Abstract
BackgroundSugarcane (Saccharum L. plant) is an important crop for sugar and bio-energy production around the world. Among sugarcane diseases, smut caused by Sporisorium scitamineum is one of the major fungal diseases causing severe losses to the sugarcane industry. The use of PCR reference genes is essential to the normalization of data on gene expression involving the sugarcane-S. scitamineum interaction system; however, no report that addresses criteria in selecting these reference genes has been published to date.ResultsIn this study, 10 sugarcane genes and eight S. scitamineum genes were selected as candidate PCR reference genes in the sugarcane-S. scitamineum interaction system. The stability and reliability of these 18 candidate genes were analyzed in smut-resistant (NCo376) and -susceptible (YC71–374) genotypes using the statistical algorithms geNorm, NormFinder, BestKeeper, and deltaCt method. Subsequently, the relative expression levels of the sugarcane chitinase I-3 gene and S. scitamineum chorismate mutase gene were determined to validate the applicability of these sugarcane and S. scitamineum PCR reference genes, respectively. We finally found that the acyl-CoA dehydrogenase gene (ACAD), serine/arginine repetitive matrix protein 1 gene (SARMp1), or their combination (ACAD + SARMp1) could be utilized as the most suitable reference genes for normalization of sugarcane gene expression in sugarcane bud tissues after S. scitamineum infection. Similarly, the inosine 5′-monophosphate dehydrogenase gene (S10), the SEC65-signal recognition particle subunit gene (S11), or their combination (S10 + S11) were suitable for normalization of S. scitamineum gene expression in sugarcane bud tissues.ConclusionsThe PCR reference genes ACAD, SARMp1, S10, and S11 may be employed in gene transcriptional studies involving the sugarcane-S. scitamineum interaction system.
Highlights
Sugarcane (Saccharum L. plant) is an important crop for sugar and bio-energy production around the world
The CV values of acyl-CoA dehydrogenase gene (ACAD), Casein kinase I isoform delta-like (CK1δ), OTU domain-containing protein 5 (OTU5), 12Oxophytodienoate reductase 7 (OPR7), Polyadenylate-binding protein 8 (PABP8), and serine/arginine repetitive matrix protein 1 gene (SARMp1) were between 2.65% and 3.88%, and their expression was higher than that of Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Table 2)
The above 10 sugarcane genes and eight S. scitamineum genes were selected as candidate PCR reference genes (Table 1) and further evaluated
Summary
Sugarcane (Saccharum L. plant) is an important crop for sugar and bio-energy production around the world. The use of PCR reference genes is essential to the normalization of data on gene expression involving the sugarcane-S. scitamineum interaction system; no report that addresses criteria in selecting these reference genes has been published to date. Gene expression analysis is essential in understanding the signaling and metabolic pathways that underly cellular and developmental processes [2]. Housekeeping genes, which were indispensable in maintaining basic metabolic activities and basic structural components, were widely used as PCR reference genes for gene expression analysis in humans [8], animals [9] and plants [10]. To reduce errors in expression quantification of pathogen genes caused by the adaptability of the pathogen, as well as nutrition and stress in host plants [11, 12], pathogen PCR reference genes have been included in investigations. There is growing evidence showing there is no single, universal gene that could be utilized in various experimental conditions [13], and the stability of reference genes should be validated before these are used for normalization of gene expression [14, 15]
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