Selection of reference genes for transcriptional analysis of edible tubers of potato (Solanum tuberosum L.)

Roberta Fogliatto Mariot; Luisa Abruzzi de Oliveira; Marleen M Voorhuijzen; Martijn Staats; Ronald C B Hutten; Jeroen P Van Dijk; Esther Kok; Jeverson Frazzon

doi:10.1371/journal.pone.0120854

Selection of reference genes for transcriptional analysis of edible tubers of potato (Solanum tuberosum L.)

PLoS One. 2015 Apr 1;10(4):e0120854. doi: 10.1371/journal.pone.0120854. eCollection 2015.

Authors

Roberta Fogliatto Mariot¹, Luisa Abruzzi de Oliveira², Marleen M Voorhuijzen³, Martijn Staats³, Ronald C B Hutten⁴, Jeroen P Van Dijk³, Esther Kok³, Jeverson Frazzon¹

Affiliations

¹ Food Science Department, Food Science and Technology Institute, Federal University of Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil.
² Genetics Department, Federal University of Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil.
³ RIKILT, Wageningen UniversityR, Wageningen, The Netherlands.
⁴ Plant Breeding, Wageningen UR, Wageningen, The Netherlands.

Abstract

Potato (Solanum tuberosum) yield has increased dramatically over the last 50 years and this has been achieved by a combination of improved agronomy and biotechnology efforts. Gene studies are taking place to improve new qualities and develop new cultivars. Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) is a bench-marking analytical tool for gene expression analysis, but its accuracy is highly dependent on a reliable normalization strategy of an invariant reference genes. For this reason, the goal of this work was to select and validate reference genes for transcriptional analysis of edible tubers of potato. To do so, RT-qPCR primers were designed for ten genes with relatively stable expression in potato tubers as observed in RNA-Seq experiments. Primers were designed across exon boundaries to avoid genomic DNA contamination. Differences were observed in the ranking of candidate genes identified by geNorm, NormFinder and BestKeeper algorithms. The ranks determined by geNorm and NormFinder were very similar and for all samples the most stable candidates were C2, exocyst complex component sec3 (SEC3) and ATCUL3/ATCUL3A/CUL3/CUL3A (CUL3A). According to BestKeeper, the importin alpha and ubiquitin-associated/ts-n genes were the most stable. Three genes were selected as reference genes for potato edible tubers in RT-qPCR studies. The first one, called C2, was selected in common by NormFinder and geNorm, the second one is SEC3, selected by NormFinder, and the third one is CUL3A, selected by geNorm. Appropriate reference genes identified in this work will help to improve the accuracy of gene expression quantification analyses by taking into account differences that may be observed in RNA quality or reverse transcription efficiency across the samples.

Publication types

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

MeSH terms

Algorithms
Computational Biology / methods*
Gene Expression Profiling / standards*
Genes, Plant / genetics*
Genetic Variation
Plant Tubers / genetics*
Reference Standards
Sequence Analysis, RNA
Solanum tuberosum / genetics*
Transcription, Genetic*

Grants and funding

This work was supported by National Council of Technological and Scientific Development of Brazil (CNPq) (J.F.grant #300912/2012-9, #473181/2013-4) and the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) of Brazil. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.