Selection of reference genes for normalization of mitochondrial gene expression by qRT-PCR in different potato tissues and during anther development

Potato is the most widely grown tuber crop worldwide and a staple food in many countries: it has become the focus of many molecular breeding studies. One topical area is breeding potato seeds, especially advancing male sterile plants, focusing on developing cytoplasmic male sterility (CMS) as a breeding tool. A major obstacle has been the identification of mitochondrial genes for CMS. Quantifying the expression of candidate CMS genes is a critical aspect needed for the validation of gene expression levels for all organisms, and quantitative real-time polymerase chain reaction (qRT-PCR) is a powerful tool for this purpose. However, selecting appropriate internal control genes for normalization of mitochondrial gene expression presents specific challenges. The aim of this study was to identify suitable reference genes best suited for analysis of mitochondrial gene expression in different tissues and developmental stages of potato, particularly in developing anthers. We assessed the expression of eighteen candidate internal control genes, including four previously studied nuclear reference genes and fourteen mitochondrial candidate reference genes. By studying gene expression in a range of tissues, the genes nad1 and nad2 were found to be the most stable reference genes, since they were expressed most consistently using four different analytical tools, GeNorm, Delta Ct, Bestkeeper and NormFinder. In contrast, expression levels of the conventional nuclear reference genes were more variable. The comprehensively ranked first candidate gene, nad2 is proposed as the preferred choice as a reference gene, especially when studying different stages of anther development. Notably, actin, the most widely used marker expression gene, worked well in some cases, but there was significant variation in its rankings, for example, using the Bestkeeper tool it was ranked sixth. The results indicate that nad1 and nad2 respectively were the most stably expressed marker genes in 8 different tissues and stages of anther development. This study provides valuable support for future research on mitochondrial gene expression in potato, specifically for identifying patterns of expression of CMS genes, and can be a valuable tool to quantify gene expression for other Solanaceae species.