Digital Absolute Gene Expression Analysis of Essential Starch-Related Genes in a Radiation Developed Amaranthus cruentus L. Variety in Comparison with Real-Time PCR

Abstract

We investigated the expression pattern of four major starch genes at different seed developmental stages in the radiation-bred amaranth variety “Pribina” (Amaranthus cruentus L.) and corresponding control genotype “Ficha” (Amaranthus cruentus L.). Two platforms were used and compared for the gene expression analysis of GBSSI, SSSI, SBE, and DBE amaranth genes, including a standard quantitative real-time PCR (qPCR) technique and relatively novel droplet digital PCR (ddPCR) assay. In our conditions, both methods showed great accuracy and revealed higher expression of the investigated genes in the mutant variety than in the control genotype. Here we report for the first time, a ddPCR gene expression assay for the cultivated grain amaranth, as the most important group of the species in the genus Amaranthus.

Digital PCR: What Relevance to Plant Studies?

“…Several technologies, such as real-time PCR, sequencing, and hybridization-based assays, are currently used for gene expression studies. The suitability of digital PCR for gene expression analysis has been demonstrated in several plant species, such as Amaranthus cruentus [45], Beta vulgaris [46], Castanea [47], and Hordeum vulgare [48]. The expression of genes involved in resistance to pathogens, in response to bioactive compounds, in starch synthesis, and in senescence developmental process have been considered.…”

Next generation multiplexing for digital PCR using a novel melt-based hairpin probe design

“…Another major advantage is that accuracy and precision of dPCR methods have been reported to be superior to other PCR methods in part because they mitigate factors that affect PCR efficiency (Simmonds et al, 1990;Sykes et al, 1992;Vogelstein and Kinzler, 1999;Hindson et al, 2013;Svec et al, 2015). For situations that require quantifying low-abundance targets present in a complex nucleic acid background, dPCR has become the method of choice (Hayden et al, 2013;Hindson et al, 2013;Doi et al, 2015;Taylor et al, 2017;Lancikova and Hricova, 2020). These improvements over NGS and other PCR approaches are attributed to the fact that in dPCR, samples are partitioned into 10 3 -10 6 reactions, preferably with each reaction containing zero or one target sequence (Hindson et al, 2011;Madic et al, 2016;Dueck et al, 2019).…”