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Selection and validation of reference genes of Paeonia lactiflora in growth development and light stress

Abstract

The stem of Paeonia lactiflora will bend when it grows in greenhouse at a low light intensity. It is important to explore causes of morphological changes of peony to improve its quality. Gene expression can be evaluated by quantitative real-time PCR, based on reference gene. However, systematic selection of reference genes under weak lighting for herbaceous peony is lacking. To address this problem, we first selected 10 candidate reference genes based on a coefficient of variation of gene expression from peony stem transcriptome data. Then, geNorm, NormFinder and BestKeeper were applied to assess the stability of the genes, and RankAggreg was used to give a comprehensive ranking. The results show that there are some differences in optimal reference genes among samples from different organs and under the two lighting conditions, and the optimal number of suitable reference genes is distinct. Two selected suitable reference genes were then used to normalize target genes, and the results were compared with transcriptome data. Consistent gene expression trends were obtained, indicating the reliability of the method. To the best of our knowledge, this is the first time reference genes for herbaceous peony were selected in different organs, developmental stages and under two kinds of lighting conditions. The findings can provide a practical method for selecting reference genes for peony under these conditions and demonstrate a useful combination of reference genes.

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Abbreviations

DFG:

Paeonia lactiflora ‘Da Fugui’

CTH:

Paeonia lactiflora ‘Chui Touhong’

qPCR:

Quantitative real-time PCR

CV:

Coefficient of variation

F-box:

F-box protein

GAPDH:

Glyceraldehyde-3-phosphate dehydrogenase

UBQ10:

Polyubiquitin

CYP:

Cyclophilin

ACT:

Actin/actin-like conserved site-containing protein

EF-α:

Elongation factor-1 alpha 3

AQU:

Probable aquaporin PIP1-2

ETI:

Eukaryotic translation initiation factor 5A-2

E3:

E3 ubiquitin protein ligase RIE1

pp2A:

Serine/threonine-protein phosphatase 2A 65 kDa regulatory subunit A beta isoform-like

Tm:

The melting temperature

Cq:

Quantification cycle

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Acknowledgements

This work was supported by Beijing Municipal Science & Technology Commission (No. D161100001916004) and National Natural Science Foundation of China (No. 31370693).

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Correspondence to Yan Liu.

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Wan, Y., Hong, A., Zhang, Y. et al. Selection and validation of reference genes of Paeonia lactiflora in growth development and light stress. Physiol Mol Biol Plants 25, 1097–1105 (2019). https://doi.org/10.1007/s12298-019-00684-2

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Keywords

  • Herbaceous peony
  • Reference gene
  • Light stress
  • F-box
  • GAPDH