Validation of Appropriate Reference Genes for qRT-PCR Normalization in Oat (Avena sativa L.) under UV-B and High-Light Stresses

Abstract

Oat is a food and forage crop species widely cultivated worldwide, and it is also an important forage grass in plateau regions of China, where there is a high level of ultraviolet radiation and sunlight. Screening suitable reference genes for oat under UV-B and high-light stresses is a prerequisite for ensuring the accuracy of real-time quantitative PCR (qRT-PCR) data used in plant adaptation research. In this study, eight candidate reference genes (sulfite oxidase, SUOX; victorin binding protein, VBP; actin-encoding, Actin1; protein PSK SIMULATOR 1-like, PSKS1; TATA-binding protein 2-like, TBP2; ubiquitin-conjugating enzyme E2, UBC2; elongation factor 1-alpha, EF1-α; glyceraldehyde-3-phosphate dehydrogenase 1, GAPDH1;) were selected based on previous studies and our oat transcriptome data. The expression stability of these reference genes in oat roots, stems, and leaves under UV-B and high-light stresses was first calculated using three frequently used statistical software (geNorm, NormFinder, and BestKeeper), and then the comprehensive stability of these genes was evaluated using RefFinder. The results showed that the most stably expressed reference genes in the roots, stems, and leaves of oat under UV-B stress were EF1-α, TBP2, and PSKS1, respectively; the most stably expressed reference genes in the roots, stems, and leaves under high-light stress were PSKS1, UBC2, and PSKS1, respectively. PSKS1 was the most stably expressed reference gene in all the samples. The reliability of the selected reference genes was further validated by analysis of the expression of the phenylalanine ammonia-lyase (PAL) gene. This study highlights reference genes for accurate quantitative analysis of gene expression in different tissues of oat under UV-B and high-light stresses.

Keywords: UV-B; high-light; oat; qRT–PCR; reference genes; stability evaluation.

Figure 1

Distribution of Ct values among eight candidate reference genes in the roots (A), stems (B), leaves (C), and all analyzed samples (D). Boxes indicate the 25th and 75th percentiles, with the line across the boxes representing the medians. The whiskers and asterisks represent the 95% confidence intervals and outliers, respectively.

Figure 2

Expression stability of eight reference genes in different oat tissues under UV-B (A–C) and high-light (D–F) stresses based on geNorm analysis.

Figure 3

geNorm analysis of the pairwise variation (V) values for eight candidate reference genes under UV-B (A) and high-light (B) stresses.

Figure 4

Relative expression level of PAL in the stems and leaves under UV-B stress (A,B) and the roots and leaves under high-light stress (C,D) when the most and least stably expressed reference genes were used for normalization.