BACKGROUND
rice  is a staple food crop for about half of the world's population.
in  <dig>  the total rice-harvested area and rough rice yield in the world were  <dig>  million hectares and  <dig>  million tons, respectively .
however, the productivity of rice is severely affected by soil nitrogen nutrient deficiency worldwide.
commercially available urea fertilizer is the most widely used resource to meet a rice crop's nitrogen requirement, of which one third is lost through emission of greenhouse gasses and leaching, causing adverse environmental impacts  <cit> .
to meet these challenges and develop environmentally sustainable rice production systems, much attention has been given to natural methods of biological nitrogen fixation   <cit>  or to increase nitrogen use efficiency   <cit> .
the genus oryza comprises  <dig> species, including  <dig> cultivated  and  <dig> wild species with diverse ecological adaptation.
these species are categorized into  <dig> recognizable genome types   <cit> .
wild rice has diversified over  <dig> million years.
wild species are tremendous gene reservoirs for domesticated rice improvement, as they possess many desirable traits, such as resistance to diseases and insect pests or tolerance to different kinds of stresses  <cit> .
oryza longistaminata chev.
, broadly distributed throughout tropical africa, is a perennial species with characteristics of long anthers, self-incompatibility, allogamy, strong rhizomes and high biomass production on poor soils.
in spite of its overall inferior appearance, o. longistaminata has furnished genes for developing perennial rice  <cit>  and for breeding blight disease resistance varieties  <cit> .
to make better use of this potential, more genomic information is required, but there are only few batches of mrnas or full-length cdnas  of o. longistaminata in public databases, and no genome sequence is available.
sequencing and analysis of expressed sequence tags  has become a primary strategy for functional genomic studies in plants including novel gene discovery, gene expression profiling, microarray and molecular marker development, and accurate genome annotation.
after completing the full genome sequence of o. sativa ssp.
japonica cv.
nipponbare and the draft genome sequence of the o. sativa ssp.
indica cv.
93- <dig> through a map-based sequencing strategy and through a whole-genome shotgun sequencing approach, respectively  <cit> , much efforts were involved into rice ests projects.
approximately  <dig>  ests and > <dig>  full-length cdna sequences of cultivated rice are currently available in public databases.
however, the genomic studies of rice wild relatives are still in their infancy with the exception of the generation of  <dig>  leaf ests from the o. minuta  and  <dig>  leaf flcdnas from the o. rufipogon   <cit> .
especially roots are organs underrepresented in est studies.
therefore, a comprehensive survey of ests in roots of o. longistaminata was undertaken to provide an overview of o. longistaminata root transcriptome and thus a molecular basis for the identification of useful genes.
as newly developed massively parallel  <dig> pyrosequencing allows rapid generation of sequence data and deep sequencing coverage with reducing labour and cost  <cit> , we here characterized the first global root transcriptome of that wild rice species o. longistaminata by  <dig> gs-flx pyrosequencing technology.
this led to the discovery of a huge amount of novel ests which will facilitate gene mining and provide a basis for comparative studies within the genus oryza.
RESULTS
sequencing and assembly of  <dig> pyrosequencing ests
in order to obtain transcripts of genes that might be required for growth under nutrient stress, o. longistaminata plants were clonally propagated and were adapted to low-nitrogen conditions in unfertilized soil for several months.
mature plants with high biomass production  were subjected to rna extraction from roots.
as soil-grown roots often yield low quality rna with inhibitory effects on enzyme activity   <cit> , several rna extraction methods were compared.
a standard extraction protocol with trizol yielded degraded rna , while rna extracted by a ctab-based method was of high quality .
pooled rna extracts from two extractions were used for normalization and sequencing of cdnas.
one gs-flx  <dig> pyrosequencing run produced a total of  <dig>  reads  with average sequence length of  <dig> bp  from root cdnas of o. longistaminata.
after removal of adaptor sequences, polya tail and low quality sequences,  <dig>  reads remained with a total length of  <dig>  mb and an average length of  <dig> ±  <dig> bases, ranging from  <dig> bp to  <dig> bp .
only sequences above  <dig> bp of length were further considered.
clustering and assembling of these sequences produced  <dig>  contigs and  <dig>  singletons.
these data were trimmed again by removing those showing homology  to sequences of bacteria, fungi or metazoa, resulting in a total of  <dig>  processed unique sequences.
the length of contigs varied from  <dig> bp to  <dig> bp with an average of  <dig> bp, and that of singlets ranged from  <dig> bp to  <dig> bp with an average of  <dig> bp .
the majority of reads was in length of 201- <dig> bp , which was consistent with the  <dig> gs-flx sequencing capacity.
the size distribution of o. longistaminata consensuses after assembly was shown in table  <dig>  revealing that 92% of them fell between  <dig> and  <dig> bp in length.
although none of singlets was longer than  <dig> bp in the whole est dataset, there were  <dig>  contigs with sequence length larger than  <dig> bp .
in addition,  <dig>  contigs  were still less than  <dig> bp.
this might be due to the short length of the sequencing read and/or the low coverage of the transcriptome represented in this dataset.
most of the contigs were derived from few reads.
<dig>   and  <dig>   consensuses were derived from  <dig> and  <dig> reads, respectively .
the sequence data obtained were in a similar range as for other plant est sequencing projects using this technology  <cit> , however with a slightly higher read length, demonstrating the power of this approach to deliver large est datasets.
mapping ests to the o. sativa genome and transcriptome revealed novel ests
comparison of the unique est sequences of o. longistaminata to chromosomal and expressed sequences of o. sativa revealed a large set of two types of novel ests, those previously not found to be expressed by rice, and those not even detected in the rice genome.
the ests were aligned to genomic sequences of two o. sativa varieties, japonica - type nipponbare http://rgp.dna.affrc.go.jp/irgsp/ and indica -type 93- <dig> http://rice.genomics.org.cn/rice/index <dig> jsp, by using the blastn program with an e-value cut-off of e- <dig>  in total,  <dig>   out of  <dig>  sequence tags were anchored in rice genome.
these sequences mapped on all the  <dig> rice chromosomes  with almost equal distribution, chromosome  <dig>   <dig>   <dig> harbouring large amount of est sites accounting for approximately 40% of a total of  <dig>  est sites.
the distribution corresponds well to the size of the chromosomes, which highlights the close relationship between these two species.
these anchored ests also had a relatively high sequence identity with o. sativa sequences ranging from 78% to 100%, with an average of 97%.
the sequence identity distribution of  <dig>  o. longistaminata ests is shown in figure  <dig>  revealing that 61% of them had a sequence similarity higher than 98%.
athe length of  <dig> nipponbare  chromosomes is indicated.
b genes not assembled into chromosomes of cv.
93- <dig>
among  <dig>  ests,  <dig> could only be aligned to indica genome sequences and  <dig> could specifically match japonica genome sequences .
among these ests,  <dig> out of  <dig> or  <dig> out of  <dig> had high similarity  to indica or japonica cdna/ests sequences, respectively.
they might be indica-specific or japonica-specific genes, or they may map to gaps in the rice genome sequences.
the latter assumption may be likely for part of these ests, as  <dig> out of the  <dig> indica-specific genes mapped to ests detected in japonica with highest score, and  <dig> out of  <dig> japonica-specific ones to indica ests.
one of the  <dig> ests, xa21_ <dig>  was selected for further analysis.
the southern blot analysis was consistent with the blast results .
our findings indicated that o. longistaminata had parallel similarity to japonica and to indica rice at the dna level.
in order to assess how many o. longistaminata ests had already been detected as expressed genes in o. sativa, the ests mapping onto the o. sativa genomes were also compared with the knowledge-based oryza molecular biological encyclopedia  cdna collection, the indica cdna database http://www.ncgr.ac.cn/ricd/, and the ncbi rice est database.
<dig> % matched to o. sativa genes found to be expressed previously.
a large amount of ests  had previously not been detected as expressed.
for most of them, we did not find homologies to predicted gene models: inspection of the  <dig> longest ests showed that 67% shared sequence similarity with o. sativa but not to predicted genes, 23% with genes of predicted functions, and 10% with genes encoding hypothetical proteins.
this was also reflected in the lack of functional assignments , as after in silico translation only for a small fraction  of these ests could be assigned according to gene ontology .
this emphasizes the power of the next generation sequencing approach to detect novel transcripts or even novel genes.
as the o. sativa genome may still contain regions that are not fully annotated, our ests might indicate as yet unpredicted genes or utrs that might be functional in o. sativa as well.
on the other hand, o. longistaminata might express a special set of genes in comparison to o. sativa, due the particular conditions - being adapted to low availability of external nitrogen sources-, or due to the interspecies differences in expression.
as another category of novel ests, in total,  <dig>   of  <dig>  unique est sequences could not be mapped to the o. sativa chromosomes by homology search against genomic sequences.
among them,  <dig> matched the publicly available o. sativa mrnas or ests.
the remaining  <dig>  sequence tags showed no significant sequence identity  with any rice genomic or expressed sequences in public database.
among these, only a very small number  had a significant hit in ncbi non-redundant  nucleotide database or ests database.
the remaining  <dig>  ests may therefore represent novel genetic material present in o. longistaminata and other root-residing eukaryotes.
functional classification of o. longistaminata ests
the consensus sequences were annotated for sequence similarities using the blastx translated sequence comparison against the ncbi non-redundant  protein database.
among the  <dig>  contigs and singlets,  <dig>   had at least a significant alignment to exisiting gene models in the nr database at an e-value cut-off of e- <dig>  a majority  of the o. longistaminata sequences did not match any known protein sequences.
most of the  <dig>  novel sequence tags  fell into this category.
this can partly be attributed to the short length of most of these uni-ests, or a large fraction of the ests might represent untranslated regions.
mapping those uni-etss to rice gene models supported this assumption.
https://www.gabipd.org/database/cgi-bin/greencards.pl.cgi.
the unique ests were further classified into molecular function, biological process and cellular components, according to the standard gene ontology terms .
only to  <dig>  o. longistaminata sequence tags go numbers were assigned, however a broad range of go categories was covered: the percentage distribution of go terms is shown in figure  <dig>  a total of  <dig>  sequences could be assigned to the molecular function.
among them, nucleotide binding  and binding   and catalytic activity  were the most dominant categories.
with regard to the category of biological process containing  <dig>  ests, cellular processes  were the most highly represented category.
the following categories were protein modification processes , metabolic processes , transport  and biosynthetic processes .
under the category of cellular component,  <dig> % of  <dig>  ests were predicted as membrane proteins , followed by plastid , nucleus  and plasma membrane  proteins.
biological validation of novel ests
approximately  <dig> % of the unique est sequences detected in the o. longistaminata root transcriptome currently are not similar to rice sequences in databases.
these may represent novel genes of o. longistaminata not present in o. sativa, or it may be possible that there are gaps existing in cultivated rice genome sequences, or a small portion of the unmapped sequences might have resulted from contamination by non-rice sources.
a total of  <dig> novel ests were randomly selected for rt-pcr to determine the portion of potential novel genes originating from o. longistaminata rather other organisms in our transcript collection.
rt-pcr experiments were conducted on rnas derived from root tissue of clonally propagated o. longistaminata plants grown in soil in the phytotron.
of the  <dig> primer pairs for pcr,  <dig> generated rt-pcr products that were of the expected size and whose sequences were confirmed by sanger sequencing.
the results demonstrated that these  <dig> novel transcripts detected among the 454-ests are indeed expressed in o. longistaminata roots grown in soil .
among another set of primer pairs for  <dig> additional ests, six yielded a positive result .
however, as conditions for pcr amplification cannot be optimized due to lack of intron-free template, these results may be an underestimation.
to test for distribution of the putatively expressed genes among different accessions of the same species, o. longistaminata grains collected at the okavango region of namibia were used for gnotobiotic cultivation of seedlings in the phytotron, and pooled for analysis.
from root rna extracts,  <dig> of the  <dig> primer pairs yielded rt-pcr products with correct size whose sequences were validated by sanger sequencing again.
this confirmed that these fragments indeed originated from this species and not e.g.
from root endophytes, and that their expression was conserved within the species.
to control occurrence in the genome, southern blot analysis was carried out from genomic dna extracted from leaves of o. longistaminata accession irgc  <dig>  and from o. sativa.
probes generated from two out of the  <dig> ests detected hybridizing fragments for wild but not for cultivated rice .
the results indicated that these  <dig> ests are indeed o. longistaminata-specific sequences.
based on these results, we estimated that a large subset of novel sequences was derived from o. longistaminata.
the remaining novel est sequences might be due to the contamination from other sources or due to the  <dig> sequencing artefacts.
CONCLUSIONS
in this study, we present a large-scale est dataset comprising  <dig>  unique est sequences derived from wild rice o. longistaminata by massively parallel pyrosequencing.
among them,  <dig>  ests matched to known gene models, and  <dig>  ests were annotated with go terms.
the comparative analysis between wild rice and two domesticated rice subspecies indicated that o. longistaminata had parallel similarity to japonica as to indica rice.
notably, a large amount of ests derived from o. longistaminata roots have not yet been detected as expressed in o. sativa, or did not show similarity to publicly available rice sequences or any other genes.
our data contribute to future annotation approaches of the o. longistaminata genome, to identification of o. longistaminata-specific genes and to the comparative study of the evolution among oryza genus.
these novel ests will particularly provide a basis for further identification of genes of o. longistaminata underlying adaptation to nutrient-limiting conditions.
all est obtained in this study is attached in the supplemental data .

 rice is a staple food crop for about half of the world's population. in the total rice-harvested area and rough rice yield in the world were million hectares and million tons, respectively. however, the productivity of rice is severely affected by soil nitrogen nutrient deficiency worldwide. commercially available urea fertilizer is the most widely used resource to meet a rice crop's nitrogen requirement, of which one third is lost through emission of greenhouse gasses and leaching, causing adverse environmental impacts. to meet these challenges and develop environmentally sustainable rice production systems, much attention has been given to natural methods of biological nitrogen fixation or to increase nitrogen use efficiency. the genus oryza comprises species, including cultivated and wild species with diverse ecological adaptation. these species are categorized into recognizable genome types. wild rice has diversified over million years. wild species are tremendous gene reservoirs for domesticated rice improvement, as they possess many desirable traits, such as resistance to diseases and insect pests or tolerance to different kinds of stresses. oryza longistaminata chev., broadly distributed throughout tropical africa, is a perennial species with characteristics of long anthers, self-incompatibility, allogamy, strong rhizomes and high biomass production on poor soils. in spite of its overall inferior appearance, o. longistaminata has furnished genes for developing perennial rice and for breeding blight disease resistance varieties. to make better use of this potential, more genomic information is required, but there are only few batches of mrnas or full-length cdnas of o. longistaminata in public databases, and no genome sequence is available. sequencing and analysis of expressed sequence tags has become a primary strategy for functional genomic studies in plants including novel gene discovery, gene expression profiling, microarray and molecular marker development, and accurate genome annotation. after completing the full genome sequence of o. sativa ssp. japonica cv. nipponbare and the draft genome sequence of the o. sativa ssp. indica cv. 93- through a map-based sequencing strategy and through a whole-genome shotgun sequencing approach, respectively, much efforts were involved into rice ests projects. approximately ests and > full-length cdna sequences of cultivated rice are currently available in public databases. however, the genomic studies of rice wild relatives are still in their infancy with the exception of the generation of leaf ests from the o. minuta and leaf flcdnas from the o. rufipogon. especially roots are organs underrepresented in est studies. therefore, a comprehensive survey of ests in roots of o. longistaminata was undertaken to provide an overview of o. longistaminata root transcriptome and thus a molecular basis for the identification of useful genes. as newly developed massively parallel pyrosequencing allows rapid generation of sequence data and deep sequencing coverage with reducing labour and cost, we here characterized the first global root transcriptome of that wild rice species o. longistaminata by gs-flx pyrosequencing technology. this led to the discovery of a huge amount of novel ests which will facilitate gene mining and provide a basis for comparative studies within the genus oryza.  sequencing and assembly of pyrosequencing ests in order to obtain transcripts of genes that might be required for growth under nutrient stress, o. longistaminata plants were clonally propagated and were adapted to low-nitrogen conditions in unfertilized soil for several months. mature plants with high biomass production were subjected to rna extraction from roots. as soil-grown roots often yield low quality rna with inhibitory effects on enzyme activity, several rna extraction methods were compared. a standard extraction protocol with trizol yielded degraded rna, while rna extracted by a ctab-based method was of high quality. pooled rna extracts from two extractions were used for normalization and sequencing of cdnas. one gs-flx pyrosequencing run produced a total of reads with average sequence length of bp from root cdnas of o. longistaminata. after removal of adaptor sequences, polya tail and low quality sequences, reads remained with a total length of mb and an average length of ± bases, ranging from bp to bp. only sequences above bp of length were further considered. clustering and assembling of these sequences produced contigs and singletons. these data were trimmed again by removing those showing homology to sequences of bacteria, fungi or metazoa, resulting in a total of processed unique sequences. the length of contigs varied from bp to bp with an average of bp, and that of singlets ranged from bp to bp with an average of bp. the majority of reads was in length of 201- bp, which was consistent with the gs-flx sequencing capacity. the size distribution of o. longistaminata consensuses after assembly was shown in table revealing that 92% of them fell between and bp in length. although none of singlets was longer than bp in the whole est dataset, there were contigs with sequence length larger than bp. in addition, contigs were still less than bp. this might be due to the short length of the sequencing read and/or the low coverage of the transcriptome represented in this dataset. most of the contigs were derived from few reads. and consensuses were derived from and reads, respectively. the sequence data obtained were in a similar range as for other plant est sequencing projects using this technology, however with a slightly higher read length, demonstrating the power of this approach to deliver large est datasets. mapping ests to the o. sativa genome and transcriptome revealed novel ests comparison of the unique est sequences of o. longistaminata to chromosomal and expressed sequences of o. sativa revealed a large set of two types of novel ests, those previously not found to be expressed by rice, and those not even detected in the rice genome. the ests were aligned to genomic sequences of two o. sativa varieties, japonica - type nipponbare http://rgp.dna.affrc.go.jp/irgsp/ and indica -type 93- http://rice.genomics.org.cn/rice/index jsp, by using the blastn program with an e-value cut-off of e- in total, out of sequence tags were anchored in rice genome. these sequences mapped on all the rice chromosomes with almost equal distribution, chromosome harbouring large amount of est sites accounting for approximately 40% of a total of est sites. the distribution corresponds well to the size of the chromosomes, which highlights the close relationship between these two species. these anchored ests also had a relatively high sequence identity with o. sativa sequences ranging from 78% to 100%, with an average of 97%. the sequence identity distribution of o. longistaminata ests is shown in figure revealing that 61% of them had a sequence similarity higher than 98%. athe length of nipponbare chromosomes is indicated. b genes not assembled into chromosomes of cv. 93- among ests, could only be aligned to indica genome sequences and could specifically match japonica genome sequences. among these ests, out of or out of had high similarity to indica or japonica cdna/ests sequences, respectively. they might be indica-specific or japonica-specific genes, or they may map to gaps in the rice genome sequences. the latter assumption may be likely for part of these ests, as out of the indica-specific genes mapped to ests detected in japonica with highest score, and out of japonica-specific ones to indica ests. one of the ests, xa21_ was selected for further analysis. the southern blot analysis was consistent with the blast results. our findings indicated that o. longistaminata had parallel similarity to japonica and to indica rice at the dna level. in order to assess how many o. longistaminata ests had already been detected as expressed genes in o. sativa, the ests mapping onto the o. sativa genomes were also compared with the knowledge-based oryza molecular biological encyclopedia cdna collection, the indica cdna database http://www.ncgr.ac.cn/ricd/, and the ncbi rice est database. % matched to o. sativa genes found to be expressed previously. a large amount of ests had previously not been detected as expressed. for most of them, we did not find homologies to predicted gene models: inspection of the longest ests showed that 67% shared sequence similarity with o. sativa but not to predicted genes, 23% with genes of predicted functions, and 10% with genes encoding hypothetical proteins. this was also reflected in the lack of functional assignments, as after in silico translation only for a small fraction of these ests could be assigned according to gene ontology. this emphasizes the power of the next generation sequencing approach to detect novel transcripts or even novel genes. as the o. sativa genome may still contain regions that are not fully annotated, our ests might indicate as yet unpredicted genes or utrs that might be functional in o. sativa as well. on the other hand, o. longistaminata might express a special set of genes in comparison to o. sativa, due the particular conditions - being adapted to low availability of external nitrogen sources-, or due to the interspecies differences in expression. as another category of novel ests, in total, of unique est sequences could not be mapped to the o. sativa chromosomes by homology search against genomic sequences. among them, matched the publicly available o. sativa mrnas or ests. the remaining sequence tags showed no significant sequence identity with any rice genomic or expressed sequences in public database. among these, only a very small number had a significant hit in ncbi non-redundant nucleotide database or ests database. the remaining ests may therefore represent novel genetic material present in o. longistaminata and other root-residing eukaryotes. functional classification of o. longistaminata ests the consensus sequences were annotated for sequence similarities using the blastx translated sequence comparison against the ncbi non-redundant protein database. among the contigs and singlets, had at least a significant alignment to exisiting gene models in the nr database at an e-value cut-off of e- a majority of the o. longistaminata sequences did not match any known protein sequences. most of the novel sequence tags fell into this category. this can partly be attributed to the short length of most of these uni-ests, or a large fraction of the ests might represent untranslated regions. mapping those uni-etss to rice gene models supported this assumption. https://www.gabipd.org/database/cgi-bin/greencards.pl.cgi. the unique ests were further classified into molecular function, biological process and cellular components, according to the standard gene ontology terms. only to o. longistaminata sequence tags go numbers were assigned, however a broad range of go categories was covered: the percentage distribution of go terms is shown in figure a total of sequences could be assigned to the molecular function. among them, nucleotide binding and binding and catalytic activity were the most dominant categories. with regard to the category of biological process containing ests, cellular processes were the most highly represented category. the following categories were protein modification processes, metabolic processes, transport and biosynthetic processes. under the category of cellular component, % of ests were predicted as membrane proteins, followed by plastid, nucleus and plasma membrane proteins. biological validation of novel ests approximately % of the unique est sequences detected in the o. longistaminata root transcriptome currently are not similar to rice sequences in databases. these may represent novel genes of o. longistaminata not present in o. sativa, or it may be possible that there are gaps existing in cultivated rice genome sequences, or a small portion of the unmapped sequences might have resulted from contamination by non-rice sources. a total of novel ests were randomly selected for rt-pcr to determine the portion of potential novel genes originating from o. longistaminata rather other organisms in our transcript collection. rt-pcr experiments were conducted on rnas derived from root tissue of clonally propagated o. longistaminata plants grown in soil in the phytotron. of the primer pairs for pcr, generated rt-pcr products that were of the expected size and whose sequences were confirmed by sanger sequencing. the results demonstrated that these novel transcripts detected among the 454-ests are indeed expressed in o. longistaminata roots grown in soil. among another set of primer pairs for additional ests, six yielded a positive result. however, as conditions for pcr amplification cannot be optimized due to lack of intron-free template, these results may be an underestimation. to test for distribution of the putatively expressed genes among different accessions of the same species, o. longistaminata grains collected at the okavango region of namibia were used for gnotobiotic cultivation of seedlings in the phytotron, and pooled for analysis. from root rna extracts, of the primer pairs yielded rt-pcr products with correct size whose sequences were validated by sanger sequencing again. this confirmed that these fragments indeed originated from this species and not e.g. from root endophytes, and that their expression was conserved within the species. to control occurrence in the genome, southern blot analysis was carried out from genomic dna extracted from leaves of o. longistaminata accession irgc and from o. sativa. probes generated from two out of the ests detected hybridizing fragments for wild but not for cultivated rice. the results indicated that these ests are indeed o. longistaminata-specific sequences. based on these results, we estimated that a large subset of novel sequences was derived from o. longistaminata. the remaining novel est sequences might be due to the contamination from other sources or due to the sequencing artefacts.  in this study, we present a large-scale est dataset comprising unique est sequences derived from wild rice o. longistaminata by massively parallel pyrosequencing. among them, ests matched to known gene models, and ests were annotated with go terms. the comparative analysis between wild rice and two domesticated rice subspecies indicated that o. longistaminata had parallel similarity to japonica as to indica rice. notably, a large amount of ests derived from o. longistaminata roots have not yet been detected as expressed in o. sativa, or did not show similarity to publicly available rice sequences or any other genes. our data contribute to future annotation approaches of the o. longistaminata genome, to identification of o. longistaminata-specific genes and to the comparative study of the evolution among oryza genus. these novel ests will particularly provide a basis for further identification of genes of o. longistaminata underlying adaptation to nutrient-limiting conditions. all est obtained in this study is attached in the supplemental data. 
the generated sequence data are publicly available and will facilitate gene discovery in o. longistaminata and rice functional genomic studies. one sequencing run using a normalized cdna library from o. longistaminata roots adapted to low n conditions generated reads, which assembled into contigs and singletons. the large number of abundant of novel ests suggests different metabolic activity in o. longistaminata roots in comparison to o. sativa roots. our results show that one run using a genome sequencer flx from life science/roche generates sufficient genomic information for adequate de novo assembly of a large number of transcripts in a wild rice species, o. longistaminata. in this study we present the first comprehensive characterization of the o. longistaminata root transcriptome using pyrosequencing. comparison with ests derived from cultivated rice collections revealed expressed genes across different plant species, however % of the o. longistaminata ests had not been detected as expressed in o. sativa. however, only limited genomic or transcriptomic data of o. longistaminata are currently available. rt-pcr and southern blot analyses confirmed the expression of selected novel transcripts in o. longistaminata.
 oryza longistaminata, an aa genome type, originates from africa and is closely related to asian cultivated rice. it contains various valuable traits with respect to tolerance to biotic and abiotic stress, qtls with agronomically important traits and high ability to use nitrogen efficiently. however, only limited genomic or transcriptomic data of o. longistaminata are currently available.  in this study we present the first comprehensive characterization of the o. longistaminata root transcriptome using pyrosequencing. one sequencing run using a normalized cdna library from o. longistaminata roots adapted to low n conditions generated reads, which assembled into contigs and singletons. by similarity search against protein databases, putative functions were assigned to over uni-ests. comparison with ests derived from cultivated rice collections revealed expressed genes across different plant species, however % of the o. longistaminata ests had not been detected as expressed in o. sativa. additionally, % had no significant similarity to known sequences. rt-pcr and southern blot analyses confirmed the expression of selected novel transcripts in o. longistaminata.  our results show that one run using a genome sequencer flx from life science/roche generates sufficient genomic information for adequate de novo assembly of a large number of transcripts in a wild rice species, o. longistaminata. the generated sequence data are publicly available and will facilitate gene discovery in o. longistaminata and rice functional genomic studies. the large number of abundant of novel ests suggests different metabolic activity in o. longistaminata roots in comparison to o. sativa roots. 