Differential LongSAGE tag abundance analysis in a barley seed germination time course and validation with relative real-time RT-PCR

A Long Serial Analysis of Gene Expression (LongSAGE) approach was employed to identify changes in mRNA transcript abundance in a time course of malting barley (Hordeum vulgare L. ). Statistical analyses confidently identified 57 LongSAGE sequence tags as having significant changes in abundance between points in the time course. Eight of the genes which correspond to these tags were targeted for validation by relative real-time reverse transcriptase PCR (RT-PCR) analysis. Each gene was analysed by SYBR® Green detection in grain sampled at each of four time points from dry seed to grain germinated to 120 h post-steeping. Among the genes examined are alpha-amylase type B, a key starch degrading enzyme involved in germination (1-3,1-4)-beta-d-glucanase, the major cell wall degrading enzyme, and cysteine proteinase EP-B, an important enzyme of proteolysis in barley seed germination. These three transcripts show significant up-regulation at 48 h post-steeping and remain significantly elevated throughout malting. These results provide transcriptional data to support the understanding of how the relative rates of protein and carbohydrate modification contribute to malting and brewing. mRNA abundance levels observed using real-time RT-PCR show good correlation with the data obtained from the LongSAGE study. This confirms the sensitivity of detection obtainable with SAGE technology and validates the patterns of change of transcript abundance exhibited by some key genes for barley seed germination.