Selection of Suitable Reference Genes for RT-qPCR Gene Expression Analysis in Siberian Wild Rye (Elymus sibiricus) under Different Experimental Conditions.

Yanrong Wang,Zongyu Zhang,Xinxuan Yu,Junchao Zhang,Yongqiang Zhao,Wengang Xie,Na Wang

doi:10.3390/genes10060451

Yanrong Wang, Zongyu Zhang + Show 5 more

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https://doi.org/10.3390/genes10060451

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Abstract

Elymus sibiricus, which is a perennial and self-pollinated grass, is the typical species of the genus Elymus, which plays an important role in forage production and ecological restoration. No reports have, so far, systematically described the selection of optimal reference genes for reverse transcriptase quantitative real-time polymerase chain reaction (RT-qPCR) analysis in E. sibiricus. The goals of this study were to evaluate the expression stability of 13 candidate reference genes in different experimental conditions, and to determine the appropriate reference genes for gene expression analysis in E. sibiricus. Five methods including Delta Ct (ΔCt), BestKeeper, NormFinder, geNorm, and RefFinder were used to assess the expression stability of 13 potential reference genes. The results of the RefFinder analysis showed that TBP2 and HIS3 were the most stable reference genes in different genotypes. TUA2 and PP2A had the most stable expression in different developmental stages. TBP2 and PP2A were suitable reference genes in different tissues. Under salt stress, ACT2 and TBP2 were identified as the most stable reference genes. ACT2 and TUA2 showed the most stability under heat stress. For cold stress, PP2A and ACT2 presented the highest degree of expression stability. DNAJ and U2AF were considered as the most stable reference genes under osmotic stress. The optimal reference genes were selected to investigate the expression pattern of target gene CSLE6 in different conditions. This study provides suitable reference genes for further gene expression analysis using RT-qPCR in E. sibiricus.

Highlights

Elymus is the largest genus of the tribe Triticeae with worldwide distribution, including approximately 150 species [1]
Specific primers for RT-qPCR were designed according to the sequences of 13 candidate reference genes
Numerous studies suggested that none of the reference genes maintain the consistent expression stability among various experimental conditions, and it is imperative to carry out reference gene screening under specific experimental conditions [11,29,48,49,50,51]

Summary

Introduction

Elymus is the largest genus of the tribe Triticeae with worldwide distribution, including approximately 150 species [1]. E. sibiricus (Siberian wild rye), which is the typical species of the genus Elymus, is a perennial, allotetraploid, and self-pollination grass [2,3]. It is one of the most important forages in Northern China due to its high protein content, strong adaptability, superior cold, and drought tolerance [4]. E. sibiricus were widely used in artificial grassland and ecological governance in recent years [5]. It is noteworthy that severe seed shattering is the main reason for seed yield losses in E. sibiricus. The degree of seed shattering in E. sibiricus

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