Selection and validation of reliable reference genes for gene expression studies from Monilinia vaccinii-corymbosi infected wild blueberry phenotypes

Jose, Sherin; Abbey, Joel; Jaakola, Laura; Percival, David

doi:10.1038/s41598-020-68597-9

Cited by 14 publications

(11 citation statements)

References 45 publications

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“…The amplification program was as follows: an initial denaturation at 95 °C for 3 min, followed by 40 cycles at 95 °C for 10 s, 60 °C for 20 s. Each run was completed with a melting curve analysis (65–95 °C with at increments of 0.5 °C) to verify the specificity of the amplification. GAPDH was selected as the reference gene for V. angustifolium f. nigrum and V. angustifolium and UBC9 for V. myrtilloides [ 46 ]. A no-template control (NTC) was included with each run for each gene to confirm the absence of non-specific products.…”

Section: Discussionmentioning

confidence: 99%

Elucidation of the molecular responses during the primary infection of wild blueberry phenotypes with Monilinia vaccinii-corymbosi under field conditions

et al. 2021

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Background Monilinia blight caused by Monilinia vaccinii-corymbosi (Reade) Honey (M.vc) is a major disease of wild blueberry that can result in severe crop losses in the absence of an integrated disease management programme. The fungus causes blight in the emerging floral and vegetative buds, but the degree of susceptibility varies among the different wild blueberry phenotypes, ranging from the highly susceptible V. a. f. nigrum to the moderately susceptible V. angustifolium and the least susceptible V. myrtilloides. Results The present study evaluated the defense responses of these major phenotypes during their primary infection (floral buds) with M.vc. The temporal expression profiles of PR genes (PR3 and PR4) and the flavonoid pathway structural genes (CHS, ANS, ANR, DFR and FLS) were analysed. The PR3 and PR4 gene expression profiles revealed that V. myrtilloides responded to M.vc infection by activating the expression of both PR genes. V. a. f. nigrum, on the other hand, failed to activate these genes, while V. angustifolium, exhibited an intermediate response. Our study with the flavonoid pathway genes indicated variability in activation of the genes during post-infection time points with ANS and ANR in V. myrtilloides, FLS in V. angustifolium and no response observed in V. a. f. nigrum. Conclusions Altogether, this study highlights that the degree of phenotype susceptibility is associated with the timely activation of host defense responsive genes. Data obtained in this study provided a starting point for a better understanding of the wild blueberry- M. vaccinii-corymbosi pathosystem.

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Section: Discussionmentioning

confidence: 99%

Elucidation of the molecular responses during the primary infection of wild blueberry phenotypes with Monilinia vaccinii-corymbosi under field conditions

et al. 2021

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“…The relative quantification of gene products was based on the ∆Ct method ( Schmittgen and Livak, 2008 ). Transcript levels were normalized to the reference genes HISTONE H4 ( H4 ) and UBIQUITIN-PROTEIN LIGASE ( UBC9 ; Zhang et al, 2018 ; Jose et al, 2020 ). All primers can be found in Supplementary Table 1 .…”

Section: Methodsmentioning

confidence: 99%

Constitutive Defense Mechanisms Have a Major Role in the Resistance of Woodland Strawberry Leaves Against Botrytis cinerea

et al. 2022

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The necrotrophic fungus Botrytis cinerea is a major threat to strawberry cultivation worldwide. By screening different Fragaria vesca genotypes for susceptibility to B. cinerea, we identified two genotypes with different resistance levels, a susceptible genotype F. vesca ssp. vesca Tenno 3 (T3) and a moderately resistant genotype F. vesca ssp. vesca Kreuzkogel 1 (K1). These two genotypes were used to identify the molecular basis for the increased resistance of K1 compared to T3. Fungal DNA quantification and microscopic observation of fungal growth in woodland strawberry leaves confirmed that the growth of B. cinerea was restricted during early stages of infection in K1 compared to T3. Gene expression analysis in both genotypes upon B. cinerea inoculation suggested that the restricted growth of B. cinerea was rather due to the constitutive resistance mechanisms of K1 instead of the induction of defense responses. Furthermore, we observed that the amount of total phenolics, total flavonoids, glucose, galactose, citric acid and ascorbic acid correlated positively with higher resistance, while H2O2 and sucrose correlated negatively. Therefore, we propose that K1 leaves are more resistant against B. cinerea compared to T3 leaves, prior to B. cinerea inoculation, due to a lower amount of innate H2O2, which is attributed to a higher level of antioxidants and antioxidant enzymes in K1. To conclude, this study provides important insights into the resistance mechanisms against B. cinerea, which highly depend on the innate antioxidative profile and specialized metabolites of woodland strawberry leaves.

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“…Both GAPDH and CYP maintained good stability in pomegranate when subjected to biotic and abiotic stresses [26]. The most consistently expressed reference genes in blueberry were UBC9 and GAPDH when attacked by Monilinia vaccinii-corymbosi [27]. In summary, no completely universal reference gene existed, and the expression stability of genes varied considerably under different experimental conditions.…”

Section: Discussionmentioning

confidence: 93%

Identification of Suitable Reference Genes for qRT-PCR Normalization in Kiwifruit

et al. 2022

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Reference genes are used for the correction of qRT-PCR data, and it is necessary to investigate the optimum reference gene under certain conditions. The expression levels of seven traditional reference genes ACT1, ACT2, GAPDH, 18S rRNA, UBQ, TUB and CYP were analyzed using qRT-PCR in different varieties, tissues, developmental stages and hormone (or pollen polysaccharide) treatments in kiwifruit. Gene expression stability was assessed with the help of three common software (geNorm, NormFinder, BestKeeper), and the minimum number of reference genes necessary for normalization was also determined. GAPDH, ACT1 and ACT2 were selected as reference genes for different genotypes of kiwifruit. GAPDH and UBQ were the best combinations of reference genes for root, stem, leaf, flower and fruit. GAPDH and ACT1 could be the preferred reference genes for normalization of qRT-PCR data during fruit development. The pairing of ACT1 and UBQ constituted the optimal combination of reference genes in kiwifruit treated with different hormones (or pollen polysaccharide). This study provides a new and reliable option for the use of reference genes in the analysis of gene expression patterns of interest in kiwifruit.

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Selection and validation of reliable reference genes for gene expression studies from Monilinia vaccinii-corymbosi infected wild blueberry phenotypes

Cited by 14 publications

References 45 publications

Elucidation of the molecular responses during the primary infection of wild blueberry phenotypes with Monilinia vaccinii-corymbosi under field conditions

Elucidation of the molecular responses during the primary infection of wild blueberry phenotypes with Monilinia vaccinii-corymbosi under field conditions

Constitutive Defense Mechanisms Have a Major Role in the Resistance of Woodland Strawberry Leaves Against Botrytis cinerea

Identification of Suitable Reference Genes for qRT-PCR Normalization in Kiwifruit

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