Cao, H.; Li, H.; Lu, L.; Ji, Y.; Ma, L.; Li, S. Screening and Validation of Internal Reference Genes for Quantitative Real-Time PCR Analysis of Leaf Color Mutants in Dendrobium officinale. Genes2023, 14, 1112.
Cao, H.; Li, H.; Lu, L.; Ji, Y.; Ma, L.; Li, S. Screening and Validation of Internal Reference Genes for Quantitative Real-Time PCR Analysis of Leaf Color Mutants in Dendrobium officinale. Genes 2023, 14, 1112.
Cao, H.; Li, H.; Lu, L.; Ji, Y.; Ma, L.; Li, S. Screening and Validation of Internal Reference Genes for Quantitative Real-Time PCR Analysis of Leaf Color Mutants in Dendrobium officinale. Genes2023, 14, 1112.
Cao, H.; Li, H.; Lu, L.; Ji, Y.; Ma, L.; Li, S. Screening and Validation of Internal Reference Genes for Quantitative Real-Time PCR Analysis of Leaf Color Mutants in Dendrobium officinale. Genes 2023, 14, 1112.
Abstract
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 both 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 will pave the way for molecular dissection of leaf color mutations in D. officinale.
Keywords
Dendrobium officinale; leaf color; qRT-PCR; reference gene; leaf color mutant
Subject
Biology and Life Sciences, Plant Sciences
Copyright:
This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
