Validation of suitable reference genes for qRT-PCR in cabbage (Brassica oleracea L.) under different abiotic stress experimental conditions

Abstract

Cabbage (Brassica oleracea) is one of the most important vegetable crops worldwide. qRT-PCR is a sensitive technique for gene expression studies and choosing the appropriate reference gene is essential to obtain reliable results. In the present work, 22 candidate reference genes were evaluated under various experimental conditions, including NaCl, drought stress treatment, temperature treatments (cold and heat) and a set of hormones stress (6-BA, NAA, and ABA) treatments, across a range of tissue types and cultivars. Gene expression data taken from 45 cabbage samples was analyzed using two algorithms, geNorm and NormFinder. Suitable combinations of reference genes for qRT-PCR normalization should be applied according to different experimental conditions. In this study, the genes EF1a, GAPC2 and SAND were verified as the suitable reference genes across all tested samples. Additionally, each experimental condition had a unique set of reference genes best suited to samples within the particular condition. To validate the suitability of the candidate reference genes, the gene expression of WSD1, a gene that may be involved in biosynthesis pathway of wax esters in cabbage, was measured across all 45 samples and normalized using the three best reference gene candidates. WSD1 displayed variation in gene expression across different tissues and cultivars, and exhibited diverse up- or down- regulated expression patterns under various treatments, which indicate that BoWSD1 may play an important role in the response to abiotic stresses in cabbage. Our results provide the foundation for gene expression analysis in Brassica oleracea and other species of Brassica vegetables.