial.  long terminal repeat retrotransposons are the major component of the oryza genomes analyzed and, along with polyploidization, are the most important contributors to the genome size variation across the oryza genus. two families of ty3-gypsy elements account for a significant portion of the genome size variations present in the oryza genus. 