Selection and Identification of a Reference Gene for Normalizing Real-Time PCR in Mangos under Various Stimuli in Different Tissues

Abstract

Real-time quantitative polymerase chain reaction (Real-Time PCR) is a rapid, highly sensitive, and highly specific technique, which is widely used to determine the relative expression of target genes in plants. It plays an indispensable role in searching for stable reference genes in different species. However, no suitable reference genes for real-time PCR normalization have been reported in mangos. In this study, 10 candidate reference genes were obtained from the ‘Carabao’ genome, and their expression stability under seven abiotic stresses (MeJA, Mannitol, NaCl, SA, ABA, heat, and cold) and in four different tissues (root, stem, leaf, and fruit) was rated using four professional reference gene scoring software packages (geNorm, NormFinder, BestKeeper, and RefFinder). The results indicated that the stability of the 10 selected genes varied significantly under different experimental conditions; moreover, TUBB is more stable than the other candidate reference genes and can be used as a suitable reference gene, since it was always ranked as one of the top three in different tissues and under multiple conditions, according to the comprehensive ranking. To ensure the applicability of the identified reference genes, the relative expression levels of Chalcone synthase 1 (CHS-1) and Chalcone synthase 2 (CHS-2) were used to confirm the accuracy of the results. The evaluation of the stability of multiple reference genes will facilitate the future accurate quantification of target genes by real-time PCR in mangos.

Keywords

• <i>Mangifera indica</i> L.
• real-time PCR
• reference gene
• gene expression
• normalization