Selection and Validation of Reference Genes for qRT-PCR Analysis in the Oil-Rich Tuber Crop Tiger Nut (Cyperus esculentus) Based on Transcriptome Data

Abstract

Tiger nut (Cyperus esculentus), a perennial C₄ plant of the Cyperaceae family, is an unconventional crop that is distinguished by its oil-rich tubers, which also possesses the advantages of strong resistance, wide adaptability, short life periods, and large biomass. To facilitate studies on gene expression in this species, we identified and validated a series of reference genes (RGs) based on transcriptome data, which can be employed as internal controls for qRT-PCR analysis in tiger nut. Fourteen putative candidate RGs were identified and evaluated across nine different tissues of two cultivars, and the RGs were analyzed using three different algorithms (geNorm, NormFinder, and BestKeeper). The stability rankings of the candidate RGs were merged into consensus lists with RankAggreg. For the below-ground storage organ of tiger nut, the optimal RGs were TUB4 and UCE2 in different developmental stages of tubers. UCE2 and UBL5 were the most stably expressed RGs among all tissues, while Rubisco and PGK exhibited the lowest expression stability. UCE2, UBL5 and Rubisco were compared to normalize the expression levels of the caleosin (CLO) and diacylglycerol acyltransferase 2-2 (DGAT2-2) genes across the same tissues. Our results showed that the RGs identified in this study, which exhibit more uniform expression patterns, may be utilized for the normalization of qRT-PCR results, promoting further research on gene expression in various tissues of tiger nut.

Keywords: Cyperus esculentus; RankAggreg; gene expression; normalization; qRT-PCR; reference gene; tuber.

Figure 1

Melt curve analysis of 14 candidate reference genes and two target genes assessed across all tissues of two cultivars, “YN” and “XJ”, of tiger nut (including young leaves, mature leaves, leaf sheathes, stem apexes, roots, rhizomes and tubers in three developmental stages) and showed a single peak for each primer pair at a specific annealing temperature. The –(δF/δT) value represents the raw fluorescence (F) versus temperature (T) values.

Figure 2

Distribution of threshold cycle (C_t) values of 14 candidate reference genes across all 18 samples. Yellow boxes represent XJ tiger nuts, and blue boxes represent YN tiger nuts. The box indicates the 25th and 75th percentiles. The line across the box indicates the median, and the cross depicts the mean.

Figure 3

Pairwise variation (V) analyses of 14 candidate reference genes to use for data normalization in two cultivars, “YN” and “XJ”, of tiger nut, as computed by geNorm. This chart contains several tissue groups, including above (aboveground), under (underground), tubers, and total samples of two cultivars.

Figure 4

Expression profiles of CLO in different tissues of YN and XJ tiger nuts. (A) Relative expressions in YN tissues by qRT-PCR. (B) Relative expressions in XJ tissues by qRT-PCR. UCE2, UBL5, and UCE2 + UBL5 were used as the one or two most stable reference genes. Rubisco was used as the least stable reference gene, which is shown in grey bars. (C) Transcript levels of CLO. The horizontal axis shows nine tissues of tiger nut (YL: young leaves, ML: mature leaves, SH: sheath, SA: shoot apexes, T-form: tubers in the formation stage, T-swell: tubers in the swelling stage, T-mature: tubers in the mature stage, RH: rhizome, R: root). Data are represented as the mean ± SD. Bars with different letters (a, b, and c) are significantly different from each other in the expression of the target gene based on three biological replications (p < 0.05, t test; n = 3).

Figure 5

Expression profiles of DGAT2-2 in different tissues of YN and XJ tiger nut. (A) Relative expressions in YN tissues by qRT-PCR. (B) Relative expressions in XJ tissues by qRT-PCR. UCE2, UBL5, and UCE2 + UBL5 were used as the one or two most stable reference genes. Rubisco was used as the least stable reference gene, which is shown in grey bars. (C) Transcript levels of DGAT2-2. The horizontal axis shows nine tissues of tiger nut (YL: young leaves, ML: mature leaves, SH: sheath, SA: shoot apexes, T-form: tubers in the formation stage, T-swell: tubers in the swelling stage, T-mature: tubers in the mature stage, RH: rhizome, R: root). Data are represented as the mean ± SD. Bars with different letters (a, b, and c) are significantly different from each other in the expression of the target gene based on three biological replications (p < 0.05, t test; n = 3).