Locating Appropriate Reference Genes in Heteroblastic Plant <i>Ottelia cordata</i> 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. <i>Ottelia cordata</i> 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 <i>actin</i> or <i>GAPDH</i>, 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 <i>O. cordata</i>, seven genes were chosen from a transcriptome database: <i>ACT7</i>, <i>EF1_α</i>, <i>GAPDH</i>, <i>BRCC36</i>, <i>PP2A</i>, <i>UBC7</i>, and <i>UBQ</i>. 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), <i>PP2A</i> and <i>UBQ</i> were identified as the most stable genes. <i>BRCC36</i> was identified as a new reference gene in plants. Finally, by contrasting the expression patterns of <i>pepc2</i>, a crucial gene connected to C₄ photosynthesis, in floating and submerged leaves, <i>PP2A</i>, <i>UBQ</i>, and <i>UBC7</i> were verified. Of these, <i>PP2A</i> and <i>UBQ</i> 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 <i>O. cordata</i> as well as the first data in Hydrocharitaceae plants. It will make it easier to do more gene function and molecular biology research on <i>O. cordata</i> and other closely related species.