BACKGROUND
noncoding intronic and intergenic dna of multicellular organisms typically comprises a large fraction of their genomes.
comparative genomic studies have revealed extensive evolutionary conservation of noncoding dna in several mammalian and other species and are beginning to reveal the extent of potentially functional noncoding dna  <cit> .
several lines of evidence have suggested that introns harbour a variety of untranslated rnas  that are involved in mrna processing, editing and transport  <cit> .
in plants, conserved noncoding sequences have been first identified in the grasses  <cit> , and evidence of regulatory elements or binding sites in these noncoding sequences has been obtained  <cit> .
interestingly, in arabidopsis thaliana, based on a well-documented recent genome duplication event, intragenomic conserved noncoding sequences have also been investigated, and a unique set of noncoding dna sequences enriched for function has been uncovered  <cit> .
the above observations indicate that at least some functional regions in introns are likely to be under the influence of natural selection in plants in general.
selective constraint  is defined here as the factor by which evolutionary divergence of a functional sequence is reduced, relative to a neutrally evolving sequence, due to the action of purifying selection  <cit> .
several methods for estimating of evolutionary constraints have been proposed, and applied to coding and noncoding dna of invertebrates and mammals  <cit> .
shabalina and kondrashov  <cit>  proposed a method to quantify the proportion of bases that are subject to strong purifying selection by comparing the genomes of distantly related species.
it is assumed that homologous segments that show significant similarity are under strong functional constraints, otherwise are evolving free from functional constraints.
another approach to identify functional regions in the genome is to compare sequences from species showing lower levels of divergence that are far from saturation  <cit> .
the basis of the method is to compare the relative divergence of putatively constrained segments of the genome with that of linked putatively neutrally evolving sequences.
in the selectively constrained segments, nucleotides are assumed to fall into two classes: neutral, which evolve at the same rate as the neutral sequence; or strongly constrained, in which mutations are eliminated unconditionally by natural selection.
selective constraint is then the proportion of new mutations that are strongly deleterious and removed by purifying selection  <cit> .
it should be noted that the presence of adaptive substitutions tends to lead to underestimation of constraint, since this leads to divergence of functional regions.
one difficulty in analyzing evolutionary constraints in noncoding dna is the inference of the correct sequence alignment.
if the sequence alignment method tends to miss genuine similarities, then functional elements could be miss-assigned as non-functional.
this uncertainty largely arises due to the unknown pattern of indels  between the pair of sequences  <cit> .
a solution to this problem is to compute probabilities of alternative alignments according to explicit models of indel evolution.
based on this method, mcalign <dig> has been developed to tackle the problem of aligning noncoding dna  <cit> .
selective constraints of introns have recently been investigated in drosophila, mammals and other animals  <cit> .
several patterns of nucleotide divergence, polymorphism, and selective constraints have been uncovered .
until recently, no such investigation has been done in plants.
the methodology chosen to study the pattern of noncoding dna evolution heavily depends on the dataset investigated.
in general, noncoding dna sequences need to be not too far diverged, so that it is not too difficult to align them.
on the other hand, sequences should not be too similar, otherwise there may be insufficient statistical power available for comparative genomics analysis.
until now, all studies of evolutionary constraints have compared different lineages, under the assumption of the same selective pressures acting on them .
here, we have compared intronic sequences from just one species, a dataset including  <dig> paralogous pairs from a recent segmental duplication in rice .
the duplication event encompasses a ~ <dig> mb segmental pair with perfect synteny between chromosome  <dig> and  <dig>  <cit> .
the duplication is estimated to have occurred about  <dig> million years ago   <cit> , although an alternative date of  <dig> mya has also been proposed  <cit> .
the evolutionary divergence is compatible with estimates for human-chimpanzee  and members of the drosophila genus , which have been previously used for noncoding dna analysis .
their average divergences are about  <dig>  between drosophila simulans and melanogaster, about  <dig>  for human-chimpanzee, whereas ours is about  <dig> .
the divergence of this segment is more suitable for noncoding dna analysis than, for example, different rice subspecies, or rice and other cereals.
rice has two cultivated subspecies, indica and japonica, for which the genomes have been sequenced.
however, the two subspecies separated within about  <dig>  mya  <cit> , so their sequence similarity is too high and power to infer constraints is low.
the divergence time of rice and other cereals is estimated to be about  <dig> mya  <cit> , and alignment of noncoding sequences between them is usually problematic.
RESULTS
compilation of intron dataset
after intron alignment and some necessary masking, a dataset of  <dig> intron pairs  was generated.
the  <dig> pairs come from  <dig> duplicated gene pairs  from a recent duplication of rice chromosomes  <dig> and  <dig> .
among the  <dig> introns, median length was  <dig> bp .
the dataset included  <dig> first introns of median length  <dig> bp , whereas non-first introns had median length  <dig> bp .
it should be noted that only first intron pairs in which both introns were first introns were considered, and the same criterion was used for non-first introns.
first introns are significantly longer than non-first introns , which parallels findings for other species investigated  <cit> .
our dataset of  <dig> duplicated gene pairs is similar to that investigated by wang et al.
<cit> , and other studies , although the identification approaches used by us are slightly different.
in this study, we employed several methods to minimize the frequency of incorrect alignments.
these included amino acid-guided methods  to anchor the coding regions of a paralogous gene pair , alignment using explicit models of indel evolution , and the use of two masking protocols for nonhomologous sites .
our finals sample size of  <dig> intron pairs from  <dig> loci is compatible with other similar studies.
for example, 200– <dig> loci were used by keightley and gaffney  <cit> ,  <dig> loci by halligan et al.
<cit>  and  <dig> intron segments by haddrill et al.
<cit> .
intron length
a negative correlation between intron length and nucleotide divergence is present in our dataset  .
this result therefore suggests that regulatory elements may be more common in long than short introns.
a significant negative correlation between divergence and intron length has also been observed in other species that have been investigated   <cit> .
intron ordinal position
to further investigate the negative correlation between divergence and intron length described above, we divided our dataset into two subsets of first and non-first introns, and calculated correlation coefficients between length and divergence for each subset separately.
the results indicate that the negative correlation between divergence and intron length is significant in first introns, while the test statistic for non-first introns is marginally significant .
if introns are divided into two different sets according their length, there is a significant difference in divergence between short and long introns for first introns, whereas the difference is non-significant for non-first introns .
in some other taxa, first introns appear to have a higher frequency of regulatory elements  <cit> .
it has thus been suggested that a relationship between intron size and divergence might only be expected for first introns  <cit> .
our results in rice seem to support this point.
we further studied the relationship between the ordinal position  of introns in a gene and divergence .
the global correlation between intron order and ki is non-significant .
we also divided the dataset into two subsets based on first or non-first introns.
similarly, no significant difference in divergence was found between first and non-first introns  .
this indicates that divergence does not decay slowly and regularly with the intronic ordinal position in a gene, which contrasts with the trends observed in the human-chimpanzee comparison  <cit> .
in addition to single nucleotide mutations, we also investigate the frequency distribution of indels in first and non-first intron.
a total of  <dig>  indels were identified in our dataset, and no significant difference in frequencies of indel lengths between first and non-first intron was observed .
however, significant differences between indel numbers and lengths per base or gene pair were observed , with more indels in first than non-first introns.
this result indicates that the evolutionary pattern of indels seems to be somewhat different from nucleotide divergence in introns in rice.
whether this trend exists in other plants or animal species need further investigation.
in summary, selective constraints seem not to be specific to first intron in rice, so our results are similar to those previously reported in drosophila.
in a comparison of two species of drosophila , haddrill et al.
<cit>  found that first introns evolve at similar rates to other introns.
in rodents and mammals, however, it has been reported that divergence varies along introns and depend on their ordinal position within gene.
gaffney and keightley  <cit>  observed a negative correlation between mean intronic selective constraint and intron ordinal number in rodents, implying that first introns are more conserved other introns.
level of intronic divergence between humans and closely related species suggest that divergence also depends on intronic ordinal number  <cit> .
the above results indicate that the rule of high constraint at first introns is not common to all taxonomic groups.
whether the phenomenon is present in other plants needs further investigation.
splice control sites
we next examined constraints near the 5' and 3' ends of introns, which contain splice control motifs  <cit> .
as expected, there is a strong signal of purifying selection in the sequences within  <dig> bp of the 5' and 3' ends, particularly at the dinucleotides adjacent to the 5' and 3' splice sites .
similar observation has been reported in rodents  <cit>  and drosophila  <cit> .
the distribution of constraints in introns moving away from the splice sites, however, indicates that the regions under strong constraints in rice are quite short, only about  <dig> bp at the 5' end and even shorter at the 3' end .
this situation is similar to what has been inferred in drosophila  <cit> .
gc content
the base composition of gramineae genes is distinct from that of dicot genes.
for example, in gramineae genes gc content is relative high, and there is a gradient of gc content along the direction of transcription  <cit> .
in our previous study, we investigated gc content evolution in coding regions  <cit> .
here we focused on gc content evolution of intronic regions.
gc content shows a significant difference between first introns and non-first introns, even in subgroups with different length .
there is also a negative gradient of gc content with intronic ordinal position, which is similar to that seen in coding sequence with transcriptional direction.
these results suggest that a mechanism involving base mutation may act on first introns to elevate their gc content.
although we observed a specific pattern of nucleotide substitution in first introns , in contrast, no significant relationship between gc content and divergence  or intron length  was observed .
we also calculated the relationship between gc content and divergence and intron length in the two datasets .
similarly, no significant relationships were detected .
this result suggests that intron length and divergence are not a confounding effect of gc content in rice.
in other words, gc content is dependent of the ordinal position of introns, but not divergence and length.
this result is dissimilar to studies on drosophila and mammals  <cit> , in whichdivergence is negatively correlated with gc content.
mammalian first introns are richer in gc content and higher in divergence than other introns.
in rice, first introns are also gc-rich, but do not have a significantly higher divergence than other introns.
substitution pattern
we used nucleotides from the fastest evolving intronic  sites as putatively neutral standards to calculate constraint.
although exonic four-fold degenerate  sites are often used as a standard against which to test for deviations from neutrality, sites in short introns evolve faster in our data set , so are more appropriate as a neutral standard .
the fei sites refer to those nucleotides not close to exon boundaries  and outside of first introns.
similar regions have previously been used to quantify functional constraints in noncoding dna  <cit> .
in general, fractions of nucleotide differences at fei sites are consistently higher than 4-fold sites and first introns.
the transition events a↔g and t↔c changes are expected to be the most common substitutional changes in all categories of sites .
the situation at 4-fold sites has previously been observed in rice coding sequences, where the two changes a↔g and t↔c are predominantly from a/t to g/c, and thereby increase gc content  <cit> .
beside of transition t↔c, the fractions of transversion c↔g change are relatively higher than other four types of nucleotide changes in first introns compared to introns in general.
CONCLUSIONS
we investigate selective constraints in a recent segmental duplication that includes  <dig> paralogous intron pairs that occurred about  <dig> million years ago in rice.
our observation of stronger selective constraints in long introns suggests that functional elements subject to purifying selection may be concentrated within long introns.
our results are consistent with the presence of strong purifying selection at splicing control sites.
selective constraints are not significantly stronger in first introns of rice, as they are in other species.

 noncoding intronic and intergenic dna of multicellular organisms typically comprises a large fraction of their genomes. comparative genomic studies have revealed extensive evolutionary conservation of noncoding dna in several mammalian and other species and are beginning to reveal the extent of potentially functional noncoding dna. several lines of evidence have suggested that introns harbour a variety of untranslated rnas that are involved in mrna processing, editing and transport. in plants, conserved noncoding sequences have been first identified in the grasses, and evidence of regulatory elements or binding sites in these noncoding sequences has been obtained. interestingly, in arabidopsis thaliana, based on a well-documented recent genome duplication event, intragenomic conserved noncoding sequences have also been investigated, and a unique set of noncoding dna sequences enriched for function has been uncovered. the above observations indicate that at least some functional regions in introns are likely to be under the influence of natural selection in plants in general. selective constraint is defined here as the factor by which evolutionary divergence of a functional sequence is reduced, relative to a neutrally evolving sequence, due to the action of purifying selection. several methods for estimating of evolutionary constraints have been proposed, and applied to coding and noncoding dna of invertebrates and mammals. shabalina and kondrashov proposed a method to quantify the proportion of bases that are subject to strong purifying selection by comparing the genomes of distantly related species. it is assumed that homologous segments that show significant similarity are under strong functional constraints, otherwise are evolving free from functional constraints. another approach to identify functional regions in the genome is to compare sequences from species showing lower levels of divergence that are far from saturation. the basis of the method is to compare the relative divergence of putatively constrained segments of the genome with that of linked putatively neutrally evolving sequences. in the selectively constrained segments, nucleotides are assumed to fall into two classes: neutral, which evolve at the same rate as the neutral sequence; or strongly constrained, in which mutations are eliminated unconditionally by natural selection. selective constraint is then the proportion of new mutations that are strongly deleterious and removed by purifying selection. it should be noted that the presence of adaptive substitutions tends to lead to underestimation of constraint, since this leads to divergence of functional regions. one difficulty in analyzing evolutionary constraints in noncoding dna is the inference of the correct sequence alignment. if the sequence alignment method tends to miss genuine similarities, then functional elements could be miss-assigned as non-functional. this uncertainty largely arises due to the unknown pattern of indels between the pair of sequences. a solution to this problem is to compute probabilities of alternative alignments according to explicit models of indel evolution. based on this method, mcalign has been developed to tackle the problem of aligning noncoding dna. selective constraints of introns have recently been investigated in drosophila, mammals and other animals. several patterns of nucleotide divergence, polymorphism, and selective constraints have been uncovered. until recently, no such investigation has been done in plants. the methodology chosen to study the pattern of noncoding dna evolution heavily depends on the dataset investigated. in general, noncoding dna sequences need to be not too far diverged, so that it is not too difficult to align them. on the other hand, sequences should not be too similar, otherwise there may be insufficient statistical power available for comparative genomics analysis. until now, all studies of evolutionary constraints have compared different lineages, under the assumption of the same selective pressures acting on them. here, we have compared intronic sequences from just one species, a dataset including paralogous pairs from a recent segmental duplication in rice. the duplication event encompasses a ~ mb segmental pair with perfect synteny between chromosome and. the duplication is estimated to have occurred about million years ago, although an alternative date of mya has also been proposed. the evolutionary divergence is compatible with estimates for human-chimpanzee and members of the drosophila genus, which have been previously used for noncoding dna analysis. their average divergences are about between drosophila simulans and melanogaster, about for human-chimpanzee, whereas ours is about. the divergence of this segment is more suitable for noncoding dna analysis than, for example, different rice subspecies, or rice and other cereals. rice has two cultivated subspecies, indica and japonica, for which the genomes have been sequenced. however, the two subspecies separated within about mya, so their sequence similarity is too high and power to infer constraints is low. the divergence time of rice and other cereals is estimated to be about mya, and alignment of noncoding sequences between them is usually problematic.  compilation of intron dataset after intron alignment and some necessary masking, a dataset of intron pairs was generated. the pairs come from duplicated gene pairs from a recent duplication of rice chromosomes and. among the introns, median length was bp. the dataset included first introns of median length bp, whereas non-first introns had median length bp. it should be noted that only first intron pairs in which both introns were first introns were considered, and the same criterion was used for non-first introns. first introns are significantly longer than non-first introns, which parallels findings for other species investigated. our dataset of duplicated gene pairs is similar to that investigated by wang et al., and other studies, although the identification approaches used by us are slightly different. in this study, we employed several methods to minimize the frequency of incorrect alignments. these included amino acid-guided methods to anchor the coding regions of a paralogous gene pair, alignment using explicit models of indel evolution, and the use of two masking protocols for nonhomologous sites. our finals sample size of intron pairs from loci is compatible with other similar studies. for example, 200– loci were used by keightley and gaffney, loci by halligan et al. and intron segments by haddrill et al.. intron length a negative correlation between intron length and nucleotide divergence is present in our dataset. this result therefore suggests that regulatory elements may be more common in long than short introns. a significant negative correlation between divergence and intron length has also been observed in other species that have been investigated. intron ordinal position to further investigate the negative correlation between divergence and intron length described above, we divided our dataset into two subsets of first and non-first introns, and calculated correlation coefficients between length and divergence for each subset separately. the results indicate that the negative correlation between divergence and intron length is significant in first introns, while the test statistic for non-first introns is marginally significant. if introns are divided into two different sets according their length, there is a significant difference in divergence between short and long introns for first introns, whereas the difference is non-significant for non-first introns. in some other taxa, first introns appear to have a higher frequency of regulatory elements. it has thus been suggested that a relationship between intron size and divergence might only be expected for first introns. our results in rice seem to support this point. we further studied the relationship between the ordinal position of introns in a gene and divergence. the global correlation between intron order and ki is non-significant. we also divided the dataset into two subsets based on first or non-first introns. similarly, no significant difference in divergence was found between first and non-first introns. this indicates that divergence does not decay slowly and regularly with the intronic ordinal position in a gene, which contrasts with the trends observed in the human-chimpanzee comparison. in addition to single nucleotide mutations, we also investigate the frequency distribution of indels in first and non-first intron. a total of indels were identified in our dataset, and no significant difference in frequencies of indel lengths between first and non-first intron was observed. however, significant differences between indel numbers and lengths per base or gene pair were observed, with more indels in first than non-first introns. this result indicates that the evolutionary pattern of indels seems to be somewhat different from nucleotide divergence in introns in rice. whether this trend exists in other plants or animal species need further investigation. in summary, selective constraints seem not to be specific to first intron in rice, so our results are similar to those previously reported in drosophila. in a comparison of two species of drosophila, haddrill et al. found that first introns evolve at similar rates to other introns. in rodents and mammals, however, it has been reported that divergence varies along introns and depend on their ordinal position within gene. gaffney and keightley observed a negative correlation between mean intronic selective constraint and intron ordinal number in rodents, implying that first introns are more conserved other introns. level of intronic divergence between humans and closely related species suggest that divergence also depends on intronic ordinal number. the above results indicate that the rule of high constraint at first introns is not common to all taxonomic groups. whether the phenomenon is present in other plants needs further investigation. splice control sites we next examined constraints near the 5' and 3' ends of introns, which contain splice control motifs. as expected, there is a strong signal of purifying selection in the sequences within bp of the 5' and 3' ends, particularly at the dinucleotides adjacent to the 5' and 3' splice sites. similar observation has been reported in rodents and drosophila. the distribution of constraints in introns moving away from the splice sites, however, indicates that the regions under strong constraints in rice are quite short, only about bp at the 5' end and even shorter at the 3' end. this situation is similar to what has been inferred in drosophila. gc content the base composition of gramineae genes is distinct from that of dicot genes. for example, in gramineae genes gc content is relative high, and there is a gradient of gc content along the direction of transcription. in our previous study, we investigated gc content evolution in coding regions. here we focused on gc content evolution of intronic regions. gc content shows a significant difference between first introns and non-first introns, even in subgroups with different length. there is also a negative gradient of gc content with intronic ordinal position, which is similar to that seen in coding sequence with transcriptional direction. these results suggest that a mechanism involving base mutation may act on first introns to elevate their gc content. although we observed a specific pattern of nucleotide substitution in first introns, in contrast, no significant relationship between gc content and divergence or intron length was observed. we also calculated the relationship between gc content and divergence and intron length in the two datasets. similarly, no significant relationships were detected. this result suggests that intron length and divergence are not a confounding effect of gc content in rice. in other words, gc content is dependent of the ordinal position of introns, but not divergence and length. this result is dissimilar to studies on drosophila and mammals, in whichdivergence is negatively correlated with gc content. mammalian first introns are richer in gc content and higher in divergence than other introns. in rice, first introns are also gc-rich, but do not have a significantly higher divergence than other introns. substitution pattern we used nucleotides from the fastest evolving intronic sites as putatively neutral standards to calculate constraint. although exonic four-fold degenerate sites are often used as a standard against which to test for deviations from neutrality, sites in short introns evolve faster in our data set, so are more appropriate as a neutral standard. the fei sites refer to those nucleotides not close to exon boundaries and outside of first introns. similar regions have previously been used to quantify functional constraints in noncoding dna. in general, fractions of nucleotide differences at fei sites are consistently higher than 4-fold sites and first introns. the transition events a↔g and t↔c changes are expected to be the most common substitutional changes in all categories of sites. the situation at 4-fold sites has previously been observed in rice coding sequences, where the two changes a↔g and t↔c are predominantly from a/t to g/c, and thereby increase gc content. beside of transition t↔c, the fractions of transversion c↔g change are relatively higher than other four types of nucleotide changes in first introns compared to introns in general.  we investigate selective constraints in a recent segmental duplication that includes paralogous intron pairs that occurred about million years ago in rice. our observation of stronger selective constraints in long introns suggests that functional elements subject to purifying selection may be concentrated within long introns. our results are consistent with the presence of strong purifying selection at splicing control sites. selective constraints are not significantly stronger in first introns of rice, as they are in other species. 
our observation of stronger selective constraints in long introns suggests that functional elements subject to purifying selection may be concentrated within long introns. our principal findings are: intronic divergence is negatively correlated with intron length, a pattern that has previously been described in drosophila and mammals; there is a signature of strong purifying selection at splice control sites; first introns are significantly longer and have a higher gc content than other introns; the divergences of first and non-first introns are not significantly different from one another, a pattern that differs from drosophila and mammals; and short introns are more diverged than four-fold degenerate sites suggesting that selection reduces divergence at four-fold sites. here, we investigate selective constraints in a recent segmental duplication that includes paralogous intron pairs that occurred about million years ago in rice. several studies have investigated the relationships between selective constraints in introns and their length, gc content and location within genes. our results are consistent with the presence of strong purifying selection at splicing control sites. studies of selective constraints in noncoding dna have generally involved interspecific comparisons, under the assumption of the same selective pressures acting in each lineage. selective constraints are not significantly stronger in first introns of rice, as they are in other species. to date, however, no such investigation has been done in plants.
 several studies have investigated the relationships between selective constraints in introns and their length, gc content and location within genes. to date, however, no such investigation has been done in plants. studies of selective constraints in noncoding dna have generally involved interspecific comparisons, under the assumption of the same selective pressures acting in each lineage. such comparisons are limited to cases in which the noncoding sequences are not too strongly diverged so that reliable sequence alignments can be obtained. here, we investigate selective constraints in a recent segmental duplication that includes paralogous intron pairs that occurred about million years ago in rice.  our principal findings are: intronic divergence is negatively correlated with intron length, a pattern that has previously been described in drosophila and mammals; there is a signature of strong purifying selection at splice control sites; first introns are significantly longer and have a higher gc content than other introns; the divergences of first and non-first introns are not significantly different from one another, a pattern that differs from drosophila and mammals; and short introns are more diverged than four-fold degenerate sites suggesting that selection reduces divergence at four-fold sites.  our observation of stronger selective constraints in long introns suggests that functional elements subject to purifying selection may be concentrated within long introns. our results are consistent with the presence of strong purifying selection at splicing control sites. selective constraints are not significantly stronger in first introns of rice, as they are in other species. 