Suitable Reference Genes/miRNAs for qRT-PCR Normalization of Expression Analysis in Sugarcane Under Sorghum mosaic virus Infection

Abstract

Sugarcane mosaic disease poses a serious threat to the sugarcane industry. Many studies have aimed to unravel the molecular mechanism related to sugarcane and mosaic virus interaction. Quantitative reverse transcription (qRT)-PCR in combination with suitable internal reference genes has been widely used for gene expression analysis. In this study, the expression of 33 candidate reference genes and 12 candidate reference miRNAs was analyzed for first time in the leaf samples of three sugarcane genotype infected with Sorghum mosaic virus using the geNorm, NormFinder and deltaCt (deltaCq) algorithms. A comparison of the expression of eIF-4E and three virus-derived siRNA (vsiR9230S, vsiR9058A and miR16) with the normalized unstable and stable reference genes or miRNA indicated that PP2A and miR159 constituted the best single reference gene/miRNA under SrMV infection. We also suggested that both CUL\u2009+\u2009CAC and miR171+\u2009miR1520 could serve as the most suitable reference gene/miRNA combination. The use of reliable reference genes and miRNAs should improve the accuracy of gene expression analysis in sugarcane leaves under SrMV stress.