Stable reference genes for RT-qPCR analysis of gene expression in the Musa acuminata-Pseudocercospora musae interaction

Rego, Erica C.S.; Pinheiro, Tatiana David Miranda; Antonino, José Dijair; Alves, Gabriel Sergio Costa; Cotta, Michelle G.; Fonseca, Fernando Campos de Assis; Miller, Robert G.

doi:10.1038/s41598-019-51040-z

Cited by 18 publications

(11 citation statements)

References 62 publications

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“…Reference genes for this pathosystem, namely Ubiquitin 2 and GTP–binding nuclear protein, were employed according to Ref. [ 144 ]. The qPCR reaction mixture contained 2 μL of template cDNA diluted 1:20, 0.2 μM of each primer and Platinum™ SYBR™ Green qPCR SuperMix-UDG w/ROX kit (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions.…”

Section: Methodsmentioning

confidence: 99%

Transcriptome Profiling of the Resistance Response of Musa acuminata subsp. burmannicoides, var. Calcutta 4 to Pseudocercospora musae

Pinheiro

Rego

Alves

et al. 2022

IJMS

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Banana (Musa spp.), which is one of the world’s most popular and most traded fruits, is highly susceptible to pests and diseases. Pseudocercospora musae, responsible for Sigatoka leaf spot disease, is a principal fungal pathogen of Musa spp., resulting in serious economic damage to cultivars in the Cavendish subgroup. The aim of this study was to characterize genetic components of the early immune response to P. musae in Musa acuminata subsp. burmannicoides, var. Calcutta 4, a resistant wild diploid. Leaf RNA samples were extracted from Calcutta 4 three days after inoculation with fungal conidiospores, with paired-end sequencing conducted in inoculated and non-inoculated controls using lllumina HiSeq 4000 technology. Following mapping to the reference M. acuminata ssp. malaccensis var. Pahang genome, differentially expressed genes (DEGs) were identified and expression representation analyzed on the basis of gene ontology enrichment, Kyoto Encyclopedia of Genes and Genomes orthology and MapMan pathway analysis. Sequence data mapped to 29,757 gene transcript models in the reference Musa genome. A total of 1073 DEGs were identified in pathogen-inoculated cDNA libraries, in comparison to non-inoculated controls, with 32% overexpressed. GO enrichment analysis revealed common assignment to terms that included chitin binding, chitinase activity, pattern binding, oxidoreductase activity and transcription factor (TF) activity. Allocation to KEGG pathways revealed DEGs associated with environmental information processing, signaling, biosynthesis of secondary metabolites, and metabolism of terpenoids and polyketides. With 144 up-regulated DEGs potentially involved in biotic stress response pathways, including genes involved in cell wall reinforcement, PTI responses, TF regulation, phytohormone signaling and secondary metabolism, data demonstrated diverse early-stage defense responses to P. musae. With increased understanding of the defense responses occurring during the incompatible interaction in resistant Calcutta 4, these data are appropriate for the development of effective disease management approaches based on genetic improvement through introgression of candidate genes in superior cultivars.

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Section: Methodsmentioning

confidence: 99%

Transcriptome Profiling of the Resistance Response of Musa acuminata subsp. burmannicoides, var. Calcutta 4 to Pseudocercospora musae

Pinheiro

Rego

Alves

et al. 2022

IJMS

Self Cite

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“…However, in order to use ANOVA model, assumptions concerning homogeneity of variance and normality of data must be made 51 . In this analysis at the subgroup level, SV of all tested RGs was below the default limit of 0.5 indicating relatively high expression stability 52 . In the total dataset, the highest variation in expression was reported for GAPDH , CYP and UBC , however the value of SV above 0.5 was reported only for GAPDH .…”

Section: Resultsmentioning

confidence: 78%

Validation of reference genes as an internal control for studying Avena sativa–Puccinia coronata interaction by RT-qPCR

Sowa

Sozoniuk

Toporowska

et al. 2022

Sci Rep

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In this study we evaluated eleven candidate reference genes in Avena sativa during compatible and incompatible interactions with two different pathotypes of Puccinia coronata f. sp. avenae in six time points post-inoculation. The identification of genes with high expression stability was performed by four algorithms (geNorm, NormFinder, BestKeeper and ΔCt method). The results obtained confirmed that the combination of two genes would be sufficient for reliable normalization of the expression data. In general, the most stable in the tested plant-pathogen system were HNR (heterogeneous nuclear ribonucleoprotein 27C) and EF1A (elongation factor 1-alpha). ARF (ADP-ribosylation factor) and EIF4A (eukaryotic initiation factor 4A-3) could also be considered as exhibiting high expression stability. CYP (cyclophilin) was shown by all assessment methods to be the worst candidate for normalization in this dataset. To date, this is the first report of reference genes selection in A. sativa–P. coronata interaction system. Identified reference genes enable reliable and comprehensive RT-qPCR analysis of oat gene expression in response to crown rust infection. Understanding the molecular mechanisms involved in the host–pathogen interactions may expand knowledge of durable resistance strategies beneficial to modern oat breeding.

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“…The optimal candidate reference genes were determined based on the average expression stability between samples and the results of variance analysis. The BestKeeper program was used to identify stable reference genes, which calculates pairwise correlations based on values of standard deviation (SD) and percentage covariance (Cov) 30 .…”

Section: Methodsmentioning

confidence: 99%

Identification of RT-qPCR reference genes suitable for gene function studies in the pitaya canker disease pathogen Neoscytalidium dimidiatum

Wang

Sang

et al. 2022

Sci Rep

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Neoscytalidium dimidiatum is the main causal agent of pitaya canker. Most studies of virulence and pathogenicity genes have measured expression levels using real-time quantitative polymerase chain reaction (RT-qPCR). Suitable reference genes are essential for ensuring that estimates of gene expression levels by RT-qPCR are accurate. However, no reference genes can be robustly applied across all contexts and species. No studies to date have evaluated the most effective reference genes for normalizing gene expression levels estimated by RT-qPCR in N. dimidiatum. In this study, RT-qPCR data for individual candidate reference genes were analyzed using four different methods: the delta Ct method and the geNorm, NormFinder, and BestKeeper algorithms. We evaluated the utility of eight candidate reference genes (18S rRNA, Actin (1), Actin (2), Actin, GAPDH (1), GAPDH (2), UBQ, and Tubulin) for normalizing expression levels estimated by RT-qPCR in N. dimidiatum at different developmental stages, at different temperatures, and during interaction with pitaya. All candidate reference genes were suitable for gene expression analysis except for Actin (2). Tubulin and Actin (1) were the most stably expressed reference genes under different temperatures. Actin (1) and Actin were the most stably expressed reference genes in N. dimidiatum at different developmental stages. Tubulin and UBQ were the most stably expressed reference genes during interaction with pitaya. Actin and 18s rRNA were the most stably expressed across all experimental conditions. Subsequently, Tubulin and UBQ were further investigated in analyses of pectinase expression during the pitaya–N. dimidiatum interaction. Our results provide insights that will aid future RT-qPCR studies of gene expression in N. dimidiatum.

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Stable reference genes for RT-qPCR analysis of gene expression in the Musa acuminata-Pseudocercospora musae interaction

Cited by 18 publications

References 62 publications

Transcriptome Profiling of the Resistance Response of Musa acuminata subsp. burmannicoides, var. Calcutta 4 to Pseudocercospora musae

Transcriptome Profiling of the Resistance Response of Musa acuminata subsp. burmannicoides, var. Calcutta 4 to Pseudocercospora musae

Validation of reference genes as an internal control for studying Avena sativa–Puccinia coronata interaction by RT-qPCR

Identification of RT-qPCR reference genes suitable for gene function studies in the pitaya canker disease pathogen Neoscytalidium dimidiatum

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