Identification of reference genes for gene expression normalization in safflower (Carthamus tinctorius)

Safflower (Carthamus tinctorius L., Asteraceae) is an important oil crop and medicinal plant. Gene expression analysis is gaining importance in the research of safflower. Quantitative PCR has become a powerful method for gene study. Reference genes are one of the major qualification requirements of qPCR because they can reduce the variability. To identify the reference genes in safflower, nine candidate genes of the housekeeping genes were selected from the EST library of safflower constructed by our lab: CtACT (actin), CtGAPDH (glyceraldehyde 3-phosphate dehydrogenase), CtElF4A (elongation factor 1 alpha), CtTUA (alpha-tubulin), CtTUB (beta-tubulin), Ct13132A (serine/threonine-protein phosphatase), CtElF4A (eukaryotic initiation factor 4A), CtUBI (Ubiquitin), and Ct6OS (60S acidic ribosomal protein). Expression stability was examined by qPCR across 54 samples, representing tissues at different flowering stages and two chemotype of safflower lines. We assessed the expression stability of these candidate genes by employing four different algorithms (geNorm, NormFinder, Delta Ct approach, and BestKeeper) and found that CtUBI and Ct6OS were the highly ranked candidate genes. CtUBI and Ct6OS were used as reference genes to evaluate the expression of CtFAD2-10 and CtKASII. Our data suggest CtUBI and Ct6OS could be used as internal controls to normalize gene expression in safflower. (C) 2016 Sociedade Brasileira de Farmacognosia. Published by Elsevier Editora Ltda. This is an open access article under the CC BY-NC-ND license.