Selection of Novel Reference Genes by RNA-Seq and Their Evaluation for Normalising Real-Time qPCR Expression Data of Anthocyanin-Related Genes in Lettuce and Wild Relatives

Medina-Lozano, Inés; Arnedo, María Soledad; Grimplet, Jérôme; Díaz, Aurora

doi:10.3390/ijms24033052

Open AccessArticle

Selection of Novel Reference Genes by RNA-Seq and Their Evaluation for Normalising Real-Time qPCR Expression Data of Anthocyanin-Related Genes in Lettuce and Wild Relatives

by

Inés Medina-Lozano

^1,2

,

María Soledad Arnedo

³,

Jérôme Grimplet

^1,2

and

Aurora Díaz

^1,2,*

¹

Department of Plant Sciences, Agrifood Research and Technology Centre of Aragon (CITA), Avd. Montañana 930, 50059 Zaragoza, Spain

²

AgriFood Institute of Aragon–IA2, CITA-University of Zaragoza, 50013 Zaragoza, Spain

³

Ramiro Arnedo S.A. Paraje La Molina 54, Las Norias de Daza, 04716 Almería, Spain

^*

Author to whom correspondence should be addressed.

Int. J. Mol. Sci. 2023, 24(3), 3052; https://doi.org/10.3390/ijms24033052

Submission received: 30 November 2022 / Revised: 23 January 2023 / Accepted: 26 January 2023 / Published: 3 February 2023

(This article belongs to the Section Molecular Plant Sciences)

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Abstract

Lettuce is a popular vegetable source of bioactive compounds, like anthocyanins, powerful antioxidants present in red and semi-red varieties. Selection of reliable reference genes (RGs) for the normalization of real-time quantitative PCR (qPCR) data is crucial to obtain accurate gene expression results. Among the genes with totally unrelated biological functions, six candidate RGs (ADF2, CYB5, iPGAM, SCL13, TRXL3-3, and VHA-H) with low variation in expression according to RNA-seq analyses, were selected for future expression studies of anthocyanin-related genes in three different experiments: leaf colour comparison (green vs. red) in commercial varieties; tissue comparison (leaf vs. stem) in a wild relative; and drought stress experiment in commercial and traditional varieties, and a wild relative. Expression profiles of the candidate RGs were obtained by qPCR and their stability was assessed by four different analytical tools, geNorm, NormFinder, BestKeeper, and Delta Ct method, all integrated in RefFinder. All results considered, we recommend CYB5 to be used as RG for the leaf colour experiment and TRXL3-3 for the tissue and drought stress ones, as they were the most stable genes in each case. RNA-seq is useful to preselect novel RGs although validation by qPCR is still advisable. These results provide helpful information for gene expression studies in Lactuca spp. under the described conditions.

Keywords: BestKeeper; Delta Ct method; drought stress; geNorm; Lactuca sativa L.; NormFinder; leaf colour; RefFinder; tissues; transcriptomics

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MDPI and ACS Style

Medina-Lozano, I.; Arnedo, M.S.; Grimplet, J.; Díaz, A. Selection of Novel Reference Genes by RNA-Seq and Their Evaluation for Normalising Real-Time qPCR Expression Data of Anthocyanin-Related Genes in Lettuce and Wild Relatives. Int. J. Mol. Sci. 2023, 24, 3052. https://doi.org/10.3390/ijms24033052

AMA Style

Medina-Lozano I, Arnedo MS, Grimplet J, Díaz A. Selection of Novel Reference Genes by RNA-Seq and Their Evaluation for Normalising Real-Time qPCR Expression Data of Anthocyanin-Related Genes in Lettuce and Wild Relatives. International Journal of Molecular Sciences. 2023; 24(3):3052. https://doi.org/10.3390/ijms24033052

Chicago/Turabian Style

Medina-Lozano, Inés, María Soledad Arnedo, Jérôme Grimplet, and Aurora Díaz. 2023. "Selection of Novel Reference Genes by RNA-Seq and Their Evaluation for Normalising Real-Time qPCR Expression Data of Anthocyanin-Related Genes in Lettuce and Wild Relatives" International Journal of Molecular Sciences 24, no. 3: 3052. https://doi.org/10.3390/ijms24033052

APA Style

Medina-Lozano, I., Arnedo, M. S., Grimplet, J., & Díaz, A. (2023). Selection of Novel Reference Genes by RNA-Seq and Their Evaluation for Normalising Real-Time qPCR Expression Data of Anthocyanin-Related Genes in Lettuce and Wild Relatives. International Journal of Molecular Sciences, 24(3), 3052. https://doi.org/10.3390/ijms24033052

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Selection of Novel Reference Genes by RNA-Seq and Their Evaluation for Normalising Real-Time qPCR Expression Data of Anthocyanin-Related Genes in Lettuce and Wild Relatives

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