Locating Appropriate Reference Genes in Heteroblastic Plant Ottelia cordata for Quantitative Real-Time PCR Normalization

Selecting the right reference genes for data normalization is the only way to ensure the precision and reproducibility of gene expression measurement using qRT-PCR. Ottelia cordata is a member of the Hydrocharitaceae family in aquatic plants that exhibits both floating and submerged leaf forms. It has recently drawn interest as a possible model plant for research into non-KRANZ C4 photosynthesis and heteroblastic leaves. Our earlier research has demonstrated bias in gene expression analysis when actin or GAPDH, two common reference genes, are used for normalization. Furthermore, there has been no study on the Hydrocharitaceae family reference gene selection published to date. To standardize qRT-PCR in O. cordata, seven genes were chosen from a transcriptome database: ACT7, EF1_α, GAPDH, BRCC36, PP2A, UBC7, and UBQ. We conducted qRT-PCR experiments in various tissues, leaves in different developmental stages, leaves in high/low carbon treatment, and leaves in high/low temperature treatment. After analyzing the stability using five statistical methods (geNorm, normFinder, comparative ΔCt, bestKeeper, and comprehensive analysis), PP2A and UBQ were identified as the most stable genes. BRCC36 was identified as a new reference gene in plants. Finally, by contrasting the expression patterns of pepc2, a crucial gene connected to C4 photosynthesis, in floating and submerged leaves, PP2A, UBQ, and UBC7 were verified. Of these, PP2A and UBQ were shown to be the superior gene for the precise qRT-PCR data normalization. The results of this study offer the initial information concerning reference gene identification for O. cordata as well as the first data in Hydrocharitaceae plants. It will make it easier to do more gene function and molecular biology research on O. cordata and other closely related species.