Expression Stabilities of Candidate Reference Genes for RT-qPCR in Chinese Jujube (<i>Ziziphus jujuba</i> Mill.) under a Variety of Conditions

<div><p>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. <i>Actin-depolymerizing factor 1</i> (<i>ACT1</i>), <i>Histone-H3</i> (<i>His3</i>), and <i>Polyadenylate-binding protein-interacting protein</i> (<i>PAIP</i>) were determined to be the most stably expressed genes during five stages of fruit development and <i>ACT1</i>, <i>SiR-Fd</i>, <i>BTF3</i>, and <i>Tubulin alpha chain</i> (<i>TUA</i>) across different tissues/organs. Whereas <i>ACT1</i>, <i>Basic Transcription factor 3</i> (<i>BTF3</i>), <i>Glyceraldehyde-3-phosphate dehydrogenase</i> (<i>GADPH</i>), and <i>PAIP</i> were the most stable under dark conditions. <i>ACT1</i>, <i>PAIP</i>, <i>BTF3</i>, and <i>Elongation factor 1- gamma</i> (<i>EF1γ</i>) were the most stably expressed genes under phytoplasma infection. Among these genes, <i>SiR-Fd</i> and <i>PAIP</i> 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.</p></div

Primer sequences and related information for each candidate reference gene.

<p>Primer sequences and related information for each candidate reference gene.</p

Relative expression using validated reference genes for normalization under different experimental conditions.

<p>(A) and (B): Relative expression of <i>GPP</i> and <i>GGP</i> in five fruit ripening stages, normalized by <i>ACT1</i>, <i>His3</i>, <i>GAPDH</i>, or <i>TUA</i>; Y: young fruit, PW: pre-white ripening stage, W: white ripening stage, HR: half-red ripening stage, WR: whole-red ripening stage. (C) and (D): Relative expression of <i>Rubisco</i> and <i>RCA1</i> under phytoplasma infection, normalized by <i>ACT1</i>, <i>PAIP</i>, <i>EF1α</i>, or <i>His3</i>. HL: healthy leaves, ANL: apparent normal leaves, P: phyllody leaves, WBL: witches’ broom leaves.</p

Average expression stability values (M values) calculated by geNorm.

<p>(A) Different fruit ripening stages, (B) Dark treatment, (C) Phytoplasma infection, (D) Different tissue/organs.</p

Ranking of candidate reference genes in order of M value as calculated by NormFinder.

<p>Ranking of candidate reference genes in order of M value as calculated by NormFinder.</p

Pairwise variation (V) calculated by geNorm to determine the optimal number of reference genes.

<p>The average pairwise variations V_n/V_n+1 was analyzed between the normalization factors NF_n and NF_n+1 to indicate the optimal number of reference genes required for RT-qPCR data normalization in different samples. T/O: Different tissue/organs, D: Dark conditions, P: Phytoplasma infection, FRS: Fruit ripening stages.</p

Ct values for nine candidate reference genes in all samples.

<p>The final Ct value of each sample was the mean of three biological and technical replicates. The boxes represent the 25th and 75th percentiles of data. A line across the box showed as the median. Whiskers represent the maximum and minimum values.</p