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Reference genes for transcriptional analysis of flowering and fruit ripening stages in apple (Malus × domestica Borkh.)

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An Erratum to this article was published on 13 May 2014

Abstract

Apple (Malus × domestica Borkh.) is the most important deciduous tree fruit crop grown around the world. Comparisons of gene expression profiles from different tissues, conditions or cultivars are valuable scientific tools to better understand the gene expression changes behind important silvicultural and nutritional traits. However, the accuracy of techniques employed to access gene expression is dependent on the evaluation of stable reference genes for data normalization to avoid statistical significance undue or incorrect conclusions. The objective of this work was to select the best genes to be used as references for gene expression studies in apple trees by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Vegetative and reproductive tissues of the apple “Gala” cultivar were evaluated during their seasonal cycle of growth and dormancy. The expression of 23 traditional housekeeping genes or genes suggested as constitutive by microarray data was investigated. Tested combinations of primers allowed the specific amplification and the generation of suitable efficiency curves for gene expression studies by RT-qPCR. Gene stability was determined by two different statistical descriptors, geNorm and NormFinder. The known variable PAL gene expression was used to validate selected normalizers. Results obtained allowed us to conclude that MDH, SAND, THFS, TMp1 and WD40 are the best reference genes to accurately normalize the relative transcript abundances using RT-qPCR in various tissues of apple.

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Abbreviations

ACT2:

Actin 2

ACT11:

Actin 11

ACTfam:

Actin family

ARC5:

Accumulation and replication of chloroplast 5

C3HC4:

Ring C3HC4 zinc finger protein

CDC48:

Cell division cycle protein 48 homolog

CKL:

Casein kinase 1 isoform delta like

Ct:

Cycle threshold

DFCI:

Dana Farber Cancer Institute and the Harvard School of Public Health

DLD:

Dihydrolipoamide dehydrogenase

E:

Efficiencies

EF1α:

Elongation factor 1 alpha

EF1β:

Elongation factor 1 beta

EST:

Expressed sequence tag

GAPDH:

Glyceraldehyde 3-phosphate dehydrogenase

KEA1:

K+ efflux antiporter 1

M:

Expression stability

MDH:

Malate dehydrogenase

miRNAs:

MicroRNAs

NF:

Normalization factor

PAL:

Phenylalanine ammonia-lyase

PCS:

Phytochelatin synthetase-like protein

PP2A-1:

Serine/threonine-protein phosphatase 2A-1

PP2A-A3:

Serine/threonine-protein phosphatase 2A subunit A3

R 2 :

Correlation coefficient

RT-qPCR:

Reverse transcription-quantitative polymerase chain reaction

SAGE:

Serial analysis of gene expression

SAND:

Protein of unknown function SAND family

THFS:

Formate-tetrahydrofolate ligase

T m :

Melting temperatures

TMp1:

Type 1 membrane protein like

TUBα5:

Tubulin alpha 5

TUBβ6:

Tubulin beta 6

UBC10:

Ubiquitin-conjugating enzyme 10

V:

Pairwise variation

WD40:

Transcription factor WD40-like repeat domain

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Acknowledgments

We thank Vanessa Buffon for technical assistance, and Diogo Denardi Porto and Vítor da Silveira Falavigna for helpful contributions on the manuscript. We also gratefully acknowledge our colleague Dr. João Caetano Fioravanço for his assistance in the sampling strategy and for the management of the experimental apple orchards. This work was supported by “Financiadora de Estudos e Projetos” (FINEP, Ministry of Science and Technology—MCT [grant number 01.10.0303.00]) and “Empresa Brasileira de Pesquisa Agropecuária” (EMBRAPA grant number 02.07.07.007.00.03), Brazil. P. Perini was recipient of a M.Sc. fellowship from the “Coordenação de Aperfeiçoamento de Pessoal de Nível Superior” (CAPES, Ministry of Education), Brazil. M. Margis-Pinheiro and G. Pasquali are recipients of research fellowships from the “Conselho Nacional de Desenvolvimento Científico e Tecnológico” (CNPq, MCT [Grant Numbers 306945/2009-6 and 311361/2009-9, respectively).

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Authors and Affiliations

  1. Programa de Pós-graduação em Biologia Celular e Molecular, Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul (UFRGS), P.O. Box 15005, Porto Alegre, RS, CEP 91501-970, Brazil

    Pâmela Perini, Giancarlo Pasquali & Márcia Margis-Pinheiro

  2. Departamento de Genética, Núcleo de Genômica Funcional de Plantas, UFRGS, P.O. Box 15053, Porto Alegre, RS, CEP 91501-970, Brazil

    Márcia Margis-Pinheiro

  3. Laboratório de Genética Molecular Vegetal, Embrapa Uva e Vinho, Rua Livramento 515, P.O. Box 130, Bento Gonçalves, RS, CEP 95700-000, Brazil

    Paulo Ricardo Dias de Oliviera & Luís Fernando Revers

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  1. Pâmela Perini
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  2. Giancarlo Pasquali
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  3. Márcia Margis-Pinheiro
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  4. Paulo Ricardo Dias de Oliviera
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  5. Luís Fernando Revers
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Correspondence to Luís Fernando Revers.

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Perini, P., Pasquali, G., Margis-Pinheiro, M. et al. Reference genes for transcriptional analysis of flowering and fruit ripening stages in apple (Malus × domestica Borkh.). Mol Breeding 34, 829–842 (2014). https://doi.org/10.1007/s11032-014-0078-3

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