Reference Gene Selection for Quantitative Real-Time Reverse-Transcriptase PCR in Annual Ryegrass (Lolium multiflorum) Subjected to Various Abiotic Stresses.

Qiuxu Liu,Linkai Huang,Xinquan Zhang,Haidong Yan,Gang Nie,Xiao Qi

doi:10.3390/molecules23010172

Qiuxu Liu, Linkai Huang + Show 4 more

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https://doi.org/10.3390/molecules23010172

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Abstract

To select the most stable reference genes in annual ryegrass (Lolium multiflorum), we studied annual ryegrass leaf tissues exposed to various abiotic stresses by qRT-PCR and selected 11 candidate reference genes, i.e., 18S rRNA, E2, GAPDH, eIF4A, HIS3, SAMDC, TBP-1, Unigene71, Unigene77, Unigene755, and Unigene14912. We then used GeNorm, NormFinder, and BestKeeper to analyze the expression stability of these 11 genes, and used RefFinder to comprehensively rank genes according to stability. Under different stress conditions, the most suitable reference genes for studies of leaf tissues of annual ryegrass were different. The expression of the eIF4A gene was the most stable under drought stress. Under saline-alkali stress, Unigene14912 has the highest expression stability. Under acidic aluminum stress, SAMDC expression stability was highest. Under heavy metal stress, Unigene71 expression had the highest stability. According to the software analyses, Unigene14912, HIS3, and eIF4A were the most suitable for analyses of abiotic stress in tissues of annual ryegrass. GAPDH was the least suitable reference gene. In conclusion, selecting appropriate reference genes under abiotic stress not only improves the accuracy of annual ryegrass gene expression analyses, but also provides a theoretical reference for the development of reference genes in plants of the genus Lolium.

Highlights

Annual ryegrass (Lolium multiflorum) is cultivated in temperate and subtropical regions worldwide and is used in forage and livestock systems as silage and green fodder owing to its high palatability and digestibility [1,2]
The expression levels of 11 candidate reference genes under various types of stress were analyzed in annual ryegrass
HIS3+TBP-1 under saline-alkali stress; the expression of HIS3 is best suited for the reference gene under acidic aluminum stress; Unigene71 + HIS3 is the optimal choice of reference gene under heavy metal stress

Summary

Introduction

Annual ryegrass (Lolium multiflorum) is cultivated in temperate and subtropical regions worldwide and is used in forage and livestock systems as silage and green fodder owing to its high palatability and digestibility [1,2]. It is one of the most widely cultivated plants in the world, and grows in warm and humid climates. Quantitative reverse transcription PCR (qRT-PCR) is widely used to determine the expression of genes in different plant tissues owing to its high sensitivity, specificity, accuracy, and repeatability [4]; it is a powerful technical method to detect gene expression levels.

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