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

Xue Bai,Tao Chen,Mingyong Tang,Zeng-Fu Xu,Yuan Wu

doi:10.3390/ijms22052569

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.

Highlights

To pursue a better quality of life and a healthier diet for humans, many economically important crops have been intensively studied
Fourteen candidate reference genes (RGs) (18S, ACT, actin-depolymerizing factor 7 (ADF7), CYC, EF1α, elongation factor 2 (EF2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), malate dehydrogenase (MDH), phosphoglycerate kinase (PGK), ribosomal protein L11 (RPL11), ribulose bisphosphate carboxylase/oxygenase (Rubisco), tubulin beta-4 (TUB4), ubiquitin-conjugating enzyme 2 (UCE2), and ubiquitin-like protein 5 (UBL5)) and two target genes (CLO and diacylglycerol acyltransferase 2-2 (DGAT2-2)) of tiger nut were selected for Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) normalization
The qRT-PCR efficiency for all 14 candidate RGs ranged from 92.54% to 118.22%, and the correlation coefficients varied from 0.9914 to 0.9998 (Table 1)

Summary

Introduction

To pursue a better quality of life and a healthier diet for humans, many economically important crops have been intensively studied. Tiger nut (Cyperus esculentus) is a perennial herb of Cyperaceae that is widely distributed in almost all middle- and low-latitude areas [8]; this plant stockpiles oil in its tubers as an underground storage organ [9]. Tiger nut is an unconventional crop that is distinguished by its oil-rich tubers, and it is known as yellow nutsedge and chufa. Oleic acid (C18:1) is abundantly contained in the tuber oil of this plant and is a kind of monounsaturated fatty acids (MUFAs) [10], which has positive effects on human health, especially by reducing the blood lipids [11], blood pressure [12], and the incidence of type 2 diabetes mellitus [13]. To facilitate the studies of new genes and pathways related to lipid accumulation in tiger nut, it is necessary to select optimal reference genes (RGs)

Methods

Results

Discussion

Conclusion