Abstract
Reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) is a powerful method for evaluating patterns of gene expression. Jujube whole-genome sequencing has been completed, and analysis of gene function, an important part of any follow-up study, requires the appropriate selection of reference genes. Indeed, suitable reference gene selection for RT-qPCR is critical for accurate normalization of target gene expression. In this study, the software packages geNorm and NormFinder were employed to examine the expression stabilities of nine candidate reference genes under a variety of conditions. Actin-depolymerizing factor 1 (ACT1), Histone-H3 (His3), and Polyadenylate-binding protein-interacting protein (PAIP) were determined to be the most stably expressed genes during five stages of fruit development and ACT1, SiR-Fd, BTF3, and Tubulin alpha chain (TUA) across different tissues/organs. Whereas ACT1, Basic Transcription factor 3 (BTF3), Glyceraldehyde-3-phosphate dehydrogenase (GADPH), and PAIP were the most stable under dark conditions. ACT1, PAIP, BTF3, and Elongation factor 1- gamma (EF1γ) were the most stably expressed genes under phytoplasma infection. Among these genes, SiR-Fd and PAIP are here first reported as stable reference genes. When normalized using these most stable reference genes, the expression patterns of four target genes were found to be in accordance with physiological data, indicating that the reference genes selected in our study are suitable for use in such analyses. This study provides appropriate reference genes and corresponding primers for further RT-qPCR studies in Chinese jujube and emphasizes the importance of validating reference genes for gene expression analysis under variable experimental conditions.
Highlights
Chinese jujube (Ziziphus jujuba Mill.), a member of the genus Ziziphus in the family Rhamnaceae, is widely distributed in temperate and subtropical areas of the Northern Hemisphere, especially the inland region of Northern China [1]
ZjH3 is reportedly the most suitable gene for evaluating gene expression in early-growth fruit-bearing shoots, shoot apices, and different organs by semi-quantitative reverse transcription PCR (RT-PCR) [3], and recently UBQ, ACTIN9, UBQ2 and CYP were validated as stable genes at some restricted conditions by reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) [4]
The Cycle threshold (Ct) values of the nine tested genes were collected under different experimental conditions (Fig 1), and the results presented a relatively wide range, from 21.16 for Actin-depolymerizing factor 1 (ACT1) to 36.30 for EF1α
Summary
Chinese jujube (Ziziphus jujuba Mill.), a member of the genus Ziziphus in the family Rhamnaceae, is widely distributed in temperate and subtropical areas of the Northern Hemisphere, especially the inland region of Northern China [1]. The identification and validation of additional jujube reference genes by RTqPCR will further contribute to related studies at the transcription level. Housekeeping genes, such as Actin-depolymerizing factor (ACT), glyceraldehyde-3- phosphate dehydrogenase (GAPDH), α-Tubulin (TUA), and elongation factor 1-alpha (EF1a), are often selected as reference genes. Ideal reference genes should exhibit stable expression levels under different experimental conditions, such as during different fruit developmental stages, in different tissues, and under various stress treatments [5,6]. Screening for suitable reference genes has been performed in many plants, including banana [10], Apricot [11], Pyrus pyrifolia [12], litchi [13], Vitis vinifera [14], and citrus [15]
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