BACKGROUND
the most widely cultivated member of the apiaceae, carrot  is a diploid  with a relatively small genome of  <dig> mb.
it has a history of cultivation as a root crop that dates back about  <dig> years to afghanistan  <cit> .
today, carrot is the largest single source of provitamin a carotenoids in the u.s. diet  <cit> , and it is among the top ten vegetable crops globally in term of area of production and market value .
world carrot production is increasing and this has, in part, been attributed to an increased awareness of health benefits associated to carrot consumption by consumers  <cit> .
phenotypic and molecular diversity of carrot is expansive  <cit>  and this diversity has been important in improving nutritional value and consumer quality; disease and pest resistance; and yield characteristics important for growers.
carrot genetic linkage maps have primarily been developed with anonymous aflps and rapds that require no prior genomic information, but these techniques yield primarily dominant markers  <cit> .
sequence-tagged codominant markers have been developed to facilitate selection for several traits, including major genes for carotenoid accumulation  <cit> , sugar type  <cit> , root-knot nematode resistance  <cit> , and for  <dig> genes in the carotenoid biosynthetic pathway targeted for candidate gene analysis  <cit> .
although the carrot plastome has been sequenced  <cit> , a 17-fold bacterial artificial chromosome library  has been developed and characterized  <cit> , and microsatellite markers are being developed , only relatively modest genomic resources have been developed for carrot  <cit> .
ests have been a valuable resource to develop snp  and ssr  markers for many plants and animals, but as of feb  <dig>   <dig>  only  <dig>  non-organellar dna sequences of daucus carota are available in genbank.
an expansion of genomic sequence resources for carrot will be useful for a broad range of carrot genomic applications including the development of co-dominant markers such as snps and ssrs for genome mapping and diversity assessment.
the development of ests has historically relied upon sanger sequence with some recent efforts also using longer-read next-generation sequencing technology such as roche  <dig>  next generation sequence  technologies are revolutionizing molecular biology by lowering the cost per sequenced nucleotide and increasing the throughput  <cit> .
short reads platforms such as illumina and solid produce higher coverage and lower cost per sequenced nucleotide, but due to their shorter reads the use of those platforms has usually been restricted to resequencing applications which rely on a reference sequence for assisting the assembly.
in absence of a reference sequence, a computational de novo assembly approach is required.
with increased read length from technologies such as illumina, and the development of new computational tools, short reads can be assembled and used for transcriptome analysis  <cit> .
recently three de novo assemblies using sequence reads from illumina technology have been developed and described for plants  <cit> .
currently there are few genomic sequence or ests available for carrot.
the objectives of this study were to sequence carrot ests using the illumina platform and to characterize the carrot transcriptome to develop a molecular resource for marker development and gene identification.
this represents an application of short read sequence technology for transcriptome assembly of a plant lacking extensive genomic molecular resources.
this est collection will provide a valuable resource for genetic, diversity, structural and functional genomic studies in carrot.
RESULTS
sequencing and assembly
to develop an overview of the carrot transcriptome and obtain an initial comparison of cultivated and wild carrot transcripts, normalized cdna libraries were constructed from four sources: two orange unrelated inbred lines of european origin, b <dig> and b <dig> with imperator and nantes root shapes, respectively,  <cit> ; a purple/yellow inbred line b <dig> derived from an intercross between purple turkish and orange danvers  carrots  <cit> ; and a pool of f <dig> rils derived from a cross between b <dig> and qal, a wild carrot from north america.
these pooled f4's are referred to as b <dig> × qal and were derived from a single b <dig> × qal f <dig> plant.
therefore at most two haplotypes are represented among these transcripts.
a total of  <dig>  <dig> high quality sanger sequences were obtained from the b <dig> library to generate a total of  <dig> , <dig> nt with an average length of  <dig> nt.
the three other libraries, b <dig>  b <dig> and b <dig> × qal were sequenced with illumina gaii platform with  <dig> cycles to yield from  <dig> m to  <dig> m usable reads of  <dig> nt or longer for each genotype.
a cap <dig>  <cit>  assembly of b <dig> sanger sequences produced  <dig> contigs plus  <dig> singletons .
a multiple step assembly strategy was used to produce a de novo assembly of the three illumina sequence sets .
for each genotype two separate assemblies were produced using either velvet  <cit>  combined with cap <dig>  or abyss  <cit>  .
the velvet+cap <dig> assembly gave  <dig> ,  <dig> , and  <dig>  contigs for b <dig>  b <dig>  and b <dig> × qal, respectively.
the number of contigs produced by abyss assembly was higher, ranging from  <dig>  in b <dig> to  <dig>  <dig> for b <dig> × qal.
to combine the four sequences sources , a combined cap <dig> assembly was created of contigs ≥ <dig> nt.
this cut off was selected based on annotation frequency vs. contig length .
the resulting sequence assembly produced  <dig>  contigs plus  <dig> sanger singletons with a total sequence length of about  <dig> mb .
the average length of the contigs and singletons was  <dig>  nt and the n <dig>  was  <dig> nt.
out of the  <dig>  contigs and singletons,  <dig>   contained b <dig> sanger sequences .
among the illumina-sequenced genotypes, b <dig> sequences were most common in contigs, represented in  <dig>  contigs .
comparing illumina-sequenced transcriptomes, a total of  <dig>  contigs  contained reads from only two genotypes, with  <dig> % of the contigs having reads from b <dig> × qal and b <dig>   <dig> % from b <dig> × qal and b <dig>  and  <dig> % from b <dig> and b <dig> .
more than 50% of the assembled contigs contained sequences from all three genotypes.
b <dig> had the highest number of genotype specific contigs , and b <dig> had the lowest with  <dig>   genotype specific genes.
to test the quality of the assembly, we compared  <dig> full-length carrot mrna sequences available from ncbi as references .
corresponding de novo contigs were located using a blastn search, and a single best match for each de novo contig was found for each of the  <dig> reference genes.
raw illumina and sanger reads from each genotype were mapped onto each reference sequence and its corresponding de novo contig.
all reference sequences were well covered by raw reads except at the ends, with 3' and 5' regions having relatively low coverage.
five of these  <dig> sequences were partially covered  by b <dig> sanger reads .
the average coverage among three illumina sequenced-genotypes ranged from  <dig>  to  <dig>  reads.
fifteen of the  <dig> sanger-derived reference cdnas were 100% covered by illumina reads.
of those, four were somewhat longer  than reference sequences, perhaps due to differences in trimming of reads and coverage.
the remaining five were partially covered by raw illumina reads.
est annotation
consensus sequences were compared to the ncbi non-redundant nucleotide database using blastx with a cutoff e-value of e- <dig> and a length of coverage ≥ <dig> aa.
in total,  <dig>   of the contigs had significant hits with known genes.
furthermore, we searched our est collection using tblastn against all available daucus protein sequences greater than  <dig> aa in genbank to find contigs covering 80% of protein length.
out of  <dig> of the proteins,  <dig>  had a match with one or more of our contigs.
blast2go was used to assign gene ontology  annotation.
in total,  <dig>  ests  were annotated and classified into the three main go categories: molecular functions, biological processes, and cellular components.
as a result,  <dig>  contigs were grouped under molecular functions,  <dig>  under biological processes and  <dig>  under cellular components.
among molecular functions, binding represented the most abundant category  followed by catalytic activity  and transport activity  .
out of  <dig>  contigs annotated as biological processes,  <dig> % were annotated in the cellular process category,  <dig> % in the metabolic process category,  <dig> % in the response to stimulus category and  <dig> % in biological regulation .
contigs associated with cell  and organelle  categories represented the most dominant group of cellular component .
as purple specialty carrots are a rich source of anthocyanins  <cit> , and our est collection included expressed genes from a purple carrot , we examined the expression of candidate genes in the anthocyanin pathway.
twelve gene families, represented by  <dig> published sequences, were compared to our assembly using blastn.
this search revealed  <dig> contigs with significant matches to  <dig> gene families and to all the carrot anthocyanin reference genes from genbank , .
in addition, five contigs in three gene families represent genes previously not described in carrot.
two phenylalanine ammoniam-lyase genes  described in petroselinum crispum aligned with contig <dig> and contig <dig>  with identities of  <dig> and 91%, respectively .
a cinnamic acid 4-hydroxylase  gene from ammi majus aligned with contig <dig>  with an identity of 89%.
in addition, two 4-coumarate coenzyme a ligase genes  from p. crispum aligned with contig <dig> and contig <dig> .
these results emphasize the depth of our est database.
* based on information from hummer and schreirer and boss et al.
<cit>
transposable elements  play an important role in shaping plant phenotypes, including an invertase mutant in carrot that conditions the type of sugar in storage roots  <cit> .
in order to identify ests potentially originating from tes, we selected  <dig> contigs annotated by blast2go as te-related.
they were queried against repbase ver.
<dig>   using censor  <cit> .
the resulting file  was manually searched for contigs with over  <dig> nt similarity to a particular superfamily and these contigs were subsequently assigned to that superfamily based on the classification system proposed by  <cit> .
this resulted in the classification of  <dig> te-related contigs.
transcripts related to class i elements were four times more abundant than those of class ii .
ests related to ltr retrotransposons, gypsy-derived transcripts were the most abundant.
transcripts originating from non-ltr retroelements and dna transposons of the tir order were also identified, while no helitron-related transcripts were observed.
ests attributed to hat, pif/harbinger, and cacta superfamilies were three times more abundant than those from tc1/mariner and mutator superfamilies.
we also searched est sequences for regions derived from known carrot class ii transposable elements, namely dcmaster-like, including krak mites  <cit> , a family of stowaway-like mites - dcsto , non-autonomous hat elements - dc-hat <dig> , and a family of cacta elements - tdc  <cit>  .
only for the latter group of tes could transposase-specific transcripts be detected.
three ests represented tdca <dig> transposase, while five others were likely chimeric transcripts containing portions of the transposase fused with other coding sequences .
only one of the tdc <dig> transposase-specific ests  was identified as cacta-derived in the general screen described above.
in contrast, dcmaster transposase-specific transcripts were not detected, even though in the general screen  <dig> ests were attributed to the pif/harbinger superfamily.
all hits found for dcmaster/krak, dcsto, and dc-hat <dig> elements originated from non-coding regions of non-autonomous elements.
dcsto elements were the most abundantly represented in the ests, including three transcripts containing complete copies of the mites.
in a number of ests, regions derived from the non-autonomous tes were present near the 5' or 3' end of the functional protein-coding transcript , suggesting that they were inserted in the 5' or 3'utrs.
polymorphism detection
in total, 92% of the contigs contained sequences from at least two genotypes, which make the assembly a suitable resource for detecting candidate polymorphic markers such as snps, ssrs, and indels .
we identified  <dig> putative ssrs in  <dig>  contigs  with  <dig>  contigs containing two or more ssrs.
a total of  <dig> ssrs were categorized as compound repeats.
repeat numbers ranged from  <dig> to  <dig> and length ranged from  <dig> to  <dig> nt.
trimers were the most abundant repeat motif accounting for  <dig>   of the ssrs.
hexamers were the least common motifs with  <dig>  of the ssrs .
based on sequence alignments, we identified in silico polymorphic ssrs among the four genotypes.
we detected  <dig> candidate polymorphic markers with ssr allele difference ranging from  <dig> nt to  <dig> nt.
trimers were confirmed to be the most abundant motif with  <dig>  of the polymorphic ssrs .
pentamers were the least common motif with  <dig> polymorphic ssr.
mosaik software was used to detect snps.
due to the lower coverage of b <dig> sanger sequences, detection of snps was performed using only into b <dig> × qal, b <dig> and b <dig> datasets.
at the level of stringency described in materials and methods, computational analysis allowed the detection of  <dig>  snps in  <dig>  contigs  with  <dig>  contigs containing two or more snps and an average number of  <dig>  snps per contig and  <dig>  snps per kb.
of the  <dig>  snps,  <dig>   were transitions  and  <dig>   were transversions .
allele variation within each genotype was categorized by "m" for intra-sample monomorphic, inter-sample polymorphic snps, or "p" for snps that were polymorphic in both intra- and inter-sample comparisons.
intra-genotype polymorphism was highest for cultivated × wild carrot  rils, and inter-genotype snp polymorphism was greatest in comparison of b <dig> × qal to the  <dig> cultivated carrot inbreds evaluated .
expressing snp categories relative to the total number of contigs,  <dig> %  of b493xqal contigs contain intra and inter polymorphic snps, b <dig> contains  <dig> % , and b <dig>  <dig> %  .
this confirms the lower level of heterozygosity in inbred lines compared with a pool of the cultivated x wild carrot rils.
considering the distribution of intra and inter-sample polymorphisms, the most abundant class of snps  were those contrasting b <dig> × qal and the two cultivated genotypes  .
those represent an important snp resource to further evaluate polymorphism between wild and cultivated germplasm.
marker validation
marker validation was carried out on genomic dna of the b <dig> × qal wild carrot derivatives and three cultivated genotypes, b <dig>  b <dig> and b <dig>  primers were designed and tested for  <dig> ssrs  identified computationally as polymorphic ssrs.
of these,  <dig> primers  produced an amplification product , with  <dig> of these producing a single product, and the remaining  <dig> ssrs have multiple products based on agarose gel resolution.
out of those  <dig> single products,  <dig> were of the expected size, and  <dig> were larger than the expected size.
to verify the expected polymorphisms,  <dig> ssr products of expected size generated from the genotypes used in this study were resolved in a capillary system using fluorescent detection, and  <dig> of them were polymorphic while  <dig> were monomorphic.
*out of  <dig> tested.
to validate snps,  <dig> primer pairs were designed and tested, of which  <dig> snps  produced an amplification product .
of these,  <dig> primer pairs produced a single product, and  <dig> gave multiple products.
out of those  <dig> single products,  <dig> were of the expected size, and  <dig> were larger than expected size.
snps were verified by sequencing all  <dig> single products with expected size, and  <dig> of those single products of larger size, but shorter than  <dig> nt.
among these  <dig> putative snp sequences,  <dig> had the expected snps.
thus, 60% of the snps tested and 82% of those that amplified a single product shorter than  <dig> nt were polymorphic and useful for mapping.
*considering  <dig> single expected product and  <dig> single larger product whit amplicon size < <dig> nt
intron predictions
primer pairs that amplify a larger than expected products are presumed to be the result of an intron between the two primer sites.
to evaluate how we could reduce the number of markers in this category which complicate snp primer design, we predicted introns for the  <dig>  snps using the solanaceae genomics network intron finder, which uses an arabidopsis database .
the number of snps with no predicted nearby intron was  <dig>  , in  <dig>  contigs.
we compared our snp pcr validation rate with this intron prediction.
of the  <dig> designed and tested primers, 46%  gave a single expected product  .
computational intron prediction found  <dig> sep primer pairs, which would be candidate for primer design .
of this  <dig>  were validated by pcr.
that prediction would also have rejected  <dig>  useful sep category.
nevertheless, in our case, the use of this intron-prediction tool would have increased success rate of single expected products from 46% to 76%.
using this approach, primer design and contig detection can be improved to optimize snp discovery.
snps polymorphism within mapping populations
the  <dig> polymorphic snp markers were screened in two mapping populations; b <dig> × qal  <cit> , which had alleles identified from this est project, and an unrelated mapping population  <dig> .
ten genotypes from each mapping population were screened by pcr and all amplicons sequenced, thus allowing identification of polymorphic markers.
in total,  <dig> polymorphic markers  were identified in the b <dig> × qal population and  <dig>  were polymorphic in the  <dig> mapping population , with  <dig> polymorphic markers common to both populations.
thus, a total of  <dig> polymorphic markers were identified.
overall, 41%  of the snp markers that were polymorphic among the genotypes used for developing the est datasets, and 28%  of the primer pairs that produced amplicons were polymorphic in the two mapping populations.
these markers have potential for mapping and as anchor points for carrot map merging.
discussion
next generation sequencing technologies are revolutionizing molecular biology by lowering the cost per sequenced nucleotide, increasing the throughput and eliminating laborious bacterial cloning.
despite of the shorter sequence reads, compared to sanger sequencing, the lower cost and high coverage of ngs are the two main factors that drive researchers to use these new technologies.
due to the longer reads produced by the roche gs flx technology, this ngs platform is most commonly used for de novo transcriptome sequencing.
this platform has been used for transcriptome sequencing of pine  <cit> , oats  <cit> , aegilops  <cit>  and buckwheat  <cit> .
in contrast, short read-length platforms such as illumina and solid, which produce higher coverage and lower cost per sequenced nucleotide, have been relegated to resequencing applications which usually depend, for their assembly, on a reference sequence.
with the improvement of read length for technologies such as illumina, and the development of new computational tools, we have demonstrated that short reads can be assembled and used for transcriptome analysis.
indeed, other recently de novo transcriptome assemblies using illumina sequences have been successfully developed and described in ipomoea  <cit> , whitefly  <cit> , eucalyptus  <cit>  chickpea  <cit>  and orchids  <cit> .
consistent with previous work, our results demonstrate that short reads can be assembled and used for transcriptome analysis, gene identification and marker development in carrot.
we assembled  <dig>  contigs and singletons from  <dig> m illumina reads and  <dig>  sanger sequences from four different genotypes.
quality of the de novo assembly was confirmed by sequence comparison, annotation and marker validation.
comparison of assembled contigs with full-length cloned carrot gene sequences confirmed the high quality of the assembly.
seventy-five percent of the contigs aligned nearly completely with mrna reference sequences.
these results were similar to those previously obtained by mizrachi and colleagues  <cit>  in eucalyptus.
distributions of genotype-specific contigs in the different est datasets of carrot were similar, with b <dig> × qal and b <dig> showing the highest number of reads in common.
in addition, only  <dig> % of all the est contigs were unique to the wild x cultivated  dataset.
altogether, these results suggest that the wild carrot transcriptome is not significantly different from the cultivated carrot transcriptome, which is consistent with cross-ability among wild and cultivated carrot in d. carota.
about 67% and 55% of the contigs exhibited homology using blastx and blast2go, respectively, indicating that contiguity of the sequence is consistent for most of the assembled transcriptome.
blast2go annotation indicated that a wide range of transcriptome diversity was included in the ests we evaluated.
contigs without significant matches to the existing databases could reflect either novel, carrot-specific genes or could reflect a poor representation of apiales sequences in genbank.
manual annotation confirmed expression of  <dig> known carrot anthocyanin genes and allowed identification of five new ones.
in addition to the three known carrot phenylalanine ammonia-lyase  genes, identification of two new pal genes suggests that multiple -and diverse- members comprise this gene family in carrot.
the five previously-unreported anthocyanin biosynthesis genes discovered in this study confirms the usefulness of this new molecular resource for discovering genes of carrot.
transcripts related to transposable elements constituted  <dig> % of the assembled contigs.
as te-related transcripts were initially identified on the basis of the blast2go annotation, that estimate should be viewed as conservative.
for example, no helitron-related contigs were found in the carrot transcriptome, which might be due to the fact that few transcripts originating from helitrons have been annotated in the genbank database.
our observation of a range of functional te transcripts suggests that members of many te families could potentially be active in carrot.
earlier reports indicated that tdc elements were activated in the course of long term in vitro cultures  <cit>  and this along with our observations that dcmaster and related mites were highly polymorphic, likely suggests their very recent activity  <cit> .
several contigs contained mite-derived regions, usually located close to the 5' or 3' ends.
this was observed for both tourist-like , stowaway-like , and hat-like  elements, indicating that mites in the carrot genome were associated with non-coding regions of genes, similar to their reported occurrence in grasses  <cit> .
in contrast, cacta elements did not show this pattern of insertion preference.
verification of assembled contigs through pcr amplification represents a good test of quality of the assembly.
the goals of this study were to use a multi-genotype based assembly to develop co-dominant molecular markers, such as simple sequence repeats  and single nucleotide polymorphisms .
ssr trimers were the most abundant repeat motif, confirming previous observations by cavagnaro and colleagues .
use of multiple genetic backgrounds in our est analysis allowed us to identify  <dig> computational polymorphic ssrs and  <dig>  snps at a depth of coverage of at least  <dig>  most of the polymorphic snps found in carrot inbred lines were polymorphic when compared against other lines but were monomorphic within lines.
this observation indicates that transcriptome comparison is an efficient strategy to identify snp markers for molecular genetic mapping and diversity analysis.
within-sample snp polymorphism in the cultivated × wild carrot  derivatives was highest .
as qal is expected to be heterozygous this sample was designed as a pool of rils to represent alleles segregating in the b <dig> x qal mapping population.
snp polymorphism  between b <dig> × qal and the two cultivated genotypes and among cultivated genotypes used in this study was also high.
although the gene content in wild and cultivated carrot was highly similar, there is a high degree of allelic variation among them.
primers were designed and tested for  <dig> computational polymorphic ssrs and  <dig> snp loci.
of these, we were able to amplify  <dig> ssrs  and  <dig> snps , with  <dig> and  <dig> markers, respectively, amplifying a product larger than expected, suggesting the presence of intron within the amplicon.
validation rate  showed that our results were similar or higher than what was previously obtained in cajanus  <cit> , iris  <cit> , epimedium  <cit> , pinus  <cit> , chickpea  <cit> , cryptomeria  <cit> , apple  <cit> , bean  <cit>  and oat  <cit>  where sanger,  <dig>  and illumina platforms were used for sequencing.
to evaluate how intron prediction could affect snp validation rate we predicted introns using the sol genomics network intron finder arabidopsis database.
based on our snp validation data, intron prediction would increase the yield of single expected size pcr products from 46% to 76%.
in contrast, due to the genetic distance between carrot and arabidopsis, carrot specific regions would be excluded and decrease the total number of useful snps by about 20%.
our data suggests that for species unrelated to arabidopsis it would be better to use both introns predicted and empirical data for assay design to maximize validation rate and evaluate genetic diversity.
in our evaluation of two mapping populations, the b <dig> × qal population had alleles identified directly from the ests, whereas the second mapping population,  <dig> was unrelated to our est sequence data.
interestingly, about a 25% of the  <dig> snps evaluated were polymorphic in both mapping populations.
about 13% of the snps were polymorphic in both mapping populations, the remainder being polymorphic in one population but not the other.
this small-scale assay provides important information useful in predicting the number of markers to screen in designing high throughput molecular assays.
CONCLUSIONS
in this study we confirmed the potential of using a short read sequencing platform for de novo assembly producing the first large-scale transcriptome sequence set of carrot a species lacking genomic resources.
est characterization provided evidence of the usefulness of this resource for gene detection and mapping of carrot.
in addition we demonstrated that transcriptome comparisons provide an efficient strategy for marker development allowing detection and validation of computational polymorphic ssrs and a large set of snps.

 the most widely cultivated member of the apiaceae, carrot is a diploid with a relatively small genome of mb. it has a history of cultivation as a root crop that dates back about years to afghanistan. today, carrot is the largest single source of provitamin a carotenoids in the u.s. diet, and it is among the top ten vegetable crops globally in term of area of production and market value. world carrot production is increasing and this has, in part, been attributed to an increased awareness of health benefits associated to carrot consumption by consumers. phenotypic and molecular diversity of carrot is expansive and this diversity has been important in improving nutritional value and consumer quality; disease and pest resistance; and yield characteristics important for growers. carrot genetic linkage maps have primarily been developed with anonymous aflps and rapds that require no prior genomic information, but these techniques yield primarily dominant markers. sequence-tagged codominant markers have been developed to facilitate selection for several traits, including major genes for carotenoid accumulation, sugar type, root-knot nematode resistance, and for genes in the carotenoid biosynthetic pathway targeted for candidate gene analysis. although the carrot plastome has been sequenced, a 17-fold bacterial artificial chromosome library has been developed and characterized, and microsatellite markers are being developed, only relatively modest genomic resources have been developed for carrot. ests have been a valuable resource to develop snp and ssr markers for many plants and animals, but as of feb only non-organellar dna sequences of daucus carota are available in genbank. an expansion of genomic sequence resources for carrot will be useful for a broad range of carrot genomic applications including the development of co-dominant markers such as snps and ssrs for genome mapping and diversity assessment. the development of ests has historically relied upon sanger sequence with some recent efforts also using longer-read next-generation sequencing technology such as roche next generation sequence technologies are revolutionizing molecular biology by lowering the cost per sequenced nucleotide and increasing the throughput. short reads platforms such as illumina and solid produce higher coverage and lower cost per sequenced nucleotide, but due to their shorter reads the use of those platforms has usually been restricted to resequencing applications which rely on a reference sequence for assisting the assembly. in absence of a reference sequence, a computational de novo assembly approach is required. with increased read length from technologies such as illumina, and the development of new computational tools, short reads can be assembled and used for transcriptome analysis. recently three de novo assemblies using sequence reads from illumina technology have been developed and described for plants. currently there are few genomic sequence or ests available for carrot. the objectives of this study were to sequence carrot ests using the illumina platform and to characterize the carrot transcriptome to develop a molecular resource for marker development and gene identification. this represents an application of short read sequence technology for transcriptome assembly of a plant lacking extensive genomic molecular resources. this est collection will provide a valuable resource for genetic, diversity, structural and functional genomic studies in carrot.  sequencing and assembly to develop an overview of the carrot transcriptome and obtain an initial comparison of cultivated and wild carrot transcripts, normalized cdna libraries were constructed from four sources: two orange unrelated inbred lines of european origin, b and b with imperator and nantes root shapes, respectively,; a purple/yellow inbred line b derived from an intercross between purple turkish and orange danvers carrots; and a pool of f rils derived from a cross between b and qal, a wild carrot from north america. these pooled f4's are referred to as b × qal and were derived from a single b × qal f plant. therefore at most two haplotypes are represented among these transcripts. a total of high quality sanger sequences were obtained from the b library to generate a total of, nt with an average length of nt. the three other libraries, b b and b × qal were sequenced with illumina gaii platform with cycles to yield from m to m usable reads of nt or longer for each genotype. a cap assembly of b sanger sequences produced contigs plus singletons. a multiple step assembly strategy was used to produce a de novo assembly of the three illumina sequence sets. for each genotype two separate assemblies were produced using either velvet combined with cap or abyss. the velvet+cap assembly gave,, and contigs for b b and b × qal, respectively. the number of contigs produced by abyss assembly was higher, ranging from in b to for b × qal. to combine the four sequences sources, a combined cap assembly was created of contigs ≥ nt. this cut off was selected based on annotation frequency vs. contig length. the resulting sequence assembly produced contigs plus sanger singletons with a total sequence length of about mb. the average length of the contigs and singletons was nt and the n was nt. out of the contigs and singletons, contained b sanger sequences. among the illumina-sequenced genotypes, b sequences were most common in contigs, represented in contigs. comparing illumina-sequenced transcriptomes, a total of contigs contained reads from only two genotypes, with % of the contigs having reads from b × qal and b % from b × qal and b and % from b and b. more than 50% of the assembled contigs contained sequences from all three genotypes. b had the highest number of genotype specific contigs, and b had the lowest with genotype specific genes. to test the quality of the assembly, we compared full-length carrot mrna sequences available from ncbi as references. corresponding de novo contigs were located using a blastn search, and a single best match for each de novo contig was found for each of the reference genes. raw illumina and sanger reads from each genotype were mapped onto each reference sequence and its corresponding de novo contig. all reference sequences were well covered by raw reads except at the ends, with 3' and 5' regions having relatively low coverage. five of these sequences were partially covered by b sanger reads. the average coverage among three illumina sequenced-genotypes ranged from to reads. fifteen of the sanger-derived reference cdnas were 100% covered by illumina reads. of those, four were somewhat longer than reference sequences, perhaps due to differences in trimming of reads and coverage. the remaining five were partially covered by raw illumina reads. est annotation consensus sequences were compared to the ncbi non-redundant nucleotide database using blastx with a cutoff e-value of e- and a length of coverage ≥ aa. in total, of the contigs had significant hits with known genes. furthermore, we searched our est collection using tblastn against all available daucus protein sequences greater than aa in genbank to find contigs covering 80% of protein length. out of of the proteins, had a match with one or more of our contigs. blast2go was used to assign gene ontology annotation. in total, ests were annotated and classified into the three main go categories: molecular functions, biological processes, and cellular components. as a result, contigs were grouped under molecular functions, under biological processes and under cellular components. among molecular functions, binding represented the most abundant category followed by catalytic activity and transport activity. out of contigs annotated as biological processes, % were annotated in the cellular process category, % in the metabolic process category, % in the response to stimulus category and % in biological regulation. contigs associated with cell and organelle categories represented the most dominant group of cellular component. as purple specialty carrots are a rich source of anthocyanins, and our est collection included expressed genes from a purple carrot, we examined the expression of candidate genes in the anthocyanin pathway. twelve gene families, represented by published sequences, were compared to our assembly using blastn. this search revealed contigs with significant matches to gene families and to all the carrot anthocyanin reference genes from genbank,. in addition, five contigs in three gene families represent genes previously not described in carrot. two phenylalanine ammoniam-lyase genes described in petroselinum crispum aligned with contig and contig with identities of and 91%, respectively. a cinnamic acid 4-hydroxylase gene from ammi majus aligned with contig with an identity of 89%. in addition, two 4-coumarate coenzyme a ligase genes from p. crispum aligned with contig and contig. these results emphasize the depth of our est database. * based on information from hummer and schreirer and boss et al. transposable elements play an important role in shaping plant phenotypes, including an invertase mutant in carrot that conditions the type of sugar in storage roots. in order to identify ests potentially originating from tes, we selected contigs annotated by blast2go as te-related. they were queried against repbase ver. using censor. the resulting file was manually searched for contigs with over nt similarity to a particular superfamily and these contigs were subsequently assigned to that superfamily based on the classification system proposed by. this resulted in the classification of te-related contigs. transcripts related to class i elements were four times more abundant than those of class ii. ests related to ltr retrotransposons, gypsy-derived transcripts were the most abundant. transcripts originating from non-ltr retroelements and dna transposons of the tir order were also identified, while no helitron-related transcripts were observed. ests attributed to hat, pif/harbinger, and cacta superfamilies were three times more abundant than those from tc1/mariner and mutator superfamilies. we also searched est sequences for regions derived from known carrot class ii transposable elements, namely dcmaster-like, including krak mites, a family of stowaway-like mites - dcsto, non-autonomous hat elements - dc-hat, and a family of cacta elements - tdc. only for the latter group of tes could transposase-specific transcripts be detected. three ests represented tdca transposase, while five others were likely chimeric transcripts containing portions of the transposase fused with other coding sequences. only one of the tdc transposase-specific ests was identified as cacta-derived in the general screen described above. in contrast, dcmaster transposase-specific transcripts were not detected, even though in the general screen ests were attributed to the pif/harbinger superfamily. all hits found for dcmaster/krak, dcsto, and dc-hat elements originated from non-coding regions of non-autonomous elements. dcsto elements were the most abundantly represented in the ests, including three transcripts containing complete copies of the mites. in a number of ests, regions derived from the non-autonomous tes were present near the 5' or 3' end of the functional protein-coding transcript, suggesting that they were inserted in the 5' or 3'utrs. polymorphism detection in total, 92% of the contigs contained sequences from at least two genotypes, which make the assembly a suitable resource for detecting candidate polymorphic markers such as snps, ssrs, and indels. we identified putative ssrs in contigs with contigs containing two or more ssrs. a total of ssrs were categorized as compound repeats. repeat numbers ranged from to and length ranged from to nt. trimers were the most abundant repeat motif accounting for of the ssrs. hexamers were the least common motifs with of the ssrs. based on sequence alignments, we identified in silico polymorphic ssrs among the four genotypes. we detected candidate polymorphic markers with ssr allele difference ranging from nt to nt. trimers were confirmed to be the most abundant motif with of the polymorphic ssrs. pentamers were the least common motif with polymorphic ssr. mosaik software was used to detect snps. due to the lower coverage of b sanger sequences, detection of snps was performed using only into b × qal, b and b datasets. at the level of stringency described in materials and methods, computational analysis allowed the detection of snps in contigs with contigs containing two or more snps and an average number of snps per contig and snps per kb. of the snps, were transitions and were transversions. allele variation within each genotype was categorized by "m" for intra-sample monomorphic, inter-sample polymorphic snps, or "p" for snps that were polymorphic in both intra- and inter-sample comparisons. intra-genotype polymorphism was highest for cultivated × wild carrot rils, and inter-genotype snp polymorphism was greatest in comparison of b × qal to the cultivated carrot inbreds evaluated. expressing snp categories relative to the total number of contigs, % of b493xqal contigs contain intra and inter polymorphic snps, b contains %, and b %. this confirms the lower level of heterozygosity in inbred lines compared with a pool of the cultivated x wild carrot rils. considering the distribution of intra and inter-sample polymorphisms, the most abundant class of snps were those contrasting b × qal and the two cultivated genotypes. those represent an important snp resource to further evaluate polymorphism between wild and cultivated germplasm. marker validation marker validation was carried out on genomic dna of the b × qal wild carrot derivatives and three cultivated genotypes, b b and b primers were designed and tested for ssrs identified computationally as polymorphic ssrs. of these, primers produced an amplification product, with of these producing a single product, and the remaining ssrs have multiple products based on agarose gel resolution. out of those single products, were of the expected size, and were larger than the expected size. to verify the expected polymorphisms, ssr products of expected size generated from the genotypes used in this study were resolved in a capillary system using fluorescent detection, and of them were polymorphic while were monomorphic. *out of tested. to validate snps, primer pairs were designed and tested, of which snps produced an amplification product. of these, primer pairs produced a single product, and gave multiple products. out of those single products, were of the expected size, and were larger than expected size. snps were verified by sequencing all single products with expected size, and of those single products of larger size, but shorter than nt. among these putative snp sequences, had the expected snps. thus, 60% of the snps tested and 82% of those that amplified a single product shorter than nt were polymorphic and useful for mapping. *considering single expected product and single larger product whit amplicon size < nt intron predictions primer pairs that amplify a larger than expected products are presumed to be the result of an intron between the two primer sites. to evaluate how we could reduce the number of markers in this category which complicate snp primer design, we predicted introns for the snps using the solanaceae genomics network intron finder, which uses an arabidopsis database. the number of snps with no predicted nearby intron was, in contigs. we compared our snp pcr validation rate with this intron prediction. of the designed and tested primers, 46% gave a single expected product. computational intron prediction found sep primer pairs, which would be candidate for primer design. of this were validated by pcr. that prediction would also have rejected useful sep category. nevertheless, in our case, the use of this intron-prediction tool would have increased success rate of single expected products from 46% to 76%. using this approach, primer design and contig detection can be improved to optimize snp discovery. snps polymorphism within mapping populations the polymorphic snp markers were screened in two mapping populations; b × qal, which had alleles identified from this est project, and an unrelated mapping population. ten genotypes from each mapping population were screened by pcr and all amplicons sequenced, thus allowing identification of polymorphic markers. in total, polymorphic markers were identified in the b × qal population and were polymorphic in the mapping population, with polymorphic markers common to both populations. thus, a total of polymorphic markers were identified. overall, 41% of the snp markers that were polymorphic among the genotypes used for developing the est datasets, and 28% of the primer pairs that produced amplicons were polymorphic in the two mapping populations. these markers have potential for mapping and as anchor points for carrot map merging. discussion next generation sequencing technologies are revolutionizing molecular biology by lowering the cost per sequenced nucleotide, increasing the throughput and eliminating laborious bacterial cloning. despite of the shorter sequence reads, compared to sanger sequencing, the lower cost and high coverage of ngs are the two main factors that drive researchers to use these new technologies. due to the longer reads produced by the roche gs flx technology, this ngs platform is most commonly used for de novo transcriptome sequencing. this platform has been used for transcriptome sequencing of pine, oats, aegilops and buckwheat. in contrast, short read-length platforms such as illumina and solid, which produce higher coverage and lower cost per sequenced nucleotide, have been relegated to resequencing applications which usually depend, for their assembly, on a reference sequence. with the improvement of read length for technologies such as illumina, and the development of new computational tools, we have demonstrated that short reads can be assembled and used for transcriptome analysis. indeed, other recently de novo transcriptome assemblies using illumina sequences have been successfully developed and described in ipomoea, whitefly, eucalyptus chickpea and orchids. consistent with previous work, our results demonstrate that short reads can be assembled and used for transcriptome analysis, gene identification and marker development in carrot. we assembled contigs and singletons from m illumina reads and sanger sequences from four different genotypes. quality of the de novo assembly was confirmed by sequence comparison, annotation and marker validation. comparison of assembled contigs with full-length cloned carrot gene sequences confirmed the high quality of the assembly. seventy-five percent of the contigs aligned nearly completely with mrna reference sequences. these results were similar to those previously obtained by mizrachi and colleagues in eucalyptus. distributions of genotype-specific contigs in the different est datasets of carrot were similar, with b × qal and b showing the highest number of reads in common. in addition, only % of all the est contigs were unique to the wild x cultivated dataset. altogether, these results suggest that the wild carrot transcriptome is not significantly different from the cultivated carrot transcriptome, which is consistent with cross-ability among wild and cultivated carrot in d. carota. about 67% and 55% of the contigs exhibited homology using blastx and blast2go, respectively, indicating that contiguity of the sequence is consistent for most of the assembled transcriptome. blast2go annotation indicated that a wide range of transcriptome diversity was included in the ests we evaluated. contigs without significant matches to the existing databases could reflect either novel, carrot-specific genes or could reflect a poor representation of apiales sequences in genbank. manual annotation confirmed expression of known carrot anthocyanin genes and allowed identification of five new ones. in addition to the three known carrot phenylalanine ammonia-lyase genes, identification of two new pal genes suggests that multiple -and diverse- members comprise this gene family in carrot. the five previously-unreported anthocyanin biosynthesis genes discovered in this study confirms the usefulness of this new molecular resource for discovering genes of carrot. transcripts related to transposable elements constituted % of the assembled contigs. as te-related transcripts were initially identified on the basis of the blast2go annotation, that estimate should be viewed as conservative. for example, no helitron-related contigs were found in the carrot transcriptome, which might be due to the fact that few transcripts originating from helitrons have been annotated in the genbank database. our observation of a range of functional te transcripts suggests that members of many te families could potentially be active in carrot. earlier reports indicated that tdc elements were activated in the course of long term in vitro cultures and this along with our observations that dcmaster and related mites were highly polymorphic, likely suggests their very recent activity. several contigs contained mite-derived regions, usually located close to the 5' or 3' ends. this was observed for both tourist-like, stowaway-like, and hat-like elements, indicating that mites in the carrot genome were associated with non-coding regions of genes, similar to their reported occurrence in grasses. in contrast, cacta elements did not show this pattern of insertion preference. verification of assembled contigs through pcr amplification represents a good test of quality of the assembly. the goals of this study were to use a multi-genotype based assembly to develop co-dominant molecular markers, such as simple sequence repeats and single nucleotide polymorphisms. ssr trimers were the most abundant repeat motif, confirming previous observations by cavagnaro and colleagues. use of multiple genetic backgrounds in our est analysis allowed us to identify computational polymorphic ssrs and snps at a depth of coverage of at least most of the polymorphic snps found in carrot inbred lines were polymorphic when compared against other lines but were monomorphic within lines. this observation indicates that transcriptome comparison is an efficient strategy to identify snp markers for molecular genetic mapping and diversity analysis. within-sample snp polymorphism in the cultivated × wild carrot derivatives was highest. as qal is expected to be heterozygous this sample was designed as a pool of rils to represent alleles segregating in the b x qal mapping population. snp polymorphism between b × qal and the two cultivated genotypes and among cultivated genotypes used in this study was also high. although the gene content in wild and cultivated carrot was highly similar, there is a high degree of allelic variation among them. primers were designed and tested for computational polymorphic ssrs and snp loci. of these, we were able to amplify ssrs and snps, with and markers, respectively, amplifying a product larger than expected, suggesting the presence of intron within the amplicon. validation rate showed that our results were similar or higher than what was previously obtained in cajanus, iris, epimedium, pinus, chickpea, cryptomeria, apple, bean and oat where sanger, and illumina platforms were used for sequencing. to evaluate how intron prediction could affect snp validation rate we predicted introns using the sol genomics network intron finder arabidopsis database. based on our snp validation data, intron prediction would increase the yield of single expected size pcr products from 46% to 76%. in contrast, due to the genetic distance between carrot and arabidopsis, carrot specific regions would be excluded and decrease the total number of useful snps by about 20%. our data suggests that for species unrelated to arabidopsis it would be better to use both introns predicted and empirical data for assay design to maximize validation rate and evaluate genetic diversity. in our evaluation of two mapping populations, the b × qal population had alleles identified directly from the ests, whereas the second mapping population, was unrelated to our est sequence data. interestingly, about a 25% of the snps evaluated were polymorphic in both mapping populations. about 13% of the snps were polymorphic in both mapping populations, the remainder being polymorphic in one population but not the other. this small-scale assay provides important information useful in predicting the number of markers to screen in designing high throughput molecular assays.  in this study we confirmed the potential of using a short read sequencing platform for de novo assembly producing the first large-scale transcriptome sequence set of carrot a species lacking genomic resources. est characterization provided evidence of the usefulness of this resource for gene detection and mapping of carrot. in addition we demonstrated that transcriptome comparisons provide an efficient strategy for marker development allowing detection and validation of computational polymorphic ssrs and a large set of snps. 
a challenge now is to develop efficient strategies to use short-read length platforms for de novo assembly and marker development. the scope of this study was to develop a de novo assembly of carrot ests from multiple genotypes using the illumina platform, and to identify polymorphisms. a de novo assembly of transcriptome sequence from four genetic backgrounds produced contigs and singletons. among next generation sequence technologies, platforms such as illumina and solid produce short reads but with higher coverage and lower cost per sequenced nucleotide than or sanger. in addition we produced the first large-scale transcriptome of carrot, a species lacking genomic resources. over 50% of these assembled sequences were annotated allowing detection of transposable elements and new carrot anthocyanin genes. out of verified ssr and snp markers 84% and 82% were polymorphic. presence of multiple genetic backgrounds in our assembly allowed the identification of computationally polymorphic ssrs, and snps at a depth of coverage of 20× or more.
 among next generation sequence technologies, platforms such as illumina and solid produce short reads but with higher coverage and lower cost per sequenced nucleotide than or sanger. a challenge now is to develop efficient strategies to use short-read length platforms for de novo assembly and marker development. the scope of this study was to develop a de novo assembly of carrot ests from multiple genotypes using the illumina platform, and to identify polymorphisms.  a de novo assembly of transcriptome sequence from four genetic backgrounds produced contigs and singletons. over 50% of these assembled sequences were annotated allowing detection of transposable elements and new carrot anthocyanin genes. presence of multiple genetic backgrounds in our assembly allowed the identification of computationally polymorphic ssrs, and snps at a depth of coverage of 20× or more. polymorphisms were predominantly between inbred lines except for the cultivated x wild ril pool which had high intra-sample polymorphism. about 90% and 88% of tested ssr and snp primers amplified a product, of which 70% and 46%, respectively, were of the expected size. out of verified ssr and snp markers 84% and 82% were polymorphic. about 25% of snps genotyped were polymorphic in two diverse mapping populations.  this study confirmed the potential of short read platforms for de novo est assembly and identification of genetic polymorphisms in carrot. in addition we produced the first large-scale transcriptome of carrot, a species lacking genomic resources. 