BACKGROUND
the origin  of asian cultivated rice has intrigued the science community for decades and is still hotly debated.
at dna level, origin means the beginning of the sequences of agricultural important loci.
it is well established that besides vertical transmission of genetic material from parent to offspring, horizontal process that genetic information move across mating barriers plays important role in the genome evolution  <cit> .
in rice species, it is widely accepted that horizontal process such as gene flow and introgression have occurred among o. sativa, o. nivara and o. rufipogon; and many studies of population genetics, e.g.
see reviews in  <cit> , have explored horizontal process between cultivated and wild rice.
however, few researches have focused on globally investigating, from the angle of genome comparison, the horizontal process between indica and japonica, the two reproductively isolated subspecies of o. sativa.
the difficulties come from at least two aspects: firstly, although horizontal process makes target sites showing higher conservation than it should be, observed high conservation does not necessarily the result of horizontal process because low substitution rate will also lead to that, as in the case of ultraconserved sequences in animals  <cit> .
secondly, two subspecies have relatively short history, thus the statistical analysis of slowly evolving sites such as genes may brings biased results because of too small number of substitutions.
therefore it is important to find proper material to perform the investigation.
tes are mobile repetitive dna that have been found in all eukaryotic genomes investigated so far  <cit> .
ltr retrotransposons are class i te that transpose in a "copy and paste" mode via intermediate rna.
the typical structural characters of an ltr retrotransposon include:  two highly similar ltr sequences;  4– <dig> bp target site repeats  at 5' and 3' ends;  primer binding site  downstream of 5' ltr and polypurine tract  upstream of 3' ltr;  protein domains important for transposition  <cit> .
they are predominant components of large plant genomes and their amplification and deletion have been taken as one of the major forces underlying the remarkable variation of plant genome size  <cit> .
historical information in full-length ltr elements allows one to trace the birth date and substitutions in elements.
if a ltr element inserts in a non-conserved region prior to the divergence of two genomes but its descendants show significant higher conservation than expected, the region must be involved in recently recombination that made alleles in two genome identical .
in addition, because of relatively high evolutionary speed, the amplification of ltr retrotransposons in short period makes them ideal markers to study short-term evolution.
with the two features, ltr elements can be used to test recent horizontal process between closely related genomes.
this report is designed to explore important events in the evolution of asian cultivated rice  from the angle of whole-genome comparison of ltr retrotransposons, with emphasis on revealing the isnr between indica and japonica.
we have developed ltr_insert to discover ltr retrotransposons in two related genomes.
unlike other methods, besides reporting the structure and location, ltr_insert tells whether an element inserts prior or posterior to the divergence of two genomes.
by applying ltr_insert to indica and japonica genome  <cit> , we identified  <dig> full-length ltr elements, annotated  <dig> copies related with them, and discovered at least  <dig> novel ltr families.
we found that many of these elements had significantly higher degree of sequence conservation than expected and that such high conservation was caused by isnr.
the subsequent large-scale sampling of protein-coding genes and random genomic sites showed that the phenomenon was not restricted to ltr retrotransposons and at least 15% of the genome was involved in isnr in the recent past.
in addition, ltr elements provided two independent evidences to confirm that two genomes diverged about  <dig>  years ago.
RESULTS
overview of ltr retrotransposons identified by ltr_insert
a pair of allelic shared ltr retrotransposons is two highly similar elements that are found at the same locus in two closely related genomes.
in contrast, if an element is present in one genome but its counterpart absent in the other, it is called an allelic specific element .
usually, allelic shared ltr retrotransposons derive from insertions prior to the divergence of two genomes , while specific ones though insertions posterior to that .
for simplicity, we use specific and shared to replace allelic specific and allelic shared sometimes in the following description.
ltr_insert scanned the indica-japonica comparative map  and identified  <dig> full-length elements, including  <dig>  shared and  <dig> specific ones.
among them,  <dig> specific elements were discovered in japonica and  <dig> in indicia.
ltr_insert also identified  <dig> allelic specific solo-ltrs  related with them.
with well-aligned flanking sequences and tsrs, most of the solo-ltrs must derive from intra-element unequal recombination of specific full-length elements.
they also represent post-divergence insertions because recombination between different elements usually results in solo-ltr without tsr and well-aligned flanking sequences  <cit> .
the subsequent blast search  retrieved more than  <dig>  potential ltr copies related with the full-length elements.
discarding short ones , we obtained a total of  <dig>  ltr copies in the two genomes.
in them, we extracted  <dig>  pairs of shared copies  that did not overlap with annotated gene models.
representing ltr-related intergenic alleles in two subspecies, these copies were used to test isnr between two genomes .
because the description of ltr elements themselves is not the main purpose of this report, we put in appendix  the detailed analysis of the rice ltr families, including the criteria of family, the phylogenetic analysis and the dynamics of elements.
here, we only give a brief account of major results.
we identified  <dig> rice ltr families  with  <dig> being reported for the first time .
<dig> of the  <dig> novel families  had no hit with any known elements in genbank  <cit> , tigr plant repeat database  <cit> , repbase  <cit>  and retroryza  <cit> .
although most of the  <dig> families lacked reliable long orfs similar to know domains, some of them were quite active.
we detected the rt domains in  <dig> families and the phylogenetic analysis based on the domains supported previous classification of rice ltr retrotransposons into copia and gypsy superfamilies .
the investigation of the amplification pattern of rice
ltr families in time and spacial dimensions reveals several features :  ltr retrotransposons have been active since the divergence of two genomes.
although there was five-fold more full-length specific elements identified in japonica than in indica, the analysis of all the ltr copies showed that their abundance was relative balance between two lineages.
80% of the post-divergence insertions were driven by 20% of highly active families .
these predominant families had been active in the common ancestor and the divergence event did not significantly change their activity.
the distribution of ltr elements  was non-random across the rice genome: the ltr density tends to decrease from centromeric regions to 5' and 3' ends of chromosomes.
besides centromeric neighborhoods, 5' ends of chromosomes were also ltr-dense regions.
ltr retrotransposons reveal recent extensive inter-species nonreciprocal recombination in cultivated rice
history inside ltr retrotransposons and isnr
full length ltr elements store information of their transposition history in the ltrs  <cit> .
when transposing, two identical ltrs synthesize from the same template, then they diverge with nucleotide mutations cumulating as time elapses.
given the average rate of nucleotide substitution  and sequence distance , insertion date  can be estimated by t = d/2r.
in this study, we use r =  <dig>  × 10-8/site/yr, as suggested in  <cit> .
further more, the comparison of specific and shared ltr elements in related species provides more information.
specific elements are mainly generated through post-divergence transposition events .
ideally, the distance between their two ltrs  should be less than the distance corresponding to the divergence time .
besides post-divergence insertion, the accurate deletion of a shared element in one genome  also makes its counterpart a specific element.
however, the probability is rather low because this requires that recombination occurs at the 4– <dig> bp tsrs.
in fact, just like insertions of lines and sines, transposition of ltr retrotransposons is almost irreversible.
even though such recombination occurs, it can be recognized by the age of this element: its dintra value is greater than d now.
in short, most specific elements represent post-divergence transposition events.
sources of shared elements can also be traced by analyzing history information inside full-length elements.
when two elements are orthologs, the distance between them  is just d and because the insertion occurs prior to the divergence, dintra > d holds.
overall one has dinter = d <dintra for orthologous shared elements .
indeed, when no isnr occurs, this is almost the unique source of shared elements because the probability that two independent transposition events in two species just insert into the same locus is low, the rather that they are highly similar.
shared elements may be converted by isnr and are not orthologs any more .
in this process, an allele in one genome is converted by its counterpart in the other through nonreciprocal homologous recombination.
we use the words "nonreciprocal homologous recombination" according to the suggestion in li   <cit> , to denote a type of recombination that "one sequence is changed whereas the other is not".
when isnr occurs, two alleles become identical and the history before isnr is removed.
as a result one observes that their pairwise distance is less than d. specifically, if the two alleles cover a pair of shared elements, one expect to see dinter <d <dintra because isnr, as a type of recombination, does not destroy the structure of elements and dintra still reflects the date of insertion, which occurs in the common ancestor.
ltr retrotransposons usually have relative high evolutionary speed, so a pair of elements is highly probable converted by isnr if its dinter value is significantly less than d.
theoretically, there are other possible ways to generate shared elements, e.g.
at a loci where one allele  contains a specific element but its counterpart  dose not, recombination converts x' by x. in this case, the shared pair shows dinter <dintra <d.
we found that two pairs of elements in rice  and  <dig>  see below) had such time pattern.
although small in sample size, the two examples indicate that such type of recombination might occur in the past.
in short, most shared and specific elements reflect pre- and post- divergence transposition events, respectively.
the relationships among dinter, d and dintra give evolutionary events that generate and modify elements.
we summarized the above discussion in table  <dig>  in practice, we infer that one of the relations hold and corresponding event took place in a group of elements based on high statistical significance.
many ltr retrotransposon-contained loci have been converted by isnr
we discarded elements of which ltrs were shorter than  <dig> bp and selected  <dig>  full-length elements for further analyses.
each pair of elements has two dintra values, one from the indica member, the other from its japonica counterpart.
as can be seen from figure  <dig>  the two values are overall equal or close to each other in pairs.
statistical analysis also supports this observation .
therefore, in the following discussion, we use the average of two values to represent dintra of each pair.
since distribution a fitted the normal distribution n well , we identified elements belong to it  by selecting pairs of which dinter values were greater than  <dig>  .
a total of  <dig> pairs were classified as group-a elements and the left  <dig> pairs as group-b elements ).
unlike some previous researches to estimate divergence date based on dintra  <cit> , we directly estimate the divergence time from dinter of orthologous elements: firstly, d = dinter holds in them because of normality of distribution a. secondly, we find that dinter <dintra in almost all pairs.
q <dig> and q <dig> in figure  <dig> are two execptions, but they can be explained by random fluctuation .
these observations support that group-a pairs are orthologs and two genomes diverged about  <dig>  mya .
distribution b is a sharp spike at dinter =  and the following characters are found in corresponding elements:  all of them show dinter <dintra.
dinter values of the  <dig> pairs are significantly  less than d.  most of them  have dintra values greater than d and 2/ <dig> greater than  <dig> my, the date corresponding to μ + 2σ ).
in summary, the inequality dinter <d <dintra holds in most of them.
further more, as can be seen from the insert figure in figure  <dig>  the great majority  of group-b pairs have dinter values less than  <dig>  , coincident with the time of the earliest evidence of modern human activities in south and east asia, which are thought to be potential regions of rice domestication.
most group-b elements are not located in conserved genomic regions because they have great dintra values, which reflect that great number of mutations cumulated in these regions.
therefore it is hard to explain their small dinter values by conservation of loci.
because the above analyses have shown that the majority of group-b elements meet the features of isnr, we propose that they were involved in such process.
providing more information than frequency histogram, scatterplot allows us to see more things.
figure  <dig> shows dinter versus dintra of the  <dig> pairs of shared elements.
according to the above discussions, the solid horizontal line  separates pairs into group-a  and group-b .
q <dig> and q <dig> are two pairs that lie above the dinter = dintra line .
that is, their dinter > dintra.
since their dintra values are small, dinter are close to μ and the phenomenon is only observed in two pairs, it is reasonable to explain the 3% outliers by random fluctuation.
p <dig> and p <dig> are two pairs located on the left of the vertical solid line, which means they have d > dintra significantly.
they also have dintra > dinter since they are located below the dashed line, but this relationship is not statistically significant because the sample size is rather low.
therefore, p <dig> and p <dig> might derive from inter-genome recombination of post-divergence insertions , but the current work lacks enough samples to confirm this beyond doubt.
whole-genome samplings reveal that at least 15% of rice genome has experienced isnr
large-scale sampling further confirmed that the same bimodal pattern of dinter existed in the entire genome, including non-ltr-related regions.
dinter is now defined as the distance between any pair of alleles and no longer restricted in shared ltr elements.
firstly, we investigated the  <dig>  intergenic ltr regions .
comparing to the shared full-length elements, the sample capacity increased  <dig> folds.
the distribution and percentage of group-b elements , table 2) are clearly consistent with figure 2: a peak is located at  <dig> – <dig>  and the two distributions separate at  <dig> – <dig> .
next we calculated pairwise distance of intron regions of  <dig>  pairs of cdna verified genes and  <dig>  pairs of bgf annotated gene models respectively  and found the bimodal pattern as well  and 3): the peak of distribution a is located at  <dig> – <dig>  and two distributions separate at  <dig> – <dig> .
the investigation of coding regions gave similar results .
a cdna verified genes.
b bgf predicted genes.
c different values result from varied window sizes.
genes and ltr elements may not accurately tell the boundaries of isnr loci because they are only subregions of the isnr loci .
we estimated the percentage of genomic sequences involved in isnr by random sampling of sites in the comparative map: windows of a certain length are randomly placed in the map and for each window, the mean mismatches/kb value  is calculated.
not preferring certain loci, the sampling provided less biased estimation of the percentage.
the calculation of mismatch density in window of 5– <dig> kb in  <dig>  random samplings confirmed the bimodal distribution: distribution a and b separate at  <dig> – <dig>  mismatches/kb and about  <dig> %– <dig> % of genomic sequences belong to group-b  and table 2).
by using a sliding window scanning the comparative map, we obtained the distribution of presumable isnr regions  in the rice genome.
as can been seen from figure  <dig>  instead of evenly distributed along the twelve pseudochromosomes, isnr regions are found to be more dense in chromosome 3– <dig>  while sparse in chromosome 9– <dig>  our investigation has shown that the rice ltr elements tend to be more dence at the centromeric regions in all chromosomes .
by contrast, isnr regions do not occur in the centromeric regions of eleven chromosomes except chromosome  <dig>  these results are consistent with that centromeric regions are rich in retroelements but contain regions with suppressed recombination.
although isnr regions seem to appear in the centromeric region of chromosome  <dig>  it should be caution that these highly conserved regions may actually not be in but near to that region because the centromeric regions are hard to sequence and we only obtained the approximate location information of centromeres from tigr rice genome annotation  <cit> .
in summary, the same bimodal pattern of the distribution of dinter appears in all cases investigated, including full-length ltr elements, genes, intergenic and random genomic regions with the sample capacity ranging from one hundred to tens of thousands.
the consistency indicates that a significant portion of rice genomes have experienced isnr.
the percentage is more than 15% of the rice genome and isnr took place with high frequency in recent past.
as the time is overlapping with the rising of modern human, it is highly probable that isnr is related with the process of domestication.
it is important to emphasize that isnr may take place directly between two subspecies or via other related genomes, e.g.
those of wild rice.
as a type of horizontal process, isnr provides a mechanism to spread alleles among cultivated rice and their wild relatives.
the present research confirms that it is an important force in the evolution of rice.
because of more than 20% protein-coding genes are belong to distribution b  and  <dig>  table 2), isnr might have greatly influenced the domestication of important traits.
besides evidences of this study, a recent population genetics analysis has shown that sh <dig>  the grain shattering gene, is once origin and spread in all cultivated rice via horizontal gene flow  <cit> .
these results further indicate that isnr, as well as other horizontal forces, may have had impact on the domestication important traits.
two genomes diverged about  <dig>  years ago
the above normality and orthology analysis of dinter for the group-a elements gave that peak-a reflected the divergence of two genomes and it occurred about  <dig>  mya.
this argument can be further supported by dintra of elements.
because most of the specific elements insert posterior to the divergence of two subspecies and most shared ones insert prior to that, so the distributions of dintra of specific and shared elements give the upper and lower limit of the divergence date, respectively.
as shown in figure  <dig> and  <dig>  the insertion dates of specific and shared elements naturally belonged to two periods.
dintra values are less than  <dig>  in 90% of specific elements while greater than  <dig>  in the same percentage of shared pairs.
this result well supports that most specific and shared elements represent post- and pre- divergence insertions, respectively.
since dintra =  corresponds to  <dig> – <dig>  mya, the fact that peak-a is located in the period is consistent with that distribution a reflects orthologous loci and two genomes diverged about  <dig>  mya, a greater value than some recent estimations  <cit> .
evolutionary speed is 5– <dig> folds faster in ltr regions than in genes
analogously, the distribution a of other cases should also represent distribution of orthologous loci.
by comparing figure  <dig> and  <dig> to figure  <dig> and  <dig>  it is easy to observe that the distance values corresponding to peak-a, peak-b, and the separation of two distribution are systematically 5– <dig> times less in gene regions.
we also find that mean dinter value of introns and ds value of coding sequences have no significant difference in group-a sequences .
these results indicate that mean speed of substitution in ltr-related loci is 5– <dig> times faster than in genic regions and the value is higher than the estimation of  <cit> .
extensive isnr in rice add puzzles to the origin of cultivated rice
traditionally, there are two models about the domestication of rice: the "single origin" hypothesis claims that the cultivated rice was only domesticated once and the differentiation of two subspecies was latter than the domestication of important traits, which took place no earlier than the beginning of agriculture  <cit> .
on the other hand, the multiple origin" hypothesis argues that domestication occurred at least twice; two subspecies were cultivated from diverged wild populations and their progenitors had already diverged by the beginning of agriculture  <cit> .
to date, the divergence time  of the two rice genomes has been widely used as the decisive criterion for this question.
recently, researchers have tried to estimate the distance from varied loci and the "multiple origin" model seems to be supported by different molecular materials  <cit> .
however, this criterion is valid only when the influence of horizontal process is trivial.
if horizontal process was important in the early stage of domestication, as has been shown in this report, indica and japonica may have ancient diverged genomic "background" yet the domestication of important traits only took place once.
in other words, traits might be domesticated first in one type of rice and the other subspecies originated from some far-related wild rice which hybridized with it and acquired these traits through isnr.
besides this picture, isnr can provide multiple possibilities on the rice domestication.
for instance, groups of traits might be independently domesticated with host genomes and subsequently combined through isnr to generate two types of cultivated rice  <cit> .
this work reveals that rice genomes are mosaic, which reconciles the once-multiple origination conflict but complicates the origin of rice: one must face distinctive histories of traits now!
to reconstruct the history of cultivated rice, isnr, as well as other horizontal process, must be considered.
only when both vertical and horizontal forces are well understood, the domestication can be understood in-depth.
CONCLUSIONS
we have introduced a new method, ltr_insert, to detect ltr retrotransposons in two related genomes and use them to explore the history of asian cultivated rice.
by discriminating between pre- and post-divergence transposition events, ltr_insert provides rich information on the evolution of both rice and ltr elements themselves.
through ltr elements and whole-genome scale loci samplings, we find that at least 15% of the rice genome has experienced isnr in very recent past.
in addition, the analyses of ltr elements also confirm that the two rice genomes diverged about  <dig>  years b.p.. these results clearly show that isnr has actively participated in shaping of the cultivated rice genome.

 the origin of asian cultivated rice has intrigued the science community for decades and is still hotly debated. at dna level, origin means the beginning of the sequences of agricultural important loci. it is well established that besides vertical transmission of genetic material from parent to offspring, horizontal process that genetic information move across mating barriers plays important role in the genome evolution. in rice species, it is widely accepted that horizontal process such as gene flow and introgression have occurred among o. sativa, o. nivara and o. rufipogon; and many studies of population genetics, e.g. see reviews in, have explored horizontal process between cultivated and wild rice. however, few researches have focused on globally investigating, from the angle of genome comparison, the horizontal process between indica and japonica, the two reproductively isolated subspecies of o. sativa. the difficulties come from at least two aspects: firstly, although horizontal process makes target sites showing higher conservation than it should be, observed high conservation does not necessarily the result of horizontal process because low substitution rate will also lead to that, as in the case of ultraconserved sequences in animals. secondly, two subspecies have relatively short history, thus the statistical analysis of slowly evolving sites such as genes may brings biased results because of too small number of substitutions. therefore it is important to find proper material to perform the investigation. tes are mobile repetitive dna that have been found in all eukaryotic genomes investigated so far. ltr retrotransposons are class i te that transpose in a "copy and paste" mode via intermediate rna. the typical structural characters of an ltr retrotransposon include: two highly similar ltr sequences; 4– bp target site repeats at 5' and 3' ends; primer binding site downstream of 5' ltr and polypurine tract upstream of 3' ltr; protein domains important for transposition. they are predominant components of large plant genomes and their amplification and deletion have been taken as one of the major forces underlying the remarkable variation of plant genome size. historical information in full-length ltr elements allows one to trace the birth date and substitutions in elements. if a ltr element inserts in a non-conserved region prior to the divergence of two genomes but its descendants show significant higher conservation than expected, the region must be involved in recently recombination that made alleles in two genome identical. in addition, because of relatively high evolutionary speed, the amplification of ltr retrotransposons in short period makes them ideal markers to study short-term evolution. with the two features, ltr elements can be used to test recent horizontal process between closely related genomes. this report is designed to explore important events in the evolution of asian cultivated rice from the angle of whole-genome comparison of ltr retrotransposons, with emphasis on revealing the isnr between indica and japonica. we have developed ltr_insert to discover ltr retrotransposons in two related genomes. unlike other methods, besides reporting the structure and location, ltr_insert tells whether an element inserts prior or posterior to the divergence of two genomes. by applying ltr_insert to indica and japonica genome, we identified full-length ltr elements, annotated copies related with them, and discovered at least novel ltr families. we found that many of these elements had significantly higher degree of sequence conservation than expected and that such high conservation was caused by isnr. the subsequent large-scale sampling of protein-coding genes and random genomic sites showed that the phenomenon was not restricted to ltr retrotransposons and at least 15% of the genome was involved in isnr in the recent past. in addition, ltr elements provided two independent evidences to confirm that two genomes diverged about years ago.  overview of ltr retrotransposons identified by ltr_insert a pair of allelic shared ltr retrotransposons is two highly similar elements that are found at the same locus in two closely related genomes. in contrast, if an element is present in one genome but its counterpart absent in the other, it is called an allelic specific element. usually, allelic shared ltr retrotransposons derive from insertions prior to the divergence of two genomes, while specific ones though insertions posterior to that. for simplicity, we use specific and shared to replace allelic specific and allelic shared sometimes in the following description. ltr_insert scanned the indica-japonica comparative map and identified full-length elements, including shared and specific ones. among them, specific elements were discovered in japonica and in indicia. ltr_insert also identified allelic specific solo-ltrs related with them. with well-aligned flanking sequences and tsrs, most of the solo-ltrs must derive from intra-element unequal recombination of specific full-length elements. they also represent post-divergence insertions because recombination between different elements usually results in solo-ltr without tsr and well-aligned flanking sequences. the subsequent blast search retrieved more than potential ltr copies related with the full-length elements. discarding short ones, we obtained a total of ltr copies in the two genomes. in them, we extracted pairs of shared copies that did not overlap with annotated gene models. representing ltr-related intergenic alleles in two subspecies, these copies were used to test isnr between two genomes. because the description of ltr elements themselves is not the main purpose of this report, we put in appendix the detailed analysis of the rice ltr families, including the criteria of family, the phylogenetic analysis and the dynamics of elements. here, we only give a brief account of major results. we identified rice ltr families with being reported for the first time. of the novel families had no hit with any known elements in genbank, tigr plant repeat database, repbase and retroryza. although most of the families lacked reliable long orfs similar to know domains, some of them were quite active. we detected the rt domains in families and the phylogenetic analysis based on the domains supported previous classification of rice ltr retrotransposons into copia and gypsy superfamilies. the investigation of the amplification pattern of rice ltr families in time and spacial dimensions reveals several features: ltr retrotransposons have been active since the divergence of two genomes. although there was five-fold more full-length specific elements identified in japonica than in indica, the analysis of all the ltr copies showed that their abundance was relative balance between two lineages. 80% of the post-divergence insertions were driven by 20% of highly active families. these predominant families had been active in the common ancestor and the divergence event did not significantly change their activity. the distribution of ltr elements was non-random across the rice genome: the ltr density tends to decrease from centromeric regions to 5' and 3' ends of chromosomes. besides centromeric neighborhoods, 5' ends of chromosomes were also ltr-dense regions. ltr retrotransposons reveal recent extensive inter-species nonreciprocal recombination in cultivated rice history inside ltr retrotransposons and isnr full length ltr elements store information of their transposition history in the ltrs. when transposing, two identical ltrs synthesize from the same template, then they diverge with nucleotide mutations cumulating as time elapses. given the average rate of nucleotide substitution and sequence distance, insertion date can be estimated by t = d/2r. in this study, we use r = × 10-8/site/yr, as suggested in. further more, the comparison of specific and shared ltr elements in related species provides more information. specific elements are mainly generated through post-divergence transposition events. ideally, the distance between their two ltrs should be less than the distance corresponding to the divergence time. besides post-divergence insertion, the accurate deletion of a shared element in one genome also makes its counterpart a specific element. however, the probability is rather low because this requires that recombination occurs at the 4– bp tsrs. in fact, just like insertions of lines and sines, transposition of ltr retrotransposons is almost irreversible. even though such recombination occurs, it can be recognized by the age of this element: its dintra value is greater than d now. in short, most specific elements represent post-divergence transposition events. sources of shared elements can also be traced by analyzing history information inside full-length elements. when two elements are orthologs, the distance between them is just d and because the insertion occurs prior to the divergence, dintra > d holds. overall one has dinter = d <dintra for orthologous shared elements. indeed, when no isnr occurs, this is almost the unique source of shared elements because the probability that two independent transposition events in two species just insert into the same locus is low, the rather that they are highly similar. shared elements may be converted by isnr and are not orthologs any more. in this process, an allele in one genome is converted by its counterpart in the other through nonreciprocal homologous recombination. we use the words "nonreciprocal homologous recombination" according to the suggestion in li, to denote a type of recombination that "one sequence is changed whereas the other is not". when isnr occurs, two alleles become identical and the history before isnr is removed. as a result one observes that their pairwise distance is less than d. specifically, if the two alleles cover a pair of shared elements, one expect to see dinter <d <dintra because isnr, as a type of recombination, does not destroy the structure of elements and dintra still reflects the date of insertion, which occurs in the common ancestor. ltr retrotransposons usually have relative high evolutionary speed, so a pair of elements is highly probable converted by isnr if its dinter value is significantly less than d. theoretically, there are other possible ways to generate shared elements, e.g. at a loci where one allele contains a specific element but its counterpart dose not, recombination converts x' by x. in this case, the shared pair shows dinter <dintra <d. we found that two pairs of elements in rice and see below) had such time pattern. although small in sample size, the two examples indicate that such type of recombination might occur in the past. in short, most shared and specific elements reflect pre- and post- divergence transposition events, respectively. the relationships among dinter, d and dintra give evolutionary events that generate and modify elements. we summarized the above discussion in table in practice, we infer that one of the relations hold and corresponding event took place in a group of elements based on high statistical significance. many ltr retrotransposon-contained loci have been converted by isnr we discarded elements of which ltrs were shorter than bp and selected full-length elements for further analyses. each pair of elements has two dintra values, one from the indica member, the other from its japonica counterpart. as can be seen from figure the two values are overall equal or close to each other in pairs. statistical analysis also supports this observation. therefore, in the following discussion, we use the average of two values to represent dintra of each pair. since distribution a fitted the normal distribution n well, we identified elements belong to it by selecting pairs of which dinter values were greater than. a total of pairs were classified as group-a elements and the left pairs as group-b elements ). unlike some previous researches to estimate divergence date based on dintra, we directly estimate the divergence time from dinter of orthologous elements: firstly, d = dinter holds in them because of normality of distribution a. secondly, we find that dinter <dintra in almost all pairs. q and q in figure are two execptions, but they can be explained by random fluctuation. these observations support that group-a pairs are orthologs and two genomes diverged about mya. distribution b is a sharp spike at dinter = and the following characters are found in corresponding elements: all of them show dinter <dintra. dinter values of the pairs are significantly less than d. most of them have dintra values greater than d and 2/ greater than my, the date corresponding to μ + 2σ ). in summary, the inequality dinter <d <dintra holds in most of them. further more, as can be seen from the insert figure in figure the great majority of group-b pairs have dinter values less than, coincident with the time of the earliest evidence of modern human activities in south and east asia, which are thought to be potential regions of rice domestication. most group-b elements are not located in conserved genomic regions because they have great dintra values, which reflect that great number of mutations cumulated in these regions. therefore it is hard to explain their small dinter values by conservation of loci. because the above analyses have shown that the majority of group-b elements meet the features of isnr, we propose that they were involved in such process. providing more information than frequency histogram, scatterplot allows us to see more things. figure shows dinter versus dintra of the pairs of shared elements. according to the above discussions, the solid horizontal line separates pairs into group-a and group-b. q and q are two pairs that lie above the dinter = dintra line. that is, their dinter > dintra. since their dintra values are small, dinter are close to μ and the phenomenon is only observed in two pairs, it is reasonable to explain the 3% outliers by random fluctuation. p and p are two pairs located on the left of the vertical solid line, which means they have d > dintra significantly. they also have dintra > dinter since they are located below the dashed line, but this relationship is not statistically significant because the sample size is rather low. therefore, p and p might derive from inter-genome recombination of post-divergence insertions, but the current work lacks enough samples to confirm this beyond doubt. whole-genome samplings reveal that at least 15% of rice genome has experienced isnr large-scale sampling further confirmed that the same bimodal pattern of dinter existed in the entire genome, including non-ltr-related regions. dinter is now defined as the distance between any pair of alleles and no longer restricted in shared ltr elements. firstly, we investigated the intergenic ltr regions. comparing to the shared full-length elements, the sample capacity increased folds. the distribution and percentage of group-b elements, table 2) are clearly consistent with figure 2: a peak is located at – and the two distributions separate at –. next we calculated pairwise distance of intron regions of pairs of cdna verified genes and pairs of bgf annotated gene models respectively and found the bimodal pattern as well and 3): the peak of distribution a is located at – and two distributions separate at –. the investigation of coding regions gave similar results. a cdna verified genes. b bgf predicted genes. c different values result from varied window sizes. genes and ltr elements may not accurately tell the boundaries of isnr loci because they are only subregions of the isnr loci. we estimated the percentage of genomic sequences involved in isnr by random sampling of sites in the comparative map: windows of a certain length are randomly placed in the map and for each window, the mean mismatches/kb value is calculated. not preferring certain loci, the sampling provided less biased estimation of the percentage. the calculation of mismatch density in window of 5– kb in random samplings confirmed the bimodal distribution: distribution a and b separate at – mismatches/kb and about %– % of genomic sequences belong to group-b and table 2). by using a sliding window scanning the comparative map, we obtained the distribution of presumable isnr regions in the rice genome. as can been seen from figure instead of evenly distributed along the twelve pseudochromosomes, isnr regions are found to be more dense in chromosome 3– while sparse in chromosome 9– our investigation has shown that the rice ltr elements tend to be more dence at the centromeric regions in all chromosomes. by contrast, isnr regions do not occur in the centromeric regions of eleven chromosomes except chromosome these results are consistent with that centromeric regions are rich in retroelements but contain regions with suppressed recombination. although isnr regions seem to appear in the centromeric region of chromosome it should be caution that these highly conserved regions may actually not be in but near to that region because the centromeric regions are hard to sequence and we only obtained the approximate location information of centromeres from tigr rice genome annotation. in summary, the same bimodal pattern of the distribution of dinter appears in all cases investigated, including full-length ltr elements, genes, intergenic and random genomic regions with the sample capacity ranging from one hundred to tens of thousands. the consistency indicates that a significant portion of rice genomes have experienced isnr. the percentage is more than 15% of the rice genome and isnr took place with high frequency in recent past. as the time is overlapping with the rising of modern human, it is highly probable that isnr is related with the process of domestication. it is important to emphasize that isnr may take place directly between two subspecies or via other related genomes, e.g. those of wild rice. as a type of horizontal process, isnr provides a mechanism to spread alleles among cultivated rice and their wild relatives. the present research confirms that it is an important force in the evolution of rice. because of more than 20% protein-coding genes are belong to distribution b and table 2), isnr might have greatly influenced the domestication of important traits. besides evidences of this study, a recent population genetics analysis has shown that sh the grain shattering gene, is once origin and spread in all cultivated rice via horizontal gene flow. these results further indicate that isnr, as well as other horizontal forces, may have had impact on the domestication important traits. two genomes diverged about years ago the above normality and orthology analysis of dinter for the group-a elements gave that peak-a reflected the divergence of two genomes and it occurred about mya. this argument can be further supported by dintra of elements. because most of the specific elements insert posterior to the divergence of two subspecies and most shared ones insert prior to that, so the distributions of dintra of specific and shared elements give the upper and lower limit of the divergence date, respectively. as shown in figure and the insertion dates of specific and shared elements naturally belonged to two periods. dintra values are less than in 90% of specific elements while greater than in the same percentage of shared pairs. this result well supports that most specific and shared elements represent post- and pre- divergence insertions, respectively. since dintra = corresponds to – mya, the fact that peak-a is located in the period is consistent with that distribution a reflects orthologous loci and two genomes diverged about mya, a greater value than some recent estimations. evolutionary speed is 5– folds faster in ltr regions than in genes analogously, the distribution a of other cases should also represent distribution of orthologous loci. by comparing figure and to figure and it is easy to observe that the distance values corresponding to peak-a, peak-b, and the separation of two distribution are systematically 5– times less in gene regions. we also find that mean dinter value of introns and ds value of coding sequences have no significant difference in group-a sequences. these results indicate that mean speed of substitution in ltr-related loci is 5– times faster than in genic regions and the value is higher than the estimation of. extensive isnr in rice add puzzles to the origin of cultivated rice traditionally, there are two models about the domestication of rice: the "single origin" hypothesis claims that the cultivated rice was only domesticated once and the differentiation of two subspecies was latter than the domestication of important traits, which took place no earlier than the beginning of agriculture. on the other hand, the multiple origin" hypothesis argues that domestication occurred at least twice; two subspecies were cultivated from diverged wild populations and their progenitors had already diverged by the beginning of agriculture. to date, the divergence time of the two rice genomes has been widely used as the decisive criterion for this question. recently, researchers have tried to estimate the distance from varied loci and the "multiple origin" model seems to be supported by different molecular materials. however, this criterion is valid only when the influence of horizontal process is trivial. if horizontal process was important in the early stage of domestication, as has been shown in this report, indica and japonica may have ancient diverged genomic "background" yet the domestication of important traits only took place once. in other words, traits might be domesticated first in one type of rice and the other subspecies originated from some far-related wild rice which hybridized with it and acquired these traits through isnr. besides this picture, isnr can provide multiple possibilities on the rice domestication. for instance, groups of traits might be independently domesticated with host genomes and subsequently combined through isnr to generate two types of cultivated rice. this work reveals that rice genomes are mosaic, which reconciles the once-multiple origination conflict but complicates the origin of rice: one must face distinctive histories of traits now! to reconstruct the history of cultivated rice, isnr, as well as other horizontal process, must be considered. only when both vertical and horizontal forces are well understood, the domestication can be understood in-depth.  we have introduced a new method, ltr_insert, to detect ltr retrotransposons in two related genomes and use them to explore the history of asian cultivated rice. by discriminating between pre- and post-divergence transposition events, ltr_insert provides rich information on the evolution of both rice and ltr elements themselves. through ltr elements and whole-genome scale loci samplings, we find that at least 15% of the rice genome has experienced isnr in very recent past. in addition, the analyses of ltr elements also confirm that the two rice genomes diverged about years b.p.. these results clearly show that isnr has actively participated in shaping of the cultivated rice genome. 
ltr_insert identifies full-length ltr elements, annotates copies related with them, and discovers at least novel ltr families in the whole-genome comparative map of two cultivated rice subspecies. because of relatively high evolutionary speed, active transposition of ltr elements in the host genomes provides rich information on their short-term history. the present research is designed to investigate important evolutionary events in the origin of asian cultivated rice through the comparison of ltr elements. we have developed ltr_insert, a new method for ltr elements discovery in two closely related genomes. from the full-length ltr elements, we estimate that a significant proportion of the rice genome has experienced inter-subspecies nonreciprocal recombination in as recent as years. in addition, ltr elements confirm that the genome of o. sativa ssp. as more and more genomes, especially those of closely related organisms, have been sequenced, it is possible to perform global comparative study of their ltr retrotransposons to reveal events in the history. our method has a distinctive feature that it is capable of judging whether an insertion occurs prior or posterior to the divergence of genomes. a new ltr retrotransposon identification method integrating both comparative genomics and ab initio algorithm is introduced and applied to asian cultivated rice genomes. they are considered to be one of the major forces underlying plant genome evolution. large-scale samplings further support that more than 15% of the rice genome has been involved in such recombination. at whole-genome level, this work confirms that recent isnr is an important factor that molds modern cultivated rice genome.
 long terminal repeats retrotransposons are ubiquitous eukaryotic transposable elements. they are considered to be one of the major forces underlying plant genome evolution. because of relatively high evolutionary speed, active transposition of ltr elements in the host genomes provides rich information on their short-term history. as more and more genomes, especially those of closely related organisms, have been sequenced, it is possible to perform global comparative study of their ltr retrotransposons to reveal events in the history.  the present research is designed to investigate important evolutionary events in the origin of asian cultivated rice through the comparison of ltr elements. we have developed ltr_insert, a new method for ltr elements discovery in two closely related genomes. our method has a distinctive feature that it is capable of judging whether an insertion occurs prior or posterior to the divergence of genomes. ltr_insert identifies full-length ltr elements, annotates copies related with them, and discovers at least novel ltr families in the whole-genome comparative map of two cultivated rice subspecies. from the full-length ltr elements, we estimate that a significant proportion of the rice genome has experienced inter-subspecies nonreciprocal recombination in as recent as years. large-scale samplings further support that more than 15% of the rice genome has been involved in such recombination. in addition, ltr elements confirm that the genome of o. sativa ssp. indica and that of japonica diverged about years ago.  a new ltr retrotransposon identification method integrating both comparative genomics and ab initio algorithm is introduced and applied to asian cultivated rice genomes. at whole-genome level, this work confirms that recent isnr is an important factor that molds modern cultivated rice genome. 