Selection and Validation of Reference Genes for Quantitative Real-Time PCR in White Clover (<i>Trifolium repens</i> L.) Involved in Five Abiotic Stresses

• Main Authors: Qi Pu, Zhou Li, Gang Nie, Jiqiong Zhou, Lin Liu, Yan Peng
• Format: Article
• Language: English
• Published: MDPI AG 2020-08-01
• Series: Plants
• Subjects: abiotic stress; gene expression; qRT-PCR; reference genes; white clover
• Online Access: https://www.mdpi.com/2223-7747/9/8/996

White clover (<i>Trifolium repens</i> L.) is a widely cultivated cool-season perennial forage legume in temperate grassland systems. Many studies have analyzed the gene expression in this grass species using quantitative real-time reverse transcription PCR (qRT-PCR). The selection of stable reference genes for qRT-PCR is crucial. However, there was no detailed study on reference genes in different tissues of white clover under various abiotic stress conditions. Herein, 14 candidate reference genes (<i>ACT7, ACT101, TUA1109, TUB, CYP, 60SrRNA, UBQ, E3, GAPDH1, GAPDH2, PP2A, BAM3, SAMDC,</i> and <i>ABC</i>) were selected and analyzed by four programs (GeNorm, NormFinder, BestKeeper, and RefFinder). Samples were taken from two tissues (leaves and roots) under five different abiotic stresses (drought, salt, heat, cold, and heavy metal stress). Our results showed that <i>60SrRNA</i> and <i>ACT101</i> were the two top-ranked genes for all samples. Under various experimental conditions, the most stable gene was different; however, <i>SAMDC, UBQ, 60SrRNA,</i> and <i>ACT101</i> were always top ranked. The most suitable reference genes should be selected according to different plant tissues and growth conditions. Validation of these reference genes by expression analysis of <i>Cyt-Cu/Zn SOD</i> and <i>CAT</i> confirmed their reliability. Our study will benefit the subsequent research of gene function in this species.