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| Screening and Stability Evaluation on qPCR Reference Genes of Camellia spp. |
| NIE Rui-Min, ZHAO Wan-Yue, CHEN Shao-Yuan, HU Yun-Chong, CHEN Sheng-Tong, CHEN Long-Qing, GENG Fang* |
| College of Landscape Architecture and Horticulture Sciences/Southwest Landscape Architecture Engineering Technology Research Center of National Forestry and Grassland Administration/Yunnan Functional Flower Resources and Industrialization Technology Engineering Research Center, Southwest Forestry University, Kunming 650224, China |
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Abstract With the increasing research on the molecular biology of Camellia spp., it is crucial to screen the reference genes suitable for qPCR analysis of Camellia spp.. 3 Camellia varieties with different ornamental values were selected as experimental materials for this study. 7 common reference genes, including elongation factor-α (EF-1α), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), Actin 7 (ACT), α- tubulin (TUA), β-tubulin (TUB), protein phosphatase 2C (PP2C), and phosphatase activator TIP41 (TIP41), were detected using reverse transcription polymerase chain reaction (RT-PCR) and qPCR in various Camellia spp. flower tissues and flower developmental periods. The stability of the candidate genes was analyzed using ΔCt, geNorm, NormFinder and BestKeeper programs, and RefFinder was used for comprehensive evaluation to screen out the candidate reference genes suitable for qPCR in Camellia spp., and to verify the stability of the reference genes by the key genes of flower color and floral aroma biosynthesis. The results showed that the average expression of EF-1α was found to be higher than that of other candidate reference genes. The analysis results from ΔCt, NormFinder and BestKeeper all showed that EF-1α was the most stable in different tissues and stages of Camellia spp. flower development, while the results from geNorm showed that GAPDH and TIP41 were more stable. Additionally, the results from all programs showed that ACT was the least stable reference gene. Based on the comprehensive evaluation of RefFinder, EF-1α and ACT were validated by the expression pattern analysis of the floral color genes, chalcone synthase (CrCHS) and chalcone isomerase (CrCHI), and the floral scent genes, 1-deoxy-D-xylulose 5-phosphate synthase (ChDXS1) and 1-deoxy-D-xylulose 5-phosphate reductoisomerase (ChDXR). The findings demonstrated that EF-1α with superior stability could be chosen as an internal reference gene in the study of Camellia spp. floral color and floral scent. The results of the study provide a theoretical basis and technical support for further research on the functional genes related to the formation process of Camellia spp. floral color and floral scent.
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Received: 14 March 2025
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Corresponding Authors:
*fanggeng20@swfu.edu.cn
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