US20070067871A1 - Agronomically elite soybeans with high beta-conglycinin content - Google Patents

Agronomically elite soybeans with high beta-conglycinin content Download PDF

Info

Publication number
US20070067871A1
US20070067871A1 US11/517,186 US51718606A US2007067871A1 US 20070067871 A1 US20070067871 A1 US 20070067871A1 US 51718606 A US51718606 A US 51718606A US 2007067871 A1 US2007067871 A1 US 2007067871A1
Authority
US
United States
Prior art keywords
plant
allele
soybean
polymorphism
plants
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Abandoned
Application number
US11/517,186
Other languages
English (en)
Inventor
Kunsheng Wu
Thomas Horejsi
Joseph Byrum
Neal Bringe
Julie Yang
Donghong Pei
Robert Reiter
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Monsanto Technology LLC
Original Assignee
Monsanto Technology LLC
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Family has litigation
First worldwide family litigation filed litigation Critical https://patents.darts-ip.com/?family=37836370&utm_source=google_patent&utm_medium=platform_link&utm_campaign=public_patent_search&patent=US20070067871(A1) "Global patent litigation dataset” by Darts-ip is licensed under a Creative Commons Attribution 4.0 International License.
Application filed by Monsanto Technology LLC filed Critical Monsanto Technology LLC
Priority to US11/517,186 priority Critical patent/US20070067871A1/en
Assigned to MONSANTO TECHNOLOGY, L.L.C. reassignment MONSANTO TECHNOLOGY, L.L.C. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: WU, KUNSHENG, BYRUM, JOSEPH R, YANG, JULIE, HOREJSI, THOMAS, REITER, ROBERT, PEI, DONGHONG, BRINGE, NEAL
Publication of US20070067871A1 publication Critical patent/US20070067871A1/en
Priority to US14/106,707 priority patent/US9554525B2/en
Priority to US15/389,038 priority patent/US20170164573A1/en
Abandoned legal-status Critical Current

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
    • A01H1/02—Methods or apparatus for hybridisation; Artificial pollination ; Fertility
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H1/00—Processes for modifying genotypes ; Plants characterised by associated natural traits
    • A01H1/04—Processes of selection involving genotypic or phenotypic markers; Methods of using phenotypic markers for selection
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
    • A01H5/10—Seeds
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
    • A01H6/54—Leguminosae or Fabaceae, e.g. soybean, alfalfa or peanut
    • A01H6/542—Glycine max [soybean]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/8245—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving modified carbohydrate or sugar alcohol metabolism, e.g. starch biosynthesis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/8247—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving modified lipid metabolism, e.g. seed oil composition
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/8251—Amino acid content, e.g. synthetic storage proteins, altering amino acid biosynthesis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8262—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield involving plant development
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8274—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for herbicide resistance
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8274—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for herbicide resistance
    • C12N15/8275—Glyphosate
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8286—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for insect resistance
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/13—Plant traits
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/172—Haplotypes
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • the present invention relates generally to the field of plant breeding and molecular biology.
  • the invention relates to agronomically elite soybean varieties with increased beta-conglycinin content and materials for making such plants.
  • Glycinin and ⁇ -Conglycinin are two major storage proteins in soybeans, accounting for approximately 70% of total proteins or 40% of total seed weight.
  • the glycinin (11s globulin) is composed of five different subunits, designated A1aB2, A2B1a, A1bB1b, A5A4B3. A3B4, respectively.
  • Each subunit is composed of two polypeptides, one acidic and one basic, covalently linked through a disulfide bond.
  • the two polypeptide chains result from post-translational cleavage of proglycinin precursors, a step that occurs after the precursor enters the protein bodies (Chrispeels et al., 1982). Five major genes have been identified to encode these polypeptide subunits.
  • Gy1, Gy2, Gy3, Gy4 and Gy5, respectively are designated as Gy1, Gy2, Gy3, Gy4 and Gy5, respectively (Nielsen et al., 1997).
  • a pseudogene, gy6, and minor gene, Gy7 were also reported (Beilinson et al., 2002). Genetic mapping of these genes has been reported by various groups (Diers et al., 1993, Chen and Shoemaker 1998, Beilinson et al., 2002). Gy1 and Gy2 were located 3 kb apart and mapped to linkage group N (Nielsen et al., 1989), Gy3 was mapped to linkage group L (Beilinson et al., 2002). Gy4 and Gy5 were mapped to linkage groups O and F, respectively. All of these genes were mapped using RFLP probes on Southern Blots.
  • ⁇ -conglycinin is composed of a ( ⁇ 67 kda), ⁇ ′ ( ⁇ 71 kDa) and ⁇ ( ⁇ 50 kDa) subunits and each subunit is processed by co- and post-translational modifications (Ladin et al., 1987; Utsumi, 1992).
  • the ⁇ -conglycinin subunits are encoded by the genes Cgy1, Cgy2 and Cgy3, respectively. Genetic analysis indicated that Cgy2 is tightly linked to Cgy3, whereas Cgy1 segregates independently of the other two.
  • the ⁇ -conglycinin gene family contains at least 15 members divided into two major groups, which encode the 2.5 kb and 1.7 kb embryo mRNA, respectively (Harada et al., 1989).
  • Soybean plants with increased ⁇ -conglycinin levels and decreased glycinin levels would provide substantial benefit.
  • ⁇ -conglycinin is a soluble protein whereas glycinin is much less soluble. It has also been found that ⁇ -conglycinin, especially the ⁇ ′ subunit, has significantly higher nutritional value and a positive impact on human health as compared to glycinin (Baba et al., 2004).
  • soybean varieties with an increased ⁇ -conglycinin content will have higher value than traditional varieties and will be suitable for use in nutrition drinks and other food products.
  • mutations in the glycinin genes have a direct impact on ⁇ -conglycinin content in soybean seeds. Mutant soybean plants with decreased glycinin content have increased ⁇ -conglycinin content. However, since multiple glycinin alleles are involved in glycinin subunit production, breeding plants with reduced expression from multiple Gy subunits has proved difficult since such plants have other attributes, such as low yield, excessive lodging and green seed that render them commercially nonviable. Previous methods for determining the inheritance of mutations resulting in decreased glycinin content did not enable high-throughput techniques required to select for these phenotypes while introducing agronomically superior characteristics.
  • Lipoxygenases are enzymes that catalyze the dioxygenation of polyunsaturated fatty acids. Soybean seeds contain three lipoxygenase isozymes—lipoxygenases 1, 2, and 3. These isozymes contribute to the production of unpleasant flavors in soybean seeds. The unpleasant flavors are absent or less pronounced in seeds deficient in these isozymes, particularly those lacking lipoxygenase-2. Accordingly, soybean seeds lacking one or more lipoxygenase isozymes are desirable for use in making drink and food products. Genetic studies of Lipoxygenase 1, 2, and 3 deficient lines demonstrated that the absence of each was due to single recessive alleles—lx1, lx2, and lx3, respectively.
  • the loci defined by lx1 and lx2 are closely linked and are not genetically linked to lx3 (Kitamura, 1984; Kitamura et al, 1985; Hajika et al., 1992; Hildebrand et al., 1982).
  • the structural genes encoding Lipoxygenases 1, 2, and 3 have been cloned and designated Lox1, Lox2, and Lox3, respectively (Shibata et al., 1987; Shibata et al., 1988; Yenofsky et al., 1988).
  • Kunitz Trypsin inhibitor is an antinutritional and allergenic factor in soybeans that interferes with digestion and absorption of proteins when present in a diet.
  • soybean varieties with a KTI-null mutant trait have a higher commercial value than traditional varieties.
  • Genetic and biochemical studies of KTI production in soybean lines have been carried out (e.g. de Moraes et al., 2006; Natarajan et al., 2006), and three related genes have been identified, with KTI3 encoding the predominant Kunitz Trypsin Inhibitor Protein in cultivated soybean genotypes (Natarajan et al., 2006).
  • Some specific DNA markers associated with loss of KTI production in certain soybean lines have been reported (de Moraes et al., 2006).
  • the current invention provides, in one embodiment, a plant of an agronomically elite soybean variety with an increased seed ⁇ -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.
  • An increased seed ⁇ -conglycinin content may be measured, for example, with respect to a plant of the same genotype as said plant but lacking the mutations.
  • the non-transgenic mutations may confer a Gy3, and Gy4 null phenotype and increased seed ⁇ -conglycinin content.
  • the plants of the current invention comprise, in one aspect, seeds with low glycinin content and high ⁇ -conglycinin content.
  • the seed ⁇ -conglycinin content for plants of the invention is about or at least about 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 percent or more of the total protein content.
  • a plant of the invention has a seed glycinin content of about or less than about 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 percent of the total protein.
  • plants of the invention may comprise a mutant Gy4 allele.
  • a mutant Gy4 allele may comprise a point mutation at nucleotide 682, such that the translation initiation codon is abrogated.
  • plants provided by the invention may comprise Gy1 and Gy2 null alleles.
  • one or more of any of the Gy1, Gy2, Gy3 and/or Gy4 alleles may be the same as the null alleles in line B2G2, a representative sample of seed of which have been deposited under ATCC Accession No. PTA-6893.
  • soybean plants are provided that further comprise a mutation that confers reduced levels of Gy1/Gy2 protein.
  • reduced levels of Gy1/Gy2 protein means seed from plants comprising the non-transgenic mutation have reduced Gy1/Gy2 protein levels as compared to plants with an identical genetic background that lack the mutation.
  • plants comprising a non-transgenic mutation that confers reduced Gy1/Gy2 may have a Gy1/Gy2 protein content of less than about 3.1% of total seed protein.
  • the mutation conferring reduced Gy1/Gy2 protein content may be a non-transgenic mutation.
  • a plant of the invention comprises a mutant Gy1 allele.
  • the mutant Gy1 allele may comprise a deletion spanning the upstream promoter region, exon I and intron I.
  • a plant of the invention may further comprise mutations conferring reduced Gy5 protein levels.
  • the mutation conferring reduced Gy5 protein content is a non-transgenic mutation.
  • plants of the invention comprise mutations conferring reduced Gy1, Gy2, Gy3, Gy4 and Gy5 protein levels.
  • plants of the invention may comprise non-transgenic mutations conferring a decreased Gy1, Gy2 Gy3, Gy4, and Gy5 phenotype.
  • the seed of these plants may have a glycinin content of about or less than about 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or 0 percent of the total protein.
  • plants of the invention comprising a decreased Gy1, Gy2, Gy3, Gy4 and Gy5 phenotype may comprise seed with a ⁇ -conglycinin content of about or at least about 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 percent or greater of the total protein content.
  • a plant of the invention may further comprise mutations conferring a lx1, lx2, and/or lx3 phenotype.
  • the mutation conferring the lx1, lx2, and/or lx3 phenotype is a non-transgenic mutation.
  • plants of the invention comprise mutations conferring a reduced Gy3 and Gy4 phenotype and mutations conferring of one or more of a lx1, lx2, and/or lx3 phenotype.
  • such plants may further comprise mutations conferring a reduced Gy1, Gy2 Gy3, and Gy5 phenotype.
  • Plant parts are also provided by the invention.
  • Parts of a plant of the invention include, but are not limited to, pollen, ovules, meristems, cells, and seed.
  • Cells of the invention may further comprise, regenerable cells, such as embryos meristematic cells, pollen, leaves, roots, root tips, and flowers. Thus, these cells could be used to regenerate plants of the invention.
  • soy meal or flour comprising crushing or grinding seed according to the invention.
  • soy flour or meal according to the invention may comprise genomic material of plants of the invention.
  • the food may be defined as comprising the genome of such a plant.
  • soy meal or flour of the invention may be defined as comprising increased ⁇ -conglycinin and decreased glycinin content, as compared to meal or flour made form seeds of a plant with an identical genetic background, but not comprising the non-transgenic, mutant Gy3 and Gy4 null alleles.
  • this method may comprise preparing a hybrid soybean seed by crossing a plant of the invention with a second, distinct, soybean plant.
  • Still yet another aspect of the invention is a method of producing a food product for human or animal consumption comprising: (a) obtaining a plant of the invention; (b) cultivating the plant to maturity; and (c) preparing a food product from the plant.
  • the food product may be protein concentrate, protein isolate, meal, oil, flour or soybean hulls.
  • the food product may comprise beverages such as soymilk and other nutritional beverages, infused foods, sauces, condiments, salad dressings, fruit juices, syrups, desserts, icings and fillings, soft frozen products, confections or intermediate foods.
  • Foods produced from the plants of the invention may comprise increased ⁇ -conglycinin content and thus be of greater nutritional value foods made with typical soybean varieties. Additionally, plants of the invention comprising decreased glycinin content may be used in food compositions requiring low amounts of insoluble protein.
  • a plant of the invention may further comprise a transgene.
  • a plant may comprise transgenes conferring herbicide tolerance, disease resistance, insect and pest resistance, altered fatty acid, protein or carbohydrate metabolism, increased grain yield, altered plant maturity and/or altered morphological characteristics.
  • a herbicide tolerance transgene may comprise a glyphosate resistance gene.
  • a plant of the invention may be defined as prepared by a method wherein a plant comprising non-transgenic mutations conferring a Gy3, and Gy4 null phenotype and increased ⁇ -conglycinin content is crossed with a plant comprising agronomically elite characteristics.
  • the progeny of this cross may be assayed for agronomically elite characteristics and Gy3 and Gy4 protein content, and progeny plants selected based on these characteristics, thereby generating the plant of the invention.
  • a plant of the invention may be produced by crossing a selected starting variety with a second soybean plant comprising agronomically elite characteristics.
  • a plant of the invention may be defined as prepared by a method wherein a plant comprising a non-transgenic mutation conferring a lx1, lx2, and/or lx3 phenotype is crossed with a plant comprising reduced Gy3 and Gy4 protein content, and increased ⁇ -conglycinin content.
  • the current invention also provides a method of plant breeding wherein a plant is assayed for the presence of a polymorphism in a soybean plant genomic region associated with Gy1/Gy2 and Gy4 alleles, comprising selecting the plant and crossing the plant with a second soybean plant to produce progeny.
  • the method of the invention may comprise selecting a progeny plant by assaying the plant for a polymorphism associated with a decreased Gy1/Gy2 or Gy4 phenotype and crossing the plant with a second soybean plant to produce further progeny plants.
  • the second soybean plant may comprise agronomically elite characteristics.
  • the method of the invention may also further comprise selecting a soybean plant comprising the polymorphism and agronomically elite characteristics.
  • the invention enables the introduction of non-transgenic mutations conferring a Gy1/Gy2 and/or Gy4 phenotype and increased seed ⁇ -conglycinin content into agronomically elite soybean plants.
  • a method of the invention may be repeated 1, 2, 3, 4, 5, 10, 15, 20, or more times as desired to select agronomically elite progeny with polymorphisms indicative of non-transgenic mutations at Gy1/Gy2 and/or Gy4 alleles at each step.
  • the first soybean plant may be a plant of line B2G2, a representative sample of seed of which have been deposited under ATCC Accession No. PTA-6893.
  • a method of the invention may further comprise selecting a plant comprising polymorphisms indicative of a non-transgenic mutation in Gy1/Gy2 and Gy4 alleles.
  • a method of the invention may further comprise selecting plants with markers indicative of reduced Gy3 and/or Gy5 content.
  • methods of marker assisted plant breeding according to the invention may be used to breed soybeans that have reduced Gy1, Gy2, Gy3, Gy4, and Gy5 content.
  • non-transgenic mutations conferring a decreased Gy1, Gy2 or Gy4 phenotype may comprise mutations in Gy1, Gy2, Gy3, or Gy4 alleles.
  • the mutant Gy alleles are detected using genetic markers comprising polymorphisms within 50 cM of a Gy allele.
  • plants with a decreased Gy1/Gy2 phenotype comprise a mutant Gy1 allele.
  • the mutant Gy1 allele comprises a deletion, such as a deletion of the promoter region, exon I and intron I.
  • mutant Gy1 alleles can be detected using markers NS0199002 or NS0199008.
  • mutant Gy1 alleles may be detected with markers of Gy2, since the two genes are closely linked.
  • mutant Gy2 alleles may be detected with markers to Gy1. It is shown herein that plants with mutant Gy1,2 alleles also may have a decreased Gy3 phenotype.
  • plants comprising a reduced Gy3 phenotype may be selected with markers for Gy1 or Gy2, since these markers have been shown to associate with a reduced Gy3 phenotype in B2G2 derived plants. Therefore, markers of Gy1 or Gy2, for example NS0199002 or NS0199008, may be used to determine the presence of mutant Gy1, Gy2, and Gy3 alleles.
  • phenotypically Gy4 null plants comprise mutant Gy4 alleles.
  • mutant Gy4 alleles comprise point mutations such as an SNP that abrogates the translation initiation codon.
  • the Gy4 null allele may be detected with the NS0199003 marker. SNP markers may be detected, for example using fluorescently labeled oligonucleotides.
  • a method of the invention may further comprise selecting plants with markers indicative of reduced lipoxygenase 1, 2, and/or 3 content.
  • methods of marker assisted plant breeding according to the invention may be used to produce soybeans that have reduced lipoxygenase 1, 2, and/or 3 content.
  • non-transgenic mutations conferring a lx1, lx2, and/or lx3 phenotype may comprise mutations in Lox1, Lox2, and/or Lox3 alleles.
  • the mutant alleles conferring a lx1, lx2, and/or lx3 phenotype are detected using genetic markers comprising polymorphisms within 50 cM of a Lox allele.
  • lx1 alleles are detected using one or more of INDEL 178-180, SNP 326, SNP 363, SNP 380, SNP 713, SNP 1196, SNP 1253, SNP 1372, SNP 1388, SNPR 1527, SNP 1554, SNP 2267, SNP 3088, SNP 3125, SNP 3139, INDEL 3832-3905, SNP 4043, SNP 4057, SNP 4193, SNP 4225, SNP 4247, SNP 4267, or SNP 4439 as shown below in Table 14.
  • lx2 alleles can be detected using one or more of SNP 323, SNP 439, SNP 1390, SNP 1431, SNP 1458, INDEL 2486-87, or SNP 2542 as shown below in Table 15.
  • SNP 2542 is also referred to as the NS0203296 marker.
  • a method of the invention may further comprise selecting plants with markers indicative of a KTI-null or KTI-reduced trait.
  • methods of marker assisted plant breeding according to the invention may be used to produce soybeans that have reduced or undetectable Kunitz Trypsin Inhibitor content.
  • mutations conferring a KTI-null phenotype may comprise mutations in a gene encoding KTI.
  • mutations conferring a KTI-null phenotype comprise mutations in the KTI3 gene, also termed “KTIA”.
  • the mutant alleles conferring a KTI-null phenotype are detected using genetic markers comprising polymorphisms within 50 cM of a KTI allele.
  • KTI alleles are detected using one or more INDELs or SNPs located within the KTI3 gene, for instance as shown below in Table 18. Such selection may thus be based on marker information (plant genotype) rather than on enzymatic analysis of Trypsin activity or analysis of KTI content.
  • Embodiments discussed in the context of a method and/or composition of the invention may be employed with respect to any other method or composition described herein. Thus, an embodiment pertaining to one method or composition may be applied to other methods and compositions of the invention as well.
  • a” or “an” may mean one or more.
  • the words “a” or “an” when used in conjunction with the word “comprising”, the words “a” or “an” may mean one or more than one.
  • another may mean at least a second or more.
  • FIG. 1 Protein extracted from the seed of indicated soybean varieties was resolved by SDS-PAGE and visualized by Coomassie staining. The mobility of acidic glycinin subunits encoded by each gene is indicated. Gel resolution was insufficient to separate Gy1 from Gy2 encoded protein subunits.
  • FIG. 2A -B Progeny plants comprising mutant Gy1 and Gy2 alleles are distributed in two phenotypic groups based Gy1 and Gy2 encoded protein content of seed.
  • FIG. 2A Graph shows the number of F2 plants (y axis) compared to the percentage of total protein that is constituted by Gy1 and Gy2 encoded acidic proteins (x axis)
  • FIG. 2B Data from F2 progeny plants indicates that mutant Gy1 and Gy2 alleles, from B2G2 soybeans, are recessive. The number of progeny plants with Gy1 and Gy2 encoded protein levels in the two classes were subjected to chi square analysis and probability values were determined in each case.
  • FIG. 3A -B Progeny of “11S null” plants are distributed into two populations based on the percentage of total protein in the seed that is Gy3.
  • FIG. 3A Graph shows the number of F2 plants (y axis) compared to the percentage of total protein that is constituted by Gy3 encoded proteins (x axis).
  • FIG. 3B Data from progeny plants indicates that the mutant Gy3 allele from B2G2 soybeans is recessive. The number of progeny plants with Gy3 encoded protein levels in the two classes were subjected to chi square analysis and probability values were determined in each case.
  • FIG. 4 The amount of Gy3 encoded protein is positively correlated with the amount of Gy1 and Gy2 encoded protein.
  • FIG. 4A Graph plots the observed amount of Gy1 and Gy2 encoded acidic protein (x axis) versus the amount of Gy3 encoded protein (y axis).
  • FIG. 4B Table shows correlation coefficients between the expression levels of the Gy1, Gy2, Gy3, Gy4 and Gy5 encoded proteins.
  • FIG. 5 Progeny of 11S null plants are distributed into two populations based on the percentage of total protein in the seed that is Gy4.
  • FIG. 5A Graph shows the number of F2 plants (y axis) compared to the percentage of total protein that is constituted by Gy4 encoded acidic protein (x axis).
  • FIG. 5B Data from progeny plants indicates that the mutant Gy4 allele from B2G2 soybeans is recessive. The number of progeny plants with Gy4 encoded protein levels in the two classes were subjected to chi square analysis and probability values were determined in each case.
  • FIG. 6A -B Decreased expression of glycinin in soybean seed correlates with increased expression of ⁇ -conglycinin.
  • FIG. 6A Graph plots the percentage of total protein encoded by Gy alleles (x axis) versus the percentage of total protein encoded by Cgy alleles (y axis).
  • FIG. 6B Table shows the correlation coefficients between expression of Gy encoded proteins and the expression of Cgy1-4 encoded proteins.
  • FIG. 7A -B Genomic Gy markers effectively selected conventional plants that produce seed with high ⁇ -conglycinin expression.
  • FIG. 7A Graph plots total ⁇ -conglycinin protein content of seed (y axis) versus total seed glycinin content (x axis). Diamonds indicate plants selected by protein analysis of glycinin subunits.
  • FIG. 7B Graph is the same as in FIG. 7A , however diamonds indicate plants selected by markers NS0199002, NS0199003, and NS0199008.
  • FIG. 8 Allelogram for the Taqman assay designed for the SNP at position 2542 (Marker NS0203296). As shown in FIG. 8 , this marker allowed a clear distinction between the “A” allele from the lx2 mutant and the “T” allele from wild type as described in Example 13 and Table 15.
  • FIG. 9 Alignment of sequences for Kunitz Trypsin inhibitor showing deletion/insertion in Kunitz null mutant lines.
  • the present invention provides plants and methods for producing plants comprising non-transgenic mutations that confer a Gy3, and Gy4 null phenotype and agronomically elite characteristics. These mutations confer low glycinin and high ⁇ -conglycinin content to seed of mutant plants. Thus, plants of the invention will be of great value as ⁇ -conglycinin provides improved nutritional characteristics and solubility as compared to glycinin. Additionally, plants provided herein comprise agronomically elite characteristics, enabling a commercially significant yield of high ⁇ -conglycinin, low glycinin, soybeans.
  • plants with increased ⁇ -conglycinin contents comprise non-transgenic null alleles for Gy3, and/or Gy4 and therefore have the additional advantage of reduced governmental regulation as compared to soybean varieties containing corresponding transgenic alleles at these loci. Also provided are plants that further comprise non-transgenic Gy1 and Gy2 null alleles and also provide such benefits.
  • the invention also provides plants and methods for producing plants comprising non-transgenic mutations that confer a lipoxygenase-2 null phenotype.
  • the combination of a lipoxygenase-2 null and glycinin null phenotype provides an increased content of the highly functional and healthful ⁇ -conglycinin protein.
  • ⁇ -conglycinin in particular contains bioactive peptides that are responsible for cholesterol-lowering and weight management (through satiety effects and reduction in fat deposits) benefits.
  • Another valuable combination in accordance with the invention is a lipoxygenase-2 null and mid-oleic content (e.g., 40-65% oleic).
  • This soybean will produce low levels of off-flavors because it lacks the main catalyst of lipid oxidation (lipoxygenase-2) and has much lower levels of the substrate (linoleic acid).
  • lipoxygenase-2 the main catalyst of lipid oxidation
  • such soybeans will have an improved (lower) ratio of omega-6 to omega-3 fatty acids, a benefit for cardiovascular health.
  • Also provided herein is a method for breeding soybean plants that comprise non-transgenic mutations conferring a decreased Gy1, Gy2, Gy3 and Gy4 phenotype and agronomically elite characteristics.
  • Studies detailed below identify polymorphisms that can be used to identify plants with decreased Gy1, Gy2, Gy3 and Gy4 protein content.
  • Three of the markers identified herein, NS0199002, NS0199003, and NS0199008, can be used to accurately predict a decreased Gy1, Gy2, Gy3, and Gy4 phenotype of soybean plants.
  • the inheritance of Gy1 and Gy2 is genetically linked, thus markers to either Gy1 (NS0199008) or Gy2 (NS0199002) may be used to track the inheritance of the decreased Gy1 and Gy2 phenotype.
  • markers to Gy1,2 may additionally be used to select for the decreased Gy3 phenotype of progeny plants. It is also shown that markers can be used to identify plants that are phenotypically null for Gy4 encoded proteins. For example, the NS0199003 marker was used in studies to accurately determine the Gy4 phenotype of soybean plants.
  • the invention enables the high throughput screening and marker assisted breeding of plants with non-transgenic mutations conferring a decreased Gy1, Gy2, Gy3 and Gy4 phenotype, and high seed ⁇ -conglycinin content. Sequencing studies were also undertaken herein that have identified markers that may be used to determine the inheritance of the decreased Gy5 phenotype and to directly select for a decreased Gy3 phenotype.
  • studies described below identified sequence variations associated with the lipoxygenase-2 null (lx2) phenotype in soybeans.
  • Molecular markers have been developed out of these sequence variations for the lx2 phenotypes.
  • breeders can make selections based on marker information, or genotypes, rather than on lipoxygenase analysis by SDS-PAGE. Marker data is more cost effective, faster and reliable, enabling one to test greater numbers and identify elite lines with multiple traits (e.g. Lipoxygenase-2 null and glycinin-null).
  • the invention provides, for the first time, plants and derivatives thereof of soybean varieties that combine non-transgenic mutations conferring a Gy3, and Gy4 null phenotype and increased ⁇ -conglycinin content with an agronomically elite phenotype.
  • such plants may further comprise non-transgenic Gy1 and Gy2 null alleles.
  • Such plants may be defined as having a commercially significant yield, for example, that is defined as a yield of at least 103% of the check lines AG2703 and DKB23-51.
  • plants are provided comprising the non-transgenic Gy1-4 mutant alleles and increased beta-conglycinin content and a grain yield of at least about 90%, 94%, 98%, 100%, 105% or about 110% of these lines.
  • Such plants may be defined, in certain embodiments of the invention, as having a yield a yield in excess of about 35, 37, 39, 41, 43 or 45 bushels per acre over at least 10 environments.
  • the ⁇ -conglycinin content of the seeds of plants of the invention may be greater than about 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, or even 50% or any range derivable therein.
  • the plants of the invention may further comprise mutations conferring a lx1, lx2, and/or lx3 phenotype.
  • Plant parts include, but are not limited to, pollen, an ovule and a cell.
  • the invention further provides tissue cultures of regenerable cells of these plants, which cultures regenerate soybean plants capable of expressing all the physiological and morphological characteristics of the starting variety.
  • regenerable cells may include embryos, meristematic cells, pollen, leaves, roots, root tips or flowers, or protoplasts or callus derived therefrom.
  • soybean plants regenerated from such a tissue culture wherein the plants are capable of expressing all the physiological and morphological characteristics of the starting plant variety from which the regenerable cells were obtained.
  • the current invention provides genetic markers and methods for the introduction of non-transgenic, mutant Gy alleles into agronomically elite soybean plants.
  • the invention therefore allows, for the first time, the creation of plants that combine these mutant Gy alleles that confer high seed ⁇ -conglycinin content with a commercially significant yield and an agronomically elite genetic background.
  • loci conferring the “11S null” phenotype may be introduced into a desired soybean genetic background, for example, in the production of new varieties with commercially significant yield and high seed ⁇ -conglycinin content.
  • Marker assisted introgression involves the transfer of a chromosome region defined by one or more markers from one germplasm to a second germplasm.
  • the initial step in that process is the localization of the trait by gene mapping, which is the process of determining the position of a gene relative to other genes and genetic markers through linkage analysis.
  • the basic principle for linkage mapping is that the closer together two genes are on the chromosome, the more likely they are to be inherited together. Briefly, a cross is generally made between two genetically compatible but divergent parents relative to traits under study. Genetic markers can then be used to follow the segregation of traits under study in the progeny from the cross, often a backcross (BC1), F 2 , or recombinant inbred population.
  • BC1 backcross
  • F 2 F 2
  • recombinant inbred population recombinant inbred population.
  • the term quantitative trait loci is used to describe regions of a genome showing quantitative or additive effects upon a phenotype.
  • the Gy loci represent exemplary QTL since multiple mutant Gy alleles result in increasing reduction in total seed glycinin content and important concomitant increases in ⁇ -conglycinin content.
  • genetic markers for non-transgenic, mutant Gy alleles that enable breeding of soybean plants comprising the non-transgenic, mutant Gy alleles with agronomically superior plants, and selection of progeny that inherited the mutant Gy alleles.
  • the invention allows the use of molecular tools to combine these QTLs with desired agronomic characteristics.
  • Soybean plants having reduced lipoxygenase are useful in themselves and are useful in combination with other agronomic characteristics. For example, reduced lipoxygenase and reduced glycinin is a valuable trait combination. Another valuable combination is reduced lipoxygenase and mid-oleic content (e.g., 40-65% oleic).
  • the invention allows the use of molecular tools to combine lx alleles with, for example, mutant Gy alleles in agronomically superior plants as described above.
  • a sample first plant population may be genotyped for an inherited genetic marker to form a genotypic database.
  • an “inherited genetic marker” is an allele at a single locus.
  • a locus is a position on a chromosome, and allele refers to conditions of genes; that is, different nucleotide sequences, at those loci.
  • the marker allelic composition of each locus can be either homozygous or heterozygous.
  • the marker In order for information to be gained from a genetic marker in a cross, the marker must be polymorphic; that is, it must exist in different forms so that the chromosome carrying the mutant gene can be distinguished from the chromosome with the normal gene by the form of the marker it also carries.
  • Formation of a phenotypic database can be accomplished by making direct observations of one or more traits on progeny derived from artificial or natural self-pollination of a sample plant or by quantitatively assessing the combining ability of a sample plant.
  • a plant line may be crossed to, or by, one or more testers.
  • Testers can be inbred lines, single, double, or multiple cross hybrids, or any other assemblage of plants produced or maintained by controlled or free mating, or any combination thereof. For some self-pollinating plants, direct evaluation without progeny testing is preferred.
  • the marker genotypes may be determined in the testcross generation and the marker loci mapped. To map a particular trait by the linkage approach, it is necessary to establish a positive correlation in inheritance of a specific chromosomal locus with the inheritance of the trait. In the case of complex inheritance, such as with quantitative traits, including specifically glycinin content and yield, linkage will generally be much more difficult to discern. In this case, statistical procedures may be needed to establish the correlation between phenotype and genotype. This may further necessitate examination of many offspring from a particular cross, as individual loci may have small contributions to an overall phenotype.
  • Coinheritance, or genetic linkage, of a particular trait and a marker suggests that they are physically close together on the chromosome. Linkage is determined by analyzing the pattern of inheritance of a gene and a marker in a cross. The unit of genetic map distance is the centimorgan (cM), which increases with increasing recombination. Two markers are one centimorgan apart if they recombine in meiosis about once in every 100 opportunities that they have to do so. The centimorgan is a genetic measure, not a physical one. Those markers located less then 50 cM from a second locus are said to be genetically linked, because they are not inherited independently of one another. Thus, the percent of recombination observed between the loci per generation will be less than 50%.
  • a marker used may be defined as located less than about 45, 35, 25, 15, 10, 5, 4, 3, 2, or 1 or less cM apart from a locus.
  • markers may be used for detecting polymorphisms within the contributing loci themselves and thus located at 0 cM respective to the loci, for example, comprising a mutation within a Gy1, Gy2, Gy3, Gy3, Gy4, or Gy5 coding sequence or regulatory element.
  • This ratio expresses the odds for (and against) that degree of linkage, and because the logarithm of the ratio is used, it is known as the logarithm of the odds, e.g. an lod score.
  • a lod score equal to or greater than 3, for example, is taken to confirm that gene and marker are linked. This represents 1000:1 odds that the two loci are linked. Calculations of linkage is greatly facilitated by use of statistical analysis employing programs.
  • the genetic linkage of marker molecules can be established by a gene mapping model such as, without limitation, the flanking marker model reported by Lander and Botstein (1989), and the interval mapping, based on maximum likelihood methods described by Lander and Botstein (1989), and implemented in the software package MAPMAKER/QTL. Additional software includes Qgene, Version 2.23 (1996) (Department of Plant Breeding and Biometry, 266 Emerson Hall, Cornell University, Ithaca, N.Y.).
  • Genetic markers comprise detected differences (polymorphisms) in the genetic information carried by two or more plants. Genetic mapping of a locus with genetic markers typically requires two fundamental components: detectably polymorphic alleles and recombination or segregation of those alleles. In plants, the recombination measured is virtually always meiotic, and therefore, the two inherent requirements of plant gene mapping are polymorphic genetic markers and one or more plants in which those alleles are segregating.
  • Markers are preferably inherited in codominant fashion so that the presence of both alleles at a diploid locus is readily detectable, and they are free of environmental variation, i.e., their heritability is 1.
  • a marker genotype typically comprises two marker alleles at each locus in a diploid organism such as soybeans.
  • the marker allelic composition of each locus can be either homozygous or heterozygous.
  • Homozygosity is a condition where both alleles at a locus are characterized by the same nucleotide sequence. Heterozygosity refers to different conditions of the gene at a locus.
  • exemplary genetic marker types are available for use in genetic mapping.
  • Exemplary genetic marker types for use with the invention include, but are not limited to, restriction fragment length polymorphisms (RFLPs), simple sequence length polymorphisms (SSLPs), amplified fragment length polymorphisms (AFLPs), single nucleotide polymorphisms (SNPs), nucleotide insertions and/or deletions (INDELs) and isozymes.
  • RFLPs restriction fragment length polymorphisms
  • SSLPs simple sequence length polymorphisms
  • AFLPs amplified fragment length polymorphisms
  • SNPs single nucleotide polymorphisms
  • INDELs nucleotide insertions and/or deletions
  • isozymes Polymorphisms comprising as little as a single nucleotide change can be assayed in a number of ways.
  • detection can be made by electrophoretic techniques including a single strand conformational polymorphism (Orita et al., 1989), denaturing gradient gel electrophoresis (Myers et al., 1985), or cleavage fragment length polymorphisms (Life Technologies, Inc., Gathersberg, Md. 20877), but the widespread availability of DNA sequencing machines often makes it easier to just sequence amplified products directly.
  • rapid assays can be designed for progeny testing, typically involving some version of PCR amplification of specific alleles (PASA, Sommer, et al., 1992), or PCR amplification of multiple specific alleles (PAMSA, Dutton and Sommer, 1991).
  • One method for detection of SNPs in DNA samples is by use of PCR in combination with fluorescent probes for the polymorphism, as described in Livak et al., 1995 and U.S. Pat. No. 5,604,099, incorporated herein by reference. Briefly, two probe oligonucleotides, one of which anneals to the SNP site and the other which anneals to the wild type sequence, are synthesized. It is preferable that the site of the SNP be near the 5′ terminus of the probe oligonucleotides.
  • Each probe is then labeled on the 3′ end with a non-fluorescent quencher and a minor groove binding moiety which lower background fluorescence and lower the T m of the oligonucleotide, respectively.
  • the 5′ ends of each probe are labeled with a different fluorescent dye wherein fluorescence is dependent upon the dye being cleaved from the probe.
  • Some non-limiting examples of such dyes include VICTM and 6-FAMTM.
  • test DNA that contains only wild type sequence will exhibit fluorescence associated with the label on the wild type probe.
  • DNA containing only the SNP sequence will have fluorescent activity from the label on the SNP probe.
  • This type of indirect genotyping at known SNP sites enables high throughput, inexpensive screening of DNA samples. Thus such a system is ideal for the identification of progeny soybean plants comprising mutant Gy alleles.
  • Restriction fragment length polymorphisms are genetic differences detectable by DNA fragment lengths, typically revealed by agarose gel electrophoresis, after restriction endonuclease digestion of DNA. There are large numbers of restriction endonucleases available, characterized by their nucleotide cleavage sites and their source, e.g., EcoRI. RFLPs result from both single-bp polymorphisms within restriction site sequences and measurable insertions or deletions within a given restriction fragment. RFLPs are easy and relatively inexpensive to generate (require a cloned DNA, but no sequence) and are co-dominant.
  • RFLPs have the disadvantage of being labor-intensive in the typing stage, although this can be alleviated to some extent by multiplexing many of the tasks and reutilization of blots. Most RFLP are biallelic and of lesser polymorphic content than microsatellites. For these reasons, the use of RFLP in plant genetic maps has waned.
  • markers are useful as tools to monitor genetic inheritance and are not limited to RFLPs, SSRs and SNPs, and one of skill would also understand that a variety of detection methods may be employed to track the various molecular markers.
  • markers of different types may be used for mapping, especially as technology evolves and new types of markers and means for identification are identified.
  • the absolute values of the scores are not important. What is important is the additive nature of the numeric designations.
  • the above scores relate to codominant markers. A similar scoring system can be given that is consistent with dominant markers.
  • the invention provides soybean plants with increased ⁇ -conglycinin content in combination with a commercially significant yield and agronomically elite characteristics. Such plants may be produced in accordance with the invention by marker assisted selection methods comprising assaying genomic DNA for the presence of markers that are genetically linked to the non-transgenic, mutant Gy1, Gy2, Gy3, Gy4, or Gy5 alleles, including all possible combinations thereof.
  • the invention also provides soybean plants with reduced lipoxygenase content.
  • Such plants may be produced in accordance with the invention by marker assisted selection methods comprising assaying genomic DNA for the presence of markers that are genetically linked to the non-transgenic, mutant Lox1, Lox2, or Lox3 alleles, including all possible combinations thereof.
  • RIL Recombinant inbred lines
  • Information obtained from dominant markers can be maximized by using RIL because all loci are homozygous or nearly so. It may also be desired to obtain additional markers linked to Lox alleles.
  • This may be carried out, for example, by first preparing an F 2 population by selfing an F 1 hybrid produced by crossing inbred varieties only one of which comprises a mutant Lox allele conferring decreased lipoxygenase content. As above, recombinant inbred lines can then be prepared and used as a mapping population, and information obtained from dominant markers can be maximized by using RIL because all loci are homozygous or nearly so.
  • the invention also provides soybean plants with reduced content of KTI, such as a KTI-null trait, which may be obtained by marker assisted selection and provided in combination with commercially significant yield and agronomically elite characteristics. Such plants may be defined, in certain embodiments of the invention, as having a yield in excess of about 35, 37, 39, 41, 43 or 45 bushels per acre over at least 10 environments.
  • the markers used for such marker assisted selection may include SNPs or INDELs.
  • the one or more INDEL(s) may be found in a gene encoding Kunitz Trypsin inhibitor.
  • the marker may comprise a deletion at position 622-623 and/or an insertion at position 664 in a gene encoding Kunitz Trypsin Inhibitor (SEQ ID NO:167) as shown in FIG. 9 , and the plants may be produced in accordance with the invention by marker assisted selection methods comprising assaying genomic DNA for the presence of such markers.
  • SEQ ID NO:167 Kunitz Trypsin Inhibitor
  • Backcross populations (e.g., generated from a cross between a desirable variety (recurrent parent) and another variety (donor parent)) carrying a trait not present in the former can also be utilized as a mapping population.
  • a series of backcrosses to the recurrent parent can be made to recover most of its desirable traits.
  • a population is created consisting of individuals similar to the recurrent parent but each individual carries varying amounts of genomic regions from the donor parent.
  • Backcross populations can be useful for mapping dominant markers if all loci in the recurrent parent are homozygous and the donor and recurrent parent have contrasting polymorphic marker alleles (Reiter et al., 1992).
  • NIL near-isogenic lines
  • NILs are created by many backcrosses to produce an array of individuals that are nearly identical in genetic composition except for the desired trait or genomic region can be used as a mapping population.
  • mapping with NILs only a portion of the polymorphic loci are expected to map to a selected region. Mapping may also be carried out on transformed plant lines.
  • Certain aspects of the invention provide methods for marker assisted breeding of plants that enable the introduction of non-transgenic, mutant Gy alleles into a heterologous soybean genetic background. Certain aspects of the invention also provide methods for marker assisted breeding of plants that enable the introduction of non-transgenic, mutant Lox alleles into a heterologous soybean genetic background.
  • breeding techniques take advantage of a plant's method of pollination. There are two general methods of pollination: self-pollination which occurs if pollen from one flower is transferred to the same or another flower of the same plant, and cross-pollination which occurs if pollen comes to it from a flower on a different plant. Plants that have been self-pollinated and selected for type over many generations become homozygous at almost all gene loci and produce a uniform population of true breeding progeny, homozygous plants.
  • pedigree breeding may be used.
  • the pedigree breeding method for specific traits involves crossing two genotypes. Each genotype can have one or more desirable characteristics lacking in the other; or, each genotype can complement the other. If the two original parental genotypes do not provide all of the desired characteristics, other genotypes can be included in the breeding population. Superior plants that are the products of these crosses are selfed and are again advanced in each successive generation. Each succeeding generation becomes more homogeneous as a result of self-pollination and selection.
  • this method of breeding involves five or more generations of selfing and selection: S1 ⁇ S2; S2 ⁇ S3; S3 ⁇ S4; S4 ⁇ S5, etc.
  • a selfed generation (S) may be considered to be a type of filial generation (F) and may be named F as such. After at least five generations, the inbred plant is considered genetically pure.
  • Each breeding program should include a periodic, objective evaluation of the efficiency of the breeding procedure. Evaluation criteria vary depending on the goal and objectives. Promising advanced breeding lines are thoroughly tested and compared to appropriate standards in environments representative of the commercial target area(s) for generally three or more years. Identification of individuals that are genetically superior is difficult because genotypic value can be masked by confounding plant traits or environmental factors. One method of identifying a superior plant is to observe its performance relative to other experimental plants and to one or more widely grown standard varieties. Single observations can be inconclusive, while replicated observations provide a better estimate of genetic worth.
  • Mass and recurrent selections can be used to improve populations of either self-or cross-pollinating crops.
  • a genetically variable population of heterozygous individuals is either identified or created by intercrossing several different parents. The best plants are selected based on individual superiority, outstanding progeny, or excellent combining ability. The selected plants are intercrossed to produce a new population in which further cycles of selection are continued.
  • Descriptions of other breeding methods that are commonly used for different traits and crops can be found in one of several reference books (e.g., Allard, 1960; Simmonds, 1979; Sneep et al., 1979; Fehr, 1987 a, b).
  • the effectiveness of selecting for genotypes with traits of interest in a breeding program will depend upon: 1) the extent to which the variability in the traits of interest of individual plants in a population is the result of genetic factors and is thus transmitted to the progenies of the selected genotypes; and 2) how much the variability in the traits of interest among the plants is due to the environment in which the different genotypes are growing.
  • the inheritance of traits ranges from control by one major gene whose expression is not influenced by the environment (i.e., qualitative characters) to control by many genes whose effects are greatly influenced by the environment (i.e., quantitative characters).
  • Breeding for quantitative traits such as yield is further characterized by the fact that: 1) the differences resulting from the effect of each gene are small, making it difficult or impossible to identify them individually; 2) the number of genes contributing to a character is large, so that distinct segregation ratios are seldom if ever obtained; and 3) the effects of the genes may be expressed in different ways based on environmental variation. Therefore, the accurate identification of transgressive segregates or superior genotypes with the traits of interest is extremely difficult and its success is dependent on the plant breeder's ability to minimize the environmental variation affecting the expression of the quantitative character in the population.
  • the likelihood of identifying a transgressive segregant is greatly reduced as the number of traits combined into one genotype is increased. For example, if a cross is made between cultivars differing in three complex characters, such as yield, ⁇ -conglycinin content and at least a first agronomic trait, it is extremely difficult without molecular tools to recover simultaneously by recombination the maximum number of favorable genes for each of the three characters into one genotype. Consequently, all the breeder can generally hope for is to obtain a favorable assortment of genes for the first complex character combined with a favorable assortment of genes for the second character into one genotype in addition to a selected gene.
  • Backcrossing is an efficient method for transferring specific desirable traits. This can be accomplished, for example, by first crossing a superior variety inbred (A) (recurrent parent) to a donor inbred (non-recurrent parent), which carries the appropriate gene(s) for the trait in question (Fehr, 1987). The progeny of this cross are then mated back to the superior recurrent parent (A) followed by selection in the resultant progeny for the desired trait to be transferred from the non-recurrent parent. Such selection can be based on genetic assays, as mentioned below, or alternatively, can be based on the phenotype of the progeny plant.
  • the progeny are heterozygous for loci controlling the characteristic being transferred, but are like the superior parent for most or almost all other genes.
  • the last generation of the backcross is selfed, or sibbed, to give pure breeding progeny for the gene(s) being transferred, for example, loci providing the plant with decreased seed glycinin content.
  • the process of backcross conversion may be defined as a process including the steps of:
  • Introgression of a particular DNA element or set of elements into a plant genotype is defined as the result of the process of backcross conversion.
  • a plant genotype into which a DNA sequence has been introgressed may be referred to as a backcross converted genotype, line, inbred, or hybrid.
  • a plant genotype lacking the desired DNA sequence may be referred to as an unconverted genotype, line, inbred, or hybrid.
  • the genetic markers linked to decreased glycinin content may be used to assist in breeding for the purpose of producing soybean plants with decreased glycinin content and preferably increased ⁇ -conglycinin content.
  • Backcrossing and marker assisted selection in particular can be used with the present invention to introduce the decreased glycinin content trait in accordance with the current invention into any variety by conversion of that variety with non-transgenic, mutant Gy1, Gy2, Gy3, Gy4, and/or Gy5 alleles associated.
  • a suitable recurrent parent is an important step for a successful backcrossing procedure.
  • the goal of a backcross protocol is to alter or substitute a trait or characteristic in the original inbred.
  • one or more loci of the recurrent inbred is modified or substituted with the desired gene from the nonrecurrent parent, while retaining essentially all of the rest of the desired genetic, and therefore the desired physiological and morphological, constitution of the original inbred.
  • the choice of the particular nonrecurrent parent will depend on the purpose of the backcross, which in the case of the present invention may be to add one or more allele(s) conferring decreased glycinin content.
  • the exact backcrossing protocol will depend on the characteristic or trait being altered to determine an appropriate testing protocol.
  • a recessive allele may also be transferred. In this instance it may be necessary to introduce a test of the progeny to determine if the desired characteristic has been successfully transferred.
  • one may test the glycinin content of progeny lines generated during the backcrossing program, for example by SDS-PAGE/Coomassie staining as well as using the marker system described herein to select lines based upon markers rather than visual traits.
  • Soybean plants can be crossed by either natural or mechanical techniques (see, e.g., Fehr, 1980). Natural pollination occurs in soybeans either by self pollination or natural cross pollination, which typically is aided by pollinating organisms. In either natural or artificial crosses, flowering and flowering time are an important consideration. Soybean is a short-day plant, but there is considerable genetic variation for sensitivity to photoperiod (Hamner, 1969; Criswell and Hume, 1972). The critical day length for flowering ranges from about 13 h for genotypes adapted to tropical latitudes to 24 h for photoperiod-insensitive genotypes grown at higher latitudes (Shibles et al., 1975).
  • hand pollination can be carried out by removing the stamens and pistil with a forceps from a flower of the male parent and gently brushing the anthers against the stigma of the female flower.
  • Access to the stamens can be achieved by removing the front sepal and keel petals, or piercing the keel with closed forceps and allowing them to open to push the petals away. Brushing the anthers on the stigma causes them to rupture, and the highest percentage of successful crosses is obtained when pollen is clearly visible on the stigma. Pollen shed can be checked by tapping the anthers before brushing the stigma.
  • Several male flowers may have to be used to obtain suitable pollen shed when conditions are unfavorable, or the same male may be used to pollinate several flowers with good pollen shed.
  • soybeans Genetic male sterility is available in soybeans and may be useful to facilitate hybridization in the context of the current invention, particularly for recurrent selection programs (Brim and Stuber, 1973).
  • the distance required for complete isolation of a crossing block is not clear; however, outcrossing is less than 0.5% when male-sterile plants are 12 m or more from a foreign pollen source (Boerma and Moradshahi, 1975). Plants on the boundaries of a crossing block probably sustain the most outcrossing with foreign pollen and can be eliminated at harvest to minimize contamination.
  • pods are typically air-dried at not more than 38° C. until the seeds contain 13% moisture or less, then the seeds are removed by hand. Seed can be stored satisfactorily at about 25° C. for up to a year if relative humidity is 50% or less. In humid climates, germination percentage declines rapidly unless the seed is dried to 7% moisture and stored in an air-tight container at room temperature. Long-term storage in any climate is best accomplished by drying seed to 7% moisture and storing it at 110° C. or less in a room maintained at 50% relative humidity or in an air-tight container.
  • a soybean plant provided by the invention may comprise one or more transgene(s).
  • a transgene confers herbicide resistance.
  • Common herbicide resistance genes include an EPSPS gene conferring glyphosate resistance, a neomycin phosphotransferase II (nptII) gene conferring resistance to kanamycin (Fraley et al., 1983), a hygromycin phosphotransferase gene conferring resistance to the antibiotic hygromycin (Vanden Elzen et al., 1985), genes conferring resistance to glufosinate or broxynil (Comai et al., 1985; Gordon-Kamm et al., 1990; Stalker et al., 1988) such as dihydrofolate reductase and acetolactate synthase (Eichholtz et al., 1987, Shah et al., 1986, Charest et al., 1990).
  • mutant ALS and AHAS enzymes conferring resistance to imidazalinone or a sulfonylurea (Lee et al., 1988; Miki et al., 1990), a phosphinothricin-acetyl-transferase gene conferring phosphinothricin resistance (European Appln.
  • genes conferring resistance to phenoxy proprionic acids and cycloshexones such as sethoxydim and haloxyfop (Marshall et al., 1992); and genes conferring resistance to triazine (psbA and gs+genes) and benzonitrile (nitrilase gene) (Przibila et al., 1991).
  • a plant of the invention may also comprise a gene that confers resistance to insect, pest, viral or bacterial attack.
  • a gene conferring resistance to a pest such as soybean cyst nematode was described in PCT Application WO96/30517 and PCT Application WO93/19181.
  • Jones et al., (1994) describe cloning of the tomato Cf-9 gene for resistance to Cladosporium fulvum );
  • Martin et al., (1993) describe a tomato Pto gene for resistance to Pseudomonas syringae pv.
  • Mindrinos et al. (1994) describe an Arabidopsis RSP2 gene for resistance to Pseudomonas syringae.
  • Bacillus thuringiensis endotoxins may also be used for insect resistance. (See, for example, Geiser et al., (1986).
  • a vitamin-binding protein such as avidin may also be used as a larvicide (PCT application US93/06487).
  • viral coat proteins in transformed plant cells are known to impart resistance to viral infection and/or disease development affected by the virus from which the coat protein gene is derived, as well as by related viruses.
  • Coat protein-mediated resistance has been conferred upon transformed plants against alfalfa mosaic virus, cucumber mosaic virus, tobacco streak virus, potato virus X, potato virus Y, tobacco etch virus, tobacco rattle virus and tobacco mosaic virus.
  • Developmental-arrestive proteins produced in nature by a pathogen or a parasite may also be used. For example, Logemann et al., (1992), have shown that transgenic plants expressing the barley ribosome-inactivating gene have an increased resistance to fungal disease.
  • Transgenes may also be used conferring increased nutritional value or another value-added trait.
  • modified fatty acid metabolism for example, by transforming a plant with an antisense gene of stearoyl-ACP desaturase to increase stearic acid content of the plant. (See Knutzon et al., 1992).
  • a sense desaturase gene may also be introduced to alter fatty acid content.
  • Phytate content may be modified by introduction of a phytase-encoding gene to enhance breakdown of phytate, adding more free phosphate to the transformed plant.
  • Modified carbohydrate composition may also be affected, for example, by transforming plants with a gene coding for an enzyme that alters the branching pattern of starch.
  • Transgenes may also be used to alter protein metabolism.
  • U.S. Pat. No. 5,545,545 describes lysine-insensitive maize dihydrodipicolinic acid synthase (DHPS), which is substantially resistant to concentrations of L-lysine which otherwise inhibit the activity of native DHPS.
  • DHPS lysine-insensitive maize dihydrodipicolinic acid synthase
  • EP 0640141 describes sequences encoding lysine-insensitive aspartokinase (AK) capable of causing a higher than normal production of threonine, as well as a subfragment encoding anti sense lysine ketoglutarate reductase for increasing lysine.
  • AK lysine-insensitive aspartokinase
  • a transgene may be employed that alters plant carbohydrate metabolism.
  • fructokinase genes are known for use in metabolic engineering of fructokinase gene expression in transgenic plants and their fruit (see U.S. Pat. No. 6,031,154).
  • a further example of transgenes that may be used are genes that alter grain yield.
  • U.S. Pat. No. 6,486,383 describes modification of starch content in plants with subunit proteins of adenosine diphosphoglucose pyrophosphorylase (“ADPG PPase”).
  • transgenic plants are discussed in which the introduction and expression of particular DNA molecules results in the formation of easily mobilized phosphate pools outside the vacuole and an enhanced biomass production and/or altered flowering behavior. Still further known are genes for altering plant maturity.
  • U.S. Pat. No. 6,774,284 describes DNA encoding a plant lipase and methods of use thereof for controlling senescence in plants.
  • U.S. Pat. No. 6,140,085 discusses FCA genes for altering flowering characteristics, particularly timing of flowering.
  • U.S. Pat. No. 5,637,785 discusses genetically modified plants having modulated flower development such as having early floral meristem development and comprising a structural gene encoding the LEAFY protein in its genome.
  • U.S. Pat. No. 6,184,440 discusses genetically engineered plants which display altered structure or morphology as a result of expressing a cell wall modulation transgene.
  • cell wall modulation transgenes include a cellulose binding domain, a cellulose binding protein, or a cell wall modifying protein or enzyme such as endoxyloglucan transferase, xyloglucan endo-transglycosylase, an expansin, cellulose synthase, or a novel isolated endo-1,4- ⁇ -glucanase.
  • Methods for introduction of a transgene are well known in the art and include biological and physical, plant transformation protocols. See, for example, Miki et al. (1993).
  • transgene Once a transgene is introduced into a variety it may readily be transferred by crossing. By using backcrossing, essentially all of the desired morphological and physiological characteristics of a variety are recovered in addition to the locus transferred into the variety via the backcrossing technique. Backcrossing methods can be used with the present invention to improve or introduce a characteristic into a plant (Poehlman et al., 1995; Fehr, 1987 a, b).
  • tissue cultures of a soybean variety of the invention indicates a composition comprising isolated cells of the same or a different type or a collection of such cells organized into parts of a plant.
  • tissue cultures are protoplasts, calli and plant cells that are intact in plants or parts of plants, such as embryos, pollen, flowers, leaves, roots, root tips, anthers, and the like.
  • the tissue culture comprises embryos, protoplasts, meristematic cells, pollen, leaves or anthers.
  • tissue culture An important ability of a tissue culture is the capability to regenerate fertile plants. This allows, for example, transformation of the tissue culture cells followed by regeneration of transgenic plants. For transformation to be efficient and successful, DNA must be introduced into cells that give rise to plants or germ-line tissue.
  • Soybeans typically are regenerated via two distinct processes; shoot morphogenesis and somatic embryogenesis (Finer, 1996).
  • Shoot morphogenesis is the process of shoot meristem organization and development. Shoots grow out from a source tissue and are excised and rooted to obtain an intact plant.
  • somatic embryogenesis an embryo (similar to the zygotic embryo), containing both shoot and root axes, is formed from somatic plant tissue. An intact plant rather than a rooted shoot results from the germination of the somatic embryo.
  • Plant morphogenesis and somatic embryogenesis are different processes and the specific route of regeneration is primarily dependent on the explant source and media used for tissue culture manipulations. While the systems are different, both systems show variety-specific responses where some lines are more responsive to tissue culture manipulations than others. A line that is highly responsive in shoot morphogenesis may not generate many somatic embryos. Lines that produce large numbers of embryos during an ‘induction’ step may not give rise to rapidly-growing proliferative cultures. Therefore, it may be desired to optimize tissue culture conditions for each soybean line. These optimizations may readily be carried out by one of skill in the art of tissue culture through small-scale culture studies. In addition to line-specific responses, proliferative cultures can be observed with both shoot morphogenesis and somatic embryogenesis. Proliferation is beneficial for both systems, as it allows a single, transformed cell to multiply to the point that it will contribute to germ-line tissue.
  • Somatic embryogenesis in soybean was first reported by Christianson et al. (1983) as a system in which embryogenic tissue was initially obtained from the zygotic embryo axis. These embryogenic cultures were proliferative but the repeatability of the system was low and the origin of the embryos was not reported. Later histological studies of a different proliferative embryogenic soybean culture showed that proliferative embryos were of apical or surface origin with a small number of cells contributing to embryo formation. The origin of primary embryos (the first embryos derived from the initial explant) is dependent on the explant tissue and the auxin levels in the induction medium (Hartweck et al., 1988). With proliferative embryonic cultures, single cells or small groups of surface cells of the ‘older’ somatic embryos form the ‘newer’ embryos.
  • Embryogenic cultures can also be used successfully for regeneration, including regeneration of transgenic plants, if the origin of the embryos is recognized and the biological limitations of proliferative embryogenic cultures are understood. Biological limitations include the difficulty in developing proliferative embryogenic cultures and reduced fertility problems (culture-induced variation) associated with plants regenerated from long-term proliferative embryogenic cultures. Some of these problems are accentuated in prolonged cultures. The use of more recently cultured cells may decrease or eliminate such problems.
  • a soybean plant provided by the invention may be used for any purpose deemed of value. Common uses include the preparation of food for human consumption, feed for non-human animal consumption and industrial uses. As used herein, “industrial use” or “industrial usage” refers to non-food and non-feed uses for soybeans or soy-based products.
  • Soybeans are commonly processed into two primary products, soybean protein (meal) and crude soybean oil. Both of these products are commonly further refined for particular uses. Refined oil products can be broken down into glycerol, fatty acids and sterols. These can be for food, feed or industrial usage. Edible food product use examples include coffee creamers, margarine, mayonnaise, pharmaceuticals, salad dressings, shortenings, bakery products, and chocolate coatings.
  • Soy protein products can be divided into soy flour concentrates and isolates which have both food/feed and industrial use.
  • Soy flour and grits are often used in the manufacturing of meat extenders and analogs, pet foods, baking ingredients and other food products.
  • Food products made from soy flour and isolate include baby food, candy products, cereals, food drinks, noodles, yeast, beer, ale, etc.
  • Soybean meal in particular is commonly used as a source of protein in livestock feeding, primarily swine and poultry. Feed uses thus include, but are not limited to, aquaculture feeds, bee feeds, calf feed replacers, fish feed, livestock feeds, poultry feeds and pet feeds, etc.
  • Whole soybean products can also be used as food or feed.
  • Common food usage includes products such as the seed, bean sprouts, baked soybean, full fat soy flour used in various products of baking, roasted soybean used as confectioneries, soy nut butter, soy coffee, and other soy derivatives of oriental foods.
  • hulls are commonly removed from the soybean and used as feed.
  • Soybeans additionally have many industrial uses.
  • One common industrial usage for soybeans is the preparation of binders that can be used to manufacture composites.
  • wood composites may be produced using modified soy protein, a mixture of hydrolyzed soy protein and PF resins, soy flour containing powder resins, and soy protein containing foamed glues.
  • Soy-based binders have been used to manufacture common wood products such as plywood for over 70 years.
  • urea-formaldehyde and phenol-formaldehyde resins has decreased the usage of soy-based adhesives in wood products, environmental concerns and consumer preferences for adhesives made from a renewable feedstock have caused a resurgence of interest in developing new soy-based products for the wood composite industry.
  • soy adhesives include soy hydrolyzate adhesives and soy flour adhesives.
  • Soy hydrolyzate is a colorless, aqueous solution made by reacting soy protein isolate in a 5 percent sodium hydroxide solution under heat (120° C.) and pressure (30 psig). The resulting degraded soy protein solution is basic (pH 11) and flowable (approximately 500 cps) at room temperature.
  • Soy flour is a finely ground, defatted meal made from soybeans.
  • Various adhesive formulations can be made from soy flour, with the first step commonly requiring dissolving the flour in a sodium hydroxide solution. The strength and other properties of the resulting formulation will vary depending on the additives in the formulation. Soy flour adhesives may also potentially be combined with other commercially available resins.
  • Soybean oil may find application in a number of industrial uses. Soybean oil is the most readily available and one of the lowest-cost vegetable oils in the world. Common industrial uses for soybean oil include use as components of anti-static agents, caulking compounds, disinfectants, fungicides, inks, paints, protective coatings, wallboard, anti-foam agents, alcohol, margarine, paint, ink, rubber, shortening, cosmetics, etc. Soybean oils have also for many years been a major ingredient in alkyd resins, which are dissolved in carrier solvents to make oil-based paints. The basic chemistry for converting vegetable oils into an alkyd resin under heat and pressure is well understood to those of skill in the art.
  • Soybean oil in its commercially available unrefined or refined, edible-grade state is a fairly stable and slow-drying oil.
  • Soybean oil can also be modified to enhance its reactivity under ambient conditions or, with the input of energy in various forms, to cause the oil to copolymerize or cure to a dry film.
  • Some of these forms of modification have included epoxidation, alcoholysis or tranesterification, direct esterification, metathesis, isomerization, monomer modification, and various forms of polymerization, including heat bodying.
  • the reactive linoleic-acid component of soybean oil with its double bonds may be more useful than the predominant oleic- and linoleic-acid components for many industrial uses.
  • Solvents can also be prepared using soy-based ingredients.
  • soy-based ingredients For example, methyl soyate, a soybean-oil based methyl ester, is gaining market acceptance as an excellent solvent replacement alternative in applications such as parts cleaning and degreasing, paint and ink removal, and oil spill remediation. It is also being marketed in numerous formulated consumer products including hand cleaners, car waxes and graffiti removers. Methyl soyate is produced by the transesterification of soybean oil with methanol. It is commercially available from numerous manufacturers and suppliers. As a solvent, methyl soyate has important environmental- and safety-related properties that make it attractive for industrial applications.
  • methyl soyate It is lower in toxicity than most other solvents, is readily biodegradable, and has a very high flash point and a low level of volatile organic compounds (VOCs).
  • VOCs volatile organic compounds
  • the compatibility of methyl soyate is excellent with metals, plastics, most elastomers and other organic solvents.
  • Current uses of methyl soyate include cleaners, paint strippers, oil spill cleanup and bioremediation, pesticide adjuvants, corrosion preventives and biodiesel fuels additives.
  • kits may thus comprise, in suitable container means, a probe or primer for detection of the polymorphism and/or an additional agent of the present invention.
  • the kit will allow detection of at least one non-transgenic Gy null allele, and may further provide for the detection of a lipoxygenase and/or KTI null allele, for example, by detection of polymorphisms in such alleles and/or otherwise in linkage disequilibrium with the allele(s).
  • kits may comprise a suitably aliquoted agent composition(s) of the present invention, whether labeled or unlabeled for any assay format desired to detect such alleles.
  • the components of the kits may be packaged either in aqueous media or in lyophilized form.
  • the container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which a component may be placed, and preferably, suitably aliquoted. Where there are more than one component in the kit, the kit also will generally contain a second, third or other additional container into which the additional components may be separately placed. However, various combinations of components may be comprised in a vial.
  • the kits of the present invention also will typically include a means for containing the detection composition and any other reagent containers in close confinement for commercial sale. Such containers may include injection or blow-molded plastic containers into which the desired vials are retained.
  • the liquid solution may be an aqueous solution, with a sterile aqueous solution being particularly preferred.
  • the components of the kit may be provided as dried powder(s).
  • the powder can be reconstituted by the addition of a suitable solvent.
  • the solvent may also be provided in another container means.
  • the container means will generally include at least one vial, test tube, flask, bottle, syringe and/or other container means, into which the composition for detecting a null allele are placed, preferably, suitably allocated.
  • the kits may also comprise a second container means for containing a sterile buffer and/or other diluent.
  • kits of the present invention will also typically include a means for containing the vials in close confinement for commercial sale, such as, e.g., injection and/or blow-molded plastic containers into which the desired vials are retained. Irrespective of the number and/or type of containers, the kits of the invention may also comprise, and/or be packaged with, an instrument for assisting with the use of the detection compositions.
  • Agronomically Elite means a genotype that has a culmination of many distinguishable traits such as seed yield, emergence, vigor, vegetative vigor, disease resistance, seed set, standability and threshability which allows a producer to harvest a product of commercial significance.
  • Allele Any of one or more alternative forms of a gene locus, all of which alleles relate to a trait or characteristic. In a diploid cell or organism, the two alleles of a given gene occupy corresponding loci on a pair of homologous chromosomes.
  • Backcrossing A process in which a breeder repeatedly crosses hybrid progeny, for example a first generation hybrid (F 1 ), back to one of the parents of the hybrid progeny. Backcrossing can be used to introduce one or more single locus conversions from one genetic background into another.
  • F 1 first generation hybrid
  • Crossing The mating of two parent plants.
  • Cross-pollination Fertilization by the union of two gametes from different plants.
  • Down-regulatory mutation For the purposes of this application a down regulatory mutation is defined as a mutation that reduces the expression levels of a protein from a given gene. Thus a down-regulatory mutation comprises null mutations.
  • F 1 , Hybrid The first generation progeny of the cross of two nonisogenic plants.
  • Genotype The genetic constitution of a cell or organism.
  • INDEL Genetic mutations resulting from insertion or deletion of nucleotide sequence.
  • soybean plant includes plant parts and derivatives of a soybean plant.
  • Linkage A phenomenon wherein alleles on the same chromosome tend to segregate together more often than expected by chance if their transmission was independent.
  • Marker A readily detectable phenotype, preferably inherited in codominant fashion (both alleles at a locus in a diploid heterozygote are readily detectable), with no environmental variance component, i.e., heritability of 1.
  • Non-transgenic mutation A mutation that is naturally occurring, or induced by conventional methods (e.g. exposure of plants to radiation or mutagenic compounds), not including mutations made using recombinant DNA techniques.
  • Null phenotype as used herein means that a given protein is not expressed at levels that can be detected. In the case of the Gy subunits, expression levels are determined by SDS-PAGE and Coomassie staining.
  • Phenotype The detectable characteristics of a cell or organism, which characteristics are the manifestation of gene expression.
  • Quantitative Trait Loci Quantitative trait loci (QTL) refer to genetic loci that control to some degree numerically representable traits that are usually continuously distributed.
  • SNP refers to single nucleotide polymorphisms, or single nucleotide mutations when comparing two homologous sequences.
  • Stringent Conditions Refers to nucleic acid hybridization conditions of 5 ⁇ SSC, 50% formamide and 42° C.
  • Tissue Culture A composition comprising isolated cells of the same or a different type or a collection of such cells organized into parts of a plant.
  • Transgene A genetic locus comprising a sequence which has been introduced into the genome of a soybean plant by transformation.
  • the B2G2 or “11S null” soybean variety has a unique seed composition including high level of ⁇ -conglycinin and low amount of glycinin. However, the B2G2 variety exhibits agronomically inferior characteristics such as low yield, excessive lodging and green seed.
  • a number of breeding lines were developed, which carried all or parts of the mutations present in the B2G2 lines. Fifteen such lines together with B2G2 lines were used as mutant lines in resequencing panel. Eight wild types were used for comparison in this study. Table 1 lists all the lines used in the sequencing panel.
  • JB4 AH_(A3244/(B2G2/A1923:.077.):0001.0097.0011.)/DAK2501A0R:@.0314.0009.@.
  • JB5 AH_(A3244/(B2G2/A1923:.077.):0001.0097.0015.)/DJW2500C0R:@.0042.0012.@.
  • JB6 AH_(A3244/(B2G2/A1923:.077.):0001.0011.0008.)/AG2402:@.0028.0010.@.
  • JB7 AH_(A3244/(B2G2/A1923:.077.):0001.0008.0016.)/DBL3201A0X:@.0256.0014.@.
  • JB8 AH_(A3244/(B2G2/A1923:.077.):0001.0097.0015.)/DJW2500C0R:@.0234.0019.@.
  • AH_704416- JB9 24/((A3244/(B2G2/A1923:.077.):0001.0097.0015.)/DJW2500C0R:@.0005.):@.0067.0016.
  • JB10 AH_704416-24/((A3244/(B2G2/A1923:.077.):0001.0097.0015.)/DJW2500C0R:@.0005.):@.0067.0007.
  • JB11 AH_704416-24/((A3244/(B2G2/A1923:.077.):0001.0097.0015.)/DJW2500C0R:@.0005.):@.0067.0003.
  • JB12 AH_704416-24/((A3244/(B2G2/A1923:.077.):0001.0097.0015.)/DJW2500C0R:@.0005.):@.0067.0002.
  • JB13 AH_DAK2301A1R/((A3244/(B2G2/A1923:.077.):0001.0097.0015.)/DJW2500C0R:@.0013.):@.0018.0001.
  • JB14 AH_DAK2301A1R/((A3244/(B2G2/A1923:.077.):0001.0097.0015.)/DJW2500C0R:@.0013.):@.0114.0011.
  • JB15 AH_DAK2301A1R/((A3244/(B2G2/A1923:.077.):0001.0097.0015.)/DJW2500C0R:@.0013.):@.0130.0001.
  • DNA sequences for all the glycinin genes are available in GenBank (NCBI). These sequences were used as queries to blast against a Monsanto sequence database. Using “blastn” programs, a number of high score hits were obtained. The resulting sequences from the blast search were aligned to provide a high quality consensus sequences for use in primer design. Nested primers were designed to completely cover the entire gene at each locus and amplicons were generated from different lines. Sequences of these amplicons were aligned to identify SNPs and INDELs associated with high ⁇ -conglycinin phenotypes. Initially 10 pairs of primers were designed for Gy1, 7 pairs each for Gy2 and Gy3, 14 pairs for Gy4, 10 pairs for Gy5 and 11 pairs for Gy7. Additional primers were designed once their sequences were known from this study. Table 2 lists the primers used in the study.
  • DNA was isolated with Qiagen Plant DNA kits, and PCR was performed with KOD protocol (EMD Biosciences, Inc, Madison, Wis.).
  • the reaction mix included 3.4 ⁇ l 5M Betaine, 2 ⁇ l 10 ⁇ KOD buffer, 2 ⁇ l of 2 mM dNTPs, 0.8 ⁇ l 25 mM MgSO 4 , 0.2 ⁇ l KOD enzyme (1 U/ ⁇ l), 1.6 ⁇ l primers (5 ⁇ M) and 10 ⁇ l DNA template (2 ng/ ⁇ l).
  • PCR cycles were as follows: 94° C. 5 mins; 8 cycles of 94° C. 40 sec, 62° C. 40 sec, 72° C. 1 min, 94° C. 40 sec, 60° C. 40 sec, 72° C.
  • PCR products were analyzed by electrophoresis in 1% agarose gels. For sequencing, 5 ⁇ l PCR products were removed to a new tube and 1 ⁇ l Exonuclease I (1:10 diluted) and 1 ⁇ l Shrimp Alkaline Phosphatase (1:100 diluted). The mix was incubated at 37° C. for 20 min and then 80° C.
  • End point SNP/Taqman® assays were designed and manufactured by Applied Biosystems based on the SNPs sequences provided to them. SNP detection was carried out according to supplied instructions (Applied Biosystems).
  • Taqman® assays or Real Time PCR, detect the accumulation of a specific PCR product by hybridization and cleavage of a double-labeled fluorogenic probe during the amplification reaction.
  • a Taqman assay includes four oligonucleotides, two of which serve as PCR primers and generate a PCR product encompassing the polymorphism to be detected.
  • the other two are allele-specific fluorescence-resonance-energy-transfer (FRET) probes; each probe has a unique fluorophore that is released upon probe degradation by Taq DNA polymerase, effectively signalling the amount of each allele present in the sample.
  • FRET allele-specific fluorescence-resonance-energy-transfer
  • Protein analysis was carried out as follows: Eight soybean seeds were pooled and ground using the CAT Mega-Grinder (SOP Asci-01-0002). Ground samples were stored at 4° C. For analysis, ⁇ 30 mg of flour from each was weighed into one well of a 96 well 2 ml microtiter plate. Protein was extracted for 1 hour with shaking in 1.0 ml 1X Laemmli SDS buffer pH 6.8 containing 0.1M dithiothreitol (DTT) as a reductant. Following centrifugation, a portion of each extract was further diluted in SDS buffer to yield 0.2-0.5 ⁇ g/ ⁇ L total protein, heated to 90-100° C. for 10 min, and cooled.
  • SOP Asci-01-0002 the CAT Mega-Grinder
  • F2 progeny plants were analyzed for total content of Gy1 and Gy2 encoded protein. As shown in FIG. 2 , the plants were distributed into two phenotypic groups, one group with less than 3% Gy1,2 encoded protein and another with 3.1% greater Gy1,2 encoded protein. Chi square analysis ( FIG. 2B ) was consistent with the linked mutant Gy1,2 alleles as recessive traits.
  • F2 progeny plants were analyzed for total content of Gy3 encoded protein. As shown in FIG. 3 , the plants were distributed into two phenotypic groups, one group with less than 1% Gy3 encoded protein and another with 1.1% or greater Gy3 encoded protein. Chi square analysis ( FIG. 3B ) was consistent with the reduced Gy3 expression as a recessive trait.
  • Progeny plants were subjected to protein analysis to determine the amount of Gy4 encoded polypeptides that were expressed. As shown in FIG. 5 , the plants were distributed in two phenotypic groups, one group that was null for Gy4 and another group that demonstrated expression of Gy4 encoded polypeptides. Chi square analysis ( FIG. 5B ) was consistent with the Gy4 null allele as a recessive trait.
  • SNPs/INDELs appear to be in linkage disequilibrium and are associated with the “11s null” phenotypes. It is still unknown if these SNPs or INDELs actually caused the loss of A3 subunit in B2G2 as shown on FIG. 1 . Since these are all sequence variations detected in the coding region there may be some other variations residing in the promoter regions which caused the loss of the A3 band as seen on the protein gel.
  • PCR analyses such as Taqman® assays, were designed for the SNPs or INDELs identified above. Table 7 lists the primer and probe sequences of each assay as well as the marker name assigned to each marker. Two assays were designed for Gy1 and Gy3 respectively using SNPs at different positions. These assays were first run on the standard panel used in resequencing in this study and then used in segregating populations.
  • FIG. 6A shows total glycinin protein versus total beta-conglycinin protein for each plant.
  • the data indicates that lower expression of glycinin correlates with higher expression of beta-conglycinin (Cgy) subunits ( FIG. 6B ).
  • plants with the mutant allele at Gy1 always contain lower glycinin A1a, A1b and A2 subunits, 2.5 and 2.4%, respectively in both populations while those with “TT” allele contain higher glycinin A1a, A1b and A2 subunits, 7.6 and 8.1% respectively.
  • the correlation is highly significant (P-values of 1.7 ⁇ 10 ⁇ 55 and 4.1 ⁇ 10 ⁇ 62 respectively) and a similar correlation was observed in the case of Gy2.
  • Agronomically elite soybean plants comprising non-transgenic mutations conferring a null Gy3 and Gy4 phenotype and a reduced Gy1/Gy2 phenotype were analyzed for total content of various glycinin and ⁇ -conglycinin subunits.
  • Gy1 Segregation of Gy1(NS0199008), Gy2(NS0199002) and Gy4(NS0199003) markers was analyzed on two F2 populations. As shown in Table 9, both Gy1 and Gy2 are dominant markers, and each segregates in a 3:1 ratio. Gy4 is a co-dominant marker.
  • FIG. 7 illustrates the glycinin and ⁇ -conglycinin content of F2 plants. Plants were selected based on either protein analysis of subunit expression ( FIG. 7A ) or using the three mutant Gy markers ( FIG. 7B ). The data shows that the selection based on markers misidentified less than 1% ( 7/754) F2 plants.
  • Green seed color is often viewed as less desirable by soybean farmers and consumers. Eliminating the green seed color of a line would therefore be desirable.
  • An analysis was carried out on low glycinin soybeans produced as described herein to analyze the extent to which the green seed color was eliminated. Color analysis was carried out on whole soybeans using the ColorFlexTM Reflectance Spectrophotometer (Model 45/0). The spectrometer was standardized using black glass and white tile. The standardization was checked using a green tile having color values certified by the manufacturer, Hunter Associates Laboratory, Inc. (Reston, Va., USA).
  • the spectrophotometer measures the CIE Tristimulus Color Scale Values X, Y and Z and from these values and calculates the CIELAB Color Scale Values L*, a* and b*.
  • the CIELAB Color Scale allows the specification of color perception in terms of three-dimensional space (CIE, Colorimetry, Publication 15.2, Second Edition, Vienna, 1986), with a CIELAB Color Space organized in a cube.
  • the L*-axis runs from top to bottom and is known as the lightness value, which extends from 0 (black) to 100 (white).
  • the a* and the b*axes have no specific numerical limits.
  • the coordinate a* represents redness when positive, gray when zero and greenness when negative.
  • the coordinate b* represents yellowness when positive, gray when zero and blueness when negative.
  • a multi-layer of whole soybeans filled the 35 ⁇ 10 mm style polystyrene tissue culture dish with the lid on the bottom of the dish in order to protect the reading surface.
  • the lid was removed from the bottom of the tissue culture dish before it was placed on the spectrophotometer port.
  • the tissue culture dish with the whole soybeans was placed on the spectrometer port with the side to be measured towards the port.
  • the light trap was placed over the sample in order to restrict any external light interference.
  • the CIE Tristimulus Color Scale screen was displayed on the ColorFlex.
  • the Read Key was pressed and the CIE Tristimulus Color Scale Values were measured for the whole soybean sample.
  • the color capture program recorded the values into an Excel spread-sheet.
  • the view was toggled to the CIELAB Color Scale screen, the Read Key was pressed, and the spectrophotometer was used to calculate the CIELAB Color Scale Values L*, a* and b* for the whole soybean sample.
  • the Lox2 sequence from GenBank, G1505137 was used as a query to blast against a Monsanto sequence database. Sixty high hits were downloaded and assembled using the SeqMan program (DNASTAR, INC, Madison, Wis.). Two distinct transcripts were identified in the Monsanto DNA sequence collection. One of the transcripts corresponded to a lipoxygenase-1 (Lox1) gene in GenBank and was thus named lx1 (SEQ ID NO:157), and the other corresponded to the Lox2 (lx2) gene (SEQ ID NO:158). Gene-specific primers were designed and used to generate amplicons from a panel of eight lines. The panel consisted of six mutants and two wild types. Table 13 lists the lines used in the sequencing panel.
  • Polymorphisms at the Lox2 locus are shown on Table 15. Six SNPs and a 2 bp INDEL were detected and two distinct haplotypes are formed in these eight lines. The haplotypes are clearly associated with lx2 phenotypes (Table 13). All of these polymorphisms except the one at position 2542 are located in introns. The SNP at position 2542 is a missense mutation, causing a change in genetic codon from CAT encoding for Histidine to CAA encoding for Glutamine.
  • a Taqman assay was designed from the SNP at position 2542.
  • the assay information is given in Table 16.
  • the allelogram of this assay is shown on FIG. 8 . This marker allowed a clear distinction between the “A” allele from the mutant at Lox2 and the “T” allele from wild type.
  • KTI Kunitz Trypsin Inhibitor Protein
  • a PCR-based TAQMAN marker-assisted assay employing primers e.g. SEQ ID NOs: 168-171
  • primers e.g. SEQ ID NOs: 168-171
  • compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Biotechnology (AREA)
  • Organic Chemistry (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Molecular Biology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • General Health & Medical Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Botany (AREA)
  • Cell Biology (AREA)
  • Plant Pathology (AREA)
  • Developmental Biology & Embryology (AREA)
  • Environmental Sciences (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Analytical Chemistry (AREA)
  • Nutrition Science (AREA)
  • Physiology (AREA)
  • Mycology (AREA)
  • Immunology (AREA)
  • Natural Medicines & Medicinal Plants (AREA)
  • Oil, Petroleum & Natural Gas (AREA)
  • Pest Control & Pesticides (AREA)
  • Insects & Arthropods (AREA)
  • Hematology (AREA)
  • Obesity (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Medicinal Chemistry (AREA)
US11/517,186 2005-09-07 2006-09-07 Agronomically elite soybeans with high beta-conglycinin content Abandoned US20070067871A1 (en)

Priority Applications (3)

Application Number Priority Date Filing Date Title
US11/517,186 US20070067871A1 (en) 2005-09-07 2006-09-07 Agronomically elite soybeans with high beta-conglycinin content
US14/106,707 US9554525B2 (en) 2005-09-07 2013-12-13 Agronomically elite soybeans with high β-conglycinin content
US15/389,038 US20170164573A1 (en) 2005-09-07 2016-12-22 Agronomically elite soybeans with high beta-conglycinin content

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US71477905P 2005-09-07 2005-09-07
US72249305P 2005-09-30 2005-09-30
US11/517,186 US20070067871A1 (en) 2005-09-07 2006-09-07 Agronomically elite soybeans with high beta-conglycinin content

Related Child Applications (1)

Application Number Title Priority Date Filing Date
US14/106,707 Division US9554525B2 (en) 2005-09-07 2013-12-13 Agronomically elite soybeans with high β-conglycinin content

Publications (1)

Publication Number Publication Date
US20070067871A1 true US20070067871A1 (en) 2007-03-22

Family

ID=37836370

Family Applications (3)

Application Number Title Priority Date Filing Date
US11/517,186 Abandoned US20070067871A1 (en) 2005-09-07 2006-09-07 Agronomically elite soybeans with high beta-conglycinin content
US14/106,707 Active 2027-06-27 US9554525B2 (en) 2005-09-07 2013-12-13 Agronomically elite soybeans with high β-conglycinin content
US15/389,038 Abandoned US20170164573A1 (en) 2005-09-07 2016-12-22 Agronomically elite soybeans with high beta-conglycinin content

Family Applications After (2)

Application Number Title Priority Date Filing Date
US14/106,707 Active 2027-06-27 US9554525B2 (en) 2005-09-07 2013-12-13 Agronomically elite soybeans with high β-conglycinin content
US15/389,038 Abandoned US20170164573A1 (en) 2005-09-07 2016-12-22 Agronomically elite soybeans with high beta-conglycinin content

Country Status (11)

Country Link
US (3) US20070067871A1 (xx)
EP (1) EP1942722A2 (xx)
JP (2) JP5952994B2 (xx)
CN (1) CN103975841B (xx)
AR (1) AR055155A1 (xx)
AU (1) AU2006287647B2 (xx)
BR (1) BRPI0615947B1 (xx)
CA (1) CA2621778C (xx)
HK (1) HK1198100A1 (xx)
UY (1) UY29784A1 (xx)
WO (1) WO2007030429A2 (xx)

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20080193587A1 (en) * 2007-02-09 2008-08-14 Vermeire Drew A Composition and method of feeding a young livestock animal
US20080260895A1 (en) * 2007-04-17 2008-10-23 Vermeire Drew A Milk replacer composition and product and method for producing the same
US20090068337A1 (en) * 2007-09-11 2009-03-12 Neal Bringe Increased alpha-prime beta-conglycinin soybeans
WO2010075860A3 (en) * 2008-12-30 2011-07-14 Carlsberg Breweries A/S Barley with reduced lipoxygenase activity and beverage prepared therefrom
WO2011109624A1 (en) * 2010-03-03 2011-09-09 Schillinger Genetics, Inc. Ultra-low trypsin inhibitor soybean
US20140366219A1 (en) * 2011-12-28 2014-12-11 University Of Tennessee Research Foundation Increasing Soybean Defense Against Pests
CN114501985A (zh) * 2019-10-01 2022-05-13 孟山都技术公司 通过液体介导将花粉递送到来自受体植物的花的封闭柱头上进行异花授粉
CN116716428A (zh) * 2023-06-07 2023-09-08 吉林省农业科学院 鉴定β-伴大豆球蛋白亚基缺失性状的标记Gm20_38690822、引物及应用

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8640525B2 (en) 2009-12-30 2014-02-04 Monsanto Technology Llc Methods and systems for differentiating soybeans
CN111411098B (zh) * 2020-05-07 2022-03-04 海南波莲水稻基因科技有限公司 水稻ALS突变基因、含有该基因的植物转基因筛选载体pCALSm2及其应用
KR102641019B1 (ko) 2021-08-31 2024-03-04 대한민국 콩 비린내 제거 유전형을 포함하는 계통 또는 품종 판별용 분자마커 세트 및 이의 용도
CN117625627B (zh) * 2024-01-26 2024-03-29 湖南工程学院 一种诱导型ProPgJOX4启动子及其在人参皂苷生物合成中的应用

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6171640B1 (en) * 1997-04-04 2001-01-09 Monsanto Company High beta-conglycinin products and their use
US20060062894A1 (en) * 2004-09-23 2006-03-23 Smith Houston S 7S/2S-rich soy protein globulin fraction composition and process for making same
US7186425B2 (en) * 2000-09-29 2007-03-06 Fuji Oil Company, Ltd. Composition for diminishing neutral fat in blood

Family Cites Families (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
BR0312609A (pt) * 2002-07-11 2005-07-26 Monsanto Technolgoy Llc Plantas de grão com alto rendimento com proteìna de semente mais óleo aumentados
JP4701665B2 (ja) * 2003-09-30 2011-06-15 独立行政法人農業・食品産業技術総合研究機構 高遊離アミノ酸含有ダイズ
US20050138681A1 (en) * 2003-09-30 2005-06-23 Inc Admin Agcy Natl Agric And Bio-Oriented Res Org Soybean containing high levels of free amino acids
JP4001239B2 (ja) 2004-01-23 2007-10-31 国立大学法人京都大学 ダイズ由来ペプチド混合物およびその利用
NZ585893A (en) 2004-07-09 2011-09-30 Monsanto Technology Llc Soy compositions having improved organoleptic properties and methods of generation
WO2006039136A2 (en) 2004-09-20 2006-04-13 Wake Forest University Health Sciences Methods and compositions for beta conglycinin fraction of soy protein

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6171640B1 (en) * 1997-04-04 2001-01-09 Monsanto Company High beta-conglycinin products and their use
US6566134B2 (en) * 1997-04-04 2003-05-20 Monsanto Company High Beta-Conglycinin products and their use
US20040037905A1 (en) * 1997-04-04 2004-02-26 Bringe Neal A. High beta-conglycinin products and their use
US7094751B2 (en) * 1997-04-04 2006-08-22 Monsanto Corporation High beta-conglycinin products and their use
US7186425B2 (en) * 2000-09-29 2007-03-06 Fuji Oil Company, Ltd. Composition for diminishing neutral fat in blood
US20060062894A1 (en) * 2004-09-23 2006-03-23 Smith Houston S 7S/2S-rich soy protein globulin fraction composition and process for making same

Cited By (14)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20080193587A1 (en) * 2007-02-09 2008-08-14 Vermeire Drew A Composition and method of feeding a young livestock animal
US20080260895A1 (en) * 2007-04-17 2008-10-23 Vermeire Drew A Milk replacer composition and product and method for producing the same
US9049823B2 (en) * 2007-09-11 2015-06-09 Monsanto Technology Llc Increased alpha-prime beta-conglycinin soybeans
US20090068337A1 (en) * 2007-09-11 2009-03-12 Neal Bringe Increased alpha-prime beta-conglycinin soybeans
US10231400B2 (en) 2007-09-11 2019-03-19 Monsanto Technology Llc Increased alpha-prime beta-conglycinin soybeans
US9668502B2 (en) 2007-09-11 2017-06-06 Monsanto Technology Llc Increased alpha-prime beta-conglycinin soybeans
WO2010075860A3 (en) * 2008-12-30 2011-07-14 Carlsberg Breweries A/S Barley with reduced lipoxygenase activity and beverage prepared therefrom
US9363959B2 (en) 2008-12-30 2016-06-14 Carlsberg Breweries A/S Barley with reduced lipoxygenase activity and beverage prepared therefrom
EA028532B1 (ru) * 2008-12-30 2017-11-30 Карлсберг Брюириз А/С Ячмень с уменьшенной липоксигеназной активностью
US20120317675A1 (en) * 2010-03-03 2012-12-13 Schillinger Genetics, Inc. Ultra-low trypsin inhibitor soybean
WO2011109624A1 (en) * 2010-03-03 2011-09-09 Schillinger Genetics, Inc. Ultra-low trypsin inhibitor soybean
US20140366219A1 (en) * 2011-12-28 2014-12-11 University Of Tennessee Research Foundation Increasing Soybean Defense Against Pests
CN114501985A (zh) * 2019-10-01 2022-05-13 孟山都技术公司 通过液体介导将花粉递送到来自受体植物的花的封闭柱头上进行异花授粉
CN116716428A (zh) * 2023-06-07 2023-09-08 吉林省农业科学院 鉴定β-伴大豆球蛋白亚基缺失性状的标记Gm20_38690822、引物及应用

Also Published As

Publication number Publication date
CN103975841B (zh) 2018-03-30
EP1942722A2 (en) 2008-07-16
AU2006287647B2 (en) 2011-11-17
WO2007030429A8 (en) 2008-07-31
US20140259196A1 (en) 2014-09-11
BRPI0615947A2 (pt) 2011-05-31
US20170164573A1 (en) 2017-06-15
CA2621778C (en) 2016-04-19
US9554525B2 (en) 2017-01-31
AR055155A1 (es) 2007-08-08
CN103975841A (zh) 2014-08-13
AU2006287647A2 (en) 2008-04-17
HK1198100A1 (en) 2015-03-13
JP5952994B2 (ja) 2016-07-13
JP2015128438A (ja) 2015-07-16
UY29784A1 (es) 2007-07-31
AU2006287647A1 (en) 2007-03-15
WO2007030429A2 (en) 2007-03-15
WO2007030429A3 (en) 2007-09-07
CA2621778A1 (en) 2007-03-15
JP2009506786A (ja) 2009-02-19
BRPI0615947B1 (pt) 2023-12-19

Similar Documents

Publication Publication Date Title
US9554525B2 (en) Agronomically elite soybeans with high β-conglycinin content
US9668439B2 (en) High yielding soybean plants with low linolenic acid
US10231400B2 (en) Increased alpha-prime beta-conglycinin soybeans
US20110252490A1 (en) Methods and Compositions for Increased Alpha-Prime Beta-Conglycinin Soybeans
US20110010793A1 (en) Methods and Compositions for Increased Yield
CN101312644A (zh) 农学上优良的具有高β-伴大豆球蛋白含量的大豆

Legal Events

Date Code Title Description
AS Assignment

Owner name: MONSANTO TECHNOLOGY, L.L.C., MISSOURI

Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:WU, KUNSHENG;HOREJSI, THOMAS;BYRUM, JOSEPH R;AND OTHERS;REEL/FRAME:018557/0085;SIGNING DATES FROM 20061027 TO 20061114

STCB Information on status: application discontinuation

Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION