Exploring reliable reference genes for gene expression normalization in Karenia mikimotoi using real-time PCR

The dinoflagellate Karenia mikimotoi is one of the most noxious species that can form harmful algal blooms in coastal areas worldwide, causing serious damage to aquaculture and fisheries. In the present study, twelve commonly reported candidate genes (cob, cyc, eif4e, ef2, mdh, sam, alpha-tub, beta-tub, actin, cal, 18s, and gapdh) were selected to evaluate their expression stability under different temperatures, salinities, phosphorus and nitrogen limitation, and different time points over a diel cycle in K. mikimotoi. In addition, three algorithms (geNorm, NormFinder, and BestKeeper) were used to evaluate and select the most suitable reference genes. It was observed that alpha-tub and eif4e were the most suitable reference genes for all samples. In contrast, ef2 and 18s genes showed least stability. Further, a list of stable genes, including alpha-tub, eif4e, gapdh, and mdh was generated that can be used as reference genes in specific environmental condition in K. mikimotoi. The gene expression profile of KFcp gene was evaluated over a diel cycle using the most stable reference genes. Results revealed that the expression of KFcp with transcripts abundances was higher in light period and lower in dark period, indicating similar gene expression profile of this gene in typically reported algae. This study provides suitable reference genes for further research on the expression and regulation of genes related to important biochemical processes in K. mikimotoi, improving our understanding of the molecular mechanism of its behind toxin secretion and bloom formation