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. 2023 Dec 27:14:1287589.
doi: 10.3389/fpls.2023.1287589. eCollection 2023.

Selection of suitable reference genes for qPCR normalization in different developmental stages of Oenanthe javanica

Kai Feng #  1 , Zhi-Yuan Yang #  1 , Ya-Jie Yan  1 , Nan Sun  1 , Zi-Qi Zhou  1 , Jia-Lu Liu  1 , Shu-Ping Zhao  1 , Peng Wu  1 , Liang-Jun Li  1   2
Affiliations

Selection of suitable reference genes for qPCR normalization in different developmental stages of Oenanthe javanica

Kai Feng et al. Front Plant Sci. .

Abstract

Gene expression analysis is widely used to unravel molecular regulatory mechanisms and identify key genes in plants. Appropriate reference gene is an important prerequisite to ensure the accuracy and reliability of qPCR analysis results. Water dropwort is a plant of the Oenanthe genus in the Apiaceae family, which has high economic benefits. However, the underlying molecular regulatory mechanisms in the growth and development of water dropwort have not been fully understood and the appropriate reference genes in different developmental stages of water dropwort not yet reported. In this study, 10 candidate reference genes (ACTIN, PP2A, SAND, EF-1α, GAPDH, UBQ, MIP, TBP, RPS-18, eIF-4α) were identified and cloned from Oenanthe javanica. The qPCR primers of candidate reference genes were designed and verified. Four statistical algorithms, geNorm, NormFinder, BestKeeper and RefFinder were used to evaluate the expression stability of 10 candidate reference genes in different developmental stages of water dropwort. The results showed that TBP and UBQ were the most stable genes in different developmental stages of water dropwort, while GAPDH was the most unstable gene. The normalization of EXP1 genes at different developmental stages further confirmed the reliability of internal reference genes. The results of this study provide a theoretical basis for selecting appropriate internal reference genes in different developmental stages of water dropwort. This study also provides technical support and reliable basis for the expression analysis of key genes in different developmental stages of water dropwort.

Keywords: RT-qPCR; development stages; growth stages; reference genes; water dropwort.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Growth status of water dropwort at seven developmental stages. Stage 1, Water dropwort callus; Stage 2, Water dropwort regenerate seedlings; Stage 3, Growth and development for 30 days; Stage 4, Growth and development for 45 days; Stage 5, Growth and development for 60 days; Stage 6, Growth and development for 75 days; Stage 7,Growth and development for 90 days.
Figure 2
Figure 2
Cq values of candidate reference genes in all samples of water dropwort. The line across the box depicts median. The inside box depicts mean. The outside box is determined by the 25th and 75th percentiles. The whiskers are determined by the 5th and 95th percentiles.
Figure 3
Figure 3
Statistic analysis (maximum, minimum, mean, and median) of Cq values of ten candidate reference genes in all samples.
Figure 4
Figure 4
Measurement(M) of expression stability of 10 candidate reference genes in samples at different developmental stages using geNorm. The most unstable genes are on the left and the most stable genes are on the right. S1-S7: The seven stages of water dropwort development. Total represents samples from the above seven experimental conditions.
Figure 5
Figure 5
geNorm analysis of paired variation V values of ten candidate reference genes. Vn/n + 1 values are used to determine the optimal number of reference genes. S1-S7: The seven stages of water dropwort development. Total represents samples from the above seven experimental conditions.
Figure 6
Figure 6
The relative expression levels of EXP1 gene normalized by different internal reference genes. (A) The relative expression of EXP1 when UBQ is used as an internal reference gene. (B) The relative expression of EXP1 when TBP is used as an internal reference gene. (C) The relative expression of EXP1 when UBQ and TBP are used as internal reference genes. (D) The relative expression of EXP1 when GAPDH is used as an internal reference gene. (E) The relative expression of EXP1 when UBQ and GAPDH are used as internal reference genes. (F) The relative expression of EXP1 when TBP and GAPDH are used as internal reference genes. S1-S7: The seven stages of water dropwort development.
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