BACKGROUND
the advances in high-throughput molecular biology technologies of the last two decades have not only led to a drastic change in our ability to study genomes, but also brought together novel mechanisms for structuring, storing and sharing information.
concerning gene annotation, previous scenarios of nomenclature diversity and large efforts for gathering all available information on a given species have been replaced by the availability of standard vocabularies extensively used by the scientific community.
this, along with the availability of user-friendly bioinformatic tools, allows the feasible evaluation, functional annotation and classification of a high number of expressed sequences in a great variety of organisms.
such characterization would be useful for functional genomics research in plants, particularly in the emergent field of systems biology.
with the progress of plant genome sequencing projects, in-depth knowledge about molecules, such as nucleic acids and deduced proteins, gene regulatory networks, and metabolic pathways becomes possible.
hierarchically structured ontological terms can now be adopted to query sequences and to describe genes and their products at different levels of knowledge and specificity  <cit> .
the gene ontology  project began in  <dig> with the integration of three model organism databases, i.e.
yeast, drosophila and mouse, and represents today the most widely used schema for the functional characterization of plant, animal and microbial genes and gene products  <cit> .
the go project has developed three structured vocabularies  describing genes, transcripts and proteins of any organism in terms of their associated cellular component, biological process and molecular function in a species-independent manner.
the use of go terms by collaborating databases facilitates uniform retrievals across them.
moreover, the go vocabularies can be queried at different levels, allowing annotators to assign properties to genes or gene products, depending on the depth of knowledge and specificity about that entity  <cit> .
in plant species for which the genome sequence is not available, aflp and cdna-aflp are two of the most commonly used methods for genome- and transcriptome-wide level analysis , respectively, capable of discovering genes not yet been cloned or even predicted, based upon their polymorphisms or differential expression patterns.
aflp markers  <cit>  represent the genomic tool with the highest polymorphism information content, and combine the reliability of the rflp technique with the power of the pcr technique.
both aflp and aflp-derived markers, such as m-aflps  and s-saps , have been widely used for whole genome scanning and fingerprinting  <cit> , characterizing single chromosomes and mapping specific genes  <cit> , as well as for transcriptome profiling and gene cloning by means of cdna-aflp  <cit> , and to generate quantitative gene expression patterns for eqtl mapping, as recently demonstrated by vuylsteke et al.
<cit> .
transcriptome differential profiling of plants with antagonistic phenotypes  is theoretically one of the most powerful strategies for identifying and cloning candidate genes not only for model organisms, but also in remarkably complex genomes such as polyploids  <cit> .
the mrna fingerprinting based on aflp technology does not require pre-existing genome or est sequence knowledge, therefore it is being widely used in less well investigated systems.
since a few genomes of agriculturally important species  have been sequenced and even fewer have been well annotated, the data provided by cdna-aflp experiments may represent a valuable resource for functional genomics and genetics in non-model plants.
compared to microarrays, cdna-aflp increases the resolution of expression patterns detection using smaller amounts of mrna  <cit> .
this feature is essential when an rna fingerprinting is applied to tissues for which it is hard to isolate stage-specific messengers, such as flowers, fruits and seeds.
both reliability and sensitivity of amplification products proved to be very high, and expression patterns visualized by cdna-aflp showed to well correlate with northern blot analyses  <cit> .
moreover, the redundancy of the technique  <cit>  proved to be very informative in cases of alternative splicing and multigene family member displaying  <cit> , for distinguishing highly homologous genes  <cit> .
after 10-year-use of aflps for dna fingerprinting and mrna profiling, large sequence collections retrieved with this technology are available in public databases for several crop and model species.
the evaluation, annotation and classification of aflp-derived genomic markers and expressed transcripts would be very useful for both functional genomics and systems biology research in plants, and crucial to allow the scientific community to promptly retrieve this pre-existing information from gene banks.
annotations could be periodically revised and implemented, and should allow to optimize genomic aflp and cdna-aflp experiments in plant species to give specific information about the targeted biological processes and molecular functions.
in the present study, we retrieved a total of  <dig>  cdna-aflp sequences related to roots, leaves, stems, flowers, fruits and seeds from both ncbi databases and unpublished repertories.
all these entries belong to  <dig> different species distributed among seven botanic families: solanaceae, fabaceae, poaceae, salicaceae, rosaceae, brassicaceae and vitaceae.
redundant sequences were clustered and contigs assembled.
functional analysis was then performed using blast2go  <cit> .
blast2go is a bioinformatic tool for the go-based annotation and data mining of sequence sets for which no go annotation is yet available, which has proven to be effective in the functional characterization of plant sequence data  <cit> .
the cdna-aflp-derived sequences were then grouped according to the go vocabularies.
experimental steps and statistical parameters adopted for the in silico analysis were critical for obtaining reliable gene ontology data.
annotation results for the whole sequence dataset and also for botanic families, single species and plant organs are presented, and the main features of genes and gene products detectable in plants by aflp technology discussed.
RESULTS
gene ontology of aflp-derived cdna sequences
the total number of plant cdna sequences derived from aflp technology deposited in ncbi databases or recovered from private unpublished collections was as high as  <dig> .
entries belonged to  <dig> different model, crop and tree species distributed among seven botanic families: solanaceae , salicaceae , fabaceae , poaceae , rosaceae , brassicaceae  and vitaceae .
additional  <dig> cdna-aflp-derived sequences of salix spp.
were analyzed separately once they became publicly available because although deposited they were not released at the time of entries downloading.
the most abundant taxonomic group included in the present study was the genus nicotiana, followed by populus, medicago, oryza and malus.
the list of organisms with their corresponding number of cdna-aflp sequences deposited in ncbi databases is reported in the methods section .
redundancy information on cdna-aflp sequences assessed on the basis of the number and proportion of contigs and singlets.
for each sequence subset, the number of retrieved sequences and the percentage of the total, the number of contigs, the number of singlets and the percentage of the total, the redundancy, and the gc content are reported.
<dig> sequences deposited in genbank but not yet publicly released; 2all  <dig> sequences were released after february 1th, 2007;  <dig> sequences are not yet publicly available.
redundant cdna-aflp sequences for each of the  <dig> organisms were clustered into a total of  <dig> contigs, whereas the remaining  <dig>  records  were singlets.
the overall estimate of redundancy was about 23%, ranging from 0% in fragaria and lolium, to  <dig> % of solanum spp, whereas the gc content of cdna-aflp sequences was found to be 45%, with a standard deviation over all organisms as low as  <dig> %.
redundancy statistics and gc contents for all organisms are summarized in table  <dig>
with the exception of the  <dig> salix entries , the  <dig>  cdna-aflp sequences equal to  <dig> contigs plus  <dig>  singlets and belonging to  <dig> angiospermae were used as blastx queries to search for structural homologies and significant similarities.
a total of  <dig>  sequences  revealed significant similarity with deposited records, showing an average nucleotide similarity estimate of 80% and a median e-value of  <dig> e- <dig> over all organisms .
the same statistics were also computed for each single species or genus .
go term mapping allowed the identification of  <dig>  go terms based upon  <dig>  matches retrieved by blastx.
the distribution of annotations among go vocabularies and the number of cdna-aflp sequences with go terms belonging to only one, a combination of two and all three vocabularies was organized in a venn diagram .
<dig>  sequences were annotated according to all the three go subvocabularies  and annotations simplified using a plant-specific goslim following the approach suggested by similar papers with comparative purposes  <cit> .
as many as  <dig>   <dig> and  <dig> sequences were annotated with a combination of bp and mf terms, bp and cc terms, and cc and mf terms, respectively.
basic statistics for all the cdna-aflp annotated sequences sorted by botanic family and by single organism, including mean length and length range for a 68% confidence interval, mean similarity and similarity range for a 68% confidence interval, median e-value with minimum and maximum e-values are reported in tables  <dig> and  <dig>
statistics on cdna-aflp annotated sequences sorted by botanic family.
†refers to the number of sequences included in the 68% confidence interval.
‡refers to the total number of sequences.
abbreviations: conf.
int., confidence interval; cv, coefficient of variability; min, minimum; max, maximum).
statistics on cdna-aflp annotated sequences sorted by organism.
†refers to the number of sequences included in the 68% confidence interval.
‡refers to the total number of sequences.
abbreviations: conf.
int., confidence interval; cv, coefficient of variability; min, minimum; max, maximum.
among the botanic families, vitaceae showed the highest proportion of annotated sequences  and rosaceae the lowest without blast matches .
on the whole, 36% of sequences did not retrieve any blast result within the set e-value threshold.
mapping of go terms and annotation were not possible for 20% and 3% of sequences, respectively, whereas the remaining 41% was annotated.
overall data distribution of cdna-aflp sequences grouped according to the organ, tissue or part of the plant, and botanic family showed a large variation for the percentage of sequences either with no blast results or annotated, whereas the percentage of sequences neither with mapping nor annotation was quite constant .
fruits were the organs with the highest percentage of annotated sequences and the lowest percentage without blast matches, whereas the opposite was for seeds.
similar properties were observed for roots, stems and leaves, and flowers.
level  <dig> goslim analysis of aflp-derived cdna sequences
the go terms obtained by the annotation procedure were mapped to a plant specific goslim to generate a more concise annotation to be used for comparative analyses as reported in similar researches  <cit> .
in the present study the 'goslim_plant.obo' developed by mundodi s. the ftp directory of the genome databases group, department of genetics, stanford university school of medicine was used   <cit> .
the goslim classification of annotated cdna-aflp sequences is reported in figures  <dig> and  <dig>  level  <dig> annotation was chosen because representative of all retrieved annotation, and a comparison with the annotations of arabidopsis and rice ests was possible.
among the annotated sequences,  <dig> were attributable to roots,  <dig> to stems plus leaves,  <dig> to flowers,  <dig> to fruits and  <dig> to seeds.
for the remaining  <dig> , no information on the plant tissue or organ source was available among the features of the database entries.
concerning the annotations of sequences grouped by organs and tissues , it is worth noting that the proportion of fruit records with catalytic and transporter activities was significantly higher  than that observed for other organs , whereas the proportion of sequences related to 'binding' was much lower  than that calculated for roots, leaves and stems, flowers and seeds .
the annotation of sequences according to the botanic family was also performed .
the relative proportions of sequences associated with the two most abundant categories of cc , bp  and mf  proved to be very similar each other, with relative differences smaller than 5%.
these findings indicate that go annotation data gained for aflp-derived cdna sequences in plants are highly concordant, when compared among botanic families, but also significantly divergent, when referred to different plant organs or tissues.
the classification of annotated cdna-aflp sequences at level  <dig> is summarized in figure  <dig> for each of the  <dig> taxonomic entities/organisms analyzed in this study and sorted by genus or species.
the proportion of annotated sequences for each organism varied from  <dig> % of nicotiana spp.
to  <dig> % of lolium multiflorum.
consistent differences were pointed out among species for go terms belonging to the three subvocabularies.
go multilevel analysis of aflp-derived cdna sequences
all cdna-aflp sequences sorted by organs and families were also analyzed using a multilevel procedure.
this further investigation enabled to find out the lowest node per branch of the dag  that fulfils the filter condition, e.g.
will find all the lowest nodes with the given number of sequences or score value .
the most represented terms among the  <dig>  belonging to cc for plant organs were 'membrane' for fruits  and roots , and 'plastid' for seeds , flowers , and leaves and stems .
sequences associated with 'cytoplasmic membrane-bound vesicle', 'intracellular organelle part' and 'membrane part' were only found in leaves and stems.
the most represented bp terms among the  <dig>  for plant organs were 'transport' in seeds , fruits  and roots , 'secretory pathway'  in leaves and stems, 'amino acid and derivative metabolism'  in flowers.
finally, the most represented terms among the  <dig>  belonging to mf for plant organs were 'hydrolase activity' in roots  and seeds , 'oxidoreductase activity'  in leaves and stems, 'nucleotide binding' in flowers  and fruits .
sequences associated with 'oxidoreductase activity', 'atp binding', 'transition metal ion binding', 'protein kinase activity' and 'peptidase activity' were only recorded for leaves and stems.
conversely, sequences related to 'hydrolase activity' and 'nucleotide binding' were found over all organs, except for leaves and stems .
regarding the by-family functional analysis at multiple levels, again an overall agreement in most represented go terms was observed.
concerning the cc, 'membrane' was the most abundant term in all families, except for the solanaceae where 'protein complex' was the most frequent.
'plastid' and 'mitochondrion' were also abundant in the fabaceae and poaceae.
for the bp ontology, the most numerically represented categories related to 'transport' over all families, varying between  <dig> % of brassicaceae to  <dig> % of poaceae.
among the other go terms, the most abundant within family were 'cell organization and biogenesis' in solanaceae, 'protein modification' in fabaceae, rosaceae and solanaceae, 'biosynthesis' in fabaceae, 'carbohydrate metabolism' in rosaceae and salicaceae, 'amino acid and derivative metabolism' in fabaceae, poaceae and salicaceae.
among the mf terms, 'hydrolase activity' and 'nucleotide binding' included the highest proportion of sequences over all families.
in particular, sequences associated with the former spanned from  <dig> % of solanaceae to  <dig> % of fabaceae, whereas those associated with the latter were around 25% in most families.
moreover, cdna-aflp profiling proved to assay expressed sequences related to 'kinase activity' with very similar efficiency  in each of the seven families analyzed in this study .
annotation index  estimates of aflp-derived cdna sequences
an annotation index  was developed to describe the information content of aflp-derived cdna sequence collections on the basis of the go annotations.
computational simulations performed with random sets of  <dig>  cdna-aflp sequences revealed that ai values can range between  <dig>  and  <dig>  , depending on the proportion of sequences associated with combinations of terms belonging to one, two or all the three go vocabularies.
annotations indexes were estimated for plant organs/tissues and botanic families in order to perform comparisons.
sequences related to seeds and fruits were those displaying, respectively, the lowest and highest ai with almost a two-fold difference, i.e.
<dig>  vs.  <dig>  .
concerning botanic families, ai ranged from  <dig>  of solanaceae to  <dig>  of vitaceae, with an average value for the total set of sequences of  <dig> .
annotation index  estimates for the groups of cdna-aflp-derived sequences analyzed.
go comparison between dicots and monocots aflp-derived cdna sequences
in order to study the functional content of cdna-aflp sequences in relation to expressed sequences in model organisms, a level  <dig> goslim classification of all the cdna-aflp records was performed by splitting the sequence dataset into monocots and dicots and by comparing the two subgroups with all annotated ests  of oryza sativa and arabidopsis thaliana, respectively .
the comparison between dicot cdna-aflps and arabidopsis est sequences revealed substantial differences for the relative frequencies of the most represented go terms associated with cc and mf ontologies.
in particular, the cc categories of 'cell', 'organelle' and 'protein complex' included  <dig> %,  <dig> % and  <dig> % of arabidopsis est sequences against  <dig> %,  <dig> % and  <dig> % of dicot cdna-aflp sequences, respectively .
even the go terms related to 'organelle part' and 'envelope' were strongly differentiated between arabidopsis and dicot species.
the two major go categories associated with bp, which were 'physiological process' and 'cellular process', showed to be similarly represented in dicot species and arabidopsis, being their proportions equal to  <dig> % vs.  <dig> % and  <dig> % vs.  <dig> %, respectively.
a large difference was observed for the term 'response to stimulus', which was assigned to  <dig> % of arabidopsis sequences and only to  <dig> % in dicot cdna-aflps .
four additional go term categories, 'regulation of biological process', 'development', 'reproduction' and 'growth' were found at  <dig> %,  <dig> %,  <dig> % and  <dig> %, respectively, in arabidopsis and at much lower frequencies in the dicot species .
regarding mf terms, highly significant differences were found for the two major categories, 'catalytic activity' and 'binding': the former was represented by  <dig> % and  <dig> % of the sequences, respectively, in arabidopsis and dicot species, whereas the latter was 28% in arabidopsis and  <dig> % in the dicots .
a marked difference was also found for the category 'transcription regulator activity' .
the comparison between monocot cdna-aflps and rice est sequences revealed also consistent differences for some of the most frequent go terms.
while in the cc branch the two major categories of 'cell' and 'organelle' were similarly represented, with  <dig> % vs.  <dig> % and  <dig> % vs.  <dig> %, respectively , a marked difference was observed for 'protein complex', being the frequency of sequences associated with this category equal to  <dig> % in rice and  <dig> % in the monocot sequences .
regarding the bp, the three major go terms proved to be much more frequent in rice ests than in monocot cdna-aflp sequences, being equal to  <dig> % vs.  <dig> % for 'physiological process',  <dig> % vs.  <dig> % for 'cellular process' and  <dig> % vs.  <dig> % for 'response to stimulus', with highly significant differences ,  <dig> % vs. 0% for 'development', and  <dig> % vs. 0% for 'regulation of biological process'.
non-significant differences were observed for the go terms belonging to the mf vocabulary, except for the category of 'binding'  .
discussion
experimental procedures adopted for go analysis of aflp-derived sequences
the experimental steps and statistical parameters adopted for aflp-derived sequence analysis are critical for obtaining reliable ontology data and deserve a specific mention.
four main steps were followed to achieve a reliable go classification of aflp-derived sequences: i. data recovery from sequence databases; ii.
preliminary selection of nucleotide records; iii.
basic annotation; iv.
enrichment and refinement of annotation terms .
the retrieval of aflp-related sequences from ncbi was performed by searching for the keywords "aflp and viridiplantae" and "cdna-aflp and viridiplantae" and subsequent manual scoring to verify the results.
the manual step was required because the keywords indicating the aflp origin were not always present in the record fields.
for our purpose, this seemed to be a noticeable limitation of the genbank database, probably due both to an unintentional inaccuracy in submitting sequences and an insufficient stringency in accepting a new record without any precise indication of its origin.
as a general remark, nucleotide ncbi database curators should consider requesting additional and more precise information about the origin of the records to be submitted.
in the case of aflp-derived sequences, at least the restriction enzymes and selective primer combinations used in the experiments should be added in the 'features' or 'comment' fields, as already done by several submitters.
the presence of redundant sequences  was a further known limitation, caused mainly by a poor evaluation of the records already present in the public databases at the time of a new submission.
however, possible polymorphisms, even at the single nucleotide level , have to be always considered as highly informative and carefully evaluated for their reliability.
the basic annotation obtained by retrieving go terms from blastx matches was enriched and refined using three main strategies integrated in the blast2go software: i) interproscan, ii) annex, and iii) goslim simplified annotation.
databases of protein domains and functional sites have become vital resources for the prediction of protein functions.
interproscan  <cit>  combines different protein signature recognition methods allowing searches against independent databases and an easy recovery of the corresponding codes to be automatically coupled to go terms annotation.
this tool allowed an average 8% increase in the overall annotated sequences .
this value is in the range of observed improvement in blast-based go annotation by interpro  and shows the important contribution of motif-based annotation in go terms enrichment as well as the basic importance of an integrated approach to the annotation of unknown nucleotide records.
a refinement of the go terms was obtained by means of the annex function .
using the original go structure, annex locates parent-offspring relationships between the annotations.
afterwards, annex suggests new bp and cc annotations as well as implicit ones deduced from univocal relationships between go terms from the different go categories providing on average 10–15% increase in functional terms.
in other words, given a molecular function, this tool identifies biological processes where the molecular functions are involved and the cellular components where they are active.
for a detailed description of the annex procedure, see  <cit> .
in the last step of the annotation procedure, a simplified version of the full ontologies  was adopted  <cit> .
goslims are cut-down versions of the full ontologies composed by high-level selected terms, or nodes, each one including subsets of the terms of the whole go.
they give a broad overview of the ontology content without the detail of the specific fine-grained terms.
goslims are created by users according to their needs, and may be specific to species or to particular areas of the ontologies.
go provides a generic goslim which, like the go itself, is not species-specific, and which should be suitable for most purposes such as the comparisons made in this research.
the adoption of the 'goslim_plant.obo' goslim allowed a visible improvement of the annotation in terms of a reduced fragmentation of the go categories and a more plant-specific terminology.
indeed, all the go terms specifically related to cc, mf and bp typical of mammals and/or unicellular organisms were removed or replaced with plant-specific terms.
the blast2go software was shown to be very effective in every step of the annotation procedure particularly with respect to the integrated tools that allowed a great time saving during the enrichment and refinement steps.
this tool offers a suitable platform for functional genomics research in non-model species and it also allows monitoring and comprehension of the whole annotation and analysis steps.
some minimal limitations were found during the elaboration of results, in particular concerning the generation of graphics.
however, future implementations may allow the blast2go software to be one of the most powerful tools for the annotation of unknown sequence pools.
gene expression analysis by means of cdna-aflp technique
the generation of transcriptional profiles has numerous applications in plant biology, including the identification of tissue-specific or developmental stage-specific, reproduction-related and stress-induced transcripts  <cit> .
many new techniques have been designed in recent years for this purpose, such as microarrays  <cit> , allowing a serial genome-wide expression profile of thousands of genes to be performed in a single experiment.
though powerful, this approach is often really expensive and can be readily applied only to model species for which significant information about the coding sequences is available.
moreover, because rarely expressed transcripts are usually missing from cdna libraries due to over-representation of abundant messengers, microarrays could fail to detect transcripts that are rare but fundamental for certain traits.
library enrichment approaches, such as library subtraction or normalization  <cit> , could also be adopted to catch low-copy mrnas, although time-consuming and cost-effective.
on the other hand, the cdna-aflp technique has become one of the most robust solutions for differential display and it has been successfully applied in several quantitative gene expression studies  <cit> , even in less investigated species for which sequence information is not available.
therefore, after ten years from its first applications in plants, cdna-aflp represents an irreplaceable tool for transcriptome profiling analysis that can be utilized for multifactorial genomics of any species.
the utility of cdna-aflp in functional genomics and systems biology research in plants can be further implemented by serial annotation analyses of species/organ/tissue-specific repertories of transcript-derived fragments, starting from the data provided in the present study.
moreover, the annotations have to be continuously updated, since further information is available by functional characterization of the most interesting sequences.
in this way, annotation data may allow to set up a forecasting model according to which the cdna-aflp experiments can be tuned to target specific gene classes, and therefore, retrieving gene sequences enriched with the desired functional categories.
blastx querying, go terms mapping and annotation of transcript sequences recovered by cdna-aflp analysis
the relatively high number of specific studies carried out in recent years using cdna-aflp as a differential display technique does not match a correspondingly high number of sequences retrievable from public databases.
the total number of plant aflp-derived est entries was shown to be as low as  <dig> , successively reduced to  <dig>  because of the 23% of redundancy.
moreover, only  <dig> plant species/genera were represented over all records, with solanaceae being the most numerous family.
a total of  <dig>  cdna-aflp sequences revealed structural homology and significant similarity with deposited records, allowing to retrieve as many as  <dig>  go terms.
the subsequent validation performed with the integrated function of the blast2go software enabled the annotation of  <dig>  cdna-aflp sequences, each with  <dig> to  <dig> go terms.
despite the important role of cereals and related research, the highest number  of aflp-derived ests with blast matches was found in the rosaceae family, immediately followed by the vitaceae , the latter being mostly represented by grape sequences.
these two taxonomic groups have been subjected to intense research activities in the last few years, supported by worldwide initiatives such as the gdr  at clemson university , estree consortium in italy  which is focused mainly on peach, international grape genome program  in united states and the franco-italian grape genome sequencing project .
the enrichment of database information resulting from such collaborative researches may be in part responsible of the availability of blast matches for rosaceae and vitaceae sequences.
for a parametric description of sequence annotability, a dedicated index  was developed in the present research and calculated for the most interesting sequence subsets.
therefore, the ai computed for rosaceae and vitaceae was as high as  <dig>  and  <dig> , respectively .
taking into account the other botanic families, some interesting results were pointed out concerning the annotability of sequences.
in particular, solanaceae family strangely showed the lowest percentage  of annotated sequences, with ai as low as  <dig>  , despite the high number of entries present in the databases and the fact that species belonging to this taxonomic group, such as tomato, tobacco and petunia, are widely used as models.
considering the information content of sequences classified according to the organ, tissue or part of the plant from which they were previously isolated, some interesting results were evidenced from the analysis of data distribution .
the highest percentage  of sequences with blast results was found for cdna-aflp entries isolated from fruits, that were also the plant organs with the highest percentage of annotated sequences, with an ai as high as  <dig>  .
this result agrees with those found for the rosaceae family, to which most of the fruits such as peach, apple, pear, plum, apricot, and cherry belong, as well as for the species included in the vitaceae taxonomic group, such as grape.
moreover, the abundance of go terms associated with carbohydrate, protein and amino acid metabolism, and catalytic as well as transporter activities may reflect the cellular and biochemical events occurring during fruit ripening.
despite the importance of seed development in cereals and legumes, and the amount of studies dedicated to its understanding, sequences related to seeds were those with the lowest percentage of entries with blast hits  and annotation , displaying an ai =  <dig>  .
on the whole, our findings suggest that the annotability of a set of sequences from a specific plant organ or a particular taxonomic group is not a direct consequence of the amount of research efforts dedicated to studying that organ or group.
in fact, the go annotations of genes are very often transferred from very phylogenetically distant organisms.
the go characterization and annotation of aflp-derived sequences allowed us to retrieve basic information on the gene function/s in crop species and their organs/tissues.
in particular, examination of thousands of est clones enabled certain inferences to be made on the potentials and drawbacks of aflp technology for mrna profiling and differential display gene cloning.
although the different number of sequences retrieved in databases for plants and organs might have biased some of the descriptive statistics, the overall go is consistent with the existence of aflp technology features exploitable across angiosperm.
the representativeness of sequence samples and goodness of statistical results over all taxonomic groups are supported by bootstrap analysis.
the calculations were performed using the  <dig>  singlets of nicotiana spp.
with a number of non-overlapping random replicates of  <dig>   <dig> and  <dig> each formed by  <dig>   <dig> and  <dig> sequences, respectively.
the bootstrap test was carried out on nicotiana because it was the most numerous genus.
the proportions of annotated sequences for each go vocabulary and over all go terms per vocabulary proved to be very similar in all replicates with standard deviations lower than 5% in most cases .
this finding demonstrates the high reliability and reproducibility of gene annotation results for the  <dig> organisms analyzed in this study.
as a consequence, est repertories equal to or larger than  <dig> cdna-aflp clones can be considered sufficient to obtain a sequence information content representative of the main go categories.
in this context, it is worth noting that cdna-aflp profiling proved to assay expressed sequences related to 'kinase activity' with very similar efficiency in each of the seven families analyzed in this study.
it may be deduced that this gene family shows distinctive conserved characteristics so that its members can be repeatedly detected by the cdna-aflp technique with a quite constant probability in all plant species.
a go classification of all cdna-aflp records was performed by splitting the sequence dataset into monocots and dicots and by comparing the two subgroups with all annotated ests of arabidopsis thaliana and oryza sativa.
although available molecular data may be biased by the experimental methodologies and plant materials, our overall go results suggest that in a given species and for a given organ or tissue est repertories developed by construction and screening of cdna libraries include a sequence pool information content different from and/or complementary to that of tdf  collections generated by cdna-aflp profiling and differential display.
since functional data supplied by aflp-derived sequences do not fully overlap the functional data obtained by est projects, the two technologies most likely allow to probe distinct target genes and to capture distinct transcript subsets from a given part of the plant.
alternatively, the functional information obtained by blast2go may have been biased in comparison to the available annotation for rice and arabidopsis.
the comparison between monocot cdna-aflps and rice est sequences also revealed interesting results.
in this case, an explanation for the deviations documented in specific go branches and categories might be found in the different goals driving basic research projects and applied breeding activities in model and crop plants.
CONCLUSIONS
in the last ten years, the cdna-aflp mrna profiling was largely adopted and considerable repertories of organ-specific and differentially expressed transcripts are now available in public databases for model, crop and tree species.
the evaluation, annotation and classification of aflp-derived sequences would therefore become crucial for both functional genomics and systems biology research in plants.
the possibility of using aflp-derived tags on cdna fragments produced directly by sequencing-by-synthesis technologies opens up the possibility of not only identifying very large numbers of expressed genes, but also retrieving large-scale snp collections.
our study suggests that a reliable go characterization of aflp-derived sequences is based on the optimization of experimental steps and statistical parameters adopted for go analysis.
the blast2go software was shown to represent a comprehensive bioinformatics solution for functionally characterizing sequences and data mining on the correspondent annotations based on the go vocabularies.
an exhaustive annotation based on gene products similarity blast searches would offer a suitable platform for functional genomics, particularly useful in non-model plant species.
therefore, the utility of aflp technology in structural and functional genomics in plants can be implemented by go annotation analyses species/organ/tissue-specific repertories of transcriptome-derived fragments.
our suggestion is that aflp-derived sequences should be systematically subjected to go annotations before their submission to ncbi databases so that a publicly available information based on yearly larger plant est collections could be periodically released to the go consortium and retrieved by other researchers when searching the genbank.

 the advances in high-throughput molecular biology technologies of the last two decades have not only led to a drastic change in our ability to study genomes, but also brought together novel mechanisms for structuring, storing and sharing information. concerning gene annotation, previous scenarios of nomenclature diversity and large efforts for gathering all available information on a given species have been replaced by the availability of standard vocabularies extensively used by the scientific community. this, along with the availability of user-friendly bioinformatic tools, allows the feasible evaluation, functional annotation and classification of a high number of expressed sequences in a great variety of organisms. such characterization would be useful for functional genomics research in plants, particularly in the emergent field of systems biology. with the progress of plant genome sequencing projects, in-depth knowledge about molecules, such as nucleic acids and deduced proteins, gene regulatory networks, and metabolic pathways becomes possible. hierarchically structured ontological terms can now be adopted to query sequences and to describe genes and their products at different levels of knowledge and specificity. the gene ontology project began in with the integration of three model organism databases, i.e. yeast, drosophila and mouse, and represents today the most widely used schema for the functional characterization of plant, animal and microbial genes and gene products. the go project has developed three structured vocabularies describing genes, transcripts and proteins of any organism in terms of their associated cellular component, biological process and molecular function in a species-independent manner. the use of go terms by collaborating databases facilitates uniform retrievals across them. moreover, the go vocabularies can be queried at different levels, allowing annotators to assign properties to genes or gene products, depending on the depth of knowledge and specificity about that entity. in plant species for which the genome sequence is not available, aflp and cdna-aflp are two of the most commonly used methods for genome- and transcriptome-wide level analysis, respectively, capable of discovering genes not yet been cloned or even predicted, based upon their polymorphisms or differential expression patterns. aflp markers represent the genomic tool with the highest polymorphism information content, and combine the reliability of the rflp technique with the power of the pcr technique. both aflp and aflp-derived markers, such as m-aflps and s-saps, have been widely used for whole genome scanning and fingerprinting, characterizing single chromosomes and mapping specific genes, as well as for transcriptome profiling and gene cloning by means of cdna-aflp, and to generate quantitative gene expression patterns for eqtl mapping, as recently demonstrated by vuylsteke et al.. transcriptome differential profiling of plants with antagonistic phenotypes is theoretically one of the most powerful strategies for identifying and cloning candidate genes not only for model organisms, but also in remarkably complex genomes such as polyploids. the mrna fingerprinting based on aflp technology does not require pre-existing genome or est sequence knowledge, therefore it is being widely used in less well investigated systems. since a few genomes of agriculturally important species have been sequenced and even fewer have been well annotated, the data provided by cdna-aflp experiments may represent a valuable resource for functional genomics and genetics in non-model plants. compared to microarrays, cdna-aflp increases the resolution of expression patterns detection using smaller amounts of mrna. this feature is essential when an rna fingerprinting is applied to tissues for which it is hard to isolate stage-specific messengers, such as flowers, fruits and seeds. both reliability and sensitivity of amplification products proved to be very high, and expression patterns visualized by cdna-aflp showed to well correlate with northern blot analyses. moreover, the redundancy of the technique proved to be very informative in cases of alternative splicing and multigene family member displaying, for distinguishing highly homologous genes. after 10-year-use of aflps for dna fingerprinting and mrna profiling, large sequence collections retrieved with this technology are available in public databases for several crop and model species. the evaluation, annotation and classification of aflp-derived genomic markers and expressed transcripts would be very useful for both functional genomics and systems biology research in plants, and crucial to allow the scientific community to promptly retrieve this pre-existing information from gene banks. annotations could be periodically revised and implemented, and should allow to optimize genomic aflp and cdna-aflp experiments in plant species to give specific information about the targeted biological processes and molecular functions. in the present study, we retrieved a total of cdna-aflp sequences related to roots, leaves, stems, flowers, fruits and seeds from both ncbi databases and unpublished repertories. all these entries belong to different species distributed among seven botanic families: solanaceae, fabaceae, poaceae, salicaceae, rosaceae, brassicaceae and vitaceae. redundant sequences were clustered and contigs assembled. functional analysis was then performed using blast2go. blast2go is a bioinformatic tool for the go-based annotation and data mining of sequence sets for which no go annotation is yet available, which has proven to be effective in the functional characterization of plant sequence data. the cdna-aflp-derived sequences were then grouped according to the go vocabularies. experimental steps and statistical parameters adopted for the in silico analysis were critical for obtaining reliable gene ontology data. annotation results for the whole sequence dataset and also for botanic families, single species and plant organs are presented, and the main features of genes and gene products detectable in plants by aflp technology discussed.  gene ontology of aflp-derived cdna sequences the total number of plant cdna sequences derived from aflp technology deposited in ncbi databases or recovered from private unpublished collections was as high as. entries belonged to different model, crop and tree species distributed among seven botanic families: solanaceae, salicaceae, fabaceae, poaceae, rosaceae, brassicaceae and vitaceae. additional cdna-aflp-derived sequences of salix spp. were analyzed separately once they became publicly available because although deposited they were not released at the time of entries downloading. the most abundant taxonomic group included in the present study was the genus nicotiana, followed by populus, medicago, oryza and malus. the list of organisms with their corresponding number of cdna-aflp sequences deposited in ncbi databases is reported in the methods section. redundancy information on cdna-aflp sequences assessed on the basis of the number and proportion of contigs and singlets. for each sequence subset, the number of retrieved sequences and the percentage of the total, the number of contigs, the number of singlets and the percentage of the total, the redundancy, and the gc content are reported. sequences deposited in genbank but not yet publicly released; 2all sequences were released after february 1th, 2007; sequences are not yet publicly available. redundant cdna-aflp sequences for each of the organisms were clustered into a total of contigs, whereas the remaining records were singlets. the overall estimate of redundancy was about 23%, ranging from 0% in fragaria and lolium, to % of solanum spp, whereas the gc content of cdna-aflp sequences was found to be 45%, with a standard deviation over all organisms as low as %. redundancy statistics and gc contents for all organisms are summarized in table with the exception of the salix entries, the cdna-aflp sequences equal to contigs plus singlets and belonging to angiospermae were used as blastx queries to search for structural homologies and significant similarities. a total of sequences revealed significant similarity with deposited records, showing an average nucleotide similarity estimate of 80% and a median e-value of e- over all organisms. the same statistics were also computed for each single species or genus. go term mapping allowed the identification of go terms based upon matches retrieved by blastx. the distribution of annotations among go vocabularies and the number of cdna-aflp sequences with go terms belonging to only one, a combination of two and all three vocabularies was organized in a venn diagram. sequences were annotated according to all the three go subvocabularies and annotations simplified using a plant-specific goslim following the approach suggested by similar papers with comparative purposes. as many as and sequences were annotated with a combination of bp and mf terms, bp and cc terms, and cc and mf terms, respectively. basic statistics for all the cdna-aflp annotated sequences sorted by botanic family and by single organism, including mean length and length range for a 68% confidence interval, mean similarity and similarity range for a 68% confidence interval, median e-value with minimum and maximum e-values are reported in tables and statistics on cdna-aflp annotated sequences sorted by botanic family. †refers to the number of sequences included in the 68% confidence interval. ‡refers to the total number of sequences. abbreviations: conf. int., confidence interval; cv, coefficient of variability; min, minimum; max, maximum). statistics on cdna-aflp annotated sequences sorted by organism. †refers to the number of sequences included in the 68% confidence interval. ‡refers to the total number of sequences. abbreviations: conf. int., confidence interval; cv, coefficient of variability; min, minimum; max, maximum. among the botanic families, vitaceae showed the highest proportion of annotated sequences and rosaceae the lowest without blast matches. on the whole, 36% of sequences did not retrieve any blast result within the set e-value threshold. mapping of go terms and annotation were not possible for 20% and 3% of sequences, respectively, whereas the remaining 41% was annotated. overall data distribution of cdna-aflp sequences grouped according to the organ, tissue or part of the plant, and botanic family showed a large variation for the percentage of sequences either with no blast results or annotated, whereas the percentage of sequences neither with mapping nor annotation was quite constant. fruits were the organs with the highest percentage of annotated sequences and the lowest percentage without blast matches, whereas the opposite was for seeds. similar properties were observed for roots, stems and leaves, and flowers. level goslim analysis of aflp-derived cdna sequences the go terms obtained by the annotation procedure were mapped to a plant specific goslim to generate a more concise annotation to be used for comparative analyses as reported in similar researches. in the present study the'goslim_plant.obo' developed by mundodi s. the ftp directory of the genome databases group, department of genetics, stanford university school of medicine was used. the goslim classification of annotated cdna-aflp sequences is reported in figures and level annotation was chosen because representative of all retrieved annotation, and a comparison with the annotations of arabidopsis and rice ests was possible. among the annotated sequences, were attributable to roots, to stems plus leaves, to flowers, to fruits and to seeds. for the remaining, no information on the plant tissue or organ source was available among the features of the database entries. concerning the annotations of sequences grouped by organs and tissues, it is worth noting that the proportion of fruit records with catalytic and transporter activities was significantly higher than that observed for other organs, whereas the proportion of sequences related to'binding' was much lower than that calculated for roots, leaves and stems, flowers and seeds. the annotation of sequences according to the botanic family was also performed. the relative proportions of sequences associated with the two most abundant categories of cc, bp and mf proved to be very similar each other, with relative differences smaller than 5%. these findings indicate that go annotation data gained for aflp-derived cdna sequences in plants are highly concordant, when compared among botanic families, but also significantly divergent, when referred to different plant organs or tissues. the classification of annotated cdna-aflp sequences at level is summarized in figure for each of the taxonomic entities/organisms analyzed in this study and sorted by genus or species. the proportion of annotated sequences for each organism varied from % of nicotiana spp. to % of lolium multiflorum. consistent differences were pointed out among species for go terms belonging to the three subvocabularies. go multilevel analysis of aflp-derived cdna sequences all cdna-aflp sequences sorted by organs and families were also analyzed using a multilevel procedure. this further investigation enabled to find out the lowest node per branch of the dag that fulfils the filter condition, e.g. will find all the lowest nodes with the given number of sequences or score value. the most represented terms among the belonging to cc for plant organs were'membrane' for fruits and roots, and'plastid' for seeds, flowers, and leaves and stems. sequences associated with'cytoplasmic membrane-bound vesicle','intracellular organelle part' and'membrane part' were only found in leaves and stems. the most represented bp terms among the for plant organs were'transport' in seeds, fruits and roots,'secretory pathway' in leaves and stems,'amino acid and derivative metabolism' in flowers. finally, the most represented terms among the belonging to mf for plant organs were'hydrolase activity' in roots and seeds,'oxidoreductase activity' in leaves and stems,'nucleotide binding' in flowers and fruits. sequences associated with'oxidoreductase activity','atp binding','transition metal ion binding','protein kinase activity' and'peptidase activity' were only recorded for leaves and stems. conversely, sequences related to'hydrolase activity' and'nucleotide binding' were found over all organs, except for leaves and stems. regarding the by-family functional analysis at multiple levels, again an overall agreement in most represented go terms was observed. concerning the cc,'membrane' was the most abundant term in all families, except for the solanaceae where'protein complex' was the most frequent.'plastid' and'mitochondrion' were also abundant in the fabaceae and poaceae. for the bp ontology, the most numerically represented categories related to'transport' over all families, varying between % of brassicaceae to % of poaceae. among the other go terms, the most abundant within family were'cell organization and biogenesis' in solanaceae,'protein modification' in fabaceae, rosaceae and solanaceae,'biosynthesis' in fabaceae,'carbohydrate metabolism' in rosaceae and salicaceae,'amino acid and derivative metabolism' in fabaceae, poaceae and salicaceae. among the mf terms,'hydrolase activity' and'nucleotide binding' included the highest proportion of sequences over all families. in particular, sequences associated with the former spanned from % of solanaceae to % of fabaceae, whereas those associated with the latter were around 25% in most families. moreover, cdna-aflp profiling proved to assay expressed sequences related to'kinase activity' with very similar efficiency in each of the seven families analyzed in this study. annotation index estimates of aflp-derived cdna sequences an annotation index was developed to describe the information content of aflp-derived cdna sequence collections on the basis of the go annotations. computational simulations performed with random sets of cdna-aflp sequences revealed that ai values can range between and, depending on the proportion of sequences associated with combinations of terms belonging to one, two or all the three go vocabularies. annotations indexes were estimated for plant organs/tissues and botanic families in order to perform comparisons. sequences related to seeds and fruits were those displaying, respectively, the lowest and highest ai with almost a two-fold difference, i.e. vs.. concerning botanic families, ai ranged from of solanaceae to of vitaceae, with an average value for the total set of sequences of. annotation index estimates for the groups of cdna-aflp-derived sequences analyzed. go comparison between dicots and monocots aflp-derived cdna sequences in order to study the functional content of cdna-aflp sequences in relation to expressed sequences in model organisms, a level goslim classification of all the cdna-aflp records was performed by splitting the sequence dataset into monocots and dicots and by comparing the two subgroups with all annotated ests of oryza sativa and arabidopsis thaliana, respectively. the comparison between dicot cdna-aflps and arabidopsis est sequences revealed substantial differences for the relative frequencies of the most represented go terms associated with cc and mf ontologies. in particular, the cc categories of'cell','organelle' and'protein complex' included %, % and % of arabidopsis est sequences against %, % and % of dicot cdna-aflp sequences, respectively. even the go terms related to'organelle part' and'envelope' were strongly differentiated between arabidopsis and dicot species. the two major go categories associated with bp, which were'physiological process' and'cellular process', showed to be similarly represented in dicot species and arabidopsis, being their proportions equal to % vs. % and % vs. %, respectively. a large difference was observed for the term'response to stimulus', which was assigned to % of arabidopsis sequences and only to % in dicot cdna-aflps. four additional go term categories,'regulation of biological process','development','reproduction' and'growth' were found at %, %, % and %, respectively, in arabidopsis and at much lower frequencies in the dicot species. regarding mf terms, highly significant differences were found for the two major categories,'catalytic activity' and'binding': the former was represented by % and % of the sequences, respectively, in arabidopsis and dicot species, whereas the latter was 28% in arabidopsis and % in the dicots. a marked difference was also found for the category'transcription regulator activity'. the comparison between monocot cdna-aflps and rice est sequences revealed also consistent differences for some of the most frequent go terms. while in the cc branch the two major categories of'cell' and'organelle' were similarly represented, with % vs. % and % vs. %, respectively, a marked difference was observed for'protein complex', being the frequency of sequences associated with this category equal to % in rice and % in the monocot sequences. regarding the bp, the three major go terms proved to be much more frequent in rice ests than in monocot cdna-aflp sequences, being equal to % vs. % for'physiological process', % vs. % for'cellular process' and % vs. % for'response to stimulus', with highly significant differences, % vs. 0% for'development', and % vs. 0% for'regulation of biological process'. non-significant differences were observed for the go terms belonging to the mf vocabulary, except for the category of'binding'. discussion experimental procedures adopted for go analysis of aflp-derived sequences the experimental steps and statistical parameters adopted for aflp-derived sequence analysis are critical for obtaining reliable ontology data and deserve a specific mention. four main steps were followed to achieve a reliable go classification of aflp-derived sequences: i. data recovery from sequence databases; ii. preliminary selection of nucleotide records; iii. basic annotation; iv. enrichment and refinement of annotation terms. the retrieval of aflp-related sequences from ncbi was performed by searching for the keywords "aflp and viridiplantae" and "cdna-aflp and viridiplantae" and subsequent manual scoring to verify the results. the manual step was required because the keywords indicating the aflp origin were not always present in the record fields. for our purpose, this seemed to be a noticeable limitation of the genbank database, probably due both to an unintentional inaccuracy in submitting sequences and an insufficient stringency in accepting a new record without any precise indication of its origin. as a general remark, nucleotide ncbi database curators should consider requesting additional and more precise information about the origin of the records to be submitted. in the case of aflp-derived sequences, at least the restriction enzymes and selective primer combinations used in the experiments should be added in the'features' or'comment' fields, as already done by several submitters. the presence of redundant sequences was a further known limitation, caused mainly by a poor evaluation of the records already present in the public databases at the time of a new submission. however, possible polymorphisms, even at the single nucleotide level, have to be always considered as highly informative and carefully evaluated for their reliability. the basic annotation obtained by retrieving go terms from blastx matches was enriched and refined using three main strategies integrated in the blast2go software: i) interproscan, ii) annex, and iii) goslim simplified annotation. databases of protein domains and functional sites have become vital resources for the prediction of protein functions. interproscan combines different protein signature recognition methods allowing searches against independent databases and an easy recovery of the corresponding codes to be automatically coupled to go terms annotation. this tool allowed an average 8% increase in the overall annotated sequences. this value is in the range of observed improvement in blast-based go annotation by interpro and shows the important contribution of motif-based annotation in go terms enrichment as well as the basic importance of an integrated approach to the annotation of unknown nucleotide records. a refinement of the go terms was obtained by means of the annex function. using the original go structure, annex locates parent-offspring relationships between the annotations. afterwards, annex suggests new bp and cc annotations as well as implicit ones deduced from univocal relationships between go terms from the different go categories providing on average 10–15% increase in functional terms. in other words, given a molecular function, this tool identifies biological processes where the molecular functions are involved and the cellular components where they are active. for a detailed description of the annex procedure, see. in the last step of the annotation procedure, a simplified version of the full ontologies was adopted. goslims are cut-down versions of the full ontologies composed by high-level selected terms, or nodes, each one including subsets of the terms of the whole go. they give a broad overview of the ontology content without the detail of the specific fine-grained terms. goslims are created by users according to their needs, and may be specific to species or to particular areas of the ontologies. go provides a generic goslim which, like the go itself, is not species-specific, and which should be suitable for most purposes such as the comparisons made in this research. the adoption of the'goslim_plant.obo' goslim allowed a visible improvement of the annotation in terms of a reduced fragmentation of the go categories and a more plant-specific terminology. indeed, all the go terms specifically related to cc, mf and bp typical of mammals and/or unicellular organisms were removed or replaced with plant-specific terms. the blast2go software was shown to be very effective in every step of the annotation procedure particularly with respect to the integrated tools that allowed a great time saving during the enrichment and refinement steps. this tool offers a suitable platform for functional genomics research in non-model species and it also allows monitoring and comprehension of the whole annotation and analysis steps. some minimal limitations were found during the elaboration of results, in particular concerning the generation of graphics. however, future implementations may allow the blast2go software to be one of the most powerful tools for the annotation of unknown sequence pools. gene expression analysis by means of cdna-aflp technique the generation of transcriptional profiles has numerous applications in plant biology, including the identification of tissue-specific or developmental stage-specific, reproduction-related and stress-induced transcripts. many new techniques have been designed in recent years for this purpose, such as microarrays, allowing a serial genome-wide expression profile of thousands of genes to be performed in a single experiment. though powerful, this approach is often really expensive and can be readily applied only to model species for which significant information about the coding sequences is available. moreover, because rarely expressed transcripts are usually missing from cdna libraries due to over-representation of abundant messengers, microarrays could fail to detect transcripts that are rare but fundamental for certain traits. library enrichment approaches, such as library subtraction or normalization, could also be adopted to catch low-copy mrnas, although time-consuming and cost-effective. on the other hand, the cdna-aflp technique has become one of the most robust solutions for differential display and it has been successfully applied in several quantitative gene expression studies, even in less investigated species for which sequence information is not available. therefore, after ten years from its first applications in plants, cdna-aflp represents an irreplaceable tool for transcriptome profiling analysis that can be utilized for multifactorial genomics of any species. the utility of cdna-aflp in functional genomics and systems biology research in plants can be further implemented by serial annotation analyses of species/organ/tissue-specific repertories of transcript-derived fragments, starting from the data provided in the present study. moreover, the annotations have to be continuously updated, since further information is available by functional characterization of the most interesting sequences. in this way, annotation data may allow to set up a forecasting model according to which the cdna-aflp experiments can be tuned to target specific gene classes, and therefore, retrieving gene sequences enriched with the desired functional categories. blastx querying, go terms mapping and annotation of transcript sequences recovered by cdna-aflp analysis the relatively high number of specific studies carried out in recent years using cdna-aflp as a differential display technique does not match a correspondingly high number of sequences retrievable from public databases. the total number of plant aflp-derived est entries was shown to be as low as, successively reduced to because of the 23% of redundancy. moreover, only plant species/genera were represented over all records, with solanaceae being the most numerous family. a total of cdna-aflp sequences revealed structural homology and significant similarity with deposited records, allowing to retrieve as many as go terms. the subsequent validation performed with the integrated function of the blast2go software enabled the annotation of cdna-aflp sequences, each with to go terms. despite the important role of cereals and related research, the highest number of aflp-derived ests with blast matches was found in the rosaceae family, immediately followed by the vitaceae, the latter being mostly represented by grape sequences. these two taxonomic groups have been subjected to intense research activities in the last few years, supported by worldwide initiatives such as the gdr at clemson university, estree consortium in italy which is focused mainly on peach, international grape genome program in united states and the franco-italian grape genome sequencing project. the enrichment of database information resulting from such collaborative researches may be in part responsible of the availability of blast matches for rosaceae and vitaceae sequences. for a parametric description of sequence annotability, a dedicated index was developed in the present research and calculated for the most interesting sequence subsets. therefore, the ai computed for rosaceae and vitaceae was as high as and, respectively. taking into account the other botanic families, some interesting results were pointed out concerning the annotability of sequences. in particular, solanaceae family strangely showed the lowest percentage of annotated sequences, with ai as low as, despite the high number of entries present in the databases and the fact that species belonging to this taxonomic group, such as tomato, tobacco and petunia, are widely used as models. considering the information content of sequences classified according to the organ, tissue or part of the plant from which they were previously isolated, some interesting results were evidenced from the analysis of data distribution. the highest percentage of sequences with blast results was found for cdna-aflp entries isolated from fruits, that were also the plant organs with the highest percentage of annotated sequences, with an ai as high as. this result agrees with those found for the rosaceae family, to which most of the fruits such as peach, apple, pear, plum, apricot, and cherry belong, as well as for the species included in the vitaceae taxonomic group, such as grape. moreover, the abundance of go terms associated with carbohydrate, protein and amino acid metabolism, and catalytic as well as transporter activities may reflect the cellular and biochemical events occurring during fruit ripening. despite the importance of seed development in cereals and legumes, and the amount of studies dedicated to its understanding, sequences related to seeds were those with the lowest percentage of entries with blast hits and annotation, displaying an ai =. on the whole, our findings suggest that the annotability of a set of sequences from a specific plant organ or a particular taxonomic group is not a direct consequence of the amount of research efforts dedicated to studying that organ or group. in fact, the go annotations of genes are very often transferred from very phylogenetically distant organisms. the go characterization and annotation of aflp-derived sequences allowed us to retrieve basic information on the gene function/s in crop species and their organs/tissues. in particular, examination of thousands of est clones enabled certain inferences to be made on the potentials and drawbacks of aflp technology for mrna profiling and differential display gene cloning. although the different number of sequences retrieved in databases for plants and organs might have biased some of the descriptive statistics, the overall go is consistent with the existence of aflp technology features exploitable across angiosperm. the representativeness of sequence samples and goodness of statistical results over all taxonomic groups are supported by bootstrap analysis. the calculations were performed using the singlets of nicotiana spp. with a number of non-overlapping random replicates of and each formed by and sequences, respectively. the bootstrap test was carried out on nicotiana because it was the most numerous genus. the proportions of annotated sequences for each go vocabulary and over all go terms per vocabulary proved to be very similar in all replicates with standard deviations lower than 5% in most cases. this finding demonstrates the high reliability and reproducibility of gene annotation results for the organisms analyzed in this study. as a consequence, est repertories equal to or larger than cdna-aflp clones can be considered sufficient to obtain a sequence information content representative of the main go categories. in this context, it is worth noting that cdna-aflp profiling proved to assay expressed sequences related to'kinase activity' with very similar efficiency in each of the seven families analyzed in this study. it may be deduced that this gene family shows distinctive conserved characteristics so that its members can be repeatedly detected by the cdna-aflp technique with a quite constant probability in all plant species. a go classification of all cdna-aflp records was performed by splitting the sequence dataset into monocots and dicots and by comparing the two subgroups with all annotated ests of arabidopsis thaliana and oryza sativa. although available molecular data may be biased by the experimental methodologies and plant materials, our overall go results suggest that in a given species and for a given organ or tissue est repertories developed by construction and screening of cdna libraries include a sequence pool information content different from and/or complementary to that of tdf collections generated by cdna-aflp profiling and differential display. since functional data supplied by aflp-derived sequences do not fully overlap the functional data obtained by est projects, the two technologies most likely allow to probe distinct target genes and to capture distinct transcript subsets from a given part of the plant. alternatively, the functional information obtained by blast2go may have been biased in comparison to the available annotation for rice and arabidopsis. the comparison between monocot cdna-aflps and rice est sequences also revealed interesting results. in this case, an explanation for the deviations documented in specific go branches and categories might be found in the different goals driving basic research projects and applied breeding activities in model and crop plants.  in the last ten years, the cdna-aflp mrna profiling was largely adopted and considerable repertories of organ-specific and differentially expressed transcripts are now available in public databases for model, crop and tree species. the evaluation, annotation and classification of aflp-derived sequences would therefore become crucial for both functional genomics and systems biology research in plants. the possibility of using aflp-derived tags on cdna fragments produced directly by sequencing-by-synthesis technologies opens up the possibility of not only identifying very large numbers of expressed genes, but also retrieving large-scale snp collections. our study suggests that a reliable go characterization of aflp-derived sequences is based on the optimization of experimental steps and statistical parameters adopted for go analysis. the blast2go software was shown to represent a comprehensive bioinformatics solution for functionally characterizing sequences and data mining on the correspondent annotations based on the go vocabularies. an exhaustive annotation based on gene products similarity blast searches would offer a suitable platform for functional genomics, particularly useful in non-model plant species. therefore, the utility of aflp technology in structural and functional genomics in plants can be implemented by go annotation analyses species/organ/tissue-specific repertories of transcriptome-derived fragments. our suggestion is that aflp-derived sequences should be systematically subjected to go annotations before their submission to ncbi databases so that a publicly available information based on yearly larger plant est collections could be periodically released to the go consortium and retrieved by other researchers when searching the genbank. 
this may be achieved by means of the gene ontology, consisting in three structured vocabularies describing genes, transcripts and proteins of any organism in terms of their associated cellular component, biological process and molecular function in a species-independent manner. the evaluation, annotation and classification of genomic markers and expressed transcripts would be of great utility for both functional genomics and systems biology research in plants. such an exhaustive annotation may offer a suitable platform for functional genomics, particularly useful in non-model species. the utility of this technology for structural and functional genomics in plants can be implemented by serial annotation analyses of genome-anchored fragments and organ/tissue-specific repertories of transcriptome-derived fragments. on the whole, the statistical parameters adopted for the in silico aflp-derived transcriptome-anchored sequence analysis proved to be critical for obtaining reliable go results. descriptive statistics on the type, size and nature of gene sequences obtained by means of aflp technology were calculated. reliable go annotations of aflp-derived sequences can be gathered through the optimization of the experimental steps and the statistical parameters adopted. in this paper, the functional annotation of about aflp-derived ests retrieved in the ncbi databases was carried out by using go terminology. after 10-year-use of aflp technology for dna fingerprinting and mrna profiling, large repertories of genome- and transcriptome-derived sequences are available in public databases for model, crop and tree species. a comparison of the functional content of cdna-aflp records was also performed by splitting the sequence dataset into monocots and dicots and by comparing them to all annotated ests of arabidopsis and rice, respectively. the blast2go software was shown to represent a comprehensive bioinformatics solution for an annotation-based functional analysis. the gene products associated with mrna transcripts were then classified according to the three main go vocabularies. according to the whole set of go annotations, the aflp technology generates thorough information for angiosperm gene products and shares common features across angiosperm species and families.
 after 10-year-use of aflp technology for dna fingerprinting and mrna profiling, large repertories of genome- and transcriptome-derived sequences are available in public databases for model, crop and tree species. aflp marker systems have been and are being extensively exploited for genome scanning and gene mapping, as well as cdna-aflp for transcriptome profiling and differentially expressed gene cloning. the evaluation, annotation and classification of genomic markers and expressed transcripts would be of great utility for both functional genomics and systems biology research in plants. this may be achieved by means of the gene ontology, consisting in three structured vocabularies describing genes, transcripts and proteins of any organism in terms of their associated cellular component, biological process and molecular function in a species-independent manner. in this paper, the functional annotation of about aflp-derived ests retrieved in the ncbi databases was carried out by using go terminology.  descriptive statistics on the type, size and nature of gene sequences obtained by means of aflp technology were calculated. the gene products associated with mrna transcripts were then classified according to the three main go vocabularies. a comparison of the functional content of cdna-aflp records was also performed by splitting the sequence dataset into monocots and dicots and by comparing them to all annotated ests of arabidopsis and rice, respectively. on the whole, the statistical parameters adopted for the in silico aflp-derived transcriptome-anchored sequence analysis proved to be critical for obtaining reliable go results. such an exhaustive annotation may offer a suitable platform for functional genomics, particularly useful in non-model species.  reliable go annotations of aflp-derived sequences can be gathered through the optimization of the experimental steps and the statistical parameters adopted. the blast2go software was shown to represent a comprehensive bioinformatics solution for an annotation-based functional analysis. according to the whole set of go annotations, the aflp technology generates thorough information for angiosperm gene products and shares common features across angiosperm species and families. the utility of this technology for structural and functional genomics in plants can be implemented by serial annotation analyses of genome-anchored fragments and organ/tissue-specific repertories of transcriptome-derived fragments. 