Identification of suitable reference genes for normalization of real-time quantitative PCR data in pecan (Carya illinoinensis)

Key message This study proposed the combination ofPR26SandPP1as a good choice for RT-qPCR normalization in pecan under abiotic stress, developing kernels, grafting, and various tissues. Reference gene selection is an essential pre-requisite to generate reliable results in real-time quantitative polymerase chain reaction (RT-qPCR) analysis. However, studies regarding systematic validation of suitable reference genes in pecan are still lacking. In this study, 17 candidate reference genes were selected and evaluated for their expression stabilities in pecan under various experimental conditions, including various tissues, developing kernels, grafting, and two plant tissues (leaves and roots) subjected to three abiotic stresses (salt, drought, and Zn deficiency). The stability of the candidate genes was assessed by geNorm, NormFinder, and BestKeeper, and their outputs were integrated to obtain a final comprehensive rank of stability based on the geometric mean. The results indicated that samples under different experimental conditions possessed their own best reference genes, and using two reference genes for RT-qPCR normalization was recommended for the tested experiments. Overall, the combination of26S protease regulatory subunit 7A(PR26S) andserine/threonine-protein phosphatase-1(PP1) was recognized as a good choice for RT-qPCR normalization in pecan across all the treatments. More importantly, the widely usedalpha-tubulin(alpha-TUB),ubiquitin(UBQ), andactin(ACT) genes were not the best suitable reference genes in most of our experiments. Our results will be helpful for future gene-expression studies in pecan.