Abstract
Leaf pathogens are limiting factors in banana (Musa spp.) production, with Pseudocercospora spp. responsible for the important Sigatoka disease complex. In order to investigate cellular processes and genes involved in host defence responses, quantitative real-time PCR (RT-qPCR) is an analytical technique for gene expression quantification. Reliable RT-qPCR data, however, requires that reference genes for normalization of mRNA levels in samples are validated under the conditions employed for expression analysis of target genes. We evaluated the stability of potential reference genes ACT1, α-TUB, UBQ1, UBQ2, GAPDH, EF1α, APT and RAN. Total RNA was extracted from leaf tissues of Musa acuminata genotypes Calcutta 4 (resistant) and Cavendish Grande Naine (susceptible), both subjected to P. musae infection. Expression stability was determined with NormFinder, BestKeeper, geNorm and RefFinder algorithms. UBQ2 and RAN were the most stable across all M. acuminata samples, whereas when considering inoculated and non-inoculated leaf samples, APT and UBQ2 were appropriate for normalization in Calcutta 4, with RAN and α-TUB most stable in Cavendish Grande Naine. This first study of reference genes for relative quantification of target gene expression in the M. acuminata-P. musae interaction will enable reliable analysis of gene expression in this pathosystem, benefiting elucidation of disease resistance mechanisms.
Highlights
Leaf pathogens are important limiting factors in global banana production, with reduced photosynthetic capacity causing negative impact on crop yield, especially in tropical growing regions[1]
Specificity of each of the primers to a single gene locus was confirmed by dissociation curve analysis, with a single peak observed for each primer pair at the expected primer annealing temperature (Fig. 1), together with an absence of any signal in negative controls lacking template cDNA
Genes encoding proteins involved in basal cell activities in plants were evaluated as potential reference genes for normalization of gene expression data in M. acuminata leaf material through a multi-algorithm based analysis of expression stability
Summary
Leaf pathogens are important limiting factors in global banana production, with reduced photosynthetic capacity causing negative impact on crop yield, especially in tropical growing regions[1]. In accordance with the Minimum Information for Publication of Real-Time Quantitative PCR Experiments (MIQE) guidelines[14], normalization of expression of each target gene to that of a reference gene is required to correct data for variations in samples that occur during cDNA preparation, with reference genes subject to the same effect of cDNA quality[15,16] For such normalization, the employment of appropriate reference genes for each organism, tissue type and time point is essential. Employed genes have included actin and tubulin, given their role in the cell cytoskeleton, genes involved in protein synthesis (elongation factor) and protein degradation (ubiquitin), and genes involved in glucose metabolism (glyceraldeide-3-phosphate dehydrogenase) With such housekeeping genes, a stable transcript response is not necessarily guaranteed across different experimental conditions, as observed in numerous plant species[18,19,20] and can lead to inaccuracies in results, masking true interpretation of gene expression[16,21]
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