Comparative analysis of gene expression among species of different ploidy.

Interspecific comparative studies require that expression data be comparable among species, and when species with different levels of ploidy are contemplated the relative expression per cell should be obtained for accurate comparisons to be made. Quantitative reverse-transcription-PCR is the most popular and sensitive technique for the detection and quantification of mRNA in gene expression analysis. In recent years it has become clear that the choice of reference genes for the normalization of expression data is very important. Several studies have shown that the expression of the traditional housekeeping genes varies under certain situations; their use as reference genes in quantitative PCR assays can therefore lead to errors when interpreting the relative expression of target genes. Normalizing with respect to endogenous genes showing a constant level of expression per cell across species, however, provides an easy way of obtaining comparable expression data for other genes in those species. In this work, the validity of several candidate genes was examined across four diploid and polyploid species of the genera Triticum and Aegilops. Candidate reference genes were chosen among the traditional housekeeping genes used in quantitative PCR analysis, as well as others found to have stable levels of expression under different conditions in other studies. After the analyses, candidate genes were gathered into two groups according to the different levels of expression per cell seen in polyploid species. For the four species studied, two genes suitable for normalization procedures in interspecific studies were identified: cell division control protein and malate dehydrogenase. Both showed a constant number of transcripts per cell, independent of the level of ploidy.