BACKGROUND
polyploidization is an ancient and ongoing evolutionary process that promotes plant evolution by reshaping genomes  <cit> .
the majority of flowering plants have undergone polyploidization  and chromosome rearrangement  <cit> .
generally, polyploids are divided into allopolyploids and autopolyploids  <cit> .
many major crops, including wheat, cotton, oat, coffee, and oilseed rape, are fundamentally allopolyploids  <cit> .
recently, many studies have revealed that methylation status, gene expression profile, chromosomal rearrangements, deletions, insertions, and sequence substitutions in many allopolyploids differ to their progenitor  <cit> .
birchler and veitia  <cit>  proposed the gene balance theory with regard to quantitative traits and gene duplication following polyploidy  <cit> .
angiosperm genome plasticity in polyploids is always related to changes in gene expression, which are largely controlled by epigenetic profiles  <cit> .
gaeta and pires  <cit>  concluded that these genomic changes occur in both natural and resynthesized polyploids  <cit> .
molecular analyses of natural and resynthesized allopolyploids indicate that genetic and epigenetic changes are common results of polyploidization in different species  <cit> .
discovering the structural and functional evolution of genomes during polyploidization is of great importance in plant biology  <cit> .
cultivated brassica species include important economical crops mostly closely related to arabidopsis thaliana and provide great chances for studying the effects of polyploidization  <cit> .
the availability of the arabidopsis genome effectively facilitates studies on brassica polyploidization  <cit> .
soon after the arabidopsis and brassica lineage diverged ~ <dig>  million years ago , the triplicated brassica subgenomes divergence was estimated to be  <dig>  mya  <cit> .
b. rapa  and b. oleracea  descended from a common hexaploid ancestor of a. thaliana.
moreover, the lineage of b. rapa and b. oleracea diverged around  <dig>  mya  <cit> , and a recent segmental duplication in b. rapa occurred ~ <dig>  mya  <cit> .
studies on a- and c-genome mapping, genome comparison, and genome evolution have been performed during the past decades.
additional genome duplication aside from triplication, as well as complex chromosomal changes was revealed in b. oleracea <cit> .
b. rapa, ~529 mb per haploid, was first launched for complete genome sequencing.
the allopolyploid b. napus  was spontaneously derived from hybridization of a and c progenitors  <cit> .
the genome of natural b. napus was confirmed intact without rearrangement, but resynthesized b. napus underwent rapid changes in the early generations, including genetic changes and methylation changes  <cit> .
non-additive proteins and additive proteins were completely identified in resynthesized b. napus, indicating the early steps of allopolyploidization repatterning are controlled by nonstochastic mechanisms  <cit> .
xiong et al.
<cit>  indicated that aneuploidy, gross chromosomal rearrangements, and dosage balance maintain the genomic stability of synthesized b. napus <cit> .
considering microarrays enable the comparison of gene expression at the transcriptome level, a set of brassica unigenes assembled using brassica expressed sequence tags  was developed and assigned to discriminate paralogous genes, but not homologous genes, between the a- and the c-genome  <cit> .
recently emerged next generation sequencing  technology is an alternative method for better genome, epigenome, and transcriptome study  <cit> .
many plant species have benefited from this technology, including b. rapa and b. napus.
the draft genome sequence of b. rapa accession chiifu-401- <dig> was newly released by illumina ga ii technology and annotated  <cit> , which provides an important resource for studying the evolution of polyploid genomes.
leaf transcriptome of b. napus had been dissected by sequencing  <cit> .
in the present research, we conducted a digital gene expression  analysis on resynthesized b. napus across the f1–f <dig> generations to address the transcriptome changes after polyploidization, which were also compared with their genetic progenitors  and natural b. napus.
previous studies on proteomic changes in this population revealed that differentially expressed proteins in f <dig> differed from the progenitors, exhibiting non-additive repatterning.
furthermore, gene silencing during polyploidization induces differences in protein expression in different generations of synthesized b. napus <cit> .
we report the expression profile of genes in resynthesized b. napus, show the upregulation of essential pathways and genes in f <dig>  and compare them with progenitors, which are downregulated in the f2–f <dig> generations.
this is the first comprehensive transcriptomic research that identifies degs and the pathways involved in first four generations of synthesized b. napus after polyploidization.
RESULTS
digital gene expression  profile
to investigate global transcriptome during the polyploidization of b. napus, we performed a dge analysis on the seedling stage of resynthesized b. napus  and its diploid parents, as well as natural b. napus.
finally, dge libraries from the leaves of four-week-old plants were generated and sequenced by illumina technology.
the sequence data are available from the geo repository, accession number gse <dig>  the statistics of the dge tags is shown in table  <dig>  approximately six million raw tags were generated for each library, and more than 97% of the raw tags were clean tags.
a total of  <dig> raw tags were obtained from b. rapa ,  <dig> from b. oleracea ,  <dig> from b. napus-f <dig> ,  <dig> from b. napus-f <dig> ,  <dig> from b. napus-f <dig> ,  <dig> from b. napus-f <dig> , and  <dig> from natural b. napus .
after removing the low-quality sequences and adapter sequences,  <dig>   <dig>   <dig>   <dig>   <dig>   <dig>  and  <dig> clean tags were obtained with  <dig> nt in length in the corresponding species.
unambiguous tags were counted and normalized to the number of transcripts per million tags  to evaluate the gene expression level.
the results show that the mrna transcribed from major genes had fewer than ten copies and only a small proportion of genes were highly expressed.
the distribution of clean tags in the seven libraries was determined to evaluate the normality of the dataset.
the results show a consistent pattern, with most of the tags coming from highly expressed genes.
in this analysis, the total number of clean tags is the sum of all clean tags and the number of distinct clean tags is the number of different clean tags .
we found that the percentage of distinct tags with high counts dropped dramatically, and the distinct tags with more than  <dig> copies accounted less than 10%.
however, more than 70% of total clean tags have accounts above  <dig> in each library.
by contrast, 50% of the distinct clean tags had copy numbers between two and five, but they only represented around 4% of the total number of clean tags, which indicates only a small number of mrna were expressed at high abundance and the majority were expressed at very low levels  <cit> .
the clean tags were then mapped onto the b. rapa genome  <cit>  and the numbers of tags that could be mapped onto genes with a maximum of one base pair mismatch in br, bo, bn-f <dig>  bn-f <dig>  bn-f <dig>  bn-f <dig>  and bn-n were  <dig>   <dig>   <dig>   <dig>   <dig>   <dig>  and  <dig>  respectively .
statistical analysis of clean tag alignment was conducted, including analysis of total clean tags and distinct clean tags .
we found that around 70% of total clean tags were mapped onto the b. rapa genome with perfect match or with 1 bp mismatch to sense or anti-sense genes, and approximately 65% of the distinct clean tags were successfully mapped.
finally, the tag mapping onto the b. rapa genome generated  <dig> tag-mapped genes for br,  <dig> for bo,  <dig> for bn-f <dig>   <dig> for bn-f <dig>   <dig> for bn-f <dig>   <dig> for bn-f <dig>  and  <dig> for bn-n. in total,  <dig> genes were identified from the seven libraries, which accounted for approximately 63% of genes in the annotated b. rapa genome .
clean tags are tags after filtering dirty tags from raw data.
distinct tags are different kinds of tags and unambiguous tags are the reminder clean tags after removing tags mapped to reference genome.
to determine whether the sequencing depth was sufficient for the transcriptome coverage, a saturation analysis was performed to check whether the number of detected genes increased with sequencing amount until the number of reads reached  <dig> million .
we also analyzed the distribution of the ratio of distinct tag copy numbers in each pair of libraries and found that more than 90% of the distinct tags had ratios up to 5-fold .
antisense genes play an important role in gene expression and regulation, and about 36%  of the genes were matched by distinct tags in antisense orientations.
up to  <dig> genes were mapped by dge tags in both sense and antisense orientations in the br library,  <dig> in the bo library,  <dig> in the bn-f <dig> library,  <dig> bn-f <dig>   <dig> in the bn-f <dig> library,  <dig> in the bn-f <dig> library, and  <dig> in the bn-n library .
in total, more than 56% of the genes  were transcribed from both strands, which indicate the importance of rna-mediated gene regulation in polyploidization.
differentially expressed genes in early generations of resynthesized b. napus
to identify genes that were differentially expressed in the early generations of synthesized b. napus after polyploidization, we compared pairs of dge profiles of the seven libraries  to analyze gene expression variations .
the comparison of b. napus-f <dig> with b. rapa revealed that  <dig> degs were significantly upregulated and  <dig> degs were downregulated in b. napus-f <dig> in comparison with b. rapa.
by contrast,  <dig> degs were downregulated and  <dig> degs were upregulated in b. napus-f <dig> compared with b. oleracea.
the number of upregulated degs in b. napus-f <dig> was more than that downregulated after polyploidization, which might indicate heterosis.
apart from the degs among resynthesized b. napus-f <dig> and its progenitors, the number of the identified degs differed in the early generations of synthesized b. napus, and most of the degs showed downregulation in the f2–f <dig> generations compared with the f <dig> generation.
up to  <dig> degs were downregulated and  <dig> were upregulated in b. napus-f <dig> compared with b. napus-f1;  <dig> degs were downregulated and  <dig> were upregulated in b. napus-f <dig> compared with b. napus-f1;  <dig> degs were downregulated and  <dig> degs were upregulated in b. napus-f <dig> and b. napus-f <dig>  comparison of resynthesized and natural b. napus showed  <dig> tags were downregulated and  <dig> tags were upregulated in b. napus-f <dig> in comparison with natural b. napus.
by comparison, the lowest number of degs was found in the f <dig> generation, which was in a relatively stable stage after polyploidization.
figure  <dig> shows the distribution of genes commonly expressed in b. rapa, b. oleracea, b. napus-f <dig>  and natural b. napus, which indicates that a number of conserved genes were identified apart from the degs.
distribution of genes commonly and specifically expressed in bn-f <dig>  f <dig>  f <dig> and f <dig> was also analyzed .
among the degs, the  <dig> most abundantly expressed genes that were upregulated or downregulated during polyploidization are listed in additional file 8: s <dig>  we found that the genes that encode dna binding/transcription factor  were more prominent, which were downregulated in f <dig> compared with its progenitors and then upregulated in the f2–f <dig> generations compared with the f <dig> generation.
genes encoding cyclin-dependent protein kinase , epoxycarotenoid dioxygenase , and glycine-rich protein  were upregulated in f <dig> during polyploidization, and then downregulated in f2–f <dig> compared with f <dig>
functional annotation of degs
annotation of the sequences using go database yielded good results for  <dig> unambiguously mapped genes identified using the bn-f <dig> dge tags.
these well-annotated sequences belonged to three main categories  and distributed into  <dig> categories, including the most dominant pathways such as ‘binding and catalytic activity,’ ‘cell and cell part,’ ‘organelle,’ ‘cellular and metabolic processes,’ and ‘response to stimulus’ .
a similar go distribution was revealed in the other six libraries .
however, we did not find any genes in the transporter cluster in bo.
genes were not found in the extracellular region cluster in bn-f <dig>  enzyme regulators and translation regulators were not observed in bn-f <dig>
to identify the biological pathways active in the dge libraries, we mapped all the annotated genes to terms in the kegg database to search for significantly enriched genes involved in metabolic or signal transduction pathways .
among all the genes with kegg annotation, degs were identified in bn-f <dig> in comparison with br.
in total, we assigned  <dig> degs to  <dig> kegg pathways.
up to  <dig> of these pathways were significantly enriched, with q values ≤  <dig>  , including metabolic pathways, ribosome, and carbon fixation in photosynthetic organisms.
similar pathway enrichment was revealed in pair comparison of each libraries  .
the  <dig> degs identified in bn-f <dig> in comparison with bo were assigned to  <dig> kegg pathways,  <dig> of which were significantly enriched.
the  <dig> degs identified in bn-f <dig> in comparison with bn-f <dig> were assigned to  <dig> kegg pathways,  <dig> degs identified in bn-f <dig> in comparison with bn-f <dig> were assigned to  <dig> pathways,  <dig> degs identified in bn-f <dig> in comparison with bn-f <dig> were assigned to  <dig> pathways, and  <dig> degs identified in bn-f <dig> in comparison with bn-n were assigned to  <dig> pathways.
genome-wide non-additive gene regulation in synthesized bn-f1
we compared the transcript abundance in synthesized bn-f <dig> with the relative mid-parent value  to identify genes that showed differential expression pattern in bn-f <dig>  up to  <dig> genes in bn-f <dig> showed differences in expression from mpv,  <dig>  of them showed higher expression levels than mpv, and  <dig>  of these genes showed lower expressions than mpv.
among these non-additively expressed genes,  <dig>  genes were expressed at higher levels in br than in bo, and  <dig>  genes were expressed at lower levels in br than bo .
the data suggest that the orthologous genes in polyploids are frequently expressed in a non-additive pattern.
cluster analysis of degs
cluster analysis of degs in the early generations of synthesized b. napus was performed with the correlated expression profile .
generally,  <dig> degs in all the comparisons were clustered as the union of degs.
up to  <dig> degs in all the comparisons were clustered as the deg intersections .
among the  <dig> major clusters, the upregulated transcripts were enriched in clusters a–k in the comparisons of bn-n vs. bn-f <dig>  br vs. bn-f <dig>  and bo vs. bn-f <dig>  the downregulated transcripts were enriched in clusters a–k in the comparisons of bn-f <dig> vs. bn-f <dig>  bn-f <dig> vs. bn-f <dig>  and bn-f <dig> vs. bn-f <dig>  only one exception indicated that cluster l showed gene upregulation in bn-f <dig>  bn-f <dig> and bn-f <dig> compared with bn-f <dig>  and downregulation in bn-f <dig> compared with bn-n, br and bo.
we also identified the enrichment of degs in each cluster in different functional categories and found that the genes involved in the metabolism category were enriched significantly in all clusters except cluster h. those in the protein with binding function or cofactor requirement category were enriched significantly in clusters a–h, whereas those in the subcellular localization category were enriched in all clusters except for cluster d. all the clustered degs are listed in additional file 11: s <dig>  multiple genes represented for proteins with binding function, such as zinc ion binding , atp binding , glutathione binding , selenium binding , and fad/nadp/nadph binding , showed different expression values in different generations of resynthesized b. napus and its progenitors.
some genes that encode ribosomal proteins were also changed during polyploidization.
discussion
differences in gene expression between synthesized b. napus-f <dig> and its progenitors
based on analysis of the leaf transcriptome data for synthesized b. napus-f <dig> and its progenitors, as well as the natural b. napus generated in this study, we found that the majority of the b. napus-f <dig> transcripts were conserved with the parental sequences.
up to  <dig>   <dig>  and  <dig> sequences were mapped by b. napus-f <dig> and its progenitors b. rapa and b. oleracea.
<dig> genes with same tpm were expressed in bn-f <dig> and br, and  <dig> genes with same tpm were expressed in bn-f <dig> and bo, which indicate that these genes might be significantly inherited by bn-f <dig> from one of its progenitors.
the genes with different tpm expressed in b. napus-f <dig> and its progenitors, which indicate that the genome combination results duplicated genes in the resynthesized polyploid.
however, only  <dig> of these commonly expressed genes showed tpm values in bn-f <dig> equal to the combination of its progenitors.
furthermore, most of the commonly expressed genes in the progenitors were non-additively expressed in bn-f <dig>  which might be responsible for gene repression and activation  <cit> .
the majority of the non-additively expressed genes in bn-f <dig> displayed expression values different to its progenitors, indicating that the transcriptome was reconciled during polyploidization  <cit> .
non-additive gene regulation should be involved in various biological pathways, which may lead to subfunctionalization of duplicated genes  <cit> .
the gene expression in the resynthesized bn-f <dig> was more complicated than the simple combination of two genomes, which might be due to the homologous recombination between closely related genomes   <cit> .
xiong et al.
<cit>  reported the variable chromosome instability of synthesized b. napus, and chromosome loss was compensated by the gain of homologous chromosomes  <cit> .
szadkowski et al.
<cit>  reported the genome blender of synthesized b. napus during first meiosis, which resulted in the rearrangement of two genomes and the restructuring in further generations  <cit> .
thus, resynthesized polyploids derived from closely related species result in changes in the non-additive pattern of multiple gene expression, which explains the molecular bases for hybrid vigor  <cit> .
besides the commonly expressed genes in polyploids and diploid progenitors, there are some species specific genes.
as shown in figure  <dig>   <dig>   <dig>   <dig> and  <dig> genes were specifically expressed in br, bo, bn-f <dig> and bn-n, respectively.
the number of bn-f <dig> specific genes was much higher than its progenitors, indicating many new transcripts emerged during polyploidization.
this was consistent with xu et al.
<cit>  that  <dig> novel transcripts were detected in leaves of resynthesized bn-f <dig> <cit> .
based on the clustering analysis of degs, we found  <dig> out of the  <dig> interspecific degs were up-regulated in bn-f <dig> compared with br and bo.
these up-regulated genes express proteins with different biological function, including salt tolerance protein, mitochondria/chloroplast membrance binding proteins, kinds of protein kinases, etc.
confirmation of these expression differences in further study is necessary for deep into the genetic causes of interest characteristics and improved qualities.
whereas  <dig> out of the  <dig> interspecific degs were down-regulated in bn-f <dig> compared with br and bo.
only two genes  were up-regulated in bn-f <dig> compared with br, whereas down-regulated in bn-f <dig> compared with bo.
five genes  were up-regulated in bn-f <dig> compared with bo, whereas down-regulated in bn-f <dig> compared with br .
all these gene expression differences are of great interest for studying the genome polyploidization of brassica species.
gene expression differences among early generations of synthesized b. napus  and natural b. napus
hitherto, studies on polyploidy mechanism were conducted on natural and resynthesized b. napus using proteomic, transcriptome, and cytogenetic analyses  <cit> .
however, few studies that trace genomic changes  and transcriptome changes in different generations of synthesized b. napus have been reported  <cit> .
as mentioned above, many novel transcripts emerged in bn-f <dig> compared its progenitors.
the number of bn-f <dig> specific genes was also higher than bn-f <dig>  bn-f <dig>  bn-f <dig> .
many of these new transcripts emerged in the first generation of resynthesized b. napus disappeared after successive self hybridizations, indicating the instability of resynthesized polyploids.
based on the clustering of degs found in the comparisons of br vs. bn-f <dig>  bo vs. bn-f <dig>  bn-f <dig> vs. bn-f <dig>  bn-f <dig> vs. bn-f <dig>  bn-f <dig> vs. bn-f <dig>  and bn-n vs. bn-f <dig>  we easily found that most degs are significantly downregulated in bn-f <dig>  bn-f <dig>  and bn-f <dig> compared with bn-f <dig>  but these degs were upregulated in bn-f <dig> compared with its progenitors and natural b. napus.
this phenomenon also indicated the genomic instability after polyploidization, and the discovery of multiple gene expression differences may compensate for the limitation of proteomic analysis  <cit> .
the upregulation of degs in synthesized bn-f <dig> during polyploidization could explain its improved characteristics compared with its progenitors.
for example, the expression pattern of bra <dig>  which encodes stress-responsive protein and bra <dig>  which encodes defense-related protein, were changed during polyploidization.
some of these gene expression differences might be related to the dna methylation or gene fragment losses during the combination of two genomes  <cit> .
for example, bra <dig>  which encodes o-methyltransferase  <dig>  and bra <dig>  which encodes an n2-dimethylguanosine trna methyltransferase family protein, were upregulated in bn-f <dig> during polyploidization and downregulated in the f2–f <dig> generations.
in the pathway analysis, we found bra <dig> participated in the flavone and flavonol biosynthesis, which functioned in the transformation of kaempferide into kaempferol.
this may indicate that kaempferol level was different in the synthesized b. napus.
the changes in expression include many transcription factors, binding proteins, and ribosomal proteins, must be important for the merging of the a- and c-genomes in synthesized polyploids.
for the  <dig> degs, which were down-regulated in bn-f <dig> compared with br and bo, we found they were up-regulated in bn-f <dig>  bn-f <dig> and bn-f <dig> compared with bn-f <dig>  this indicated that transcriptome recovery of genome balance functioned after polyploidization.
further research is needed to verify and determine the causes of these transcriptome changes and how they correlate with the phenotypic divergence and fertility of synthesized polyploids.
CONCLUSIONS
by applying the dge deep sequencing, this study investigated the transcriptome profile of resynthesized b. napus and its diploid progenitors, as well as natural b. napus, with an aim to illustrate the gene expression differences during polyploidization.
the amounts of transcripts obtained provided a foundation for future research on polyploidy mechanism of b. napus.
globally identified degs and putative biological pathways revealed the gene expression difference between synthesized b. napus and progenitors, and differential expression of genes across generations of synthesized b. napus.
generally, the gene expression in the resynthesized b. napus was more complicated than the simple combination of two genomes, and non-additive gene regulation was also detected in synthesized b. napus.
these findings provided a contribution to the existing sequence resources for brassica and would certainly facilitate polyploidy research of this genus.

 polyploidization is an ancient and ongoing evolutionary process that promotes plant evolution by reshaping genomes. the majority of flowering plants have undergone polyploidization and chromosome rearrangement. generally, polyploids are divided into allopolyploids and autopolyploids. many major crops, including wheat, cotton, oat, coffee, and oilseed rape, are fundamentally allopolyploids. recently, many studies have revealed that methylation status, gene expression profile, chromosomal rearrangements, deletions, insertions, and sequence substitutions in many allopolyploids differ to their progenitor. birchler and veitia proposed the gene balance theory with regard to quantitative traits and gene duplication following polyploidy. angiosperm genome plasticity in polyploids is always related to changes in gene expression, which are largely controlled by epigenetic profiles. gaeta and pires concluded that these genomic changes occur in both natural and resynthesized polyploids. molecular analyses of natural and resynthesized allopolyploids indicate that genetic and epigenetic changes are common results of polyploidization in different species. discovering the structural and functional evolution of genomes during polyploidization is of great importance in plant biology. cultivated brassica species include important economical crops mostly closely related to arabidopsis thaliana and provide great chances for studying the effects of polyploidization. the availability of the arabidopsis genome effectively facilitates studies on brassica polyploidization. soon after the arabidopsis and brassica lineage diverged ~ million years ago, the triplicated brassica subgenomes divergence was estimated to be mya. b. rapa and b. oleracea descended from a common hexaploid ancestor of a. thaliana. moreover, the lineage of b. rapa and b. oleracea diverged around mya, and a recent segmental duplication in b. rapa occurred ~ mya. studies on a- and c-genome mapping, genome comparison, and genome evolution have been performed during the past decades. additional genome duplication aside from triplication, as well as complex chromosomal changes was revealed in b. oleracea. b. rapa, ~529 mb per haploid, was first launched for complete genome sequencing. the allopolyploid b. napus was spontaneously derived from hybridization of a and c progenitors. the genome of natural b. napus was confirmed intact without rearrangement, but resynthesized b. napus underwent rapid changes in the early generations, including genetic changes and methylation changes. non-additive proteins and additive proteins were completely identified in resynthesized b. napus, indicating the early steps of allopolyploidization repatterning are controlled by nonstochastic mechanisms. xiong et al. indicated that aneuploidy, gross chromosomal rearrangements, and dosage balance maintain the genomic stability of synthesized b. napus. considering microarrays enable the comparison of gene expression at the transcriptome level, a set of brassica unigenes assembled using brassica expressed sequence tags was developed and assigned to discriminate paralogous genes, but not homologous genes, between the a- and the c-genome. recently emerged next generation sequencing technology is an alternative method for better genome, epigenome, and transcriptome study. many plant species have benefited from this technology, including b. rapa and b. napus. the draft genome sequence of b. rapa accession chiifu-401- was newly released by illumina ga ii technology and annotated, which provides an important resource for studying the evolution of polyploid genomes. leaf transcriptome of b. napus had been dissected by sequencing. in the present research, we conducted a digital gene expression analysis on resynthesized b. napus across the f1–f generations to address the transcriptome changes after polyploidization, which were also compared with their genetic progenitors and natural b. napus. previous studies on proteomic changes in this population revealed that differentially expressed proteins in f differed from the progenitors, exhibiting non-additive repatterning. furthermore, gene silencing during polyploidization induces differences in protein expression in different generations of synthesized b. napus. we report the expression profile of genes in resynthesized b. napus, show the upregulation of essential pathways and genes in f and compare them with progenitors, which are downregulated in the f2–f generations. this is the first comprehensive transcriptomic research that identifies degs and the pathways involved in first four generations of synthesized b. napus after polyploidization.  digital gene expression profile to investigate global transcriptome during the polyploidization of b. napus, we performed a dge analysis on the seedling stage of resynthesized b. napus and its diploid parents, as well as natural b. napus. finally, dge libraries from the leaves of four-week-old plants were generated and sequenced by illumina technology. the sequence data are available from the geo repository, accession number gse the statistics of the dge tags is shown in table  approximately six million raw tags were generated for each library, and more than 97% of the raw tags were clean tags. a total of raw tags were obtained from b. rapa, from b. oleracea, from b. napus-f, from b. napus-f, from b. napus-f, from b. napus-f, and from natural b. napus. after removing the low-quality sequences and adapter sequences, and clean tags were obtained with nt in length in the corresponding species. unambiguous tags were counted and normalized to the number of transcripts per million tags to evaluate the gene expression level. the results show that the mrna transcribed from major genes had fewer than ten copies and only a small proportion of genes were highly expressed. the distribution of clean tags in the seven libraries was determined to evaluate the normality of the dataset. the results show a consistent pattern, with most of the tags coming from highly expressed genes. in this analysis, the total number of clean tags is the sum of all clean tags and the number of distinct clean tags is the number of different clean tags. we found that the percentage of distinct tags with high counts dropped dramatically, and the distinct tags with more than copies accounted less than 10%. however, more than 70% of total clean tags have accounts above in each library. by contrast, 50% of the distinct clean tags had copy numbers between two and five, but they only represented around 4% of the total number of clean tags, which indicates only a small number of mrna were expressed at high abundance and the majority were expressed at very low levels. the clean tags were then mapped onto the b. rapa genome and the numbers of tags that could be mapped onto genes with a maximum of one base pair mismatch in br, bo, bn-f bn-f bn-f bn-f and bn-n were and respectively. statistical analysis of clean tag alignment was conducted, including analysis of total clean tags and distinct clean tags. we found that around 70% of total clean tags were mapped onto the b. rapa genome with perfect match or with 1 bp mismatch to sense or anti-sense genes, and approximately 65% of the distinct clean tags were successfully mapped. finally, the tag mapping onto the b. rapa genome generated tag-mapped genes for br, for bo, for bn-f for bn-f for bn-f for bn-f and for bn-n. in total, genes were identified from the seven libraries, which accounted for approximately 63% of genes in the annotated b. rapa genome. clean tags are tags after filtering dirty tags from raw data. distinct tags are different kinds of tags and unambiguous tags are the reminder clean tags after removing tags mapped to reference genome. to determine whether the sequencing depth was sufficient for the transcriptome coverage, a saturation analysis was performed to check whether the number of detected genes increased with sequencing amount until the number of reads reached million. we also analyzed the distribution of the ratio of distinct tag copy numbers in each pair of libraries and found that more than 90% of the distinct tags had ratios up to 5-fold. antisense genes play an important role in gene expression and regulation, and about 36% of the genes were matched by distinct tags in antisense orientations. up to genes were mapped by dge tags in both sense and antisense orientations in the br library, in the bo library, in the bn-f library, bn-f in the bn-f library, in the bn-f library, and in the bn-n library. in total, more than 56% of the genes were transcribed from both strands, which indicate the importance of rna-mediated gene regulation in polyploidization. differentially expressed genes in early generations of resynthesized b. napus to identify genes that were differentially expressed in the early generations of synthesized b. napus after polyploidization, we compared pairs of dge profiles of the seven libraries to analyze gene expression variations. the comparison of b. napus-f with b. rapa revealed that degs were significantly upregulated and degs were downregulated in b. napus-f in comparison with b. rapa. by contrast, degs were downregulated and degs were upregulated in b. napus-f compared with b. oleracea. the number of upregulated degs in b. napus-f was more than that downregulated after polyploidization, which might indicate heterosis. apart from the degs among resynthesized b. napus-f and its progenitors, the number of the identified degs differed in the early generations of synthesized b. napus, and most of the degs showed downregulation in the f2–f generations compared with the f generation. up to degs were downregulated and were upregulated in b. napus-f compared with b. napus-f1; degs were downregulated and were upregulated in b. napus-f compared with b. napus-f1; degs were downregulated and degs were upregulated in b. napus-f and b. napus-f comparison of resynthesized and natural b. napus showed tags were downregulated and tags were upregulated in b. napus-f in comparison with natural b. napus. by comparison, the lowest number of degs was found in the f generation, which was in a relatively stable stage after polyploidization. figure  shows the distribution of genes commonly expressed in b. rapa, b. oleracea, b. napus-f and natural b. napus, which indicates that a number of conserved genes were identified apart from the degs. distribution of genes commonly and specifically expressed in bn-f f f and f was also analyzed. among the degs, the most abundantly expressed genes that were upregulated or downregulated during polyploidization are listed in additional file 8: s we found that the genes that encode dna binding/transcription factor were more prominent, which were downregulated in f compared with its progenitors and then upregulated in the f2–f generations compared with the f generation. genes encoding cyclin-dependent protein kinase, epoxycarotenoid dioxygenase, and glycine-rich protein were upregulated in f during polyploidization, and then downregulated in f2–f compared with f functional annotation of degs annotation of the sequences using go database yielded good results for unambiguously mapped genes identified using the bn-f dge tags. these well-annotated sequences belonged to three main categories and distributed into categories, including the most dominant pathways such as ‘binding and catalytic activity,’ ‘cell and cell part,’ ‘organelle,’ ‘cellular and metabolic processes,’ and ‘response to stimulus’. a similar go distribution was revealed in the other six libraries. however, we did not find any genes in the transporter cluster in bo. genes were not found in the extracellular region cluster in bn-f enzyme regulators and translation regulators were not observed in bn-f to identify the biological pathways active in the dge libraries, we mapped all the annotated genes to terms in the kegg database to search for significantly enriched genes involved in metabolic or signal transduction pathways. among all the genes with kegg annotation, degs were identified in bn-f in comparison with br. in total, we assigned degs to kegg pathways. up to of these pathways were significantly enriched, with q values ≤, including metabolic pathways, ribosome, and carbon fixation in photosynthetic organisms. similar pathway enrichment was revealed in pair comparison of each libraries. the degs identified in bn-f in comparison with bo were assigned to kegg pathways, of which were significantly enriched. the degs identified in bn-f in comparison with bn-f were assigned to kegg pathways, degs identified in bn-f in comparison with bn-f were assigned to pathways, degs identified in bn-f in comparison with bn-f were assigned to pathways, and degs identified in bn-f in comparison with bn-n were assigned to pathways. genome-wide non-additive gene regulation in synthesized bn-f1 we compared the transcript abundance in synthesized bn-f with the relative mid-parent value to identify genes that showed differential expression pattern in bn-f up to genes in bn-f showed differences in expression from mpv, of them showed higher expression levels than mpv, and of these genes showed lower expressions than mpv. among these non-additively expressed genes, genes were expressed at higher levels in br than in bo, and genes were expressed at lower levels in br than bo. the data suggest that the orthologous genes in polyploids are frequently expressed in a non-additive pattern. cluster analysis of degs cluster analysis of degs in the early generations of synthesized b. napus was performed with the correlated expression profile. generally, degs in all the comparisons were clustered as the union of degs. up to degs in all the comparisons were clustered as the deg intersections. among the major clusters, the upregulated transcripts were enriched in clusters a–k in the comparisons of bn-n vs. bn-f br vs. bn-f and bo vs. bn-f the downregulated transcripts were enriched in clusters a–k in the comparisons of bn-f vs. bn-f bn-f vs. bn-f and bn-f vs. bn-f only one exception indicated that cluster l showed gene upregulation in bn-f bn-f and bn-f compared with bn-f and downregulation in bn-f compared with bn-n, br and bo. we also identified the enrichment of degs in each cluster in different functional categories and found that the genes involved in the metabolism category were enriched significantly in all clusters except cluster h. those in the protein with binding function or cofactor requirement category were enriched significantly in clusters a–h, whereas those in the subcellular localization category were enriched in all clusters except for cluster d. all the clustered degs are listed in additional file 11: s multiple genes represented for proteins with binding function, such as zinc ion binding, atp binding, glutathione binding, selenium binding, and fad/nadp/nadph binding, showed different expression values in different generations of resynthesized b. napus and its progenitors. some genes that encode ribosomal proteins were also changed during polyploidization. discussion differences in gene expression between synthesized b. napus-f and its progenitors based on analysis of the leaf transcriptome data for synthesized b. napus-f and its progenitors, as well as the natural b. napus generated in this study, we found that the majority of the b. napus-f transcripts were conserved with the parental sequences. up to and sequences were mapped by b. napus-f and its progenitors b. rapa and b. oleracea. genes with same tpm were expressed in bn-f and br, and genes with same tpm were expressed in bn-f and bo, which indicate that these genes might be significantly inherited by bn-f from one of its progenitors. the genes with different tpm expressed in b. napus-f and its progenitors, which indicate that the genome combination results duplicated genes in the resynthesized polyploid. however, only of these commonly expressed genes showed tpm values in bn-f equal to the combination of its progenitors. furthermore, most of the commonly expressed genes in the progenitors were non-additively expressed in bn-f which might be responsible for gene repression and activation. the majority of the non-additively expressed genes in bn-f displayed expression values different to its progenitors, indicating that the transcriptome was reconciled during polyploidization. non-additive gene regulation should be involved in various biological pathways, which may lead to subfunctionalization of duplicated genes. the gene expression in the resynthesized bn-f was more complicated than the simple combination of two genomes, which might be due to the homologous recombination between closely related genomes. xiong et al. reported the variable chromosome instability of synthesized b. napus, and chromosome loss was compensated by the gain of homologous chromosomes. szadkowski et al. reported the genome blender of synthesized b. napus during first meiosis, which resulted in the rearrangement of two genomes and the restructuring in further generations. thus, resynthesized polyploids derived from closely related species result in changes in the non-additive pattern of multiple gene expression, which explains the molecular bases for hybrid vigor. besides the commonly expressed genes in polyploids and diploid progenitors, there are some species specific genes. as shown in figure  and genes were specifically expressed in br, bo, bn-f and bn-n, respectively. the number of bn-f specific genes was much higher than its progenitors, indicating many new transcripts emerged during polyploidization. this was consistent with xu et al. that novel transcripts were detected in leaves of resynthesized bn-f. based on the clustering analysis of degs, we found out of the interspecific degs were up-regulated in bn-f compared with br and bo. these up-regulated genes express proteins with different biological function, including salt tolerance protein, mitochondria/chloroplast membrance binding proteins, kinds of protein kinases, etc. confirmation of these expression differences in further study is necessary for deep into the genetic causes of interest characteristics and improved qualities. whereas out of the interspecific degs were down-regulated in bn-f compared with br and bo. only two genes were up-regulated in bn-f compared with br, whereas down-regulated in bn-f compared with bo. five genes were up-regulated in bn-f compared with bo, whereas down-regulated in bn-f compared with br. all these gene expression differences are of great interest for studying the genome polyploidization of brassica species. gene expression differences among early generations of synthesized b. napus and natural b. napus hitherto, studies on polyploidy mechanism were conducted on natural and resynthesized b. napus using proteomic, transcriptome, and cytogenetic analyses. however, few studies that trace genomic changes and transcriptome changes in different generations of synthesized b. napus have been reported. as mentioned above, many novel transcripts emerged in bn-f compared its progenitors. the number of bn-f specific genes was also higher than bn-f bn-f bn-f. many of these new transcripts emerged in the first generation of resynthesized b. napus disappeared after successive self hybridizations, indicating the instability of resynthesized polyploids. based on the clustering of degs found in the comparisons of br vs. bn-f bo vs. bn-f bn-f vs. bn-f bn-f vs. bn-f bn-f vs. bn-f and bn-n vs. bn-f we easily found that most degs are significantly downregulated in bn-f bn-f and bn-f compared with bn-f but these degs were upregulated in bn-f compared with its progenitors and natural b. napus. this phenomenon also indicated the genomic instability after polyploidization, and the discovery of multiple gene expression differences may compensate for the limitation of proteomic analysis. the upregulation of degs in synthesized bn-f during polyploidization could explain its improved characteristics compared with its progenitors. for example, the expression pattern of bra which encodes stress-responsive protein and bra which encodes defense-related protein, were changed during polyploidization. some of these gene expression differences might be related to the dna methylation or gene fragment losses during the combination of two genomes. for example, bra which encodes o-methyltransferase and bra which encodes an n2-dimethylguanosine trna methyltransferase family protein, were upregulated in bn-f during polyploidization and downregulated in the f2–f generations. in the pathway analysis, we found bra participated in the flavone and flavonol biosynthesis, which functioned in the transformation of kaempferide into kaempferol. this may indicate that kaempferol level was different in the synthesized b. napus. the changes in expression include many transcription factors, binding proteins, and ribosomal proteins, must be important for the merging of the a- and c-genomes in synthesized polyploids. for the degs, which were down-regulated in bn-f compared with br and bo, we found they were up-regulated in bn-f bn-f and bn-f compared with bn-f this indicated that transcriptome recovery of genome balance functioned after polyploidization. further research is needed to verify and determine the causes of these transcriptome changes and how they correlate with the phenotypic divergence and fertility of synthesized polyploids.  by applying the dge deep sequencing, this study investigated the transcriptome profile of resynthesized b. napus and its diploid progenitors, as well as natural b. napus, with an aim to illustrate the gene expression differences during polyploidization. the amounts of transcripts obtained provided a foundation for future research on polyploidy mechanism of b. napus. globally identified degs and putative biological pathways revealed the gene expression difference between synthesized b. napus and progenitors, and differential expression of genes across generations of synthesized b. napus. generally, the gene expression in the resynthesized b. napus was more complicated than the simple combination of two genomes, and non-additive gene regulation was also detected in synthesized b. napus. these findings provided a contribution to the existing sequence resources for brassica and would certainly facilitate polyploidy research of this genus. 
almost million clean tags were generated using illumina technology to produce the expression data for genes, which account for 63% of the annotated b. rapa genome. thousands of differentially expressed genes were identified and revealed the substantial changes in f1–f among the most degs are dna binding/transcription factor, cyclin-dependent protein kinase, epoxycarotenoid dioxygenase, and glycine-rich protein. we examined the global gene expression patterns of the first four generations of resynthesized b. napus, its diploid progenitors b. rapa and b. oleracea, and natural b. napus using digital gene expression analysis. tag mapping of the b. rapa genome generated and annotated genes for the b. rapa, b. oleracea, f1–f of synthesized b. napus, and natural b. napus libraries, respectively. the systematic deep sequencing analysis provided a comprehensive understanding of the transcriptome complexity of early generations of synthesized b. napus. this information broadens our understanding of the mechanisms of b. napus polyploidization and contributes to molecular and genetic research by enriching the brassica database. polyploidy is an important evolutionary mechanism in flowering plants that often induces immediate extensive changes in gene expression through genomic merging and doubling. rna-seq is a recently developed technique for transcriptome study, which could be in choice for profiling gene expression pattern in polyploids. more than 56% of the genes were transcribed from both strands, which indicate the importance of rna-mediated gene regulation in polyploidization.
 polyploidy is an important evolutionary mechanism in flowering plants that often induces immediate extensive changes in gene expression through genomic merging and doubling. brassica napus l. is one of the most economically important polyploid oil crops and has been broadly studied as an example of polyploid crop. rna-seq is a recently developed technique for transcriptome study, which could be in choice for profiling gene expression pattern in polyploids.  we examined the global gene expression patterns of the first four generations of resynthesized b. napus, its diploid progenitors b. rapa and b. oleracea, and natural b. napus using digital gene expression analysis. almost million clean tags were generated using illumina technology to produce the expression data for genes, which account for 63% of the annotated b. rapa genome. more than 56% of the genes were transcribed from both strands, which indicate the importance of rna-mediated gene regulation in polyploidization. tag mapping of the b. rapa genome generated and annotated genes for the b. rapa, b. oleracea, f1–f of synthesized b. napus, and natural b. napus libraries, respectively. the unambiguous tag-mapped genes in the libraries were functionally categorized via gene ontological analysis. thousands of differentially expressed genes were identified and revealed the substantial changes in f1–f among the most degs are dna binding/transcription factor, cyclin-dependent protein kinase, epoxycarotenoid dioxygenase, and glycine-rich protein. the kyoto encyclopedia of genes and genomes analysis of the degs suggested approximately biological pathways.  the systematic deep sequencing analysis provided a comprehensive understanding of the transcriptome complexity of early generations of synthesized b. napus. this information broadens our understanding of the mechanisms of b. napus polyploidization and contributes to molecular and genetic research by enriching the brassica database. 