Identification of Suitable Internal Control Genes for Quantitative Real-Time PCR Expression Analyses in Peanut (Arachis hypogaea)

Abstract

Real-time qPCR is currently the most sensitive technique available for the detection of lowlevel mRNA expression. For more reliable and precise gene expression analyses, real-time PCR data for a sequence of interest must be normalized against that of a control gene, which is uniformly expressed in various tissues and during different phases of development. So far, suitable internal controls for gene expression studies in peanut have not been identified. We assessed the expression of 10 frequently used housekeeping genes, specifically ubq10, gapdh, hel1, yls8, 14-3-3, 60s, ubc, ef-1a, act7, and adh3. Using the algorithms available through the GeNorm and NormFinder programs, the stability of their expression was estimated in a set of five diverse peanut tissue samples derived from a Virginiatype peanut cultivar (Shulamit). Collectively, the gene with the most stable expression across all of the examined tissues and both programs was adh3, followed by 60s and yls8, which had minimal estimated intra-and inter-tissue variation. The stability of two stable reference genes (adh3 and yls8) compared with two less stable (14-3-3 and ubq10) reference genes was validated in unpooled tissue samples from five peanut kernel developmental stages. Finally, the effect of the use of one or more reference genes on the observed relative expression levels of an important seed oil metabolism gene, diacylglycerol acyltransferase 1 (Dgat1), during kernel development was demonstrated. Based on findings, the suggestion is that adh3, or a combination of this gene with 60s and yls8 should be considered for use in quantitative mRNA expression analyses in Arachis, particularly in studies involving seed development; whereas ubq10 and gapdh should be avoided.

Evaluation of Reference Genes for RT-qPCR Expression Studies in Hop (Humulus lupulus L.) during Infection with Vascular Pathogen Verticillium albo-atrum

“…It has been identified as a novel suitable reference in A. thaliana [7] and was also shown to be one of the most stable genes used for normalization in studies of Havea brasilensis in response to tapping, hormone and salicylic acid treatment [3]. This gene was shown to be highly stable in Arabidopsis exposed to increased metal concentrations [7] and constant in different tissues of cultivated peanut [32]. The same gene was found to be less stable in various cucumber organs exposed to heavy metal stress [33].…”

Fine-Mapping the Branching Habit Trait in Cultivated Peanut by Combining Bulked Segregant Analysis and High-Throughput Sequencing

“…Working on the RNA level was preferable to working on the DNA genomic level due to the large and relatively complex peanut genome and also facilitated the detection of candidate genes. Samples were taken of each of the ground tissues (400 mg each) and were used for RNA extraction using the hot-borate method, as described by Brand and Hovav (2010). The total RNA was used to prepare two RNA-Seq libraries, using TruSeq RNA Sample Preparation Kit v2 (Illumina) following the manufacturer's protocol as described previously (Gupta et al, 2016).…”

Identification and Molecular Characterization of Homeologous Δ9-Stearoyl Acyl Carrier Protein Desaturase 3 Genes from the Allotetraploid Peanut (Arachis hypogaea)

“…Primers were [5′-GAAGTTGGCACAAATATGTGG-3′/5′-GGCATAGC TATTTTCTTCCTC-3′], [5′-CTCCGAGGAGAATAGGC-3′/5′-TTGTTTTCAGTTTTCACATCCAC-3′] and [5′-G C AT G AT TA A C A A C C TA G AT G G -3 ′ / 5 ′ -C C AT C TAGGTTGTTAATCATGC-3′] for ahSad1/2, ahSad3, and ahSad4, respectively. The alcohol dehydrogenase class III gene (adh3) that was previously found to be the preferred control gene for quantitative PCR studies in developing peanut seeds (Brand and Hovav 2010) was used as the r e f e r e n c e g e n e ( p r i m e r s :…”

“…Total RNA was extracted using the hot borate (sodium borate decahydrate) method, as previously described (Brand and Hovav 2010). In brief, tissue samples were ground in liquid nitrogen and were combined with 8 mL of borate buffer [0.2 M sodium borate decahydrate; 30 mM ethylene glycol tetraacetic acid; 1% (wt/vol) sodium dodecyl sulfate; 1% sodium deoxycholic acid; 10 mM dithiothreitol; 1% IGEPAL CA-630 (Nonidet P-40, NP-40); 2% (wt/vol) polyvinylpyrrolidone (PVP)-40] at 65°C.…”