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Selection and validation of appropriate reference genes for gene expression studies in Forsythia

Abstract

The qRT-PCR method has been widely used to detect gene expression level in plants, helping to understand the molecular mechanisms. However, there are few researches which focus on the selection of the internal reference genes in Forsythia. To select the appropriate reference genes of Forsythia aimed at qRT-PCR normalization, twelve candidate reference genes were selected from our transcriptome data. Their expression was assessed by RT-PCR analysis in 47 Forsythia samples, including 12 species cultivars, different organs and tissues. GeNorm, NormFinder, and BestKeeper software were used to select the appropriate reference genes, AG and PSY were used to verify the accuracy of the outcome. The results showed that UKN1 was a stable reference gene in leaves of twelve Forsythia germplasms and in different developmental stages of fruits. MTP, ABCT + MTP, and ABCT + MTP + TIP were stable reference genes in different organs. ACT and SDH were stable reference genes in different flower tissues and different developmental stages of the flower buds. When Forsythia plants were stressed with PEG or ABA, SDH + UKN1 + G6PD was the stable reference gene group for qRT-PCR. The results provided the basis for investigating the physiological and biochemical processes of Forsythia related to medicinal and ornamental properties, and drought-resistance in the level of gene expression.

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Acknowledgements

The funding was supported by the World-Class Discipline Construction and Characteristic Development Guidance Funds for Beijing Forestry University (Grant No. 2019XKJS0323), Beijing Municipal Science and Technology Project (Grant No. Z181100002418006), Fundamental Research Fund for the Central University (Grant No. 2015ZCQ-YL-03), National Key Clinical Specialty Discipline Construction Program of China (CN), Hebei Provincial Department of science and Technology Project (Grant No. 18226316D), Key Technology Research and Development Program of Shandong (CN) (Grant No. HBCT2018060203).

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Correspondence to Huitang Pan.

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Figure S1

Expression stability, ranking and pairwise variation of 12 candidate reference genes in in different species, different cultivars of F. intermedia and F. koreana, and different style types by geNorm software. (A, Ranking in different Forsythia speciesl; B, Ranking in different cultivars of F. intermedia; C, Ranking in different cultivars of F.koreana; D, Ranking in species and cultivars with Style type ‘S’; E, Ranking in species and cultivars with Style type ‘L’) (TIFF 12317 kb)

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Shen, J., Wu, Y., Jiang, Z. et al. Selection and validation of appropriate reference genes for gene expression studies in Forsythia. Physiol Mol Biol Plants 26, 173–188 (2020). https://doi.org/10.1007/s12298-019-00731-y

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Keywords

  • qRT-PCR
  • Forsythia
  • Reference genes
  • Flower
  • Fruit
  • Drought stress