Suitable reference genes for accurate gene expression analysis in Papaver rhoeas under 2,4-D herbicide stress

Resistance to 2,4-D (2,4-diclorophenoxyacetic acid) herbicide is increasing in various dicotyledonous weed species, including Papaver rhoeas, a weed infesting Southern European wheat crops. Non-target-site resistance to this herbicide is governed by a range of genes involved in herbicide stress response. To enable reliable measurement of gene expression levels in herbicide-resistant and susceptible plants it is necessary to normalize qPCR data using internal control genes with stable expression. In an attempt to find the best reference genes, the stability of seven candidate reference genes was assessed in plants resistant and susceptible to 2,4-D, subjected or not to herbicide stress. Using three statistical algorithms (geNorm, BestKeeper and NormFinder), the overall results revealed that glyceraldehyde-3-phosphate dehydrogenase, actin and ubiquitin were the most stable reference genes. The normalization expression levels of GH3 (indole-3-acetic acid amido synthetase) and GST3 (glutathione S-transferase) which are two genes up-regulated following 2,4-D treatment, were determined to verify the stability of these selected reference genes. A sudden increase in GH3 and GST3 expression was already detected 5 h after herbicide application, confirming their involvement in plant response to 2,4-D. The validation results confirmed the applicability and accuracy of these reference genes. This study identified and validated reference genes in the non-model weed species P. rhoeas and these will facilitate gene expression analysis studies aimed at identifying functional genes associated with non-target-site resistance.