Selection of reference genes for flowering pathway analysis in the masting plants, Celmisia lyallii and Chionochloa pallens, under variable environmental conditions

Mast flowering is characterised by mass synchronised flowering at irregular intervals over a wide geographical area. An understanding of the molecular drivers of mast flowering requires expression analysis of key developmentally regulated gene(s). Reverse transcription-quantitative PCR is the gold standard technique used to assess expression of target gene(s) and to validate high-throughput sequencing data. Selection and validation of appropriate reference gene(s), used as normalisation factors in transcript abundance analysis, is an essential step to avoid ambiguous expression results. Eight candidate reference genes were assessed to select the best internal normalisation factors in naturally growing masting plants Chionochloa pallens and Celmisia lyallii. Statistical packages geNorm, Normfinder, BestKeeper, Delta C-t and RefFinder were used to determine the expression stability in plants translocated to different altitudes and sampled across the season. GAPDH and PP2 alpha in Celmisia and ExP and THP in Chionochloa were found to be the best pairs of reference genes for normalisation of the gene expression data. Our study revealed environmentally-induced changes in reference gene expression, information that will be utilised as we investigate flowering phenology of masting plants under global climatic change.