BMC Genomics 2009, 10:295 http://www.biomedcentral.com/1471-2164/10/295 (37,182 to 41,806 tags per million). The four A. thaliana 2S seed storage proteins share 75–91% amino acid (aa) sequence identity with each other. The four 2S seed stor- age protein genes and 7 other protease inhibitor/seed stor- age/lipid transfer protein (LTP) family protein genes were also among the genes with the highest tag counts matched in antisense orientation (see additional file 3: All_matched_tags.xls). For comparison of SAGE results with quantitative Real- time RT-PCR detection, two sets of primers and their anti- sense reverse-complemented sequences were derived from SAGE primer amplified napin 3'-termini (see above) and from assembled contigs of 8,977 B. napus ESTs align- ing with an e-value cut-off of 1e-6 to A. thaliana 2S seed storage protein 1 (AT4G27140). One primer set (napin) was designed specifically for the major cluster of B. napus assembled EST contigs most similar to the A. thaliana 2S seed storage proteins 1–4 (59% to 70% on the aa level). Another set of primers (napin-related) was designed spe- cifically for the minor cluster of B. napus assembled EST clusters grouping in-between the four A. thaliana 2S seed storage proteins and the related protease inhibitor/seed storage/lipid transfer (LTP) family protein AT5G54740 (about 47% to 57% identity on the aa level). AT5G54740 represents the next closely related protease inhibitor/seed storage/lipid transfer protein (LTP) family protein gene in A. thaliana, which like other more distantly related pro- teins of this large family (about 113 members in A. thal- iana) exhibits an AAI_SS domain and shares about 53– 57% aa identity with the A. thaliana 2S seed storage pro- teins 1–4. Figure 3 shows the expression profile from days 17 to 70 after pollination from seeds for sense and anti- sense napin and napin-related transcript expression. The coexpression of sense and antisense transcripts was con- firmed for a minor subgroup of napin-related transcripts by strand-specific Real-time RT-PCR, but not for the major napin transcript group as indicated by LongSAGE. Discussion B. napus and A. thaliana are members of the Brassicaceae family and exhibit around 87% conservation of their pro- tein-coding sequences [22]. Based on this close phyloge- netic relationship, the tag-to-gene matching strategy applied in this study for LongSAGE expression profiling in the non-model plant B. napus uses the information avail- able for the well-annotated proteome of the model species A. thaliana. Our results show that expression profiling can be achieved for B. napus seeds using publically available EST resources and the annotated proteome of A. thaliana for tag-to-gene assignment. Major factors influencing the accuracy of the first step of the SAGE analysis are the sequencing error rate, the tag length and the transcriptome complexity. The accuracy of SAGE depends on the ability to unambiguously match the tags to the genes of origin. Theoretical calculations show that >99.8% of 21 bp LongSAGE tags are expected to occur only once in large genomes such as the human genome, which is about twice the size of the B. napus genome, with the remaining tags matching duplicated genes or repeated sequences [9]. In a polyploid plant genome the propor- tion of duplicated genes is generally considerably higher, and our strategy of matching tags via ESTs to a related model plant proteome will not per se allow differentiation between closely related duplicated genes. However, matching to multiple members of gene families still allows biological inferences from the LongSAGE data, since the predicted function in each case is identical. In our case only around 90% of LongSAGE tags were assigned to a unique A. thaliana gene. This underlines the highly duplicated nature of the B. napus and A. thaliana genomes [17] and reflects the well-documented ancient hexaploidization events in major angiosperm phyla [23]. Despite this we found no example where a B. napus tag matched multiple A. thaliana genes with apparently unre- lated functions. This suggests that the matching strategy we applied is specific enough with 21 bp B. napus tags to draw biological conclusions on the expression levels of functionally related gene families in B. napus seeds. Rais- ing the blastx e-value stringency to 1e-100 for matching of Brassica ESTs to the A. thaliana proteome was found to reduce the average number of genes per gene family to which the observed tags were assigned. However, this also resulted in a higher number of tags that could no longer be assigned to A. thaliana genes and thus could not be annotated (data not shown). For LongSAGE analysis from B. napus we found an e-value of >1e-6 useful; this is also the cut-off value used by NCBI in the UniGene database for EST-to-protein matching to create clusters of transcript sequences that are expected to come from the same tran- scription locus. The development of oilseeds can be broadly divided into three phases, (1) the pattern formation/cell proliferation phase, (2) the maturation phase, with lipid and storage protein accumulation, and (3) the desiccation phase. At 23 DAP B. napus seed has reached the end of phase 1 and is beginning the transition to phase 2, while at 35 DAP the seed is in the middle of the maturation phase. The most abundantly expressed gene detected at 23 DAP, SAG12, is a cysteine protease expressed during leaf senescence in A. thaliana [24]. In B. napus SAG12 is encoded by two orthol- ogous copies, BnSAG12-1 and BnSAG12-2, involved in leaf senescence and seed development, respectively. BnSAG12-2 and BnCysP1, a cysteine protease with around 91% identity to BnSAG12-2, are expressed in the early phase of B. napus seed development within the inner integument of the testa and are predicted to function in the disposal of proteins in the inner integument cells committed to programmed cell death [25]. Other genes encoding proteases and protease inhibitor genes are also Page 11 of 16 (page number not for citation purposes)