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Selection of candidate reference genes for real-time PCR studies in lettuce under abiotic stresses

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Abstract

The process of selection and validation of reference genes is the first step in studies of gene expression by real-time quantitative polymerase chain reaction (RT-qPCR). The genome of lettuce, the most popular leaf vegetable cultivated worldwide, has recently been sequenced; therefore, suitable reference genes for reliable results in RT-qPCR analyses are required. In the present study, 17 candidate reference genes were selected, and their expression stability in lettuce leaves under drought, salt, heavy metal, and UV-C irradiation conditions and under the application of abscisic acid (ABA) was evaluated using geNorm and NormFinder software. The candidate reference genes included protein-coding traditional and novel reference genes and microRNAs (miRNAs). The results indicate that the expression stability is dependent on the experimental conditions. The novel protein-coding reference genes were more suitable than the traditional reference genes under drought, UV-C irradiation, and heavy metal conditions and under the application of ABA. Only under salinity conditions were the traditional protein-coding reference genes more stable than the novel genes. In addition, the miRNAs, mainly MIR169, MIR171/170 and MIR172, were stably expressed under the abiotic stresses evaluated, representing a suitable alternative approach for gene expression data normalization. The expression of phenylalanine ammonia lyase (PAL) and 4-hydroxyphenylpyruvate dioxygenase (HPPD) was used to further confirm the validated protein-coding reference genes, and the expression of MIR172 and MIR398 was used to confirm the validated miRNA genes, showing that the use of an inappropriate reference gene induces erroneous results. This work is the first survey of the stability of reference genes in lettuce and provides guidelines to obtain more accurate RT-qPCR results in lettuce studies.

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Abbreviations

18S :

18S ribosomal RNA

40S :

40S ribosomal RNA

ABA:

Absicic acid

ACT :

Actin

APT1 :

Adenosine phosphoribosyl transferase

EIF2A :

Elongation initiation factor gamma subunit

EIF4A1 :

Eukaryotic translation initiation factor 4A1

EXP45 :

Expansin 5 precursor

microRNA:

miRNA

HPPD :

4-Hydroxyphenylpyruvate dioxygenase

PAL :

Phenylalanine ammonia-lyase

PP2A :

Serine/threonine protein phosphatase 2A

RT-qPCR:

Real-time quantitative polymerase chain reaction

TIP41 :

TAP42-interacting protein of 41 kDa

TUB :

Tubulin

UBC21 :

Ubiquitin-protein ligase

UBQ :

Ubiquitin

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Acknowledgments

The authors gratefully acknowledge the technical and financial support of the Embrapa Clima Temperado (Embrapa), Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul (Fapergs), and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).

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Authors and Affiliations

  1. Embrapa Clima Temperado, Rodovia BR 396, Km 78, Caixa Postal 403, Pelotas, RS, CEP 96001-970, Brazil

    Joyce Moura Borowski, Vanessa Galli, Rafael da Silva Messias, Ellen Cristina Perin, Julieti Hugh Buss & Sérgio Delmar dos Anjos e Silva

  2. Faculdade de Agronomia Eliseu Maciel, Campus Universitário s/n, Universidade Federal de Pelotas, Caixa Postal 354, Pelotas, RS, CEP 96010-900, Brazil

    Joyce Moura Borowski, Rafael da Silva Messias, Ellen Cristina Perin, Julieti Hugh Buss & Cesar Valmor Rombaldi

  3. Universidade Federal do Rio Grande do Sul, Centro de Biotecnologia, Av. Bento Gonçalves 9500, Campus do Vale, Caixa Postal 15005, Porto Alegre, RS, CEP 91501-970, Brazil

    Vanessa Galli

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  1. Joyce Moura Borowski
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  2. Vanessa Galli
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Correspondence to Cesar Valmor Rombaldi.

Electronic supplementary material

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425_2014_2041_MOESM1_ESM.tif

Online resource 1 Specific amplification. RT-qPCR melting curve analysis calculated by SDS version 1.1 software in a 7500 Real time Fast thermocycler (Applied Biosystems). (TIFF 15872 kb)

425_2014_2041_MOESM2_ESM.tif

Online resource 2 Specific amplicon size. Agarose gel (3 %) showing amplification of a specific PCR product of the expected size for the twelve protein-coding reference genes, five miRNAs, PAL and HPPD genes using cDNA from lettuce leaves. Lines 1, 19 and 22: 1 kb plus (Invitrogen); Line 2: ACT; Line 3: APT1; Line 4: EIF2A; Line 5: EIF4A1; Line 6: EXP45; Line 7: PP2A; Line 8: TIP41, Line 9: TUB; Line 10: UBC21; Line 11: UBQ; Line 12: 18S; Line 13: 40S; Line 14: MIR156; Line 15: MIR169; Line 16: MIR171/170; Line 17: MIR172; Line 18: MIR398; Line 20: PAL; Line 21: HPPD. (TIFF 19361 kb)

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Borowski, J.M., Galli, V., da Silva Messias, R. et al. Selection of candidate reference genes for real-time PCR studies in lettuce under abiotic stresses. Planta 239, 1187–1200 (2014). https://doi.org/10.1007/s00425-014-2041-2

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