Selection and Validation of Reference Genes for RT-qPCR Analysis in Aegilops tauschii (Coss.) under Different Abiotic Stresses.

Adeel Abbas,Hailan Cui,Haiyan Yu,Ping Huang,Xiangju Li,Jingchao Chen

doi:10.3390/ijms222011017

Abstract

Aegilops tauschii (Coss.) is an aggressive and serious annual grass weed in China. Its DD genome is a rich source of genetic material and performs better under different abiotic stress conditions (salinity, drought, temperature, etc.). Reverse-transcribed quantitative polymerase chain reaction (RT-qPCR) is a reliable technique for reference gene selection and validation. This work aimed to evaluate the stability of reference gene expression in Ae. tauschii under different abiotic stresses (salinity, drought, hot, and cold) and developmental stages (seedling and development). The results show that the ubiquitin-conjugating enzyme E2 36-like (UBC36) and protein microrchidia 2-like (HSP) are the most stable genes under control and salinity conditions, respectively. Under drought stress conditions, UBC36 is more stable as compared with others. Glyceraldehyde-3-phosphate dehydrogenase (GADPH) is the most stable reference gene during heat stress conditions and thioredoxin-like protein (YLS) under cold stress condition. Phosphate2A serine/threonine-protein phosphatase 2A (PP2A) and eukaryotic translation initiation factor 3 (ETIF3) are the most stable genes at seedling and developmental stages. Intracellular transport protein (CAC) is recommended as the most stable gene under different abiotic stresses and at developmental stages. Furthermore, the relative expression levels of NHX1 and DREB under different levels of salinity and drought stress conditions varied with the most (HSP and UBC36) and least (YLS and ACT) stable genes. This study provides reliable reference genes for understanding the tolerance mechanisms in Ae. tauschii under different abiotic stress conditions.

Highlights

Aegilops tauschii Coss. (2n = 2x = 14) is a wild species and reported as a donor D genome in cultivate wheat (Triticum aestivum L.=AABBDD) [1,2]
We evaluated 12 reference genes—actin (ACT), glyceraldehyde3-phosphate dehydrogenase (GAPDH), thioredoxin-like protein (YLS), polypyrimidine tract-binding protein homolog 1-like (PTB), protein microrchidia 2-like (HSP), S-adenosyl methionine decarboxylase proenzyme (SAMDC), ubiquitin-conjugating enzyme E2 36like (UBC36), elongation factor 1 alpha (EF1α), serine/threonine-protein phosphatase 2A (PP2A), eukaryotic translation initiation factor 3 (ET1F3), intracellular transport protein (CAC), and peptidyl-prolyl cis-trans isomerase CYP19-4-like (CYP)—under different abiotic stresses and developmental stages
The results under different abiotic stresses and developmental stages in Ae. tauschii was based on the estimation of Cq

Summary

Introduction

(2n = 2x = 14) is a wild species and reported as a donor D genome in cultivate wheat (Triticum aestivum L.=AABBDD) [1,2] It has the widest geographic distribution in many wheat-growing countries from Turkey to west Afghanistan and Central Asia, and it has adapted to different diversified conditions, performing better against heat, drought, and salinity stresses [3]. This weed spreads in China’s main wheat production areas and competes for light, water, and resources [4]. Gene expression analysis is important in crop improvement programs and helps understand plants’ molecular mechanisms against different abiotic stresses [7]

Objectives

Methods

Results

Conclusion