Evaluation of suitable qRT-PCR normalization genes for various citrus rootstocks

Citrus rootstock can modify plant growth and enhance stress resistance. There are many genotypes and species used as citrus rootstocks. Although multiple citrus rootstocks whole genome sequence and transcriptome databases have previously been published, no suitable internal reference genes have been investigated for standardization of gene expression via quantitative real-time PCR. Here we reported the first systematic and comprehensive analysis of reference genes for quantitative real-time PCR standardization in various citrus rootstocks. The expression stability of ten candidate reference genes in diverse sample subsets of flooding, drought, alkaline and cold treatments was evaluated using four statistical algorithms, geNorm, NormFinder, BestKeeper and Delta Ct method. The results demonstrated that the expression stability of reference genes varied under different experimental conditions. In addition, normalization of gene expression of 9-cis-epoxycarotenoid dioxygenase 3 (NCED 3), involved in abscisic acid biosynthesis, was conducted to further confirm the reliability of the reference genes. Overall, EF-1 alpha, DIM 1, GAPC and UBC expressed much more stably. ACTIN and GAPDH were not recommended for normalization in given experimental conditions due to low stability. Our main contribution was to identify reference genes with suitable and stable expression in citrus rootstocks varied across selected experimental conditions. Furthermore, these results will provide valuable information for future research on citrus rootstocks.