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 (Universidad de Zaragoza) ; Díaz, Aurora (Universidad de Zaragoza)

Resumen: 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.

Idioma: Inglés

DOI: 10.3390/ijms24033052

Año: 2023

Publicado en: International Journal of Molecular Sciences 24, 3 (2023), 3052 [14 pp]
ISSN: 1661-6596

Factor impacto JCR: 4.9 (2023)

Categ. JCR: BIOCHEMISTRY & MOLECULAR BIOLOGY rank: 66 / 313 = 0.211 (2023) - Q1 - T1
Categ. JCR: CHEMISTRY, MULTIDISCIPLINARY rank: 68 / 231 = 0.294 (2023) - Q2 - T1

Factor impacto CITESCORE: 8.1 - Spectroscopy (Q1) - Computer Science Applications (Q1) - Physical and Theoretical Chemistry (Q1) - Inorganic Chemistry (Q1) - Organic Chemistry (Q1) - Molecular Biology (Q2) - Catalysis (Q2)

Factor impacto SCIMAGO: 1.179 - Medicine (miscellaneous) (Q1) - Physical and Theoretical Chemistry (Q1) - Computer Science Applications (Q1) - Inorganic Chemistry (Q1) - Spectroscopy (Q1) - Organic Chemistry (Q1) - Molecular Biology (Q2) - Catalysis (Q2)

Financiación: info:eu-repo/grantAgreement/ES/DGA-FEDER/A12-17R
Financiación: info:eu-repo/grantAgreement/ES/DGA/LMP148-21
Financiación: info:eu-repo/grantAgreement/ES/DGA/LMP164-18
Financiación: info:eu-repo/grantAgreement/ES/INIA/RTA2017-00093-00-00

Tipo y forma: Article (Published version)

Área (Departamento): Área Producción Vegetal (Dpto. CC.Agrar.y Medio Natural)