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Selection of suitable reference genes for qRT-PCR expression analysis of Codonopsis pilosula under different experimental conditions

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Abstract

Codonopsis pilosula is a well-known medicinal plant. Although its transcriptome sequence has been published, suitable reference genes have not been systematically identified for conducting expression analyses via quantitative real-time polymerase chain reaction (qRT-PCR). To screen appropriate genes for use with this species, we applied four different methods—GeNorm, NormFinder, BestKeeper, and RefFinder—to evaluate the stability of 13 candidates: CpiEF1Bb, CpiCACS, CpiF-Box, Cpiβ-Tubulin, CpiGAPDH, CpiActin2, CpiAPT1, CpiActin7, CpiActin8, CpiRPL6, CpiHAF1, CpiTubulin6, and CpiUBQ12. Expression was examined by qRT-PCR for various tissue types, chemical treatments, and developmental stages. For all tested samples, CpiGAPDH proved to be the most stable. Comprehensive analysis indicated that the most stable internal reference genes were CpiF-Box and CpiCACS in different tissues and at different developmental stages, respectively. Under NaCl stress, CpiAPT1 was the best internal reference gene. For methyl jasmonate and abscisic acid treatments, CpiGAPDH and CpiF-Box, respectively, presented the highest degree of expression stability. Based on these findings, we chose CpiSPL9 as the target gene for validating the suitability of these selected reference genes. All of these results provide a foundation for accurate quantification of expression levels by genes of interest in C. pilosula.

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Funding

This study was supported by the Major Project of Shaanxi Province, China (Grant No.2017ZDXM-SF-005) and The Youth Innovation Team of Shaanxi Universities.

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Authors and Affiliations

  1. Key Laboratory of the Ministry of Education for Medicinal Resources and Natural Pharmaceutical Chemistry, National Engineering Laboratory for Resource Development of Endangered Crude Drugs in Northwest of China, Shaanxi Normal University, Xi’an, 710062, China

    Jing Yang, Bin Li, Xiayang Lu, Xiaoyan Cao & Jiefang Kang

  2. Beijing Agro-Biotechnology Research Center, Beijing Academy of Agriculture and Forestry Sciences, Beijing, 100097, China

    Jing Yang, Xiaozeng Yang, Zheng Kuang & Xiayang Lu

  3. State Key Laboratory of Protein and Plant Gene Research, Peking-Tsinghua Center for Life Sciences, School of Life Sciences and School of Advanced Agricultural Sciences, Peking University, Beijing, 100871, China

    Zheng Kuang

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  1. Jing Yang
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  4. Bin Li
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11033_2020_5501_MOESM1_ESM.tif

Figure S1. Gel map of total RNAs in different samples. Lines 1–13: one-month-old whole seedling, two-month-old seedling, three-month-old seedling, five-month-old seedling, one-year-old plant, roots, stems, leaves, flowers, NaCl stress, MeJA treatment, ABA treatment, and control, respectively.Supplementary file1 (TIF 2025 kb)

Table S1. Nucleotide sequences of 13 candidate reference genes. Supplementary file2 (DOCX 21 kb)

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Yang, J., Yang, X., Kuang, Z. et al. Selection of suitable reference genes for qRT-PCR expression analysis of Codonopsis pilosula under different experimental conditions. Mol Biol Rep 47, 4169–4181 (2020). https://doi.org/10.1007/s11033-020-05501-8

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