Screening and Validation of Internal Reference Genes for Quantitative Real-Time PCR Analysis of Leaf Color Mutants in Dendrobium officinale

Leaf color mutants (LCMs) are important resources for studying diverse metabolic processes such as chloroplast biogenesis and differentiation, pigments’ biosynthesis and accumulation, and photosynthesis. However, in Dendrobium officinale, LCMs are yet to be fully studied and exploited due to the unavailability of reliable RGs (reference genes) for qRT-PCR (quantitative real-time reverse transcription PCR) normalization. Hence, this study took advantage of previously released transcriptome data to select and evaluate the suitability of ten candidate RGs, including Actin (Actin), polyubiquitin (UBQ), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), elongation factor 1-α (EF1α), β-tubulin (β-TUB), α-tubulin (α-TUB), 60S ribosomal protein L13-1 (RPL13AD), aquaporin PIP1-2 (PIP1-2), Intima protein (ALB3) and Cyclin (CYCB1-2) for normalizing leaf color-related genes’ expression levels via qRT-PCR. Stability rankings analysis via common software Best-Keeper, GeNorm, and NormFinder disclosed that all ten genes met the requirements of RGs. Of them, EF1α exhibited the highest stability and was selected as the most reliable. The reliability and accuracy of EF1α were confirmed through qRT-PCR analysis of fifteen chlorophyll pathway-related genes. The expression patterns of these genes via EF1α normalization were consistent with the results by RNA-Seq. Our results offer key genetic resources for the functional characterization of leaf color-related genes and will pave the way for molecular dissection of leaf color mutations in D. officinale.