Screening of Reference Genes for Quantitative Real-time PCR in Globba spp.

Objective Screening for stable reference genes that are applicable to the study of different varieties of Globba spp. bracts and different tissue parts of G. sherwoodiana 'White Dragon' provides a theoretical foundation for subsequent expression analysis of target genes in Globba spp..

Method Based on the transcriptome sequences of different varieties of Globba spp. bracts obtained in the preliminary stage, seven genes were selected as candidate reference genes according to their stability of relative expression levels: malate dehydrogenase (MDH), actin (Actin12), transcription elongation factor (EF1α2), transcriptional coactivator (TAP46), tubulin β (TUB7), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) encoding genes, and 18S ribosomal RNA gene (18S rRNA). qRT-PCR assays were carried out to assess the expression levels of seven candidate internal reference genes, utilizing bract tissues from nine color-varied varieties of Globba spp. and samples from five distinct plant parts (roots, leaves, lateral buds, bracts, and small flowers) of the G. sherwoodiana 'White Dragon'. The expression stability of these genes as internal references in bract tissues across different varieties of Globba spp. and in various plant parts of G. sherwoodiana 'White Dragon' was evaluated using BestKeeper, NormFinder, and geNorm software.

Result The PCR products of the seven candidate reference genes exhibited single and distinct bands following gel electrophoresis, with sizes consistent with the theoretically predicted fragment lengths. The melting curves displayed single peaks, and the amplification efficiencies ranged from 92.91% to 102.33%. The linear correlation coefficients (r) were all greater than 0.990, confirming the high specificity of the primers and their suitability for qRT-PCR experiments. In the reference gene screening conducted in the bracts of different Globba spp. varieties, Actin12 was ranked first according to evaluations by BestKeeper, NormFinder, and geNorm software. In the screening of reference genes across different tissue parts of G. sherwoodiana 'White Dragon', TAP46 was ranked first in stability assessments by BestKeeper and NormFinder software but placed fourth according to geNorm software.

Conclusion The comprehensive analysis results from the three software suggest that Actin12 is the most stable reference gene for qRT-PCR experiments involving bracts across different varieties of Globba spp., while TAP46 is the most stable reference gene for qRT-PCR experiments targeting different parts of G. sherwoodiana 'White Dragon'.