Validation of Reference Genes for RT-qPCR Normalization in Saccharum Genotypes During Smut Infection

Abstract

Sugarcane and energy cane (Saccharum spp. hybrids) yields are significantly threatened by smut disease, caused by the fungus Sporisorium scitamineum. Gene expression studies are essential for understanding molecular responses in this pathosystem; however, reference gene stability may vary among genotypes, plant organs, and across infection stages, highlighting the need for systematic validation under the specific conditions of each experiment. In this study, eight candidate genes were assessed in the buds of sugarcane IACSP02-1064 (intermediate resistance) and energy cane Vertix 10 (susceptible) during the course of the infection (12, 24, 48, and 120 hpi). Expression stability was assessed using geNorm, NormFinder, and BestKeeper algorithms. For IACSP02-1064, ACAD (Acyl-CoA dehydrogenase family member 10) and GAPDH (Glyceraldehyde-3-phosphate dehydrogenase) were the most stable genes, whereas GAPDH/ACAD and ACAD/UBQ2 (Ubiquitin 2) were identified as optimal pairs. For Vertix 10, GAPDH, UBQ2, and ACAD showed the highest stability, while GAPDH/UBQ2, ACAD/GAPDH, and ACAD/SARMp1 were recommended for normalization. The selected reference genes were further evaluated by analyzing ScSPL19 (SQUAMOSA promoter-binding protein-like 19 from Saccharum spp.) expression, a member of the SQUAMOSA PROMOTER-BINDING PROTEIN-LIKE (SPL) transcription factor family, whose members have previously been reported to respond to smut infection, using the most and least stable reference gene pairs. This analysis revealed differential temporal regulation of ScSPL19 when normalized with the most stable reference gene pair (GAPDH/ACAD). These results support the suitability of this reference gene combination for accurate relative gene expression analysis under the evaluated conditions.