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Identification and validation of reference genes for RT-qPCR normalization in wheat meiosis

Meiosis is a specialized type of cell division occurring in sexually reproducing organisms to generate haploid cells known as gametes. In flowering plants, male gametes are produced in anthers, being encased in pollen grains. Understanding the genetic regulation of meiosis key events such as chromos...

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Autores principales: Garrido, José, Aguilar, Miguel, Prieto, Pilar
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7026057/
https://www.ncbi.nlm.nih.gov/pubmed/32066846
http://dx.doi.org/10.1038/s41598-020-59580-5
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author Garrido, José
Aguilar, Miguel
Prieto, Pilar
author_facet Garrido, José
Aguilar, Miguel
Prieto, Pilar
author_sort Garrido, José
collection PubMed
description Meiosis is a specialized type of cell division occurring in sexually reproducing organisms to generate haploid cells known as gametes. In flowering plants, male gametes are produced in anthers, being encased in pollen grains. Understanding the genetic regulation of meiosis key events such as chromosome recognition and pairing, synapsis and recombination, is needed to manipulate chromosome associations for breeding purposes, particularly in important cereal crops like wheat. Reverse transcription-quantitative PCR (RT-qPCR) is widely used to analyse gene expression and to validate the results obtained by other transcriptomic analyses, like RNA-seq. Selection and validation of appropriate reference genes for RT-qPCR normalization is essential to obtain reproducible and accurate expression data. In this work, twelve candidate reference genes were evaluated using the mainstream algorithms geNorm, Normfinder, BestKeeper and ΔCt, then ranked from most to least suitable for normalization with RefFinder. Different sets of reference genes were recommended to normalize gene expression data in anther meiosis of bread and durum wheat, their corresponding genotypes in the absence of the Ph1 locus and for comparative studies among wheat genotypes. Comparisons between meiotic (anthers) and somatic (leaves and roots) wheat tissues were also carried out. To the best of our knowledge, our study provides the first comprehensive list of reference genes for robust RT-qPCR normalization to study differentially expressed genes during male meiosis in wheat in a breeding framework.
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spelling pubmed-70260572020-02-24 Identification and validation of reference genes for RT-qPCR normalization in wheat meiosis Garrido, José Aguilar, Miguel Prieto, Pilar Sci Rep Article Meiosis is a specialized type of cell division occurring in sexually reproducing organisms to generate haploid cells known as gametes. In flowering plants, male gametes are produced in anthers, being encased in pollen grains. Understanding the genetic regulation of meiosis key events such as chromosome recognition and pairing, synapsis and recombination, is needed to manipulate chromosome associations for breeding purposes, particularly in important cereal crops like wheat. Reverse transcription-quantitative PCR (RT-qPCR) is widely used to analyse gene expression and to validate the results obtained by other transcriptomic analyses, like RNA-seq. Selection and validation of appropriate reference genes for RT-qPCR normalization is essential to obtain reproducible and accurate expression data. In this work, twelve candidate reference genes were evaluated using the mainstream algorithms geNorm, Normfinder, BestKeeper and ΔCt, then ranked from most to least suitable for normalization with RefFinder. Different sets of reference genes were recommended to normalize gene expression data in anther meiosis of bread and durum wheat, their corresponding genotypes in the absence of the Ph1 locus and for comparative studies among wheat genotypes. Comparisons between meiotic (anthers) and somatic (leaves and roots) wheat tissues were also carried out. To the best of our knowledge, our study provides the first comprehensive list of reference genes for robust RT-qPCR normalization to study differentially expressed genes during male meiosis in wheat in a breeding framework. Nature Publishing Group UK 2020-02-17 /pmc/articles/PMC7026057/ /pubmed/32066846 http://dx.doi.org/10.1038/s41598-020-59580-5 Text en © The Author(s) 2020 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as 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 images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
spellingShingle Article
Garrido, José
Aguilar, Miguel
Prieto, Pilar
Identification and validation of reference genes for RT-qPCR normalization in wheat meiosis
title Identification and validation of reference genes for RT-qPCR normalization in wheat meiosis
title_full Identification and validation of reference genes for RT-qPCR normalization in wheat meiosis
title_fullStr Identification and validation of reference genes for RT-qPCR normalization in wheat meiosis
title_full_unstemmed Identification and validation of reference genes for RT-qPCR normalization in wheat meiosis
title_short Identification and validation of reference genes for RT-qPCR normalization in wheat meiosis
title_sort identification and validation of reference genes for rt-qpcr normalization in wheat meiosis
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7026057/
https://www.ncbi.nlm.nih.gov/pubmed/32066846
http://dx.doi.org/10.1038/s41598-020-59580-5
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