BACKGROUND
grapevine is the most cultivated fruit crop in the world, covering approximately  <dig>  million hectares in  <dig> and producing more than  <dig> million tons of berries .
the modern grapevine  was domesticated 8000 years ago in the southern caucasus region
<cit>  from its wild ancestor v.vinifera sbs.
sylvestris.
the grapevine genome is highly polymorphic
<cit>  and vegetative propagation is preferred over seed germination because the extensive heterozygosity results in erratic yields and produces offspring with diverse characteristics
<cit> .
at least  <dig>  grapevine varieties have been cataloged
<cit>  but breeding is restricted to a relatively small number of cultivars, such as pinot and traminer
<cit> .
however, the global demand for high-quality wines is increasing, awakening interest in the use of local cultivars to create premium products and in the molecular analysis of their prized organoleptic traits
<cit> .
the genetic analysis of grapevine has been hindered by the long generation time , extensive heterozygosity and phenotypic plasticity.
even berries of the same cultivar may differ markedly in their properties because of environmental factors, from which arises the concept of terroir in viticulture
<cit> .
detailed characterization of the genome is therefore necessary to separate the genetic and environmental components underlying the phenotype.
grapevine was the first fruit species to be sequenced, but the reference genome is that of a near-homozygous and non-cultivated accession, pn40024
<cit> .
this resource has facilitated the detailed phylogenetic analysis of specific gene families
<cit> , the creation of snp catalogs which can be used as genetic markers for cultivar differentiation
<cit>  and the development of microarrays for transcriptomic analysis
<cit> , but recent deep sequencing experiments have shown that relying on a single reference genome may underestimate the variability among different genotypes.
the comparison of asian and african human genomes with the reference sequence has revealed 5 mb of novel sequence in each assembly containing population-specific coding regions
<cit> .
furthermore, the assembly of genomes and transcriptomes from  <dig> different arabidopsis thaliana ecotypes led to the identification of  <dig> genes that are not present in the col- <dig> reference
<cit> .
these data indicate that the comparison of polymorphisms in a reference genotype may not represent the full genetic diversity of a species, and this is particularly relevant in grapevine because v. vinifera is more genetically diverse than both homo sapiens and a. thaliana
<cit> .
these challenges could be addressed by de novo sequencing and annotating each grapevine cultivar.
de novo assembly of a complex genome is however hindered by repetitive dna sequences and low complexity regions.
in order to address these problems multiple paired-end and mate-pair libraries with different insert sizes are necessary but require consistent efforts to be produced
<cit> .
moreover, gene annotation of de novo assembled genomes is a time and labor intensive task which includes both the use of gene prediction and annotation methods and a lengthy manual curation.
an alternative to whole-genome sequencing is the direct reconstruction of the transcriptome by de novo assembly.
the potential of this approach has been demonstrated in animals lacking reference genome sequences such as the coral acropora millepora
<cit> , the whitefly bemisia tabaci
<cit> , the butterfly melitaea cinxia
<cit> , the mosquito anopheles funestus
<cit>  and the planarian schmidtea mediterranea
<cit> .
transcript sequences generally lack the repetitive sequences that complicate genome assembly.
de novo transcriptome assembly was used to characterize the varietal diversity of v. vinifera cv.
corvina, an indigenous cultivar of the verona area in north italy which has recently been subject to comprehensive transcriptomic, proteomic and metabolomic analysis
<cit> , including an rna-seq based expression profiling of berry development
<cit> .
the direct comparison of potential polymorphisms by projection onto the reference genome indicated that up to one third of the pn <dig> proteins could be affected by disruptive mutations, suggesting that a full reconstruction and re-annotation of corvina genes is required.
the de novo transcriptome assembly strategy allowed us to identify  <dig>  novel splice isoforms among  <dig> known genes, and  <dig> potentially novel protein-coding genes in the raw pn <dig> reads but not in the assembled sequence.
we also identified  <dig> apparently private corvina genes, 27% of which are modulated during berry development and withering.
RESULTS
sequencing the corvina transcriptome and the characterization of sequence variation
to characterize the v. vinifera cv.
corvina transcriptome,  <dig> samples were collected from different organs/tissues at several developmental stages .
total rna from pooled samples was used to generate a single cdna library with a mean insert size of 310 bp estimated by mapping the reads onto the pn <dig> reference genome
<cit> .
sequencing generated  <dig> , <dig> paired-end reads 100 bp in length, equivalent to  <dig> gb of total sequence data.
the sequences were quality filtered and the resulting  <dig> , <dig> paired-end reads were aligned to the 450-mb pn <dig> reference genome  with a success rate of 89%.
this analysis allowed us to determine the pervasiveness of transcription in terms of the entire gene catalog and to estimate the extent of transcriptional overlap between corvina and the pn <dig> reference genome.
we found that  <dig> % of the reference genome was covered by at least three reads  and the proportion did not change significantly by increasing the coverage threshold to  <dig> or even  <dig> reads .
in comparison, at least 63% of the mouse genome
<cit> , 93% of the human genome
<cit>  and 38% of the rice genome is transcribed
<cit> .
however, it should be noted that for human genome only  <dig> chromosomes were used, accounting for 30% of the entire genome, while for rice genome the coverage was at least  <dig> read and thus any comparison should be treated with care.
the covered bases included approximately 123 mb of non-annotated sequence, representing 46% of the total and probably reflecting both transcriptional noise and the presence of noncoding rnas, transposable elements and non-annotated genes in the reference sequence .
the covered bases also included 106 mb of introns, representing 40% of the total and suggesting extensive differences in alternative splicing between the corvina cultivar and pn <dig> .
we identified  <dig>  polymorphisms between the corvina and pn <dig> sequences, including  <dig>  insertion/deletion polymorphisms  and  <dig>  single nucleotide polymorphisms .
approximately 13% of the indels and 29% of the snps were located in regions annotated as coding sequences in the v <dig> reference annotation .
we further filtered this dataset with a minimum frequency threshold  of the alternative polymorphism calculated on the total of read pairs aligning on the region this final set contained  <dig>  putative mutations, of which  <dig>  are snps and  <dig> indels.
putative mutations were annotated to determine their potential effect on the encoded proteins .
a simple projection of the polymorphisms onto the pn <dig> annotation showed that  <dig> corvina proteins were potentially changed by substitutions and  <dig> proteins were potentially destroyed by frameshifts, premature stop codons, stop-codon mutations and mutations at splice sites .
these data are reminiscent of the situation reported in different a. thaliana ecotypes supporting earlier claims that reference annotations cannot be transferred reliably to any cultivar/accession without the reassembly and re-annotation of the genome and/or the transcriptome
<cit> .
reconstruction of the corvina gene catalog and comparison with reference annotations
the corvina transcriptome was reconstructed without the reference sequence and annotation by using a two-step strategy involving  <dig>  million filtered high-quality paired-end reads.
first we generated a preliminary assembly using velvet, incorporating  <dig>  million reads  and generating  <dig>  contigs.
these contigs were then processed with oases to produce the final set of contigs.
having been assembled from transcriptomic sequence reads, they can be referred to as “putative transcripts”.
this analysis produced  <dig>  putative transcripts with a minimum length of 200 bp , these were clustered and the longest fragment in each cluster was retained, returning  <dig>  clusters, each representing a single putative transcript .
the assembly achieved an average length similar to that of the annotated dataset  with a slightly higher n <dig> score  suggesting that most of the reconstructed putative transcripts were essentially complete.
assembly was performed with velvet and oases.
clustering was carried out using cdhit.
our putative transcripts were aligned onto the genome to assign them to distinct genomic loci.
in this way,  <dig>  putative transcripts were mapped onto the genome with ≥ 90% identity and coverage, including  <dig> classified as chimeras.
these were excluded from subsequent analysis.
another  <dig>  putative transcripts corresponding to single exons, exon fragments or partial introns were removed from the dataset.
of the remaining  <dig> putative transcripts,  <dig>  were assigned to regions encoding known genes and  <dig> were detected in non-annotated regions .
the expression levels of all putative transcripts were calculated and  <dig>  putative transcripts were discarded as potential pre-mrnas because their expression level fell below 5% of the major isoform of the corresponding gene
<cit>  .
this filter increased the percentage of potential protein-coding genes from 83%  to 88%
<cit> .
we tested higher thresholds  but this adversely affected the recovery of potential protein-coding genes from the dataset .
our final set of putative transcripts were compared with the raw read alignments, revealing a strong reduction in the signal representing intergenic and intronic regions , but an increase in the signal representing annotated exons .
we also compared putative transcript mappings with aligned public est/cdna data , and found that  <dig>  putative transcripts overlapped at least one of the  <dig>  ests, and  <dig>  were supported by at least one of the  <dig>  available grapevine cdnas .
contigs coordinates were compared with those of public ests, cdnas and v <dig> annotation of 12x assembly and with v <dig> annotation of 8x assembly.
number of contigs with at least a 50% overlap with the features of interest is reported for each comparison.
the coordinates of the mapped putative transcripts were then compared with the current v <dig> annotation .
the genomic coordinates of the final set of  <dig>  putative transcripts corresponded to  <dig>  annotated gene loci,  <dig> known repetitive regions and  <dig> putative novel gene loci .
a similar number of putative novel gene loci  was obtained by a reference alignment and prediction approach starting from the same dataset
<cit>  .
by comparing our putative transcripts to the corresponding known genes, we identified  <dig> adjacent genes that merged into  <dig> putative loci.
we compared our  <dig> putative loci with the group of  <dig> genes that were recently shown to be erroneously split in the v <dig> annotation
<cit> , finding  <dig> of the loci in common between the two studies.
based on genome alignments of reconstructed contigs, we compared their structures with known transcripts and were able to identify  <dig> de novo reconstructed transcripts with an exon-intron structure identical to known pn <dig> transcripts.
finally, the analysis allowed us to identify  <dig>  novel isoforms representing  <dig> genes annotated in the pn <dig> reference sequence,  <dig> of which generated multiple isoforms .
among the  <dig> potential novel gene loci,  <dig> showed significant sequence similarity to transposon or retrotransposon proteins,  <dig> were identified using coding potential calculator  and  <dig> generated hits when used as blast queries against the ncbi non-redundant protein database .
the remaining  <dig> loci were considered to be putative non-coding rnas .
functional annotations were applied using gene ontology  classifications, revealing  <dig> of  <dig> loci that were associated with at least one go term  .
finally, the putative transcript coordinates of novel genes were compared with v <dig>  and 8x annotations
<cit>  .
identification of corvina private genes
of the  <dig> identified putative transcripts that could not be mapped onto the pn <dig> genome ,  <dig> were discarded as contaminants because most  appeared to be fungal in origin .
we found that  <dig> of the remaining  <dig> putative transcripts matched expressed grapevine sequences represented in the vvgi database v <dig>  or other plant proteins, and these were considered as novel grapevine transcripts potentially restricted to v. vinifera cv.
corvina .
to avoid false positives reflecting gaps in the pn <dig> gene space, we compared the  <dig> potential corvina private genes with pn <dig> raw sequence reads and found matches for another  <dig> sequences,  <dig> of which were potential coding regions that might represent previously undiscovered genes in the pn <dig> genome.
the remaining  <dig> putative transcripts appeared to be corvina-specific, and formed  <dig> clusters based on similarity to sequences present in the vvgi database v <dig>  and other plant proteins.
these  <dig> clusters correspond to  <dig> putative private genes.
cpc indicated that  <dig> of the clusters had a high coding potential
<cit>  and go classifications indicated preliminary functional classifications for  <dig> of the sequences  .
dynamic gene expression during corvina berry development and withering
we assessed the biological significance of the novel genes and corvina private genes by analyzing berry samples at two developmental stages and one withering stage.
three biological replicates were collected at each stage and were processed to generate indexed rna-seq libraries, which were sequenced using an illumina hiseq  <dig>  we obtained  <dig> million 50-bp paired-end reads , comprising  <dig>  million reads representing the post fruit-set  stage,  <dig>  million reads from the pre-ripening  stage and  <dig>  million reads from the post-harvest withering  stage .
gene expression levels at all three stages were quantified using the pn <dig> genome and the corvina private genes as reference sequences.
the abundance of each transcript was expressed as fragments per kilobase of exon model per million mapped reads  as implemented in cufflinks
<cit> .
a gene was considered to be expressed if the fpkm 95% confidence interval lower boundary was greater than zero and if the fpkm value was higher than  <dig> .
we detected  <dig>  expressed genes in at least one of the three samples, including  <dig> of the  <dig> novel genes and  <dig> of the  <dig> corvina private genes.
this represented 72% of the grapevine transcriptome .
although more genes were detected in the berry samples than in the reconstructed pool,  <dig> genes expressed in the pooled samples were not represented in any of the three berry samples.
raw counts of uniquely-mapped reads were estimated and normalized
<cit> .
we identified  <dig>  loci that were modulated in at least one of the samples  .
at each time point, we detected both unique and overlapping sets of differentially-expressed genes, e.g.
<dig> genes were differentially expressed in all three stages analyzed and  <dig> were differentially expressed specifically during withering.
interestingly,  <dig> of the  <dig> corvina private genes were differentially expressed and  <dig>  were induced specifically during withering.
similarly,  <dig> of the  <dig> novel loci were differentially expressed in at least one sample.
genes were grouped according to their expression profiles by transforming the expression data into moderate fold change estimates using a variance stabilizing transformation
<cit> .
differentially-expressed genes were thus grouped into four clusters according to their expression profiles, comprising those repressed at pr and phwii or exclusively at phwii but not pfs , those transiently repressed at pr , those transiently induced at pr  and those induced at the pr and phwii stages or at the phwii stage alone  .
to gain insight into the functions of the modulated genes in each cluster, each group was enriched with go terms  associated with  <dig>  of the  <dig>  differentially-expressed genes .
looking specifically at genes induced during the phwii phase  given that withering is peculiar to wines produced from this cultivar, the statistics show an enrichment for genes involved in stress responses such as programmed cell death and in the synthesis of flavonoids, as previously described
<cit> .
discussion
sequence diversity is usually described in comparison to a reference genome
<cit> .
given the high degree of genetic diversity among plant cultivars, this approach might fail to recognize highly polymorphic regions and will not detect the presence or absence of genes residing in private  regions of the genome
<cit> .
whole-genome sequencing and re-annotation is therefore recommended for each variety, but in predominantly heterozygous species such as grapevine the sequence diversity would make contig assembly a daunting and resource-intensive task
<cit> .
when a reference genome is available, genes and transcript isoforms are built de novo by mapping rna-seq reads, but this does not solve the problem of hypervariable sequences and private genes
<cit> .
however, the de novo assembly strategy does not depend on the genome and has been applied successfully to reconstruct the transcriptomes of non-model species lacking reference genomes.
we have demonstrated the feasibility cdna sequencing by rna-seq for the analysis of varietal diversity between a local grapevine cultivar  and the pn <dig> reference genome without genomic data.
the availability of a reference genome allows the reconstruction procedure to be validated and highlights the diversity between the two genomes.
improved annotation of the reference genome
the latest grapevine genome annotation  comprises  <dig>  genes identified by a combination of ab initio prediction and cdna mapping.
by comparing this annotation to the transcripts we identified, we found our method had detected 51% of the annotated genes, the remainder probably representing tissue/condition-specific transcripts that were not present in our pooled samples.
the genes overlapping our sample and the v <dig> annotation have a higher expression level than the v1-specific genes .
these data indicate that many of the v <dig> annotations undetected using our method were missed because of the paucity of sequencing reads generated from the corresponding loci.
a large number  of potential protein-coding genes were detected in the non-annotated parts of the genome.
a recent comparison of the 8x, 12x v <dig> and 12x v <dig> annotations showed that  <dig> genes present in either the 8x or 12x v <dig> assemblies were not present in the v <dig> annotation
<cit> .
interestingly,  <dig> of our  <dig> potential protein-coding genes  were represented in the 8x or 12x v <dig> annotations.
current annotation is therefore incomplete and insufficient to describe the full gene space of a cultivar other than the reference pinot noir clone.
our method provided experimental support for  <dig> protein-coding genes missing from the final assembly because they were excluded from the 12x consensus, and for  <dig> additional genes that appear to have been missed in the v <dig> annotation.
novel genes excluded from the v <dig> annotation appear to have meaningful biological roles, including those modulated during berry ripening and/or withering e.g.
eight disease-resistance genes , two stress-inducible genes , a heat shock protein  <dig> gene  and a senescence-associated gene .
the expression of the disease-resistance genes generally declined during berry development and withering  suggesting their role is to protect the berry from pathogens and pests during early development.
in contrast, the stress-inducible genes and heat shock protein gene were induced during ripening and withering, supporting a protective role against abiotic stress during the accumulation of sugars and secondary metabolites as previously reported
<cit> .
the rna-seq data therefore provide a comprehensive insight into the biologically-relevant landscape of gene expression during berry development and ripening.
our method not only offers a way to annotate previously uncharacterized genes but also improves the annotation of known genes by helping to define their boundaries more robustly and to identify splice variants.
our data indicate that up to 11% of the genes in the v <dig> annotation are split incorrectly, similar to the error rate in other annotated plant genomes
<cit> .
a previous in silico analysis identified  <dig> instances of erroneously split genes in the v <dig> annotation
<cit> .
we also detected  <dig> of these genes and our analysis suggested that 75% of them were split incorrectly in the v <dig> annotation.
furthermore, our data resulted in the 3′ and 5′ extension of nearly 90% of the genes we detected compared to the boundaries in the v <dig> annotation, indicating that the untranslated regions were longer than previously reported, using in silico prediction methods
<cit> .
our approach may therefore provide a useful complement to ab initio gene prediction methods to establish gene boundaries and define utrs.
finally, our de novo transcriptome assembly method detected an average of  <dig>  transcripts per locus, in line with previous reports using a reference-guided assembly of grapevine transcripts .
although beyond the scope of our investigation, the de novo reconstruction indicated alternative splice variants for  <dig> loci, providing a much more exhaustive description of the grapevine transcriptome compared to in silico predictions.
the number of studies which try to describe alternative splicing events in plants are still scarce, however many recent studies point to an extensive diffusion of the phenomenon and to its important role in modulating gene expression and stress response .
our results indicate that the transcriptional landscape in vitis is more complex than previously thought and therefore warrants further investigation.
expression of corvina private genes during berry development
recent data from the deep sequencing of human individuals and arabidopsis ecotypes revealed portions of genome that are not shared among all genotypes and the reference genome
<cit> .
interestingly, the novel genomic sequences included a set of protein-coding genes  potentially contributing to the intra-species variability.
similarly, we detected  <dig> putative protein-coding genes with a high coding potential or matches to plant ests that represent potential corvina private genes.
we identified  <dig> private genes expressed in at least one berry-sampling phase,  <dig> of which were differentially expressed between samples, and these could represent a group of genes that directly contribute to the specific characteristics of the corvina berry.
some of these private genes could have been selected by ancient breeders looking for particular berry quality traits, such as the ability to withstand the lengthy drying phase  required to make passito wines  such as amarone and recioto.
for example, we identified a heat shock protein gene  and a stress-inducible gene  induced during ripening, consistent with the ability of corvina berries to undergo dehydration for up to 100 days
<cit> .
furthermore, we detected the induction of genes involved in translation and protein metabolism during withering, including three ribosomal proteins , three elongation factors , ubiquitin , a 5-methyltetrahydropteroyltriglutamate-homocysteine methyltransferase  and a dna-binding protein .
this supports cdna-aflp data indicating the induction of genes with similar functions during withering
<cit> .
thirty-three of the corvina private genes matched homologs in other grape varieties but not the reference genome.
this is expected because the dispensable part of the genome may be partly shared among different cultivars and only a few genes may be truly unique to a particular accession
<cit> .
for example, we found two flowering locus t  genes  the first corresponding to the previously-described vvft gene found in the cultivars cabernet sauvignon
<cit>  and tempranillo
<cit> .
at least six members of the ft/tfl <dig> gene family were identified in the tempranillo genome, including vvft which appears to be the ortholog of arabidopsis ft and therefore induces precocious flowering when expressed in arabidopsis, consistent with reported expression patterns associated with seasonal floral induction in latent buds and with the development of inflorescences, flowers and fruits
<cit> .
there is no evidence for the presence of classical floral regulatory pathways in grapevine, and the expression profile of vvft suggests that it only partially corresponds to the florigen role of arabidopsis ft. we also observed the expression of vvft during berry formation, suggesting an additional and uncharacterized role of this gene during early berry formation.
CONCLUSIONS
we were able to reconstruct a substantial part of the grapevine transcriptome , improve the annotations of known genes by defining their boundaries and splice variants, add  <dig> apparently novel genes representing non-annotated or unassembled regions of the reference genome, and also add  <dig> potentially corvina-specific private genes that are not present in the reference sequence.
our results are consistent with data from other plant species showing that different genotypes share a common majority of genes but also possess smaller sets of private genes that are likely to be dispensable, that contribute to intra-specific variation and that produce unique, variety-dependent characteristics
<cit> .
given the substantial divergence among registered ecotypes and cultivars
<cit> , we argue that in plant biology a de novo transcriptome assembly approach should not be limited to species lacking reference genome  because it can improve the annotation of diverse cultivars and identify cultivar-specific private genes without embarking on a labor-intensive reconstructing of the entire genome.

 grapevine is the most cultivated fruit crop in the world, covering approximately million hectares in and producing more than million tons of berries. the modern grapevine was domesticated 8000 years ago in the southern caucasus region from its wild ancestor v.vinifera sbs. sylvestris. the grapevine genome is highly polymorphic and vegetative propagation is preferred over seed germination because the extensive heterozygosity results in erratic yields and produces offspring with diverse characteristics. at least grapevine varieties have been cataloged but breeding is restricted to a relatively small number of cultivars, such as pinot and traminer. however, the global demand for high-quality wines is increasing, awakening interest in the use of local cultivars to create premium products and in the molecular analysis of their prized organoleptic traits. the genetic analysis of grapevine has been hindered by the long generation time, extensive heterozygosity and phenotypic plasticity. even berries of the same cultivar may differ markedly in their properties because of environmental factors, from which arises the concept of terroir in viticulture. detailed characterization of the genome is therefore necessary to separate the genetic and environmental components underlying the phenotype. grapevine was the first fruit species to be sequenced, but the reference genome is that of a near-homozygous and non-cultivated accession, pn40024. this resource has facilitated the detailed phylogenetic analysis of specific gene families, the creation of snp catalogs which can be used as genetic markers for cultivar differentiation and the development of microarrays for transcriptomic analysis, but recent deep sequencing experiments have shown that relying on a single reference genome may underestimate the variability among different genotypes. the comparison of asian and african human genomes with the reference sequence has revealed 5 mb of novel sequence in each assembly containing population-specific coding regions. furthermore, the assembly of genomes and transcriptomes from different arabidopsis thaliana ecotypes led to the identification of genes that are not present in the col- reference. these data indicate that the comparison of polymorphisms in a reference genotype may not represent the full genetic diversity of a species, and this is particularly relevant in grapevine because v. vinifera is more genetically diverse than both homo sapiens and a. thaliana. these challenges could be addressed by de novo sequencing and annotating each grapevine cultivar. de novo assembly of a complex genome is however hindered by repetitive dna sequences and low complexity regions. in order to address these problems multiple paired-end and mate-pair libraries with different insert sizes are necessary but require consistent efforts to be produced. moreover, gene annotation of de novo assembled genomes is a time and labor intensive task which includes both the use of gene prediction and annotation methods and a lengthy manual curation. an alternative to whole-genome sequencing is the direct reconstruction of the transcriptome by de novo assembly. the potential of this approach has been demonstrated in animals lacking reference genome sequences such as the coral acropora millepora, the whitefly bemisia tabaci, the butterfly melitaea cinxia, the mosquito anopheles funestus and the planarian schmidtea mediterranea. transcript sequences generally lack the repetitive sequences that complicate genome assembly. de novo transcriptome assembly was used to characterize the varietal diversity of v. vinifera cv. corvina, an indigenous cultivar of the verona area in north italy which has recently been subject to comprehensive transcriptomic, proteomic and metabolomic analysis, including an rna-seq based expression profiling of berry development. the direct comparison of potential polymorphisms by projection onto the reference genome indicated that up to one third of the pn proteins could be affected by disruptive mutations, suggesting that a full reconstruction and re-annotation of corvina genes is required. the de novo transcriptome assembly strategy allowed us to identify novel splice isoforms among known genes, and potentially novel protein-coding genes in the raw pn reads but not in the assembled sequence. we also identified apparently private corvina genes, 27% of which are modulated during berry development and withering.  sequencing the corvina transcriptome and the characterization of sequence variation to characterize the v. vinifera cv. corvina transcriptome, samples were collected from different organs/tissues at several developmental stages. total rna from pooled samples was used to generate a single cdna library with a mean insert size of 310 bp estimated by mapping the reads onto the pn reference genome. sequencing generated, paired-end reads 100 bp in length, equivalent to gb of total sequence data. the sequences were quality filtered and the resulting, paired-end reads were aligned to the 450-mb pn reference genome with a success rate of 89%. this analysis allowed us to determine the pervasiveness of transcription in terms of the entire gene catalog and to estimate the extent of transcriptional overlap between corvina and the pn reference genome. we found that % of the reference genome was covered by at least three reads and the proportion did not change significantly by increasing the coverage threshold to or even reads. in comparison, at least 63% of the mouse genome, 93% of the human genome and 38% of the rice genome is transcribed. however, it should be noted that for human genome only chromosomes were used, accounting for 30% of the entire genome, while for rice genome the coverage was at least read and thus any comparison should be treated with care. the covered bases included approximately 123 mb of non-annotated sequence, representing 46% of the total and probably reflecting both transcriptional noise and the presence of noncoding rnas, transposable elements and non-annotated genes in the reference sequence. the covered bases also included 106 mb of introns, representing 40% of the total and suggesting extensive differences in alternative splicing between the corvina cultivar and pn. we identified polymorphisms between the corvina and pn sequences, including insertion/deletion polymorphisms and single nucleotide polymorphisms. approximately 13% of the indels and 29% of the snps were located in regions annotated as coding sequences in the v reference annotation. we further filtered this dataset with a minimum frequency threshold of the alternative polymorphism calculated on the total of read pairs aligning on the region this final set contained putative mutations, of which are snps and indels. putative mutations were annotated to determine their potential effect on the encoded proteins. a simple projection of the polymorphisms onto the pn annotation showed that corvina proteins were potentially changed by substitutions and proteins were potentially destroyed by frameshifts, premature stop codons, stop-codon mutations and mutations at splice sites. these data are reminiscent of the situation reported in different a. thaliana ecotypes supporting earlier claims that reference annotations cannot be transferred reliably to any cultivar/accession without the reassembly and re-annotation of the genome and/or the transcriptome. reconstruction of the corvina gene catalog and comparison with reference annotations the corvina transcriptome was reconstructed without the reference sequence and annotation by using a two-step strategy involving million filtered high-quality paired-end reads. first we generated a preliminary assembly using velvet, incorporating million reads and generating contigs. these contigs were then processed with oases to produce the final set of contigs. having been assembled from transcriptomic sequence reads, they can be referred to as “putative transcripts”. this analysis produced putative transcripts with a minimum length of 200 bp, these were clustered and the longest fragment in each cluster was retained, returning clusters, each representing a single putative transcript. the assembly achieved an average length similar to that of the annotated dataset with a slightly higher n score suggesting that most of the reconstructed putative transcripts were essentially complete. assembly was performed with velvet and oases. clustering was carried out using cdhit. our putative transcripts were aligned onto the genome to assign them to distinct genomic loci. in this way, putative transcripts were mapped onto the genome with ≥ 90% identity and coverage, including classified as chimeras. these were excluded from subsequent analysis. another putative transcripts corresponding to single exons, exon fragments or partial introns were removed from the dataset. of the remaining putative transcripts, were assigned to regions encoding known genes and were detected in non-annotated regions. the expression levels of all putative transcripts were calculated and putative transcripts were discarded as potential pre-mrnas because their expression level fell below 5% of the major isoform of the corresponding gene. this filter increased the percentage of potential protein-coding genes from 83% to 88%. we tested higher thresholds but this adversely affected the recovery of potential protein-coding genes from the dataset. our final set of putative transcripts were compared with the raw read alignments, revealing a strong reduction in the signal representing intergenic and intronic regions, but an increase in the signal representing annotated exons. we also compared putative transcript mappings with aligned public est/cdna data, and found that putative transcripts overlapped at least one of the ests, and were supported by at least one of the available grapevine cdnas. contigs coordinates were compared with those of public ests, cdnas and v annotation of 12x assembly and with v annotation of 8x assembly. number of contigs with at least a 50% overlap with the features of interest is reported for each comparison. the coordinates of the mapped putative transcripts were then compared with the current v annotation. the genomic coordinates of the final set of putative transcripts corresponded to annotated gene loci, known repetitive regions and putative novel gene loci. a similar number of putative novel gene loci was obtained by a reference alignment and prediction approach starting from the same dataset. by comparing our putative transcripts to the corresponding known genes, we identified adjacent genes that merged into putative loci. we compared our putative loci with the group of genes that were recently shown to be erroneously split in the v annotation, finding of the loci in common between the two studies. based on genome alignments of reconstructed contigs, we compared their structures with known transcripts and were able to identify de novo reconstructed transcripts with an exon-intron structure identical to known pn transcripts. finally, the analysis allowed us to identify novel isoforms representing genes annotated in the pn reference sequence, of which generated multiple isoforms. among the potential novel gene loci, showed significant sequence similarity to transposon or retrotransposon proteins, were identified using coding potential calculator and generated hits when used as blast queries against the ncbi non-redundant protein database. the remaining loci were considered to be putative non-coding rnas. functional annotations were applied using gene ontology classifications, revealing of loci that were associated with at least one go term. finally, the putative transcript coordinates of novel genes were compared with v and 8x annotations. identification of corvina private genes of the identified putative transcripts that could not be mapped onto the pn genome, were discarded as contaminants because most appeared to be fungal in origin. we found that of the remaining putative transcripts matched expressed grapevine sequences represented in the vvgi database v or other plant proteins, and these were considered as novel grapevine transcripts potentially restricted to v. vinifera cv. corvina. to avoid false positives reflecting gaps in the pn gene space, we compared the potential corvina private genes with pn raw sequence reads and found matches for another sequences, of which were potential coding regions that might represent previously undiscovered genes in the pn genome. the remaining putative transcripts appeared to be corvina-specific, and formed clusters based on similarity to sequences present in the vvgi database v and other plant proteins. these clusters correspond to putative private genes. cpc indicated that of the clusters had a high coding potential and go classifications indicated preliminary functional classifications for of the sequences. dynamic gene expression during corvina berry development and withering we assessed the biological significance of the novel genes and corvina private genes by analyzing berry samples at two developmental stages and one withering stage. three biological replicates were collected at each stage and were processed to generate indexed rna-seq libraries, which were sequenced using an illumina hiseq we obtained million 50-bp paired-end reads, comprising million reads representing the post fruit-set stage, million reads from the pre-ripening stage and million reads from the post-harvest withering stage. gene expression levels at all three stages were quantified using the pn genome and the corvina private genes as reference sequences. the abundance of each transcript was expressed as fragments per kilobase of exon model per million mapped reads as implemented in cufflinks. a gene was considered to be expressed if the fpkm 95% confidence interval lower boundary was greater than zero and if the fpkm value was higher than. we detected expressed genes in at least one of the three samples, including of the novel genes and of the corvina private genes. this represented 72% of the grapevine transcriptome. although more genes were detected in the berry samples than in the reconstructed pool, genes expressed in the pooled samples were not represented in any of the three berry samples. raw counts of uniquely-mapped reads were estimated and normalized. we identified loci that were modulated in at least one of the samples. at each time point, we detected both unique and overlapping sets of differentially-expressed genes, e.g. genes were differentially expressed in all three stages analyzed and were differentially expressed specifically during withering. interestingly, of the corvina private genes were differentially expressed and were induced specifically during withering. similarly, of the novel loci were differentially expressed in at least one sample. genes were grouped according to their expression profiles by transforming the expression data into moderate fold change estimates using a variance stabilizing transformation. differentially-expressed genes were thus grouped into four clusters according to their expression profiles, comprising those repressed at pr and phwii or exclusively at phwii but not pfs, those transiently repressed at pr, those transiently induced at pr and those induced at the pr and phwii stages or at the phwii stage alone. to gain insight into the functions of the modulated genes in each cluster, each group was enriched with go terms associated with of the differentially-expressed genes. looking specifically at genes induced during the phwii phase given that withering is peculiar to wines produced from this cultivar, the statistics show an enrichment for genes involved in stress responses such as programmed cell death and in the synthesis of flavonoids, as previously described. discussion sequence diversity is usually described in comparison to a reference genome. given the high degree of genetic diversity among plant cultivars, this approach might fail to recognize highly polymorphic regions and will not detect the presence or absence of genes residing in private regions of the genome. whole-genome sequencing and re-annotation is therefore recommended for each variety, but in predominantly heterozygous species such as grapevine the sequence diversity would make contig assembly a daunting and resource-intensive task. when a reference genome is available, genes and transcript isoforms are built de novo by mapping rna-seq reads, but this does not solve the problem of hypervariable sequences and private genes. however, the de novo assembly strategy does not depend on the genome and has been applied successfully to reconstruct the transcriptomes of non-model species lacking reference genomes. we have demonstrated the feasibility cdna sequencing by rna-seq for the analysis of varietal diversity between a local grapevine cultivar and the pn reference genome without genomic data. the availability of a reference genome allows the reconstruction procedure to be validated and highlights the diversity between the two genomes. improved annotation of the reference genome the latest grapevine genome annotation comprises genes identified by a combination of ab initio prediction and cdna mapping. by comparing this annotation to the transcripts we identified, we found our method had detected 51% of the annotated genes, the remainder probably representing tissue/condition-specific transcripts that were not present in our pooled samples. the genes overlapping our sample and the v annotation have a higher expression level than the v1-specific genes. these data indicate that many of the v annotations undetected using our method were missed because of the paucity of sequencing reads generated from the corresponding loci. a large number of potential protein-coding genes were detected in the non-annotated parts of the genome. a recent comparison of the 8x, 12x v and 12x v annotations showed that genes present in either the 8x or 12x v assemblies were not present in the v annotation. interestingly, of our potential protein-coding genes were represented in the 8x or 12x v annotations. current annotation is therefore incomplete and insufficient to describe the full gene space of a cultivar other than the reference pinot noir clone. our method provided experimental support for protein-coding genes missing from the final assembly because they were excluded from the 12x consensus, and for additional genes that appear to have been missed in the v annotation. novel genes excluded from the v annotation appear to have meaningful biological roles, including those modulated during berry ripening and/or withering e.g. eight disease-resistance genes, two stress-inducible genes, a heat shock protein gene and a senescence-associated gene. the expression of the disease-resistance genes generally declined during berry development and withering suggesting their role is to protect the berry from pathogens and pests during early development. in contrast, the stress-inducible genes and heat shock protein gene were induced during ripening and withering, supporting a protective role against abiotic stress during the accumulation of sugars and secondary metabolites as previously reported. the rna-seq data therefore provide a comprehensive insight into the biologically-relevant landscape of gene expression during berry development and ripening. our method not only offers a way to annotate previously uncharacterized genes but also improves the annotation of known genes by helping to define their boundaries more robustly and to identify splice variants. our data indicate that up to 11% of the genes in the v annotation are split incorrectly, similar to the error rate in other annotated plant genomes. a previous in silico analysis identified instances of erroneously split genes in the v annotation. we also detected of these genes and our analysis suggested that 75% of them were split incorrectly in the v annotation. furthermore, our data resulted in the 3′ and 5′ extension of nearly 90% of the genes we detected compared to the boundaries in the v annotation, indicating that the untranslated regions were longer than previously reported, using in silico prediction methods. our approach may therefore provide a useful complement to ab initio gene prediction methods to establish gene boundaries and define utrs. finally, our de novo transcriptome assembly method detected an average of transcripts per locus, in line with previous reports using a reference-guided assembly of grapevine transcripts. although beyond the scope of our investigation, the de novo reconstruction indicated alternative splice variants for loci, providing a much more exhaustive description of the grapevine transcriptome compared to in silico predictions. the number of studies which try to describe alternative splicing events in plants are still scarce, however many recent studies point to an extensive diffusion of the phenomenon and to its important role in modulating gene expression and stress response. our results indicate that the transcriptional landscape in vitis is more complex than previously thought and therefore warrants further investigation. expression of corvina private genes during berry development recent data from the deep sequencing of human individuals and arabidopsis ecotypes revealed portions of genome that are not shared among all genotypes and the reference genome. interestingly, the novel genomic sequences included a set of protein-coding genes potentially contributing to the intra-species variability. similarly, we detected putative protein-coding genes with a high coding potential or matches to plant ests that represent potential corvina private genes. we identified private genes expressed in at least one berry-sampling phase, of which were differentially expressed between samples, and these could represent a group of genes that directly contribute to the specific characteristics of the corvina berry. some of these private genes could have been selected by ancient breeders looking for particular berry quality traits, such as the ability to withstand the lengthy drying phase required to make passito wines such as amarone and recioto. for example, we identified a heat shock protein gene and a stress-inducible gene induced during ripening, consistent with the ability of corvina berries to undergo dehydration for up to 100 days. furthermore, we detected the induction of genes involved in translation and protein metabolism during withering, including three ribosomal proteins, three elongation factors, ubiquitin, a 5-methyltetrahydropteroyltriglutamate-homocysteine methyltransferase and a dna-binding protein. this supports cdna-aflp data indicating the induction of genes with similar functions during withering. thirty-three of the corvina private genes matched homologs in other grape varieties but not the reference genome. this is expected because the dispensable part of the genome may be partly shared among different cultivars and only a few genes may be truly unique to a particular accession. for example, we found two flowering locus t genes the first corresponding to the previously-described vvft gene found in the cultivars cabernet sauvignon and tempranillo. at least six members of the ft/tfl gene family were identified in the tempranillo genome, including vvft which appears to be the ortholog of arabidopsis ft and therefore induces precocious flowering when expressed in arabidopsis, consistent with reported expression patterns associated with seasonal floral induction in latent buds and with the development of inflorescences, flowers and fruits. there is no evidence for the presence of classical floral regulatory pathways in grapevine, and the expression profile of vvft suggests that it only partially corresponds to the florigen role of arabidopsis ft. we also observed the expression of vvft during berry formation, suggesting an additional and uncharacterized role of this gene during early berry formation.  we were able to reconstruct a substantial part of the grapevine transcriptome, improve the annotations of known genes by defining their boundaries and splice variants, add apparently novel genes representing non-annotated or unassembled regions of the reference genome, and also add potentially corvina-specific private genes that are not present in the reference sequence. our results are consistent with data from other plant species showing that different genotypes share a common majority of genes but also possess smaller sets of private genes that are likely to be dispensable, that contribute to intra-specific variation and that produce unique, variety-dependent characteristics. given the substantial divergence among registered ecotypes and cultivars, we argue that in plant biology a de novo transcriptome assembly approach should not be limited to species lacking reference genome because it can improve the annotation of diverse cultivars and identify cultivar-specific private genes without embarking on a labor-intensive reconstructing of the entire genome. 
the genetic components underlying phenotypic diversity in grapevine must be understood in order to disentangle genetic and environmental factors. we also discovered apparent private genes in the corvina genome which were missing from the reference genome. the private genes we discovered are likely to be nonessential but could influence certain cultivar-specific characteristics. we have shown that cdna sequencing by rna-seq is a robust approach for the characterization of varietal diversity between a local grapevine cultivar and the pn reference genome. therefore, the application of de novo transcriptome assembly should not be restricted to species lacking a reference genome because it can also improve existing reference genome annotations and identify novel, cultivar-specific genes. we detected known genes including with novel splice isoforms, and identified potentially novel protein-coding genes in non-annotated or unassembled regions of the reference genome. the de novo assembly approach allowed a substantial amount of the corvina transcriptome to be reconstructed, improving known gene annotations by robustly defining gene structures, annotating splice isoforms and detecting genes without annotations.
 plants such as grapevine display significant inter-cultivar genetic and phenotypic variation. the genetic components underlying phenotypic diversity in grapevine must be understood in order to disentangle genetic and environmental factors.  we have shown that cdna sequencing by rna-seq is a robust approach for the characterization of varietal diversity between a local grapevine cultivar and the pn reference genome. we detected known genes including with novel splice isoforms, and identified potentially novel protein-coding genes in non-annotated or unassembled regions of the reference genome. we also discovered apparent private genes in the corvina genome which were missing from the reference genome.  the de novo assembly approach allowed a substantial amount of the corvina transcriptome to be reconstructed, improving known gene annotations by robustly defining gene structures, annotating splice isoforms and detecting genes without annotations. the private genes we discovered are likely to be nonessential but could influence certain cultivar-specific characteristics. therefore, the application of de novo transcriptome assembly should not be restricted to species lacking a reference genome because it can also improve existing reference genome annotations and identify novel, cultivar-specific genes. transcriptomicsrna-seqde novo assemblygrapevarietal diversity 