Assessment of reference genes for quantitative real-time PCR gene expression normalization in periwinkle during Wheat Blue Dwarf phytoplasma infection

Quantitative real-time PCR (qRT-PCR) has becoming a widely used method for detection and characterization of gene expression in recent years. However, the accuracy of the qPCR output strongly depends on the performance of selected internal reference genes. In this study, we used periwinkle as material to select the most stably expressed reference genes during Wheat Blue Dwarf (WBD) phytoplasma infection in a spatial and temporal way. Ten candidate reference genes were selected, and their expression stability were evaluated using the algorithms geNorm and NormFinder program. Our results demonstrated that all of selected reference genes did not express stably enough across all of the experimental groups, the suitable reference genes should be selected according to the experiment conditions. In the present study, CrL23 performed well when all samples were considered. CrTUB and CrACT, CrEF1 alpha and CrACT were the most stable reference genes for samples from different tissues of periwinkle according to the outcome of geNorm and NormFinder, respectively. In addition, CrTUB and CrTUA were the most stable reference genes for flower malformation during WBD phytoplasma infection. Furthermore, to illustrate the usefulness of the most stable reference genes, the expression pattern of CrDEF involving in flower abnormality during WBD phytoplasma infection was analyzed. Taken together, the present study is valuable for future research on gene expression of periwinkle infected by WBD phytoplasma and maybe other phytoplasmas.