Transcriptome-Based Identification of Reference Genes for Expression Analysis in Cassava Under Xanthomonas phaseoli...

Reverse transcription quantitative PCR (RT-qPCR) is a powerful and widely used technique for quantifying alterations in gene expression. Cassava bacterial blight caused by Xanthomonas phaseoli pv. manihotis severely constraints cassava growth and yield. Accurate evaluation of the expression levels of genes following infection by X. phaseoli pv. manihotis is crucial for the identification of potential cassava resistance genes. In this study, thirty-two novel potential reference genes were screened from the cassava-X. phaseoli pv. manihotis transcriptome. Their expression, along with that of seven literature-reported cassava reference genes, was evaluated in two susceptible and two resistant cassava varieties at six time points post-inoculation by X. phaseoli pv. manihotis through RT-qPCR analysis. The stability of thirty-nine candidate reference genes was assessed by four algorithms: geNorm, NormFinder, Delta Ct, and RefFinder. The results demonstrated that serving as new reference genes, MehnRNPR and MePRPF38B consistently exhibited superior expression stability over seven established reference genes under X. phaseoli pv. manihotis infection, regardless of the susceptible or resistant cassava varieties. The reliability of the reference genes was validated by assessing the expression pattern of MeNAC35 and MeSWEET10a under X. phaseoli pv. manihotis infection. The findings of this study provide valuable insights for advancing the precision of the quantification of cassava candidate genes associated with disease resistance.