Molecular Biology Reports

, Volume 46, Issue 6, pp 6003–6011 | Cite as

Selection of reliable reference genes for gene expression analysis in seeds at different developmental stages and across various tissues in Paeonia ostii

  • Chaoqiong LiEmail author
  • Lizong Hu
  • Xueqin Wang
  • Hongzhan Liu
  • Huihui Tian
  • Junsheng Wang
Original Article


Paeonia ostii seeds have recently been identified as a new source of α-linolenic acid in China. Studying the gene expression patterns of unsaturated fatty acid-related genes would be helpful for understanding the mechanism of α-linolenic acid accumulation. Quantitative real-time polymerase chain reaction (qRT-PCR) is a useful method for reliably evaluating gene expression, and it is necessary to select reliable reference genes for data normalization in qRT-PCR analysis. In this study, we evaluated the expression stability of 12 candidate reference genes using four mathematical algorithms (∆Ct, BestKeeper, NormFinder, and geNorm). The web-based tool RefFinder was used to integrate the results and to provide a comprehensive ranking order. The expression stability ranking orders of reference genes were different caculated by these four algorithms, and the ranking order analyzed by the RefFinder was UBQ > Tip41 > UCE > EF-1α > α-TUB > PP2A > ACT > GAPDH > SAM > CYP > β-TUB > 18S at the different seed development stages, and UBQ > Tip41 > EF-1α > α-TUB > PP2A > UCE > GAPDH > SAM > ACT > CYP > 18S > β-TUB in P. ostii tissues. UBQ and Tip41 are the two most stable whereas 18S and β-TUB are the two least stable reference genes for gene expression in various tissues and seeds at different developmental stages in P. ostii.


Paeonia ostii Reference gene Normalization qRT-PCR Seed development 



This work was supported by the National Natural Science Foundation of China (31500534), the Startup Fund for Advanced Talents of Zhoukou Normal University (ZKNU2014108), the School-Based Program of Zhoukou Normal University (ZKUNB115203), the Natural Science Foundation of Henan province (162300410346), and the Key Scientific Research Project in Colleges and Universities of Henan Province (15A180024).

Author contributions

CL and LH designed research; CL, LH, XW, HL, HT and JW performed experiments. CL and LH wrote the paper.

Compliance with ethical standards

Conflict of interest

All authors declare that they have no conflicts of interest.

Supplementary material

11033_2019_5036_MOESM1_ESM.tif (1.7 mb)
Electronic supplementary material 1 (TIFF 1728 kb)Fig. S1 Pairwise variation analysis by geNorm to determine the optimal number of reference genes needed for accurate normalization in seeds at different developmental stages and across various tissues in Paeonia ostii. Cutoff value = 0.15
11033_2019_5036_MOESM2_ESM.docx (17 kb)
Electronic supplementary material 2 (DOCX 17 kb)
11033_2019_5036_MOESM3_ESM.docx (17 kb)
Electronic supplementary material 3 (DOCX 17 kb)
11033_2019_5036_MOESM4_ESM.docx (17 kb)
Electronic supplementary material 4 (DOCX 17 kb)


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Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  1. 1.College of Life Science and AgronomyZhoukou Normal UniversityZhoukouChina
  2. 2.Key Laboratory of Plant Genetics and Molecular BreedingZhoukou Normal UniversityZhoukouChina
  3. 3.College of Journalism and MediaZhoukou Normal UniversityZhoukouChina

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