Abstract: Chenopodium quinoa (quinoa), an orphan crop from the Andean region, adapts well to extreme abiotic stresses and its seeds are of high nutritional value. These characteristics make quinoa a promising crop as the effects of climate change and global warming threatens food security, but its agricultural value still needs to be improved. Quinoa seeds, mainly in the husk, accumulate triterpenoid saponins up to 4% of the seed mass1. These saponins are antinutrients2–6 that are mainly perceived as bitter2; therefore, they have to be removed from edible quinoa products before consumption. Jarvis et al. (2017) proposed that TSARL1 (AUR62017204), which encodes a bHLH transcription factor, may control biosynthesis of saponins in quinoa seeds via regulating the expression of enzymes in saponin biosynthetic pathway1. Recently, FIND-IT (Fast Identification of Nucleotide variants by droplet DigITal polymerase chain reaction) technology was reported as a simple, fast, sensitive, and non-GMO approach for identification of specific genetic diversity in large populations of plants7. Here we employed the FIND-IT method to identify specific point mutations in the progeny of EMS-mutagenized seeds of the bitter quinoa cultivar Titicaca, well adapted and cultivated in Europe with high yield and harvest index8,9, and could identify two independent mutants of TSARL1: sweet seed1 (sws1) containing a G2079A substitution and sws2 with a G12A substitution. The mutation in sws1 resulted in the cryptic splice site (G1875|G1876) and a truncated TSARL1 transcript, which is also observed in other tested sweet cultivars such as CHEN-125, CHEN-161, CHEN-183, CHEN-211, CHEN-262, and CHEN-345. The mutation in sws2 resulted in an early stop codon in the TSARL1 transcript (W4STOP). Afrosimetric assay and LC-MS analysis of saponin extracts from mutant seeds revealed that both mutants were deficient in the biosynthesis of saponins. Loss-of-function of TSARL1 did not affect plant development as such (e.g. plant height and biomass) and no significant difference in size and weight of seeds could be identified between mutants and wild-type (WT). RNA-seq analysis showed that the expression of enzymes in the mevalonate, terpenoid backbone biosynthetic, and triterpenoid saponin biosynthetic pathways were consistently downregulated in the two mutants compared to those in WT plants. Taken together, we have confirmed the significance of TSARL1 in regulating saponin biosynthesis and have successfully used FIND-IT to generate new sweet quinoa lines.

References
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