Identification of suitable reference genes for normalization of qPCR data in comparative transcriptomics analyses in...

Comparative transcriptomics are useful to determine the role of orthologous genes among Triticeae species. Thus they constitute an interesting tool to improve the use of wild relatives for crop breeding. Reverse transcription quantitative real-time PCR (qPCR) is the most accurate measure of gene expression but efficient normalization is required. The choice and optimal number of reference genes must be experimentally determined and the primers optimized for cross-species amplification. Our goal was to test the utility of wheat-reference genes for qPCR normalization when species carrying the following genomes (A, B, D, R, H ( v ) and H ( ch )) are compared either simultaneously or in smaller subsets of samples. Wheat/barley/rye consensus primers outperformed wheat-specific ones which indicate that consensus primers should be considered for data normalization in comparative transcriptomics. All genes tested were stable but their ranking in terms of stability differed among subsets of samples. CDC (cell division control protein, AAA-superfamily of ATPases, Ta54227) and RLI (68 kDa protein HP68 similar to Arabidopsis thaliana RNase L inhibitor protein, Ta2776) were always among the three most stable genes. The optimal number of reference genes varied between 2 and 3 depending on the subset of samples and the method used (geNorm vs. coefficient of determination between sequential normalization factors). In any case a maximum number of three reference genes would provide adequate normalization independent of the subset of samples considered. This work constitutes a substantial advance towards comparative transcriptomics using qPCR since it provides useful primers/reference genes.