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Reference genes identification for normalization of qPCR under multiple stresses in Hordeum brevisubulatum
BACKGROUND: Real-time quantitative PCR has been widely used as the most reliable method to measure gene expression, due to its high accuracy and specificity. Wild barley (Hordeum brevisubulatum (Trin.) Link) is a wild relative species in Triticeae that has strong tolerance to abiotic stresses and ex...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2018
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6297944/ https://www.ncbi.nlm.nih.gov/pubmed/30568722 http://dx.doi.org/10.1186/s13007-018-0379-3 |
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author | Zhang, Lili Zhang, Qike Jiang, Ying Li, Yang Zhang, Haiwen Li, Ruifen |
author_facet | Zhang, Lili Zhang, Qike Jiang, Ying Li, Yang Zhang, Haiwen Li, Ruifen |
author_sort | Zhang, Lili |
collection | PubMed |
description | BACKGROUND: Real-time quantitative PCR has been widely used as the most reliable method to measure gene expression, due to its high accuracy and specificity. Wild barley (Hordeum brevisubulatum (Trin.) Link) is a wild relative species in Triticeae that has strong tolerance to abiotic stresses and extremely wide adaptation. However, suitable references gene have not been documented for standardization of gene expression in wild barley under abiotic stress. RESULTS: Here we report the first systematic and comprehensive analysis of reference genes for quantitative real-time PCR standardization in wild barley. We selected 11 genes, including ACT (Actin), ADP (ADP-ribosylation factor 1), CYP2 (Cyclophilin 2), EF-1α (Elongation factor 1-alpha), GAPDH (Glyceraldehyde 3-phosphate dehydrogenase), HSP90 (Heat shock protein 90), TUBα (Alpha-tubulin), TUBβ6 (Beta-tubulin 6), UBI (Ubiquitin), 18SrRNA-1 (guanine1575-N7-methyltransferase) and 18SrRNA-3 (adenine1779-N6-dimethyltransferase) from a wild barley transcriptome database and analyzed their expression stabilities in shoots and roots of wild barley seedling under various stress conditions using comparative ΔCt, BestKeeper, Normfinder and geNorm software. The results demonstrated that ADP was the most suitable reference gene in salt stress while UBI showed peak stability under mannitol and ABA stress; EF-1α was the most appropriate reference gene for PEG, GA(3), ethylene and heat stress; 18SrRNA-3 was the best choice for cold stress; and TUBα was the first stable gene across different tissues. CONCLUSIONS: Our main contribution was to identify reference genes with suitable and stable expression in wild barley under various stress conditions and in different tissues to provide a useful resource for future studies. The results demonstrate the importance of transcriptome data as a useful resource for the screening of candidate reference genes and highlight the need for specific reference genes for specific conditions. Furthermore, these findings will provide valuable information for wild barley and relative species for future research. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1186/s13007-018-0379-3) contains supplementary material, which is available to authorized users. |
format | Online Article Text |
id | pubmed-6297944 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2018 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-62979442018-12-19 Reference genes identification for normalization of qPCR under multiple stresses in Hordeum brevisubulatum Zhang, Lili Zhang, Qike Jiang, Ying Li, Yang Zhang, Haiwen Li, Ruifen Plant Methods Research BACKGROUND: Real-time quantitative PCR has been widely used as the most reliable method to measure gene expression, due to its high accuracy and specificity. Wild barley (Hordeum brevisubulatum (Trin.) Link) is a wild relative species in Triticeae that has strong tolerance to abiotic stresses and extremely wide adaptation. However, suitable references gene have not been documented for standardization of gene expression in wild barley under abiotic stress. RESULTS: Here we report the first systematic and comprehensive analysis of reference genes for quantitative real-time PCR standardization in wild barley. We selected 11 genes, including ACT (Actin), ADP (ADP-ribosylation factor 1), CYP2 (Cyclophilin 2), EF-1α (Elongation factor 1-alpha), GAPDH (Glyceraldehyde 3-phosphate dehydrogenase), HSP90 (Heat shock protein 90), TUBα (Alpha-tubulin), TUBβ6 (Beta-tubulin 6), UBI (Ubiquitin), 18SrRNA-1 (guanine1575-N7-methyltransferase) and 18SrRNA-3 (adenine1779-N6-dimethyltransferase) from a wild barley transcriptome database and analyzed their expression stabilities in shoots and roots of wild barley seedling under various stress conditions using comparative ΔCt, BestKeeper, Normfinder and geNorm software. The results demonstrated that ADP was the most suitable reference gene in salt stress while UBI showed peak stability under mannitol and ABA stress; EF-1α was the most appropriate reference gene for PEG, GA(3), ethylene and heat stress; 18SrRNA-3 was the best choice for cold stress; and TUBα was the first stable gene across different tissues. CONCLUSIONS: Our main contribution was to identify reference genes with suitable and stable expression in wild barley under various stress conditions and in different tissues to provide a useful resource for future studies. The results demonstrate the importance of transcriptome data as a useful resource for the screening of candidate reference genes and highlight the need for specific reference genes for specific conditions. Furthermore, these findings will provide valuable information for wild barley and relative species for future research. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1186/s13007-018-0379-3) contains supplementary material, which is available to authorized users. BioMed Central 2018-12-18 /pmc/articles/PMC6297944/ /pubmed/30568722 http://dx.doi.org/10.1186/s13007-018-0379-3 Text en © The Author(s) 2018 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. |
spellingShingle | Research Zhang, Lili Zhang, Qike Jiang, Ying Li, Yang Zhang, Haiwen Li, Ruifen Reference genes identification for normalization of qPCR under multiple stresses in Hordeum brevisubulatum |
title | Reference genes identification for normalization of qPCR under multiple stresses in Hordeum brevisubulatum |
title_full | Reference genes identification for normalization of qPCR under multiple stresses in Hordeum brevisubulatum |
title_fullStr | Reference genes identification for normalization of qPCR under multiple stresses in Hordeum brevisubulatum |
title_full_unstemmed | Reference genes identification for normalization of qPCR under multiple stresses in Hordeum brevisubulatum |
title_short | Reference genes identification for normalization of qPCR under multiple stresses in Hordeum brevisubulatum |
title_sort | reference genes identification for normalization of qpcr under multiple stresses in hordeum brevisubulatum |
topic | Research |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6297944/ https://www.ncbi.nlm.nih.gov/pubmed/30568722 http://dx.doi.org/10.1186/s13007-018-0379-3 |
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