Genome-wide identification of internal reference genes for normalization of gene expression values during endosperm development in wheat

  • Junyi Mu
  • Lin Chen
  • Yunsong Gu
  • Luning Duan
  • Shichen Han
  • Yaxuan Li
  • Yueming Yan
  • Xiaohui LiEmail author
Plant Genetics • Original Paper


Internal reference genes that are stably expressed are essential for normalization in comparative expression analyses. However, gene expression varies significantly among species, organisms, tissues, developmental stages, stresses, and treatments. Therefore, identification of stably expressed reference genes in developmental endosperm of bread wheat is important for expression analysis of endosperm genes. As the first study to systematically screen for reference genes across different developmental stages of wheat endosperm, nine genes were selected from among 76 relatively stable genes based on high-throughput RNA sequencing data. The expression stability of these candidate genes and five traditional reference genes was assessed by real-time quantitative PCR combined with three independent algorithms: geNorm, NormFinder, and BestKeeper. The results showed that ATG8d was the most stable gene during wheat endosperm development, followed by Ta54227, while the housekeeping gene GAPDH, commonly used as an internal reference, was the least stable. ATG8d and Ta54227 together formed the optimal combination of reference genes. Comparative expression analysis of glutenin genes indicated that credible quantification could be achieved by normalization against ATG8d in developmental endosperm. The stably expressed gene characterized here can act as a proper internal reference for expression analysis of wheat endosperm genes, especially nutrient- and nutrient synthesis–related genes.


Wheat Endosperm Internal reference gene RNA-Seq RT-qPCR Glutenin 


Author contributions

XHL conceived and designed the experiment. JYM, LC, YSG, LND, and SCH performed the experiment. JYM and LC analyzed the data. YXL and YMY provided technical assistance and scientific discussion. JYM and XHL wrote the paper.


This research was supported by grants from National Key R&D Program of China (2016ZX08009003-004), National Natural Science Foundation of China (31571652), the Natural Science Foundation of Beijing (6162002, 6122002), and the Youth Innovative Research Team of Capital Normal University.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

This article does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

13353_2019_503_MOESM1_ESM.docx (1.9 mb)
ESM 1 (DOCX 1992 kb)


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Copyright information

© Institute of Plant Genetics, Polish Academy of Sciences, Poznan 2019

Authors and Affiliations

  1. 1.Key Laboratory of Genetics and Biotechnology, College of Life ScienceCapital Normal UniversityBeijingChina

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