BACKGROUND
diatoms are important planktons that are believed to be responsible for one-fifth of the primary productivity on earth  <cit> .
there are two major classes of diatoms, the pennates and the centrics.
with their vital role in silica cycling  <cit> , the unusual evolutionary position of secondary endosymbiotic origin  <cit> , the presence of c <dig> photosynthesis in some species  <cit> , and potential as sources of biodiesel fuel  <cit> , diatoms have attracted increasing attention.
as early as  <dig>  scala et al.
<cit>  analyzed est  data of the pennate diatom phaeodactylum tricornutum and found that some of its genes were more similar to those of animals than of photosynthetic counterparts, implying an unusual evolutionary history.
the genome of p. tricornutum and the centric diatom thalassiosira pseudonana have been sequenced, shedding light on significant features of diatom genomes, including the mosaic genome that contains 'animal-like', 'plant-like' and 'bacteria-like' genes, performing fatty acid metabolism in both peroxisomes and mitochondria, and the presence of enzymes necessary for a complete urea cycle  <cit> .
these characteristics prompted us to hypothesize that the gene expression regulators  of diatoms may show some different specificity to other photosynthetic organisms.
mirnas are important post-transcriptional regulators.
they regulate gene expression in eukaryotes by targeting mrnas for translational repression or cleavage  <cit> .
it is believed that mirnas exist extensively in eukaryotes such as animals and plants with high conservation in each kingdom  <cit> .
the expression of mirnas has a spatio-temporal pattern  <cit>  and they influence the transcription and translation of many genes  <cit> .
generally, their functions involve various processes, including developmental patterning, organ separation, cell differentiation and proliferation, tumor generation, cell death and cell apoptosis, stress resistance, auxin response, fat metabolism and mirna biogenesis  <cit> .
in higher plants and animals, mirnas have been extensively studied but rarely so in algae.
p. tricornutum is an atypical diatom with a weakly silicified outer shell, and the unusual property of being pleiomorphic with three convertible morphotypes  <cit>  , and silicification essentially restricted to one valve of the oval cells  <cit> .
with its characteristics of short life-cycle, small genome size and ease of transformation, p. tricornutum has become an attractive photosynthetic model  <cit> .
additionally, being rich in polyunsaturated fatty acid , especially in eicosapentaenoic acid , p. tricornutum has been used as a food organism and is considered a potential source of epa.
there have been many studies investigating the factors affecting its cell composition  <cit> .
there were reports that microalgae accumulated lipids under nitrogen-limited as well as silicon-limited conditions  <cit> , with similar studies conducted on p. tricornutum  <cit> .
accumulation of lipids in cells and a significant change in fatty acid composition were observed in p. tricornutum under low nitrogen conditions.
using suppression subtractive hybridization technology, tang et al.
separated a number of upregulated genes from p. tricornutum under nitrogen starvation, seven of which had high similarity with functional genes related to nitrogen utilization  <cit> .
studies of lipid metabolism of p. tricornutum under silicon-limited conditions are scarce.
notwithstanding, sapriel et al.
identified  <dig> genes regulated by silicic acid availability, including  <dig> upregulated and  <dig> downregulated genes, from p. tricornutum under silicon-limited conditions  <cit> .
interestingly, they also observed some upregulated genes coding for transporters of metabolites related to nitrogen assimilation and transfer from p. tricornutum in the complete medium compared to silicon-limited conditions.
a previous study on t. pseudonana showed that a glutamate acetyltransferase was involved in silicon metabolism  <cit> .
how are these genes regulated?
do mirnas play a role in p. tricornutum nitrogen and silicon metabolism?
there have been few studies that address these questions.
in the present study, we constructed small rna  libraries from p. tricornutum under normal, nitrogen-limited and silicon-limited conditions and then used high-throughput solexa technology to deeply sequence the srnas.
the sequencing data were analyzed and mirnas were identified from all samples studied.
RESULTS
a diverse set of endogenous small rnas
to determine the likely roles of mirnas in nitrogen and silicon metabolism in p. tricornutum, we constructed and sequenced small rna libraries from p. tricornutum grown in normal , nitrogen-free  and silicon-free  media, respectively.
after removing adaptor sequences and filtering out low quality data , we obtained small rnas with size range of 10- <dig> nt, with an enrichment in 20- <dig> nt .
after removing sequences shorter than  <dig> nt, we obtained  <dig>  <dig>  <dig>   <dig>  <dig>  <dig> and  <dig>  <dig>  <dig> total sequences, representing  <dig>  <dig>   <dig>  <dig> and  <dig>  <dig> unique, although sometimes partially overlapping, clean reads from pt <dig>  pt <dig> and pt <dig>  respectively .
of these unique sequences, about 73% , 74%  and 73%  were only sequenced once.
there were  <dig>  <dig>  <dig>   <dig>  <dig>  <dig> and  <dig>  <dig>  <dig> total; and  <dig>  <dig>   <dig>  <dig> and  <dig>  <dig> unique sequences with at least one perfect match in the p. tricornutum nuclear genome - whereas  <dig>  <dig>  <dig>   <dig>  <dig>  <dig> and  <dig>  <dig>  <dig> total; and  <dig>  <dig>   <dig>  <dig> and  <dig>  <dig> unique sequences matched the chloroplast genome, in pt <dig>  pt <dig> and pt <dig>  respectively .
it was quite unexpected that a majority of srnas were located in the minus strand of chromosome  <dig> and both strands of the chloroplast genome .
the usual preference for a u at the 5' - end of plant small rna sequences  <cit>  was not observed .
the four types of bases appeared equally in each locus.
asrnas that matched nuclear genome.
bsrnas that matched chloroplast genome.
cunique srnas that appeared once.
all clean reads were annotated according to their identities with non-coding rnas , plant mirnas , exon and intron  and sirnas .
in the case that some srna was mapped to more than one category, the following priority rule was adopted: rrna etc.
> known mirna > exon > intron  <cit> .
rrna degraded fragments were the most abundant sequences retrieved from the p. tricornutum total srna pools, boasting the highest read frequency of all small rna classes in all the samples:  <dig> ,  <dig>  and  <dig> % for pt <dig>  pt <dig> and pt <dig>  respectively .
yet in the unique srna pools, non-annotated srna represented a significant part, with  <dig> ,  <dig>  and  <dig> % in pt <dig>  pt <dig> and pt <dig>  respectively.
homologs of plant known mirnas accounted for approximately  <dig> % of the unique sequences in all the three samples, whereas in total sequences pools, the numbers were approximately  <dig> % in pt <dig> and pt <dig> and only  <dig> % in pt <dig>  srnas mapped to exons and introns in either sense or antisense directions also represented a considerable part.
the remaining srnas were snrna, snorna and trna.
common and specific sequences analysis showed that only approximately 15% of the unique sequences were shared by every two samples , suggesting a diverse set of endogenous small rnas in p. tricornutum.
anot annotated srnas.
mirnas in p. tricornutum
the identification of a great quantity of small rnas in p. tricornutum prompted us to examine whether some were mirnas.
first we compared all the non-annotated srnas with the sequences of animal mirnas and virus mirnas available from mirbase   <cit>  to identify homologs of known mirnas.
then we used the small rnas with homology to all known mirnas  and the remaining non-annotated srnas to identify candidate known and novel mirna families in p. tricornutum, respectively .
first we mapped these small rnas onto the p. tricornutum nuclear genome.
then we extracted  <dig> nt upstream and  <dig> nt downstream from those loci and examined whether they could form hairpin secondary structures, a character of known plant and animal pre-mirnas, using criteria developed previously for plant mirna prediction  <cit> .
basically, precursors with free energy ≤ - <dig> kcal/mol checking by mfold  <cit> , ≥  <dig> bp and ≤  <dig> bulges or asymmetries between mirna and mirna*, with mirna sequence length between 18-25nt and flank sequence length of  <dig>  were considered as potential p. tricornutum pre-mirnas and selected for further analysis.
secondary structural predictions identified a total of  <dig> small rna species that were derived from genomic loci whose surrounding sequences had the probability to form hairpin structures that met the requirements as a mirna precursor.
then we checked for the structure stabilities of these  <dig> sequences.
among these, five were found to have a p-value lower than  <dig> .
they were checked for 5' homogeneity using  <dig>  as cut off.
for those sequences with a p-value above  <dig> , a more stringent 5' homogeneity of  <dig>  was used.
all together we obtained  <dig> sequences for manually rechecking according to criteria made previously for mirna identification  <cit> .
finally we determined  <dig> sequences to be p. tricornutum mirnas.
they were submitted to mirbase and named pti-mir5471- <dig>  of these  <dig> small rnas, seven of pre-mirna hairpins were supported by est data.
each mirna had a single precursor.
the length of pre-mirna ranged from  <dig> to  <dig> nt, with a mean of  <dig> nt .
the mfe range was - <dig> to - <dig>  kcal/mol, with a mean of - <dig>  kcal/mol.
most pre-mirnas were located in intergenic regions and the others were mapped to genes of hypothetical protein, probably being mis-annotated.
aindicated the chromosome number, start and end site and sense  or antisense strand  that the pre-mirnas located.
bminimum free energy  of pre-mirnas, predicted by mfold.
clength of pre-mirnas.
dcomputed p-values of mirna precursors.
enumber of reads that had the same 5' end as the mature mirna and the total number reads mapped to the precursors.
fasymmetric bulges larger than  <dig> nt and mismatched mirna bases in the mirna region.
expression patterns of mirnas/candidates during nitrogen-limited and silicon-limited conditions, and target prediction
to investigate the probable roles of mirnas in nitrogen and silicon metabolism in p. tricornutum, we sequenced small rnas from p. tricornutum grown in normal, nitrogen-limited and silicon-limited media.
of the  <dig> mirnas identified, two appeared in all the three small rna libraries, one exclusively in pt <dig> and eight in pt3; and one was shared by pt <dig> and pt <dig>  and one by pt <dig> and pt <dig> .
the expression of mirnas in the samples indicated that they might play an important role under nitrogen-limited and/or silicon-limited conditions.
to determine the likely regulated genes, we predicted targets of these mirnas.
using the rules for target prediction suggested by allen  <cit> , no target was identified.
ignoring locus one and those larger than  <dig> nt and allowing four mismatches between the mirna-target duplex in positions 2- <dig>  some potential target sites were suggested .
some of these potential targets were involved in lipid metabolism, suggesting that p. tricornutum mirnas might play a role in fatty acid metabolism.
this was in accord with the report that p. tricornutum accumulated lipids under nitrogen-limited and silicon-limited conditions  <cit> .
however, as the genome of p. tricornutum is not fully annotated and the functions of many protein-coding genes are unknown, it is difficult to determine whether these mirna targets have any functional bias.
sirna in p. tricornutum
it has been reported that in arabidopsis, mirnas direct the generation of sirna , which were phased relatively with each other  <cit> .
to determine whether mirnas direct the generation of sirna in p. tricornutum, we identified potential sirnas and determined their location.
potential sirnas were found in these samples: with  <dig>   <dig> and  <dig> unique sequences; and  <dig>   <dig> and  <dig> total sequences in pt <dig>  pt <dig> and pt <dig>  respectively.
the majority of sirna were produced from a few hot-spots distributed in all the chromosomes; however, they were not phased relatively with each other.
to determine whether small rnas play a role in silencing of repetitive sequences in p. tricornutum, as for other organisms, we performed a blast search against p. tricornutum repeat sequences and found  <dig> ,  <dig>  and  <dig>  sirna derived from these regions.
this implied that small rnas might induce silencing of repetitive sequences in p. tricornutum.
mirna northern blot
mirna northern blotting was used to detect initial expression of mirnas and their precursors in p. tricornutum.
5s rna was blotted as load control.
northern blot hybridization detected precursors of expected size  in all the samples .
this provided strong evidence for their expression.
discussion
did p. tricornutum mirnas evolve independently?
we compared all p. tricornutum small rnas  with all known plant, animal and virus mirnas in mirbase, and found significant identities .
however, these identities did not pass the criteria we used to identify mirnas.
the most straightforward interpretation for the relative lack of universally conserved mirnas between p. tricornutum and other organisms is that all mirnas in p. tricornutum are rare due to its small genome size, although scenarios that p. tricornutum contains novel mirnas that have no sequence homology with all known ones cannot be ruled out.
in a study of mirnas in the unicellular green alga chlamydomonas reinhardtii, zhao et al.
<cit>  compared its mirnas with all known plant and animal mirnas, and found no homologs.
in fact, c. reinhardtii lacked homologous mirnas even with other green algae  <cit> .
thus we asked whether p. tricornutum had some specific mirnas that have no sequence homology with all known mirnas, as for c. reinhardtii.
we predicted novel mirnas from the small non-annotated rnas, using the same criteria as used to identify known mirnas.
a total of  <dig> novel mirnas were identified from p. tricornutum under normal, nitrogen-limited and/or silicon-limited conditions.
they lacked homology with all known mirnas in the mirbase, including c. reinhardtii mirnas.
thus we propose that mirnas in algae may have evolved independently to animals and plants, consistent with the suggestion of zhao et al  <cit> .
we also used the p. tricornutum chloroplast genome to identify mirnas.
two loci met all the criteria we used to identify mirnas.
interestingly, one of these mirna-like small rnas was homolog of cin-mir <dig>  and part of the potential precursor shared 74% identity  to cin-mir <dig> precursor.
est analysis of p. tricornutum showed that many of its genes were more similar to animals than photosynthetic organisms  <cit> .
complete genome sequences showed that diatoms had a mosaic genome with genes from animals, plants and bacteria  <cit> .
thus it is probable that p. tricornutum might share some common mirnas with animals, although the percentage may be relatively low.
we propose that this animal mirna-like small rna from p. tricornutum might be present in diatoms due to gene transformation, or are conserved mirnas derived from the heterotrophic secondary-host evolutionarily prior to the secondary endosymbiosis, or may be mirnas lost in the plant/red algal lineage during evolution, similar to the incorporation of animal-like genes in diatoms  <cit> .
if this small rna found in our study was genuine mirnas , then this represents a very interesting discovery.
de riso, et al.
had successfully demonstrated gene silencing in p. tricornutum  <cit> .
they analyzed molecular players involved in rna silencing in p. tricornutum and identified both dicer like proteins  and argonaute like proteins .
these argonaute like proteins in p. tricornutum clustered in a clade different from either animals or plants  <cit> , suggesting that p. tricornutum might own a special risc pathway different from that of animals and plants, and thus probably result in the lack of preference for u at the 5' of p. tricornutum srnas.
probable roles of mirnas in metabolism of p. tricornutum
mirnas have been found to play important regulatory roles in various processes in multicellular organisms as well as the unicellular green alga c. reinhardtii  <cit> .
in the present study, mirnas were sequenced from p. tricornutum under normal, nitrogen-limited and silicon-limited conditions .
this suggests that mirnas might play important roles in p. tricornutum.
mirnas expressed in all three samples
two mirnas appeared in all samples .
candidate target genes for these mirnas included dna-directed rna polymerase; glutamate synthase and Δ <dig> fatty acid desaturase .
this indicates that p. tricornutum mirnas might play important roles in a range of biological processes.
it was reported that the composition of fatty acids was significantly influenced by availability of nitrogen  <cit>  and silicon  <cit> .
some genes related to glutamate/glutamine metabolism are regulated by silicon availability  <cit> .
interestingly, we predicted that one gene involved in glutamate synthesis  was targeted by pti-mir <dig>  which was downregulated in both pt <dig> and pt <dig>  indicating that mirna might play a role in silicon-regulated glutamate metabolism.
mirnas that exclusively sequenced from pt3
there were eight mirnas exclusively sequenced from pt <dig> .
candidate target genes for these mirnas include phospholipase c isoform delta , nucleotide transporter, ornithine aminotransferase, nucleosome remodeling factor.
in p. tricornutum, silicification is restricted to one valve of the oval cells and there is no silicon requirement for growth  <cit> .
the strain used in the present study was a fusiform type whose cell wall was not silicified.
however, mirna species were most abundant in pt <dig> , and their targets involved in various processes, indicating that various biological processes might be influenced by silicon available through mirna regulation.
the enrichment of srnas originating from the minus strand of chr <dig> and both strands of the chloroplast genome
it was interesting that a majority of srnas were located in the minus strand of chromosome  <dig> and both strands of the chloroplast genome .
as reported by mcfadden and van dooren  <cit> , green algal/plant and red algal originated from a first endosymbiosis between a eukaryotic and a endosymbiont, whereas diatoms originated from the secondary endosymbiosis between a heterotrophic organism and a red alga.
the diatom chloroplast originated from the plasmid of the second endosymbionts, while nucleus of the second endosymbionts lost, living enormous numbers of their genes - typically more than 90% - house in the second host nucleus  <cit> .
we proposed that the enrichment of srnas on the minus strand of chr <dig> as well as both strands of the chloroplast genome indicated that chr <dig> might have some relative to the second endosymbionts.
e.g., chr <dig> might have originated from nucleus of the second endosymbionts or the majority of the second endosymbionts nuclear genes might have transform into chr <dig>  to test this hypothesis, we extracted the hot spot loci where most small rna derived from.
those were 39000- <dig> nt of the minus strand of chr  <dig>  63675- <dig> nt of the sense strand of chloroplast genome, and 110485- <dig> nt of the minus strand of the chloroplast genome.
we then aligned them and found that the hot spot locus of chr  <dig> had no homology with the chloroplast genome.
thus, even if chr  <dig> have some relative to the second endosymbionts, our data has little support for this hypothesis.
we also found that the two hot spot loci of the chloroplast genome in fact share 100% identity.
they are two inverted repeats, ira and irb, on the chloroplast genome.
thus, small rnas might play an important role in silencing of inverted repeat region.
the failure of detection of mature mirnas by northern blotting was probable due to their low expression
we detected precursors of expected size for pti-mir <dig> and pti-mir <dig>  in other organisms, precursors were more difficult to detect than mature mirnas in wild type samples  <cit> , probably due to their temporary summation in the cells and convert fast into mature mirnas.
we detected mirna precursors in all the three samples of p. tricornutum easily , implied that diatom might obtain different mirna processor from other organisms, which made the accumulation of mirna precursors.
expected sizes for the mature mirnas were not detected.
the most straightforward interpretation for this is the low expression of mature mirnas in the samples we detected, although scenarios that these mirnas are not real mirnas but sequencing artifacts or fragments of a longer transcript cannot be ruled out.
more sensitive technology is needed to perform further analysis.
CONCLUSIONS
our results indicated that p. tricornutum owned a complex srna processing system.
it contained novel mirnas that have no sequence homology with mirnas of other organisms and that they might play important regulator roles in p. tricornutum metabolism.

 diatoms are important planktons that are believed to be responsible for one-fifth of the primary productivity on earth. there are two major classes of diatoms, the pennates and the centrics. with their vital role in silica cycling, the unusual evolutionary position of secondary endosymbiotic origin, the presence of c photosynthesis in some species, and potential as sources of biodiesel fuel, diatoms have attracted increasing attention. as early as scala et al. analyzed est data of the pennate diatom phaeodactylum tricornutum and found that some of its genes were more similar to those of animals than of photosynthetic counterparts, implying an unusual evolutionary history. the genome of p. tricornutum and the centric diatom thalassiosira pseudonana have been sequenced, shedding light on significant features of diatom genomes, including the mosaic genome that contains'animal-like','plant-like' and'bacteria-like' genes, performing fatty acid metabolism in both peroxisomes and mitochondria, and the presence of enzymes necessary for a complete urea cycle. these characteristics prompted us to hypothesize that the gene expression regulators of diatoms may show some different specificity to other photosynthetic organisms. mirnas are important post-transcriptional regulators. they regulate gene expression in eukaryotes by targeting mrnas for translational repression or cleavage. it is believed that mirnas exist extensively in eukaryotes such as animals and plants with high conservation in each kingdom. the expression of mirnas has a spatio-temporal pattern and they influence the transcription and translation of many genes. generally, their functions involve various processes, including developmental patterning, organ separation, cell differentiation and proliferation, tumor generation, cell death and cell apoptosis, stress resistance, auxin response, fat metabolism and mirna biogenesis. in higher plants and animals, mirnas have been extensively studied but rarely so in algae. p. tricornutum is an atypical diatom with a weakly silicified outer shell, and the unusual property of being pleiomorphic with three convertible morphotypes, and silicification essentially restricted to one valve of the oval cells. with its characteristics of short life-cycle, small genome size and ease of transformation, p. tricornutum has become an attractive photosynthetic model. additionally, being rich in polyunsaturated fatty acid, especially in eicosapentaenoic acid, p. tricornutum has been used as a food organism and is considered a potential source of epa. there have been many studies investigating the factors affecting its cell composition. there were reports that microalgae accumulated lipids under nitrogen-limited as well as silicon-limited conditions, with similar studies conducted on p. tricornutum. accumulation of lipids in cells and a significant change in fatty acid composition were observed in p. tricornutum under low nitrogen conditions. using suppression subtractive hybridization technology, tang et al. separated a number of upregulated genes from p. tricornutum under nitrogen starvation, seven of which had high similarity with functional genes related to nitrogen utilization. studies of lipid metabolism of p. tricornutum under silicon-limited conditions are scarce. notwithstanding, sapriel et al. identified genes regulated by silicic acid availability, including upregulated and downregulated genes, from p. tricornutum under silicon-limited conditions. interestingly, they also observed some upregulated genes coding for transporters of metabolites related to nitrogen assimilation and transfer from p. tricornutum in the complete medium compared to silicon-limited conditions. a previous study on t. pseudonana showed that a glutamate acetyltransferase was involved in silicon metabolism. how are these genes regulated? do mirnas play a role in p. tricornutum nitrogen and silicon metabolism? there have been few studies that address these questions. in the present study, we constructed small rna libraries from p. tricornutum under normal, nitrogen-limited and silicon-limited conditions and then used high-throughput solexa technology to deeply sequence the srnas. the sequencing data were analyzed and mirnas were identified from all samples studied.  a diverse set of endogenous small rnas to determine the likely roles of mirnas in nitrogen and silicon metabolism in p. tricornutum, we constructed and sequenced small rna libraries from p. tricornutum grown in normal, nitrogen-free and silicon-free media, respectively. after removing adaptor sequences and filtering out low quality data, we obtained small rnas with size range of 10- nt, with an enrichment in 20- nt. after removing sequences shorter than nt, we obtained and total sequences, representing and unique, although sometimes partially overlapping, clean reads from pt pt and pt respectively. of these unique sequences, about 73%, 74% and 73% were only sequenced once. there were and total; and and unique sequences with at least one perfect match in the p. tricornutum nuclear genome - whereas and total; and and unique sequences matched the chloroplast genome, in pt pt and pt respectively. it was quite unexpected that a majority of srnas were located in the minus strand of chromosome and both strands of the chloroplast genome. the usual preference for a u at the 5' - end of plant small rna sequences was not observed. the four types of bases appeared equally in each locus. asrnas that matched nuclear genome. bsrnas that matched chloroplast genome. cunique srnas that appeared once. all clean reads were annotated according to their identities with non-coding rnas, plant mirnas, exon and intron and sirnas. in the case that some srna was mapped to more than one category, the following priority rule was adopted: rrna etc. > known mirna > exon > intron. rrna degraded fragments were the most abundant sequences retrieved from the p. tricornutum total srna pools, boasting the highest read frequency of all small rna classes in all the samples:, and % for pt pt and pt respectively. yet in the unique srna pools, non-annotated srna represented a significant part, with, and % in pt pt and pt respectively. homologs of plant known mirnas accounted for approximately % of the unique sequences in all the three samples, whereas in total sequences pools, the numbers were approximately % in pt and pt and only % in pt srnas mapped to exons and introns in either sense or antisense directions also represented a considerable part. the remaining srnas were snrna, snorna and trna. common and specific sequences analysis showed that only approximately 15% of the unique sequences were shared by every two samples, suggesting a diverse set of endogenous small rnas in p. tricornutum. anot annotated srnas. mirnas in p. tricornutum the identification of a great quantity of small rnas in p. tricornutum prompted us to examine whether some were mirnas. first we compared all the non-annotated srnas with the sequences of animal mirnas and virus mirnas available from mirbase to identify homologs of known mirnas. then we used the small rnas with homology to all known mirnas and the remaining non-annotated srnas to identify candidate known and novel mirna families in p. tricornutum, respectively. first we mapped these small rnas onto the p. tricornutum nuclear genome. then we extracted nt upstream and nt downstream from those loci and examined whether they could form hairpin secondary structures, a character of known plant and animal pre-mirnas, using criteria developed previously for plant mirna prediction. basically, precursors with free energy ≤ - kcal/mol checking by mfold, ≥ bp and ≤ bulges or asymmetries between mirna and mirna*, with mirna sequence length between 18-25nt and flank sequence length of were considered as potential p. tricornutum pre-mirnas and selected for further analysis. secondary structural predictions identified a total of small rna species that were derived from genomic loci whose surrounding sequences had the probability to form hairpin structures that met the requirements as a mirna precursor. then we checked for the structure stabilities of these sequences. among these, five were found to have a p-value lower than. they were checked for 5' homogeneity using as cut off. for those sequences with a p-value above, a more stringent 5' homogeneity of was used. all together we obtained sequences for manually rechecking according to criteria made previously for mirna identification. finally we determined sequences to be p. tricornutum mirnas. they were submitted to mirbase and named pti-mir5471- of these small rnas, seven of pre-mirna hairpins were supported by est data. each mirna had a single precursor. the length of pre-mirna ranged from to nt, with a mean of nt. the mfe range was - to - kcal/mol, with a mean of - kcal/mol. most pre-mirnas were located in intergenic regions and the others were mapped to genes of hypothetical protein, probably being mis-annotated. aindicated the chromosome number, start and end site and sense or antisense strand that the pre-mirnas located. bminimum free energy of pre-mirnas, predicted by mfold. clength of pre-mirnas. dcomputed p-values of mirna precursors. enumber of reads that had the same 5' end as the mature mirna and the total number reads mapped to the precursors. fasymmetric bulges larger than nt and mismatched mirna bases in the mirna region. expression patterns of mirnas/candidates during nitrogen-limited and silicon-limited conditions, and target prediction to investigate the probable roles of mirnas in nitrogen and silicon metabolism in p. tricornutum, we sequenced small rnas from p. tricornutum grown in normal, nitrogen-limited and silicon-limited media. of the mirnas identified, two appeared in all the three small rna libraries, one exclusively in pt and eight in pt3; and one was shared by pt and pt and one by pt and pt. the expression of mirnas in the samples indicated that they might play an important role under nitrogen-limited and/or silicon-limited conditions. to determine the likely regulated genes, we predicted targets of these mirnas. using the rules for target prediction suggested by allen, no target was identified. ignoring locus one and those larger than nt and allowing four mismatches between the mirna-target duplex in positions 2- some potential target sites were suggested. some of these potential targets were involved in lipid metabolism, suggesting that p. tricornutum mirnas might play a role in fatty acid metabolism. this was in accord with the report that p. tricornutum accumulated lipids under nitrogen-limited and silicon-limited conditions. however, as the genome of p. tricornutum is not fully annotated and the functions of many protein-coding genes are unknown, it is difficult to determine whether these mirna targets have any functional bias. sirna in p. tricornutum it has been reported that in arabidopsis, mirnas direct the generation of sirna, which were phased relatively with each other. to determine whether mirnas direct the generation of sirna in p. tricornutum, we identified potential sirnas and determined their location. potential sirnas were found in these samples: with and unique sequences; and and total sequences in pt pt and pt respectively. the majority of sirna were produced from a few hot-spots distributed in all the chromosomes; however, they were not phased relatively with each other. to determine whether small rnas play a role in silencing of repetitive sequences in p. tricornutum, as for other organisms, we performed a blast search against p. tricornutum repeat sequences and found, and sirna derived from these regions. this implied that small rnas might induce silencing of repetitive sequences in p. tricornutum. mirna northern blot mirna northern blotting was used to detect initial expression of mirnas and their precursors in p. tricornutum. 5s rna was blotted as load control. northern blot hybridization detected precursors of expected size in all the samples. this provided strong evidence for their expression. discussion did p. tricornutum mirnas evolve independently? we compared all p. tricornutum small rnas with all known plant, animal and virus mirnas in mirbase, and found significant identities. however, these identities did not pass the criteria we used to identify mirnas. the most straightforward interpretation for the relative lack of universally conserved mirnas between p. tricornutum and other organisms is that all mirnas in p. tricornutum are rare due to its small genome size, although scenarios that p. tricornutum contains novel mirnas that have no sequence homology with all known ones cannot be ruled out. in a study of mirnas in the unicellular green alga chlamydomonas reinhardtii, zhao et al. compared its mirnas with all known plant and animal mirnas, and found no homologs. in fact, c. reinhardtii lacked homologous mirnas even with other green algae. thus we asked whether p. tricornutum had some specific mirnas that have no sequence homology with all known mirnas, as for c. reinhardtii. we predicted novel mirnas from the small non-annotated rnas, using the same criteria as used to identify known mirnas. a total of novel mirnas were identified from p. tricornutum under normal, nitrogen-limited and/or silicon-limited conditions. they lacked homology with all known mirnas in the mirbase, including c. reinhardtii mirnas. thus we propose that mirnas in algae may have evolved independently to animals and plants, consistent with the suggestion of zhao et al. we also used the p. tricornutum chloroplast genome to identify mirnas. two loci met all the criteria we used to identify mirnas. interestingly, one of these mirna-like small rnas was homolog of cin-mir and part of the potential precursor shared 74% identity to cin-mir precursor. est analysis of p. tricornutum showed that many of its genes were more similar to animals than photosynthetic organisms. complete genome sequences showed that diatoms had a mosaic genome with genes from animals, plants and bacteria. thus it is probable that p. tricornutum might share some common mirnas with animals, although the percentage may be relatively low. we propose that this animal mirna-like small rna from p. tricornutum might be present in diatoms due to gene transformation, or are conserved mirnas derived from the heterotrophic secondary-host evolutionarily prior to the secondary endosymbiosis, or may be mirnas lost in the plant/red algal lineage during evolution, similar to the incorporation of animal-like genes in diatoms. if this small rna found in our study was genuine mirnas, then this represents a very interesting discovery. de riso, et al. had successfully demonstrated gene silencing in p. tricornutum. they analyzed molecular players involved in rna silencing in p. tricornutum and identified both dicer like proteins and argonaute like proteins. these argonaute like proteins in p. tricornutum clustered in a clade different from either animals or plants, suggesting that p. tricornutum might own a special risc pathway different from that of animals and plants, and thus probably result in the lack of preference for u at the 5' of p. tricornutum srnas. probable roles of mirnas in metabolism of p. tricornutum mirnas have been found to play important regulatory roles in various processes in multicellular organisms as well as the unicellular green alga c. reinhardtii. in the present study, mirnas were sequenced from p. tricornutum under normal, nitrogen-limited and silicon-limited conditions. this suggests that mirnas might play important roles in p. tricornutum. mirnas expressed in all three samples two mirnas appeared in all samples. candidate target genes for these mirnas included dna-directed rna polymerase; glutamate synthase and Δ fatty acid desaturase. this indicates that p. tricornutum mirnas might play important roles in a range of biological processes. it was reported that the composition of fatty acids was significantly influenced by availability of nitrogen and silicon. some genes related to glutamate/glutamine metabolism are regulated by silicon availability. interestingly, we predicted that one gene involved in glutamate synthesis was targeted by pti-mir which was downregulated in both pt and pt indicating that mirna might play a role in silicon-regulated glutamate metabolism. mirnas that exclusively sequenced from pt3 there were eight mirnas exclusively sequenced from pt. candidate target genes for these mirnas include phospholipase c isoform delta, nucleotide transporter, ornithine aminotransferase, nucleosome remodeling factor. in p. tricornutum, silicification is restricted to one valve of the oval cells and there is no silicon requirement for growth. the strain used in the present study was a fusiform type whose cell wall was not silicified. however, mirna species were most abundant in pt, and their targets involved in various processes, indicating that various biological processes might be influenced by silicon available through mirna regulation. the enrichment of srnas originating from the minus strand of chr and both strands of the chloroplast genome it was interesting that a majority of srnas were located in the minus strand of chromosome and both strands of the chloroplast genome. as reported by mcfadden and van dooren, green algal/plant and red algal originated from a first endosymbiosis between a eukaryotic and a endosymbiont, whereas diatoms originated from the secondary endosymbiosis between a heterotrophic organism and a red alga. the diatom chloroplast originated from the plasmid of the second endosymbionts, while nucleus of the second endosymbionts lost, living enormous numbers of their genes - typically more than 90% - house in the second host nucleus. we proposed that the enrichment of srnas on the minus strand of chr as well as both strands of the chloroplast genome indicated that chr might have some relative to the second endosymbionts. e.g., chr might have originated from nucleus of the second endosymbionts or the majority of the second endosymbionts nuclear genes might have transform into chr to test this hypothesis, we extracted the hot spot loci where most small rna derived from. those were 39000- nt of the minus strand of chr 63675- nt of the sense strand of chloroplast genome, and 110485- nt of the minus strand of the chloroplast genome. we then aligned them and found that the hot spot locus of chr had no homology with the chloroplast genome. thus, even if chr have some relative to the second endosymbionts, our data has little support for this hypothesis. we also found that the two hot spot loci of the chloroplast genome in fact share 100% identity. they are two inverted repeats, ira and irb, on the chloroplast genome. thus, small rnas might play an important role in silencing of inverted repeat region. the failure of detection of mature mirnas by northern blotting was probable due to their low expression we detected precursors of expected size for pti-mir and pti-mir in other organisms, precursors were more difficult to detect than mature mirnas in wild type samples, probably due to their temporary summation in the cells and convert fast into mature mirnas. we detected mirna precursors in all the three samples of p. tricornutum easily, implied that diatom might obtain different mirna processor from other organisms, which made the accumulation of mirna precursors. expected sizes for the mature mirnas were not detected. the most straightforward interpretation for this is the low expression of mature mirnas in the samples we detected, although scenarios that these mirnas are not real mirnas but sequencing artifacts or fragments of a longer transcript cannot be ruled out. more sensitive technology is needed to perform further analysis.  our results indicated that p. tricornutum owned a complex srna processing system. it contained novel mirnas that have no sequence homology with mirnas of other organisms and that they might play important regulator roles in p. tricornutum metabolism. 
to identify micrornas from p. tricornutum and to study their probable roles in nitrogen and silicon metabolism, we constructed and sequenced small rna libraries from p. tricornutum under normal, nitrogen-limited and silicon-limited conditions. they were probable p. tricornutum-specific novel mirnas. these mirnas were sequenced from p. tricornutum under normal, nitrogen-limited and/or silicon-limited conditions, and their potential targets were involved in various processes, such as signal transduction, protein amino acid phosphorylation, fatty acid biosynthetic process, regulation of transcription and so on. our results indicated that p. tricornutum contained novel mirnas that have no identifiable homologs in other organisms and that they might play important regulator roles in p. tricornutum metabolism. the genomes of the pennate diatom phaeodactylum tricornutum and the centric diatom thalassiosira pseudonana have been sequenced, revealing some characteristics of the diatoms' mosaic genome as well as some features of their fatty acid metabolism and urea cycle, and indicating their unusual properties. diatoms, which are important planktons widespread in various aquatic environments, are believed to play a vital role in primary production as well as silica cycling.
 diatoms, which are important planktons widespread in various aquatic environments, are believed to play a vital role in primary production as well as silica cycling. the genomes of the pennate diatom phaeodactylum tricornutum and the centric diatom thalassiosira pseudonana have been sequenced, revealing some characteristics of the diatoms' mosaic genome as well as some features of their fatty acid metabolism and urea cycle, and indicating their unusual properties. to identify micrornas from p. tricornutum and to study their probable roles in nitrogen and silicon metabolism, we constructed and sequenced small rna libraries from p. tricornutum under normal, nitrogen-limited and silicon-limited conditions.  a total of mirnas were identified. they were probable p. tricornutum-specific novel mirnas. these mirnas were sequenced from p. tricornutum under normal, nitrogen-limited and/or silicon-limited conditions, and their potential targets were involved in various processes, such as signal transduction, protein amino acid phosphorylation, fatty acid biosynthetic process, regulation of transcription and so on.  our results indicated that p. tricornutum contained novel mirnas that have no identifiable homologs in other organisms and that they might play important regulator roles in p. tricornutum metabolism. 