Validation of suitable reference genes for qRT-PCR in cabbage (Brassica oleracea L.) under different abiotic stress experimental conditions

Abstract

Cabbage (Brassica oleracea) is one of the most important vegetable crops worldwide. qRT-PCR is a sensitive technique for gene expression studies and choosing the appropriate reference gene is essential to obtain reliable results. In the present work, 22 candidate reference genes were evaluated under various experimental conditions, including NaCl, drought stress treatment, temperature treatments (cold and heat) and a set of hormones stress (6-BA, NAA, and ABA) treatments, across a range of tissue types and cultivars. Gene expression data taken from 45 cabbage samples was analyzed using two algorithms, geNorm and NormFinder. Suitable combinations of reference genes for qRT-PCR normalization should be applied according to different experimental conditions. In this study, the genes EF1a, GAPC2 and SAND were verified as the suitable reference genes across all tested samples. Additionally, each experimental condition had a unique set of reference genes best suited to samples within the particular condition. To validate the suitability of the candidate reference genes, the gene expression of WSD1, a gene that may be involved in biosynthesis pathway of wax esters in cabbage, was measured across all 45 samples and normalized using the three best reference gene candidates. WSD1 displayed variation in gene expression across different tissues and cultivars, and exhibited diverse up- or down- regulated expression patterns under various treatments, which indicate that BoWSD1 may play an important role in the response to abiotic stresses in cabbage. Our results provide the foundation for gene expression analysis in Brassica oleracea and other species of Brassica vegetables.

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Abbreviations

ABA:

Abscisic acid

bp:

Base pair

NAA:

1-Naphthaleneacetic acid

PEG6000:

Polyethylene Glycol 6000

qRT-PCR:

Quantitative real-time polymerase chain reaction

6-BA:

6-Benzylaminopurine

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Acknowledgements

The study was partially supported by the Natural Science Foundation of Jiangsu Province (Nos. BK20191239 and BK20190262) and the National Key Research and Development Program of China (2017YFD0101804).

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Correspondence to Yuanyuan Xu or Jiyong Yan.

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Fig. S1

Dissociation curve analysis generated for all amplicons (DOC 816 kb)

Fig. S2

Expression levels of candidate reference genes across all samples. A line across the box depicts the median. The box indicates the 25% and 75% percentiles. Whiskers represent the maximum and minimum values (DOC 10980 kb)

Fig. S3

Average expression stability values (M) of the candidate reference genes. Average expression stability values (M) of the candidate reference genes were calculated by the geNorm software in cabbage samples under different experimental conditions, including different cultivars (a) and tissues (b), temperature treatment (c), NaCl treatment (d), PEG6000 treatment (e), hormonal treatment (f), and total (g). The lowest M-value indicates the most stable gene and vice versa (DOC 571 kb)

Fig. S4

Average expression stability values (M) of the candidate reference genes. Average expression stability values (M) of the candidate reference genes were calculated by the geNorm software in cabbage samples under different experimental conditions, including heat treatment (a), cold treatment (b), ABA treatment (c), 6-BA treatment (d), and NAA treatment (e). The lowest M-value indicates the most stable gene and vice versa (DOC 456 kb)

Fig. S5

Determination of the optimal number of reference genes. Every bar represents change in normalization accuracy upon stepwise addition of more reference genes according to the ranking in Fig. 4 and Fig. 5. The pairwise variation (Vn/Vn + 1) was calculated from the normalization factors NFn and NFn + 1, with a recommended cutoff threshold of 0.150 (DOC 64 kb)

Supplementary material 6 (DOC 90 kb)

Supplementary material 7 (DOC 76 kb)

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Zeng, A., Xu, Y., Song, L. et al. Validation of suitable reference genes for qRT-PCR in cabbage (Brassica oleracea L.) under different abiotic stress experimental conditions. J. Plant Biochem. Biotechnol. 30, 184–195 (2021). https://doi.org/10.1007/s13562-020-00556-x

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Keywords

  • Cabbage
  • qRT-PCR
  • Reference genes
  • Selection
  • Validation
  • Abiotic stress