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New sources of soybean seed meal and oil composition traits identified through TILLING.

Dierking EC, Bilyeu KD - BMC Plant Biol. (2009)

Bottom Line: Four mutations in independent lines were identified in the raffinose synthase gene RS2; two mutations resulted in amino acid mutations and one resulted in an altered seed oligosaccharide phenotype.Molecular marker assays were developed to reliably detect the inheritance of the mutant alleles and can be used in efficient breeding for these desired seed phenotypes.Our results serve as the first demonstration of the identification of soybean mutants controlling seed phenotypes discovered through the reverse genetics technique TILLING.

View Article: PubMed Central - HTML - PubMed

Affiliation: University of Missouri-Columbia, Division of Plant Sciences, 110 Waters Hall, Columbia, MO 65211, USA. Emily.Dierking@mizzou.edu

ABSTRACT

Background: Several techniques are available to study gene function, but many are less than ideal for soybean. Reverse genetics, a relatively new approach, can be utilized to identify novel mutations in candidate genes; this technique has not produced an allelic variant with a confirmed phenotype in soybean. Soybean raffinose synthase genes and microsomal omega-6 fatty acid desaturase genes were screened for novel alleles in mutagenized soybean populations.

Results: Four mutations in independent lines were identified in the raffinose synthase gene RS2; two mutations resulted in amino acid mutations and one resulted in an altered seed oligosaccharide phenotype. The resulting phenotype was an increase in seed sucrose levels as well as a decrease in both raffinose and stachyose seed oligosaccharide levels. Three mutations in independent lines were identified in the omega-6 fatty acid desaturase gene FAD2-1A; all three mutations resulted in missense amino acid mutations and one resulted in an altered seed fatty acid profile that led to an increase in oleic acid and a decrease in linoleic acid in the seed oil.

Conclusion: The oligosaccharide phenotype controlled by the novel RS2 allele is similar to previously observed seed oligosaccharide phenotypes in RS2 mutant (PI 200508) allele-containing lines. Due to the anti-nutritional characteristics of raffinose and stachyose, this represents a positive change in seed composition. The fatty acid phenotype controlled by the novel FAD2-1A allele controls an increase in oleic acid in the seed oil, a phenotype also observed in a line previously characterized to have a allele of the FAD2-1A gene. Molecular marker assays were developed to reliably detect the inheritance of the mutant alleles and can be used in efficient breeding for these desired seed phenotypes. Our results serve as the first demonstration of the identification of soybean mutants controlling seed phenotypes discovered through the reverse genetics technique TILLING.

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Omega-6 fatty acid desaturase (FAD2) amino acid sequence alignments in the regions surrounding the induced mutations in the soybean FAD2-1A gene. Amino acid positions are indicated at the beginning of each alignment. The position of the polymorphic amino acid is indicated by an asterisk. Identical amino acid residues are highlighted in black while similar amino acid residues are highlighted in gray. Underlined amino acids represent the histidine-rich region Ia, a critical region for fatty acid desaturase enzyme function [24]. A. The region containing the induced mutation in line 17D which resulted in S117N. B. The region containing the induced mutation in line 615 which resulted in S238F. C. The region containing the induced mutation in line 743 which resulted in G374E. D. Weblogo output of the amino acid conservation in region Ia of omega-6 fatty acid desaturase enzymes [24] aligned as part of the BLINK feature at NCBI using GI number 197111724.
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Figure 2: Omega-6 fatty acid desaturase (FAD2) amino acid sequence alignments in the regions surrounding the induced mutations in the soybean FAD2-1A gene. Amino acid positions are indicated at the beginning of each alignment. The position of the polymorphic amino acid is indicated by an asterisk. Identical amino acid residues are highlighted in black while similar amino acid residues are highlighted in gray. Underlined amino acids represent the histidine-rich region Ia, a critical region for fatty acid desaturase enzyme function [24]. A. The region containing the induced mutation in line 17D which resulted in S117N. B. The region containing the induced mutation in line 615 which resulted in S238F. C. The region containing the induced mutation in line 743 which resulted in G374E. D. Weblogo output of the amino acid conservation in region Ia of omega-6 fatty acid desaturase enzymes [24] aligned as part of the BLINK feature at NCBI using GI number 197111724.

Mentions: The FAD2-1A mutations identified in DNA from M2 tissue were homozygous in all three cases, and the mutations were confirmed in the M3 seedlings corresponding to the original M2 plants. Line 17D contained a SNP (g350a in the coding sequence) resulting in the amino acid change S117N. Line 615 contained a SNP (c713t in the coding sequence) resulting in the amino acid change S238F. Line 743 contained a SNP (g1121a in the coding sequence) resulting in the amino acid change G374E. The missense mutation for line 17D was in a highly conserved region of the protein sequence, while the missense mutations in lines 615 and 743 were in less conserved regions (Figure 2)[24].


New sources of soybean seed meal and oil composition traits identified through TILLING.

Dierking EC, Bilyeu KD - BMC Plant Biol. (2009)

Omega-6 fatty acid desaturase (FAD2) amino acid sequence alignments in the regions surrounding the induced mutations in the soybean FAD2-1A gene. Amino acid positions are indicated at the beginning of each alignment. The position of the polymorphic amino acid is indicated by an asterisk. Identical amino acid residues are highlighted in black while similar amino acid residues are highlighted in gray. Underlined amino acids represent the histidine-rich region Ia, a critical region for fatty acid desaturase enzyme function [24]. A. The region containing the induced mutation in line 17D which resulted in S117N. B. The region containing the induced mutation in line 615 which resulted in S238F. C. The region containing the induced mutation in line 743 which resulted in G374E. D. Weblogo output of the amino acid conservation in region Ia of omega-6 fatty acid desaturase enzymes [24] aligned as part of the BLINK feature at NCBI using GI number 197111724.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2719642&req=5

Figure 2: Omega-6 fatty acid desaturase (FAD2) amino acid sequence alignments in the regions surrounding the induced mutations in the soybean FAD2-1A gene. Amino acid positions are indicated at the beginning of each alignment. The position of the polymorphic amino acid is indicated by an asterisk. Identical amino acid residues are highlighted in black while similar amino acid residues are highlighted in gray. Underlined amino acids represent the histidine-rich region Ia, a critical region for fatty acid desaturase enzyme function [24]. A. The region containing the induced mutation in line 17D which resulted in S117N. B. The region containing the induced mutation in line 615 which resulted in S238F. C. The region containing the induced mutation in line 743 which resulted in G374E. D. Weblogo output of the amino acid conservation in region Ia of omega-6 fatty acid desaturase enzymes [24] aligned as part of the BLINK feature at NCBI using GI number 197111724.
Mentions: The FAD2-1A mutations identified in DNA from M2 tissue were homozygous in all three cases, and the mutations were confirmed in the M3 seedlings corresponding to the original M2 plants. Line 17D contained a SNP (g350a in the coding sequence) resulting in the amino acid change S117N. Line 615 contained a SNP (c713t in the coding sequence) resulting in the amino acid change S238F. Line 743 contained a SNP (g1121a in the coding sequence) resulting in the amino acid change G374E. The missense mutation for line 17D was in a highly conserved region of the protein sequence, while the missense mutations in lines 615 and 743 were in less conserved regions (Figure 2)[24].

Bottom Line: Four mutations in independent lines were identified in the raffinose synthase gene RS2; two mutations resulted in amino acid mutations and one resulted in an altered seed oligosaccharide phenotype.Molecular marker assays were developed to reliably detect the inheritance of the mutant alleles and can be used in efficient breeding for these desired seed phenotypes.Our results serve as the first demonstration of the identification of soybean mutants controlling seed phenotypes discovered through the reverse genetics technique TILLING.

View Article: PubMed Central - HTML - PubMed

Affiliation: University of Missouri-Columbia, Division of Plant Sciences, 110 Waters Hall, Columbia, MO 65211, USA. Emily.Dierking@mizzou.edu

ABSTRACT

Background: Several techniques are available to study gene function, but many are less than ideal for soybean. Reverse genetics, a relatively new approach, can be utilized to identify novel mutations in candidate genes; this technique has not produced an allelic variant with a confirmed phenotype in soybean. Soybean raffinose synthase genes and microsomal omega-6 fatty acid desaturase genes were screened for novel alleles in mutagenized soybean populations.

Results: Four mutations in independent lines were identified in the raffinose synthase gene RS2; two mutations resulted in amino acid mutations and one resulted in an altered seed oligosaccharide phenotype. The resulting phenotype was an increase in seed sucrose levels as well as a decrease in both raffinose and stachyose seed oligosaccharide levels. Three mutations in independent lines were identified in the omega-6 fatty acid desaturase gene FAD2-1A; all three mutations resulted in missense amino acid mutations and one resulted in an altered seed fatty acid profile that led to an increase in oleic acid and a decrease in linoleic acid in the seed oil.

Conclusion: The oligosaccharide phenotype controlled by the novel RS2 allele is similar to previously observed seed oligosaccharide phenotypes in RS2 mutant (PI 200508) allele-containing lines. Due to the anti-nutritional characteristics of raffinose and stachyose, this represents a positive change in seed composition. The fatty acid phenotype controlled by the novel FAD2-1A allele controls an increase in oleic acid in the seed oil, a phenotype also observed in a line previously characterized to have a allele of the FAD2-1A gene. Molecular marker assays were developed to reliably detect the inheritance of the mutant alleles and can be used in efficient breeding for these desired seed phenotypes. Our results serve as the first demonstration of the identification of soybean mutants controlling seed phenotypes discovered through the reverse genetics technique TILLING.

Show MeSH
Related in: MedlinePlus