RS2 mutant allele, perfect molecular markers, and low raffinose/stachyose soybean germplasm
Inventors
Bilyeu, Kristin D. • DIERKING, EMILY C.
Assignees
US Department of Agriculture USDA • University of Missouri St Louis
Publication Number
US-8728726-B1
Publication Date
2014-05-20
Expiration Date
2031-07-06
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Abstract
A mutation in the gene encoding the raffinose synthase 2 enzyme, RS2, in soybean, Glycine max (L.) Merr., is associated with a reduced raffinose and stachyose seed phenotype. Soybean homozygous for a mutant allele of the RS2 gene which encodes an amino acid change to isoleucine at position 107 of the enzyme's amino acid sequence exhibit significantly reduced seed raffinose and stachyose content, and increased seed sucrose content. Nucleic acid samples of soybean may be assayed for the presence of this mutant allele, and soybean containing the allele may be selected for breeding to generate reduced raffinose and stachyose soybean lines. Alternatively, the mutation may be detected by analysis of the RS2 enzyme produced in the soybean to determine the presence of either an isoleucine or a threonine at amino acid 107. Molecular markers have been developed for detecting the presence or absence of the mutant allele.
Core Innovation
The invention discloses a novel mutation in the gene encoding the raffinose synthase 2 enzyme, RS2, in soybean (Glycine max (L.) Merr.). This mutant allele encodes an amino acid change to isoleucine at position 107 of the enzyme's amino acid sequence. Soybean homozygous for this mutant RS2 allele exhibit significantly reduced seed raffinose and stachyose content, alongside increased seed sucrose content. The invention provides methods for assaying nucleic acid samples for the presence of this mutant allele, allowing selection for breeding soybean lines with reduced raffinose and stachyose levels.
The problem addressed by this invention arises from the anti-nutritional effects of raffinose family oligosaccharides, including raffinose and stachyose, which accumulate during normal soybean seed development. These compounds limit the digestibility and metabolizable energy of soybean meal in monogastric animal diets, reducing the meal's value. Previous soybean lines with reduced raffinose and stachyose content have been identified, but improved soybean lines with further reduction of these oligosaccharides remain needed.
The invention further provides molecular markers for detecting the presence or absence of the mutant RS2 allele. Detection can be achieved through nucleic acid assays or by analyzing the RS2 enzyme to determine if isoleucine or threonine is present at amino acid position 107. The mutant allele serves as a marker for selecting soybean germplasm with the desired low raffinose and stachyose phenotype, facilitating breeding programs to develop improved soybean lines.
Claims Coverage
The claims describe six inventive features related to a mutant RS2 allele in soybean and methods for its detection and use in breeding.
Soybean comprising a mutant RS2 allele encoding isoleucine at position 107
A soybean seed, plant, or parts containing a mutant allele of the gene encoding the soybean raffinose synthase 2 enzyme, RS2, which encodes an isoleucine at position 107 of the enzyme amino acid sequence.
Detection of mutant RS2 allele by nucleic acid assay
A method for detecting a mutant allele of RS2 in soybean by assaying nucleic acid samples for the presence of a sequence encoding a variant RS2 enzyme comprising isoleucine at amino acid 107, including genomic DNA, cDNA, or RNA samples.
Use of molecular markers for selecting soybean with mutant RS2 allele
Methods comprising selecting soybean for breeding which possess the mutant RS2 allele encoding isoleucine at position 107 and removing soybean lacking the mutation from breeding.
Screening for RS2 alleles encoding isoleucine at position 107
A screening method comprising obtaining nucleic acid samples from soybean, assaying for a polymorphism encoding isoleucine at position 107 of RS2, and selecting soybean comprising the polymorphism for breeding.
Detection of RS2 mutation by amino acid analysis
A method for detecting a mutation in RS2 by determining the presence of either isoleucine or threonine at amino acid 107 of the RS2 enzyme, and selecting soybean having isoleucine at this position for breeding.
Homozygous soybean for mutant RS2 allele
Soybean seeds, plants or parts thereof which are homozygous for the mutant allele encoding isoleucine at amino acid position 107 of the RS2 enzyme.
The claims collectively cover soybean plants and seeds comprising a mutant RS2 allele encoding an isoleucine at position 107, methods for detecting this allele through nucleic acid or protein assays, and the use of these methods to select and breed soybean lines with reduced raffinose and stachyose seed content.
Stated Advantages
The mutant RS2 allele is associated with significantly reduced seed raffinose and stachyose content and increased seed sucrose content, improving soybean meal nutritional quality.
Molecular markers and assays for the mutant allele enable efficient selection of low raffinose and stachyose soybean lines in breeding programs.
The invention facilitates rapid incorporation of the low raffinose/stachyose trait into elite soybean cultivars using conventional breeding methods.
The use of induced mutations and molecular marker selection provides a non-transgenic approach that avoids regulatory restrictions associated with genetic modification.
Documented Applications
Selection and breeding of soybean germplasm possessing the mutant RS2 allele to develop low raffinose and stachyose soybean lines.
Assaying soybean nucleic acid samples to detect the presence of the mutant RS2 allele for breeding and germplasm selection purposes.
Analyzing RS2 enzyme amino acid sequence from soybean samples to determine the presence of isoleucine at position 107 as an indicator of the low raffinose/stachyose phenotype.
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