Characterisation of the malting barley transcriptome

The introduction of large-scale gene expression technologies such as serial analysis of gene expression (SAGE) has provided the tools to significantly increase the amount of genetic data available. To characterise the transcriptional profile of grain during the malting process, eight LongSAGE libraries from Barley var. Tallon were constructed. One library was constructed from mature un-steeped seed and seven libraries were generated from barley grain at specific time points during malting, 0, 12, 24, 48, 72, 96 and 120 hours post steeping. A total of 155,206 LongSAGE tags, representing 41,909 different tags, were analysed. The one hundred most abundant tags from each library were examined in more depth to identify the putative functional role of highly abundant transcripts in barley seed germination. The largest functional groups include transcripts coding for stress response and cell defense, protein synthesis and storage proteins. Statistical analyses were implemented to determine which tags demonstrate significant differential expression across time. Eight of these genes were targeted for analysis by quantitative real-time reverse transcriptase PCR. Alpha-amylase, (1-3,1-4)-beta-D-glucanase, Cysteine proteinase and Isocitrate lyase show the most significant changes and are up-regulated between 12 and 48 hours post steeping and except for Isocitrate lyase, remain high until 96 hours post steeping. The gene expression patterns observed by real-time PCR show strong correlations with the expression levels identified by LongSAGE analysis for the same genes. This confirms the level of sensitivity able to be detected with SAGE technology and provides greater confidence in the data obtainable by high-throughput gene expression techniques.