Evaluation of Expression Stability of Candidate References Genes among Green and Yellow Pea Cultivars (<i>Pisum sativum</i> L.) Subjected to Abiotic and Biotic Stress

Abstract

Dry pea (Pisum sativum L.) is grown as human and animal feed throughout the world. Large yield losses in pea due to biotic and abiotic stresses compel an improved understanding of mechanisms of stress tolerance and genetic determinants conditioning these tolerances. The availability of stably expressed reference genes is a prerequisite for examining differential gene expression. The objective of this study was to examine the expression profile of several candidate reference genes across a broad range of commercial pea cultivars. Expression profiles of five candidate reference genes; 18s rRNA, actin, TIF, β tubulin-2 and β tubulin-3 were examined. Relative quantifications of candidate reference genes were estimated from control plants, plants after 48 h of cold treatment, and plants 24 and 48 h after inoculation with Sclerotinia sclerotiorum, the causal agent of white mold disease of pea. RT-qPCR was performed on cDNA synthesized from three food grade spring peas, Ariel, Aragorn, and Sterling, and two spring yellow peas, Delta and Universal, which are used as animal feed. Analysis of variance (ANOVA) of C T values demonstrated significant variation between varieties and treatments under cold and disease conditions. The most abundant transcripts among tested reference genes were for 18s rRNA. Stability analysis indicated that TIF and β tubulin-3 genes were the most stably expressed candidate genes under both cold and disease stress and could serve as reference genes across a wide range of pea cultivars.

Identification of a Novel Reference Gene for Apple Transcriptional Profiling under Postharvest Conditions

“…The transcriptional stability of MdACT , a commonly employed reference gene, was observed in the majority of the investigated experimental conditions, except for experiments IV and V, which use cold storage for postharvest fruit conservation. Similarly, previous studies have demonstrated that ACT transcription in tissues submitted to cold is less stable [ 42 , 43 ], suggesting that low temperatures influence its transcription and/or turnover rates. The transcription stability of the gene MdUBC was observed for all investigated experimental conditions; thus, it can be considered a suitable reference gene for a wide range of biological samples and experimental conditions.…”

Selection of suitable reference genes for reverse transcription quantitative real-time PCR studies on different experimental systems from carrot (Daucus carota L.)

“…In the two experimental systems here tested, the highly expressed ribosomal 5.8S, 25S and 18S rRNA genes appear as well ranked genes. Also in other plant species and experimental conditions, ribosomal genes are expressed at very high levels ( Barsalobres-Cavallari et al, 2009;Jain et al, 2006;Saha and Vandemark, 2012). Furthermore, the commonly used reference gene ˇ-TUB was repeatedly ranked in the last positions, regardless the experimental system analyzed.…”

Association mapping of agronomic and quality traits in USDA pea single-plant collection

“…The MTAs identified by the diversity panel in this study helped determining SNP haplotypes associated with certain traits. Marker-trait associations of interest can be further studied or tested using quantitative PCR for the putative associations (Saha and Vandemark 2012). We report four categories of traits including morphology, disease resistance, seed concentrations of mineral nutrients, and low molecular weight carbohydrates.…”