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Selection and validation of reference genes by RT-qPCR under photoperiodic induction of flowering in sugarcane (Saccharum spp.).

ABSTRACT: 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.

SUBMITTER: da Silva Santos PH

PROVIDER: S-EPMC7907395 | biostudies-literature | 2021 Feb

REPOSITORIES: biostudies-literature

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Selection and validation of reference genes by RT-qPCR under photoperiodic induction of flowering in sugarcane (Saccharum spp.).

da Silva Santos Paulo H PH Manechini João R Vieira JRV Brito Michael S MS Romanel Elisson E Vicentini Renato R Scarpari Maximiliano M Jackson Stephen S Pinto Luciana R LR

Scientific reports 20210225 1

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 ...[more]

PMID: 33633157

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Project description:Flowering is of utmost relevance for the agricultural productivity of the sugarcane bioeconomy, but data and knowledge of the genetic mechanisms underlying its photoperiodic induction are still scarce. An understanding of the molecular mechanisms that regulate the transition from vegetative to reproductive growth in sugarcane could provide better control of flowering for breeding. This study aimed to investigate the transcriptome of +1 mature leaves of a sugarcane cultivar subjected to florally inductive and non-inductive photoperiodic treatments to identify gene expression patterns and molecular regulatory modules. We identified 7,083 differentially expressed (DE) genes, of which 5,623 showed significant identity to other plant genes. Functional group analysis showed differential regulation of important metabolic pathways involved in plant development, such as plant hormones (i.e., cytokinin, gibberellin, and abscisic acid), light reactions, and photorespiration. Gene ontology enrichment analysis revealed evidence of upregulated processes and functions related to the response to abiotic stress, photoprotection, photosynthesis, light harvesting, and pigment biosynthesis, whereas important categories related to growth and vegetative development of plants, such as plant organ morphogenesis, shoot system development, macromolecule metabolic process, and lignin biosynthesis, were downregulated. Also, out of 76 sugarcane transcripts considered putative orthologs to flowering genes from other plants (such as Arabidopsis thaliana, Oryza sativa, and Sorghum bicolor), 21 transcripts were DE. Nine DE genes related to flowering and response to photoperiod were analyzed either at mature or spindle leaves at two development stages corresponding to the early stage of induction and inflorescence primordia formation. Finally, we report a set of flowering-induced long non-coding RNAs and describe their level of conservation to other crops, many of which showed expression patterns correlated against those in the functionally grouped gene network.

Dataset Information

Selection and validation of reference genes by RT-qPCR under photoperiodic induction of flowering in sugarcane (Saccharum spp.).

Publications

Selection and validation of reference genes by RT-qPCR under photoperiodic induction of flowering in sugarcane (Saccharum spp.).

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