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Genome-Wide Identification and Evaluation of New Reference Genes in Pineapple (Ananas comosus L.) during Stamen and Ovule Development

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Abstract

Quantitative real-time PCR (qRT-PCR) is a convenient tool for gene expression analysis. However, reliability of its result is influenced largely by the selection of reference genes. Pineapple molecular breeding is limited due to lack of understanding of sexual organs’ development. Similarly, only a few reference genes have identified for qRT-PCR analysis in pineapple stamen and ovule. In this study, we initially identified 20 candidate reference genes using transcriptome data, and determined their expression stabilities in 36 ovule and stamen developmental samples using qRT-PCR. Three algorithms, GeNorm, NormFinder, and BestKeeper were used to determine the superiority of those candidate genes. Our results revealed that the use of combined RPS4 and RPL23 during ovule development, CCR and RPS4 during stamen development were sufficient for reliable normalization. These recommended reference genes were further verified by evaluating the temporal expression abundance of the meiosis-specific protein-encoding genes AcASY1 and AcASY3 in all experimental samples. Our results complement previous pineapple normalization study by providing appropriate reference genes during the entire reproductive period, and will beneficial for future studies on the pineapple stamen and ovule development. Finally, this result will help for the molecular breeding of pineapple for crop improvement.

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Abbreviations

qRT-PCR:

Quantative real-time PCR

FPKM:

Fragments Per Kilobase per Million

RE:

Relative Expression

AVE:

Average expression level

CV:

Coefficient variance

E:

Amplification efficiency

R2 :

Correlation coefficient

SD:

Standard deviation

GM:

Geometric mean

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Acknowledgements

We would like to thank the reviewers for their helpful comments on the original manuscript.

Funding

This research work was funded by the National Natural Science Foundation of China (U1605212 and 3197033) and a Guangxi Distinguished Experts Fellowship to Y.Q.

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Author notes

  1. Xingyue Jin and Zhimin Hou contributed equally to this work.

Authors and Affiliations

  1. Key Laboratory of Genetics, Breeding and Multiple Utilization of Crops, Ministry of Education, Fujian Provincial Key Laboratory of Haixia Applied Plant Systems Biology, College of Horticulture, Fujian Agriculture and Forestry University, Fuzhou, Fujian, China

    Xingyue Jin, Lihua Zhao, S. V. G. N. Priyadarshani, Youmei Huang, Fangqian Chen & Yuan Qin

  2. College of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou, Fujian, China

    Zhimin Hou, Liping Liu, Lulu Wang & Yuan Qin

  3. State Key Laboratory for Conservation and Utilization of Subtropical Agro-Bioresources, Guangxi Key Lab of Sugarcane Biology, College of Agriculture, Guangxi University, Nanning, Guangxi Province, China

    Yuan Qin

Authors
  1. Xingyue Jin
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  2. Zhimin Hou
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  3. Lihua Zhao
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  4. Liping Liu
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Contributions

XJ and ZH contributed to primer design, performed the qRT-PCR and took the co-lead role in writing the manuscript. LZ analyzed transcriptome data. LL, S.P., LW, YH and FC performed PCR, offered plant materials and reviewed the manuscript. YQ conceived the project, provided funding, and approved the final version of this paper. All authors have read and approved the published version of the manuscript.

Corresponding author

Correspondence to Yuan Qin.

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The authors declare no conflict of interest.

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Communicated by: Zhi-Liang Zheng

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Jin, X., Hou, Z., Zhao, L. et al. Genome-Wide Identification and Evaluation of New Reference Genes in Pineapple (Ananas comosus L.) during Stamen and Ovule Development. Tropical Plant Biol. 13, 371–381 (2020). https://doi.org/10.1007/s12042-020-09269-w

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  • DOI: https://doi.org/10.1007/s12042-020-09269-w

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