Abstract
Monilinia blight disease caused by Monilinia vaccinii-corymbosi (Reade) Honey (M.vc) causes severe damage and economic losses in wild blueberry growing regions. Molecular mechanisms regulating defence responses of wild blueberry phenotypes towards this causal fungus are not yet fully known. A reliable quantification of gene expression using quantitative real time PCR (qPCR) is fundamental for measuring changes in target gene expression. A crucial aspect of accurate normalisation is the choice of appropriate reference genes. This study evaluated the expression stability of seven candidate reference genes (GAPDH, UBC9, UBC28, TIP41, CaCSa, PPR and RH8) in floral tissues of diploid and tetraploid wild blueberry phenotypes challenged with M.vc. The expression stability was calculated using five algorithms: geNorm, NormFinder, BestKeeper, deltaCt and RefFinder. The results indicated that UBC9 and GAPDH were the most stable reference genes, while RH8 and PPR were the least stable ones. To further validate the suitability of the analyzed reference genes, the expression level of a pathogenesis related protein gene (i.e., PR3) was analysed for both phenotypes at four time points of infection. Our results may be beneficial for future studies involving the quantification of relative gene expression levels in wild blueberry species.
Highlights
Monilinia blight disease caused by Monilinia vaccinii-corymbosi (Reade) Honey (M.vc) causes severe damage and economic losses in wild blueberry growing regions
PR3 gene was used to validate the accuracy of identified RGs in wild blueberry phenotypes infected with M. vaccinii-corymbosi
The results showed that all the primer pairs were suitable for RT-quantitative real time PCR (qPCR) analysis
Summary
Monilinia blight disease caused by Monilinia vaccinii-corymbosi (Reade) Honey (M.vc) causes severe damage and economic losses in wild blueberry growing regions. This study evaluated the expression stability of seven candidate reference genes (GAPDH, UBC9, UBC28, TIP41, CaCSa, PPR and RH8) in floral tissues of diploid and tetraploid wild blueberry phenotypes challenged with M.vc. To further validate the suitability of the analyzed reference genes, the expression level of a pathogenesis related protein gene (i.e., PR3) was analysed for both phenotypes at four time points of infection. Commercial wild blueberry fields mostly consist of Vaccinium angustifolium (tetraploid) (~ 70–80% on a surface area basis), V. myrtilloides (diploid) (~ 10–20%) and a few h ybrids[3]. The Monilinia–Vaccinium pathosystem is an important phenomenon, a detailed molecular understanding of gene expression profile during the infection process is lacking within and between the phenotypes in wild blueberries. The selection and validation of stable reference genes for each experimental condition is a prerequisite for performing qRT-PCR studies[13,14,15]
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