Evaluation of candidate reference genes for normalization of RT-qPCR data in wheat during pre-haustorial stages of Puccinia triticina infection

Expression of five wheat (Triticum aestivum L.) housekeeping genes (HKGs), coding for ubiquitin (TaUBI, AY297059.2), elongation factor-1α (TaEF1α, BT009129.1), 18S ribosomal RNA (Ta18SrRNA, HG670306.1), glucose-6-phosphate dehydrogenase (TaG6PDH, AB029454) and actin (TaACT, AB181991), was analyzed using BestKeeper and NormFinder algorithms and genes with superior expression uniformity were identified in wheat leaves during pre-haustorial (early) stages of T. aestivum-Puccinia triticina (Pt) interaction. Results of expression stability analyses of the HKGs confirmed highly stable expressions of TaUBI and TaEF1α. The expression of TaACT was moderately stable and those of TaG6PDH and Ta18SrRNA were the least stable. Therefore, the HKGs TaUBI and TaEF1α are superior reference genes for normalization of gene expression during T. aestivum-Pt interaction studies, whereas the use of Ta18SrRNA as reference genes should be avoided in such studies. Furthermore, validation of expression stability of the best reference gene identified (TaUBI) in a cultivar exhibiting an incompatible interaction showed results similar to those obtained in a compatible interaction when plants were mock-treated and inoculated with the fungus. In summary, this study identified two superior reference genes, TaUBI and TaEF1α, useful for analyses of gene expression during pre-haustorial stages of wheat leaf rust infection.