Validation of reference genes as an internal control for studying Avena sativa-Puccinia coronata interaction by RT-qPCR

In this study we evaluated eleven candidate reference genes in Avena sativa during compatible and incompatible interactions with two different pathotypes of Puccinia coronata f. sp. avenae in six time points post-inoculation. The identification of genes with high expression stability was performed by four algorithms (geNorm, NormFinder, BestKeeper and Delta Ct method). The results obtained confirmed that the combination of two genes would be sufficient for reliable normalization of the expression data. In general, the most stable in the tested plant-pathogen system were HNR (heterogeneous nuclear ribonucleoprotein 27C) and EF1A (elongation factor 1-alpha). ARF (ADP-ribosylation factor) and EIF4A (eukaryotic initiation factor 4A-3) could also be considered as exhibiting high expression stability. CYP (cyclophilin) was shown by all assessment methods to be the worst candidate for normalization in this dataset. To date, this is the first report of reference genes selection in A. sativa-P. coronata interaction system. Identified reference genes enable reliable and comprehensive RT-qPCR analysis of oat gene expression in response to crown rust infection. Understanding the molecular mechanisms involved in the host-pathogen interactions may expand knowledge of durable resistance strategies beneficial to modern oat breeding.

Automated summary

This study evaluated eleven candidate reference genes in Avena sativa during interactions with two different pathotypes of Puccinia coronata f. sp. avenae. Four algorithms were used to identify genes with high expression stability. HNR and EF1A were found to be the most stable genes, while CYP was the least suitable for normalization. This is the first report on reference gene selection in the A. sativa-P. coronata interaction system.