Gene expression profiling via LongSAGE in a non-model plant species: A case study in seeds of Brassica napus . Brassica napus

Serial Analysis of Gene Expression (LongSAGE) was applied for gene expression profiling in seeds of oilseed rape (Brassica napus ssp. napus). The usefulness of this technique for detailed expression profiling in a non-model organism was demonstrated for the highly complex, neither fully sequenced nor annotated genome of B. napus by applying a tag-to-gene matching strategy based on Brassica ESTs and the annotated proteome of the closely related model crucifer A. thaliana. Transcripts from 3,094 genes were detected at two time-points of seed development, 23 days and 35 days after pollination (DAP). Differential expression in about 10 % of all matched genes, with a total abundance of 44 %, showed a shift from gene expression involved in diverse developmental processes including cell proliferation and seed coat formation at 23 DAP to more focussed metabolic processes including storage protein accumulation and lipid deposition at 35 DAP. The most abundant transcripts at 23 DAP were coding for diverse protease inhibitor proteins and proteases, including cysteine proteases involved in seed coat formation and a number of lipid transfer proteins involved in embryo pattern formation. At 35 DAP, transcripts encoding napin, cruciferin and oleosin storage proteins were most abundant. Over both time-points, 18.6 % of A. thaliana genes were matched by Brassica ESTs detected by LongSAGE tags in antisense orientation. This suggests a strong involvement of antisense transcript expression in regulatory processes during B. napus seed development. Overall design: Seeds from 2 developmental stages of B. napus were used to construct 2 LongSAGE libraries, 23 days after pollination (23 DAP) and 35 days after pollination (35 DAP). Biological replicates and confirmation: Cloning of tag-amplified RT-PCR products, Real-time RT-PCR