Identification of suitable reference genes for normalization of real-time quantitative PCR data in pecan ( Carya illinoi
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ORIGINAL ARTICLE
Identification of suitable reference genes for normalization of real‑time quantitative PCR data in pecan (Carya illinoinensis) Zhenghai Mo1,2 · Yaqi Chen1,2 · Wenrui Lou1,2 · Xiaodong Jia1,2 · Min Zhai1,2 · Jiping Xuan1,2 · Zhongren Guo1,2 · Yongrong Li3 Received: 17 September 2019 / Accepted: 23 May 2020 © Springer-Verlag GmbH Germany, part of Springer Nature 2020
Abstract Key message 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. Keywords Pecan · RT-qPCR · Reference gene · Abiotic stress · Kernel development Abbreviations α-TUB Alpha-tubulin β-TUB Beta-tubulin ACT Actin ADP-RF ADP-ribosylation factor Communicated by Francisco M. Cánovas . Electronic supplementary material The online version of this article (https://doi.org/10.1007/s00468-020-01993-w) contains supplementary material, which is available to authorized users. * Jiping Xuan [email protected] 1
Institute of Botany, Jiangsu Province and Chinese Academy of Science, Nanjing 210014, China
2
The Jiangsu Provincial Platform for Conservation and Utilization of Agricultural Germplasm, Nanjing 210014, China
3
Green Universe Pecan Science and Technology Co., Ltd., Nanjing 210007, China
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 phosphat
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