Identification of a novel reference gene for apple transcriptional profiling under postharvest conditions

Tatiane Timm Storch; Camila Pegoraro; Taciane Finatto; Vera Quecini; Cesar Valmor Rombaldi; César Luis Girardi

doi:10.1371/journal.pone.0120599

Identification of a novel reference gene for apple transcriptional profiling under postharvest conditions

PLoS One. 2015 Mar 16;10(3):e0120599. doi: 10.1371/journal.pone.0120599. eCollection 2015.

Authors

Tatiane Timm Storch¹, Camila Pegoraro², Taciane Finatto², Vera Quecini², Cesar Valmor Rombaldi³, César Luis Girardi²

Affiliations

¹ Empresa Brasileira de Pesquisa Agropecuária Uva e Vinho, Bento Gonçalves, Brazil; Universidade Federal de Pelotas, Pelotas, Brazil.
² Empresa Brasileira de Pesquisa Agropecuária Uva e Vinho, Bento Gonçalves, Brazil.
³ Universidade Federal de Pelotas, Pelotas, Brazil.

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.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Computational Biology
Gene Expression Profiling
Gene Expression Regulation, Plant*
Malus / genetics*
RNA Stability
Transcriptome*

Grants and funding

The work was supported by the following: Research Grant 02.10.01.002 from Embrapa/SEG to CLG (https://sistemas.sede.embrapa.br/ideare); CNPq Productivity Research Grant to CVR (http://www.cnpq.br/); CAPES Institutional Doctorate Grant to TTS (http://www.capes.gov.br/bolsas/bolsas-no-pais/ds-e-proap); and Post Doctoral Grant 02986/9 to CP (www.capes.gov.br/bolsas/bolsas-no-pais/pnpd). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Embrapa, the funding agency with which one or more of the authors is affiliated, is a government research institution (https://www.embrapa.br/en/home) similar to the United States Department of Agriculture (USDA) and provides competitive funding to research projects without playing a role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript and only provided financial support in the form of authors' salaries and/or research materials.