Dataset Information

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

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 (?Ct), 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.

SUBMITTER: Zhang J

PROVIDER: S-EPMC6627066 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature

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

Zhang Junchao J Xie Wengang W Yu Xinxuan X Zhang Zongyu Z Zhao Yongqiang Y Wang Na N Wang Yanrong Y

Genes 20190613 6

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 refere ...[more]

PMID: 31200580

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Project description:A detailed understanding of the distribution and degree of genetic variation within a species is important for determining their evolutionary potential, which in return facilitates the development of efficient conservation strategies aimed at preserving adaptive genetic variation. As an important perennial, cool-season grass in temperate Eurasia, increasing attention has been paid to Siberian wildrye (Elymus sibiricus) due to its excellent ecological utilization value and forage production potential in China, particularly in the Qinghai-Tibet Plateau (QTP) regions. In this study, we applied two chloroplast (cp) genes (matK and rbcL), three cp spacer regions (trnY-GUA∼trnD-GUC, atpH∼atpF, and rps4∼trnT-UGU), and six cpSSR markers to the genetic and phylogenetic analysis of 137 wild E. sibiricus accessions from 23 natural populations that represent the main distribution regions in China. The results show the highest genetic diversity (h = 0.913) and haplotype richness (10 haplotypes) for the QTP population, which indicates QTP as the probable diversity center and geographic origin of E. sibiricus in China. Population divergence was high, indicating a significant phylogeographic structure together with a significantly higher Nst value (Nst > Gst, P < 0.05) at the species level, QTP+XJ (combined populations from QTP and Xinjiang), QTP+NC (combined populations from QTP and North China), and XJ+NC (combined populations from Xinjiang and North China) group levels, respectively. An expansion was revealed in the distributional range of E. sibiricus in China from paleo times up to the recent past, while a dramatic range of contraction was predicted for the near future. The predicted main limiting factor for the further spread of E. sibiricus is an increasing global mean temperature. We recommend that the combination of Es-cpDNA1 and Es-cpDNA3+4+5 can be used as effective markers for phylogenetic analysis and phylogeographical history analysis of E. sibiricus. These findings shed new light on the historical population dynamics of cold-season herbs in the QTP region and the north of China and are of great significance for the future establishment of protection and collection strategies for wild E. sibiricus germplasm.

Project description:The reverse transcription quantitative polymerase chain reaction (RT-qPCR) has been widely used to determine gene functions in Laodelphax striatellus (Fallén) (small brown planthopper). Selection of suitable reference gene(s) for normalizations of RT-qPCR data is critical for reliable results. To date, reports on identification of suitable L. striatellus reference genes are still very limited. L. striatellus is a destructive rice pest and it can transmit multiple viruses, including Rice black-streaked dwarf virus (RBSDV), Rice stripe virus (RSV), and Maize rough dwarf virus (MRDV), to many important cereal crops worldwide. In this study, we examined the stablity of seven selected candidate reference genes in L. striatellus at different developmental stages, in different tissues, in RBSDV- or RSV-infected L. striatellus or in RBSDV-infected and Lssynaptojanin 1 (LsSYNJ1)-silenced L. striatellus. The RT-qPCR data representing individual candidate genes were analyzed using five different methods: the delta Ct method, geNorm, NormFinder, BestKeeper, and the RefFinder algorithm, respectively. The most stable reference gene for the specific condition was selected according to a comprehensive analysis using the RefFinder method. Ribosomal protein L5 (LsRPL5) and LsRPL8 are the most stably expressed genes in L. striatellus at different developmental stages. Alpha-1-tubulin (Lsα-TUB) is the most stably expressed reference gene in different tissues of RBSDV viruliferous (RBSDV-V) or non-viruliferous (RBSDV-NV) L. striatellus. LsRPL8 is the most stably expressed reference gene in RBSDV-V or RSV viruliferous (RSV-V) L. striatellus, while beta-tubulin (Lsβ-TUB) is the most stably expressed reference gene in RBSDV-V and LsSYNJ1-silenced L. striatellus. The selected reference genes were further investigated during analyses of RBSDV P5-1 and P10 gene expression in different tissues from RBSDV-V or RBSDV-NV L. striatellus. The stably expressed reference genes identified in this study will benefit future gene function studies using L. striatellus.

Dataset Information

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

Publications

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

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