Identification of reference genes for real-time quantitative PCR experiments in the liverwort Marchantia polymorpha

Abstract

Real-time quantitative polymerase chain reaction (qPCR) has become widely used as a method to compare gene transcript levels across different conditions. However, selection of suitable reference genes to normalize qPCR data is required for accurate transcript level analysis. Recently, Marchantia polymorpha has been adopted as a model for the study of liverwort development and land plant evolution. Identification of appropriate reference genes has therefore become a necessity for gene expression studies. In this study, transcript levels of eleven candidate reference genes have been analyzed across a range of biological contexts that encompass abiotic stress, hormone treatment and different developmental stages. The consistency of transcript levels was assessed using both geNorm and NormFinder algorithms, and a consensus ranking of the different candidate genes was then obtained. MpAPT and MpACT showed relatively constant transcript levels across all conditions tested whereas the transcript levels of other candidate genes were clearly influenced by experimental conditions. By analyzing transcript levels of phosphate and nitrate starvation reporter genes, we confirmed that MpAPT and MpACT are suitable reference genes in M. polymorpha and also demonstrated that normalization with an inappropriate gene can lead to erroneous analysis of qPCR data.

Fig 1. Range of transcript levels detected for each of the eleven candidate genes across all 22 conditions.

Average N₀ values per gene and condition (n = 3) were normalized on the smallest value then log transformed. Data were then represented for each gene as a box-plot.

Fig 2. Average variation in transcript levels for each candidate gene.

All 22 conditions tested (A, highlighted in blue), development group (B), abiotic stress group (C), and hormone group (D). Variation is represented as the average of z-scores derived from geNorm and NormFinder results. The lower the z-score, the more invariant the transcript levels between samples.

Fig 3. Estimation of the optimal number of reference genes required for accurate qPCR data normalization.

Estimation was done by calculating the pairwise variation (V_n/n+1) of the normalization factors NF_n and NF_n+1 as described in Vandesomple et al., [16].

Fig 4. Transcript levels of MpPHT1-1 and MpNRT2-1 after phosphate and nitrogen starvation.

Histograms represent the fold-change of the average transcript levels (n = 3) compared to the long-term control condition of MpPHT1-1 (A) and MpNRT2-1 (B). Data were non-normalized (‘no ref’) or normalized by a factor consisting of the geometric mean of the transcript level of MpAPT and MpACT (highlighted in blue), of MpACT and MpSAND, of MpACT, MpSAND and MpELF5, and only by the transcript level of MpGAPC1. Biological replicates are depicted superimposed with crosses, diamonds and squares, respectively for replicate one, two and three. NP stands for normal phosphate, LP for low phosphate, NN for normal nitrate, and LN for low nitrate media respectively. A ‘s’ indicates a short (24 h) treatment, a ‘l’ a long (17 days) treatment.