Identification and Validation of Reference Genes for Seashore Paspalum Response to Abiotic Stresses.

Yu Liu,Bingru Huang,Zhimin Yang,Jun Liu,Hui Lai,Lei Xu,Yu Chen

doi:10.3390/ijms18061322

Abstract

Seashore paspalum (Paspalum vaginatum) is among the most salt- and cadmium-tolerant warm-season perennial grass species widely used as turf or forage. The objective of this study was to select stable reference genes for quantitative real-time polymerase chain reaction (qRT-PCR) analysis of seashore paspalum in response to four abiotic stresses. The stability of 12 potential reference genes was evaluated by four programs (geNorm, NormFinder, BestKeeper, and RefFinder). U2AF combined with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) showed stable expression in Cd-treated leaves and cold-treated roots. U2AF and FBOX were the most stable reference genes in Cd-treated roots and cold-treated leaves. In Polyethylene Glycol (PEG)- or salt-treated roots, the reference gene U2AF paired with either ACT or CYP were stable. SAND and CACS exhibited the most stability in salt-treated leaves, and combining UPL, PP2A, and EF1a was most suitable for PEG-treated leaves. The stability of U2AF and instability of UPL and TUB was validated by analyzing the expression levels of four target genes (MT2a, VP1, PIP1, and Cor413), and were shown to be capable of detecting subtle changes in expression levels of the target genes in seashore paspalum. This study demonstrated that FBOX, U2AF, and PP2A could be used in future molecular studies that aim to understand the mechanisms of abiotic stress tolerance in seashore paspalum.

Highlights

Quantifying the level of gene expression is a critical step for gene discovery and molecular analysis [1]
Microarray and transcriptome data were used to develop new reference genes with highly stable expression levels, such as SAND family protein (SAND), F-box/kelch-repeat protein (F-box), clathrin adapter complex subunit family protein (CACS), splicing factor (U2AF), and TIP41-like family protein (TIP41), all of which were identified in Arabidopsis thaliana [10]
The quantification cycle (Cq) values of the 12 candidate reference genes were measured by quantitative real-time polymerase chain reaction (qRT-PCR) analysis and ranged from 18 to 32 (Figure 2), with the lower Cq values showing higher mRNA transcript levels

Summary

Introduction

Quantifying the level of gene expression is a critical step for gene discovery and molecular analysis [1]. A number of reference genes have been screened from many plant species, and the specific reference genes suitable for gene expression quantification vary with plant species [4,5], as a result of their having different response mechanisms These traditional reference genes include those associated with primary metabolism or other cellular processes, such as actin (ACT), tubulin (TUB), elongation factor 1a (EF1a), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and 18s ribosomal RNA (18S rRNA) [6,7]. The previous work strongly suggests that it is important to select suitable reference genes for different organs in specific plant species under various environmental conditions in order to accurately quantify expression levels of target genes using qRT-PCR

Objectives

Methods

Results