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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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Phenotype to genotype association of a homozygous soybean population derived from the mutagenized soybean line 397. The x-axis is represented by two distinct RS2 genotypes: WT represents wild-type RS2 alleles and mut represents mutant RS2 alleles from the line 397; n represents the number of individual seeds from each genotypic class. The oligosaccharide phenotype of four individual M4 seeds from each plant was determined. The data represents the mean of the ratio of extractable seed sucrose to the sum of raffinose and stachyose. Error bars represent plus and minus one standard deviation from the mean.
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Figure 4: Phenotype to genotype association of a homozygous soybean population derived from the mutagenized soybean line 397. The x-axis is represented by two distinct RS2 genotypes: WT represents wild-type RS2 alleles and mut represents mutant RS2 alleles from the line 397; n represents the number of individual seeds from each genotypic class. The oligosaccharide phenotype of four individual M4 seeds from each plant was determined. The data represents the mean of the ratio of extractable seed sucrose to the sum of raffinose and stachyose. Error bars represent plus and minus one standard deviation from the mean.

Mentions: A population consisting of plants with contrasting RS2 genotypes was then developed from line 397-derived plants that contained either homozygous wild-type (Williams 82) or homozygous mutant alleles at the RS2 locus in order to further characterize the phenotype resulting from the novel allele. Seven independent wild-type RS2 and nine independent mutant RS2 plants were selected to negate the action of unidentified genes that may contribute to the oligosaccharide content; the mutation density was previously determined to average 1/550 kilobases [4]. Four seeds from each of the plants of the homozygous population were analyzed for oligosaccharide content. For the plants that contained the T107I RS2 mutation, the average seed sucrose was increased by 28%, raffinose was reduced to 37% and stachyose was reduced to approximately 24% of 397-derived seeds which carried the wild-type allele of RS2 (Figure 4). This oligosaccharide phenotype is similar to the phenotype controlled by the previously described RS2 mutant alleles in PI 200508 which also resulted in a decrease in seed raffinose and stachyose content along with an increase in seed sucrose levels [16].


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

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

Phenotype to genotype association of a homozygous soybean population derived from the mutagenized soybean line 397. The x-axis is represented by two distinct RS2 genotypes: WT represents wild-type RS2 alleles and mut represents mutant RS2 alleles from the line 397; n represents the number of individual seeds from each genotypic class. The oligosaccharide phenotype of four individual M4 seeds from each plant was determined. The data represents the mean of the ratio of extractable seed sucrose to the sum of raffinose and stachyose. Error bars represent plus and minus one standard deviation from the mean.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Phenotype to genotype association of a homozygous soybean population derived from the mutagenized soybean line 397. The x-axis is represented by two distinct RS2 genotypes: WT represents wild-type RS2 alleles and mut represents mutant RS2 alleles from the line 397; n represents the number of individual seeds from each genotypic class. The oligosaccharide phenotype of four individual M4 seeds from each plant was determined. The data represents the mean of the ratio of extractable seed sucrose to the sum of raffinose and stachyose. Error bars represent plus and minus one standard deviation from the mean.
Mentions: A population consisting of plants with contrasting RS2 genotypes was then developed from line 397-derived plants that contained either homozygous wild-type (Williams 82) or homozygous mutant alleles at the RS2 locus in order to further characterize the phenotype resulting from the novel allele. Seven independent wild-type RS2 and nine independent mutant RS2 plants were selected to negate the action of unidentified genes that may contribute to the oligosaccharide content; the mutation density was previously determined to average 1/550 kilobases [4]. Four seeds from each of the plants of the homozygous population were analyzed for oligosaccharide content. For the plants that contained the T107I RS2 mutation, the average seed sucrose was increased by 28%, raffinose was reduced to 37% and stachyose was reduced to approximately 24% of 397-derived seeds which carried the wild-type allele of RS2 (Figure 4). This oligosaccharide phenotype is similar to the phenotype controlled by the previously described RS2 mutant alleles in PI 200508 which also resulted in a decrease in seed raffinose and stachyose content along with an increase in seed sucrose levels [16].

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