Abstract
Although reference genes have previously been used in the expression analysis of genes involved in sugarcane flowering they had not been experimentally validated for stability and consistency of expression between different samples over a wide range of experimental conditions. Here we report the analysis of candidate reference genes in different tissue types, at different temporal time-points, in both short and long day photoperiodic treatments. The stability of the candidate reference genes in all conditions was evaluated with NormFinder, BestKeeper, and RefFinder algorithms that complement each other for a more robust analysis. As the Normfinder algorithm was more appropriate for our experimental conditions, greater emphasis was placed on Normfinder when choosing the most stable genes. UBQ1 and TUB were shown to be the most stable reference genes to use for normalizing RT-qPCR gene expression data during floral induction, whilst 25SrRNA1 and GAPDH were the least stable. Their use as a reference gene pair was validated by analyzing the expression of two differentially expressed target genes (PIL5 and LHP1). The UBQ1/TUB reference genes combination was able to reveal small significant differences in gene expression of the two target genes that were not detectable when using the least stable reference gene combination. These results can be used to inform the choice of reference genes to use in the study of the sugarcane floral induction pathway. Our work also demonstrates that both PIL5 and LHP1 are significantly up-regulated in the initial stages of photoperiodic induction of flowering in sugarcane.
Highlights
Sugarcane (Saccharum spp. hybrids) is an important economic crop worldwide, and is a predominant crop in Brazil’s agribusiness mainly for sugar, ethanol, and bioelectricity production
After the assessment of the Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) data obtained from separate cDNA pools of samples from mature leaves and spindle leaves, primer specificity was confirmed by obtaining single melting curve peaks for all the candidate reference genes primer pairs together with the absence of any amplification in the negative controls (Supplementary Fig. S1 online)
The best melting curve peaks were obtained at a primer concentration of 0.25 μM for the reference genes UBIQUITIN 1(UBQ1) and TUBULIN (TUB), whilst for UBIQUITIN 2(UBQ2), GLYCERALDEHYDE-3 PHOSPHATE DEHYDROGENASE (GAPDH) and TONOPLAST INTRINSIC PROTEIN (TIPS-41) it was at 0.4 μM, and for 60S RIBOSOMAL PROTEIN L35-4 (RPL), 25S RIBOSOMAL RNA (25SrRNA1) and ELONGATION FACTOR-1Α (EF1) it was at 0.8 μM (Table 1)
Summary
Sugarcane (Saccharum spp. hybrids) is an important economic crop worldwide, and is a predominant crop in Brazil’s agribusiness mainly for sugar, ethanol, and bioelectricity production. The flowering induction process, marked by the apical meristem transition from vegetative to reproductive stage, negatively impacts sugarcane production and quality traits. Knowledge of the genes involved in the flowering induction pathway, as well as their expression patterns during the induction process, is still scarce in this high complex polyploid crop. The flowering process involves several steps governed by a complex molecular genetic network which cause changes in the apical meristem, initiating with induction up to inflorescence formation and emission. Accurate RT-qPCR analysis requires proper normalization of gene expression data through the use of reference genes whose expression should be constant in different tissues, organs and developmental stages, regardless of the experimental conditions. There are several algorithms with mutually complementary approaches that are available for the analysis of candidate reference genes that can be used to obtain information on the best reference genes to use
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