Identification of Reference Genes for Normalizing RNA Expression in Potato Roots Infected with Cyst Nematodes

Potato cyst nematodes induce changes in plant host gene expression following root invasion. For an accurate comparison of gene expression by reverse transcription quantitative real-time PCR (RT-qPCR), internal reference genes are necessary for transcript normalization. Very few experimental data on suitable reference genes are available for interactions between plant and root pathogens. In this study, we tested eight potential candidate reference genes for normalizing levels of potato gene transcripts by RT-qPCR after inoculation by nematodes. The ranking of candidate reference genes was performed using RefFinder WEB-based software. Four reference genes, RPN7 (26S proteasome regulatory subunit), UBP22 (ubiquitin-specific protease 22), OXA1 (OXA1 protein), and MST2 (mercaptopyruvate sulfurtransferase), were stably expressed in roots of susceptible or resistant potato plants, infected or uninfected by Globodera pallida. A normalization factor based on data from these four genes, highly homologous between potato and tomato, was used to normalize the expression of a chitinase gene, which was induced by nematodes in roots of potatoes carrying the resistance allele at a low-effect QTL, GpaXI (spl) .