Abstract
Reverse transcription quantitative real-time polymerase chain reaction (real-time PCR, also referred to as quantitative RT-PCR or RT-qPCR) is a highly sensitive and high-throughput method used to study gene expression. Despite the numerous advantages of RT-qPCR, its accuracy is strongly influenced by the stability of internal reference genes used for normalizations. To date, few studies on the identification of reference genes have been performed on cassava (Manihot esculenta Crantz). Therefore, we selected 26 candidate reference genes mainly via the three following channels: reference genes used in previous studies on cassava, the orthologs of the most stable Arabidopsis genes, and the sequences obtained from 32 cassava transcriptome sequence data. Then, we employed ABI 7900 HT and SYBR Green PCR mix to assess the expression of these genes in 21 materials obtained from various cassava samples under different developmental and environmental conditions. The stability of gene expression was analyzed using two statistical algorithms, namely geNorm and NormFinder. geNorm software suggests the combination of cassava4.1_017977 and cassava4.1_006391 as sufficient reference genes for major cassava samples, the union of cassava4.1_014335 and cassava4.1_006884 as best choice for drought stressed samples, and the association of cassava4.1_012496 and cassava4.1_006391 as optimal choice for normally grown samples. NormFinder software recommends cassava4.1_006884 or cassava4.1_006776 as superior reference for qPCR analysis of different materials and organs of drought stressed or normally grown cassava, respectively. Results provide an important resource for cassava reference genes under specific conditions. The limitations of these findings were also discussed. Furthermore, we suggested some strategies that may be used to select candidate reference genes.
Highlights
Analysis of mRNA expression patterns is one of the most commonly used molecular techniques to verify the function of a candidate gene
Novel superior internal controls were identified via genome-wide screening, and most of them were expressed at much lower levels than traditional reference genes (Czechowski et al, 2005)
We searched for the orthologs of the genes listed as stably expressed in a genome-wide investigation of Arabidopsis in cassava genome
Summary
Analysis of mRNA expression patterns is one of the most commonly used molecular techniques to verify the function of a candidate gene. The expressed sequence tag (EST) database, transcriptome data and whole-genome GeneChip data comprise a large set of data sources used to select candidate reference genes. Several classical reference genes were proposed as internal controls based on the relative abundance of their EST database (Coker and Davies, 2003). Novel superior internal controls were identified via genome-wide screening, and most of them were expressed at much lower levels than traditional reference genes (Czechowski et al, 2005). Based on the work of Czechowski et al (2005), these superior reference genes have been successfully employed to search for orthologs in unrelated species, such as tomato (Exposito-Rodriguez et al, 2008; Dekkers et al, 2012), Eucalyptus (Cassan-Wang et al, 2012), and desert moss Syntrichia caninervis (Li et al, 2015)
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