Agronomically elite soybeans with high beta-conglycinin content

Abstract

The invention overcomes the deficiencies of the art by providing an agronomically elite soybean plant with non-transgenic mutations of at least two of the glycinin subunits selected from the group consisting of Gy1, Gy2, Gy3, Gy4, and Gy5, such as conferring a Gy3 and Gy4 null phenotype and increased β-conglycinin content in seed. The invention also provides derivatives, and plant parts of these plants and uses thereof. Methods for marker assisted selection of soybean varieties comprising non-transgenic mutations conferring a reduced Gy1, Gy2, Gy3, Gy4, and Gy5 phenotype are also provided as part of the current invention. Methods for producing such plants that are further lipoxygenase and/or Kunitz Trypsin Inhibitor null and the plants produced thereby are also provided. The invention is significant in that soybeans from such plants are preferred dietary additives and provide important health benefits.

1 . A plant of an agronomically elite soybean variety with an increased seed β-conglycinin content, comprising non-transgenic mutations providing a null phenotype of at least two of the glycinin subunits selected from the group consisting of Gy1, Gy2, Gy3, Gy4, and Gy5.  
     
     
         2 . The plant of  claim 1 , defined as comprising non-transgenic mutations providing a Gy3 and Gy4 null phenotype conferring increased seed β-conglycinin content.  
     
     
         3 . The plant of  claim 1 , wherein the seed β-conglycinin content is at least about 30%.  
     
     
         4 . The plant of  claim 1 , wherein the seed β-conglycinin content is at least about 40%.  
     
     
         5 . The plant of  claim 3 , wherein the seed β-conglycinin content is further defined as selected from the group consisting of from about 30% to about 60%, from about 35% to about 60%, from about 40% to about 60%, from about 35% to about 55%, from about 40% to about 55%, and from about 40% to about 50%.  
     
     
         6 . The plant of  claim 1 , defined as comprising a seed acidic glycinin content of less than about 25% of total extracted protein.  
     
     
         7 . The plant of  claim 1 , defined as comprising a seed acidic glycinin content of less than about 18% of total extracted protein.  
     
     
         8 . The plant of  claim 1 , defined as comprising a seed acidic glycinin content of less than about 10% of total extracted protein.  
     
     
         9 . The plant of  claim 1 , defined as comprising a seed acidic glycinin content of less than about 8% of total extracted protein.  
     
     
         10 . The plant of  claim 2 , wherein the non-transgenic mutations providing a Gy3 and Gy4 null phenotype confer decreased Gy1 and Gy2 expression relative to a plant lacking said non-transgenic mutations.  
     
     
         11 . The plant of  claim 1 , defined as comprising the Gy1 and Gy2 mutations found in line B2G2, a representative sample of the seed of which is deposited under ATCC Accession No. PTA-6893.  
     
     
         12 . The plant of  claim 2 , wherein the mutations providing a Gy3 and Gy4 null phenotype conferring increased seed β-conglycinin content are the mutations providing a Gy3 and Gy4 null phenotype found in line Pedigree 3, a representative sample of the seed of which is deposited under ATCC Accession No. PTA-6892.  
     
     
         13 . The plant of  claim 1 , defined as comprising a deletion in the Gy1 locus spanning the upstream promoter region, Exon I and Intron I.  
     
     
         14 . The plant of  claim 1 , wherein the plant comprises a Gy3 allele containing an insertion of nucleotides TGAT corresponding to nucleotides 848-851 of SEQ ID NO:164 at the position in said allele.  
     
     
         15 . The plant of  claim 1 , wherein the plant comprises a Gy4 allele comprising an adenine at the position of said allele corresponding to nucleotide 682 of SEQ ID NO:165 that abrogates the translation initiation codon.  
     
     
         16 . The plant of  claim 1 , defined as comprising a mutation resulting in reduced Gy5 protein content.  
     
     
         17 . The plant of  claim 1 , wherein the plant comprises a Gy5 allele comprising at least a first polymorphism selected from the group consisting of a single nucleotide polymorphism (SNP) at position 363, a SNP at position 612, a deletion at positions 447-453 and a deletion at positions 519-524, wherein the position is the position in said allele corresponding to SEQ ID NO:166.  
     
     
         18 . The plant of  claim 2 , wherein the Gy3 and Gy4 null phenotype is provided by genetic means for the expression of said null phenotype found in line B2G2, a representative sample of the seed of said line B2G2 having been deposited under ATCC Accession No. PTA-6893.  
     
     
         19 . The plant of  claim 1 , defined as producing soybeans with a CIELAB Color Scale a* value of at least 5 and an L* value greater than 54.  
     
     
         20 . The plant of  claim 1 , further defined as comprising a transgene.  
     
     
         21 . The plant of  claim 20 , wherein the transgene is further defined as conferring a trait selected from the group consisting of herbicide tolerance; disease resistance; insect or pest resistance; altered fatty acid, protein or carbohydrate metabolism; increased grain yield; altered plant maturity, and altered morphological characteristics.  
     
     
         22 . The plant of  claim 21 , wherein the transgene confers tolerance to glyphosate herbicide.  
     
     
         23 . The plant of  claim 1 , further comprising a lipoxygenase null allele selected from the group consisting of lx1, lx2 and lx3.  
     
     
         24 . The plant of  claim 23 , further defined as comprising at least two of said null alleles.  
     
     
         25 . The plant of  claim 23 , further defined as comprising a lx1, lx2 null phenotype.  
     
     
         26 . The plant of  claim 23 , further defined as comprising a lx1, lx2 and lx3 null phenotype.  
     
     
         27 . The plant of  claim 23 , wherein the lipoxygenase null allele is a non-transgenic null allele.  
     
     
         28 . The plant of  claim 23 , wherein the lipoxygenase null allele is lx2.  
     
     
         29 . The plant of  claim 1 , further defined as comprising a Kunitz Trypsin Inhibitor (KTI) null allele.  
     
     
         30 . A plant part of the plant of  claim 1 .  
     
     
         31 . The plant part of  claim 30 , further defined as pollen, an ovule, a seed, a meristem or a cell.  
     
     
         32 . The plant part of  claim 30 , further comprising parts of a seed.  
     
     
         33 . A tissue culture of regenerable cells of the plant of  claim 1 .  
     
     
         34 . The tissue culture of  claim 33 , wherein the regenerable cells are embryos, meristematic cells, pollen, leaves, roots, root tips or flowers.  
     
     
         35 . The plant of  claim 1 , defined as prepared by a method comprising the steps of: 
 (a) crossing first and second soybean plants, wherein the first and second plants collectively comprise non-transgenic mutations resulting in a Gy3 and Gy4 null phenotype and increased seed β-conglycinin content, and wherein the first and second plants collectively comprise germplasm capable of conferring agronomically elite characteristics to a progeny plant of said plants; and    (b) assaying progeny soybean plants resulting from the crossing for agronomically elite characteristics and for Gy3 and Gy4 protein content; and    (c) selecting at least a first progeny plant comprising said Gy3 and Gy4 null phenotype and agronomically elite characteristics to obtain the plant of  claim 1 .    
     
     
         36 . The plant of  claim 35 , wherein one of the first or second soybean plants is variety B2G2, wherein a representative sample of seed of B2G2 has been deposited under ATCC Accession No. PTA-6893.  
     
     
         37 . The plant of  claim 35 , wherein the first and second plants collectively comprise non-transgenic mutations resulting in a Gy1-Gy4 null phenotype and increased seed β-conglycinin content, and wherein the first progeny plant comprises the non-transgenic mutations resulting in a Gy1-Gy4 null phenotype.  
     
     
         38 . A method of producing a soybean food or feed commodity product comprising obtaining the plant of  claim 1  or a part thereof and producing the food or feed therefrom.  
     
     
         39 . The method of  claim 38 , comprising obtaining seed from the plant of  claim 1  and preparing food from the seed.  
     
     
         40 . The method of  claim 38 , wherein the food is protein concentrate, protein isolate, soybean hulls, meal or flour.  
     
     
         41 . The method of  claim 38 , wherein the food is oil.  
     
     
         42 . The method of  claim 38 , wherein the food comprises beverages, infused foods, sauces, condiments, salad dressings, fruit juices, syrups, desserts, icings and fillings, soft frozen products, confections or intermediate food.  
     
     
         43 . Food produced by the method of  claim 38 , wherein the food comprises genomic material from the plant of  claim 1 .  
     
     
         44 . A method of producing soybean seed, comprising crossing the plant of  claim 1  with itself or a second soybean plant.  
     
     
         45 . The method of  claim 44 , further defined as a method of preparing hybrid soybean seed, comprising crossing the plant of  claim 1  to a second, distinct soybean plant.  
     
     
         46 . A method of predicting the phenotype of a soybean plant for glycinin and β-conglycinin content, comprising assaying the soybean plant for the presence of at least a first polymorphism in a soybean plant genomic region within 50 cM of a non-transgenic mutant Gy1, Gy2, or Gy4 allele to identify at least a first allele that confers decreased glycinin and increased β-conglycinin content.  
     
     
         47 . A method of plant breeding comprising the steps of: 
 (a) assaying soybean plants for the presence of at least a first polymorphism in a soybean plant genomic region within 50 cM of a non-transgenic mutant Gy1, Gy2, or Gy4 allele that confers decreased glycinin and increased β-conglycinin content;    (b) selecting at least a first soybean plant comprising said polymorphism to select said allele; and    (c) crossing the first soybean plant comprising said polymorphism to a second soybean plant to generate a progeny plant comprising at least a first non-transgenic mutant Gy1, Gy2, or Gy4 allele that confers decreased glycinin and increased β-conglycinin content.    
     
     
         48 . The method of  claim 47 , further comprising the step of: 
 (d) repeating steps (a)-(c) with the progeny plant of step (c) as starting material at least about 2-10 times to produce additional progeny plants.    
     
     
         49 . The method of  claim 47 , wherein the second soybean plant is of an agronomically elite variety.  
     
     
         50 . The method of  claim 47 , comprising selecting a soybean plant comprising said polymorphism and agronomically elite characteristics.  
     
     
         51 . The method of  claim 47 , wherein the mutant Gy1 allele comprises a deletion.  
     
     
         52 . The method of  claim 47 , wherein the non-transgenic mutant Gy1, Gy2, or Gy4 allele corresponds to the mutant allele found in line B2G2, a representative sample of the seed of which is deposited under ATCC Accession No. PTA-6893.  
     
     
         53 . The method of  claim 47 , wherein the mutant Gy4 allele comprises a point mutation that abrogates the translation initiation codon.  
     
     
         54 . The method of  claim 47 , further comprising selecting a first soybean plant that is homozygous for said polymorphism.  
     
     
         55 . The method of  claim 47 , further comprising selecting a first soybean plant comprising at least two polymorphisms.  
     
     
         56 . The method of  claim 47 , comprising assaying polymorphisms within 50 cM of Gy1 and Gy4 alleles.  
     
     
         57 . The method of  claim 47 , comprising assaying polymorphisms within 50 cM of Gy2 and Gy4 alleles.  
     
     
         58 . The method of  claim 47 , comprising assaying a polymorphism in the Gy4 allele corresponding to nucleotide 682 of SEQ ID NO:165.  
     
     
         59 . The method of  claim 47 , further comprising selecting a first soybean plant comprising at least three polymorphisms.  
     
     
         60 . The method of  claim 47 , further comprising selecting a first soybean plant comprising a polymorphism within 50 cM of a non-transgenic mutant Gy3 allele.  
     
     
         61 . The method of  claim 60 , wherein selecting the first soybean plant comprises detecting at least a first polymorphism in the Gy3 allele selected from the group consisting of an insertion at position 848-851, a SNP at position 1083, a SNP at position 1120, and a SNP at position or 1866; wherein the position is the position in said allele corresponding to SEQ ID NO:164.  
     
     
         62 . The method of  claim 47 , further comprising selecting a first soybean plant comprising a polymorphism within 50 cM of a non-transgenic mutant Gy5 allele.  
     
     
         63 . The method of  claim 62 , wherein selecting the plant comprises detecting at least a first polymorphism in the Gy5 allele selected from the group consisting of a single nucleotide polymorphism (SNP) at position 363, a SNP at position 612, a deletion at positions 447-453 and a deletion at positions 519-524, wherein the position is the position in said allele corresponding to SEQ ID NO:166.  
     
     
         64 . The method of  claim 47 , comprising detecting a marker selected from the group consisting of NS0199002, NS0199003 and NS0199008.  
     
     
         65 . The method of  claim 47 , further comprising selecting a first soybean plant comprising a polymorphism within 50 cM of a non-transgenic mutant lx2 allele.  
     
     
         66 . The method of  claim 63 , wherein the polymorphism comprises a polymorphism in the lx2 allele selected from the group consisting of a single nucleotide polymorphism (SNP) at position 323, a SNP at position 439, a SNP at position 1390, a SNP at position 1431, a SNP at position 1458, a deletion at positions 2486-2487 and a SNP at position 2542; wherein the position is the position in said allele corresponding to SEQ ID NO:158.  
     
     
         67 . The method of  claim 47 , wherein assaying soybean plants for a polymorphism comprises PCR.  
     
     
         68 . The method of  claim 47 , wherein the polymorphism is detected by hybridization with a labeled nucleotide probe.  
     
     
         69 . The method of  claim 47 , wherein the polymorphism is detected by DNA sequencing.  
     
     
         70 . The method of  claim 47 , wherein step (a) further comprises assaying soybean plants for the presence of at least a first polymorphism in a soybean plant genomic region within 50 cM of a Kunitz Trypsin Inhibitor (KTI) null allele.  
     
     
         71 . The method of  claim 47 , wherein selecting at least a first soybean plant in step (b) further comprises selecting a first soybean plant comprising a polymorphism within 50 cM of Kunitz Trypsin Inhibitor (KTI) null allele to select said null allele.  
     
     
         72 . The method of  claim 70 , wherein the polymorphism within 50 cM of Kunitz Trypsin Inhibitor (KTI) null allele is selected from the group consisting of a 2-bp deletion at position 622-623 and a mutation at position 624, wherein the position is the position in said allele corresponding to SEQ ID NO:167.  
     
     
         73 . The method of  claim 70 , wherein assaying soybean plants for a polymorphism comprises PCR.  
     
     
         74 . The method of  claim 70 , wherein the polymorphism is detected by hybridization with a labeled nucleotide probe.  
     
     
         75 . The method of  claim 70 , wherein the polymorphism is detected by DNA sequencing.  
     
     
         76 . A method of plant breeding comprising the steps of: 
 (a) assaying soybean plants for the presence of at least a first polymorphism in a lox2 allele selected from the group consisting of SNP 1390, SNP 1431, SNP 1458, and INDEL 2486-2487, wherein the allele is associated with decreased lipoxygenase-2 content;    (b) selecting at least a first soybean plant comprising said polymorphism to select said allele; and    (c) crossing the first soybean plant to a second soybean plant to generate a progeny plant comprising said polymorphism and said allele.    
     
     
         77 . The method of  claim 76 , further comprising the step of: 
 (d) repeating steps (a)-(c) with the progeny plant of step (c) as starting material for step (a) at least about 2-10 times to produce additional progeny plants.    
     
     
         78 . The method of  claim 76 , further comprising selecting a first soybean plant that is homozygous for said polymorphism.  
     
     
         79 . The method of  claim 76 , further comprising selecting a first soybean plant comprising at least two of said polymorphisms.  
     
     
         80 . The method of  claim 76 , further comprising selecting a first soybean plant comprising at least three of said polymorphisms.  
     
     
         81 . A kit comprising a probe or primer that amplifies and/or hybridizes to at least a first polymorphism in linkage disequilibrium with a non-transgenic soybean mutant Gy1, Gy2, or Gy4 allele conferring decreased glycinin and increased α-conglycinin content.  
     
     
         82 . A kit comprising a probe or primer that amplifies and/or hybridizes to at least a first polymorphism in linkage disequilibrium with a lox2 null allele, wherein the polymorphism is selected from the group consisting of SNP 1390, SNP 1431, SNP 1458, and INDEL 2486-2487.