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 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 germination, plant development and morphology was observed in the mutants. Our findings indicate that GmGOLS1A and GmGOLS1B contribute to soybean oligosaccharides 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 cultivar for transformation and targeted genome editing.
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Claims 10
Claim · model-written summary
The CRISPR construct induced deletions at target sites of both genes.
- Taxon
- soybean
Glycine maxt0 - Gene
- GmGOLS1A and GmGOLS1Be0
- Outcome
- various deletionse0
- Value
- two target sitese0
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“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”
t0“soybean seeds”
Claim · model-written summary
Some induced alleles passed to progeny and T2 null segregants lacked induced off-target mutations.
- Taxon
- soybeanGlycine maxt0
- Organ / stage
- T2 generatione0
- Outcome
- null segregants of single and double mutant genotypes without off-target induced mutationse0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“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”
t0“soybean seeds”
Claim · model-written summary
Double mutant seeds had 35.2% less total RFO content, falling from 64.7 to 41.95 mg/g dry weight.
- Taxon
- soybeanGlycine maxe0
- Material
- double mutant linese0
- Organ / stage
- seede0
- Treatment
- double mutant linese0
- Outcome
- total RFO contente0
- Direction
- reductione0
- Value
- 35.2%e0
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“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”
Claim · model-written summary
Stachyose content declined by 35.4% in double mutant soybean.
- Taxon
- soybeanGlycine maxe0
- Treatment
- double mutant soybeane0
- Outcome
- stachyose contente0
- Direction
- decreasede0
- Value
- 35.4%e0
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“the stachyose content, the most predominant RFO in soybean seeds, decreased by 35.4% in double mutant soybean”
Claim · model-written summary
Raffinose content rose by 41.7% in double mutant soybean.
- Taxon
- soybeanGlycine maxt0
- Treatment
- double mutant soybeane1
- Outcome
- raffinose contente0
- Direction
- increasede0
- Value
- 41.7%e0
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“the raffinose content increased by 41.7%”
t0“soybean seeds”
e1“decreased by 35.4% in double mutant soybean”
Claim · model-written summary
Verbascose content decreased slightly in mutant lines.
- Taxon
- soybeanGlycine maxt0
- Treatment
- mutant linese0
- Outcome
- verbascose contente0
- Direction
- decreasee0
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“A slight decrease in verbascose content was also observed in mutant lines”
t0“soybean seeds”
Claim · model-written summary
Some mutant lines had increased seed protein and fat contents.
- Taxon
- soybeanGlycine maxt0
- Treatment
- mutant linese0
- Outcome
- protein and fat contentse0
- Direction
- increasede0
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“some mutant lines also exhibited increased protein and fat contents”
t0“soybean seeds”
Claim · model-written summary
Mutants showed no observed differences in seed germination, development or morphology.
- Taxon
- soybeanGlycine maxt0
- Treatment
- mutantse0
- Outcome
- seed germination, plant development and morphologye0
- Direction
- no differencee0
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“no difference in seed germination, plant development and morphology was observed in the mutants”
t0“soybean seeds”
Claim · model-written summary
The authors infer that GmGOLS1A and GmGOLS1B contribute to soybean oligosaccharide profiles through RFO biosynthesis.
- Taxon
- soybeanGlycine maxe0
- Gene
- GmGOLS1A and GmGOLS1Be0
- Outcome
- soybean oligosaccharides profilee0
- Qualifier
- RFO biosynthesis pathwayse0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“GmGOLS1A and GmGOLS1B contribute to soybean oligosaccharides profile through RFO biosynthesis pathways”
Claim · model-written summary
The authors present the genes as targets for crop improvement.
- Taxon
- soybeanGlycine maxt0
- Gene
- GmGOLS1A and GmGOLS1Be1
- Outcome
- crop improvement effortse0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“are promising targets for future investigation, as well as crop improvement efforts”
t0“soybean seeds”
e1“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”