Identification of a Novel Reference Gene for Apple Transcriptional Profiling under Postharvest Conditions

Abstract

Reverse Transcription quantitative PCR (RT-qPCR) is one of the most important techniques for gene expression profiling due to its high sensibility and reproducibility. However, the reliability of the results is highly dependent on data normalization, performed by comparisons between the expression profiles of the genes of interest against those of constitutively expressed, reference genes. Although the technique is widely used in fruit postharvest experiments, the transcription stability of reference genes has not been thoroughly investigated under these experimental conditions. Thus, we have determined the transcriptional profile, under these conditions, of three genes commonly used as reference—ACTIN (MdACT), PROTEIN DISULPHIDE ISOMERASE (MdPDI) and UBIQUITIN-CONJUGATING ENZYME E2 (MdUBC)—along with two novel candidates—HISTONE 1 (MdH1) and NUCLEOSSOME ASSEMBLY 1 PROTEIN (MdNAP1). The expression profile of the genes was investigated throughout five experiments, with three of them encompassing the postharvest period and the other two, consisting of developmental and spatial phases. The transcriptional stability was comparatively investigated using four distinct software packages: BestKeeper, NormFinder, geNorm and DataAssist. Gene ranking results for transcriptional stability were similar for the investigated software packages, with the exception of BestKeeper. The classic reference gene MdUBC ranked among the most stably transcribed in all investigated experimental conditions. Transcript accumulation profiles for the novel reference candidate gene MdH1 were stable throughout the tested conditions, especially in experiments encompassing the postharvest period. Thus, our results present a novel reference gene for postharvest experiments in apple and reinforce the importance of checking the transcription profile of reference genes under the experimental conditions of interest.

Identification of stable reference genes for qPCR studies in common wheat (Triticum aestivum L.) seedlings under short-term drought stress

“…Thus, the ranking order was not identical; however, the three first genes, which were considered the most stable under the given experimental conditions, were the same. A similar variance was described for apple under postharvest conditions [39], Caragana intermedia under osmotic stress [8] and Actinidia deliciosa infected by Pseudomonas syringae pv. actinidiae [9].…”

“…Based on previous studies, we suggest that these variances are caused by the BestKeeper algorithm, which employs correlation analyses between the candidate gene Cq and an index derived from the candidate geometric mean. In contrast, the algorithms of the geNorm and NormFinder platforms use variation measures to calculate the stability of gene transcription [39].…”

Contrasting Transcriptional Programs Control Postharvest Development of Apples (Malus x domestica Borkh.) Submitted to Cold Storage and Ethylene Blockage

“…Synthesis of cDNA and qPCR conditions were performed as described. 19 Results were normalized employing the reference gene MdUBI and the genes corresponding to apple genome identifiers MDP0000217860 and MDP0000271281, which showed stable transcription in the microarray analyses. Expression profile validation for 14 genes is shown in Figure S6.…”

Molecular Basis of Flower Formation in Apple Caused by Defoliation and Gibberellins

“…Real-time PCR was then carried out on a CFX96 Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA), with PerfeCTa SYBR Green FastMix (Quantabio, Beverly, MA) in a 15-mL final volume to determine the transcript levels of the target genes. The ubiquitin C (UBC) gene (MDP0000205182) was used as an internal control for the normalization of gene transcript levels (Storch et al, 2015).…”