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

Hua Cao; Han Li; Lin Lu; Yulu Ji; Lulin Ma; Shenchong Li

doi:10.3390/genes14051112

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

Genes (Basel). 2023 May 19;14(5):1112. doi: 10.3390/genes14051112.

Authors

Hua Cao¹, Han Li¹, Lin Lu¹, Yulu Ji², Lulin Ma¹, Shenchong Li³

Affiliations

¹ Flower Research Institute of Yunnan Academy of Agricultural Sciences, Kunming 650205, China.
² Fujian Forestry Science and Technology Experimental Center, Zhangzhou 363600, China.
³ National Engineering Technology Research Center for Ornamental Horticulture, No. 2238 Beijing Road, Kunming 650204, China.

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 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.

Keywords: Dendrobium officinale; leaf color; leaf color mutant; qRT-PCR; reference gene.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Actins / genetics
Dendrobium* / genetics
Gene Expression Profiling* / methods
Plant Leaves / genetics
Real-Time Polymerase Chain Reaction
Reproducibility of Results

Substances

Actins

Grants and funding

This research was funded by grants from Yunnan Province Science and Technology Department Technology Talent and Platform Program (202005AD160014), the Green Food Brand-Build a Special Project (Floriculture) supported by Science and Technology (530000210000000013742), Major science and technology project in Yunnan Province (202002AA100007), and Yunnan Province Seed Industry Joint Laboratory Project (202205AR070001-05).