BACKGROUND
the solanaceae family comprises 1000- <dig> species that show wide morphological variability and ecological adaptability  <cit> .
this taxon includes a number of vegetable crops including fruit-bearing vegetables, tuber-bearing vegetables, and ornamental plants, many of which have economic importance.
tomato  is a member of the solanaceae, and it has served as a model system for fruit development  <cit>  and plant defense  <cit> .
a number of studies have accumulated substantial information regarding the genetics and physiology of tomato.
to understand the physiological processes of tomato at the molecular level, it is necessary to link traits to dna sequence.
whole-genome sequencing is one of the approaches to achieve this goal.
a project for sequencing the tomato genome was launched by the international solanaceae project  in the year  <dig>  and the project is currently in the final stage of sequence polishing   <cit> .
accumulation of expressed sequence tags  is an alternative approach to collecting dna sequence information.
a large number of ests have been produced to provide a resource for gene finding and expression studies  <cit> .
for tomato ests, several repositories are available worldwide, such as the dana-farber cancer institute  tomato gene index, the sol genomics network   <cit> , mibase  <cit> , plantgdb  <cit> , and tomatest  <cit> , and the number of tomato ests accumulated in the databases is  <dig>  .
to elucidate the functions of individual genes, attempts have been made to organize ests into consensus sequences, which are referred to as unigenes or tentative consensus  sequences.
however, a more direct approach to collecting dna information of individual genes is to analyze full-length cdna sequences.
major advantages of sequencing full-length cdna over the est assembly include that the data is derived from a single clone rather than the assembly of multiple ests which can generate contigs containing sequences from multiple transcripts.
another major advantage of this approach is that full-length cdna clones have complete sequences of transcripts including coding regions  and untranslated regions .
this facilitates the subsequent annotation and prediction of genomic structures.
the complete cdss in full-length cdnas allow the prediction of protein sequences, conserved domains, and conserved motifs.
furthermore, full-length cdnas are easy to use with gene transfer systems, permitting the functional analysis of individual genes through reverse-genetics approaches.
thus, full-length cdnas are a powerful genomic research tool.
in tomato genomics, a miniature tomato cultivar of s. lycopersicum, micro-tom  <cit> , is regarded as an excellent model system.
micro-tom has characteristics that make it suitable for experimental study, such as small size, short generation time, and ease of transformation  <cit> .
recently, resources such as various mutagenized lines have been developed in the micro-tom-background  <cit> .
thus, a collection of micro-tom full-length cdnas would boost the molecular bioengineering of tomato by synergistically combining with other resources.
however, no full-length cdna collection of micro-tom, or of tomato, has yet been established.
in this study, we developed a large-scale collection of micro-tom full-length cdnas.
we constructed full-length-enriched cdna libraries from micro-tom leaf, fruit, and root tissues, and we obtained full-length sequences of  <dig>  cdnas.
we then checked for redundancy between sequences, for the presence of cdss, and for the contamination of chimeric sequences, thus generating a set of  <dig>  non-redundant full-length cdna sequences  for sequence analysis.
we report the results of utr analyses, including length distribution, base composition; and the results of cds analyses, including classification of functions of deduced proteins, metabolic pathway annotations, and comparisons with genes of other plants.
we also report the results of mapping nrflcdnas onto the draft of tomato whole-genome sequence.
mapping onto genome sequence suggested that introns of tomato genes are relatively long compared to arabidopsis and rice.
we also found that the frequency of nucleotide mismatch between exon regions of micro-tom and those of the genome-sequencing cultivar, heinz  <dig>  is  <dig> %.
finally, the value of micro-tom full-length cdna information will be discussed in terms of integration with the tomato genome sequence and other tomato resources.
RESULTS
construction of full-length-enriched cdna libraries
we constructed four full-length-enriched cdna libraries from micro-tom tissues including leaves, fruits, and roots.
rna samples were prepared from plants grown under  <dig> different conditions to increase the variation of transcripts in the libraries .
leaves were treated with  <dig> pathogens,  <dig> chemical and biochemical elicitors, salicylic acid, and methyl jasmonic acid .
leaf rna samples were prepared from  <dig> tissue types under different treatment conditions .
fruit rna samples were prepared from the pericarp at four ripening stages  harvested in the year  <dig> and  <dig> .
root rna samples were prepared from plants both with and without flowers, expecting that tissues of different growth phases have different transcripts.
we also prepared rna samples from roots treated with fusarium oxysporum race  <dig> .
rna samples prepared from leaf, fruit tissues harvested in the year  <dig>  fruit tissues harvested in the year  <dig>  and root-tissues were mixed, and the respective mixtures were used as templates for four separate cdna libraries, designated lefl <dig>  fc, lefl <dig>  and lefl <dig>  the fc library was constructed by the vector-capping method  <cit> , and the lefl  <dig>  lefl <dig>  and lefl <dig> libraries were constructed by the cap-trapper method  <cit> .
cdnas derived from pathogens were excluded by hybridization to mrnas prepared from the pathogens.
the subtraction process was effective, since 5'-end sequences of only  <dig> clones out of  <dig>  randomly selected clones matched pathogen sequences in the ncbi genbank, and these were excluded from further data processing.
abbreviations: dpi, days post inoculation; hpi, hours post inoculation; hps, hour post spray; dps, days post spray; daa, days after anthesis.
more detailed dexcription of the samples is available as additional file  <dig>
full-length sequencing of cdnas
a schematic flow of the sequencing process is presented in figure 1a.
we randomly selected  <dig>  independent clones from leaf, fruit, and root libraries, and sequenced them from the 5'-end.
after trimming vector-derived sequences and low-quality sequences,  <dig>  5'-end sequences  were combined with micro-tom ests reported previously  <cit>  and tomato ests registered in the sgn database.
a total of  <dig>  ests were grouped into  <dig>  clusters.
from  <dig>  clusters,  <dig>  clusters containing fc and lefl clones were selected.
the number of fc or lefl clones in each cluster ranged from  <dig> to  <dig> .
the fc or lefl sequence having the longest 5'-end extension in each cluster was selected as the representative of that cluster.
full-length sequencing was performed using the representative clones by the clone-by-clone primer walking method.
out of  <dig>  clones, full-length inserts of  <dig>  cdna clones were sequenced .
mean of phred quality value of each base was  <dig> , and more than 70% of bases exceeded quality value  <dig>  this collection was registered to the high-throughput cdna sequence  division of the ddbj.
generation of a non-redundant set of the full-length cdnas
we checked for redundancy within the set of  <dig>  htcs, and then a set of  <dig>  htcs representing non-redundant transcripts was sent for further processing .
to check whether each of the  <dig>  htcs has a full-length cds or not, we first searched for cdnas derived from non-coding rnas using the noncode version  <dig>  dataset  <cit> .
three htcs  were identified as cdnas derived from non-coding rna  and excluded from further analysis.
next, we confirmed that all of the htcs did not match dna sequences from pathogens used to attack leaf and root tissues, suggesting that the probability of contamination with pathogen-derived transcripts was negligible .
we then checked for chimeric clones putatively generated during cdna cloning process.
according to the criteria described in methods,  <dig> htcs were regarded as chimeric clones .
we then identified sets of htcs generated by the alternative splicing event, and excluded  <dig> htcs containing retained introns .
from the remaining  <dig>  htcs, cdss were predicted by using framedp  <cit>  and genemark.hmm-e  <cit>  programs, and by selecting open reading frame encoding the longest amino acid sequence.
essentially, a cds encoding amino acid sequence that had the highest similarity to proteins registered in either the nr or the tomato sbm protein databases was selected as representative cds of the htc .
htcs with cdss shorter than nine amino acid long were excluded from further analysis, since the shortest length tomato protein registered in uniprot was  <dig> aa .
out of  <dig>  non-redundant htcs, cds encoding full-length protein was predicted for  <dig>  htcs.
full-length cds were not predicted for  <dig> htcs and no coding regions were predicted for  <dig> htcs.
finally, a set of  <dig>  htcs  was generated, hereafter referred to as nrflcdna.
this nrflcdna set was subjected to subsequent sequence analyses.
nucleotide sequences of  <dig>  nrflcdnas were compared with dfci tomato gene index release  <dig>  tentative consensus , sgn tomato unigenes , and the prerelease of tomato genome shotgun sequence  .
percentage of nrflcdnas that did not match  was  <dig> %,  <dig> %, and  <dig> % against dfci tomato tcs, sgn tomato unigenes, and tomato genome sequences, respectively.
predicted amino acid sequences of  <dig>  htcs  were compared with proteins registered in ncbi nr dataset and tomato sbm protein dataset  .
distribution of e-value was similar against both datasets.
approximately 22% of the nrflcdnas had very high similarity  to nr and sbm proteins, and more than 75% of the nrflcdnas have e-value smaller than 1e- <dig>
length distributions of utrs and cdss
figures 3a and 3b show the distributions of cdna insert length and the cds length, respectively.
the average insert length was  <dig> bp, which is shorter than those of arabidopsis  and soybean  .
the median value for the insert length was  <dig> bp, which is shorter than arabidopsis  and rice   <cit> , but longer than poplar  .
the average length of the cds was  <dig> bp, corresponding to an average polypeptide length of  <dig> aa, which was also shorter than those of arabidopsis, rice, and soybean, and longer than poplar and maize  <cit>  .
the nrflcdna set contains  <dig> sequences derived from the fc clones that were cloned by the vector-capping method, and these harbor shorter cdna inserts  than those derived from the lefl clones that were cloned by the cap-trapper method .
this probably explains why the average lengths are shorter than those reported for full-length cdnas of other plants produced by the cap-trapper method.
n.a.
: not available.
figures 3c and 3d show the distributions of the 5'- and 3'-utr lengths, respectively.
in contrast to cds length, tomato nrflcdnas had longer utrs compared to those of other plants.
the average 5'- and 3'-utr lengths were  <dig> and  <dig> bp, respectively.
the median 5'- and 3'-utr lengths were  <dig> and  <dig> bp, respectively.
on both average and median bases, the utrs of tomato are longer than those of arabidopsis, soybean, poplar, and maize, and slightly shorter than those of rice .
the relatively long utrs of tomato likely contribute to the regulation of mrna transcription, translation, and stability  <cit> .
nucleotide composition
the nucleotide composition of nrflcdnas differed among the 5'-utrs, cdss, and 3'-utrs .
the at-content of the full-length cdnas was  <dig> %, which is slightly higher than that of arabidopsis full-length cdnas   <cit> .
at richness was more pronounced in the 5'- and 3'-utrs , and less pronounced in the cdss .
the 5'-utrs had more cs than the 3'-utrs, and the number of ts was higher in the 3'-utrs than in the 5'-utrs.
these results are similar to those observed in arabidopsis full-length cdnas  <cit> .
however, nucleotide compositions of the nrflcdna was more at-rich than those of maize transcripts  <cit> .
the most frequently used stop codon is tga  followed by taa  and tag .
frequency of stop codon is similar to that occurred in arabidopsis , but different from those occurred in rice  and maize   <cit> .
alternative splicing and retained intron
as reported for arabidopsis and rice full-length cdna collections, the collection of full-length cdnas contains cdnas generated from alternatively spliced transcripts  <cit> .
to identify nrflcdnas derived from alternatively spliced transcripts, we first generated multi-sequence groups using the non-redundant  <dig>  htcs, sgn tomato unigenes, and dfci tomato tcs according to the similarity to the target non-redundant htcs .
all pairs of target non-redundant htcs and member sequences were subjected to intron detection, and we confirmed that  <dig> target non-redundant htcs had splicing variants.
the percentage of non-redundant htcs with splicing variants was 10%, which is slightly lower than that of arabidopsis   <cit>  or rice   <cit> .
out of these  <dig> non-redundant htcs,  <dig> htcs were aligned with sgn tomato bac sequences , which allowed us to classify the types of  <dig> alternative splicing events .
the retained intron-type was the most frequent .
the alternative acceptor site- and alternative donor site-types also occurred frequently, comprising 20% and 16% of all events, respectively.
the exon skip-type and other types  were rather rare.
the occurrence of alternative acceptor site- and alternative donor site-types was slightly higher than that reported previously for tomato  <cit> .
nrflcdnas with retained introns encode proteins with alternative amino acid sequences.
we found that  <dig> nrflcdnas may contain retained full- or partial-length introns .
larger scale identification of alternative splicing events using the draft of tomato whole-genome sequence is currently underway.
functional annotation of tomato full-length cdna
next, putative functions of nrflcdnas were assessed by blastp searches against the tair <dig>  rap-db, and ncbi nr protein datasets.
to identify functions of the nrflcdnas, we used gene ontology  annotations for arabidopsis genes that matched nrflcdnas.
the results from the case using arabidopsis proteins that match nrflcdnas at e-value smaller than 1e- <dig> is shown .
this revealed that nrflcdnas covered functional categories as broadly as all arabidopsis genes .
to obtain insights into the metabolism-related genes, the nrflcdnas were assigned to the lycocyc pathways http://sgn.cornell.edu/tools/solcyc/.
based on the best matches to sgn tomato unigenes,  <dig> nrflcdnas  were assigned to lycocyc pathways, and  <dig> pathway annotations were obtained.
comparison of the pathway-classification patterns with those for all sgn unigenes demonstrated that the relative abundance of annotations assigned to the pathways "amino acids degradation", "fermentation", and "tca cycle" was higher in the nrflcdnas than in all sgn unigenes .
this demonstrated that genes related to primary metabolism are slightly overrepresented in the nrflcdna set compared to genes related to secondary metabolism.
we then attempted to identify transcription factors that are crucial for the transcriptional regulation of various biological processes.
we searched for transcription factor domains described in agris attfdb  <cit>  using the interpro database by querying the cds of the nrflcdnas.
this search demonstrated that  <dig> nrflcdnas  had  <dig> interpro entries classified into  <dig> transcription factor families .
the g2-like and myb families were the most numerous , followed by the homeobox family.
this result was slightly different from those of arabidopsis  <cit>  and rice  <cit> , in which the ap2/erebp family and zinc finger family were predominant, respectively.
difference in the distribution of transcription factors between tomato nrflcdna and arabidopsis implies that the nrflcdna set failed to contain transcription factors whose expression level is low or cell type-specific.
amember nrflcdnas of myb were completely overlapped with those of g2-like.
bmember nrflcdnas of c2c2-yabby and arid completely overlapped.
comparative analysis with genes of other plants
to assess the similarity of nrflcdnas with genes of other plants, deduced peptide sequences were compared with protein databases of arabidopsis  and rice  using blastp, and with the dfci gene indices of barley, wheat, maize, pine, spruce, poplar, lotus japonicus, medicago truncatula, soybean, orange, apple, grape, tobacco, and potato using tblastn .
nrflcdnas has high similarity to two solanaceae plants, potato and tobacco .
percentages of nrflcdna that did not match to potato and tobacco tcs  were  <dig> % and  <dig> %, respectively, which are the lowest among these  <dig> species.
on the other hand, percentages of nrflcdna that did not match  to barley, rice, wheat, maize, pine, spruce, and l.japonicus were higher than the average over these  <dig> species .
distribution patterns of nrflcdnas in varied e-value range between 1e- <dig> and 1e- <dig> were classified into two groups .
distribution curves of nr flcdnas matched potato and tobacco tcs have peaks at 1e- <dig> and are similar to the distribution of nrflcdnas matched dfci tomato tcs.
on the other hand, distribution curves of nrflcdnas matched tcs of other  <dig> plants have peaks at 1e- <dig> or larger.
these results suggest that tomato transcript sequences are more closely related to transcripts from potato and tobacco than to other plant species.
to obtain insight into functions of nrflcdnas that were not found in other plants, we extracted  <dig> nrflcdnas that matched none of the other plant genes .
more than 90% of the  <dig> nrflcdnas matched the prerelease of tomato genome shotgun sequence with e-value smaller than 1e- <dig> and only  <dig> nrflcdnas did not match genome sequence with e-value threshold of 1e- <dig>  suggesting that most of the  <dig> nrflcdnas are tomato-derived transcripts.
by searching the interpro database, out of the  <dig> nrflcdnas,  <dig> nrflcdnas were assigned with  <dig> protein domains.
we also identified known protein domains by searching the similarity with the tomato sbm protein datasets.
out of  <dig> nrflcdnas matched  sbm proteins,  <dig> nrflcdna had known protein domains.
domains such as cyclin-like f-box , aldehyde dehydrogenase , and polynucleotidyl transferase, ribonuclease h fold  were identified in both searches.
however, known functional protein domains were not found in most  of the nrflcdnas that were not found in other  <dig> plants.
mapping full-length cdnas onto tomato genomic sequence
mapping of full-length cdna sequences onto the genome sequence provides insights into tomato genomic structure.
out of  <dig>  nrflcdnas, full-length regions of  <dig>  nrflcdnas  were mapped onto the prerelease of tomato genome shotgun sequence .
the rest,  <dig> nrflcdnas, failed to match genome sequence with nucleotide identity larger than 90% or with alignment length more than 90% of the nrflcdna, or failed to match in full-length.
based on this full-length mapping, the average exon number per gene was estimated to be  <dig>   .
exon length ranged from  <dig> to  <dig> bp.
the average and median exon lengths were estimated to be  <dig> bp and  <dig> bp, respectively .
intron lengths showed larger variation than exon lengths, ranging from  <dig> to  <dig> bp.
the average and median intron lengths were estimated to be  <dig> bp and  <dig> bp, respectively .
the median intron length was longer than those of arabidopsis  and rice .
to compare exon lengths with those of arabidopsis and rice, we classified exons into four types: initial, internal, terminal, and single exons.
the distribution profiles of exon lengths were similar to those observed in arabidopsis   <cit> .
the median lengths of the initial, internal, and terminal exons were  <dig>   <dig>  and  <dig> bp, respectively, which were comparable to those of arabidopsis  and rice .
finally, we estimated the entire length of each gene corresponding to nrflcdna.
the average and median lengths of tomato genes were estimated to be  <dig> bp and  <dig> bp, respectively .
to investigate the occurrence of introns in utrs, the positions of translation start sites and translation termination sites were mapped to the exons.
the frequency of the first methionine in the internal exons was  <dig> %.
on the other hand, the frequency of stop codons in the internal exons was  <dig> %.
this result indicates that intron mapping to a utr was more likely to occur in 5'-utrs than in 3'-utrs.
this mapping also provides insights into the single nucleotide polymorphism frequency between micro-tom and the genome-sequencing cultivar, heinz  <dig>  to estimate frequency of nucleotide mismatch, we used nrflcdna-scaffold pairs with identity in the blast output equal to or larger than  <dig> %.
resulting set of nrflcdna-scaffold pairs contained 90% of the total pairs.
frequency of nucleotide mismatches in exon regions was estimated to be  <dig> % .
this is nearly comparable to the previous result based on the comparison of ests between micro-tom and other s. lycopersicum cultivars  <cit> .
this result suggests that the micro-tom cdna sequence serves as a good reference for the tomato genome sequence.
we note that the results presented here will be revised when annotation of the tomato genome sequence is finished in near future.
a tomato full-length cdna database: kaftom
information regarding the <dig>  full-length cdna sequences including annotations and the results of similarity searches are distributed from the kaftom database http://www.pgb.kazusa.or.jp/kaftom/.
kaftom also provides the results of mapping full-length sequences onto sgn tomato bac sequences and predictions of exons and introns.
it directly links to the clone request site, and all of the full-length cdna clones  are available from the national bioresource project tomato http://tomato.nbrp.jp.
significance of micro-tom full-length cdna in tomato genomics
full-length cdnas serves as a valuable tool that will accelerate tomato genomics in several respects.
first, the sequence information of the full-length cdnas will be integrated into the whole-genome sequence information of tomato, and it will help genome annotation and the identification of regulatory elements in utrs.
second, sequences of the full-length cdnas are valuable information for generating molecular markers.
third, full-length cdnas help our understanding of the functions of tomato genes through gain-of-function and loss-of-function analyses.
fourth, full-length cdnas promote the use of "targeting induced local lesions in genome"  screening.
by combining the use of full-length cdna sequence information with ems-mutagenized lines of micro-tom  <cit> , the tilling approach will provide an efficient way to screen non-transgenic mutant lines as parental germplasm favorable for breeding programs.
CONCLUSIONS
we developed a set of  <dig>  full-length cdnas from the model tomato cultivar micro-tom, and then we generated a set of  <dig>  nrflcdnas, each of which represents a non-redundant transcript coding a full-length cds.
analysis of the 5'-utrs, cdss, and 3'-utrs demonstrated that tomato transcripts have longer 5'- and 3'-utrs than other plant species.
classification of functions of deduced proteins according to the go annotation revealed that the nrflcdna set covers a broad range of proteins.
comparison of the nrflcdnas with genes of other plants facilitated the identification of tomato cdnas that have very low similarity to  <dig> other plants tested, most of which did not have known protein domains.
mapping of the nrflcdnas onto tomato genome sequences revealed that tomato genes have longer introns than arabidopsis and rice.
the frequency of single nucleotide polymorphisms between exons of micro-tom and the genome-sequencing cultivar, heinz  <dig>  was estimated to be  <dig> %.
in combination with other resources, the micro-tom full-length cdnas will bridge the gap between basic and applied studies, providing a valuable tool not only for tomato whole-genome annotation but also for genomics studies and molecular breeding.

 the solanaceae family comprises 1000- species that show wide morphological variability and ecological adaptability. this taxon includes a number of vegetable crops including fruit-bearing vegetables, tuber-bearing vegetables, and ornamental plants, many of which have economic importance. tomato is a member of the solanaceae, and it has served as a model system for fruit development and plant defense. a number of studies have accumulated substantial information regarding the genetics and physiology of tomato. to understand the physiological processes of tomato at the molecular level, it is necessary to link traits to dna sequence. whole-genome sequencing is one of the approaches to achieve this goal. a project for sequencing the tomato genome was launched by the international solanaceae project in the year and the project is currently in the final stage of sequence polishing. accumulation of expressed sequence tags is an alternative approach to collecting dna sequence information. a large number of ests have been produced to provide a resource for gene finding and expression studies. for tomato ests, several repositories are available worldwide, such as the dana-farber cancer institute tomato gene index, the sol genomics network, mibase, plantgdb, and tomatest, and the number of tomato ests accumulated in the databases is. to elucidate the functions of individual genes, attempts have been made to organize ests into consensus sequences, which are referred to as unigenes or tentative consensus sequences. however, a more direct approach to collecting dna information of individual genes is to analyze full-length cdna sequences. major advantages of sequencing full-length cdna over the est assembly include that the data is derived from a single clone rather than the assembly of multiple ests which can generate contigs containing sequences from multiple transcripts. another major advantage of this approach is that full-length cdna clones have complete sequences of transcripts including coding regions and untranslated regions. this facilitates the subsequent annotation and prediction of genomic structures. the complete cdss in full-length cdnas allow the prediction of protein sequences, conserved domains, and conserved motifs. furthermore, full-length cdnas are easy to use with gene transfer systems, permitting the functional analysis of individual genes through reverse-genetics approaches. thus, full-length cdnas are a powerful genomic research tool. in tomato genomics, a miniature tomato cultivar of s. lycopersicum, micro-tom, is regarded as an excellent model system. micro-tom has characteristics that make it suitable for experimental study, such as small size, short generation time, and ease of transformation. recently, resources such as various mutagenized lines have been developed in the micro-tom-background. thus, a collection of micro-tom full-length cdnas would boost the molecular bioengineering of tomato by synergistically combining with other resources. however, no full-length cdna collection of micro-tom, or of tomato, has yet been established. in this study, we developed a large-scale collection of micro-tom full-length cdnas. we constructed full-length-enriched cdna libraries from micro-tom leaf, fruit, and root tissues, and we obtained full-length sequences of cdnas. we then checked for redundancy between sequences, for the presence of cdss, and for the contamination of chimeric sequences, thus generating a set of non-redundant full-length cdna sequences for sequence analysis. we report the results of utr analyses, including length distribution, base composition; and the results of cds analyses, including classification of functions of deduced proteins, metabolic pathway annotations, and comparisons with genes of other plants. we also report the results of mapping nrflcdnas onto the draft of tomato whole-genome sequence. mapping onto genome sequence suggested that introns of tomato genes are relatively long compared to arabidopsis and rice. we also found that the frequency of nucleotide mismatch between exon regions of micro-tom and those of the genome-sequencing cultivar, heinz is %. finally, the value of micro-tom full-length cdna information will be discussed in terms of integration with the tomato genome sequence and other tomato resources.  construction of full-length-enriched cdna libraries we constructed four full-length-enriched cdna libraries from micro-tom tissues including leaves, fruits, and roots. rna samples were prepared from plants grown under different conditions to increase the variation of transcripts in the libraries. leaves were treated with pathogens, chemical and biochemical elicitors, salicylic acid, and methyl jasmonic acid. leaf rna samples were prepared from tissue types under different treatment conditions. fruit rna samples were prepared from the pericarp at four ripening stages harvested in the year and. root rna samples were prepared from plants both with and without flowers, expecting that tissues of different growth phases have different transcripts. we also prepared rna samples from roots treated with fusarium oxysporum race. rna samples prepared from leaf, fruit tissues harvested in the year fruit tissues harvested in the year and root-tissues were mixed, and the respective mixtures were used as templates for four separate cdna libraries, designated lefl fc, lefl and lefl the fc library was constructed by the vector-capping method, and the lefl lefl and lefl libraries were constructed by the cap-trapper method. cdnas derived from pathogens were excluded by hybridization to mrnas prepared from the pathogens. the subtraction process was effective, since 5'-end sequences of only clones out of randomly selected clones matched pathogen sequences in the ncbi genbank, and these were excluded from further data processing. abbreviations: dpi, days post inoculation; hpi, hours post inoculation; hps, hour post spray; dps, days post spray; daa, days after anthesis. more detailed dexcription of the samples is available as additional file full-length sequencing of cdnas a schematic flow of the sequencing process is presented in figure 1a. we randomly selected independent clones from leaf, fruit, and root libraries, and sequenced them from the 5'-end. after trimming vector-derived sequences and low-quality sequences, 5'-end sequences were combined with micro-tom ests reported previously and tomato ests registered in the sgn database. a total of ests were grouped into clusters. from clusters, clusters containing fc and lefl clones were selected. the number of fc or lefl clones in each cluster ranged from to. the fc or lefl sequence having the longest 5'-end extension in each cluster was selected as the representative of that cluster. full-length sequencing was performed using the representative clones by the clone-by-clone primer walking method. out of clones, full-length inserts of cdna clones were sequenced. mean of phred quality value of each base was, and more than 70% of bases exceeded quality value this collection was registered to the high-throughput cdna sequence division of the ddbj. generation of a non-redundant set of the full-length cdnas we checked for redundancy within the set of htcs, and then a set of htcs representing non-redundant transcripts was sent for further processing. to check whether each of the htcs has a full-length cds or not, we first searched for cdnas derived from non-coding rnas using the noncode version dataset. three htcs were identified as cdnas derived from non-coding rna and excluded from further analysis. next, we confirmed that all of the htcs did not match dna sequences from pathogens used to attack leaf and root tissues, suggesting that the probability of contamination with pathogen-derived transcripts was negligible. we then checked for chimeric clones putatively generated during cdna cloning process. according to the criteria described in methods, htcs were regarded as chimeric clones. we then identified sets of htcs generated by the alternative splicing event, and excluded htcs containing retained introns. from the remaining htcs, cdss were predicted by using framedp and genemark.hmm-e programs, and by selecting open reading frame encoding the longest amino acid sequence. essentially, a cds encoding amino acid sequence that had the highest similarity to proteins registered in either the nr or the tomato sbm protein databases was selected as representative cds of the htc. htcs with cdss shorter than nine amino acid long were excluded from further analysis, since the shortest length tomato protein registered in uniprot was aa. out of non-redundant htcs, cds encoding full-length protein was predicted for htcs. full-length cds were not predicted for htcs and no coding regions were predicted for htcs. finally, a set of htcs was generated, hereafter referred to as nrflcdna. this nrflcdna set was subjected to subsequent sequence analyses. nucleotide sequences of nrflcdnas were compared with dfci tomato gene index release tentative consensus, sgn tomato unigenes, and the prerelease of tomato genome shotgun sequence. percentage of nrflcdnas that did not match was %, %, and % against dfci tomato tcs, sgn tomato unigenes, and tomato genome sequences, respectively. predicted amino acid sequences of htcs were compared with proteins registered in ncbi nr dataset and tomato sbm protein dataset. distribution of e-value was similar against both datasets. approximately 22% of the nrflcdnas had very high similarity to nr and sbm proteins, and more than 75% of the nrflcdnas have e-value smaller than 1e- length distributions of utrs and cdss figures 3a and 3b show the distributions of cdna insert length and the cds length, respectively. the average insert length was bp, which is shorter than those of arabidopsis and soybean. the median value for the insert length was bp, which is shorter than arabidopsis and rice, but longer than poplar. the average length of the cds was bp, corresponding to an average polypeptide length of aa, which was also shorter than those of arabidopsis, rice, and soybean, and longer than poplar and maize. the nrflcdna set contains sequences derived from the fc clones that were cloned by the vector-capping method, and these harbor shorter cdna inserts than those derived from the lefl clones that were cloned by the cap-trapper method. this probably explains why the average lengths are shorter than those reported for full-length cdnas of other plants produced by the cap-trapper method. n.a.: not available. figures 3c and 3d show the distributions of the 5'- and 3'-utr lengths, respectively. in contrast to cds length, tomato nrflcdnas had longer utrs compared to those of other plants. the average 5'- and 3'-utr lengths were and bp, respectively. the median 5'- and 3'-utr lengths were and bp, respectively. on both average and median bases, the utrs of tomato are longer than those of arabidopsis, soybean, poplar, and maize, and slightly shorter than those of rice. the relatively long utrs of tomato likely contribute to the regulation of mrna transcription, translation, and stability. nucleotide composition the nucleotide composition of nrflcdnas differed among the 5'-utrs, cdss, and 3'-utrs. the at-content of the full-length cdnas was %, which is slightly higher than that of arabidopsis full-length cdnas. at richness was more pronounced in the 5'- and 3'-utrs, and less pronounced in the cdss. the 5'-utrs had more cs than the 3'-utrs, and the number of ts was higher in the 3'-utrs than in the 5'-utrs. these results are similar to those observed in arabidopsis full-length cdnas. however, nucleotide compositions of the nrflcdna was more at-rich than those of maize transcripts. the most frequently used stop codon is tga followed by taa and tag. frequency of stop codon is similar to that occurred in arabidopsis, but different from those occurred in rice and maize. alternative splicing and retained intron as reported for arabidopsis and rice full-length cdna collections, the collection of full-length cdnas contains cdnas generated from alternatively spliced transcripts. to identify nrflcdnas derived from alternatively spliced transcripts, we first generated multi-sequence groups using the non-redundant htcs, sgn tomato unigenes, and dfci tomato tcs according to the similarity to the target non-redundant htcs. all pairs of target non-redundant htcs and member sequences were subjected to intron detection, and we confirmed that target non-redundant htcs had splicing variants. the percentage of non-redundant htcs with splicing variants was 10%, which is slightly lower than that of arabidopsis or rice. out of these non-redundant htcs, htcs were aligned with sgn tomato bac sequences, which allowed us to classify the types of alternative splicing events. the retained intron-type was the most frequent. the alternative acceptor site- and alternative donor site-types also occurred frequently, comprising 20% and 16% of all events, respectively. the exon skip-type and other types were rather rare. the occurrence of alternative acceptor site- and alternative donor site-types was slightly higher than that reported previously for tomato. nrflcdnas with retained introns encode proteins with alternative amino acid sequences. we found that nrflcdnas may contain retained full- or partial-length introns. larger scale identification of alternative splicing events using the draft of tomato whole-genome sequence is currently underway. functional annotation of tomato full-length cdna next, putative functions of nrflcdnas were assessed by blastp searches against the tair rap-db, and ncbi nr protein datasets. to identify functions of the nrflcdnas, we used gene ontology annotations for arabidopsis genes that matched nrflcdnas. the results from the case using arabidopsis proteins that match nrflcdnas at e-value smaller than 1e- is shown. this revealed that nrflcdnas covered functional categories as broadly as all arabidopsis genes. to obtain insights into the metabolism-related genes, the nrflcdnas were assigned to the lycocyc pathways http://sgn.cornell.edu/tools/solcyc/. based on the best matches to sgn tomato unigenes, nrflcdnas were assigned to lycocyc pathways, and pathway annotations were obtained. comparison of the pathway-classification patterns with those for all sgn unigenes demonstrated that the relative abundance of annotations assigned to the pathways "amino acids degradation", "fermentation", and "tca cycle" was higher in the nrflcdnas than in all sgn unigenes. this demonstrated that genes related to primary metabolism are slightly overrepresented in the nrflcdna set compared to genes related to secondary metabolism. we then attempted to identify transcription factors that are crucial for the transcriptional regulation of various biological processes. we searched for transcription factor domains described in agris attfdb using the interpro database by querying the cds of the nrflcdnas. this search demonstrated that nrflcdnas had interpro entries classified into transcription factor families. the g2-like and myb families were the most numerous, followed by the homeobox family. this result was slightly different from those of arabidopsis and rice, in which the ap2/erebp family and zinc finger family were predominant, respectively. difference in the distribution of transcription factors between tomato nrflcdna and arabidopsis implies that the nrflcdna set failed to contain transcription factors whose expression level is low or cell type-specific. amember nrflcdnas of myb were completely overlapped with those of g2-like. bmember nrflcdnas of c2c2-yabby and arid completely overlapped. comparative analysis with genes of other plants to assess the similarity of nrflcdnas with genes of other plants, deduced peptide sequences were compared with protein databases of arabidopsis and rice using blastp, and with the dfci gene indices of barley, wheat, maize, pine, spruce, poplar, lotus japonicus, medicago truncatula, soybean, orange, apple, grape, tobacco, and potato using tblastn. nrflcdnas has high similarity to two solanaceae plants, potato and tobacco. percentages of nrflcdna that did not match to potato and tobacco tcs were % and %, respectively, which are the lowest among these species. on the other hand, percentages of nrflcdna that did not match to barley, rice, wheat, maize, pine, spruce, and l.japonicus were higher than the average over these species. distribution patterns of nrflcdnas in varied e-value range between 1e- and 1e- were classified into two groups. distribution curves of nr flcdnas matched potato and tobacco tcs have peaks at 1e- and are similar to the distribution of nrflcdnas matched dfci tomato tcs. on the other hand, distribution curves of nrflcdnas matched tcs of other plants have peaks at 1e- or larger. these results suggest that tomato transcript sequences are more closely related to transcripts from potato and tobacco than to other plant species. to obtain insight into functions of nrflcdnas that were not found in other plants, we extracted nrflcdnas that matched none of the other plant genes. more than 90% of the nrflcdnas matched the prerelease of tomato genome shotgun sequence with e-value smaller than 1e- and only nrflcdnas did not match genome sequence with e-value threshold of 1e- suggesting that most of the nrflcdnas are tomato-derived transcripts. by searching the interpro database, out of the nrflcdnas, nrflcdnas were assigned with protein domains. we also identified known protein domains by searching the similarity with the tomato sbm protein datasets. out of nrflcdnas matched sbm proteins, nrflcdna had known protein domains. domains such as cyclin-like f-box, aldehyde dehydrogenase, and polynucleotidyl transferase, ribonuclease h fold were identified in both searches. however, known functional protein domains were not found in most of the nrflcdnas that were not found in other plants. mapping full-length cdnas onto tomato genomic sequence mapping of full-length cdna sequences onto the genome sequence provides insights into tomato genomic structure. out of nrflcdnas, full-length regions of nrflcdnas were mapped onto the prerelease of tomato genome shotgun sequence. the rest, nrflcdnas, failed to match genome sequence with nucleotide identity larger than 90% or with alignment length more than 90% of the nrflcdna, or failed to match in full-length. based on this full-length mapping, the average exon number per gene was estimated to be. exon length ranged from to bp. the average and median exon lengths were estimated to be bp and bp, respectively. intron lengths showed larger variation than exon lengths, ranging from to bp. the average and median intron lengths were estimated to be bp and bp, respectively. the median intron length was longer than those of arabidopsis and rice. to compare exon lengths with those of arabidopsis and rice, we classified exons into four types: initial, internal, terminal, and single exons. the distribution profiles of exon lengths were similar to those observed in arabidopsis. the median lengths of the initial, internal, and terminal exons were and bp, respectively, which were comparable to those of arabidopsis and rice. finally, we estimated the entire length of each gene corresponding to nrflcdna. the average and median lengths of tomato genes were estimated to be bp and bp, respectively. to investigate the occurrence of introns in utrs, the positions of translation start sites and translation termination sites were mapped to the exons. the frequency of the first methionine in the internal exons was %. on the other hand, the frequency of stop codons in the internal exons was %. this result indicates that intron mapping to a utr was more likely to occur in 5'-utrs than in 3'-utrs. this mapping also provides insights into the single nucleotide polymorphism frequency between micro-tom and the genome-sequencing cultivar, heinz to estimate frequency of nucleotide mismatch, we used nrflcdna-scaffold pairs with identity in the blast output equal to or larger than %. resulting set of nrflcdna-scaffold pairs contained 90% of the total pairs. frequency of nucleotide mismatches in exon regions was estimated to be %. this is nearly comparable to the previous result based on the comparison of ests between micro-tom and other s. lycopersicum cultivars. this result suggests that the micro-tom cdna sequence serves as a good reference for the tomato genome sequence. we note that the results presented here will be revised when annotation of the tomato genome sequence is finished in near future. a tomato full-length cdna database: kaftom information regarding the full-length cdna sequences including annotations and the results of similarity searches are distributed from the kaftom database http://www.pgb.kazusa.or.jp/kaftom/. kaftom also provides the results of mapping full-length sequences onto sgn tomato bac sequences and predictions of exons and introns. it directly links to the clone request site, and all of the full-length cdna clones are available from the national bioresource project tomato http://tomato.nbrp.jp. significance of micro-tom full-length cdna in tomato genomics full-length cdnas serves as a valuable tool that will accelerate tomato genomics in several respects. first, the sequence information of the full-length cdnas will be integrated into the whole-genome sequence information of tomato, and it will help genome annotation and the identification of regulatory elements in utrs. second, sequences of the full-length cdnas are valuable information for generating molecular markers. third, full-length cdnas help our understanding of the functions of tomato genes through gain-of-function and loss-of-function analyses. fourth, full-length cdnas promote the use of "targeting induced local lesions in genome" screening. by combining the use of full-length cdna sequence information with ems-mutagenized lines of micro-tom, the tilling approach will provide an efficient way to screen non-transgenic mutant lines as parental germplasm favorable for breeding programs.  we developed a set of full-length cdnas from the model tomato cultivar micro-tom, and then we generated a set of nrflcdnas, each of which represents a non-redundant transcript coding a full-length cds. analysis of the 5'-utrs, cdss, and 3'-utrs demonstrated that tomato transcripts have longer 5'- and 3'-utrs than other plant species. classification of functions of deduced proteins according to the go annotation revealed that the nrflcdna set covers a broad range of proteins. comparison of the nrflcdnas with genes of other plants facilitated the identification of tomato cdnas that have very low similarity to other plants tested, most of which did not have known protein domains. mapping of the nrflcdnas onto tomato genome sequences revealed that tomato genes have longer introns than arabidopsis and rice. the frequency of single nucleotide polymorphisms between exons of micro-tom and the genome-sequencing cultivar, heinz was estimated to be %. in combination with other resources, the micro-tom full-length cdnas will bridge the gap between basic and applied studies, providing a valuable tool not only for tomato whole-genome annotation but also for genomics studies and molecular breeding. 
classification of functions of proteins predicted from the coding sequences demonstrated that nrflcdnas covered a broad range of functions. in particular, a miniature cultivar, micro-tom, is regarded as a model system in tomato genomics, and a number of genomics resources in the micro-tom-background, such as ests and mutagenized lines, have been established by an international alliance. to accelerate the progress in tomato genomics, we developed a collection of fully-sequenced micro-tom full-length cdnas. full-length cdna sequences and their annotations are provided in the database kaftom http://www.pgb.kazusa.or.jp/kaftom/ via the website of the national bioresource project tomato http://tomato.nbrp.jp. according to a comparison of exon sequences between the nrflcdnas and the tomato genome sequences, the frequency of nucleotide mismatch in exons between micro-tom and the genome-sequencing cultivar was estimated to be %. the nrflcdna sequences will help annotation of the tomato whole-genome sequence and aid in tomato functional genomics and molecular breeding. recently, a number of tomato resources have been developed in parallel with the ongoing tomato genome sequencing project. mapping of the nrflcdnas onto currently available tomato genome sequences facilitated prediction of exon-intron structure. a comparison of nrflcdnas with genes of sixteen plants facilitated the identification of tomato genes that are not found in other plants, most of which did not have known protein domains. by checking redundant sequences, coding sequences, and chimeric sequences, a set of non-redundant full-length cdnas was generated. introns of tomato genes were longer than those of arabidopsis and rice.
 the solanaceae family includes several economically important vegetable crops. the tomato is regarded as a model plant of the solanaceae family. recently, a number of tomato resources have been developed in parallel with the ongoing tomato genome sequencing project. in particular, a miniature cultivar, micro-tom, is regarded as a model system in tomato genomics, and a number of genomics resources in the micro-tom-background, such as ests and mutagenized lines, have been established by an international alliance.  to accelerate the progress in tomato genomics, we developed a collection of fully-sequenced micro-tom full-length cdnas. by checking redundant sequences, coding sequences, and chimeric sequences, a set of non-redundant full-length cdnas was generated. analysis of untranslated regions demonstrated that tomato has longer 5'- and 3'-untranslated regions than most other plants but rice. classification of functions of proteins predicted from the coding sequences demonstrated that nrflcdnas covered a broad range of functions. a comparison of nrflcdnas with genes of sixteen plants facilitated the identification of tomato genes that are not found in other plants, most of which did not have known protein domains. mapping of the nrflcdnas onto currently available tomato genome sequences facilitated prediction of exon-intron structure. introns of tomato genes were longer than those of arabidopsis and rice. according to a comparison of exon sequences between the nrflcdnas and the tomato genome sequences, the frequency of nucleotide mismatch in exons between micro-tom and the genome-sequencing cultivar was estimated to be %.  the collection of micro-tom nrflcdnas generated in this study will serve as a valuable genomic tool for plant biologists to bridge the gap between basic and applied studies. the nrflcdna sequences will help annotation of the tomato whole-genome sequence and aid in tomato functional genomics and molecular breeding. full-length cdna sequences and their annotations are provided in the database kaftom http://www.pgb.kazusa.or.jp/kaftom/ via the website of the national bioresource project tomato http://tomato.nbrp.jp. 