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Identification of suitable reference genes for normalization of real-time quantitative PCR data in pecan (Carya illinoinensis)

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This study proposed the combination of PR26S and PP1 as a good choice for RT-qPCR normalization in pecan under abiotic stress, developing kernels, grafting, and various tissues.

Abstract

Reference gene selection is an essential pre-requisite to generate reliable results in real-time quantitative polymerase chain reaction (RT-qPCR) analysis. However, studies regarding systematic validation of suitable reference genes in pecan are still lacking. In this study, 17 candidate reference genes were selected and evaluated for their expression stabilities in pecan under various experimental conditions, including various tissues, developing kernels, grafting, and two plant tissues (leaves and roots) subjected to three abiotic stresses (salt, drought, and Zn deficiency). The stability of the candidate genes was assessed by geNorm, NormFinder, and BestKeeper, and their outputs were integrated to obtain a final comprehensive rank of stability based on the geometric mean. The results indicated that samples under different experimental conditions possessed their own best reference genes, and using two reference genes for RT-qPCR normalization was recommended for the tested experiments. Overall, the combination of 26S protease regulatory subunit 7A (PR26S) and serine/threonine-protein phosphatase-1 (PP1) was recognized as a good choice for RT-qPCR normalization in pecan across all the treatments. More importantly, the widely used alpha-tubulin (α-TUB), ubiquitin (UBQ), and actin (ACT) genes were not the best suitable reference genes in most of our experiments. Our results will be helpful for future gene-expression studies in pecan.

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Abbreviations

α-TUB:

Alpha-tubulin

β-TUB:

Beta-tubulin

ACT:

Actin

ADP-RF:

ADP-ribosylation factor

CAC:

Clathrin adaptor complex

CAD:

Cinnamyl alcohol dehydrogenase

CYP:

Cyclophilin

GAPDH:

Glyceraldehyde 3-phosphate dehydrogenase

HEL:

RNA helicase family

PEG:

Polyethylene glycol

PP1:

Serine/threonine-protein phosphatase-1

PP2A:

Serine/threonine-protein phosphatase 2A

PR26S:

26S protease regulatory subunit 7A

PTBP1:

Polypyrimidine tract-binding protein 1

RPN6:

26S proteasome non-ATPase

SAD:

Stearoyl-ACP desaturase

TLF:

Translation factor

TIP41:

TIP41-like family protein

UBQ:

Ubiquitin

60S:

60S ribosomal protein L22

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Acknowledgements

This work was financially supported by the National Natural Science Foundation of China (no. 31901347), the Basic Talent Project of Institute of Botany, Jiangsu Province and Chinese Academy of Science (JSPKLB202002), and Scientific Research Staring Foundation for the Doctor of Institute of Botany, Jiangsu Province and Chinese Academy of Science.

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Authors and Affiliations

  1. Institute of Botany, Jiangsu Province and Chinese Academy of Science, Nanjing, 210014, China

    Zhenghai Mo, Yaqi Chen, Wenrui Lou, Xiaodong Jia, Min Zhai, Jiping Xuan & Zhongren Guo

  2. The Jiangsu Provincial Platform for Conservation and Utilization of Agricultural Germplasm, Nanjing, 210014, China

    Zhenghai Mo, Yaqi Chen, Wenrui Lou, Xiaodong Jia, Min Zhai, Jiping Xuan & Zhongren Guo

  3. Green Universe Pecan Science and Technology Co., Ltd., Nanjing, 210007, China

    Yongrong Li

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  1. Zhenghai Mo
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Contributions

JX and ZG designed the research; ZM, YC, WL, and XJ performed the experiments and data analysis; ZM wrote the manuscript; MZ and YL prepared the materials. All authors both read and approved the manuscript.

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Correspondence to Jiping Xuan.

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Communicated by Francisco M. Cánovas .

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Mo, Z., Chen, Y., Lou, W. et al. Identification of suitable reference genes for normalization of real-time quantitative PCR data in pecan (Carya illinoinensis). Trees 34, 1233–1241 (2020). https://doi.org/10.1007/s00468-020-01993-w

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