BMC Genomics 2009, 10:295 http://www.biomedcentral.com/1471-2164/10/295 thesis. For many of the highly expressed genes several dif- ferent tags were found (e.g. 44 for AT5G45890 at 23 DAP) matching to a large number of different ESTs (e.g. 5,452 for AT5G45890 at 23 DAP) that were aligned to a particu- lar A. thaliana gene at <1e-6. Differentially expressed genes 620 tags showed differential expression at p ≤ 0.05 between 23 and 35 DAP. These represent only 1.7% of the accepted unique tags, but 21.7% of all normalized tag counts. 498 of 620 (80.3%) differentially expressed unique tags were successfully matched to 490 A. thaliana genes (5.9% of all 8,255 matched genes, Table 2), and these differentially expressed genes were matched by 18.2% and 38.1% of all matched tags in 23 DAP and 35 DAP libraries, respectively (see additional file 1: 490_differentially_expressed_genes.xls). This indicates that, in the 35 DAP library but not in the 23 DAP library, many of the differentially expressed transcripts are highly abundant. Of these 490 genes, 194 genes were upregu- lated at 23 DAP and 296 genes were upregulated at 35 DAP. The 20 most abundant differentially expressed genes at 23 DAP include 6 genes encoding proteases, 5 genes coding for protease inhibitor/seed storage/lipid transfer protein (LTP) family proteins and 2 genes coding for defensin-like family proteins. SAG12 is the most abun- dant differentially expressed gene at 23 DAP with 1.591% and is down-regulated about 3-fold at 35 DAP with 0.566% total abundance. The 20 most abundant differen- tially expressed genes at 35 DAP cover a narrower range of genes coding for 2S seed storage proteins, oleosins, cruci- ferins and protease inhibitor proteins. The 5 most abun- dant differentially expressed genes are the 2S seed storage proteins 1 to 4 and cruciferin 3. The 2S seed storage pro- tein 1 is the most abundant differentially expressed gene with 2.657% abundance and is up-regulated 374-fold compared to time-point 23 DAP. From all 490 genes showing a significantly different expression level at the two time-points, 23 were exclusively expressed at 23 DAP and 54 at 35 DAP (see additional file 1: 490_differentially_expressed_genes.xls). A number of cases were observed where different matched A. thaliana genes with similar functional annotations were expressed at the two different time-points, e.g. different A. thaliana genes belonging to the large cysteine protease and lipid transfer protein families were exclusively expressed at 23 DAP and 35 DAP, respectively. In contrast to cruciferin A (CRA1) and cruciferin 3 (CRU3), cruciferin 2 (CRU2) was exclusively expressed at 35 DAP. Expression data produced by microarray hybridization for 79 developmental stages of A. thaliana were downloaded from EBI ArrayExpress (accession number E-AFMX-9, [18]) and expression profiles were compared with expres- sion profiles obtained by LongSAGE for B. napus seeds using hierarchical cluster analysis and Spearman's rank correlations according to Lu et al. [19]. Both B. napus SAGE data sets showed weak correlations with expression value estimates from A. thaliana microarray data sets with the highest correlation for the seed developmental stages ATGE_77 (0.522 for 23 DAP) and ATGE_78 (0.440 for 35 DAP). Sample ATGE_77 was derived from A. thaliana seed stage 4, early to late heart stage embryos and sample ATGE_78 from seed stage 5, late heart to mid torpedo embryos. Embryos dissected from sample 23 DAP were classified to be at the mid torpedo stage and from sample 35 DAP to be at the early curled cotyledon stage. Genes that were differentially expressed between two different A. thaliana seed stages (ATGE_77 and ATGE_78) were iden- tified at different stringencies and compared with the 490 genes identified to be differentially expressed between 23 DAP and 35 DAP in B. napus as described above. From the 490 genes identified by LongSAGE 76 (15%) were not represented on the microarray chip. From the remaining 414 genes a maximum of 29% (122) overlapping genes were detected that were mainly highly abundant genes (e.g. 2S seed storage proteins). The percentage of overlap- ping genes identified here between the two different cru- cifer species during morphological similar seed developmental stages appears to be high when taking into account that the correlation of microarray hybridization and LongSAGE data analysis has been found to be low (below 0.5) even when using the same RNA preparations and LongSAGE has been characterized to be more efficient at identifying differentially expressed tags than microarray technology [19]. Gene ontology enrichment analysis A total of 7,208 of the 8,255 matched genes were expressed at 23 DAP, while 4,875 genes were expressed at 35 DAP (Table 2). 3,828 of 8,255 genes (46.4%) were expressed at both time-points. Figure 1 gives a compari- son of GO categories that are statistically over-represented for the genes expressed at 23 DAP compared to all other A. thaliana genes. From the 490 genes matched by statisti- cally significant differentially expressed tags between 23 and 35 DAP, 194 were up-regulated at 23 DAP and 296 were up-regulated at 35 DAP. Statistically enriched GO terms found for the differentially expressed genes either up-regulated at 23 or up-regulated at 35 DAP were calcu- lated using GOEAST [20] and details of the results are pro- vided in additional file 2: Enriched_GO_terms_490_differentially_expressed_genes .xls. Within the category 'Biological Process', the GO terms 'developmental process', 'localization' and 'metabolic process' are enriched at both time-points. Generally, GO terms that are statistically enriched at the highly general information level 2 are similar for the two time-points. Comparison of statistically enriched GO terms for 490 differentially expressed genes at 23 and 35 DAP reveals their involvement in seed maturation, regulation of mer- istem organization and photomorphogenesis, seed coat Page 8 of 16 (page number not for citation purposes)