BACKGROUND
endogenous non-coding small rnas  play an important role in post-transcriptional gene regulation in plants.
in general, small rnas are grouped into two major classes, micrornas  and short-interfering rnas  based on the mechanisms by which they are synthesized and function  <cit> .
mirnas are generated from a stem-loop primary transcript by the endonuclease dicer-like  <dig>  and its partners.
to date,  <dig> mirnas from  <dig> mirna families , including several natural antisense mirnas  and a mitron, have been identified in rice  <cit> .
sirnas derive from double-stranded rna precursors that are processed mainly by the endonucleases dcl <dig>  dcl <dig> and/or dcl <dig>  <cit> .
several types of sirnas, such as trans-acting sirnas , natural antisense transcript-derived sirnas , repeat-associated sirnas  and mirna-like long hairpin sirnas, have been identified in rice and other plants  <cit> .
mrnas are regulated by several classes of small rnas through base complementarity leading to cleavage or repression of translation of the mrna.
it has been observed that single nucleotide polymorphisms  within a mirna binding site can cause significant phenotypic changes  <cit> .
genome-wide investigation of nucleotide variation at small rna loci can determine the prevalence of functional variations in a species; this was first assessed in humans  <cit> .
in plants, a large-scale survey of mirna polymorphism has been performed in arabidopsis  <cit> .
this survey showed that mutations in mature mirna sequences are rare and that the level of polymorphism in mirnas is lower than in their flanking regions, suggesting purifying selection in the mirna sequences.
evidence of positive selection on some mirna loci has been found in arabidopsis  <cit> .
our previous studies on rice mirnas and mirna binding sites suggested that positive selection and nucleotide mutations play an important role in co-evolution of mirnas and their targets  <cit> .
despite the importance of small rnas in regulation of development in crops, investigation of polymorphism of mirnas and other small rnas in rice and other crops is still in its infancy.
the extent of sequence variation at functional regions of small rnas and their potential contribution to phenotypic evolution during crop domestication are unclear.
unlike arabidopsis, rice has experienced domestication and subsequent artificial genetic improvement and therefore has been shaped by an additional bottleneck effect.
rice is believed to have been domesticated approximately  <dig>  years ago  <cit> .
several protein-coding genes controlling domestication traits have been identified and a notable feature of these domestication genes is that they generally encode transcription factors that regulate other protein-coding genes by direct binding to their dna  <cit> .
most conserved mirnas target transcription factors with an important role in plant development  <cit> , but whether mirnas and other types of non-coding small rnas are targets of domestication selection in crops is yet to be determined.
in this study, we carried out an investigation of sequence variation and selection of small rnas in domesticated rice by two steps .
firstly, we selected  <dig> small rna generating loci  and analyzed their sequence variation and neutrality within a domesticated rice population of  <dig> accessions, and secondly, we further investigated  <dig> of the  <dig> small rna loci in populations of  <dig> cultivated rice accessions and  <dig> wild rice accessions to determine how many of these loci might have experienced domestication selection.
our results suggest a non-neutral evolutionary pattern of small rnas in the cultivated rice and that several of them are putative targets of selection during rice domestication.
RESULTS
nucleotide divergence and neutrality of small rnas
a total of  <dig> small rna loci, including  <dig> mirnas from  <dig> mirna families, four members of the tas <dig> family and two mirna-like long hairpins, were selected for a polymorphism survey of rice small rnas .
with the exception of the two mirna-like long hairpins, genomic fragments containing the intact precursor sequences of the small rnas were amplified and sequenced from a population of  <dig> domesticated rice accessions , which were collected from diverse geographic locations .
for the two mirna-like long hairpins which have large loop regions, the two stem regions were amplified and sequenced.
fragments from each rice accession were aligned to each other for each small rna locus.
the average fragment length of all alignments was  <dig>  bp, which covered the precursor sequences of the investigated small rnas .
of the  <dig> investigated mirna genes,  <dig>  showed snps or insertion/deletions  within their mature mirna sequences in the cultivated rice population , leading to an average number of snps per  <dig> sites of  <dig>  ±  <dig>   or an average pairwise nucleotide diversity  of  <dig>  ±  <dig>  .
as an example, the polymorphic mature sequence of the mir <dig> family is presented in figure  <dig> .
a snp was observed at the fourth position from the 5' end of the mature sequences of mir166e.
out of the  <dig> cultivated indica and japonica accessions investigated,  <dig> including indica cultivar 93- <dig> and wild rice had the same mir166e sequence  as in mirbase http://microrna.sanger.ac.uk/sequences/, while  <dig> had an a at the fourth position .
in all other members of the mir <dig> family the fourth base is g .
these results suggest that the fourth position a could be a recent mutation.
it would be interesting to determine whether this mutation has an impact on the interaction between mir166e and its targets because it results in a perfect match with the predicted targets at the fourth position.
from our sequencing data for mir166e target genes and the recently released rice snp data http://www.oryzasnp.org <cit> , a snp was found at the tenth position  of the mir166e binding site in os <dig> g <dig>  one of the predicted mir166e targets, in 20% of accessions investigated.
mirna-mediated cleavage takes place between positions  <dig> and  <dig>  a mutation from c to g at the tenth position might abolish mir166e-mediated cleavage of os <dig> g <dig> in these accessions, but whether this change has a consequence on rice depends on the specific interaction between mir166e and os <dig> g <dig> because os <dig> g <dig> could still be regulated by other members of the mir <dig> family.
in addition to the mature mirna sequences, many snps were also observed in the flanking regions of mirnas.
compared to the mature mirna sequences, significantly higher snp densities or diversities were observed in the precursor mirna  sequences, but the highest polymorphism was found in the upstream or downstream region of pre-mirnas .
these results suggest that purifying selection was the predominant evolutionary force acting on mirna sequences in rice as reported for arabidopsis  <cit> .
some mirnas are conserved in both dicot and monocot plants while others are species-specific.
of the  <dig> mirna genes investigated in this study,  <dig> are conserved and  <dig> are rice-specific .
a dramatic difference in nucleotide diversities in the mature mirnas and their flanking regions was observed between the conserved and rice-specific mirna loci .
the nucleotide diversity of the rice-specific mirnas was nearly twice that of the conserved mirnas.
however, no significant difference in nucleotide diversity could be found in the mirnas from the two subspecies of cultivated rice although the average number of snps per site of the indica subgroup was slightly lower than that of the japonica subgroup in both the mature mirna and its flanking regions .
polymorphisms were also found in the functional regions of sirna-generating loci in rice, for example, at phases p5'_ <dig> and p5'_ <dig> of a mirna-like long hairpin , and in ta-siarf and the 3' mir <dig> binding site of tas3a <dig> .
to examine neutrality of small rnas in rice, tajima's d  <cit> , a widely used neutrality test, was employed.
tajima's d test measures the frequency distribution of polymorphisms and selection is expected to skew the population frequency of genetic variants relative to the neutral equilibrium model .
under ne, the mean tajima's d is expected to be zero and a negative value indicates an excess of rare sequence variants relative to ne expectation and a recent positive selection is thus inferred.
in contrast to positive selection, balanced selection retains genetic differences and elevates the tajima's d statistic towards a positive value  <cit> .
for each small rna locus, tajima's d was calculated for the mirna- or sirna-containing sequence fragment.
a slightly skewed distribution of tajima's d values was observed in domesticated rice , which is similar to that observed in arabidopsis  <cit> .
significant probabilities of non-neutral patterns of sequence variation were detected at several loci in one or both subgroups of the cultivated rice .
significantly negative values were found for several small rnas, such as mir395a/b and tas3a <dig>  in the indica subgroup, suggesting that these small rna loci could have experienced positive selection during rice evolution.
on the other hand, some small rna loci possessed an extreme positive tajima's d value , indicating a balanced selection for these loci.
the above results suggest that polymorphisms at or linked to the small rna loci might be a result of selection.
potential domestication selection
indica and japonica rice were domesticated independently and their domestication genes usually show different evolutionary histories of selection between cultivated and wild rice populations  <cit> .
to find candidate small rna loci that experienced domestication selection,  <dig> loci were selected from the set of  <dig> small rna loci for further analysis  using a cultivated rice population including  <dig> indica and  <dig> japonica cultivars, a wild population including  <dig> accessions of o. rufipogon and an outgroup  .
of the  <dig> loci selected, three  had significant negative tajima's d values and one  had extremely low divergence according to the above neutral test results ; the remaining loci were randomly chosen from the small rna list shown in additional file  <dig>
as previously mentioned, the extent of nucleotide diversity of selected genes tends to be reduced.
our analyses indicated that nucleotide diversity  of the four neutral genes  reduced less than  <dig>  fold in the cultivated rice population compared to their wild progenitors.
of the  <dig> small rna loci, eight  had a nucleotide diversity reduction greater than  <dig>  fold  in the domesticated rice compared to their wild progenitors .
for example, nucleotide diversity of mir <dig> reduced  <dig>  and  <dig>  fold in the two domesticated subgroups relative to the wild population, and nucleotide diversities of the mirna-like long hairpin  reduced  <dig>  and  <dig>  fold.
this result suggests that these eight small rna loci probably underwent selection during rice evolution.
n, number of accessions sequenced d:tajima' d; hka-p: p value of multi-locus hka test; na: not available; nac, nac domain transcription factor; tas <dig>  ta-sirna gene using mir <dig> as the processing guide; aps, atp-sulfurylase; ast, sulfate transcription factor; arf, auxin response factor; hd-zip, homeodomain leucine-zipper transcription factor; grl, grl transcription factor; pt: phosphate transporter; ef: ef hand proteins; nd, not determined.
the significant  test results were labeled bold and * .
for details, also see additional file  <dig>
to further investigate positive selection in these small rna loci, in addition to the tajima's d test, another independent test, the hudson-kreitman-aguade  test  <cit> , was performed.
the hka test is able to measure the degree of nucleotide diversity reduced by positive selection, a feature of the sequence data that cannot be measured by the tajima's d test.
rejection of the multi-locus hka test that measures the difference in diversity within species relative to divergence between species is often considered as evidence of a positive selection.
the multi-locus hka test requires an outgroup and reference loci that are believed to have not been affected by selection.
for this purpose, african wild rice  that is the nearest phylogenetic node to o. sativa and o. rufipogon  <cit>  was used as the outgroup and the aforementioned four neutral genes were used as the reference loci.
of the  <dig> small rna loci, four mirna genes  and two sirna genes  had signatures of positive selection in indica and/or japonica subgroup according to tajima's d or hka test .
no positive selection was detected by the two tests for other  <dig> mirna loci.
in the wild rice population, mir399d, mir1862d and the mirna-like long hairpin  presented evidence of positive selection , suggesting the non-neutral evolution of these three genes in the wild rice population.
a gene can be considered as a candidate domestication gene if a significantly positive selection signal is detected in the cultivated population but not in the wild population  <cit> .
based on this criterion, our results thus suggest that mir164e, mir <dig>  mir395a/b and tas3a <dig> are potential candidates of small rna loci that have experienced direct selection during rice domestication.
discussion
to date, large-scale studies of nucleotide variation at mirnas have only been carried out in human  <cit>  and arabidopsis  <cit> .
overall, our results in rice support the findings in human and arabidopsis that purifying selection is one of the main evolutionary forces acting on rice mirna genes, maintaining lower levels of sequence divergence in mature mirnas than in their flanking regions.
in arabidopsis, only a few substitution events were observed in the mature mirna sequences  in a population including  <dig> diverse wild accessions.
however, nucleotide mutations in the mature mirna sequences were found in  <dig> of the  <dig> investigated rice mirnas in this study.
we also found polymorphisms in at least seven mirnas in the wild rice population .
the higher percentage of snps or indels found in rice mirnas implies that the polymorphism levels of mirnas and other mirna-like small rnas may vary among species.
our results suggest selection, and probably domestication selection, on some small rna loci .
small rnas regulate genes that control a wide range of traits; the functions of these small rna regulated genes might provide clues for the traits under selection via small rna.
one interesting result in this study is that both mir <dig> and tas <dig> are potential candidates of selection.
mir <dig> acts as a guide for processing of tas <dig> and biogenesis of ta-sirnas, including the functional ta-siarf  <cit> .
arfs  are encoded by a large gene family  and are involved in regulation of a wide range of biological functions in rice  <cit> .
ta-siarf downregulates arf <dig>  arf <dig> and arf <dig>  <cit>  that affect gynoecium patterning  <cit> , proper timing of vegetative shoot development and establishment of leaf polarity  <cit> .
at least two arfs have been detected as selected genes in maize  <cit> .
it is unclear whether rice arf genes have experienced selection during evolution.
our results suggest that genes involved in the same regulatory pathway could all be targeted by domestication selection for enhanced growth response and productivity.
mir164e targets nac domain transcription factors that play an important role in development, stress-tolerance and disease-resistance in rice  <cit> .
mir <dig> targets atp-sulfurylases that are involved in sulfate assimilation.
expression of mir <dig> is greatly increased under sulfate starvation conditions  <cit> .
sulfur is an essential macronutrient required by plants.
mir <dig> regulated sulfate metabolism might have been selected for high sulfate usage efficiency.
our results suggest that mirnas and other small rna loci should not be ignored in the endeavor to identify the molecular basis of domestication and improvement of crops.
note that there are three limitations in our effort to identify candidate small rna loci experienced domestication selection.
first, a bottleneck effect or demographic effect can also cause significant reduction of genetic diversity and it could not be excluded in our analysis.
second, the positive selection detected in this study could have arisen by selective sweep rather than direct selection on the small rna loci themselves.
third, although we identified several mutations in the functional regions  of the candidate small rna genes in domesticated rice, such as in the mature mir395a/b sequences and in tasi-arf and the mir <dig> binding sites of tas <dig> , and found distinct expression levels of mir164e in the cultivated and wild rice , it does not necessarily indicate a direct consequence of the domestication event.
further experiments are needed to reveal the consequence of positive selection, such as phenotypic changes.
our sequencing results revealed sequence polymorphism in mir166e and its target gene os <dig> g <dig> .
by checking the recently released rice snp dataset , we found a similar situation with mir <dig> and the target gene os <dig> g <dig> .
we also observed snps in the mirna binding sites of targets of mir164d/e and mir <dig>  but no snp was observed in the mature sequences of the two mirnas.
these results suggest that evolution of mirnas and their targets could be independent, although a co-evolution relationship between mirnas and their target sequences does happen in cultivated rice as demonstrated previously  <cit> .
at least one member each of four mirna families  were found to have experienced positive selection based on tajima's d test in the natural arabidopsis populations  <cit> .
these four mirna families are conserved in dicot and monocot plants.
in our study, significant signals of positive selection were also detected by tajima's d test in at least one member of the mir <dig>  mir <dig> and mir <dig> family in indica or japonica subgroup , implying a potential conservation of adaptive evolution between rice and arabidopsis for these mirna families.
a common feature of these three families is that they have relatively large number of family members .
these homologous genes might have experienced rapid functional divergence after gene expansion.
it has been suggested that positive selection acts on gene copy number variations  <cit> .
positive selection could promote adaptive evolution but whether plants enhance their fitness in the changing environment by adding copy number of mirnas requires further investigation.
CONCLUSIONS
our survey of polymorphisms in  <dig> small rna loci in rice, the first such effort in crops, revealed a higher level of sequence variations in mirnas in domesticated rice than in arabidopsis.
in agreement with previous studies, our investigation suggests that purifying selection dominated the evolution of small rnas in rice.
a neutrality test revealed non-neutral evolution of small rna loci in which both positive selection and balanced selection are involved.
our results suggest that domestication selection on small rna loci could play an important role in rice domestication and/or improvement.

 endogenous non-coding small rnas play an important role in post-transcriptional gene regulation in plants. in general, small rnas are grouped into two major classes, micrornas and short-interfering rnas based on the mechanisms by which they are synthesized and function. mirnas are generated from a stem-loop primary transcript by the endonuclease dicer-like and its partners. to date, mirnas from mirna families, including several natural antisense mirnas and a mitron, have been identified in rice. sirnas derive from double-stranded rna precursors that are processed mainly by the endonucleases dcl dcl and/or dcl. several types of sirnas, such as trans-acting sirnas, natural antisense transcript-derived sirnas, repeat-associated sirnas and mirna-like long hairpin sirnas, have been identified in rice and other plants. mrnas are regulated by several classes of small rnas through base complementarity leading to cleavage or repression of translation of the mrna. it has been observed that single nucleotide polymorphisms within a mirna binding site can cause significant phenotypic changes. genome-wide investigation of nucleotide variation at small rna loci can determine the prevalence of functional variations in a species; this was first assessed in humans. in plants, a large-scale survey of mirna polymorphism has been performed in arabidopsis. this survey showed that mutations in mature mirna sequences are rare and that the level of polymorphism in mirnas is lower than in their flanking regions, suggesting purifying selection in the mirna sequences. evidence of positive selection on some mirna loci has been found in arabidopsis. our previous studies on rice mirnas and mirna binding sites suggested that positive selection and nucleotide mutations play an important role in co-evolution of mirnas and their targets. despite the importance of small rnas in regulation of development in crops, investigation of polymorphism of mirnas and other small rnas in rice and other crops is still in its infancy. the extent of sequence variation at functional regions of small rnas and their potential contribution to phenotypic evolution during crop domestication are unclear. unlike arabidopsis, rice has experienced domestication and subsequent artificial genetic improvement and therefore has been shaped by an additional bottleneck effect. rice is believed to have been domesticated approximately years ago. several protein-coding genes controlling domestication traits have been identified and a notable feature of these domestication genes is that they generally encode transcription factors that regulate other protein-coding genes by direct binding to their dna. most conserved mirnas target transcription factors with an important role in plant development, but whether mirnas and other types of non-coding small rnas are targets of domestication selection in crops is yet to be determined. in this study, we carried out an investigation of sequence variation and selection of small rnas in domesticated rice by two steps. firstly, we selected small rna generating loci and analyzed their sequence variation and neutrality within a domesticated rice population of accessions, and secondly, we further investigated of the small rna loci in populations of cultivated rice accessions and wild rice accessions to determine how many of these loci might have experienced domestication selection. our results suggest a non-neutral evolutionary pattern of small rnas in the cultivated rice and that several of them are putative targets of selection during rice domestication.  nucleotide divergence and neutrality of small rnas a total of small rna loci, including mirnas from mirna families, four members of the tas family and two mirna-like long hairpins, were selected for a polymorphism survey of rice small rnas. with the exception of the two mirna-like long hairpins, genomic fragments containing the intact precursor sequences of the small rnas were amplified and sequenced from a population of domesticated rice accessions, which were collected from diverse geographic locations. for the two mirna-like long hairpins which have large loop regions, the two stem regions were amplified and sequenced. fragments from each rice accession were aligned to each other for each small rna locus. the average fragment length of all alignments was bp, which covered the precursor sequences of the investigated small rnas. of the investigated mirna genes, showed snps or insertion/deletions within their mature mirna sequences in the cultivated rice population, leading to an average number of snps per sites of ± or an average pairwise nucleotide diversity of ±. as an example, the polymorphic mature sequence of the mir family is presented in figure. a snp was observed at the fourth position from the 5' end of the mature sequences of mir166e. out of the cultivated indica and japonica accessions investigated, including indica cultivar 93- and wild rice had the same mir166e sequence as in mirbase http://microrna.sanger.ac.uk/sequences/, while had an a at the fourth position. in all other members of the mir family the fourth base is g. these results suggest that the fourth position a could be a recent mutation. it would be interesting to determine whether this mutation has an impact on the interaction between mir166e and its targets because it results in a perfect match with the predicted targets at the fourth position. from our sequencing data for mir166e target genes and the recently released rice snp data http://www.oryzasnp.org, a snp was found at the tenth position of the mir166e binding site in os g one of the predicted mir166e targets, in 20% of accessions investigated. mirna-mediated cleavage takes place between positions and a mutation from c to g at the tenth position might abolish mir166e-mediated cleavage of os g in these accessions, but whether this change has a consequence on rice depends on the specific interaction between mir166e and os g because os g could still be regulated by other members of the mir family. in addition to the mature mirna sequences, many snps were also observed in the flanking regions of mirnas. compared to the mature mirna sequences, significantly higher snp densities or diversities were observed in the precursor mirna sequences, but the highest polymorphism was found in the upstream or downstream region of pre-mirnas. these results suggest that purifying selection was the predominant evolutionary force acting on mirna sequences in rice as reported for arabidopsis. some mirnas are conserved in both dicot and monocot plants while others are species-specific. of the mirna genes investigated in this study, are conserved and are rice-specific. a dramatic difference in nucleotide diversities in the mature mirnas and their flanking regions was observed between the conserved and rice-specific mirna loci. the nucleotide diversity of the rice-specific mirnas was nearly twice that of the conserved mirnas. however, no significant difference in nucleotide diversity could be found in the mirnas from the two subspecies of cultivated rice although the average number of snps per site of the indica subgroup was slightly lower than that of the japonica subgroup in both the mature mirna and its flanking regions. polymorphisms were also found in the functional regions of sirna-generating loci in rice, for example, at phases p5'_ and p5'_ of a mirna-like long hairpin, and in ta-siarf and the 3' mir binding site of tas3a. to examine neutrality of small rnas in rice, tajima's d, a widely used neutrality test, was employed. tajima's d test measures the frequency distribution of polymorphisms and selection is expected to skew the population frequency of genetic variants relative to the neutral equilibrium model. under ne, the mean tajima's d is expected to be zero and a negative value indicates an excess of rare sequence variants relative to ne expectation and a recent positive selection is thus inferred. in contrast to positive selection, balanced selection retains genetic differences and elevates the tajima's d statistic towards a positive value. for each small rna locus, tajima's d was calculated for the mirna- or sirna-containing sequence fragment. a slightly skewed distribution of tajima's d values was observed in domesticated rice, which is similar to that observed in arabidopsis. significant probabilities of non-neutral patterns of sequence variation were detected at several loci in one or both subgroups of the cultivated rice. significantly negative values were found for several small rnas, such as mir395a/b and tas3a in the indica subgroup, suggesting that these small rna loci could have experienced positive selection during rice evolution. on the other hand, some small rna loci possessed an extreme positive tajima's d value, indicating a balanced selection for these loci. the above results suggest that polymorphisms at or linked to the small rna loci might be a result of selection. potential domestication selection indica and japonica rice were domesticated independently and their domestication genes usually show different evolutionary histories of selection between cultivated and wild rice populations. to find candidate small rna loci that experienced domestication selection, loci were selected from the set of small rna loci for further analysis using a cultivated rice population including indica and japonica cultivars, a wild population including accessions of o. rufipogon and an outgroup. of the loci selected, three had significant negative tajima's d values and one had extremely low divergence according to the above neutral test results; the remaining loci were randomly chosen from the small rna list shown in additional file as previously mentioned, the extent of nucleotide diversity of selected genes tends to be reduced. our analyses indicated that nucleotide diversity of the four neutral genes reduced less than fold in the cultivated rice population compared to their wild progenitors. of the small rna loci, eight had a nucleotide diversity reduction greater than fold in the domesticated rice compared to their wild progenitors. for example, nucleotide diversity of mir reduced and fold in the two domesticated subgroups relative to the wild population, and nucleotide diversities of the mirna-like long hairpin reduced and fold. this result suggests that these eight small rna loci probably underwent selection during rice evolution. n, number of accessions sequenced d:tajima' d; hka-p: p value of multi-locus hka test; na: not available; nac, nac domain transcription factor; tas ta-sirna gene using mir as the processing guide; aps, atp-sulfurylase; ast, sulfate transcription factor; arf, auxin response factor; hd-zip, homeodomain leucine-zipper transcription factor; grl, grl transcription factor; pt: phosphate transporter; ef: ef hand proteins; nd, not determined. the significant test results were labeled bold and *. for details, also see additional file to further investigate positive selection in these small rna loci, in addition to the tajima's d test, another independent test, the hudson-kreitman-aguade test, was performed. the hka test is able to measure the degree of nucleotide diversity reduced by positive selection, a feature of the sequence data that cannot be measured by the tajima's d test. rejection of the multi-locus hka test that measures the difference in diversity within species relative to divergence between species is often considered as evidence of a positive selection. the multi-locus hka test requires an outgroup and reference loci that are believed to have not been affected by selection. for this purpose, african wild rice that is the nearest phylogenetic node to o. sativa and o. rufipogon was used as the outgroup and the aforementioned four neutral genes were used as the reference loci. of the small rna loci, four mirna genes and two sirna genes had signatures of positive selection in indica and/or japonica subgroup according to tajima's d or hka test. no positive selection was detected by the two tests for other mirna loci. in the wild rice population, mir399d, mir1862d and the mirna-like long hairpin presented evidence of positive selection, suggesting the non-neutral evolution of these three genes in the wild rice population. a gene can be considered as a candidate domestication gene if a significantly positive selection signal is detected in the cultivated population but not in the wild population. based on this criterion, our results thus suggest that mir164e, mir mir395a/b and tas3a are potential candidates of small rna loci that have experienced direct selection during rice domestication. discussion to date, large-scale studies of nucleotide variation at mirnas have only been carried out in human and arabidopsis. overall, our results in rice support the findings in human and arabidopsis that purifying selection is one of the main evolutionary forces acting on rice mirna genes, maintaining lower levels of sequence divergence in mature mirnas than in their flanking regions. in arabidopsis, only a few substitution events were observed in the mature mirna sequences in a population including diverse wild accessions. however, nucleotide mutations in the mature mirna sequences were found in of the investigated rice mirnas in this study. we also found polymorphisms in at least seven mirnas in the wild rice population. the higher percentage of snps or indels found in rice mirnas implies that the polymorphism levels of mirnas and other mirna-like small rnas may vary among species. our results suggest selection, and probably domestication selection, on some small rna loci. small rnas regulate genes that control a wide range of traits; the functions of these small rna regulated genes might provide clues for the traits under selection via small rna. one interesting result in this study is that both mir and tas are potential candidates of selection. mir acts as a guide for processing of tas and biogenesis of ta-sirnas, including the functional ta-siarf. arfs are encoded by a large gene family and are involved in regulation of a wide range of biological functions in rice. ta-siarf downregulates arf arf and arf that affect gynoecium patterning, proper timing of vegetative shoot development and establishment of leaf polarity. at least two arfs have been detected as selected genes in maize. it is unclear whether rice arf genes have experienced selection during evolution. our results suggest that genes involved in the same regulatory pathway could all be targeted by domestication selection for enhanced growth response and productivity. mir164e targets nac domain transcription factors that play an important role in development, stress-tolerance and disease-resistance in rice. mir targets atp-sulfurylases that are involved in sulfate assimilation. expression of mir is greatly increased under sulfate starvation conditions. sulfur is an essential macronutrient required by plants. mir regulated sulfate metabolism might have been selected for high sulfate usage efficiency. our results suggest that mirnas and other small rna loci should not be ignored in the endeavor to identify the molecular basis of domestication and improvement of crops. note that there are three limitations in our effort to identify candidate small rna loci experienced domestication selection. first, a bottleneck effect or demographic effect can also cause significant reduction of genetic diversity and it could not be excluded in our analysis. second, the positive selection detected in this study could have arisen by selective sweep rather than direct selection on the small rna loci themselves. third, although we identified several mutations in the functional regions of the candidate small rna genes in domesticated rice, such as in the mature mir395a/b sequences and in tasi-arf and the mir binding sites of tas, and found distinct expression levels of mir164e in the cultivated and wild rice, it does not necessarily indicate a direct consequence of the domestication event. further experiments are needed to reveal the consequence of positive selection, such as phenotypic changes. our sequencing results revealed sequence polymorphism in mir166e and its target gene os g. by checking the recently released rice snp dataset, we found a similar situation with mir and the target gene os g. we also observed snps in the mirna binding sites of targets of mir164d/e and mir but no snp was observed in the mature sequences of the two mirnas. these results suggest that evolution of mirnas and their targets could be independent, although a co-evolution relationship between mirnas and their target sequences does happen in cultivated rice as demonstrated previously. at least one member each of four mirna families were found to have experienced positive selection based on tajima's d test in the natural arabidopsis populations. these four mirna families are conserved in dicot and monocot plants. in our study, significant signals of positive selection were also detected by tajima's d test in at least one member of the mir mir and mir family in indica or japonica subgroup, implying a potential conservation of adaptive evolution between rice and arabidopsis for these mirna families. a common feature of these three families is that they have relatively large number of family members. these homologous genes might have experienced rapid functional divergence after gene expansion. it has been suggested that positive selection acts on gene copy number variations. positive selection could promote adaptive evolution but whether plants enhance their fitness in the changing environment by adding copy number of mirnas requires further investigation.  our survey of polymorphisms in small rna loci in rice, the first such effort in crops, revealed a higher level of sequence variations in mirnas in domesticated rice than in arabidopsis. in agreement with previous studies, our investigation suggests that purifying selection dominated the evolution of small rnas in rice. a neutrality test revealed non-neutral evolution of small rna loci in which both positive selection and balanced selection are involved. our results suggest that domestication selection on small rna loci could play an important role in rice domestication and/or improvement. 
endogenous non-coding small rnas play an important role in post-transcriptional gene regulation in plants. like protein-coding genes, non-coding small rna loci could be targets of domestication selection and play an important role in rice domestication and improvement. the extent of polymorphism at small rna loci in domesticated rice and whether small rna loci are targets of domestication selection have not yet been determined. a polymorphism survey of small rna loci was conducted in domesticated rice, generating mb of sequence data. many mutations were observed at small rna loci in domesticated rice, e.g. several small rna loci showed significant signals for positive selection and/or potential domestication selection. domestication selection is the most important evolutionary force in shaping crop genomes. mutation sites were observed in the mature mirna sequences of mirnas. sequence variation at mirnas and other small rnas is higher than expected in domesticated rice.
 endogenous non-coding small rnas play an important role in post-transcriptional gene regulation in plants. domestication selection is the most important evolutionary force in shaping crop genomes. the extent of polymorphism at small rna loci in domesticated rice and whether small rna loci are targets of domestication selection have not yet been determined.  a polymorphism survey of small rna loci was conducted in domesticated rice, generating mb of sequence data. many mutations were observed at small rna loci in domesticated rice, e.g. mutation sites were observed in the mature mirna sequences of mirnas. several small rna loci showed significant signals for positive selection and/or potential domestication selection.  sequence variation at mirnas and other small rnas is higher than expected in domesticated rice. like protein-coding genes, non-coding small rna loci could be targets of domestication selection and play an important role in rice domestication and improvement. 