Abstract
Raffinose family oligosaccharides (RFOs) are major soluble carbohydrates in soybean seeds that cannot be digested by human and other monogastric animals. Hence, a major goal is to reduce RFO levels to improve the nutritional quality of soybean. In this study, we utilized a dual gRNAs CRISPR/Cas9 system to induce knockouts in two soybean galactinol synthase (GOLS) genes, GmGOLS1A and its homeolog GmGOLS1B. Genotyping of T0 plants showed that the construct design was efficient in inducing various deletions in the target sites or sequences spanning the two target sites of both GmGOLS1A and GmGOLS1B genes. A subset of induced alleles was successfully transferred to progeny and, at the T2 generation, we identified null segregants of single and double mutant genotypes without off-target induced mutations. The seed carbohydrate analysis of double mutant lines showed a reduction in the total RFO content of soybean seed from 64.7 mg/g dry weight to 41.95 mg/g dry weight, a 35.2% decrease. On average, the stachyose content, the most predominant RFO in soybean seeds, decreased by 35.4% in double mutant soybean, while the raffinose content increased by 41.7%. A slight decrease in verbascose content was also observed in mutant lines. Aside from changes in soluble carbohydrate content, some mutant lines also exhibited increased protein and fat contents. Otherwise, no difference in seed weight, seed germination, plant development and morphology was observed in the mutants. Our findings indicate that GmGOLS1A and GmGOLS1B contribute to the soybean oligosaccharide profile through RFO biosynthesis pathways, and are promising targets for future investigation, as well as crop improvement efforts. Our results also demonstrate the potential in using elite soybean cultivars for transformation and targeted genome editing.
Highlights
Soybean [Glycine max (L.) Merr.] is a major crop grown worldwide with 341 million metric tons produced in 2019 (The American Soybean Association, 2019)
We found that Glyma.03G222000 (GmGOLS1A) expression increases sharply as the soybean seed matures and shows the highest expression level in seeds among all GmGOLS genes (Supplementary Figure 3)
The GmGOLS1A homeolog, Glyma.19G219100 (GmGOLS1B) gene, was initially expressed at a similar level to the GmGOLS1A gene at early stages of seed development, i.e., up to seed stage S6, but expression decreased during later stages
Summary
Soybean [Glycine max (L.) Merr.] is a major crop grown worldwide with 341 million metric tons produced in 2019 (The American Soybean Association, 2019). Soybean seeds can be used for human consumption. The raffinose family oligosaccharides (RFOs) are a major class of watersoluble carbohydrates present in soybean seeds. Previous studies of soybean seed lines with low stachyose and/or raffinose showed no significant differences in stress tolerance or germination compared to wild-type (Neus et al, 2005; Valentine et al, 2017). The fermentation process produces gases including carbon dioxide and methane, which disturb digestive activity and possibly result in flatulence and diarrhea in monogastric animals (Coon et al, 1990). Efforts to reduce raffinose and stachyose accumulation in soybean seeds may improve the usefulness of soybean for consumption by humans and monogastric animals, while other seed qualities may remain uncompromised
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