Reference genes for normalization of qPCR assays in sugarcane plants under water deficit

Abstract

BackgroundSugarcane (Saccharum spp.) is the main raw material for sugar and ethanol production. Among the abiotic stress, drought is the main one that negatively impact sugarcane yield. Although gene expression analysis through quantitative PCR (qPCR) has increased our knowledge about biological processes related to drought, gene network that mediates sugarcane responses to water deficit remains elusive. In such scenario, validation of reference gene is a major requirement for successful analyzes involving qPCR. ResultsIn this study, candidate genes were tested for their suitable as reference genes for qPCR analyses in two sugarcane cultivars with varying drought tolerance. Eight candidate reference genes were evaluated in leaves sampled in plants subjected to water deficit in both field and greenhouse conditions. In addition, five genes were evaluated in shoot roots of plants subjected to water deficit by adding PEG8000 to the nutrient solution. NormFinder and RefFinder algorithms were used to identify the most stable gene(s) among genotypes and under different experimental conditions. Both algorithms revealed that in leaf samples, UBQ1 and GAPDH genes were more suitable as reference genes, whereas GAPDH was the best reference one in shoot roots.ConclusionReference genes suitable for sugarcane under water deficit were identified, which would lead to a more accurate and reliable analysis of qPCR. Thus, results obtained in this study may guide future research on gene expression in sugarcane under varying water conditions.Electronic supplementary materialThe online version of this article (doi:10.1186/s13007-017-0178-2) contains supplementary material, which is available to authorized users.

Selection of the Reference Gene for Expression Normalization in Papaver somniferum L. under Abiotic Stress and Hormone Treatment

“…Therefore, we recommend the top three candidate genes-NCBP2, GAPC2, and TIP41 as reference genes. Interestingly, although a commonly used reference gene, expression of GAPDH in P. somniferum was not stable enough for accurate normalization, but was recommended as one of the best reference genes in Barber [41], E. konishii Hayata [42], and sugarcane under a water deficit environment [27]. This difference demonstrates that no reference gene is universally available, so the selection and validation of suitable reference genes remain indispensable.…”

“…For example, GAPDH, β-TUB, CYP2, and 18s rRNA are often selected as reference genes. However, the expression levels of HKGs may vary between different species, and experimental conditions [21][22][23][24][25][26][27]. Thus, the selection of a stable reference gene has become increasingly important.…”

Identification of candidate reference genes in tropical bamboos stable across species, tissues, and developmental stages

“…However, the accuracy of this method is heavily dependent on data normalization done by stably expressed reference genes. It is now well accepted that a single or a pair of reference genes that are useful for one species or experimental settings may not be equally useful for another species or even for the same species but under different experimental conditions (Morgante et al 2011, De Andrade et al 2017, Niu et al 2017.…”

“…However, in order to avoid obtaining differential expression due to technical discrepancies, proper data normalization using a set of stable reference genes is mandatory. Therefore, extensive studies have been performed over the years to identify suitable reference genes that are stable across species (Iskandar et al 2004, Morgante et al 2011, developmental stages (Jain et al 2006, Li et al 2016, tissues (Jian et al 2008, Reddy et al 2016, different seasons (Kim et al 2015), and even diverse environmental conditions (Janská et al 2013, Silva et al 2014, De Andrade et al 2017.…”

Expression Analysis of XTH in Stem Swelling of Stem Mustard and Selection of Reference Genes

“…In garlic, UBQ and ACTIN are the most reliable reference genes and therefore recommended for analysis of different developmental stages and abiotic stress management, respectively [7]. UBQ gene was the most stable reference gene in sugarcane leaves under drought stress, while PP2A was the best reference gene under sorghum Mosaic virus [8]. Moreover, in Sorghum, PP2A was found to be the most stable gene in analysis of abiotic stress, while UBC showed the least stability [9].…”