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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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Raffinose synthase amino acid sequence alignments in the regions surrounding the induced mutations in the RS2 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. A. The exon one region containing the induced mutation in line 165 which resulted in S150F. B. The exon one region containing the induced mutation in line 397 which resulted in T107I. C. Weblogo output of the amino acid conservation of raffinose synthase enzymes aligned as part of the BLINK feature at NCBI  using GI number 187610414. Amino acid positions within the protein are listed on the X axis. The overall height for each amino acid column stack indicates the sequence conservation at that position while the height of one-letter amino acid symbols within the column stack indicates the relative frequency of each amino acid in that position [23].
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Figure 1: Raffinose synthase amino acid sequence alignments in the regions surrounding the induced mutations in the RS2 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. A. The exon one region containing the induced mutation in line 165 which resulted in S150F. B. The exon one region containing the induced mutation in line 397 which resulted in T107I. C. Weblogo output of the amino acid conservation of raffinose synthase enzymes aligned as part of the BLINK feature at NCBI using GI number 187610414. Amino acid positions within the protein are listed on the X axis. The overall height for each amino acid column stack indicates the sequence conservation at that position while the height of one-letter amino acid symbols within the column stack indicates the relative frequency of each amino acid in that position [23].

Mentions: Previously, mutations in the soybean raffinose synthase gene, RS2, have been shown to result in an increase in seed sucrose and a decrease in raffinose and stachyose [16]. Reverse genetics screening of the EMS mutagenized populations created the potential to find additional mutations in RS2 and confirm the contribution of this gene to the seed oligosaccharide phenotype in soybean. A portion of the RS2 gene [GenBank: EU651888] was screened for mutations utilizing the TILLING strategy [4]; four lines were identified which contained single nucleotide polymorphisms (SNPs). These lines were subsequently confirmed by sequence analysis to contain independent RS2 mutations. The four identified lines all contained a SNP typical of EMS mutagenesis, G/C to A/T transitions. Two of the mutations did not result in amino acid changes and therefore were not considered candidates for phenotypic characterization. The other two lines, designated 165 and 397, contained mutations which resulted in missense amino acid changes (Figure 1)[23].


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

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

Raffinose synthase amino acid sequence alignments in the regions surrounding the induced mutations in the RS2 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. A. The exon one region containing the induced mutation in line 165 which resulted in S150F. B. The exon one region containing the induced mutation in line 397 which resulted in T107I. C. Weblogo output of the amino acid conservation of raffinose synthase enzymes aligned as part of the BLINK feature at NCBI  using GI number 187610414. Amino acid positions within the protein are listed on the X axis. The overall height for each amino acid column stack indicates the sequence conservation at that position while the height of one-letter amino acid symbols within the column stack indicates the relative frequency of each amino acid in that position [23].
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Raffinose synthase amino acid sequence alignments in the regions surrounding the induced mutations in the RS2 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. A. The exon one region containing the induced mutation in line 165 which resulted in S150F. B. The exon one region containing the induced mutation in line 397 which resulted in T107I. C. Weblogo output of the amino acid conservation of raffinose synthase enzymes aligned as part of the BLINK feature at NCBI using GI number 187610414. Amino acid positions within the protein are listed on the X axis. The overall height for each amino acid column stack indicates the sequence conservation at that position while the height of one-letter amino acid symbols within the column stack indicates the relative frequency of each amino acid in that position [23].
Mentions: Previously, mutations in the soybean raffinose synthase gene, RS2, have been shown to result in an increase in seed sucrose and a decrease in raffinose and stachyose [16]. Reverse genetics screening of the EMS mutagenized populations created the potential to find additional mutations in RS2 and confirm the contribution of this gene to the seed oligosaccharide phenotype in soybean. A portion of the RS2 gene [GenBank: EU651888] was screened for mutations utilizing the TILLING strategy [4]; four lines were identified which contained single nucleotide polymorphisms (SNPs). These lines were subsequently confirmed by sequence analysis to contain independent RS2 mutations. The four identified lines all contained a SNP typical of EMS mutagenesis, G/C to A/T transitions. Two of the mutations did not result in amino acid changes and therefore were not considered candidates for phenotypic characterization. The other two lines, designated 165 and 397, contained mutations which resulted in missense amino acid changes (Figure 1)[23].

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