Selection and validation of reference genes by RT-qPCR under photoperiodic induction of flowering in sugarcane (Saccharum spp.)
Autor(a) principal: | |
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Data de Publicação: | 2021 |
Outros Autores: | , , , , , , |
Tipo de documento: | Artigo |
Idioma: | eng |
Título da fonte: | Repositório Institucional da UNESP |
Texto Completo: | http://dx.doi.org/10.1038/s41598-021-83918-2 http://hdl.handle.net/11449/205977 |
Resumo: | Although reference genes have previously been used in the expression analysis of genes involved in sugarcane flowering they had not been experimentally validated for stability and consistency of expression between different samples over a wide range of experimental conditions. Here we report the analysis of candidate reference genes in different tissue types, at different temporal time-points, in both short and long day photoperiodic treatments. The stability of the candidate reference genes in all conditions was evaluated with NormFinder, BestKeeper, and RefFinder algorithms that complement each other for a more robust analysis. As the Normfinder algorithm was more appropriate for our experimental conditions, greater emphasis was placed on Normfinder when choosing the most stable genes. UBQ1 and TUB were shown to be the most stable reference genes to use for normalizing RT-qPCR gene expression data during floral induction, whilst 25SrRNA1 and GAPDH were the least stable. Their use as a reference gene pair was validated by analyzing the expression of two differentially expressed target genes (PIL5 and LHP1). The UBQ1/TUB reference genes combination was able to reveal small significant differences in gene expression of the two target genes that were not detectable when using the least stable reference gene combination. These results can be used to inform the choice of reference genes to use in the study of the sugarcane floral induction pathway. Our work also demonstrates that both PIL5 and LHP1 are significantly up-regulated in the initial stages of photoperiodic induction of flowering in sugarcane. |
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Selection and validation of reference genes by RT-qPCR under photoperiodic induction of flowering in sugarcane (Saccharum spp.)Although reference genes have previously been used in the expression analysis of genes involved in sugarcane flowering they had not been experimentally validated for stability and consistency of expression between different samples over a wide range of experimental conditions. Here we report the analysis of candidate reference genes in different tissue types, at different temporal time-points, in both short and long day photoperiodic treatments. The stability of the candidate reference genes in all conditions was evaluated with NormFinder, BestKeeper, and RefFinder algorithms that complement each other for a more robust analysis. As the Normfinder algorithm was more appropriate for our experimental conditions, greater emphasis was placed on Normfinder when choosing the most stable genes. UBQ1 and TUB were shown to be the most stable reference genes to use for normalizing RT-qPCR gene expression data during floral induction, whilst 25SrRNA1 and GAPDH were the least stable. Their use as a reference gene pair was validated by analyzing the expression of two differentially expressed target genes (PIL5 and LHP1). The UBQ1/TUB reference genes combination was able to reveal small significant differences in gene expression of the two target genes that were not detectable when using the least stable reference gene combination. These results can be used to inform the choice of reference genes to use in the study of the sugarcane floral induction pathway. Our work also demonstrates that both PIL5 and LHP1 are significantly up-regulated in the initial stages of photoperiodic induction of flowering in sugarcane.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Biotechnology and Biological Sciences Research CouncilCoordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Faculdade de Ciências Agrárias e Veterinárias UNESP, Campus JaboticabalUniversidade Estadual de Campinas UNICAMPInstituto de Ciência e Tecnologia da Universidade Federal de São PauloDepartamento de Biotecnologia Escola de Engenharia de Lorena/Universidade de São Paulo EEL/USPInstituto Agronômico de Campinas Centro de CanaSchool of Life Sciences University of Warwick, Gibbet HillFaculdade de Ciências Agrárias e Veterinárias UNESP, Campus JaboticabalFAPESP: 2016/02679-9Biotechnology and Biological Sciences Research Council: 2018/08380-0CAPES: Finance Code 001Universidade Estadual Paulista (Unesp)Universidade Estadual de Campinas (UNICAMP)Universidade Federal de São Paulo (UNIFESP)Universidade de São Paulo (USP)Centro de CanaUniversity of Warwickda Silva Santos, Paulo H. [UNESP]Manechini, João R. VieiraBrito, Michael S.Romanel, ElissonVicentini, RenatoScarpari, MaximilianoJackson, StephenPinto, Luciana R.2021-06-25T10:24:35Z2021-06-25T10:24:35Z2021-12-01info:eu-repo/semantics/publishedVersioninfo:eu-repo/semantics/articlehttp://dx.doi.org/10.1038/s41598-021-83918-2Scientific Reports, v. 11, n. 1, 2021.2045-2322http://hdl.handle.net/11449/20597710.1038/s41598-021-83918-22-s2.0-85101813884Scopusreponame:Repositório Institucional da UNESPinstname:Universidade Estadual Paulista (UNESP)instacron:UNESPengScientific Reportsinfo:eu-repo/semantics/openAccess2021-10-22T20:18:53Zoai:repositorio.unesp.br:11449/205977Repositório InstitucionalPUBhttp://repositorio.unesp.br/oai/requestopendoar:29462021-10-22T20:18:53Repositório Institucional da UNESP - Universidade Estadual Paulista (UNESP)false |
dc.title.none.fl_str_mv |
Selection and validation of reference genes by RT-qPCR under photoperiodic induction of flowering in sugarcane (Saccharum spp.) |
title |
Selection and validation of reference genes by RT-qPCR under photoperiodic induction of flowering in sugarcane (Saccharum spp.) |
spellingShingle |
Selection and validation of reference genes by RT-qPCR under photoperiodic induction of flowering in sugarcane (Saccharum spp.) da Silva Santos, Paulo H. [UNESP] |
title_short |
Selection and validation of reference genes by RT-qPCR under photoperiodic induction of flowering in sugarcane (Saccharum spp.) |
title_full |
Selection and validation of reference genes by RT-qPCR under photoperiodic induction of flowering in sugarcane (Saccharum spp.) |
title_fullStr |
Selection and validation of reference genes by RT-qPCR under photoperiodic induction of flowering in sugarcane (Saccharum spp.) |
title_full_unstemmed |
Selection and validation of reference genes by RT-qPCR under photoperiodic induction of flowering in sugarcane (Saccharum spp.) |
title_sort |
Selection and validation of reference genes by RT-qPCR under photoperiodic induction of flowering in sugarcane (Saccharum spp.) |
author |
da Silva Santos, Paulo H. [UNESP] |
author_facet |
da Silva Santos, Paulo H. [UNESP] Manechini, João R. Vieira Brito, Michael S. Romanel, Elisson Vicentini, Renato Scarpari, Maximiliano Jackson, Stephen Pinto, Luciana R. |
author_role |
author |
author2 |
Manechini, João R. Vieira Brito, Michael S. Romanel, Elisson Vicentini, Renato Scarpari, Maximiliano Jackson, Stephen Pinto, Luciana R. |
author2_role |
author author author author author author author |
dc.contributor.none.fl_str_mv |
Universidade Estadual Paulista (Unesp) Universidade Estadual de Campinas (UNICAMP) Universidade Federal de São Paulo (UNIFESP) Universidade de São Paulo (USP) Centro de Cana University of Warwick |
dc.contributor.author.fl_str_mv |
da Silva Santos, Paulo H. [UNESP] Manechini, João R. Vieira Brito, Michael S. Romanel, Elisson Vicentini, Renato Scarpari, Maximiliano Jackson, Stephen Pinto, Luciana R. |
description |
Although reference genes have previously been used in the expression analysis of genes involved in sugarcane flowering they had not been experimentally validated for stability and consistency of expression between different samples over a wide range of experimental conditions. Here we report the analysis of candidate reference genes in different tissue types, at different temporal time-points, in both short and long day photoperiodic treatments. The stability of the candidate reference genes in all conditions was evaluated with NormFinder, BestKeeper, and RefFinder algorithms that complement each other for a more robust analysis. As the Normfinder algorithm was more appropriate for our experimental conditions, greater emphasis was placed on Normfinder when choosing the most stable genes. UBQ1 and TUB were shown to be the most stable reference genes to use for normalizing RT-qPCR gene expression data during floral induction, whilst 25SrRNA1 and GAPDH were the least stable. Their use as a reference gene pair was validated by analyzing the expression of two differentially expressed target genes (PIL5 and LHP1). The UBQ1/TUB reference genes combination was able to reveal small significant differences in gene expression of the two target genes that were not detectable when using the least stable reference gene combination. These results can be used to inform the choice of reference genes to use in the study of the sugarcane floral induction pathway. Our work also demonstrates that both PIL5 and LHP1 are significantly up-regulated in the initial stages of photoperiodic induction of flowering in sugarcane. |
publishDate |
2021 |
dc.date.none.fl_str_mv |
2021-06-25T10:24:35Z 2021-06-25T10:24:35Z 2021-12-01 |
dc.type.status.fl_str_mv |
info:eu-repo/semantics/publishedVersion |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/article |
format |
article |
status_str |
publishedVersion |
dc.identifier.uri.fl_str_mv |
http://dx.doi.org/10.1038/s41598-021-83918-2 Scientific Reports, v. 11, n. 1, 2021. 2045-2322 http://hdl.handle.net/11449/205977 10.1038/s41598-021-83918-2 2-s2.0-85101813884 |
url |
http://dx.doi.org/10.1038/s41598-021-83918-2 http://hdl.handle.net/11449/205977 |
identifier_str_mv |
Scientific Reports, v. 11, n. 1, 2021. 2045-2322 10.1038/s41598-021-83918-2 2-s2.0-85101813884 |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.relation.none.fl_str_mv |
Scientific Reports |
dc.rights.driver.fl_str_mv |
info:eu-repo/semantics/openAccess |
eu_rights_str_mv |
openAccess |
dc.source.none.fl_str_mv |
Scopus reponame:Repositório Institucional da UNESP instname:Universidade Estadual Paulista (UNESP) instacron:UNESP |
instname_str |
Universidade Estadual Paulista (UNESP) |
instacron_str |
UNESP |
institution |
UNESP |
reponame_str |
Repositório Institucional da UNESP |
collection |
Repositório Institucional da UNESP |
repository.name.fl_str_mv |
Repositório Institucional da UNESP - Universidade Estadual Paulista (UNESP) |
repository.mail.fl_str_mv |
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1799965372347056128 |