Reference gene selection for qPCR gene expression analysis of rust-infected wheat

Real-time PCR (qPCR) is an effective method to quantify mRNA levels, but requires validated reference genes for data normalisation. The GeNorm-Plus algorithm was used to examine the expression stability of six candidate reference genes in resistant Avocet Yr1 wheat infected with Puccinia triticina, Puccinia striiformis and Puccinia graminis f.sp. tritici respectively. Results indicated that within the first 48 h after inoculation, the expression stability of the candidate reference genes differed between the three incompatible interactions. The geometric mean of ARF and RLI showed the best stability in P. triticina-infected wheat, CDC and RLI in P. striiformis-infected wheat and CDC, 18S and TUBB in P. graminis f.sp. tritici-infected wheat respectively. This clearly emphasised the need for reference gene validation for each different plant–pathogen interaction.

► Wheat was infected with three different Puccinia species. ► Examined the stability of six candidate reference genes in infected wheat. ► Appropriate reference genes differed between infections.