BACKGROUND
sweetpotato  lam.)
belongs to the convolvulaceae .
with an annual global production of  <dig> million tons, it is an important crop in developing countries and has an abundance of uses, ranging from consumption of fresh roots or leaves to processing into animal feed, starch, flour, candy, and alcohol.
sweetpotato produces more food per hectare than wheat, rice, or cassava, which makes it an important food security crop.
the high pro-vitamin a content of orange-fleshed sweetpotato varieties plays a crucial role in mitigating vitamin a deficiency that is still widespread among the poor in developing countries.
adding orange-fleshed sweetpotato to the daily diet could prevent vitamin a deficiency-related blindness and maternal mortality  <cit> .
african farmers produce about  <dig> million tons of sweetpotato annually with yields of about  <dig>  t/ha.
yields in africa amount to about a fifth of those in china, indicating a huge potential for future growth  <cit> .
main reasons for low yields in africa are, besides lack of high-quality planting material, fertilizers and irrigation, prevailing insect pests, and viral diseases  <cit> .
genomic tools such as molecular markers could support breeding efforts to produce varieties with high nutritional value and improved virus and insect resistance that would ensure food security of resource-poor farmers in developing countries.
sweetpotato breeding is constrained by the complexity of the genetics of this out-crossing hexaploid crop and by the lack of genomic resources.
up to now, sweetpotato sequence information has been limited to some  <dig>  expressed sequence tag  sequences and ~  <dig>  genbank sequences and one bacterial artificial chromosome sequence deposited in public databases.
at the plant gene database http://www.plantgdb.org/download/download.php?dir=/sequence/estcontig/ipomoea_batatas/current_version and at tigr plant transcript assemblies http://plantta.jcvi.org/index.shtml, assemblies of genbank-deposited ipomoea batatas ests are available.
more assembled est sequences are available from morning glory , a wild relative of cultivated sweetpotato http://compbio.dfci.harvard.edu/tgi/cgi-bin/tgi/gimain.pl?gudb=morning_glory that might be of use as a model plant for some aspects of sweetpotato genomics.
sweetpotato genomics tools are currently restricted to a medium-density cdna microarray http://www.picme.at/index.php/products/arrays/?task=details&species=sweetpotato%20microarray%20picme_swp_15k_ <dig>  linkage maps  <cit> , and some  <dig> published microsatellite  markers  <cit> .
to prepare the path for using the large biodiversity of this crop in molecular breeding of improved cultivars, more genomic resources such as sweetpotato gene sequences and markers are urgently needed.
cip holds in trust  <dig>  sweetpotato accessions, including breeding lines, improved varieties, landraces, and wild accessions from  <dig> countries.
a subset of this collection, consisting of  <dig> accessions that represent the diversity of this crop with respect to agronomical and resistance traits as well as nutritional quality--referred to as a "composite genotype set"--is available for international distribution in the form of disease-free in vitro plants to facilitate access of breeders to sweetpotato biodiversity http://gcpcr.grinfo.net/files/cr_files/gcpcr_file <dig> xls.
the sweetpotato clone tanzania, a member of the composite genotype set, is a high-yielding, stress-tolerant, and broadly cultivated african landrace that is used in various breeding programs due to its high dry matter content and drought tolerance.
this clone has been submitted to transcriptome sequencing in the present work, with the aim to augment the available gene sequence and marker information for this crop.
<dig> pyrosequencing has become a popular method for high throughput sequencing.
at low cost, it provides a huge amount of sequence information at relative low error frequency  <cit> .
it is widely used for genome resequencing  <cit> , de novo sequencing of small genomes , snp detection  <cit> , or transcriptome sequencing  <cit> .
specialized software for de novo assembly of the relative short  <dig> sequences, ranging from around  <dig> bp with the  <dig> flx system but now approaching  <dig> bp with the  <dig> flx titanium system, is available.
however, only a few software packages claim to be suitable for hybrid assembly of the relatively short  <dig> reads with sanger sequences  <cit> .
the choice of optimal assembly parameters to significantly reduce redundancy of an est collection and, at the same time, maintain paralogous sequences separated in different contigs remains a challenging task.
particularly in a highly heterozygote hexaploid plant such as sweetpotato, discrimination between paralogs and allelic variants on whole transcriptome level remains difficult.
for an est assembly, it might therefore be preferable to accept an increased level of redundancy instead of risking the merging of different members of a gene family into one contig by using too loose assembly parameters.
the quality of an assembly generally augments with increasing sequence information.
therefore assemblies with a limited number of ests represent a work in progress rather than a finished transcriptome assembly.
as more sequence information becomes available, reassembly can lead to splitting or rearrangement of contigs, thus leading to an optimized representation of the transcriptome.
here we present the de novo assembly of  <dig> sequencing reads from two normalized sweetpotato libraries, together with publically available ests, which resulted in a gene index for hexaploid sweetpotato.
this gene index facilitates the access to sweetpotato gene sequences and has allowed for the design of gene-based microsatellite markers.
RESULTS
<dig> sequencing
a total of  <dig>  raw reads comprising  <dig> , <dig> bases were obtained with a  <dig> flx quarter run of a normalized cdna library of leaves from drought-stressed plants of the sweetpotato clone tanzania.
another quarter run of  <dig> flx titanium resulted in  <dig>  raw reads and  <dig> , <dig> bases for a normalized cdna library of stems of the same clone.
the average length per read amounted to  <dig>  bp for the  <dig> flx run and to  <dig>  for the  <dig> flx titanium run.
cdna synthesis primer sequences present in the reads were removed and very short reads with less than  <dig> bp and low-quality sequences were eliminated.
the remaining  <dig>  raw reads were blasted against vector-cleaned sweetpotato ests from the genbank.
blast hits at > 80% coverage and > 80% identity were found in  <dig> % of the  <dig> sequences, suggesting that the remaining two-thirds  of the  <dig> reads represent new est sequences.
sequence assembly
the  <dig>  raw reads were assembled together with  <dig>  ests from the genbank.
we attempted to optimize the assembly parameters by stepwise variation of the minimum match percent  parameter from  <dig> to 90% and testing for good representativeness and minimal redundancy of the gene index .
the contig number obtained changed linearly and slightly when the mmp parameter was increased from  <dig> to 80%.
from  <dig> mmp on, the increment of singleton number became steeper and the number of contigs augmented stronger from  <dig> mmp on .
redundancy in the gene index was assessed by self-megablast and blastx with a a. thaliana protein database.
at the nucleic acid level, we performed a self-megablast test to assess similarity between contig and singleton sequences produced at different mmps.
sequences sharing 80% identity over 80% of their length with another sequence were considered as redundant.
as expected, self-megablast hits augmented with increasing mmps, indicating that sequences representing allelic variants increasingly remained unassembled or were resolved into different contigs.
the increase in the number of self-megablast hits went in parallel with the number of sequences and became steeper at mmps above 80% .
additionally, we have assessed the redundancy of the assemblies produced at different mmp levels by counting the number of total and unique blastx hits of the contig and singleton sequences with a protein database.
at this stage, in order to make redundancy analysis quicker and less demanding with respect to computer power, we used the a. thaliana protein database instead of the very large uniref <dig> protein set for blastx analysis.
predictably, the total number of blastx hits increased with rising mmp and the increase became steeper above 80% mmp, whereas unique blastx hits increased linearly over the whole range of mmp and at a much lower path than the total hit number.
the number of hits increased mainly for singletons, as more sequences remained unassembled at higher mmp values .
to investigate whether assembly at low mmp results in merging of reads of paralogous sequences into one contig, we have analyzed the number of total and different members of  <dig> randomly chosen gene families in the assemblies at different mmp .
in a simple text search using uniprot identifiers and text annotation, we have looked for sequences annotated as zinc finger a <dig> and an <dig> domain-containing stress-associated protein, xyloglucan endotransglucosylase/hydrolase protein, α-tubulin, β-tubulin, myb, bhlh, thioredoxin, syntaxin, superoxide dismutase, sulfate transporter, na+/h+ exchanger, scarecrow, serine carboxypeptidase, actin, ferritin, pra <dig> family protein, enolase, β-amylase, bzip, and glutaredoxin according to sequence comparison with proteins of the a. thaliana proteome.
although the total count of members over the  <dig> gene families increased from  <dig> at  <dig> mmp to  <dig>  at  <dig> mmp, the number of different gene family members varied little over the different mmp--from  <dig> to 296--indicating that the risk of merging paralogs into one contig at low mmp exists but remains low.
therefore, to ensure both good representativeness and low redundancy and, at the same time, to mitigate the risk that reads derived from paralogous sequences are assembled together into one contig, we chose the medium stringent  <dig> mmp as assembly parameter for the sweetpotato gene index.
at  <dig> mmp the assembly still had relative low redundancy, while the representativeness was better than at lower mmp values, indicated by  <dig> more unique blastx hits with the a. thaliana proteome than at  <dig> mmp.
hybrid assembly of our  <dig> reads with est sequences obtained from the genbank at  <dig> mmp produced  <dig>  contigs and  <dig>  reads remained as singletons.
contigs ranged from  <dig> to  <dig>  bp in size, with an average of  <dig> bp.
the cumulative length of the contigs was  <dig> , <dig> bp.
the size distribution for the contigs is shown in figure 3a.
a substantial number of large contigs was obtained:  <dig>  contigs were >  <dig> bp in length, and  <dig>  were >  <dig> kb.
sequencing coverage ranged from  <dig> to  <dig>  reads per contig, with an average coverage of  <dig>  .
reciprocal blastn queries with previous ipomoea est assemblies
an assembly of genbank-available est and other sequences of i. batatas consisting of  <dig>  contig and  <dig>  singletons has been released previously  <cit> .
sequence comparison to this assembly gives an estimate of the amount of novel sequences in the present gene index.
our expectation was that, in addition to new sequences, our gene index should cover all contigs and singletons of the previous assembly, as the est sequences used in the previous assembly also were co-assembled with the new  <dig> reads.
and indeed, all but  <dig> out of  <dig>  sequences of the previous assembly had hits in our gene index.
of the  <dig> no-hit sequences,  <dig> corresponded to genbank mrna sequences that were not included as input for our assembly, and  <dig> other sequences corresponded to singletons of genbank-derived ests of bad quality or with highly repetitive sequences that were filtered out by our sequence cleaning process.
a total of  <dig>  sequences of the present gene index had significant similarity to  <dig>  contigs and singletons of the plant est assembly.
this indicates that redundancy in our gene index is about  <dig>  times higher than in the previous assembly.
no significant similarity to the previous est assembly was found in  <dig>  gene index sequences that are considered to represent new sequences .
similarly, only  <dig> sequences of the plantgdb sweetpotato est assembly were not contained in our gene index .
assuming a redundancy of  <dig> -fold of our gene index, the present gene index would contain about three times more sequence information for sweetpotato genes than the previous est assemblies.
for i. nil, a near relative of sweetpotato, a gene index has been established based on  <dig>  est and  <dig> mature transcripts , resulting in  <dig>  tentative consensus sequences ,  <dig>  est-singletons, and  <dig> et singletons  <cit> , http://compbio.dfci.harvard.edu/cgi-bin/tgi/gimain.pl?gudb=morning_glory.
the present gene index had  <dig>  significant  blastn hits with the i. nil database, while  <dig>  sequences remained without a hit and can be considered as specific for sweetpotato.
about 19% of the sequences of the i. nil gene index  had no significant hits with the present sweetpotato gene index.
these reciprocal blastn tests indicate good coverage of the present gene index for sweetpotato, but also a relatively high redundancy compared to previous est assemblies.
sequence annotation
of the  <dig>  sequences,  <dig>   of the gene index sequences had significant blastx matches  with sequences of the uniref <dig> protein database.
these matches corresponded to  <dig>  different unique uniref <dig> accession numbers.
the frequency of sequences with significant blastx hits was higher in contigs than in singletons .
from  <dig>  contigs without significant hits to proteins of the uniref <dig> database,  <dig>  contained a putative poly-a tail and thus most probably represent a part of a transcribed gene.
from the remaining  <dig>  contigs,  <dig>  had an open reading frame  encoding at least  <dig> amino acids.
this suggests that most contigs without blastx hits are in fact derived from protein coding genes.
the lower hit frequency of singletons in blastx searches might be caused by the smaller size of these sequences compared to contigs .
another possibility for decreased hit frequency of singleton sequences would be contamination of the cdna preparation with genomic dna, resulting in reads derived from genomic dna that remained unassembled.
analogous to the contig sequences, we also have searched for the presence of poly-a tails and orfs in singletons without significant blastx hits.
from  <dig>  singletons without significant similarity to any protein of the uniref <dig> database,  <dig>  had a putative poly-a tail.
only  <dig> singletons of the no-hit singletons, including  <dig> ests from the genbank database, did not encode any orf larger than  <dig> amino acids, while the remaining no-hit singletons had orfs from  <dig> to  <dig> bp.
the presence of orfs in more than 90% of the un-annotated singletons suggests that these sequences also are derived from protein coding genes.
the sweetpotato gene index including the annotation of the contigs and singletons can be viewed, searched, and downloaded at http://www.cipotato.org/sweetpotato_gene_index.
not all contigs and singletons, however, were derived from sweetpotato: several sequences were clearly identified as viral sequences .
these viral sequences were derived from sweetpotato feathery mottle virus, sweetpotato chlorotic stunt virus, sweetpotato leaf curl virus, or viruses with similarity to potato virus a and petunia vain clearing virus that apparently have been present in the experimental plants.
also, two singleton sequences derived from sanger ests downloaded from the genbank represented viral transcripts .
a total of  <dig>  out of the  <dig>  blastx-annotated sequences gene ontology  terms could be associated.
we annotated the gene index sequences at level  <dig> for the three main go vocabularies: cellular component, biological process, and molecular function .
microsatellite marker identification and testing
microsatellites for sweetpotato have been identified in ests previously  <cit> .
to ensure identification of new microsatellite motifs only, sequences similar to genbank ests were excluded from the search.
a total of  <dig>  new ssr motifs have been identified in the gene index, including  <dig> di-,  <dig> tri-, and  <dig> tetra-nucleotide ssr motifs with a minimum of  <dig>   <dig>  and  <dig> contiguous repeating units, respectively.
for ssr testing, we have taken into account the sequence redundancy of the gene index and selected only those ssrs that were present either in a single contig or singleton or in a group of homologous sequences that were derived most probably from a single locus.
this analysis resulted in  <dig> ssr loci, for which high-quality primers were designed.
amplification of the  <dig> ssr loci was tested in eight sweetpotato clones, consisting of six hexaploid and two diploid accessions .
from the  <dig> selected ssr loci,  <dig> yielded amplification products in the predicted size,  <dig> ssrs revealed to be polymorphic in the eight-clone test set, and  <dig> were monomorphic representing in total  <dig> alleles .
none of the ssrs showed more than  <dig> alleles in any of the diploid test accessions but up to six alleles in hexaploid sweetpotato.
for  <dig> ssr primer pairs that successfully amplified fragments from genomic dna, polymorphism tests have not been performed.
CONCLUSIONS
the present gene index http://www.cipotato.org/sweetpotato_gene_index strongly augments the available sequence information on sweetpotato genes.
owing to the heterozygous and hexaploid nature of this plant, the assembly contains a relatively high level of redundancy compared to previous assemblies to prevent merging of paralogous sequences into contigs.
fifty-nine percent of the obtained sequences could be functionally annotated.
the index has been successfully mined for microsatellite sequences and  <dig> out of  <dig> selected loci could be successfully amplified.

 sweetpotato lam.) belongs to the convolvulaceae. with an annual global production of million tons, it is an important crop in developing countries and has an abundance of uses, ranging from consumption of fresh roots or leaves to processing into animal feed, starch, flour, candy, and alcohol. sweetpotato produces more food per hectare than wheat, rice, or cassava, which makes it an important food security crop. the high pro-vitamin a content of orange-fleshed sweetpotato varieties plays a crucial role in mitigating vitamin a deficiency that is still widespread among the poor in developing countries. adding orange-fleshed sweetpotato to the daily diet could prevent vitamin a deficiency-related blindness and maternal mortality. african farmers produce about million tons of sweetpotato annually with yields of about t/ha. yields in africa amount to about a fifth of those in china, indicating a huge potential for future growth. main reasons for low yields in africa are, besides lack of high-quality planting material, fertilizers and irrigation, prevailing insect pests, and viral diseases. genomic tools such as molecular markers could support breeding efforts to produce varieties with high nutritional value and improved virus and insect resistance that would ensure food security of resource-poor farmers in developing countries. sweetpotato breeding is constrained by the complexity of the genetics of this out-crossing hexaploid crop and by the lack of genomic resources. up to now, sweetpotato sequence information has been limited to some expressed sequence tag sequences and ~ genbank sequences and one bacterial artificial chromosome sequence deposited in public databases. at the plant gene database http://www.plantgdb.org/download/download.php?dir=/sequence/estcontig/ipomoea_batatas/current_version and at tigr plant transcript assemblies http://plantta.jcvi.org/index.shtml, assemblies of genbank-deposited ipomoea batatas ests are available. more assembled est sequences are available from morning glory, a wild relative of cultivated sweetpotato http://compbio.dfci.harvard.edu/tgi/cgi-bin/tgi/gimain.pl?gudb=morning_glory that might be of use as a model plant for some aspects of sweetpotato genomics. sweetpotato genomics tools are currently restricted to a medium-density cdna microarray http://www.picme.at/index.php/products/arrays/?task=details&species=sweetpotato%20microarray%20picme_swp_15k_ linkage maps, and some published microsatellite markers. to prepare the path for using the large biodiversity of this crop in molecular breeding of improved cultivars, more genomic resources such as sweetpotato gene sequences and markers are urgently needed. cip holds in trust sweetpotato accessions, including breeding lines, improved varieties, landraces, and wild accessions from countries. a subset of this collection, consisting of accessions that represent the diversity of this crop with respect to agronomical and resistance traits as well as nutritional quality--referred to as a "composite genotype set"--is available for international distribution in the form of disease-free in vitro plants to facilitate access of breeders to sweetpotato biodiversity http://gcpcr.grinfo.net/files/cr_files/gcpcr_file xls. the sweetpotato clone tanzania, a member of the composite genotype set, is a high-yielding, stress-tolerant, and broadly cultivated african landrace that is used in various breeding programs due to its high dry matter content and drought tolerance. this clone has been submitted to transcriptome sequencing in the present work, with the aim to augment the available gene sequence and marker information for this crop. pyrosequencing has become a popular method for high throughput sequencing. at low cost, it provides a huge amount of sequence information at relative low error frequency. it is widely used for genome resequencing, de novo sequencing of small genomes, snp detection, or transcriptome sequencing. specialized software for de novo assembly of the relative short sequences, ranging from around bp with the flx system but now approaching bp with the flx titanium system, is available. however, only a few software packages claim to be suitable for hybrid assembly of the relatively short reads with sanger sequences. the choice of optimal assembly parameters to significantly reduce redundancy of an est collection and, at the same time, maintain paralogous sequences separated in different contigs remains a challenging task. particularly in a highly heterozygote hexaploid plant such as sweetpotato, discrimination between paralogs and allelic variants on whole transcriptome level remains difficult. for an est assembly, it might therefore be preferable to accept an increased level of redundancy instead of risking the merging of different members of a gene family into one contig by using too loose assembly parameters. the quality of an assembly generally augments with increasing sequence information. therefore assemblies with a limited number of ests represent a work in progress rather than a finished transcriptome assembly. as more sequence information becomes available, reassembly can lead to splitting or rearrangement of contigs, thus leading to an optimized representation of the transcriptome. here we present the de novo assembly of sequencing reads from two normalized sweetpotato libraries, together with publically available ests, which resulted in a gene index for hexaploid sweetpotato. this gene index facilitates the access to sweetpotato gene sequences and has allowed for the design of gene-based microsatellite markers.  sequencing a total of raw reads comprising, bases were obtained with a flx quarter run of a normalized cdna library of leaves from drought-stressed plants of the sweetpotato clone tanzania. another quarter run of flx titanium resulted in raw reads and, bases for a normalized cdna library of stems of the same clone. the average length per read amounted to bp for the flx run and to for the flx titanium run. cdna synthesis primer sequences present in the reads were removed and very short reads with less than bp and low-quality sequences were eliminated. the remaining raw reads were blasted against vector-cleaned sweetpotato ests from the genbank. blast hits at > 80% coverage and > 80% identity were found in % of the sequences, suggesting that the remaining two-thirds of the reads represent new est sequences. sequence assembly the raw reads were assembled together with ests from the genbank. we attempted to optimize the assembly parameters by stepwise variation of the minimum match percent parameter from to 90% and testing for good representativeness and minimal redundancy of the gene index. the contig number obtained changed linearly and slightly when the mmp parameter was increased from to 80%. from mmp on, the increment of singleton number became steeper and the number of contigs augmented stronger from mmp on. redundancy in the gene index was assessed by self-megablast and blastx with a a. thaliana protein database. at the nucleic acid level, we performed a self-megablast test to assess similarity between contig and singleton sequences produced at different mmps. sequences sharing 80% identity over 80% of their length with another sequence were considered as redundant. as expected, self-megablast hits augmented with increasing mmps, indicating that sequences representing allelic variants increasingly remained unassembled or were resolved into different contigs. the increase in the number of self-megablast hits went in parallel with the number of sequences and became steeper at mmps above 80%. additionally, we have assessed the redundancy of the assemblies produced at different mmp levels by counting the number of total and unique blastx hits of the contig and singleton sequences with a protein database. at this stage, in order to make redundancy analysis quicker and less demanding with respect to computer power, we used the a. thaliana protein database instead of the very large uniref protein set for blastx analysis. predictably, the total number of blastx hits increased with rising mmp and the increase became steeper above 80% mmp, whereas unique blastx hits increased linearly over the whole range of mmp and at a much lower path than the total hit number. the number of hits increased mainly for singletons, as more sequences remained unassembled at higher mmp values. to investigate whether assembly at low mmp results in merging of reads of paralogous sequences into one contig, we have analyzed the number of total and different members of randomly chosen gene families in the assemblies at different mmp. in a simple text search using uniprot identifiers and text annotation, we have looked for sequences annotated as zinc finger a and an domain-containing stress-associated protein, xyloglucan endotransglucosylase/hydrolase protein, α-tubulin, β-tubulin, myb, bhlh, thioredoxin, syntaxin, superoxide dismutase, sulfate transporter, na+/h+ exchanger, scarecrow, serine carboxypeptidase, actin, ferritin, pra family protein, enolase, β-amylase, bzip, and glutaredoxin according to sequence comparison with proteins of the a. thaliana proteome. although the total count of members over the gene families increased from at mmp to at mmp, the number of different gene family members varied little over the different mmp--from to 296--indicating that the risk of merging paralogs into one contig at low mmp exists but remains low. therefore, to ensure both good representativeness and low redundancy and, at the same time, to mitigate the risk that reads derived from paralogous sequences are assembled together into one contig, we chose the medium stringent mmp as assembly parameter for the sweetpotato gene index. at mmp the assembly still had relative low redundancy, while the representativeness was better than at lower mmp values, indicated by more unique blastx hits with the a. thaliana proteome than at mmp. hybrid assembly of our reads with est sequences obtained from the genbank at mmp produced contigs and reads remained as singletons. contigs ranged from to bp in size, with an average of bp. the cumulative length of the contigs was, bp. the size distribution for the contigs is shown in figure 3a. a substantial number of large contigs was obtained: contigs were > bp in length, and were > kb. sequencing coverage ranged from to reads per contig, with an average coverage of. reciprocal blastn queries with previous ipomoea est assemblies an assembly of genbank-available est and other sequences of i. batatas consisting of contig and singletons has been released previously. sequence comparison to this assembly gives an estimate of the amount of novel sequences in the present gene index. our expectation was that, in addition to new sequences, our gene index should cover all contigs and singletons of the previous assembly, as the est sequences used in the previous assembly also were co-assembled with the new reads. and indeed, all but out of sequences of the previous assembly had hits in our gene index. of the no-hit sequences, corresponded to genbank mrna sequences that were not included as input for our assembly, and other sequences corresponded to singletons of genbank-derived ests of bad quality or with highly repetitive sequences that were filtered out by our sequence cleaning process. a total of sequences of the present gene index had significant similarity to contigs and singletons of the plant est assembly. this indicates that redundancy in our gene index is about times higher than in the previous assembly. no significant similarity to the previous est assembly was found in gene index sequences that are considered to represent new sequences. similarly, only sequences of the plantgdb sweetpotato est assembly were not contained in our gene index. assuming a redundancy of -fold of our gene index, the present gene index would contain about three times more sequence information for sweetpotato genes than the previous est assemblies. for i. nil, a near relative of sweetpotato, a gene index has been established based on est and mature transcripts, resulting in tentative consensus sequences, est-singletons, and et singletons, http://compbio.dfci.harvard.edu/cgi-bin/tgi/gimain.pl?gudb=morning_glory. the present gene index had significant blastn hits with the i. nil database, while sequences remained without a hit and can be considered as specific for sweetpotato. about 19% of the sequences of the i. nil gene index had no significant hits with the present sweetpotato gene index. these reciprocal blastn tests indicate good coverage of the present gene index for sweetpotato, but also a relatively high redundancy compared to previous est assemblies. sequence annotation of the sequences, of the gene index sequences had significant blastx matches with sequences of the uniref protein database. these matches corresponded to different unique uniref accession numbers. the frequency of sequences with significant blastx hits was higher in contigs than in singletons. from contigs without significant hits to proteins of the uniref database, contained a putative poly-a tail and thus most probably represent a part of a transcribed gene. from the remaining contigs, had an open reading frame encoding at least amino acids. this suggests that most contigs without blastx hits are in fact derived from protein coding genes. the lower hit frequency of singletons in blastx searches might be caused by the smaller size of these sequences compared to contigs. another possibility for decreased hit frequency of singleton sequences would be contamination of the cdna preparation with genomic dna, resulting in reads derived from genomic dna that remained unassembled. analogous to the contig sequences, we also have searched for the presence of poly-a tails and orfs in singletons without significant blastx hits. from singletons without significant similarity to any protein of the uniref database, had a putative poly-a tail. only singletons of the no-hit singletons, including ests from the genbank database, did not encode any orf larger than amino acids, while the remaining no-hit singletons had orfs from to bp. the presence of orfs in more than 90% of the un-annotated singletons suggests that these sequences also are derived from protein coding genes. the sweetpotato gene index including the annotation of the contigs and singletons can be viewed, searched, and downloaded at http://www.cipotato.org/sweetpotato_gene_index. not all contigs and singletons, however, were derived from sweetpotato: several sequences were clearly identified as viral sequences. these viral sequences were derived from sweetpotato feathery mottle virus, sweetpotato chlorotic stunt virus, sweetpotato leaf curl virus, or viruses with similarity to potato virus a and petunia vain clearing virus that apparently have been present in the experimental plants. also, two singleton sequences derived from sanger ests downloaded from the genbank represented viral transcripts. a total of out of the blastx-annotated sequences gene ontology terms could be associated. we annotated the gene index sequences at level for the three main go vocabularies: cellular component, biological process, and molecular function. microsatellite marker identification and testing microsatellites for sweetpotato have been identified in ests previously. to ensure identification of new microsatellite motifs only, sequences similar to genbank ests were excluded from the search. a total of new ssr motifs have been identified in the gene index, including di-, tri-, and tetra-nucleotide ssr motifs with a minimum of and contiguous repeating units, respectively. for ssr testing, we have taken into account the sequence redundancy of the gene index and selected only those ssrs that were present either in a single contig or singleton or in a group of homologous sequences that were derived most probably from a single locus. this analysis resulted in ssr loci, for which high-quality primers were designed. amplification of the ssr loci was tested in eight sweetpotato clones, consisting of six hexaploid and two diploid accessions. from the selected ssr loci, yielded amplification products in the predicted size, ssrs revealed to be polymorphic in the eight-clone test set, and were monomorphic representing in total alleles. none of the ssrs showed more than alleles in any of the diploid test accessions but up to six alleles in hexaploid sweetpotato. for ssr primer pairs that successfully amplified fragments from genomic dna, polymorphism tests have not been performed.  the present gene index http://www.cipotato.org/sweetpotato_gene_index strongly augments the available sequence information on sweetpotato genes. owing to the heterozygous and hexaploid nature of this plant, the assembly contains a relatively high level of redundancy compared to previous assemblies to prevent merging of paralogous sequences into contigs. fifty-nine percent of the obtained sequences could be functionally annotated. the index has been successfully mined for microsatellite sequences and out of selected loci could be successfully amplified. 
the sweetpotato gene index is a useful source for functionally annotated sweetpotato gene sequences that contains three times more gene sequence information for sweetpotato than previous est assemblies. the availability of genomic resources for sweetpotato is in striking contrast to its importance for human nutrition. on the basis of this gene index, we have identified gene-based microsatellite sequences, of which were selected for testing and were successfully amplified in a test panel of hexaploid and diploid accessions. two quarter pyrosequencing runs used two normalized cdna collections from stems and leaves from drought-stressed sweetpotato clone tanzania and yielded reads, which were assembled together with publically available expressed sequence tags into sets of overlapping dna segments and unassembled sequences. a searchable version of the gene index, including a blastn function, is available at http://www.cipotato.org/sweetpotato_gene_index.
 sweetpotato lam.), a hexaploid outcrossing crop, is an important staple and food security crop in developing countries in africa and asia. the availability of genomic resources for sweetpotato is in striking contrast to its importance for human nutrition. previously existing sequence data were restricted to around expressed sequence tag sequences and ~ genbank sequences. we have used pyrosequencing to augment the available gene sequence information to enhance functional genomics and marker design for this plant species.  two quarter pyrosequencing runs used two normalized cdna collections from stems and leaves from drought-stressed sweetpotato clone tanzania and yielded reads, which were assembled together with publically available expressed sequence tags into sets of overlapping dna segments and unassembled sequences. blastx comparisons with the uniref database allowed annotation of contigs and singletons resulting in putatively unique genes. further, sequences had no match to protein sequences of uniref100database. on the basis of this gene index, we have identified gene-based microsatellite sequences, of which were selected for testing and were successfully amplified in a test panel of hexaploid and diploid accessions.  the sweetpotato gene index is a useful source for functionally annotated sweetpotato gene sequences that contains three times more gene sequence information for sweetpotato than previous est assemblies. a searchable version of the gene index, including a blastn function, is available at http://www.cipotato.org/sweetpotato_gene_index. 