WO2011094205A1 - Hppd-inhibitor herbicide tolerance - Google Patents

Hppd-inhibitor herbicide tolerance Download PDF

Info

Publication number
WO2011094205A1
WO2011094205A1 PCT/US2011/022395 US2011022395W WO2011094205A1 WO 2011094205 A1 WO2011094205 A1 WO 2011094205A1 US 2011022395 W US2011022395 W US 2011022395W WO 2011094205 A1 WO2011094205 A1 WO 2011094205A1
Authority
WO
WIPO (PCT)
Prior art keywords
tolerance
marker
plant
herbicide
markers
Prior art date
Application number
PCT/US2011/022395
Other languages
French (fr)
Inventor
Mark Bogner
Julian Chaky
Jennifer A. Klaiber
Donald Kyle
Mark D. Vogt
Original Assignee
Pioneer Hi-Bred International, Inc.
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
Application filed by Pioneer Hi-Bred International, Inc. filed Critical Pioneer Hi-Bred International, Inc.
Priority to CA2788198A priority Critical patent/CA2788198C/en
Priority to BR112012018618A priority patent/BR112012018618A2/en
Priority to EP11701602A priority patent/EP2529027A1/en
Publication of WO2011094205A1 publication Critical patent/WO2011094205A1/en

Links

Classifications

    • 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
    • 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
    • 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
    • A01H1/045—Processes of selection involving genotypic or phenotypic markers; Methods of using phenotypic markers for selection using molecular 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
    • C12Q2600/13—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

Definitions

  • This invention relates generally to the detection of genetic differences among soybeans.
  • Soybeans (Glycine max L. Merr.) are a major cash crop and investment commodity in North America and elsewhere. Soybean oil is one of the most widely used edible oils, and soybeans are used worldwide both in animal feed and in human food production. Additionally, soybean utilization is expanding to industrial, manufacturing, and pharmaceutical applications. Weed management in soybean fields is important to maximizing yields. A recent development in soybean technology has been the development of herbicide-tolerant soybean varieties. Glyphosate tolerant soybeans were commercially introduced in 1996 and accounted for more than 85% percent of U.S.
  • This invention relates generally to the detection of genetic differences among soybeans. More particularly, the invention relates to soybean quantitative trait loci (QTL) for tolerance or sensitivity to mesotrione and/or isoxazole herbicides, to soybean plants possessing these QTLs, which map to a novel chromosomal region, and to genetic markers that are indicative of phenotypes associated with mesotrione and/or isoxazole herbicide tolerance. Methods and compositions for use of these markers in genotyping, screening, and selection of soybean are also disclosed.
  • QTL quantitative trait loci
  • a navel method for determining the presence or absence in soybean germplastn of a QTL associated with tolerance or susceptibility to HPPD-inhibitor herbicides, including mesotrione and/or isoxazole herbicides.
  • the trait has been found to be closely linked to a number of molecular markers that map to linkage group L, on chromosome 19 (Gml9). Soybean plants, seeds, tissue cultures, variants and mutants having tolerance or susceptibility to HPPD-inhibitor herbicides, including mesotrione and/or isoxazole herbicides, produced by the foregoing methods are also provided.
  • the QTL associated with tolerance or sensitivity to mesotrione and/or isoxazole herbicides maps to soybean linkage group L.
  • the QTL may be mapped by one or more molecular markers, including SATT495, P10649C-3, S ATT 182, SATT388, SATO 13, SATT613, and markers closely linked thereto.
  • Other markers of linkage group L may also be used to identify the presence or absence of the gene, including other markers above marker SATT613.
  • Additional relevant markers on linkage group L include S03859-1-A, S08103-1-Q1, S08104-1-Q1, S08106-1-Q1, S08110-1-Q1, S08111-1-Q1, S08115-2-Q1, S08117-1-Q1, S08119-1-Q1, S08118-1-Q1, S08116-1-Q1, S08114-1-Q1, S08113-1-Q1, S08112-1-Q1, S08108-1-Q1, S08101-2-Q1, S08101-3-Q1, S08101-4-Q1, S08105-1-Q1, S08102-1-Q1, S08107-1-Q1, S08109-1-Q1, and S08101-1 -Ql, or markers closely linked thereto (see, e.g., Figure 1). Other markers of linkage group L may also be used to identify the presence or absence of the gene, including other markers above marker SATT613.
  • the information disclosed herein regarding the QTL for tolerance or sensitivity to mesotrione and/or isoxazole herbicides which maps to soybean linkage group L is used to aid in the selection of breeding plants, lines, and populations containing tolerance or sensitivity to mesotrione and/or isoxazole herbicides for use in introgression of this trait into elite soybean germplasm, or germplasm of proven genetic superiority suitable for variety release.
  • nucleic acid markers mapping the QTL are used to select soybean plants containing the QTL. Plants so selected have a high probability of expressing the trait tolerance or sensitivity to mesotrione and/or isoxazole herbicides. Plants so selected can be used in a soybean breeding program.
  • the QTL associated with tolerance or sensitivity to mesotrione and/or isoxazole herbicides is introduced from plants identified using marker-assisted selection to other plants.
  • agronomically desirable plants and seeds can be produced containing the QTL associated with tolerance or sensitivity to mesotrione and/or isoxazole herbicides from germplasm containing the QTL.
  • Sources of tolerance or sensitivity to mesotrione and/or isoxazole herbicides are disclosed below.
  • donor soybean plants for a parental line containing the tolerance QTL are selected.
  • selection can be accomplished via nucleic acid marker-associated selection as explained herein.
  • Selected plant material may represent, among others, an inbred line, a hybrid, a heterogeneous population of soybean plants, or simply an individual plant.
  • this donor parental line is crossed with a second parental line.
  • the second parental line is a high yielding line. This cross produces a segregating plant population composed of genetically heterogeneous plants.
  • Plants of the segregating plant population are screened for the tolerance QTL and are subjected to further breeding.
  • This further breeding may include, among other techniques, additional crosses with other lines, hybrids, backcrossing, or self- crossing.
  • the result is a line of soybean plants that is tolerant to mesotrione and/or isoxazole herbicides, and also has other desirable traits, such as yield, from one or more other soybean lines.
  • nucleic acid markers mapping the QTL are used to select soybean plants containing the QTL. Plants so selected have a high probability of expressing the trait tolerance or sensitivity to mesotrione and/or isoxazole herbicides. Plants so selected can be used in a soybean breeding program.
  • the QTL associated with tolerance or sensitivity to mesotrione and/or isoxazole herbicides is introduced from plants identified using marker-assisted selection to other plants.
  • agronomically desirable plants and seeds can be produced containing the QTL associated with tolerance or sensitivity to mesotrione and/or isoxazole herbicides from germplasm containing the QTL.
  • Sources of tolerance or sensitivity to mesotrione and/or isoxazole herbicides are disclosed below. Soybean plants, seeds, tissue cultures, variants and mutants having tolerance or sensitivity to mesotrione and/or isoxazole herbicides produced by the foregoing methods are also provided.
  • compositions useful in the disclosed methods including polynucleotide primers and probes useful for detecting relevant markers, as well as kits containing the same.
  • Panel A provides an integrated genetic map of soybean markers on linkage group L (chromosome 19), including the marker type (SS or ASH/SNP).
  • the genetic map positions of the markers are indicated in centiMorgans (cM), typically with position zero being the first (most distal) marker on the chromosome.
  • the map includes relative positions for some markers for which higher resolution genetic mapping data was not available; no position in cM is provided for such markers.
  • Panel B provides a table listing genetic markers that are linked to the mesotrione and isoxazole herbicide tolerance and sensitivity markers identified on linkage group L. These markers are from the soybean public composite map of June 18, 2008 for linkage group L.
  • Figure 2 provides examples of primer and probe nucleic acid sequences that are useful for detecting SNP markers associated with tolerance, improved tolerance, or susceptibility/sensitivity to mesotrione and/or isoxazole herbicides.
  • Figure 3 provides examples of cultivars with vastly different mesotrione and isoxazole herbicide tolerance or sensitivity phenotype. Shown are field samples, with a non-tolerant variety in the center and tolerant varieties on the left and right (normal growth). SUMMARY OF THE SEQUENCES
  • SEQ ID NOs: 1-5 comprise nucleotide sequences of regions of the Soybean genome, each capable of being used as a probe or primer, either alone or in combination, for the detection of marker locus P10649C-3 on LG-L.
  • SEQ ID NOs: 1 and 2 are used as primers while SEQ ID NOs: 3-5 are used as probes.
  • SEQ ID NOs: 6-9 comprise nucleotide sequences of regions of the Soybean genome, each capable of being used as a probe or primer, either alone or in combination, for the detection of marker locus S00224-1 on LG-L.
  • SEQ ID NOs: 6 and 7 are used as primers while SEQ ID NOs: 8 and 9 are used as probes.
  • SEQ ID NOs: 10-13 comprise nucleotide sequences of regions of the Soybean genome, each capable of being used as a probe or primer, either alone or in combination, for the detection of marker locus P5467-1 on LG-N.
  • SEQ ID NOs: 10 and 11 are used as primers while SEQ ID NOs: 12 and 13 are used as probes.
  • SEQ ID NOs: 14-17 comprise nucleotide sequences of regions of the Soybean genome, each capable of being used as a probe or primer, either alone or in combination, for the detection of marker locus S08101-1 -Ql on LG-L.
  • SEQ ID NOs: 14 and 15 are used as primers while SEQ ID NOs: 16 and 17 are used as probes.
  • SEQ ID NOs: 18-21 comprise nucleotide sequences of regions of the Soybean genome, each capable of being used as a probe or primer, either alone or in combination, for the detection of marker locus S08101-2-Q1 on LG-L.
  • SEQ ID NOs: 18 and 19 are used as primers while SEQ ID NOs: 20 and 21 are used as probes.
  • SEQ ID NOs: 22-25 comprise nucleotide sequences of regions of the Soybean genome, each capable of being used as a probe or primer, either alone or in combination, for the detection of marker locus S08101-3-Q1 on LG-L.
  • SEQ ID NOs: 22 and 23 are used as primers while SEQ ID NOs: 24 and 25 are used as probes.
  • SEQ ID NOs: 26-29 comprise nucleotide sequences of regions of the Soybean genome, each capable of being used as a probe or primer, either alone or in combination, for the detection of marker locus S08101-4-Q1 on LG-L.
  • SEQ ID NOs: 26 and 27 are used as primers while SEQ ID NOs: 28 and 29 are used as probes.
  • SEQ ID NOs: 30-33 comprise nucleotide sequences of regions of the Soybean genome, each capable of being used as a probe or primer, either alone or in combination, for the detection of marker locus S08102-1-Q1 on LG-L.
  • SEQ ID NOs: 30 and 31 are used as primers while SEQ ID NOs: 32 and 33 are used as probes.
  • SEQ ID NOs: 34-37 comprise nucleotide sequences of regions of the Soybean genome, each capable of being used as a probe or primer, either alone or in combination, for the detection of marker locus S08103-1-Q1 on LG-L.
  • SEQ ID NOs: 34 and 35 are used as primers while SEQ ID NOs: 36 and 37 are used as probes.
  • SEQ ID NOs: 38-41 comprise nucleotide sequences of regions of the Soybean genome, each capable of being used as a probe or primer, either alone or in combination, for the detection of marker locus S08104-1-Q1 on LG-L.
  • SEQ ID NOs: 38 and 39 are used as primers while SEQ ID NOs: 40 and 41 are used as probes.
  • SEQ ID NOs: 42-45 comprise nucleotide sequences of regions of the Soybean genome, each capable of being used as a probe or primer, either alone or in combination, for the detection of marker locus S08105-1-Q1 on LG-L.
  • SEQ ID NOs: 42 and 43 are used as primers while SEQ ID NOs: 44 and 45 are used as probes.
  • SEQ ID NOs: 46-49 comprise nucleotide sequences of regions of the Soybean genome, each capable of being used as a probe or primer, either alone or in combination, for the detection of marker locus S08106-1-Q1 on LG-L.
  • SEQ ID NOs: 46 and 47 are used as primers while SEQ ID NOs: 48 and 49 are used as probes.
  • SEQ ID NOs: 50-53 comprise nucleotide sequences of regions of the Soybean genome, each capable of being used as a probe or primer, either alone or in combination, for the detection of marker locus S08107-1-Q1 on LG-L.
  • SEQ ID NOs: 50 and 51 are used as primers while SEQ ID NOs: 52 and 53 are used as probes.
  • SEQ ID NOs: 54-57 comprise nucleotide sequences of regions of the Soybean genome, each capable of being used as a probe or primer, either alone or in combination, for the detection of marker locus S08108-1-Q1 on LG-L.
  • SEQ ID NOs: 54 and 55 are used as primers while SEQ ID NOs: 56 and 57 are used as probes.
  • SEQ ID NOs: 58-61 comprise nucleotide sequences of regions of the Soybean genome, each capable of being used as a probe or primer, either alone or in combination, for the detection of marker locus S08109-1-Q1 on LG-L.
  • SEQ ID NOs: 58 and 59 are used as primers while SEQ ID NOs: 60 and 61 are used as probes.
  • SEQ ID NOs: 62-65 comprise nucleotide sequences of regions of the Soybean genome, each capable of being used as a probe or primer, either alone or in combination, for the detection of marker locus S08110-1-Q1 on LG-L.
  • SEQ ID NOs: 62 and 63 are used as primers while SEQ ID NOs: 64 and 65 are used as probes.
  • SEQ ID NOs: 66-69 comprise nucleotide sequences of regions of the Soybean genome, each capable of being used as a probe or primer, either alone or in combination, for the detection of marker locus S0813 1 -1-Ql on LG-L.
  • SEQ ID NOs: 66 and 67 are used as primers while SEQ ID NOs: 68 and 69 are used as probes.
  • SEQ ID NOs: 70-73 comprise nucleotide sequences of regions of the Soybean genome, each capable of being used as a probe or primer, either alone or in combination, for the detection of marker locus S08112-1-Ql on LG-L.
  • SEQ ID NOs: 70 and 71 are used as primers while SEQ ID NOs: 72 and 73 are used as probes.
  • SEQ ID NOs: 74-77 comprise nucleotide sequences of regions of the Soybean genome, each capable of being used as a probe or primer, either alone or in combination, for the detection of marker locus S08115-2-Q1 on LG-L.
  • SEQ ID NOs: 74 and 75 are used as primers while SEQ ID NOs: 76 and 77 are used as probes.
  • SEQ ID NOs: 78-81 comprise nucleotide sequences of regions of the Soybean genome, each capable of being used as a probe or primer, either alone or in combination, for the detection of marker locus S08116-1-Ql on LG-L.
  • SEQ ID NOs: 78 and 79 are used as primers while SEQ ID NOs: 80 and 81 are used as probes.
  • SEQ ID NOs: 82-85 comprise nucleotide sequences of regions of the Soybean genome, each capable of being used as a probe or primer, either alone or in combination, for the detection of marker locus S08117-1-Ql on LG-L.
  • SEQ ID NOs: 82 and 83 are used as primers while SEQ ID NOs: 84 and 85 are used as probes.
  • SEQ ID NOs: 86-89 comprise nucleotide sequences of regions of the Soybean genome, each capable of being used as a probe or primer, either alone or in combination, for the detection of marker locus S08118-1-Ql on LG-L.
  • SEQ ID NOs: 86 and 87 are used as primers while SEQ ID NOs: 88 and 89 are used as probes.
  • SEQ ID NOs: 90-93 comprise nucleotide sequences of regions of the Soybean genome, each capable of being used as a probe or primer, either alone or in combination, for the detection of marker locus S08119-1-Ql on LG-L.
  • SEQ ID NOs: 90 and 91 are used as primers while SEQ ID NOs: 92 and 93 are used as probes.
  • SEQ ID NOs: 94-97 comprise nucleotide sequences of regions of the Soybean genome, each capable of being used as a probe or primer, either alone or in combination, for the detection of marker locus S04867-1-A on LG-L.
  • SEQ ID NOs: 94 and 95 are used as primers while SEQ ID NOs: 96 and 97 are used as probes.
  • SEQ ID NOs: 98-101 comprise nucleotide sequences of regions of the Soybean genome, each capable of being used as a probe or primer, either alone or in combination, for the detection of marker locus S03859-1-A on LG-L.
  • SEQ ID NOs: 98 and 99 are used as primers while SEQ ID NOs: 100 and 101 are used as probes.
  • SEQ ID NOs: 102-105 comprise nucleotide sequences of regions of the Soybean genome, each capable of being used as a probe or primer, either alone or in combination, for the detection of marker locus S08010-1-Q1 on LG-L.
  • SEQ ID NOs: 102 and 103 are used as primers while SEQ ID NOs: 104 and 105 are used as probes.
  • SEQ ID NOs: 106-109 comprise nucleotide sequences of regions of the Soybean genome, each capable of being used as a probe or primer, either alone or in combination, for the detection of marker locus S08010-2-Q1 on LG-L.
  • SEQ ID NOs: 106 and 107 are used as primers while SEQ ID NOs: 108 and 109 are used as probes.
  • SEQ ED NOs: 110-113 comprise nucleotide sequences of regions of the Soybean genome, each capable of being used as a probe or primer, either alone or in combination, for the detection of marker locus S08114-1-Ql on LG-L.
  • SEQ ID NOs: 110 and 111 are used as primers while SEQ ID NOs: 112 and 113 are used as probes.
  • SEQ ID NOs: 114-117 comprise nucleotide sequences of regions of the Soybean genome, each capable of being used as a probe or primer, either alone or in combination, for the detection of marker locus S08113-1-Q1 on LG-L.
  • SEQ ID NOs: 114 and 115 are used as primers while SEQ ID NOs: 116 and 117 are used as probes.
  • SEQ ID Nos: 118-121 comprise nucleotide sequences of regions of the Soybean genome, each capable of being used as a probe or primer, either alone or in combination, for the detection of marker locus S08007-1-Q1 on LG-L.
  • SEQ ID Nos: 118-121 comprise nucleotide sequences of regions of the Soybean genome, each capable of being used as a probe or primer, either alone or in combination, for the detection of marker locus S08007-1-Q1 on LG-L.
  • SEQ ID Nos: 118-121 comprise nucleotide sequences of regions of the Soybean genome, each capable of being used as a probe or primer, either alone or in combination, for the detection of marker locus S08007-1-Q1 on LG-L.
  • SEQ ID Nos: 118-121 comprise nucleotide sequences of regions of the Soybean genome, each capable of being used as a probe or primer, either alone or in combination, for the detection of
  • SEQ ID NOs: 118 and 119 are used as primers while SEQ ID NOs: 120 and 121 are used as probes.
  • kits comprising one pair of oligonucleotide primers may have two or more pairs of oligonucleotide primers.
  • Agronomics refers to the traits (and underlying genetic elements) of a given plant variety that contribute to yield over the course of a growing season.
  • Individual agronomic traits include emergence vigor, vegetative vigor, stress tolerance, disease resistance or tolerance, insect resistance or tolerance, herbicide resistance or tolerance, branching, flowering, seed set, seed size, seed density, standability, threshability, and the like.
  • Allele means any of one or more alternative forms of a genetic sequence. In a diploid cell or organism, the two alleles of a given sequence typically occupy
  • allele refers to the specific nucleotide base present at that SNP locus in that individual plant.
  • amplifying in the context of nucleic acid amplification is any process whereby additional copies of a selected nucleic acid (or a transcribed form thereof) are produced.
  • Typical amplification methods include various polymerase based replication methods, including the polymerase chain reaction (PCR), ligase mediated methods, such as the ligase chain reaction (LCR), and RNA polymerase based amplification (e.g., by transcription) methods.
  • An "amplicon” is an amplified nucleic acid, e.g., a nucleic acid that is produced by amplifying a template nucleic acid by any available amplification method (e.g., PCR, LCR, transcription, or the like).
  • An "ancestral line” is a parent line used as a source of genes, e.g., for the development of elite lines.
  • An "ancestral population” is a group of ancestors that have contributed the bulk of the genetic variation that was used to develop elite lines.
  • Backcrossing is a process in which a breeder crosses a progeny variety back to one of the parental genotypes one or more times.
  • chromosome segment designates a contiguous linear span of genomic DNA that resides in planta on a single chromosome.
  • crossed means the fusion of gametes via pollination to produce progeny (e.g., cells, seeds or plants).
  • progeny e.g., cells, seeds or plants.
  • the term encompasses both sexual crosses (the pollination of one plant by another) and selfing (self-pollination, e.g., when the pollen and ovule are from the same plant).
  • Crop and “variety” are used synonymously and mean a group of plants within a species (e.g. , Glycine max) that share certain genetic traits that separate them from other possible varieties within that species. Soybean cultivars are inbred lines produced after several generations of self-pollinations. Individuals within a soybean cultivar are homogeneous, nearly genetically identical, with most loci in the homozygous state.
  • An "elite line” is an agronomically superior line that has resulted from many cycles of breeding and selection for superior agronomic performance. Numerous elite lines are available and known to those of skill in the art of soybean breeding. An "elite population” is an assortment of elite individuals or lines that can be used to represent the state of the art in terms of agronomically superior genotypes of a given crop species, such as soybean.
  • an "equivalent position" in a polynucleotide and/or polypeptide sequence is a position that correlates to a position in the reference sequence when the sequences are aligned for a maximum correspondence.
  • the sequences are aligned across their whole length using a global alignment program.
  • a portion of the sequence or sequences may be aligned using a local alignment program or a global alignment program, for example a sequence may comprise exons and introns, conserved motifs or domains, or functional motifs or domains which may be aligned to the reference sequence(s) to identify equivalent positions.
  • Equivalent positions in polynucleotides encoding a polypeptide can be determined using the encoded amino acid, and/or using a FrameAlign program to align the polynucleotide and polypeptide for maximal
  • polynucleotides or polypeptides refer to molecules that have been artificially supplied to a biological system (e.g., a plant cell, a plant gene, a particular plant species or a plant chromosome under study) and are not native to that particular biological system.
  • a biological system e.g., a plant cell, a plant gene, a particular plant species or a plant chromosome under study
  • the terms indicate that the relevant material originated from a source other than the naturally occurring source, or refers to molecules having a non-natural configuration, genetic location or arrangement of parts.
  • exogenous polynucleotides include polynucleotides from another organism or from the same organism which have been modified by linkage to a distinct non-endogenous polynucleotide and/or inserted to a distinct non-endogenous locus.
  • a “native” or “endogenous” gene is a gene that does not contain nucleic acid elements encoded by sources other than the chromosome or other genetic element on which it is normally found in nature.
  • An endogenous gene, transcript or polypeptide is encoded by its natural chromosomal locus, and not artificially supplied to the cell.
  • suitable vector e.g., naked linear DNA, plasmid, plastid, or virion
  • transiently expressed e.g., transfected mRNA
  • host cell means a cell that contains an exogenous nucleic acid, such as a vector, and supports the replication and/or expression of the nucleic acid.
  • Host celfs may be prokaryotic cells such as E. colt, or eukaryotic cells such as yeast, insect, amphibian or mammalian cells.
  • host cells are plant cells, including, but not limited to, dicot and monocot cells.
  • an “exotic soybean strain” or an “exotic soybean germplasm” is a strain or germplasm derived from a soybean not belonging to an available elite soybean line or strain of germplasm.
  • an exotic germplasm is not closely related by descent to the elite germplasm with which it is crossed. Most commonly, the exotic germplasm is not derived from any known elite line of soybean, but rather is selected to introduce novel genetic elements (typically novel alleles) into a breeding program.
  • a "genetic map” is a description of genetic linkage relationships among loci on one or more chromosomes (or linkage groups) within a given species, generally depicted in a diagrammatic or tabular form.
  • Gene refers to the genetic constitution of a cell or organism.
  • Germplasm means the genetic material that comprises the physical foundation of the hereditary qualities of an organism. As used herein, germplasm includes seeds and living tissue from which new plants may be grown; or, another plant part, such as leaf, stem, pollen, or cells, that may be cultured into a whole plant. Germplasm resources provide sources of genetic traits used by plant breeders to improve commercial cultivars.
  • An individual is “homozygous” if the individual has only one type of allele at a given locus (e.g., a diploid individual has a copy of the same allele at a locus for each of two homologous chromosomes).
  • An individual is “heterozygous” if more than one allele type is present at a given locus (e.g., a diploid individual with one copy each of two different alleles).
  • the term “homogeneity” indicates that members of a group have the same genotype at one or more specific loci. In contrast, the term “heterogeneity” is used to indicate that individuals within the group differ in genotype at one or more specific loci.
  • Haplotype means a combination of sequence polymorphisms that are located closely together on the same chromosome and that can discriminate between different genotypes.
  • the combination represented by the haplotype tends to be inherited together, and this combination may represent sequence differences or alleles within a region.
  • the region may contain one gene, or more than one gene.
  • homologous refers to nucleic acid sequences that are derived from a common ancestral gene through natural or artificial processes (e.g., are members of the same gene family), and thus, typically share sequence similarity.
  • homologous nucleic acids have sufficient sequence identity that one of the sequences or a subsequence thereof or its complement is able to selectively hybridize to the other under selective (e.g. , stringent) hybridization conditions.
  • the term "selectively hybridizes" includes reference to hybridization, under stringent hybridization conditions, of a nucleic acid sequence to a specified nucleic acid target sequence to a detectably greater degree (e.g., at least 2-fold over background) than its hybridization to non-target nucleic acid sequences and to the substantial exclusion of non-target nucleic acids.
  • Selectively hybridizing nucleic acid sequences typically have about at least 70% sequence identity, at least 80% sequence identity, or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.75%, or 100% sequence identity with each other.
  • a nucleic acid that exhibits at least some degree of homology to a reference nucleic acid can be unique or identical to the reference nucleic acid or its complementary sequence.
  • introgression refers to the transmission of a desired allele of a genetic locus from one genetic background to another by sexual crossing, transgenic means, or any other means known in the art.
  • introgression of a desired allele at a specified locus can be transmitted to at least one progeny plant via a sexual cross between two parent plants, at least one of the parent plants having the desired allele within its genome.
  • transmission of an allele can occur by recombination between two donor genomes, e.g., in a fused protoplast, where at least one of the donor protoplasts has the desired allele in its genome.
  • the desired allele can be, e.g. , a transgene or a gene allele that imparts resistance to a plant pathogen.
  • isolated refers to material, such as polynucleotides or polypeptides, which are identified and separated from at least one contaminant with which it is ordinarily associated in its natural or original source. Furthermore, an isolated polynucleotide or polypeptide is typically present in a form or setting that is different from the form or setting that is normally found in nature. In some examples, the isolated molecule is substantially free from components that normally accompany or interact with it in its naturally occurring environment. In some embodiments, the isolated material optionally comprises material not found with the material in its natural environment, e.g., in a cell.
  • a “line” or “strain” is a group of individuals of identical parentage that are generally inbred to some degree and that are generally homozygous and homogeneous at most loci (isogenic or near isogenic).
  • a “subline” refers to an inbred subset of descendents that are genetically distinct from other similarly inbred subsets descended from the same progenitor. Traditionally, a subline has been derived by inbreeding the seed from an individual soybean plant selected at the F3 to F5 generation until the residual segregating loci are "fixed” or homozygous across most or all loci.
  • soybean varieties are typically produced by aggregating ("bulking") the self-pollinated progeny of a single F3 to F5 plant from a controlled cross between two genetically different parents. While the variety typically appears uniform, the self-pollinating variety derived from the selected plant eventually (e.g., F8) becomes a mixture of homozygous plants that can vary in genotype at any locus that was heterozygous in the originally selected F3 to F5 plant.
  • Marker-based sublines that differ from each other based on qualitative polymorphism at the DNA level at one or more specific marker loci are derived by genotyping a sample of seed derived from individual self-pollinated progeny derived from a selected F3-F5 plant.
  • the seed sample can be genotyped directly as seed, or as plant tissue grown from such a seed sample.
  • seed sharing a common genotype at the specified locus (or loci) are bulked providing a subline that is genetically homogenous at identified loci important for a trait of interest (e.g., yield, tolerance, etc.).
  • Linkage refers to a phenomenon wherein alleles on the same chromosome tend to segregate together more often than expected by chance if their transmission was independent. Genetic recombination occurs with an assumed random frequency over the entire genome. Genetic maps are constructed by measuring the frequency of recombination between pairs of traits or markers. The closer the traits or markers lie to each other on the chromosome, the lower the frequency of recombination, and the greater the degree of linkage. Traits or markers are considered herein to be linked if they generally co-segregate. A 1/100 probability of recombination per generation is defined as a map distance of 1.0 centiMorgan (1.0 cM).
  • 1 cM correlates, on average, to about 400,000 base pairs (400 Kb).
  • the genetic elements or genes located on a single chromosome segment are physically linked.
  • the two loci are located in close proximity such that recombination between homologous chromosome pairs does not occur between the two loci during meiosis with high frequency, e.g., such that linked loci co-segregate at least about 90% of the time, e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.75%, or more of the time.
  • the genetic elements located within a chromosome segment are also genetically linked, typically within a genetic recombination distance of less than or equal to 50 centimorgans (cM), e.g., about 49, 40, 30, 20, 10, 5, 4, 3, 2, 1, 0.75, 0.5, or 0.25 cM or less. That is, two genetic elements within a single chromosome segment undergo recombination during meiosis with each other at a frequency of less than or equal to about 50%, e.g., about 49%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, 1%, 0.75%, 0.5%, or 0.25% or less. Closely linked markers display a cross over frequency with a given marker of about 10% or less (the given marker is within about lOcM of a closely linked marker). Put another way, closely linked loci co-segregate at least about 90% of the time.
  • cM centimorgans
  • coupling phase linkage indicates the state where the "favorable” allele at the resistance locus is physically associated on the same chromosome strand as the "favorable” allele of the respective linked marker locus.
  • both favorable alleles are inherited together by progeny that inherit that chromosome strand.
  • the "favorable” allele at the locus of interest e.g., a QTL for resistance
  • the two "favorable” alleles are not inherited together (i.e., the two loci are "out of phase” with each other).
  • Linkage disequilibrium refers to a phenomenon wherein alleles tend to remain together in linkage groups when segregating from parents to offspring, with a greater frequency than expected from their individual frequencies.
  • Linkage group refers to traits or markers that generally co-segregate.
  • a linkage group generally corresponds to a chromosomal region containing genetic material that encodes the traits or markers.
  • Locus is a defined segment of DNA.
  • a “map location” is an assigned location on a genetic map relative to linked genetic markers where a specified marker can be found within a given species. Markers are frequently described as being “above” or “below” other markers on the same linkage group; a marker is “above” another marker if it appears earlier on the linkage group, whereas a marker is “below” another marker if it appears later on the linkage group.
  • Mapping is the process of defining the linkage relationships of loci through the use of genetic markers, populations segregating for the markers, and standard genetic principles of recombination frequency.
  • Marker or “molecular marker” is a term used to denote a nucleic acid or amino acid sequence that is sufficiently unique to characterize a specific locus on the genome. Examples include Restriction Fragment Length Polymorphisms (RFLPs), Single Sequence Repeats (SSRs), Target Region Amplification Polymorphisms (TRAPs), Isozyme
  • Electrophoresis Randomly Amplified Polymorphic DNAs (RAPDs), Arbitrarily Primed Polymerase Chain Reaction (AP-PCR), DNA Amplification Fingerprinting (DAF), Sequence Characterized Amplified Regions (SCARs), Amplified Fragment Length Polymorphisms (AFLPs), and Single Nucleotide Polymorphisms (SNPs).
  • RAPDs Randomly Amplified Polymorphic DNAs
  • AP-PCR Arbitrarily Primed Polymerase Chain Reaction
  • DAF DNA Amplification Fingerprinting
  • SCARs Sequence Characterized Amplified Regions
  • AFLPs Amplified Fragment Length Polymorphisms
  • SNPs Single Nucleotide Polymorphisms
  • the map positions provide a measure of the relative positions of particular markers with respect to one another.
  • a trait is stated to be linked to a given marker it will be understood that the actual DNA segment whose sequence affects the trait generally co-segregates with the marker. More precise and definite localization of a trait can be obtained if markers are identified on both sides of the trait.
  • the existence of the trait can be detected by relatively simple molecular tests without actually evaluating the appearance of the trait itself, which can be difficult and time-consuming because the actual evaluation of the trait requires growing plants to a stage and/or under specific conditions where the trait can be expressed.
  • Molecular markers have been widely used to determine genetic composition in soybeans.
  • Marker assisted selection refers to the process of selecting a desired trait or desired traits in a plant or plants by detecting one or more molecular markers from the plant, where the molecular marker is linked to the desired trait.
  • plant includes reference to an immature or mature whole plant, including a plant from which seed or grain or anthers have been removed. Seed or embryo that will produce the plant is also considered to be the plant.
  • plant cell includes, without limitation, cells within or derived from, for example and without limitation, plant seeds, plant tissue suspension cultures, plant tissue, plant tissue explants, plant embryos, meristematic tissue, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen and microspores.
  • Plant parts means any portion or piece of a plant, including leaves, stems, buds, roots, root tips, anthers, seed, grain, embryo, pollen, ovules, flowers, cotyledons, hypocotyls, pods, flowers, shoots, stalks, tissues, tissue cultures, cells and the like.
  • Polymorphism means a change or difference between two related nucleic acids.
  • a “nucleotide polymorphism” refers to a nucleic acid comprising at least one nucleotide difference when compared to a related sequence when the two nucleic acids are aligned for maximal correspondence.
  • a “genetic nucleotide polymorphism” refers to a nucleic acid comprising at least one nucleotide difference when compared to a related sequence when the two nucleic acids are aligned for maximal correspondence, where the two nucleic acids are genetically related, i.e., homologous, for example, where the nucleic acids are isolated from different strains of a soybean plant, or from different alleles of a single strain, or the like.
  • Polynucleotide “polynucleotide sequence,” “nucleic acid sequence,” “nucleic acid fragment,” and “oligonucleotide” are used interchangeably herein. These terms encompass nucleotide sequences and the like.
  • a polynucleotide may be a polymer of RNA or DNA. that is single- or double-stranded, that optionally contains synthetic, non-natural, or altered nucleotide bases.
  • a polynucleotide in the form of a polymer of DNA may be comprised of one or more strands of cDNA, genomic DNA, synthetic DNA, or mixtures thereof:
  • “Positional cloning” is a cloning procedure in which a target nucleic acid is identified and isolated by its genomic proximity to marker nucleic acid.
  • a genomic nucleic acid clone can include part or all of two more chromosomal regions that are proximal to one another. If a marker can be used to identify the genomic nucleic acid clone from a genomic library, standard methods such as sub-cloning or sequencing can be used to identify and or isolate subsequences of the clone that are located near the marker.
  • Primer refers to an oligonucleotide (synthetic or occurring naturally), which is capable of acting as a point of initiation of nucleic acid synthesis or replication along a complementary strand when placed under conditions in which synthesis of a
  • primers are any organic radicals that are complementary to a polymerase.
  • primers are organic radicals.
  • oligonucleotides from 10 to 30 nucleic acids in length, but longer or shorter sequences can be employed.
  • Primers may be provided in double-stranded form, though the single- stranded form is preferred.
  • a primer can further contain a detectable label, for example a 5' end label.
  • Probe refers to an oligonucleotide (synthetic or occurring naturally) that is complementary (though not necessarily fully complementary) to a polynucleotide of interest and forms a duplexed structure by hybridization with at least one strand of the polynucleotide of interest.
  • probes are oligonucleotides from 10 to 50 nucleic acids in length, but longer or shorter sequences can be employed.
  • a probe can further contain a detectable label.
  • label and “detectable label” refer to a molecule capable of detection, including, but not limited to, radioactive isotopes, fluorescers, chemiluminescers, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, chromophores, dyes, metal ions, metal sols, semiconductor nanocrystals, ligands (e.g., biotin, avidin, streptavidin, or haptens), and the like.
  • a detectable label can also include a combination of a reporter and a quencher, such as are employed in FRET probes or
  • reporter refers to a substance or a portion thereof which is capable of exhibiting a detectable signal, which signal can be suppressed by a quencher.
  • the detectable signal of the reporter is, e.g., fluorescence in the detectable range.
  • quencher refers to a substance or portion thereof which is capable of suppressing, reducing, inhibiting, etc., the detectable signal produced by the reporter.
  • quenching and “fluorescence energy transfer” refer to the process whereby, when a reporter and a quencher are in close proximity, and the reporter is excited by an energy source, a substantial portion of the energy of the excited state nonradiatively transfers to the quencher where it either dissipates nonradiatively or is emitted at a different emission wavelength than that of the reporter.
  • RAPD marker means random amplified polymorphic DNA marker. Chance pairs of sites complementary to single octa- or decanucleotides may exist in the correct orientation and close enough to one another for PCR amplification. With some randomly chosen decanucleotides no sequences are amplified. With others, the same length products are generated from DNAs of different individuals. With still others, patterns of bands are not the same for every individual in a population. The variable bands are commonly called random amplified polymorphic DNA (RAPD) bands.
  • RAPD random amplified polymorphic DNA
  • recombinant indicates that the material (e.g., a recombinant nucleic acid, gene, polynucleotide or polypeptide) has been altered by human intervention.
  • the arrangement of parts of a recombinant molecule is not a native
  • the alteration to yield the recombinant material can be performed on the material within or removed from its natural environment or state.
  • a naturally occurring nucleic acid becomes a recombinant nucleic acid if it is altered, or if it is transcribed from DNA which has been altered, by means of human intervention performed within the cell from which it originates.
  • a gene sequence open reading frame is recombinant if that nucleotide sequence has been removed from it natural text and cloned into any type of artificial nucleic acid vector.
  • Protocols and reagents to produce recombinant molecules, especially recombinant nucleic acids are common and routine in the art (see, e.g., aniatis et al. (eds.), Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY, [1982]; Sambrook et al. (eds.), Molecular Cloning: A Laboratory Manual, Second Edition, Volumes 1-3, Cold Spring Harbor Laboratory Press, NY, [1989]; and Ausubel et al. (eds.), Current Protocols in Molecular Biology, Vol. 1-4, John Wiley & Sons, Inc., New York [1994]).
  • recombinant can also refer to an organism that harbors a recombinant material, e.g., a plant that comprises a recombinant nucleic acid is considered a recombinant plant.
  • a recombinant organism is a transgenic organism.
  • Recombination frequency is the frequency of a crossing over event
  • a marker locus is "associated with" another marker locus or some other locus (for example, a tolerance locus), when the relevant loci are part of the same linkage group and are in linkage disequilibrium. This occurs when the marker locus and a linked locus are found together in progeny plants more frequently than if the two loci segregated randomly.
  • a marker locus can also be associated with a trait, e.g., a marker locus can be "associated with tolerance or improved tolerance,” when the marker locus is in linkage disequilibrium with the trait.
  • RFLP restriction fragment length polymorphism. Any sequence change in DNA, including a single base substitution, insertion, deletion or inversion, can result in loss or gain of a restriction endonuclease recognition site. The size and number of fragments generated by one such enzyme is therefore altered. A probe that hybridizes specifically to DNA in the region of such an alteration can be used to rapidly and specifically identify a region of DNA that displays allelic variation between two plant varieties. Isozyme Electrophoresis and RFLPs have been widely used to determine genetic composition
  • SNP single nucleotide polymorphism
  • A, T, C, or G single nucleotide sequence
  • SNP markers exist when SNPs are mapped to sites on the soybean genome. Many techniques for detecting SNPs are known in the art, including allele specific hybridization, primer extension, direct sequencing, and real-time PCR, such as the TaqManTM assay.
  • SSR short sequence repeats.
  • SSR markers are genetic markers based on polymorphisms in repeated nucleotide sequences, such as microsatellites. A marker system based on SSRs can be highly informative in linkage analysis relative to other marker systems in that multiple alleles may be present.
  • the PCR detection is done by use of two oligonucleotide primers flanking the polymorphic segment of repetitive DNA. Repeated cycles of heat denaturation of the DNA followed by annealing of the primers to their complementary sequences at low temperatures, and extension of the annealed primers with DNA polymerase, comprise the major part of the methodology.
  • Transgenic plant refers to a plant that comprises within its cells an exogenous polynucleotide, e.g., a polynucleotide from another organism (including a polynucleotide from another soybean plant).
  • exogenous polynucleotide e.g., a polynucleotide from another organism (including a polynucleotide from another soybean plant).
  • the exogenous polynucleotide is stably integrated within a genome such that the polynucleotide is passed on to successive generations.
  • the exogenous polynucleotide may be integrated into the genome alone or as part of a recombinant expression cassette.
  • Transgenic is used herein to refer to any cell, cell line, callus, tissue, plant part, or plant, the genotype of which has been altered by the presence of exogenous nucleic acid including those transgenic organisms or cells initially so altered, as well as those created by crosses or asexual propagation from the initial transgenic organism or cell.
  • the term "transgenic” as used herein does not encompass the alteration of the genome (chromosomal or extra-chromosomal) by conventional plant breeding methods (e.g., crosses) or by naturally occurring events such as random cross-fertilization, non- recombinant viral infection, non-recombinant bacterial transformation, non-recombinant transposition, or spontaneous mutation.
  • TRAP marker means target region amplification polymorphism marker.
  • TRAP technique employs one fixed primer of known sequence in combination with a random primer to amplify genomic fragments. The differences in fragments between alleles can be detected by gel electrophoresis.
  • vector is used in reference to polynucleotide or other molecules that transfer nucleic acid segment(s) into a cell.
  • a vector optionally comprises parts which mediate vector maintenance and enable its intended use (e.g., sequences necessary for replication, genes imparting drug or antibiotic resistance, a multiple cloning site, operably linked promoter/enhancer elements which enable the expression of a cloned gene, etc.).
  • Vectors are often derived from plasmids, bacteriophages, or plant or animal viruses.
  • Isoxazole is used as a pre-plant herbicide while mesotrione is used as either a pre-plant or post-emergent herbicide.
  • Isoxazole is member of the isoxazole chemical family.
  • isoxazole is rapidly converted to a diketonitrile derivative (2-cyclopropyI-3 -(2-mesyl-4-trifluoromethylphenyl)-3 -oxopropanenitrile) by opening of the isoxazole ring.
  • This diketonitrile undergoes degradation to a benzoic acid derivative (2-mesyl-4-trifiuoromethyl benzoic acid) in treated plants and the extent of this degradation is correlated to the degree of susceptibility, being most rapid in tolerant plants and slowest in susceptible plants.
  • Mesotrione belongs to the triketone family of herbicides, which are chemically derived from a natural phytotoxin produced by the bottlebrush plant Callistemon citrinus. Mesotrione works by inhibiting HPPD (p-hydroxyphenylpyruvate dioxygenase), an essential enzyme in the biosynthesis of carotenoids. Carotenoids protect chlorophyll from excess light energy.
  • HPPD p-hydroxyphenylpyruvate dioxygenase
  • Linkage analysis is the well-characterized descriptive framework of how traits are grouped together based upon the frequency with which they segregate together. Because chromosomal distance is approximately proportional to the frequency of crossing over events between traits, there is an approximate physical distance that correlates with recombination frequency.
  • Marker loci are traits, and can be assessed according to standard linkage analysis by tracking the marker loci during segregation. Thus, one cM is equal to a 1% chance that a marker locus will be separated from another locus (which can be any other trait, e.g., another marker locus, or another trait locus that encodes a QTL), due to crossing over in a single generation. Any detectible polymorphic trait can be used as a marker so long as it is inherited differentially and exhibits linkage disequilibrium with a phenotypic trait of interest. A number of soybean markers have been mapped and linkage groups created, as described in Cregan, P.B.
  • QTL quantitative trait loci
  • the genetic distance is also typically related to the physical distance between the marker and the QTL; however, certain biological phenomenon (including recombinational "hot spots") can affect the relationship between physical distance and genetic distance.
  • certain biological phenomenon including recombinational "hot spots"
  • the usefulness of a molecular marker is determined by the genetic and physical distance between the marker and the selectable trait of interest.
  • the method for determining the presence or absence of a QTL associated with tolerance or sensitivity to mesotrione and/or isoxazole herbicides in soybean germplasm comprises analyzing genomic DNA from a soybean germplasm for the presence of at least one molecular marker, wherein at least one molecular marker is linked to the QTL, and wherein the QTL maps to soybean major linkage group L and is associated with tolerance or sensitivity to mesotrione and/or isoxazole herbicides.
  • the term "is associated with” in this context means that the QTL associated with tolerance or sensitivity to mesotrione and/or isoxazole herbicides has been found to be present in soybean plants showing tolerance or sensitivity to mesotrione and/or isoxazole herbicides as described herein.
  • Marker loci are especially useful when they are closely linked to target loci ⁇ e.g., QTL for tolerance, or, alternatively, simply other marker loci, such as those identified herein, that are linked to such QTL) for which they are being used as markers.
  • a marker more closely linked to a target locus is a better indicator for the target locus (due to the reduced cross-over frequency between the target locus and the marker).
  • closely linked loci such as a marker locus and a second locus ⁇ e.g., a given marker or a QTL) display an inter-locus cross-over frequency of about 10% or less, about 9% or less, about 8% or less, about 7% or less, about 6% or less, about 5% or less, about 4% or less, about 3% or less, or about 2% or less.
  • the relevant loci e.g. , a marker locus and a target locus such as a QTL
  • the loci are about 10 cM, 9 cM, 8 cM, 7 cM, 6 cM, 5 cM, 4 cM, 3 cM, 2cM, lcM, 0.75 cM, 0.5 cM or 0.25 cM or less apart.
  • two loci that are localized to the same chromosome, and at such a distance that recombination between the two loci occurs at a frequency of no more than 10% (e.g., about 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.75%, 0.5%, 0.25%, or less) are said to be proximal to each other.
  • marker alleles can be detected or selected for or against.
  • one, two, three, or more marker allele(s) can be identified in or introgressed into the plant. Plants or germplasm frequently are identified that have at least one favorable allele that positively correlates with tolerance or improved tolerance. However, it is useful for exclusionary purposes during breeding to also identify alleles that negatively correlate with tolerance, to eliminate such plants or germplasm from subsequent rounds of breeding.
  • the identification of favorable marker alleles may be germplasm-specific. The determination of which marker alleles correlate with tolerance (or non-tolerance) is determined for the particular germplasm under study.
  • tolerance or non-tolerance
  • markers disclosed herein have been found to be associated with or to correlate with tolerance, improved tolerance, or susceptibility/sensitivity to mesotrione and/or isoxazole herbicides in soybean. Generally, markers that map closer to the QTL mapped to linkage group L and associated with tolerance or sensitivity to mesotrione and/or isoxazole herbicides are superior to markers that map farther from the QTL.
  • Additional useful and/or relevant markers include S03859-1-A, S08103-1-Q1, S08104-1-Q1, S08106-1-Q1, S08110-1-Q1, S08111-1-Q1, S08U5-2-Q1, S08117-1-Q1, S081 19-1-Ql, S08118-1-Q1, S08116-1-Q1, S08114-1-Q1, S08113-1-Q1, S08112-1-Q1, S08108-1-Q1, S08101-2-Q1, S08101-3-Q1, S08101-4-Q1, S08105-1-Q1, S08102-1-Q1, S08107-1-Q1, S08109-1-Q1, and S08101-1-Q1.
  • Any marker assigned to soybean linkage group L and linked or closely linked to a marker disclosed herein as associated with tolerance or sensitivity to mesotrione and/or isoxazole herbicides may be used.
  • a linked marker is within 50 cM of the referenced marker or trait
  • a closely linked marker is within 10 cM of the referenced marker or trait.
  • Updated information regarding markers assigned to soybean linkage group L may be found on the USDA's Soybase website. Further, linkage group L is now formally referred to as chromosome #19.
  • Intervals defined by markers flanking the QTL associated with tolerance or sensitivity to mesotrione and/or isoxazole herbicides are useful, as well.
  • the genomic DNA of soybean germplasm is typically tested for the presence of at least two of the foregoing molecular markers, one marker on each side of the QTL.
  • intervals include the interval flanked by and including SATT613 and above on LG-L, the interval flanked by and including markers SATT495 and SATT613, the interval flanked by and including SATT313 and above on LG-L, the interval flanked by and including markers SATT495 and SATT313, the interval flanked by and including markers SATT495 and SATT388, the interval flanked by and including markers P10649C- 3 and S ATT 182, the interval flanked by and including markers S04867-1-A and S03859-1- A, the interval flanked by and including markers S08110-1-Ql and S08010-1-Q1, the interval flanked by and including markers S08117-1-Ql and S08010-1-Q1, the interval flanked by and including markers S08110-1-Q1 and S08105-1-Q1, the interval flanked by and including markers S08117-1-Ql and S08105-1-Q1, and the interval flanked by and including markers S08117-1-
  • Initial fine mapping isolated the location of the QTL associated with herbicide tolerance/sensitivity to a -56 kb interval between marker S08117-1-Ql and S08105-1-Q1 on linkage group L. Further fine mapping refined the location of the QTL to a -44 kb interval between marker S08113-1-Q1 and S08105-1-Q1 on linkage group L.
  • markers that map within the interval defined by and including these markers are particularly useful for selecting for this QTL.
  • These markers include S08117-1-Ql, S08119-1-Q1, S08118-1-Q1, S08U6-1-Q1, S08101-1-Q1, S08112-1-Q1, S08108-1-Q1, S08101-1-Q1, S08101-2-Q1, S08101-3-Q1, S08101-4-Q1, and S08105-1-Q1.
  • the markers are S08112-1-Q1, S08108-1-Q1, S08101-2-Q1, S08101-3-Q1, and S08101-4-Q1.
  • Methods of introgressing tolerance to mesotrione and/or isoxazole herbicides into non-tolerant or less-tolerant soybean germplasm are provided. Any method for introgressing QTLs into soybean plants can be used.
  • a first soybean germplasm that contains tolerance or sensitivity to mesotrione and/or isoxazole herbicides derived from the QTL mapped to linkage group L which is associated with tolerance or sensitivity to mesotrione and/or isoxazole herbicides and a second soybean germplasm that lacks tolerance or sensitivity to mesotrione and/or isoxazole herbicides derived from the QTL mapped to linkage group L are provided.
  • the first soybean plant may be crossed with the second soybean plant to provide progeny soybeans.
  • Phenotypic and/or marker screening is performed on the progeny plants to determine the presence of tolerance or sensitivity to mesotrione and/or isoxazole herbicides derived from the QTL mapped to linkage group L. Progeny that test positive for the presence of tolerance or sensitivity to mesotrione and/or isoxazole herbicides derived from the QTL mapped to linkage group L can be selected.
  • Amplification primers for amplifying marker loci and suitable marker probes to detect marker loci or to genotype SNP alleles are provided, for example, in Figure 2 and the related sequence listing (SEQ ID NOs: 1-121).
  • other sequences to either side of the given primers can be used in place of the given primers, so long as the primers can amplify a region that includes the allele to be detected.
  • the precise probe to be used for detection can vary, e.g., any probe that can identify the region of a marker amplicon to be detected can be substituted for those examples provided herein.
  • the configuration of the amplification primers and detection probes can, of course, vary. Thus, the invention is not limited to the primers and probes specifically recited herein.
  • Systems including automated systems for selecting plants that comprise a marker of interest and/or for correlating presence of the marker with tolerance are also provided. These systems can include probes relevant to marker locus detection, detectors for detecting labels on the probes, appropriate fluid handling elements and temperature controllers that mix probes and templates and/or amplify templates, and systems and/or instructions that correlate label detection to the presence of a particular marker locus or allele.
  • Kits are also provided.
  • a kit can include appropriate primers or probes for detecting tolerance associated marker loci and instructions for using the primers or probes for detecting the marker loci and correlating the loci with predicted tolerance to mesotrione and/or isoxazole herbicides.
  • the kits can further include packaging materials for packaging the probes, primers, or instructions; controls, such as control amplification reactions that include probes, primers, or template nucleic acids for amplifications;
  • molecular size markers or the like.
  • Isolated nucleic acid fragments comprising a nucleic acid sequence coding for soybean tolerance or sensitivity to mesotrione and/or isoxazole herbicides.
  • the nucleic acid fragment comprises at least a portion of a nucleic acid belonging to linkage group L.
  • the nucleic acid fragment is capable of hybridizing under stringent conditions to a nucleic acid of a soybean cultivar tolerant to mesotrione and/or isoxazole.
  • Seed of a soybean produced by crossing a soybean variety having mesotrione and/or isoxazole herbicide tolerance QTL located on major linkage group L in its genome with another soybean variety, and progeny thereof, are provided.
  • Any suitable detection method known in the art can be used to detect the markers, QTL, or traits discussed herein. ⁇ some examples, the presence of marker loci is directly detected in unamplified genomic DNA by performing a Southern blot on a sample of genomic DNA using probes to the marker loci. In other examples, amplification based techniques are employed. PCR, RT-PCR, and LCR are in particularly broad use as amplification and amplification-detection methods for amplifying nucleic acids of interest, thus facilitating detection of markers.
  • molecular markers are detected by any established method available, including, without limitation, allele specific hybridization (ASH), real-time PCR assays for detecting single nucleotide polymorphisms (SNP), amplified fragment length
  • ASH allele specific hybridization
  • SNP single nucleotide polymorphisms
  • AFLP amplified variable sequence detection
  • RAPD randomly amplified polymorphic DNA
  • RFLP restriction fragment length polymorphism
  • SSR simple sequence repeat
  • SSCP single-strand conformation polymorphisms
  • isozyme markers detection or the like. While the exemplary markers provided in the tables herein are either SSR or SNP markers, any of the aforementioned marker types can be employed to identify chromosome segments encompassing genetic element that contribute to superior agronomic performance (e.g., tolerance or improved tolerance).
  • the presence or absence of a molecular marker is determined by nucleotide sequencing of the polymorphic marker region.
  • This method is readily adapted to high throughput analysis, as are the other methods noted above, e.g. , using available high throughput sequencing methods such as sequencing by hybridization.
  • the majority of genetic markers rely on one or more properties of nucleic acids for their detection.
  • some techniques for detecting genetic markers utilize hybridization of a probe nucleic acid to nucleic acids corresponding to the genetic marker (e.g., amplified nucleic acids produced using genomic soybean DNA as a template).
  • Hybridization formats including but not limited to solution phase, solid phase, mixed phase, or in situ hybridization assays are useful for allele detection. An extensive guide to the hybridization of nucleic acids is found in Tijssen (1993) Laboratory
  • markers that comprise restriction fragment length polymorphisms are described.
  • RFLP RFLP
  • the restriction enzyme is selected to provide restriction fragments of at least two alternative (or polymorphic) lengths in different individuals or populations. Determining one or more restriction enzymes that produce informative fragments for each cross is a simple procedure.
  • the labeled probe After separation by length in an appropriate matrix (e.g., agarose, polyacrylamide, etc.) and transfer to a membrane (e.g., nitrocellulose, nylon, etc.), the labeled probe is hybridized under conditions which result in equilibrium binding of the probe to the target followed by removal of excess probe by washing.
  • an appropriate matrix e.g., agarose, polyacrylamide, etc.
  • a membrane e.g., nitrocellulose, nylon, etc.
  • molecular markers are detected using a suitable PCR-based detection method.
  • a suitable PCR-based detection method This includes methods where the size or sequence of the PCR amplicon is indicative of the absence or presence of the marker (e.g., a particular marker allele), as well as methods where a labeled allele-specific probe is used for detection (e.g., a TaqMan® assay).
  • PCR primers and, optionally, probes are hybridized to the conserved regions flanking the polymorphic marker region.
  • Suitable primers can be designed using any suitable method. It is not intended that the invention be limited to any particular primer or primer pair.
  • primers can be designed using any suitable software program, such as LASERGENE ® .
  • primers are labeled by any suitable means (e.g., using a nonradioactive fluorescent tag) to allow for rapid visualization of the different size amplicons following an amplification reaction without any additional labeling step or visualization step.
  • the primers are not labeled, and the amplicons are visualized following their size resolution, e.g., following agarose gel electrophoresis.
  • ethidium bromide staining of the PCR amplicons following size resolution allows visualization of the different size amplicons.
  • marker amplification produces an amplicon at least 20 nucleotides in length, or alternatively, at least 50 nucleotides in length, or alternatively, at least 100 nucleotides in length, or alternatively, at least 200 nucleotides in length, or up to and including the full length of the amplicon.
  • Nucleic acid probes to the marker loci can also be cloned and/or synthesized. Any suitable label can be used with a probe.
  • Detectable labels suitable for use with nucleic acid probes include, for example, any composition detectable by spectroscopic, radioisotopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means.
  • Useful labels include biotin for staining with labeled streptavidin conjugate, magnetic beads, fluorescent dyes, radiolabels, enzymes, and colorimetric labels.
  • Other labels include ligands, which bind to antibodies labeled with fluorophores, chemiluminescent agents, and enzymes.
  • a probe can also constitute radiolabelled PCR primers that are used to generate a radiolabelled amplicon.
  • Separate detection probes can also be omitted in amplification/detection methods, e.g., by performing a real time amplification reaction that detects product formation by modification of the relevant amplification primer upon incorporation into a product, incorporation of labeled nucleotides into an amplicon, or by monitoring changes in molecular rotation properties of amplicons as compared to unamplified precursors (e.g., by fluorescence polarization).
  • in silico methods can be used to detect the marker loci of interest.
  • the sequence of a nucleic acid comprising the marker locus of interest can be stored in a computer.
  • the desired marker locus sequence or its homolog can be identified using an appropriate nucleic acid search algorithm as provided by, for example, in such readily available programs as BLAST, or even simple word processors.
  • a molecular beacon is an oligonucleotide or peptide nucleic acid (PNA) which, under appropriate hybridization conditions, self-hybridizes to form a stem and loop structure.
  • the MB has a label and a quencher at the termini of the oligonucleotide or PNA; thus, under conditions that permit intra-molecular hybridization, the label is typically quenched (or at least altered in its fluorescence) by the quencher.
  • the MB label is unquenched and signal is detected.
  • Standard methods of making and using MBs are known and MBs and reagents are commercially available. See also, e.g., Leone et al. (1995) "Molecular beacon probes combined with amplification by NASBA enable homogenous real-time detection of RNA.” Nucleic Acids Res.
  • PCR detection and quantification using dual-labeled fluorogenic oligonucleotide probes can be done, using, for example, TaqMan® probes. These probes are composed of short (e.g., 10-40 bases) oligodeoxynucleotides that are labeled with two different fluorescent dyes. On the 5' terminus of each probe is a reporter dye, and on the 3' terminus of each probe a quenching dye is found.
  • the oligonucleotide probe sequence is complementary to an internal target sequence present in a PGR amplicon. When the probe is intact, energy transfer occurs between the two fluorophores and emission from the reporter is quenched by the quencher via FRET.
  • the probe is cleaved by 5' nuclease activity of the polymerase used in the reaction, thereby releasing the reporter from the oligonucleotide-quencher and producing an increase in reporter emission intensity.
  • TaqMan® probes are oligonucleotides that have a label and a quencher, where the label is released during amplification by the exonuclease action of the polymerase used in amplification. This provides a real time measure of amplification during synthesis.
  • TaqMan® reagents are commercially available, e.g., from Applied Biosystems (Division Headquarters in Foster City, CA) as well as from a variety of specialty vendors such as Biosearch Technologies (e.g., black hole quencher probes).
  • oligonucleotides In general, synthetic methods for making oligonucleotides, including probes, primers, molecular beacons, PNAs, LNAs (locked nucleic acids), etc., are well known.
  • oligonucleotides can be synthesized chemically according to the solid phase phosphoramidite triester method described by Beaucage and Caruthers (1981), Tetrahedron Letts 22: 1 859-1862, e.g., using a commercially available automated synthesizer.
  • Oligonucleotides including modified oligonucleotides and PNAs, can also be ordered from a variety of commercial sources known to persons of skill.
  • Amplified variable sequences refer to amplified sequences of the plant genome, which exhibit high nucleic acid residue variability between members of the same species. All organisms have variable genomic sequences and each organism (with the exception of a clone) has a different set of variable sequences. Once identified, the presence of specific variable sequence can be used to predict phenotypic traits. Typically, DNA from the plant serves as a template for amplification with primers that flank a variable sequence of DNA. The variable sequence is amplified and then sequenced.
  • self-sustained sequence replication can be used to identify genetic markers.
  • Self-sustained sequence replication refers to a method of nucleic acid
  • Amplified fragment length polymorphisms which are amplified before or after cleavage by a restriction endonuclease, can also be used as genetic markers (V os et al. (1995) Nucl Acids Res 23:4407).
  • the amplification step allows easier detection of specific restriction fragments.
  • AFLP allows the detection large numbers of polymorphic markers and has been used for genetic mapping of plants (Becker et al. (1995) Mol Gen Genet 249:65; and Meksem et al (1995) Mol Gen Genet 249:74).
  • oligonucleotide probe to a completely complementary single-strand target nucleic acid. Detection is via an isotopic or non-isotopic label attached to the probe.
  • two or more different ASH probes are designed to have identical DNA sequences except at the polymorphic nucleotides. Each probe will have exact homology with one allele sequence so that the range of probes can distinguish all the known alternative allele sequences. Each probe is hybridized to the target DNA. With appropriate probe design and hybridization conditions, a single-base mismatch between the probe and target DNA will prevent hybridization. In this manner, only one of the alternative probes will hybridize to a target sample that is homozygous or homogenous for an allele. Samples that are heterozygous or heterogeneous for two alleles will hybridize to both of two alternative probes.
  • ASH markers are used as dominant markers where the presence or absence of only one allele is determined from hybridization or lack of hybridization by only one probe. The alternative allele may be inferred from the lack of hybridization.
  • ASH probe and target molecules are optionally RNA or DNA; the target molecules are any length of nucleotides beyond the sequence that is complementary to the probe; the probe is designed to hybridize with either strand of a DNA target; the probe ranges in size to conform to variously stringent hybridization conditions, etc.
  • PCR allows the target sequence for ASH to be amplified from low concentrations of nucleic acid in relatively small volumes. Otherwise, the target sequence from genomic DNA is digested with a restriction endonuclease and size separated by gel electrophoresis. Hybridizations typically occur with the target sequence bound to the surface of a membrane or, as described in U.S. Patent 5,468,613, the ASH probe sequence may be bound to a membrane.
  • ASH data are typically obtained by amplifying nucleic acid fragments (amplicons) from genomic DNA using PCR, transferring the amplicon target DNA to a membrane in a dot-blot format, hybridizing a labeled oligonucleotide probe to the amplicon target, and observing the hybridization dots by autoradiography.
  • amplicons nucleic acid fragments
  • Single nucleotide polymorphi sms are markers that consist of a shared sequence differentiated on the basis of a single nucleotide. Typically, this distinction is detected by differential migration patterns of an amplicon comprising the SNP on, e.g., an acrylamide gel.
  • SNP Single nucleotide polymorphi sms
  • alternative modes of detection such as hybridization, e.g., ASH, or RFLP analysis are also appropriate.
  • Isozyme markers can be employed as genetic markers, e.g. , to track markers other than the tolerance markers herein, or to track isozyme markers linked to the markers herein.
  • Isozymes are multiple forms of enzymes that differ from one another in their amino acid sequence, and therefore their nucleic acid sequences. Some isozymes are multimeric enzymes containing slightly different subunits. Other isozymes are either multimeric or monomeric but have been cleaved from the proenzyme at different sites in the amino acid sequence. Isozymes can be characterized and analyzed at the protein level, or alternatively, isozymes, which differ at the nucleic acid level, can be determined. In such cases any of the nucleic acid based methods described herein can be used to analyze isozyme markers.
  • the identification of markers associated with a particular phenotypic trait can allow for selection of plants possessing that trait, for example, via marker assisted selection (MAS).
  • MAS marker assisted selection
  • the application of MAS uses the identification of a population of tolerant plants and genetic mapping of the tolerance trait. Polymorphic loci in the vicinity of the mapped tolerance trait are chosen as potential tolerance markers. Typically, a marker locus closest to the tolerance locus is a preferred marker. Linkage analysis is then used to determine which polymorphic marker allele sequence demonstrates a statistical likelihood of co-segregation with the tolerant phenotype (thus, a "tolerance marker allele").
  • Tissue samples can be taken, for example, from the first leaf of the plant and screened with the appropriate molecular marker, and within days it is determined which progeny will advance.
  • Linked markers also remove the impact of environmental factors that can often influence phenotypic expression.
  • the polymorphic marker locus can be used to select for marker alleles that segregate with the desired tolerance phenotype.
  • This general process is typically called marker-assisted selection (MAS).
  • MAS marker-assisted selection
  • This detection can take the form of hybridization of a probe nucleic acid to a marker allele or amplicon thereof, e.g., using allele-specific hybridization, Southern analysis, northern analysis, in situ hybridization, hybridization of primers followed by PCR amplification of a region of the marker, or the like. After the presence (or absence) of a particular marker in the biological sample is verified, the plant is selected, e.g., used to make progeny plants by selective breeding.
  • Soybean plant breeders desire combinations of tolerance loci with genes for high yield and other desirable traits to develop improved soybean varieties. Screening large numbers of samples by non-molecular methods (e.g., trait evaluation in soybean plants) can be expensive, time consuming, and unreliable.
  • Use of the polymorphic markers described herein genetically linked to tolerance loci provide effective methods for selecting tolerant varieties in breeding programs. For example, one advantage of marker-assisted selection over field evaluations for tolerance is that MAS can be done at any time of year, regardless of the growing season. Moreover, environmental effects are largely irrelevant to marker-assisted selection.
  • relevant markers include: SATT495, P10649C-3, S ATT 182, S03859-1, S00224-1, SATT388, SATT313, and SATT613 (or other markers above SATT613).
  • markers on linkage group L include S03859-1-A, S08103-1-Q1, S08104-1-Q1, S08106-1-Q1, S08110-1-Q1, S081 1 1-1-Q1, S08115-2-Q1, S08117-1-Q1, S08119-1-Q1, S08118-1-Q1, S08116-1-Q1, S081 14-1-Q1, S08113-1-Q1, S08112-1-Q1, S08108-1-Q1, S08101-2-Q1, S08101-3-Q1, S08101-4-Q1, S08105-1-Q1, S08102-1-Q1, S08107-1-Q1, S08109-1-Q1, and S08101-1- Ql, , and markers for other traits, transgenes, and/or loci can be assayed simultaneously or sequentially in a single sample or population of samples. Markers for other traits, transgenes, and/or loci can be assayed simultaneously or sequentially in a single sample or population of samples.
  • MAS is a use of MAS in plant breeding.
  • Backcross breeding is the process of crossing a progeny back to one of its parents or parent lines. Backcrossing is usually done for the purpose of introgressing one or a few loci from a donor parent (e.g. , a parent comprising desirable tolerance marker loci) into an otherwise desirable genetic background from the recurrent parent (e.g., an otherwise high yielding soybean line). The more cycles of backcrossing that are done, the greater the genetic contribution of the recurrent parent to the resulting introgressed variety. This is often necessary, because tolerant plants may be otherwise undesirable, e.g., due to low yield, low fecundity, or the like. In contrast, strains which are the result of intensive breeding programs may have excellent yield, fecundity or the like, merely being deficient in one desired trait, such as tolerance or sensitivity to mesotrione and/or isoxazole herbicides.
  • the plant can be self fertilized to create a true breeding line with the same genotype, or it can be crossed with a plant with the same marker or with other desired characteristics to create a sexually crossed hybrid generation.
  • One application of MAS is to use the tolerance or improved tolerance markers to increase the efficiency of an introgression or backcrossing effort aimed at introducing a tolerance QTL into a desired (typically high yielding) background.
  • marker assisted backcrossing of specific markers (and associated QTL) from a donor source e.g., to an elite genetic background, one selects among progeny or backcross progeny for the donor trait.
  • the markers and methods can be utilized to guide marker assisted selection or breeding of soybean varieties with the desired complement (set) of allelic forms of chromosome segments associated with herbicide tolerance as well as markers associated with superior agronomic performance (tolerance, along with any other available markers for yield, disease tolerance, etc.).
  • Any of the disclosed marker alleles can be introduced into a soybean line via, for example, introgression, traditional breeding, or transformation, or a combination thereof, to yield a soybean plant with superior agronomic performance.
  • the number of alleles associated with mesotrione and/or isoxazole tolerance that can be introduced or be present in a soybean plant ranges from 1 to the number of alleles disclosed herein, each integer of which is incorporated herein as if explicitly recited.
  • Methods of making a progeny soybean plant and these progeny soybean plants having tolerance or susceptibility to mesotrione and/or isoxazole, are provided. These methods comprise crossing a first parent soybean plant with a second soybean plant and growing the female soybean plant under plant growth conditions to yield soybean plant progeny. Such soybean plant progeny can be assayed for alleles associated with tolerance and, thereby, the desired progeny selected. Such progeny plants or seed can be sold commercially for soybean production, used for food, processed to obtain a desired constituent of the soybean, or further utilized in subsequent rounds of breeding. At least one of the first or second soybean plants is a soybean plant comprising at least one of the allelic forms of the markers provided, such that the progeny are capable of inheriting the allele.
  • Inheritance of the desired tolerance allele can be traced, such as from progenitor or descendant lines in the subject soybean plants pedigree such that the number of generations separating the soybean plants being subject to the methods will generally be from 1 to 20, commonly 1 to 5, and typically 1, 2, or 3 generations of separation, and quite often a direct descendant or parent of the soybean plant will be subject to the method (i.e., 1 generation of separation).
  • the molecular marker loci and alleles associated with tolerance or susceptibility to mesotrione and/or isoxazole e.g., SATT495, P10649C-3, SATT182, S03859-1, S00224-1, SATT388, SATT313, SATT613 (including markers above SATT613), S03859-1-A, S08103-1-Q1, S08104-1-Q1, S08106-1-Q1, S081 10-I-Q1, S08111-1-Q1, S08115-2-Q1, S08117-1-Q1, S08119-1-Q1, S08118-1-Q1, S08116-1-Q1, S08114-1-Q1, S08113-1-Q1, S08112-1-QL S08108-1-Q1, S08101-2-Q1, S08101-3-Q1, S08101-4-Q1, S08105-1-Q1, S08102-1-Q1 , S08107-1-Q1, S08109-1-Q1, and S08101-1 -Ql, can be
  • tolerance clones are first identified by their genetic linkage to markers provided herein. Isolation of a nucleic acid of interest is achieved by any number of methods as discussed in detail in such references as Ausubel, Berger and Sambrook, herein, and Clark, ed. (1997) Plant Molecular Biology: A Laboratory Manual Springer- Verlag, Berlin.
  • positional gene cloning uses the proximity of a tolerance marker to physically define an isolated chromosomal fragment containing a tolerance QTL gene.
  • the isolated chromosomal fragment can be produced by such well known methods as digesting chromosomal DNA with one or more restriction enzymes, or by amplifying a chromosomal region in a polymerase chain reaction (PCR), or any suitable alternative amplification reaction.
  • PCR polymerase chain reaction
  • the digested or amplified fragment is typically ligated into a vector suitable for replication, and, e.g., expression, of the inserted fragment.
  • Markers that are adjacent to an open reading frame (ORF) associated with a phenotypic trait can hybridize to a DNA clone (e.g., a clone from a genomic DNA library), thereby identifying a clone on which an ORF (or a fragment of an ORF) is located. If the marker is more distant, a fragment containing the open reading frame is identified by successive rounds of screening and isolation of clones which together comprise a contiguous sequence of DNA, a process termed
  • Variant sequences have a high degree of sequence similarity.
  • conservative variants include those sequences that, because of the degeneracy of the genetic code, encode the amino acid sequence of one of the native recombinase polypeptides.
  • Variants such as these can be identified with the use of well-known molecular biology techniques, as, for example, with polymerase chain reaction (PCR) and hybridization techniques.
  • Variant polynucleotides also include synthetically derived nucleotide sequences, such as those generated, for example, by using site-directed mutagenesis but which still encode a recombinase protein.
  • variants of a particular polynucleotide will have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to that particular polynucleotide as determined by known sequence alignment programs and parameters.
  • Variants of a particular polynucleotide can also be evaluated by comparison of the percent sequence identity between the polypeptide encoded by a variant polynucleotide and the polypeptide encoded by the reference polynucleotide. Percent sequence identity between any two polypeptides can be calculated using sequence alignment programs and parameters described.
  • the percent sequence identity between the two encoded polypeptides is at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity.
  • Variant proteins include proteins derived from the native protein by deletion, addition, and/or substitution of one or more amino acids to the N-terminal, internal region(s), and/or C-terminal end of the native protein.
  • Variant proteins can be biologically active, that is they continue to possess the desired biological activity of the native protein, for example a variant recombinase can implement a recombination event between appropriate recombination sites. Such variants may result from, for example, genetic polymorphism or from human manipulation.
  • a biologically active variant of a protein may differ from that protein by as few as 1-15 amino acid residues, as few as 1-10, such as 6- 10, as few as 5, as few as 4, 3, 2, or even 1 amino acid residue.
  • Sequence relationships can be analyzed and described using computer-implemented algorithms.
  • the sequence relationship between two or more polynucleotides or two or more polypeptides can be determined by generating the best alignment of the sequences, and scoring the matches and the gaps in the alignment, which yields the percent sequence identity, and the percent sequence similarity.
  • Polynucleotide relationships can also be described based on a comparison of the polypeptide each encodes. Many programs and algorithms for the comparison and analysis of sequences are available.
  • sequence identity/similarity values provided herein refer to the value obtained using GAP Version 10 (GCG, Accelrys, San Diego, CA) using the following parameters: % identity and % similarity for a nucleotide sequence using a gap creation penalty weight of 50 and a gap length extension penalty weight of 3, and the nwsgapdnaxmp scoring matrix; % identity and % similarity for an amino acid sequence using a GAP creation penalty weight of 8 and a gap length extension penalty of 2, and the BLOSUM62 scoring matrix (Henikoff & Henikoff (1989) Proc Natl Acad Sci USA 89:10915).
  • GAP uses the algorithm of Needleman & Wunsch (1970) J Mol Biol 48:443-453, to find an alignment of two complete sequences that maximizes the number of matches and minimizes the number of gaps. GAP considers all possible alignments and gap positions and creates the alignment with the largest number of matched bases and the fewest gaps. It allows for the provision of a gap creation penalty and a gap extension penalty in units of matched bases. GAP must make a profit of gap creation penalty number of matches for each gap it inserts. If a gap extension penalty greater than zero is chosen, GAP must, in addition, make a profit for each gap inserted of the length of the gap times the gap extension penalty. GAP presents one member of the family of best alignments.
  • Sequence identity is a measure of the residues in the two sequences that are the same when aligned for maximum correspondence. Sequences, particularly polypeptides, that differ by conservative substitutions are said to have sequence similarity or similarity. Means for making this adjustment are known, and typically involve scoring a conservative substitution as a partial rather than a full mismatch. For example, where an identical amino acid is given a score of 1 and a non-conser ative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated using the selected scoring matrix (BLOSUM62 by default for GAP).
  • Equivalent positions between two or more polynucleotides, and/or polypeptides can be identified using any searching, sequence assembly, and/or alignment tool including, but not limited to, BLAST, GAP, PILEUP, FrameAIign, Sequencher, or similar tools.
  • GAP alignment can be used to identify equivalent positions, using the following parameters: for a nucleotide sequence using a gap creation penalty weight of 50 and a gap length extension penalty weight of 3, and the nwsgapdna.cmp scoring matrix; for an amino acid sequence using a gap creation penalty weight of 8 and a gap length extension penalty of 2, and the BLOSUM62 scoring matrix (Henikoff & Henikoff (1989) Proc Natl Acad Sci USA 89:10915).
  • PILEUP can be used to identify equivalent positions, using the following parameters for a nucleotide sequence: a gap weight of 5 and a gap length weight of 1, and the pileupdna.cmp scoring matrix; for an amino acid sequence using a gap weight of 8 and a gap length weight of 2, and the BLOSUM62 scoring matrix (Henikoff & Henikoff (1989) Proc Natl Acad Sci USA 89:10915).
  • Proteins may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known. Methods for mutagenesis and nucleotide sequence alterations are described, for example, in Kunkel (1 85) Proc Natl Acad Sci USA 82:488-492; Kunkel et al. (1987) Methods in Enzymol 154:367-382; U.S. Patent 4,873,192; Walker & Gaastra, eds. (1983) Techniques in Molecular Biology (MacMillan Publishing Company, New York) and the references cited therein. Guidance as to appropriate amino acid substitutions that do not affect biological activity of the protein of interest may be found in the model of Dayhoff et al. (1978) Atlas of Protein Sequence and Structure (Natl Biomed Res Found, Washington, D.C.). Conservative substitutions, such as exchanging one amino acid with another having similar properties, may be preferable. GENERATION OF TRANSGENIC CELLS AND PLAN
  • the present invention also relates to host cells and organisms which are transformed with nucleic acids corresponding to the tolerance, improved tolerance, or susceptibility/sensitivity markers, traits, or QTLs identified herein.
  • nucleic acids include chromosome intervals (e.g., genomic fragments), ORFs, and/or cDNAs that encode a tolerance or improved tolerance trait.
  • production of polypeptides that provide tolerance or improved tolerance by recombinant techniques are provided.
  • Host cells are genetically engineered ⁇ e.g., transduced, transfected, transformed, etc.) with the vectors ⁇ e.g., vectors, such as expression vectors which comprise an ORF derived from or related to a tolerance QTL) which can be, for example, a cloning vector, a shuttle vector, or an expression vector.
  • vectors are, for example, in the form of a plasmid, a phagemid, an agrobacterium, a virus, a naked polynucleotide (linear or circular), or a conjugated polynucleotide.
  • Vectors can be introduced into bacteria, especially for the purpose of propagation and expansion.
  • the vectors are also introduced into plant tissues, cultured plant cells, or plant protoplasts by a variety of standard methods known in the art, including but not limited to electroporation (From et al. (1985) Proc. Natl. Acad. Sci. USA 82; 5824), infection by viral vectors such as cauliflower mosaic virus (CaMV) (Hohn et al. (1982) Molecular Biology of Plant Tumors (Academic Press, New York, pp. 549-560; Howell U.S. Pat. No. 4,407,956), high velocity ballistic penetration by small particles with the nucleic acid either within the matrix of small beads or particles or on the surface (Klein et al. (1987) Nature 327;70), use of pollen as vector (WO 85/01856), or use of
  • the T-DNA plasmid is transmitted to plant cells upon infection by Agrobacterium tumefaciens, and a portion is stably integrated into the plant genome (Horsch et al. (1984) Science 233:496; Fraley et al. (1983) Proc. Natl. Acad. Sci. USA 80:4803). Additional details regarding nucleic acid introduction methods are found in Sambrook, Berger and Ausubel.
  • the method of introducing a nucleic acid into a host cell is not critical, and therefore should not be limited to any particular method for introducing exogenous genetic material into a host cell.
  • any suitable method that provides for effective introduction of a nucleic acid into a cell or protoplast can be employed.
  • the engineered host cells can be cultured in conventional nutrient media modified as appropriate for such activities as, for example, activating promoters or selecting transformants. These cells can optionally be cultured into transgenic plants.
  • Plant regeneration from cultured protoplasts is described in Evans et al. (1983) "Protoplast Isolation and Culture,” Handbook of Plant Cell Cultures 1, 124-176 (MacMillan Publishing Co., New York; Davey (1983) "Recent Developments in the Culture and Regeneration of Plant Protoplasts," Protoplasts, pp.
  • transgenic organisms which may be bacteria, yeast, fungi, animals or plants, transduced with the nucleic acids (e.g., nucleic acids comprising the marker loci and/or QTL noted herein).
  • nucleic acids e.g., nucleic acids comprising the marker loci and/or QTL noted herein.
  • Bacterial cells can be used to amplify the number of plasmids containing DNA constructs. The bacteria are grown to log phase and the plasmids within the bacteria can be isolated by a variety of methods known in the art (see, for instance, Sambrook). In addition, a plethora of kits are commercially available for the purification of plasmids from bacteria.
  • the isolated and purified plasmids are then further manipulated to produce other plasmids, used to transfect plant cells or incorporated into Agrobacterium tumefaciens related vectors to infect plants.
  • Typical vectors contain transcription and translation terminators, transcription and translation initiation sequences, and promoters useful for regulation of the expression of the particular target nucleic acid.
  • the vectors optionally comprise generic expression cassettes containing at least one independent terminator sequence, sequences permitting replication of the cassette in eukaryotes, or prokaryotes, or both, (e.g., shuttle vectors), and selection markers for both prokaryotic and eukaryotic systems.
  • Vectors are suitable for replication and integration in prokaryotes, eukaryotes, or both. See, Giliman & Smith (1979) Gene 8:81; Roberts et al. (1987) Nature 328:731 ; Schneider et al. (1995) Protein Expr. Purif. 6435:10; Ausubel, Sambrook, Berger (all infra).
  • one or more of the herbicide-tolerant polynucleotides employed in the methods and compositions can be provided in an expression cassette for expression in the plant or other organism of interest
  • the cassette will include 5' and 3' regulatory sequences operably linked to an herbicide-tolerance polynucleotide.
  • "Operably linked” is intended to mean a functional linkage between two or more elements.
  • an operable linkage between a polynucleotide of interest and a regulatory sequence is functional link that allows for expression of the
  • Operably linked elements may be contiguous or noncontiguous.
  • “operably linked” is intended that the coding regions are in the same reading frame.
  • “operably linked” indicates that the enhancer increases the expression of a particular polynucleotide or polynucleotides of interest. Where the polynucleotide or polynucleotides of interest encode a polypeptide, the encoded polypeptide is produced at a higher level.
  • the cassette may additionally contain at least one additional gene to be co- transformed into the organism.
  • the additional gene(s) can be provided on multiple expression cassettes.
  • Such an expression cassette is provided with a plurality of restriction sites and/or recombination sites for insertion of the herbicide-tolerance polynucleotide to be under the transcriptional regulation of the regulatory regions.
  • the expression cassette may additionally contain other genes, including other selectable marker genes. Where a cassette contains more than one polynucleotide, the polynucleotides in the cassette may be transcribed in the same direction or in different directions (also called "divergent" transcription).
  • An expression cassette comprising an herbicide-tolerance polynucleotide will include, in the 5'-3' direction of transcription, a transcriptional and translational initiation region (i.e., a promoter), an herbicide-tolerance polynucleotide, and a transcriptional and translational termination region (i.e., termination region) functional in plants or the other organism of interest. Accordingly, plants having such expression cassettes are also provided.
  • the regulatory regions (i.e., promoters, transcriptional regulatory regions, and translational termination regions) and/or the herbicide-tolerance polynucleotide may be native (i.e., analogous) to the host cell or to each other. Alternatively, the regulatory regions and/or the herbicide-tolerance polynucleotide may be heterologous to the host cell or to each other.
  • native promoter sequences may be used. Such constructs can change expression levels and/or expression patterns of the encoded polypeptide in the plant or plant cell. Expression levels and/or expression patterns of the encoded polypeptide may also be changed as a result of an additional regulatory element that is part of the construct, such as, for example, an enhancer. Thus, the phenotype of the plant or cell can be altered even though a native promoter is used.
  • the termination region may be native with the transcriptional initiation region, may be native with the operably linked herbicide-tolerance polynucleotide of interest, may be native with the plant host, or may be derived from another source (i.e., foreign or heterologous) to the promoter, the herbicide-tolerance polynucleotide of interest, the plant host, or any combination thereof.
  • Convenient termination regions are available from the Ti-plasmid of A. tumefaciens, such as the octopine synthase and nopaline synthase termination regions, or can be obtained from plant genes such as the Solanum tuberosum proteinase inhibitor II gene. See Guerineau et al. (1991) Mol. Gen. Genet.
  • a number of promoters can be used, including the native promoter of the polynucleotide sequence of interest.
  • the promoters can be selected based on the desired outcome.
  • the polynucleotides of interest can be combined with constitutive, tissue- preferred, or other promoters for expression in plants.
  • Such constitutive promoters include, for example, the core promoter of the Rsyn7 promoter and other constitutive promoters disclosed in WO 99/43838 and U.S. Pat. No. 6,072,050; the core CaMV 35S promoter (Odell et al (1985) Nature 313:810-812); rice actin (McElroy et al. (1990) Plant Cell 2:163-171); the maize actin promoter; the ubiquitin promoter (see, e.g., Christensen et al. (1989) Plant Mol. Biol. 12:619-632; Christensen et al. (1992) Plant Mol. Biol. 18:675-689; Callis et al.
  • Some promoters show improved expression when they are used in conjunction with a native 5' untranslated region and/or other elements such as, for example, an intron.
  • the maize ubiquitin promoter is often placed upstream of a polynucleotide of interest along with at least a portion of the 5' untranslated region of the ubiquitin gene, including the first intron of the maize ubiquitin gene.
  • Chemical-regulated promoters can be used to modulate the expression of a gene in a plant through the application of an exogenous chemical regulator.
  • the promoter may be a chemical-inducible promoter for which application of the chemical induces gene expression or the promoter may be a chemical-repressible promoter for which application of the chemical represses gene expression.
  • Chemical-inducible promoters are known in the art and include, but are not limited to, the maize In2-2 promoter, which is activated by benzenesulfonamide herbicide safeners, the maize GST promoter, which is activated by hydrophobic electrophilic compounds that are used as pre- emergent herbicides, and the tobacco PR-la promoter, which is activated by salicylic acid.
  • Other chemical-regulated promoters of interest include steroid-responsive promoters (see, e.g., the glucocorticoid-inducible promoter in Schena et al. (1991) Proc. Natl. Acad. Sci. USA 88:10421-10425 and McNellis et al. (1998) Plant J.
  • Tissue-preferred promoters can be utilized to target enhanced herbicide-tolerance polypeptide expression within a particular plant tissue.
  • Tissue-preferred promoters include Yamamoto et al. (1997) Plant J. 12:255-265; Kawamata et al. (1997) Plant Cell Physiol. 38:792-803; Hansen et al. (1997) Mol. Gen Genet. 254:337-343; Russell et al. (1997) Transgenic Res. 6:157-168; Rinehart et al. (1996) Plant Physiol. 112:1331-1341 ; Van Camp et al. (1996) Plant Physiol. 112:525-535; Canevascini et al. (1996) Plant Physiol.
  • Leaf-preferred promoters are known in the art. See, e.g., Yamamoto et al. (1997) Plant J. 12:255-265; Kwon et al. (1994) Plant Physiol. 105:357-67; Yamamoto et al (1994) Plant Cell Physiol. 35:773-778; Gotor et al. (1993) Plant J. 3:509-18; Orozco et al. (1993) Plant Mol. Biol. 23:1129-1138; and Matsuoka et al. (1993) Proc. Natl. Acad. Sci. USA 90(20):9586-9590.
  • Root-preferred promoters are known and can be selected from the many available from the literature or isolated de novo from various compatible species. See, e.g., Hire et al. (1992) Plant Mol. Biol. 20:207-218 (soybean root-specific glutamine synthetase gene); Keller and Baumgartner (1991) Plant Cell 3:1051-1061 (root-specific control element in the GRP 1.8 gene of French bean); Sanger et al. (1990) Plant Mol. Biol. 14(3):433-443 (root-specific promoter of the mannopine synthase (MAS) gene of Agrobacterium tumefaciens); and Miao et al.
  • MAS mannopine synthase
  • Seed-preferred promoters include both seed-specific promoters (those promoters active during seed development such as promoters of seed storage proteins) as well as seed-germinating promoters (those promoters active during seed germination). See Thompson et al. (1989) BioEssays 10: 108, herein incorporated by reference.
  • Such seed- preferred promoters include, but are not limited to, Ciml (cytokinin-induced message); cZ19Bl (maize 19 kDa zein); milps (myo-inositol-1 -phosphate synthase) (see WO 00/11177 and U.S. Pat. No. 6,225,529; herein incorporated by reference).
  • Gamma-zein is an endosperm-specific promoter.
  • Globulin 1 (GIb-1) is a representative embryo-specific promoter.
  • seed-specific promoters include, but are not limited to, bean ⁇ - phaseolin, napin, ⁇ -conglycinin, soybean lectin, cruciferin, and the like.
  • seed-specific promoters include, but are not limited to, maize 1 kDa zein, 22 kDa zein, 27 kDa zein, gamma-zein, waxy, shrunken 1, shrunken 2, globulin 1, etc. See also WO 00/12733, where seed-preferred promoters from endl and end2 genes are disclosed; herein incorporated by reference.
  • Additional promoters of interest include the SCP1 promoter (U.S. Pat. No.
  • promoters can be used with these promoters including, for example, the ubiquitin intron ⁇ i.e., the maize ubiquitin intron 1 ⁇ see, e.g., NCBI sequence S94464), the omega enhancer or the omega prime enhancer (Gallie et al. (1989) Molecular Biology of RNA ed. Cech (Liss, N.Y.) 237-256 and Gallie et al. Gene (1987) 60:217-25), or the 35S enhancer; each of which is incorporated by reference.
  • the expression cassette can also comprise a selectable marker gene for the selection of transformed cells.
  • Selectable marker genes are utilized for the selection of transformed cells or tissues.
  • Marker genes include genes encoding antibiotic resistance, such as those encoding neomycin phosphotransferase II (NEO) and hygromycin phosphotransferase (HPT), as well as genes conferring resistance to herbicidal compounds, such as glufosinate ammonium, bromoxynil, imidazolinones, and 2,4-dichlorophenoxyacetate (2,4-D).
  • Additional selectable markers include phenotypic markers such as beta-galactosidase and fluorescent proteins such as green fluorescent protein (GFP) (Su et al. (2004) Biotechnol Bioeng 85:610-9 and Fetter et al. (2004) Plant Cell 16:215-28), cyan florescent protein (CYP) (Bolte et al. (2004) J. Cell Science 117:943-54 and Kato et al. (2002) Plant Physiol 129:913-42), and yellow fluorescent protein (PhiYFP from Evrogen, see, Bolte et al (2004) J. Cell Science 117:943-54).
  • GFP green fluorescent protein
  • CYP cyan florescent protein
  • PhiYFP yellow fluorescent protein
  • selectable marker genes are not meant to be limiting. Any selectable marker gene can be used, including the GAT gene and or H A gene.
  • Methods are known in the art of increasing the expression level of a polypeptide in a plant or plant cell, for example, by inserting into the polypeptide coding sequence one or two G/C-rich codons (such as GCG or GCT) immediately adjacent to and downstream of the initiating methionine ATG codon.
  • the polynucleotides may be modified for increased expression in the transformed plant. That is, the polynucleotides can be synthesized substituting in the polypeptide coding sequence one or more codons which are less frequently utilized in plants for codons encoding the same amino acid(s) which are more frequently utilized in plants, and introducing the modified coding sequence into a plant or plant cell and expressing the modified coding sequence.
  • Additional sequence modifications are known to enhance gene expression in a cellular host. These include elimination of sequences encoding spurious polyadenylation signals, exon-intron splice site signals, transposon-like repeats, and other such well- characterized sequences that may be deleterious to gene expression.
  • the G-C content of the sequence may be adjusted to levels average for a given cellular host, as calculated by reference to known genes expressed in the host cell. When possible, the sequence is modified to avoid predicted hairpin secondary mRNA structures. Enhancers such as the CaMV 35S enhancer may also be used (see, e.g., Benfey et al. (1990) EMBO J. 9:1685- 96), or other enhancers may be used.
  • an enhancer when operably linked to an appropriate promoter, will modulate the level of transcription of an operably linked polynucleotide of interest.
  • Biologically active fragments and variants of the enhancer domain may retain the biological activity of modulating (increase or decrease) the level of transcription when operab y linked to an appropriate promoter.
  • variants of a particular polynucleotides will have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to another polynucleotides as determined by sequence alignment programs and parameters.
  • Variants of a particular polynucleotides also include those encoding a polypeptide having at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference polypeptide as determined by sequence alignment programs and parameters.
  • Polypeptide variants include those encoded by variant polynucleotides, and those having at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference polypeptide as determined by sequence alignment programs and parameters.
  • the level and/or activity of a polypeptide of interest may be modulated by employing a polynucleotide that is not capable of directing, in a transformed plant, the expression of a protein or an RNA.
  • a polynucleotide that is not capable of directing, in a transformed plant, the expression of a protein or an RNA.
  • polynucleotides may be used to design polynucleotide constructs that can be employed in methods for altering or mutating a genomic nucleotide sequence in an organism.
  • Such polynucleotide constructs include, but are not limited to, RNA:DNA vectors, RNArDNA mutational vectors, RNA:DNA repair vectors, mixed-duplex oligonucleotides, self- complementary RNA:DNA oligonucleotides, and recombinogenic oligonucleobases.
  • RNA:DNA vectors include, but are not limited to, RNA:DNA vectors, RNArDNA mutational vectors, RNA:DNA repair vectors, mixed-duplex oligonucleotides, self- complementary RNA:DNA oligonucleotides, and recombinogenic oligonucleobases.
  • Such nucleotide constructs and methods of use are known in the art. See, U.S. Pat. Nos.
  • the expression cassette may additionally contain 5' leader sequences.
  • leader sequences can act to enhance translation.
  • Translation leaders are known in the art and include: picornavirus leaders, for example, EMCV leader (Encephalomyocarditis 5' noncoding region) (Elroy-Stein et al. (1989) Proc. Natl. Acad. Sci. USA 86: 6126-61 0); potyvirus leaders, for example, TEV leader (Tobacco Etch Virus) (Gallie et al. (1995) Gene 165(2): 233-238), MDMV leader (Maize Dwarf Mosaic Virus) (Kong et al.
  • the various polynucleotide fragments may be manipulated, so as to provide for sequences to be in the proper orientation and, as appropriate, in the proper reading frame.
  • adapters or linkers may be employed to join the fragments or other manipulations may be involved to provide for convenient restriction sites, removal of superfluous material such as the removal of restriction sites, or the like.
  • in vitro mutagenesis, primer repair, restriction, annealing, resubstitutions, e.g., transitions and transversions may be involved.
  • Standard recombinant DNA and molecular cloning techniques used herein are well known in the art and are described more fully, for example, in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor) (also known as "Maniatis").
  • Chloroplast targeting sequences are known in the art and include the chloroplast small subunit of ribulose-l,5-bisphosphate carboxylase (Rubisco) (de Castro Silva Filho et al. (1996) Plant Mol. Biol. 30: 769-780; Schnell et al. (1991) J. Biol. Chem. 266(5): 3335- 3342); 5-(enolpyruvyl)shikimate-3-phosphate synthase (EPSPS) (Archer et al. (1990) J. Bioenerg Biomemb. 22(6): 789-810); tryptophan synthase (Zhao et al. (1995) J. Biol. Chem.
  • EPSPS 5-(enolpyruvyl)shikimate-3-phosphate synthase
  • plastid transformation can be accomplished by transactivation of a silent plastid-borne transgene by tissue-preferred expression of a nuclear-encoded and plastid-directed RNA polymerase.
  • tissue-preferred expression of a nuclear-encoded and plastid-directed RNA polymerase Such a system has been reported in McBride et al. (1994) Proc. Natl. Acad. Sci. USA 91: 7301-7305.
  • the polynucleotides of interest to be targeted to the chloroplast may be optimized for expression in the chloroplast to account for differences in codon usage between the plant nucleus and this organelle. In this manner, the polynucleotide of interest may be synthesized using chloroplast-preferred codons. See, e.g., U.S. Pat. No. 5,380,831, herein incorporated by reference. Introducing Nucleic Acids into Plants:
  • transgenic plants comprising the cloned nucleic acids, e.g., isolated ORFs and cDNAs encoding tolerance genes.
  • Techniques for transforming plant cells with nucleic acids are widely available and can be readily adapted.
  • useful general references for plant cell cloning, culture and regeneration include Jones (ed) (1995) Plant Gene Transfer and Expression Protocols—Methods in Molecular Biology, Volume 49 Humana Press Towata N. J.; Payne et al. (1992) Plant Cell and Tissue Culture in Liquid Systems John Wiley & Sons, Inc. New York, N.Y.
  • nucleic acid constructs e.g., DNA molecules plasmids, cosmids, artificial chromosomes, DNA, and RNA polynucleotides
  • sequence is expressed, the sequence is optionally combined with transcriptional and translational initiation regulatory sequences which direct the transcription or translation of the sequence from the exogenous DNA in the intended tissues of the transformed plant.
  • Isolated nucleic acid acids can be introduced into plants according to any of a variety of techniques known in the art. Techniques for transforming a wide variety of higher plant species are also well known and described in widely available technical, scientific, and patent literature. See, e.g., Weising et al. (1988) Ann. Rev. Genet. 22:421- 477.
  • Such methods for introducing polynucleotide or polypeptides into plants include stable transformation methods, transient transformation methods, virus-mediated methods, and breeding.
  • Stable transformation is intended to mean that the nucleotide construct introduced into a plant integrates into the genome of the plant and is capable of being inherited by the progeny thereof.
  • Transient transformation is intended to mean that a polynucleotide is introduced into the plant and does not integrate into the genome of the plant or a polypeptide is introduced into a plant.
  • DNA constructs for example DNA fragments, plasmids, phagemids, cosmids, phage, naked or variously conjugated-DNA polynucleotides, (e.g., polylysine-conjugated DNA, peptide-conjugated DNA, lipo some-conjugated DNA, etc.), or artificial
  • chromosomes can be introduced directly into the genomic DNA of the plant cell using techniques such as electroporation and microinjection of plant cell protoplasts, or the DN A constructs can be introduced directly to plant cells using ballistic methods, such as DNA particle bombardment.
  • Transformation protocols as well as protocols for introducing polypeptides or polynucleotide sequences into plants may vary depending on the type of plant or plant cell (i.e., monocot or dicot) targeted for transformation. Suitable methods of introducing polypeptides and polynucleotides into plant cells include microinjection (Crossway et al. (1986) Biotechniques 4:320-334), electroporation (Riggs et al. (1986) Proc. Natl. Acad. Sci. USA 83: 5602-5606, Agrobacterium-mediated transformation (U.S. Pat. No.
  • Microinjection techniques for injecting plant e.g., cells, embryos, callus, and protoplasts, are known in the art and well described in the scientific and patent literature. For example, a number of methods are described in Jones (ed) (1995) Plant Gene Transfer and Expression Protocols—Methods in Molecular Biology, Volume 49 Humana Press, Towata, N.J., as well as in the other references noted herein and available in the literature.
  • Agrobacterium-mediated transformation is employed to generate transgenic plants.
  • Agrobacterium-mediated transformation techniques including disarming and use of binary vectors, are also well described in the scientific literature. See, e.g., Horsch, et al (1984) Science 233:496; and Fraley et al. (1984) Proc. Natl. Acad. Sci. USA 80:4803 and recently reviewed in Hansen and Chilton (1998) Current Topics in
  • DNA constructs are optionally combined with suitable T-DNA flanking regions and introduced into a conventional Agrobacterium tumefaciens host vector.
  • the virulence functions of the Agrobacterium tumefaciens host will direct the insertion of the construct and adjacent marker into the plant cell DNA when the cell is infected by the bacteria. See, U.S. Pat. No. 5,591,616.
  • Agrobacterium is useful primarily in dicots, certain monocots can be transformed by Agrobacterium. For instance, Agrobacterium
  • tumefaciens vectors pARC8 or pARC16 (2) liposome-mediated DNA uptake (see, e.g., Freeman et al (1984) Plant Cell Physiol. 25:1353), (3) the vortexing method (see, e.g., Kindle (1990) Proc. Natl. Acad. Sci., (USA) 87:1228.
  • DNA can also be introduced into plants by direct DNA transfer into pollen as described by Zhou et al. (1983) Methods in Enzymology, 101 :433; D. Hess (1987) Intern Rev. Cytol. 107:367; Luo et al. (1988) Plant Mol. Biol. Reporter 6: 165.
  • polypeptide coding genes can be obtained by injection of the DNA into reproductive organs of a plant as described by Pena et al. (1987) Nature 325:274. DNA can also be injected directly into the cells of immature embryos and the desiccated embryos rehydrated as described by Neuhaus et al. (1987) Theor. Appl. Genet. 75:30; and Benbrook et al. (1986) in Proceedings Bio Expo Butterworth, Stoneham, Mass., pp. 27-54.
  • plant viruses that can be employed as vectors are known in the art and include cauliflower mosaic virus (CaMV), geminivirus, brome mosaic virus, and tobacco mosaic virus.
  • polynucleotide at a desired genomic location is achieved using a site-specific
  • Transformed plant cells which are derived by any of the above transformation techniques can be cultured to regenerate a whole plant that possesses the transformed genotype and thus the desired phenotype. Such regeneration techniques rely on
  • Regeneration can also be obtained from plant callus, explants, somatic embryos (Dandekar et al. (1989) J. Tissue Cult. Meth. 12:145; McGranahan, et al. (1990) Plant Cell Rep. 8:512) organs, or parts thereof. Such regeneration techniques are described generally in Klee et al. (1987)., Ann. Rev. of Plant Phys. 38:467-486.
  • This regeneration and growth process includes the steps of selection of transformant cells and shoots, rooting the transformant shoots and growth of the plantlets in soil. These methods are adapted to produce transgenic plants bearing QTLs and other genes isolated according to the methods.
  • the regeneration of plants containing the polynucleotides and introduced by Agrobacterium into cells of leaf explants can be achieved as described by Horsch et al. (1985) Science 227:1229-1231.
  • transformants are grown in the presence of a selection agent and in a medium that induces the regeneration of shoots in the plant species being transformed as described by Fraley et al (1983) Proc. Natl. Acad. Sci. (U.S.A.) 80:4803.
  • This procedure typically produces shoots within two to four weeks and these transformant shoots are then transferred to an appropriate root-inducing medium containing the selective agent and an antibiotic to prevent bacterial growth.
  • Transgenic plants may be fertile or sterile.
  • polypeptides that provide herbicide tolerance can also provide a similar phenotype when transformed and expressed in other plants.
  • plant genuses and species of interest include, but are not limited to, monocots and dicots such as corn (Tea mays), Brassica sp. (e.g., B. napus, B. rapa, B.junced), particularly those Brassica species useful as sources of seed oil, alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secale cereale), sorghum
  • Vegetables include tomatoes (Lycopersicon esculentum), lettuce (e.g., Lactuca sativa), green beans (Phaseolus vulgaris), lima beans (Phaseolus limensis), peas (Lathyrus spp.), and members of the genus Cucumis such as cucumber (C. sativus), cantaloupe (C. cantalupensis), and musk melon (C. melo).
  • tomatoes Locopersicon esculentum
  • lettuce e.g., Lactuca sativa
  • green beans Phaseolus vulgaris
  • lima beans Phaseolus limensis
  • peas Lathyrus spp.
  • members of the genus Cucumis such as cucumber (C. sativus), cantaloupe (C. cantalupensis), and musk melon (C. melo).
  • Ornamentals include azalea (Rhododendron spp.), hydrangea (Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosa spp.), tulips (Tulipa spp.), daffodils (Narcissus spp.), petunias (Petunia hybrida), carnation (Dianthus caryophyllus), poinsettia (Euphorbia pulch rrimd), and chrysanthemum.
  • Conifers include, for example, pines such as loblolly pine (Pinus taeda), slash pine (Pinus elliotii), ponderosa pine (Pinus ponderosd), lodgepole pine (Pinus contorta), and Monterey pine (Pinus radiata); Douglas-fir (Pseudotsuga menziesii) Western hemlock (Tsuga canadensis); Sitka spruce (Picea glauca); redwood (Sequoia sempervirens); true firs such as silver fir (Abies amabilis) and balsam fir (Abies balsamea); and cedars such as Western red cedar (Thuja plicata) and Alaska yellow-cedar (Chamaecyparis nootkatensis).
  • pines such as loblolly pine (Pinus taeda), slash pine (Pinus elliotii),
  • Promoters of bacterial origin that operate in plants include the octopine synthase promoter, the nopaline synthase promoter and other promoters derived from native Ti plasmids. See, Herrara-Estrella et al. (1983), Nature, 303:209. Viral promoters include the 35S and 19S RNA promoters of cauliflower mosaic virus. See, Odell et al (1985) Nature, 313:810. Other plant promoters include Kunitz trypsin inhibitor promoter ( ⁇ ), SCP1, SUP, UCD3, the ribulose-l,3-bisphosphate carboxylase small subunit promoter and the phaseolin promoter. The promoter sequence from the E8 gene and other genes may also be used.
  • E8 promoter The isolation and sequence of the E8 promoter is described in detail in Deikman and Fischer (1988) EMBO J. 7:3315. Many other promoters are in current use and can be coupled to an exogenous DNA sequence to direct expression of the nucleic acid.
  • a polyadenylation region at the 3 '-end of the coding region is typically included.
  • the polyadenylation region can be derived from the natural gene, from a variety of other plant genes, or from, e.g., T-DNA.
  • the vector comprising the sequences (e.g., promoters or coding regions) from genes encoding expression products and transgenes will typically include a nucleic acid subsequence, a marker gene which confers a selectable, or alternatively, a screenable, phenotype on plant cells.
  • the marker can encode biocide tolerance, particularly antibiotic tolerance, such as tolerance to kanamycin, G418, bleomycin, hygromycin, or herbicide tolerance, such as tolerance to chlorsulforon, or phosphinothricin (the active ingredient in the herbicides bialaphos or Basta). See, e.g., Padgette et at.
  • transgenic plants can be introduced into other plants by sexual crossing. Any of a number of standard breeding techniques can be used, depending upon the species to be crossed.
  • vegetatively propagated crops mature transgenic plants can be propagated by the taking of cuttings or by tissue culture techniques to produce multiple identical plants. Selection of desirable transgenics is made and new varieties are obtained and propagated vegetatively for commercial use.
  • seed propagated crops mature transgenic plants can be self crossed to produce a homozygous inbred plant. The inbred plant produces seed containing the newly introduced heterologous nucleic acid. These seeds can be grown to produce plants that would produce the selected phenotype.
  • Parts obtained from the regenerated plant such as flowers, seeds, leaves, branches, fruit, and the like are included, provided that these parts comprise cells comprising the isolated nucleic acid. Progeny and variants, and mutants of the regenerated plants are also included, provided that these parts comprise the introduced nucleic acid sequences.
  • Transgenic or introgressed plants expressing a polynucleotide can be screened for transmission of the nucleic acid by, for example, standard nucleic acid detection methods or by immunoblot protocols. Expression at the RNA level can be determined to identify and quantitate expression-positive plants.
  • immunocytochemistry can be done using heterologous nucleic acid specific polynucleotide probes and antibodies, respectively, to localize sites of expression within transgenic tissue.
  • a number of transgenic lines are usually screened for the incorporated nucleic acid to identify and select plants with the most appropriate expression profiles.
  • a transgenic plant that is homozygous for the added heterologous nucleic acid e.g. , a transgenic plant that contains two added nucleic acid sequence copies, such as a gene at the same locus on each chromosome of a homologous chromosome pair
  • a homozygous transgenic plant can be obtained by sexually mating (self- fertilizing) a heterozygous transgenic plant that contains a single added heterologous nucleic acid, germ inating some of the seed produced and analyzing the resulting plants produced for altered expression of a polynucleotide relative to a control plant (e.g., a native, non-transgenic plant).
  • Back-crossing to a parental plant and out-crossing with a non-trans genie plant can be used to introgress the heterologous nucleic acid into a selected background (e.g., an elite or exotic soybean line).
  • Plants may be produced by any suitable method, including breeding.
  • Plant breeding can be used to introduce desired characteristics (e.g., a stably incorporated transgene or a genetic variant or genetic alteration of interest) into a particular plant line of interest, and can be performed in any of several different ways.
  • Pedigree breeding starts with the crossing of two genotypes, such as an elite line of interest and one other elite inbred line having one or more desirable characteristics (i.e., having stably incorporated a polynucleotide of interest, having a modulated activity and/or level of the polypeptide of interest, etc.) which complements the elite plant line of interest. If the two original parents do not provide all the desired characteristics, other sources can be included in the breeding population.
  • the inbred line comprises homozygous alleles at about 95% or more of its loci.
  • breeding e.g., backcrossing
  • techniques known in the art can be used to facilitate and accelerate the breeding (e.g., backcrossing) process, including, for example, the use of a greenhouse or growth chamber with accelerated day/night cycles, the analysis of molecular markers to identify desirable progeny, and the like.
  • backcrossing can also be used in combination with pedigree breeding to modify an elite line of interest and a hybrid that is made using the modified elite line.
  • backcrossing can be used to transfer one or more specifically desirable traits from one line, the donor parent, to an inbred called the recurrent parent, which has overall good agronomic characteristics yet lacks that desirable trait or traits.
  • the same procedure can be used to move the progeny toward the genotype of the recurrent parent but at the same time retain many components of the non-recurrent parent by stopping the backcrossing at an early stage and proceeding with selfing and selection. For example, an Fl, such as a commercial hybrid, is created.
  • This commercial hybrid may be backcrossed to one of its parent lines to create a BCl or BC2.
  • Progeny are selfed and selected so that the newly developed inbred has many of the attributes of the recurrent parent and yet several of the desired attributes of the nonrecurrent parent. This approach leverages the value and strengths of the recurrent parent for use in new hybrids and breeding.
  • a method of making a backcross conversion of an inbred line of interest comprising the steps of crossing a plant from the inbred line of interest with a donor plant comprising at least one mutant gene or transgene conferring a desired trait (e.g., herbicide tolerance), selecting an Fl progeny plant comprising the mutant gene or transgene conferring the desired trait, and backcrossing the selected Fl progeny plant to a plant of the inbred line of interest.
  • This method may further comprise the step of obtaining a molecular marker profile of the inbred line of interest and using the molecular marker profile to select for a progeny plant with the desired trait and the molecular marker profile of the inbred line of interest.
  • this method may be used to produce an Fl hybrid seed by adding a final step of crossing the desired trait conversion of the inbred line of interest with a different plant to make Fl hybrid seed comprising a mutant gene or transgene conferring the desired trait.
  • Recurrent selection is a method used in a plant breeding program to improve a population of plants.
  • the method entails individual plants cross pollinating with each other to form progeny.
  • the progeny are grown and the superior progeny selected by any number of selection methods, which include individual plant, half-sib progeny, full-sib progeny, selfed progeny and topcrossing.
  • the selected progeny are cross-pollinated with each other to form progeny for another population. This population is planted and again superior plants are selected to cross pollinate with each other.
  • Recurrent selection is a cyclical process and therefore can be repeated as many times as desired.
  • the objective of recurrent selection is to improve the traits of a population.
  • the improved population can then be used as a source of breeding material to obtain inbred lines to be used in hybrids or used as parents for a synthetic cultivar.
  • a synthetic cultivar is the resultant progeny formed by the intercrossing of several selected inbreds.
  • Mass selection is a useful technique when used in conjunction with molecular marker enhanced selection.
  • seeds from individuals are selected based on phenotype and/or genotype. These selected seeds are then bulked and used to grow the next generation.
  • Bulk selection requires growing a population of plants in a bulk plot, allowing the plants to self-pollinate, harvesting the seed in bulk and then using a sample of the seed harvested in bulk to plant the next generation. Instead of self pollination, directed pollination could be used as part of the breeding program.
  • Mutation breeding is one of many methods that could be used to introduce new traits into an elite line. Mutations that occur spontaneously or are artificially induced can be useful sources of variability for a plant breeder. The goal of artificial mutagenesis is to increase the rate of mutation for a desired characteristic.
  • Mutation rates can be increased by many different means including temperature, long-term seed storage, tissue culture conditions, radiation such as X-rays, gamma rays (e.g., cobalt 60 or cesium 137), neutrons, (product of nuclear fission of uranium 235 in an atomic reactor), Beta radiation (emitted from radioisotopes such as phosphorus 32 or carbon 14), or ultraviolet radiation (typically from 2500 to 2900 ran), or chemical mutagens (such as base analogues (5-bromo-uracil), related compounds (8-ethoxy caffeine), antibiotics (streptonigrin), alkylating agents (sulfur mustards, nitrogen mustards, epoxides, ethylenamines, sulfates, sulfonates, sulfones, lactones), azide, hydroxylamine, nitrous acid, or acridines.
  • radiation such as X-rays, gamma rays (e.g
  • the activity and/or level of the polypeptide is modulated (i.e., increased or decreased).
  • An increase in the level and/or activity of the polypeptide can be achieved by providing the polypeptide to the plant.
  • many methods are known the art for providing a polypeptide to a plant including, but not limited to, direct introduction of the polypeptide into the plant, introducing into the plant (transiently or stably) a polynucleotide construct encoding a polypeptide having the desired activity.
  • tolerant soybean plants can recognize tolerant soybean plants in the field, and can select the tolerant individuals or populations for breeding purposes or for propagation.
  • the plant breeder recognizes tolerant, and non-tolerant soybean plants.
  • the tolerance is observed in the context of herbicide carryover from the previous crop season.
  • the screening and selection may also be performed by exposing plants containing said progeny germplasm to mesotrione and/or isoxazole in an assay and selecting those plants showing tolerance or sensitivity to mesotrione and/or isoxazole herbicides as containing soybean germplasm into which germplasm having tolerance or sensitivity to mesotrione and/or isoxazole herbicides derived from the QTL mapped to linkage group L has been introgressed.
  • the live assay may be any such assay known to the art, e.g., Taylor- Lovell et al. (2001) Weed Tech 15:95-102.
  • plant tolerance is a phenotypic spectrum consisting of extremes of high tolerance to non-tolerance with a continuum of intermediate tolerance phenotypes.
  • the methods include an automated system for detecting markers and or correlating the markers with a desired phenotype (e.g. , tolerance or susceptibility).
  • a typical system can include a set of marker probes or primers configured to detect at least one favorable allele of one or more marker locus associated with tolerance or improved tolerance or sensitivity to mesotrione and/or isoxazole herbicides.
  • These probes or primers are configured to detect the marker alleles noted in the tables and examples herein, e.g., using any available allele detection format, e.g., solid or liquid phase array based detection, microfluidic-based sample detection, etc.
  • markers involving linkage group L are used.
  • the marker locus is SATT495, P10649C-3, S ATT 182, S03859-1, S00224-1, SATT388, SATT313, SATT613 (or another marker above SATT613), S03859-1-A, S08103-1-Q1, S08104-1-Q1 , S08106-1-Q1, S08110-1-Q1, S08111-1-Q1, S08115-2-Q1, S08117-1-Q1, S08119-1 -Q1, S08118-1-Q1, S08116-1-Q1, S08114-1-Q1, S08113-1-Q1, S08112-1-Q1, S08108-1-Q1, S08101-2-Q1, S08101-3-Q1, S08101-4-Q1, S08105-1-Q1, S08102-1-Q1, S08107-1-Q1, S08109-1 -Ql, and S08101-1-Q1, and the probe set is configured to detect the locus.
  • alleles of SATT495, P10649C-3, S ATT 182, S03859-1, S00224-1, SATT388, SATT313, SATT613 (or another marker above SATT613), S03859-1-A, S081O3-1-Q1, S08104-1-Q1, S08106-1-Q1, S08110-1-Ql, S08111-1-Ql, S08115-2-Q1, S081 17-1-Ql, S081 19-1-Q1, S08118-1-Q1, S08116-1-Q1, S08114-1-Q1, S08113-1-Q1, S08112-1 -Q1, S081O8-1-Q1, S08101-2-Q1, S08101-3-Q1, S08101-4-Q1, S08105-1-Q1, S08102-1-Q1, S08107-1-Q1, S08109-1-Q1, and S08101-1 -Ql can be detected.
  • the typical system includes a detector that is configured to detect one or more signal outputs from the set of marker probes or primers, or amplicon thereof, thereby identifying the presence or absence of the allele.
  • a detector that is configured to detect one or more signal outputs from the set of marker probes or primers, or amplicon thereof, thereby identifying the presence or absence of the allele.
  • signal detection apparatus including photo multiplier tubes, spectrophotometers, CCD arrays, arrays and array scanners, scanning detectors, phototubes and photodiodes, microscope stations, galvo-scans, microfluidic nucleic acid amplification detection appliances and the like.
  • the precise configuration of the detector will depend, in part, on the type of label used to detect the marker allele, as well as the instrumentation that is most conveniently obtained for the user.
  • Detectors that detect fluorescence, phosphorescence, radioactivity, pH, charge, absorbance, luminescence, temperature, magnetism or the like can be used.
  • Typical detector examples include light (e.g., fluorescence) detectors or radioactivity detectors.
  • detection of a light emission (e.g., a fluorescence emission) or other probe label is indicative of the presence or absence of a marker allele.
  • Fluorescent detection is generally used for detection of amplified nucleic acids (however, upstream and/or downstream operations can also be performed on amplicons, which can involve other detection methods).
  • the detector detects one or more label (e.g., light) emission from a probe label, which is indicative of the presence or absence of a marker allele.
  • the detector(s) optionally monitors one or a plurality of signals from an amplification reaction. For example, the detector can monitor optical signals which correspond to "real time" amplification assay results.
  • System instructions that correlate the presence or absence of the favorable allele with the predicted tolerance are also provided.
  • the instructions can include at least one look-up table that includes a correlation between the presence or absence of the favorable alleles and the predicted tolerance or improved tolerance.
  • the precise form of the instructions can vary depending on the components of the system, e.g., they can be present as system software in one or more integrated unit of the system (e.g., a microprocessor, computer or computer readable medium), or can be present in one or more units ⁇ e.g., computers or computer readable media) operably coupled to the detector.
  • the system instructions include at least one look-up table that includes a correlation between the presence or absence of the favorable alleles and predicted tolerance or improved tolerance.
  • the instructions also typically include instructions providing a user interface with the system, e.g., to permit a user to view results of a sample analysis and to input parameters into the system.
  • the system typically includes components for storing or transmitting computer readable data representing or designating the alleles detected, e.g., in an automated system.
  • the computer readable media can include cache, main, and storage memory and/or other electronic data storage components (hard drives, floppy drives, storage drives, etc.) for storage of computer code.
  • Data representing alleles detected can also be electronically, optically, magnetically o transmitted in a computer data signal embodied in a transmission medium over a network such as an intranet or internet or combinations thereof.
  • the system can also or alternatively transmit data via wireless, I , or other available transmission alternatives.
  • the system typically comprises a sample that is to be analyzed, such as a plant tissue, or material isolated from the tissue such as genomic DNA, amplified genomic DNA, cDNA, amplified cDNA, RNA, amplified RNA, or the like.
  • allele detection/correlation system refers to a system in which data entering a computer corresponds to physical objects or processes external to the computer, e.g., a. marker allele, and a process that, within a computer, causes a physical
  • the input data e.g., amplification of a particular marker allele
  • output data e.g., the identification of the allelic form of a chromosome segment.
  • the process within the computer is a set of instructions, or "program,” by which positive amplification or hybridization signals are recognized by the integrated system and attributed to individual samples as a genotype. Additional programs correlate the identity of individual samples with phenotypic values or marker alleles, e.g., statistical methods.
  • tolerance marker allele values assigned to a population of progeny descending from crosses between elite lines are recorded in a computer readable medium, thereby establishing a database corresponding tolerance alleles with unique identifiers for members of the population of progeny.
  • Any file or folder, whether custom-made or commercially available (e.g., from Oracle or Sybase) suitable for recording data in a computer readable medium is acceptable as a database.
  • Data regarding genotype for one or more molecular markers e.g., ASH, SSR, RFLP, RAPD, AFLP, SNP, isozyme markers or other markers as described herein, are similarly recorded in a computer accessible database.
  • marker data is obtained using an integrated system that automates one or more aspects of the assay (or assays) used to determine marker(s) genotype.
  • input data corresponding to genotypes for molecular markers are relayed from a detector, e.g., an array, a scanner, a CCD, or other detection device directly to files in a computer readable medium accessible to the central processing unit.
  • a set of system instructions typically embodied in one or more programs
  • encoding the correlations between tolerance and the alleles of the invention is then executed by the computational device to identify correlations between marker alleles and predicted trait phenotypes.
  • the system also includes a user input device, such as a keyboard, a mouse, a touchscreen, or the like, for, e.g., selecting files, retrieving data, reviewing tables of maker information, etc., and an output device (e.g., a monitor, a printer, etc.) for viewing or recovering the product of the statistical analysis.
  • a user input device such as a keyboard, a mouse, a touchscreen, or the like
  • an output device e.g., a monitor, a printer, etc.
  • Integrated systems comprising a computer or computer readable medium comprising set of files and/or a database with at least one data set that corresponds to the marker alleles herein are provided.
  • the systems optionally also includes a user interface allowing a user to selectively view one or more of these databases.
  • the systems optionally include components for sample manipulation, e.g., incorporating robotic devices.
  • a robotic liquid control armature for transferring solutions (e.g., plant cell extracts) from a source to a destination, e.g., from a microtiter plate to an array substrate, is optionally operably linked to the digital computer (or to an additional computer in the integrated system).
  • An input device for entering data to the digital computer to control high throughput liquid transfer by the robotic liquid control armature and, optionally, to control transfer by the armature to the solid support is commonly a feature of the integrated system. Many such automated robotic fluid handling systems are commercially available.
  • a variety of automated systems are available from Caliper Technologies (Hopkinton, MA), which utilize various Zymate systems, which typically include, e.g., robotics and fluid handling modules.
  • the common O CA ® robot which is used in a variety of laboratory systems, e.g. , for microtiter tray manipulation, is also commercially available, e.g., from Beckman Coulter, Inc. (Fullerton, CA).
  • microfluidic systems for performing fluid handling and detection are now widely available, e.g. , from Caliper Technologies Corp. (Hopkinton, MA) and Agilent technologies (Palo Alto, CA).
  • Systems for molecular marker analysis can include a digital computer with one or more of high-throughput liquid control software, image analysis software for analyzing data from marker labels, data interpretation software, a robotic liquid control armature for transferring solutions from a source to a destination operably linked to the digital computer, an input device (e.g., a computer keyboard) for entering data to the digital computer to control high throughput liquid transfer by the robotic liquid control armature and, optionally, an image scanner for digitizing label signals from labeled probes hybridized, e.g., to markers on a solid support operably linked to the digital computer.
  • an input device e.g., a computer keyboard
  • an image scanner for digitizing label signals from labeled probes hybridized, e.g., to markers on a solid support operably linked to the digital computer.
  • the image scanner interfaces with the image analysis software to provide a measurement of, e.g., nucleic acid probe label intensity upon hybridization to an arrayed sample nucleic acid population (e.g., comprising one or more markers), where the probe label intensity measurement is interpreted by the data interpretation software to show whether, and to what degree, the labeled probe hybridizes to a marker nucleic acid (e.g., an amplified marker allele).
  • a marker nucleic acid e.g., an amplified marker allele.
  • the data so derived is then correlated with sample identity, to determine the identity of a plant with a particular genotype(s) for particular markers or alleles, e.g., to facilitate marker assisted selection of soybean plants with favorable allelic forms of chromosome segments involved in agronomic performance (e.g., tolerance or improved tolerance).
  • Optical images e.g., hybridization patterns viewed (and, optionally, recorded) by a camera or other recording device (e.g., a photodiode and data storage device) are optionally further processed in any of the embodiments herein, e.g., by digitizing the image and/or storing and analyzing the image on a computer.
  • a camera or other recording device e.g., a photodiode and data storage device
  • a variety of commercially available peripheral equipment and software is available for digitizing, storing and analyzing a digitized video or digitized optical image.
  • the polynucleotide conferring the tolerance in the plants are engineered into a molecular stack with at least one additional polynucleotide.
  • the additional polynucleotide may confer any additional trait of interest, such as tolerance to an additional herbicide, insects, disease, or any other desirable trait.
  • a trait refers to the phenotype derived from a particular sequence or groups of sequences.
  • herbicide-tolerance polynucleotides may be stacked with any other
  • polynucleotides encoding polypeptides having pesticidal and/or insecticidal activity, such as Bacillus thuringiensis toxic proteins (described in U.S. Pat. Nos. 5,366,892; 5,747,450; 5,737,514; 5,723,756; 5,593,881; Geiser et al. (1986) Gene 48:109; Lee et al. (2003) Appl. Environ. Microbiol. 69:4648-4657 (Vip3A); Galitzky et al. (2001) Acta Crystallogr. D. Biol. Crystallogr. 57:1101-1109 (Cry3Bbl); and Herman et al. (2004) J. Agric. Food Chem.
  • the combinations generated can also include multiple copies of any one of the polynucleotides of interest.
  • an herbicide-tolerance polynucleotide described herein may be stacked with other herbicide-tolerance traits to create a transgenic plant with further improved properties.
  • Other herbicide-tolerance polynucleotides that could be used in such embodiments include those conferring tolerance to the same herbicide by other modes of action, or a different herbicide.
  • Other traits that could be combined with herbicide- tolerance polynucleotides include those derived from polynucleotides that confer on the plant the capacity to produce a higher level of 5-enolpyravylshikimate-3-phosphate synthase (EPSPS), for example, as more fully described in U.S. Pat. Nos.
  • EPSPS 5-enolpyravylshikimate-3-phosphate synthase
  • herbicide-tolerance polynucleotides of the plants may be stacked with, for example, hydroxyphenylpyruvatedioxygenases which are enzymes that catalyze the reaction in which para-hydroxyphenylpyruvate (HPP) is transformed into homogentisate.
  • HPP para-hydroxyphenylpyruvate
  • Molecules which inhibit this enzyme and which bind to the enzyme in order to inhibit transformation of the HPP into homogentisate are useful as herbicides. Traits conferring tolerance to such herbicides in plants are described in U.S. Pat. Nos. 6,245,968 Bl; 6,268,549; and 6,069,115; and WO 99/23886.
  • herbicide-tolerance traits that could be stacked with herbicide-tolerance polynucleotides include aryloxyalkanoate dioxygenase polynucleotides (which reportedly confer tolerance to 2,4-D and other phenoxy auxin herbicides as well as to aryloxyphenoxypropionate herbicides as described, for example, in WO2005/107437) and dicamba-tolerance polynucleotides as described, for example, in Herman et al (2005) J. Biol. Chem. 280: 24759-24767.
  • aryloxyalkanoate dioxygenase polynucleotides which reportedly confer tolerance to 2,4-D and other phenoxy auxin herbicides as well as to aryloxyphenoxypropionate herbicides as described, for example, in WO2005/107437
  • dicamba-tolerance polynucleotides as described, for example, in Herman et al (2005) J. Biol
  • herbicide-tolerance traits that could be combined with herbicide- tolerance polynucleotides include those conferred by polynucleotides encoding an exogenous phosphinothricin acetyltransferase, as described in U.S. Pat. Nos. 5,969,213; 5,489,520; 5,550,318; 5,874,265; 5,919,675; 5,561,236; 5,648,477; 5,646,024; 6,177,616; and 5,879,903. Plants containing an exogenous phosphinothricin acetyltransferase can exhibit improved tolerance to glufosinate herbicides, which inhibit the enzyme glutamine synthase.
  • herbicide-tolerance traits that could be combined with the herbicide-tolerance polynucleotides include those conferred by polynucleotides conferring altered protoporphyrinogen oxidase (protox) activity, as described in U.S. Pat. Nos.
  • Nicosulfuron and Mesotrione poster presented at the WSSA Annual Meeting in Kansas City, Mo., Feb. 9-12, 2004; Green (1998) Weed Technology 12:474-477; Green and Ulrich (1993) Weed Science 41:508-516.
  • the trait(s) responsible for these tolerances can be combined by breeding or via other methods with herbicide-tolerance polynucleotides to provide a plant as well as methods of use thereof.
  • methods for growing a crop ⁇ i.e., for selectively controlling weeds in an area of cultivation
  • treating an area of interest ⁇ e.g., a field or area of cultivation
  • methods further comprise treatment with additional herbicides to which the plant is tolerant.
  • the methods permit selective control of weeds without significantly damaging the crop.
  • an "area of cultivation" comprises any region in which one desires to grow a plant.
  • Such areas of cultivations include, but are not limited to, a field in which a plant is cultivated (such as a crop field, a sod field, a tree field, a managed forest, a field for culturing fruits and vegetables, etc), a greenhouse, a growth chamber, etc.
  • a field in which a plant is cultivated such as a crop field, a sod field, a tree field, a managed forest, a field for culturing fruits and vegetables, etc
  • a greenhouse a growth chamber, etc.
  • Herbicide-tolerant traits can also be combined with at least one other trait to produce plants that further comprise a variety of desired trait combinations including, but not limited to, traits desirable for animal feed such as high oil content ⁇ e.g., U.S. Pat. No. 6,232,529); increased digestibility ⁇ e.g., modified storage proteins (U.S. application Ser. No. 10/053,410, filed Nov. 7, 2001); and thioredoxins (U.S. application Ser. No.
  • Desired trait combinations also include LLNC (low linolenic acid content; see, e.g., Dyer et al. (2002) Appl. Microbiol. Biotechnol. 59:224-230) and OLCH (high oleic acid content; see, e.g., Fernandez-Moya et al. (2005) J. Agric. Food Chem. 53:5326-5330).
  • LLNC low linolenic acid content
  • OLCH high oleic acid content
  • Herbicide-tolerant traits of interest can also be combined with other desirable traits such as, for example, fumonisim detoxification genes (U.S. Pat. No. 5,792,931), avirulence and disease resistance genes (Jones et al. (1994) Science 266:789; Martin et al. (1993) Science 262: 1432; Mindrinos et al. (1994) Cell 78: 1089), and traits desirable for processing or process products such as modified oils ⁇ e.g., fatty acid desaturase genes (U.S. Pat. No.
  • modified starches ⁇ e.g., ADPG yrophosphorylases (AGPase), starch synthases (SS), starch branching enzymes (SBE), and starch debranching enzymes (SDBE)); and polymers or bioplastics (e.g., U.S. Pat. No. 5,602,321; beta- ketothiolase, polyhydroxybutyrate synthase, and acetoacetyl-CoA reductase (Schubert et al. (1988) J. Bacteriol. 170:5837-5847) facilitate expression of polyhydroxyalkanoates (PHAs)); the disclosures of which are herein incorporated by reference.
  • PHAs polyhydroxyalkanoates
  • agronomic traits such as male sterility (e.g. , see U.S. Pat. No. 5,583,210), stalk strength, flowering time, or transformation technology traits such as cell cycle regulation or gene targeting (e.g., WO 99/61619, WO 00/17364, and WO 99/25821); the disclosures of which are herein incorporated by reference.
  • the herbicide-tolerant traits of interest can also be combined with the Rcgl sequence or biologically active variant or fragment thereof.
  • the Rcgl sequence is an anthracnose stalk rot resistance gene in com. See, e.g., U.S. patent application Ser. No. 11 /397, 153, 11/397,275, and 11/397,247, each of which is herein incorporated by reference.
  • stacked combinations can be created by any method including, but not limited to, breeding plants by any conventional or TopCross methodology, or genetic transformation. If the sequences are stacked by genetically transforming the plants, the polynucleotide sequences of interest can be combined at any time and in any order. For example, a transgenic plant comprising one or more desired traits can be used as the target to introduce further traits by subsequent transformation. The traits can be introduced simultaneously in a co-transformation protocol with the polynucleotides of interest provided by any combination of transformation cassettes. For example, if two sequences will be introduced, the two sequences can be contained in separate transformation cassettes (trans) or contained on the same transformation cassette (cis). Expression of the sequences can be driven by the same promoter or by different promoters.
  • polynucleotide sequences can be stacked at a desired genomic location using a site-specific recombination system. See, e.g.,
  • W099/25821, W099/25854, WO99/25840, W099/25855, and W099/25853 all of which are herein incorporated by reference.
  • Insect resistance genes may encode resistance to pests that have great yield drag such as rootworm, cutworm, European Corn Borer, and the like.
  • Such genes include, for example, Bacillus thuringiensis toxic protein genes (U.S. Pat. Nos. 5,366,892; 5,747,450; 5,736,514; 5,723,756; 5,593,881; and Geiser et al. (1986) Gene 48: 109); and the like.
  • Genes encoding disease resistance traits include detoxification genes, such as against fumonosin (U.S. Pat. No. 5,792,931); avirulence (avr) and disease resistance (R) genes (Jones et al. (1994) Science 266: 789; Martin et al. (1993) Science 262: 1432; and Mindrinos et al. (1994) Cell 78: 1089); and the like.
  • Sterility genes can also be encoded in an expression cassette and provide an alternative to physical detasseling. Examples of genes used in such ways include male tissue-preferred genes and genes with male sterility phenotypes such as QM, described in U.S. Pat. No. 5,583,210. Other genes include kinases and those encoding compounds toxic to either male or female gametophytic development.
  • controlling weeds in an area of cultivation preventing the development or the appearance of herbicide resistant weeds in an area of cultivation, producing a crop, and increasing crop safety.
  • controlling weeds refers to one or more of inhibiting the growth, germination, reproduction, and/or proliferation of; and/or killing, removing, destroying, or otherwise diminishing the occurrence and/or activity of a weed.
  • the mesotrione and/or isoxazole tolerant plants display a modified tolerance to herbicides and therefore allow for the application of one or more herbicides at rates that would significantly damage control plants and further allow for the application of combinations of herbicides at lower concentrations than normally applied which still continue to selectively control weeds.
  • the mesotrione and/or isoxazole tolerant plants may also display tolerance to HPPD-inhibitor herbicide carryover from a previous crop growing season.
  • the mesotrione and/or isoxazole tolerant plants can be used in combination with herbicide blends technology and thereby make the application of chemical pesticides more convenient, economical, and effective for the producer.
  • the methods comprise planting the area of cultivation with mesotrione and/or isoxazole tolerant crop seeds or plants, and applying to any crop, crop part, weed or area of cultivation thereof an effective amount of a mesotrione and/or isoxazole herbicide of interest. It is recognized that the herbicide can be applied before or after the crop is planted in the area of cultivation. Such herbicide applications can include an application of a mesotrione and/or an isoxazole chemistry, or any combination thereof.
  • the combination of herbicides comprises a glyphosate, a glufosinate, a dicamba, a bialaphos, a phosphinothricin, a protox inhibitor, a sulfonylurea, an imidazolinone, a chlorsulfuron, an imazapyr, a chlorimuron-etbyl, a quizalofop, an HPPD, a PPO, and/or a fomesafen, or combinations thereof, wherein said effective amount is tolerated by the crop and controls weeds.
  • any effective amount of these herbicides can be applied, wherein the effective amount is any amount that differentiates between plant cells, plants, and/or seed comprising a mesotrione and/or isoxazole tolerance allele, a mesotrione and/or isoxazole tolerance polynucleotide, and/or a polynucleotide encoding an ABC transporter protein that confers tolerance to herbicide formulations comprising a mesotrione and/or isoxazole.
  • the herbicides are applied simultaneously, in some examples the herbicides are applied sequentially, in some examples the herbicides are applied as pre-emergent treatments, in some examples the herbicides are applied as post-emergent treatments, in some examples the herbicides are applied as a combination of pre- and post-emergent treatments.
  • the method of controlling weeds comprises planting the area with mesotrione and/or isoxazole tolerant crop seeds or plants and applying to the crop, crop part, seed of said crop or the area under cultivation, an effective amount of a herbicide, wherein said effective amount comprises an amount that is not tolerated by a control crop when applied to the control crop, crop part, seed or the area of cultivation, wherein the control crop does not express a polynucleotide that encodes an herbicide- tolerance polypeptide.
  • combinations of herbicides may be used, such as when an additional tolerance trait is incorporated into the plant.
  • the method of controlling weeds comprises planting the area with a mesotrione and/or isoxazole tolerant crop seeds or plant and applying to the crop, crop part, seed of said crop or the area under cultivation, an effective amount of a herbicide, wherein said effective amount comprises a level that is above the recommended label use rate for the crop, wherein said effective amount is tolerated when applied to the mesotrione and/or isoxazole tolerant crop, crop part, seed, or the area of cultivation thereof.
  • Any herbicide can be applied to the tolerant crop, crop part, or the area of cultivation containing said crop plant.
  • Classifications of herbicides i.e., the grouping of herbicides into classes and subclasses
  • HRAC Herbicide Resistance Action Committee
  • WSSA the Weed Science Society of America
  • Herbicides can be classified by their mode of action and/or site of action and can also be classified by the time at which they are applied (e.g., pre-emergent or post- emergent), by the method of application (e.g., foliar application or soil application), or by how they are taken up by or affect the plant.
  • Mode of action generally refers to the metabolic or physiological process within the plant that the herbicide inhibits or otherwise impairs
  • site of action generally refers to the physical location or biochemical site within the plant where the herbicide acts or directly interacts.
  • Herbicides can be classified in various ways, including by mode of action and/or site of action.
  • a transgenic plant is tolerant to more than one herbicide or chemical in the same class or subclass, such as, for example, an inhibitor of PPO, a sulfonylurea, a glyphosate, or a synthetic auxin.
  • the plant is transgenic for one or more of the herbicide tolerance traits, non-transgenic for one of more of the tolerance traits, or any combination thereof
  • the plants provided can tolerate treatment with different types of herbicides (i.e., herbicides having different modes of action and/or different sites of action) as well as with higher amounts of herbicides than previously known plants, thereby permitting improved weed management strategies that are recommended in order to reduce the incidence and prevalence of herbicide-tolerant weeds.
  • Specific herbicide combinations can be employed to effectively control weeds.
  • a transgenic crop plant which can be selected for use in crop production based on the prevalence of herbicide-tolerant weed species in the area where the transgenic crop is to be grown is provided.
  • Methods are known in the art for assessing the herbicide tolerance of various weed species.
  • Weed management techniques are also known in the art, such as for example, crop rotation using a crop that is tolerant to an herbicide to which the local weed species are not tolerant.
  • a number of entities monitor and publicly report the incidence and characteristics of herbicide-tolerant weeds, including the Herbicide
  • HRAC Resistance Action Committee
  • various state agencies see, e.g., herbicide tolerance scores for various broadleaf weeds from the 2004 Illinois Agricultural Pest Management Handbook
  • one of skill in the art would be able to use this information to determine which crop and herbicide combinations should be used in a particular location.
  • These entities also publish advice and guidelines for preventing the development and/or appearance of and controlling the spread of herbicide tolerant weeds (see, e.g., Owen and Hartzler (2004), 2005 Herbicide Manual for Agricultural Professionals, Pub.
  • plant cells, plants, and/or seeds produced by any of the foregoing methods.
  • Example 1 Identification of Isoxafutole Tolerant and Sensitive Soybean Lines - herbicide screening bioassay and intergroup association marker based diagnostic
  • Two soybean mapping populations were used to confirm significant QTLs related to tolerance and susceptibility to mesotrione and/or isoxazole herbicides, to identify any potential QTLs associated with the tolerance or susceptibility to these herbicides, and to identify any varietal variation due to differences between the two herbicide chemistries used in the study.
  • the mesotrione herbicide used in this study was Callisto ® (referred to as Herbicide B); the isoxazole herbicide used in this study was Balance Pro ® (referred to as Herbicide A).
  • Herbicide A and B were applied as a pre-plant incorporated herbicide.
  • the application rate for Herbicide A was based on soil organic matter and was applied at half the recommended labeled rate.
  • Herbicide B was applied at half the pre-plant incorporated label rate.
  • Both herbicides were applied using an ATV sprayer outfitted with a 10-foot boom, GPS and a Raven control system. The herbicides were applied at a rate of 30 gallons of water per acre and a spray pressure of 35-40 psi.
  • the herbicides were incorporated into the soil to a depth of 1-2 inches using a field cultivator with rolling baskets 2-5 days following application (Table 2). Incorporation was performed in two directions to ensure even distribution of the herbicide in the soil.
  • the soybeans were planted into the soil to a depth of 1-1.5 inches using an Almaco 4-row index planter set on 30-inch row spacing. All plots were planted as single row plots with 25 seeds for 4.5 feet of planted row with a three-foot alleyway. The planted population was approximately 90,000 seeds per acre. Both Princeton and Johnston locations were planted on June 4. The herbicide application, planting, and rating dates for both Princeton and Johnston locations are presented in Table 2.
  • Soybean varietal herbicide reactions were evaluated using visual scores for plant growth reduction (STNT) and crop injury rating (HERSC) using descriptions defined below. Two ratings were conducted at both locations; the initial rating (V3) was based off of the clearest distinction of symptoms across the experiments. A second rating (V5 or V6) was conducted to ensure accuracy and note any varietal variation over time. An untreated check was used as a guide for the expected plant growth and development over time. Plant Growth Reduction Rating (STNT)
  • HERSC Crop Injury Rating
  • mapping populations were used that contained known susceptible and tolerant parents that were fixed and carried different alleles for two QTLs identified on linkage group L.
  • the mapping populations were screened using herbicide A and the herbicide screening protocol described above.
  • Two populations (Pop A and Pop B) of 90 randomly selected F3:F5 lines were used in the study.
  • Four replications of the populations were placed in a row by column design to help adjust means due to field variation.
  • the parents of each population were replicated 3 times per replication for a total of 12 times per location.
  • An analysis of variance (ANOVA) was conducted to identify significant differences between the varieties within the populations.
  • a variety trial was conducted using 144 varieties with herbicides A and B and the herbicide screening protocol described above. Four replications of the varieties were placed in a row by column design to help adjust means due to field variation for both herbicides.
  • the 144 lines included 52 susceptible and 61 tolerant lines identified previously as well as lines identified as moderate but containing the susceptible or tolerant allele for the QTLs.
  • the Czek Coeff which reports the top 10 and 20 percent of the entries that were the same for both the RCB and ICB designs was 73 and 83% for the top 10% of entries and 86 and 90% for the top 20% of the entries for the variety trials using herbicides A and B, respectfully.
  • the underlying logic is that markers with significantly different allele distributions between the tolerant and non-tolerant groups (z. e. , non-random distributions) might be associated with the trait and can be used to separate them for purposes of marker assisted selection of soybean lines with previously uncharacterized tolerance or non-tolerance or sensitivity to mesotrione and/or isoxazole herbicides.
  • the present analysis examined one marker locus at a time and determined if the allele distribution within the tolerant group is significantly different from the allele distribution within the non-tolerant group.
  • a statistically different allele distribution is an indication that the marker is linked to a locus that is associated with tolerance or non-tolerance or sensitivity to mesotrione and/or isoxazole herbicides.
  • map data for example, a composite soybean genetic map
  • map data is relevant in that multiple significant markers that are also genetically linked can be considered as collaborating evidence that a given chromosomal region is associated with the trait of interest.
  • the analysis identified 275 markers (PO.05), of these 275 markers identified, 243 of the markers had been previously mapped to a particular genomic position. This allowed for further analysis to identify potential genomic regions for genes and to eliminate marker regions that are likely not associated with the native tolerance to the herbicide. There were a total of 6 regions identified where multiple markers were pointing to a particular genomic region (Table 5). The regions identified included regions on B2, D2, E, G, L and 7 unmapped (UM) markers. Of the original 275 markers identified, 1 12 markers were used to identify the 6 genomic regions.
  • Table 8 shows the allele distribution for marker S03859, which is closely linked to this region, among the 144 lines analyzed; 32 tolerant lines, 37 non-tolerant (susceptible) lines, 67 moderate, and 8 segregating lines analyzed. Marker calls for the S03859 locus were available for 1 11 of the 144 lines.

Landscapes

  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Analytical Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Physics & Mathematics (AREA)
  • Molecular Biology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Botany (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • Biomedical Technology (AREA)
  • Immunology (AREA)
  • Mycology (AREA)
  • Developmental Biology & Embryology (AREA)
  • Environmental Sciences (AREA)
  • Spectroscopy & Molecular Physics (AREA)
  • Plant Pathology (AREA)
  • Cell Biology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
  • Agricultural Chemicals And Associated Chemicals (AREA)
  • Catching Or Destruction (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Medicinal Chemistry (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

This invention relates generally to the detection of genetic differences among soybeans. More particularly, the invention relates to soybean quantitative trait loci (QTL) for tolerance or sensitivity to HPPD-inhibitor herbicides, such as mesotrione and isoxazole herbicides, to soybean plants possessing these QTLs, which map to a novel chromosomal region, and to genetic markers that are indicative of phenotypes associated with tolerance, improved tolerance, susceptibility, or increased susceptibility. Methods and compositions for use of these markers in genotyping of soybean and selection are also disclosed, as are methods and compositions for use of these markers in selection and use of herbicides for weed control. Also disclosed are isolated polynucleotides and polypeptides relating to such tolerance or sensitivity and methods of introgressing such tolerance into a plant by breeding or transgenically or by a combination thereof. Plant cells, plants, and seeds produced are also provided.

Description

HPPD-INHIBITOR HERBICIDE TOLERANCE
FIELD OF THE INVENTION
This invention relates generally to the detection of genetic differences among soybeans.
BACKGROUND OF THE INVENTION
Soybeans (Glycine max L. Merr.) are a major cash crop and investment commodity in North America and elsewhere. Soybean oil is one of the most widely used edible oils, and soybeans are used worldwide both in animal feed and in human food production. Additionally, soybean utilization is expanding to industrial, manufacturing, and pharmaceutical applications. Weed management in soybean fields is important to maximizing yields. A recent development in soybean technology has been the development of herbicide-tolerant soybean varieties. Glyphosate tolerant soybeans were commercially introduced in 1996 and accounted for more than 85% percent of U.S.
soybean acreage in 2007.
Some weeds are starting to show increased tolerance to glyphosate. This increased tolerance decreases the effectiveness of glyphosate application and results in lower yields for farmers. As a result there is a need in the art for soybean varieties that are tolerant to other herbicide chemistry.
SUMMARY OF THE INVENTION
This invention relates generally to the detection of genetic differences among soybeans. More particularly, the invention relates to soybean quantitative trait loci (QTL) for tolerance or sensitivity to mesotrione and/or isoxazole herbicides, to soybean plants possessing these QTLs, which map to a novel chromosomal region, and to genetic markers that are indicative of phenotypes associated with mesotrione and/or isoxazole herbicide tolerance. Methods and compositions for use of these markers in genotyping, screening, and selection of soybean are also disclosed.
A navel method is provided for determining the presence or absence in soybean germplastn of a QTL associated with tolerance or susceptibility to HPPD-inhibitor herbicides, including mesotrione and/or isoxazole herbicides. The trait has been found to be closely linked to a number of molecular markers that map to linkage group L, on chromosome 19 (Gml9). Soybean plants, seeds, tissue cultures, variants and mutants having tolerance or susceptibility to HPPD-inhibitor herbicides, including mesotrione and/or isoxazole herbicides, produced by the foregoing methods are also provided.
The QTL associated with tolerance or sensitivity to mesotrione and/or isoxazole herbicides maps to soybean linkage group L. The QTL may be mapped by one or more molecular markers, including SATT495, P10649C-3, S ATT 182, SATT388, SATO 13, SATT613, and markers closely linked thereto. Other markers of linkage group L may also be used to identify the presence or absence of the gene, including other markers above marker SATT613. Additional relevant markers on linkage group L include S03859-1-A, S08103-1-Q1, S08104-1-Q1, S08106-1-Q1, S08110-1-Q1, S08111-1-Q1, S08115-2-Q1, S08117-1-Q1, S08119-1-Q1, S08118-1-Q1, S08116-1-Q1, S08114-1-Q1, S08113-1-Q1, S08112-1-Q1, S08108-1-Q1, S08101-2-Q1, S08101-3-Q1, S08101-4-Q1, S08105-1-Q1, S08102-1-Q1, S08107-1-Q1, S08109-1-Q1, and S08101-1 -Ql, or markers closely linked thereto (see, e.g., Figure 1). Other markers of linkage group L may also be used to identify the presence or absence of the gene, including other markers above marker SATT613.
The information disclosed herein regarding the QTL for tolerance or sensitivity to mesotrione and/or isoxazole herbicides which maps to soybean linkage group L is used to aid in the selection of breeding plants, lines, and populations containing tolerance or sensitivity to mesotrione and/or isoxazole herbicides for use in introgression of this trait into elite soybean germplasm, or germplasm of proven genetic superiority suitable for variety release.
Also provided is a method for introgressing a soybean QTL associated with tolerance or sensitivity to mesotrione and/or isoxazole herbicides into non-tolerant soybean germplasm or less tolerant soybean germplasm. According to the method, nucleic acid markers mapping the QTL are used to select soybean plants containing the QTL. Plants so selected have a high probability of expressing the trait tolerance or sensitivity to mesotrione and/or isoxazole herbicides. Plants so selected can be used in a soybean breeding program. Through the process of introgression, the QTL associated with tolerance or sensitivity to mesotrione and/or isoxazole herbicides is introduced from plants identified using marker-assisted selection to other plants. According to the method, agronomically desirable plants and seeds can be produced containing the QTL associated with tolerance or sensitivity to mesotrione and/or isoxazole herbicides from germplasm containing the QTL. Sources of tolerance or sensitivity to mesotrione and/or isoxazole herbicides are disclosed below.
Also provided herein is a method for producing a soybean plant adapted for conferring tolerance or sensitivity to mesotrione and/or isoxazole herbicides. First, donor soybean plants for a parental line containing the tolerance QTL are selected. According to the method, selection can be accomplished via nucleic acid marker-associated selection as explained herein. Selected plant material may represent, among others, an inbred line, a hybrid, a heterogeneous population of soybean plants, or simply an individual plant. According to techniques well known in the art of plant breeding, this donor parental line is crossed with a second parental line. Typically, the second parental line is a high yielding line. This cross produces a segregating plant population composed of genetically heterogeneous plants. Plants of the segregating plant population are screened for the tolerance QTL and are subjected to further breeding. This further breeding may include, among other techniques, additional crosses with other lines, hybrids, backcrossing, or self- crossing. The result is a line of soybean plants that is tolerant to mesotrione and/or isoxazole herbicides, and also has other desirable traits, such as yield, from one or more other soybean lines.
Also provided is a method for introgressing a soybean QTL associated with tolerance or sensitivity to mesotrione and/or isoxazole herbicides into non-tolerant soybean germplasm or less tolerant soybean germplasm. According to the method, nucleic acid markers mapping the QTL are used to select soybean plants containing the QTL. Plants so selected have a high probability of expressing the trait tolerance or sensitivity to mesotrione and/or isoxazole herbicides. Plants so selected can be used in a soybean breeding program. Through the process of introgression, the QTL associated with tolerance or sensitivity to mesotrione and/or isoxazole herbicides is introduced from plants identified using marker-assisted selection to other plants. According to the method, agronomically desirable plants and seeds can be produced containing the QTL associated with tolerance or sensitivity to mesotrione and/or isoxazole herbicides from germplasm containing the QTL. Sources of tolerance or sensitivity to mesotrione and/or isoxazole herbicides are disclosed below. Soybean plants, seeds, tissue cultures, variants and mutants having tolerance or sensitivity to mesotrione and/or isoxazole herbicides produced by the foregoing methods are also provided. Also provided herein are methods for controlling weeds in a crop by applying to the crop and any weeds affecting such crop an effective amount of such herbicide(s), either pre-emergent or post-emergent, such that the weeds are substantially controlled without substantially negatively impacting the crop. Also provided are compositions useful in the disclosed methods, including polynucleotide primers and probes useful for detecting relevant markers, as well as kits containing the same.
BRIEF DESCRIPTION OF THE DRAWINGS
The invention can be more fully understood from the following detailed description and the accompanying drawings and Sequence Listing, which form a part of this application.
Figure 1. Panel A provides an integrated genetic map of soybean markers on linkage group L (chromosome 19), including the marker type (SS or ASH/SNP). The genetic map positions of the markers are indicated in centiMorgans (cM), typically with position zero being the first (most distal) marker on the chromosome. The map includes relative positions for some markers for which higher resolution genetic mapping data was not available; no position in cM is provided for such markers. Panel B provides a table listing genetic markers that are linked to the mesotrione and isoxazole herbicide tolerance and sensitivity markers identified on linkage group L. These markers are from the soybean public composite map of June 18, 2008 for linkage group L.
Figure 2 provides examples of primer and probe nucleic acid sequences that are useful for detecting SNP markers associated with tolerance, improved tolerance, or susceptibility/sensitivity to mesotrione and/or isoxazole herbicides.
Figure 3 provides examples of cultivars with vastly different mesotrione and isoxazole herbicide tolerance or sensitivity phenotype. Shown are field samples, with a non-tolerant variety in the center and tolerant varieties on the left and right (normal growth). SUMMARY OF THE SEQUENCES
SEQ ID NOs: 1-5 comprise nucleotide sequences of regions of the Soybean genome, each capable of being used as a probe or primer, either alone or in combination, for the detection of marker locus P10649C-3 on LG-L. In certain examples, SEQ ID NOs: 1 and 2 are used as primers while SEQ ID NOs: 3-5 are used as probes.
SEQ ID NOs: 6-9 comprise nucleotide sequences of regions of the Soybean genome, each capable of being used as a probe or primer, either alone or in combination, for the detection of marker locus S00224-1 on LG-L. In certain examples, SEQ ID NOs: 6 and 7 are used as primers while SEQ ID NOs: 8 and 9 are used as probes.
SEQ ID NOs: 10-13 comprise nucleotide sequences of regions of the Soybean genome, each capable of being used as a probe or primer, either alone or in combination, for the detection of marker locus P5467-1 on LG-N. In certain examples, SEQ ID NOs: 10 and 11 are used as primers while SEQ ID NOs: 12 and 13 are used as probes.
SEQ ID NOs: 14-17 comprise nucleotide sequences of regions of the Soybean genome, each capable of being used as a probe or primer, either alone or in combination, for the detection of marker locus S08101-1 -Ql on LG-L. In certain examples, SEQ ID NOs: 14 and 15 are used as primers while SEQ ID NOs: 16 and 17 are used as probes.
SEQ ID NOs: 18-21 comprise nucleotide sequences of regions of the Soybean genome, each capable of being used as a probe or primer, either alone or in combination, for the detection of marker locus S08101-2-Q1 on LG-L. In certain examples, SEQ ID NOs: 18 and 19 are used as primers while SEQ ID NOs: 20 and 21 are used as probes.
SEQ ID NOs: 22-25 comprise nucleotide sequences of regions of the Soybean genome, each capable of being used as a probe or primer, either alone or in combination, for the detection of marker locus S08101-3-Q1 on LG-L. In certain examples, SEQ ID NOs: 22 and 23 are used as primers while SEQ ID NOs: 24 and 25 are used as probes.
SEQ ID NOs: 26-29 comprise nucleotide sequences of regions of the Soybean genome, each capable of being used as a probe or primer, either alone or in combination, for the detection of marker locus S08101-4-Q1 on LG-L. In certain examples, SEQ ID NOs: 26 and 27 are used as primers while SEQ ID NOs: 28 and 29 are used as probes.
SEQ ID NOs: 30-33 comprise nucleotide sequences of regions of the Soybean genome, each capable of being used as a probe or primer, either alone or in combination, for the detection of marker locus S08102-1-Q1 on LG-L. In certain examples, SEQ ID NOs: 30 and 31 are used as primers while SEQ ID NOs: 32 and 33 are used as probes.
SEQ ID NOs: 34-37 comprise nucleotide sequences of regions of the Soybean genome, each capable of being used as a probe or primer, either alone or in combination, for the detection of marker locus S08103-1-Q1 on LG-L. In certain examples, SEQ ID NOs: 34 and 35 are used as primers while SEQ ID NOs: 36 and 37 are used as probes.
SEQ ID NOs: 38-41 comprise nucleotide sequences of regions of the Soybean genome, each capable of being used as a probe or primer, either alone or in combination, for the detection of marker locus S08104-1-Q1 on LG-L. In certain examples, SEQ ID NOs: 38 and 39 are used as primers while SEQ ID NOs: 40 and 41 are used as probes.
SEQ ID NOs: 42-45 comprise nucleotide sequences of regions of the Soybean genome, each capable of being used as a probe or primer, either alone or in combination, for the detection of marker locus S08105-1-Q1 on LG-L. In certain examples, SEQ ID NOs: 42 and 43 are used as primers while SEQ ID NOs: 44 and 45 are used as probes.
SEQ ID NOs: 46-49 comprise nucleotide sequences of regions of the Soybean genome, each capable of being used as a probe or primer, either alone or in combination, for the detection of marker locus S08106-1-Q1 on LG-L. In certain examples, SEQ ID NOs: 46 and 47 are used as primers while SEQ ID NOs: 48 and 49 are used as probes.
SEQ ID NOs: 50-53 comprise nucleotide sequences of regions of the Soybean genome, each capable of being used as a probe or primer, either alone or in combination, for the detection of marker locus S08107-1-Q1 on LG-L. In certain examples, SEQ ID NOs: 50 and 51 are used as primers while SEQ ID NOs: 52 and 53 are used as probes.
SEQ ID NOs: 54-57 comprise nucleotide sequences of regions of the Soybean genome, each capable of being used as a probe or primer, either alone or in combination, for the detection of marker locus S08108-1-Q1 on LG-L. In certain examples, SEQ ID NOs: 54 and 55 are used as primers while SEQ ID NOs: 56 and 57 are used as probes.
SEQ ID NOs: 58-61 comprise nucleotide sequences of regions of the Soybean genome, each capable of being used as a probe or primer, either alone or in combination, for the detection of marker locus S08109-1-Q1 on LG-L. In certain examples, SEQ ID NOs: 58 and 59 are used as primers while SEQ ID NOs: 60 and 61 are used as probes.
SEQ ID NOs: 62-65 comprise nucleotide sequences of regions of the Soybean genome, each capable of being used as a probe or primer, either alone or in combination, for the detection of marker locus S08110-1-Q1 on LG-L. In certain examples, SEQ ID NOs: 62 and 63 are used as primers while SEQ ID NOs: 64 and 65 are used as probes.
SEQ ID NOs: 66-69 comprise nucleotide sequences of regions of the Soybean genome, each capable of being used as a probe or primer, either alone or in combination, for the detection of marker locus S0813 1 -1-Ql on LG-L. In certain examples, SEQ ID NOs: 66 and 67 are used as primers while SEQ ID NOs: 68 and 69 are used as probes.
SEQ ID NOs: 70-73 comprise nucleotide sequences of regions of the Soybean genome, each capable of being used as a probe or primer, either alone or in combination, for the detection of marker locus S08112-1-Ql on LG-L. In certain examples, SEQ ID NOs: 70 and 71 are used as primers while SEQ ID NOs: 72 and 73 are used as probes.
SEQ ID NOs: 74-77 comprise nucleotide sequences of regions of the Soybean genome, each capable of being used as a probe or primer, either alone or in combination, for the detection of marker locus S08115-2-Q1 on LG-L. In certain examples, SEQ ID NOs: 74 and 75 are used as primers while SEQ ID NOs: 76 and 77 are used as probes.
SEQ ID NOs: 78-81 comprise nucleotide sequences of regions of the Soybean genome, each capable of being used as a probe or primer, either alone or in combination, for the detection of marker locus S08116-1-Ql on LG-L. In certain examples, SEQ ID NOs: 78 and 79 are used as primers while SEQ ID NOs: 80 and 81 are used as probes.
SEQ ID NOs: 82-85 comprise nucleotide sequences of regions of the Soybean genome, each capable of being used as a probe or primer, either alone or in combination, for the detection of marker locus S08117-1-Ql on LG-L. In certain examples, SEQ ID NOs: 82 and 83 are used as primers while SEQ ID NOs: 84 and 85 are used as probes.
SEQ ID NOs: 86-89 comprise nucleotide sequences of regions of the Soybean genome, each capable of being used as a probe or primer, either alone or in combination, for the detection of marker locus S08118-1-Ql on LG-L. In certain examples, SEQ ID NOs: 86 and 87 are used as primers while SEQ ID NOs: 88 and 89 are used as probes.
SEQ ID NOs: 90-93 comprise nucleotide sequences of regions of the Soybean genome, each capable of being used as a probe or primer, either alone or in combination, for the detection of marker locus S08119-1-Ql on LG-L. In certain examples, SEQ ID NOs: 90 and 91 are used as primers while SEQ ID NOs: 92 and 93 are used as probes.
SEQ ID NOs: 94-97 comprise nucleotide sequences of regions of the Soybean genome, each capable of being used as a probe or primer, either alone or in combination, for the detection of marker locus S04867-1-A on LG-L. In certain examples, SEQ ID NOs: 94 and 95 are used as primers while SEQ ID NOs: 96 and 97 are used as probes.
SEQ ID NOs: 98-101 comprise nucleotide sequences of regions of the Soybean genome, each capable of being used as a probe or primer, either alone or in combination, for the detection of marker locus S03859-1-A on LG-L. In certain examples, SEQ ID NOs: 98 and 99 are used as primers while SEQ ID NOs: 100 and 101 are used as probes.
SEQ ID NOs: 102-105 comprise nucleotide sequences of regions of the Soybean genome, each capable of being used as a probe or primer, either alone or in combination, for the detection of marker locus S08010-1-Q1 on LG-L. In certain examples, SEQ ID NOs: 102 and 103 are used as primers while SEQ ID NOs: 104 and 105 are used as probes.
SEQ ID NOs: 106-109 comprise nucleotide sequences of regions of the Soybean genome, each capable of being used as a probe or primer, either alone or in combination, for the detection of marker locus S08010-2-Q1 on LG-L. In certain examples, SEQ ID NOs: 106 and 107 are used as primers while SEQ ID NOs: 108 and 109 are used as probes.
SEQ ED NOs: 110-113 comprise nucleotide sequences of regions of the Soybean genome, each capable of being used as a probe or primer, either alone or in combination, for the detection of marker locus S08114-1-Ql on LG-L. In certain examples, SEQ ID NOs: 110 and 111 are used as primers while SEQ ID NOs: 112 and 113 are used as probes.
SEQ ID NOs: 114-117 comprise nucleotide sequences of regions of the Soybean genome, each capable of being used as a probe or primer, either alone or in combination, for the detection of marker locus S08113-1-Q1 on LG-L. In certain examples, SEQ ID NOs: 114 and 115 are used as primers while SEQ ID NOs: 116 and 117 are used as probes.
SEQ ID NOs: 118-121 comprise nucleotide sequences of regions of the Soybean genome, each capable of being used as a probe or primer, either alone or in combination, for the detection of marker locus S08007-1-Q1 on LG-L. In certain examples, SEQ ID
NOs: 118 and 119 are used as primers while SEQ ID NOs: 120 and 121 are used as probes.
DETAILED DESCRIPTION
It is to be understood that this invention is not limited to particular embodiments or examples, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. Further, all publications referred to herein are incorporated by reference herein for the purpose cited to the same extent as if each was specifically and individually indicated to be incorporated by reference herein.
Definitions:
As used in this specification and the appended claims, terms in the singular and the singular forms "a," "an" and "the," for example, include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "plant," "the plant" or "a plant" also includes a plurality of plants; also, depending on the context, use of the term "plant" can also include genetically similar or identical progeny of that plant; use of the term "a nucleic acid" optionally includes, as a practical matter, many copies of that nucleic acid molecule; similarly, the term "probe" optionally (and typically) encompasses many similar or identical probe molecules.
Additionally, as used herein, "comprising" is to be interpreted as specifying the presence of the stated features, integers, steps, or components as referred to, but does not preclude the presence or addition of one or more features, integers, steps, or components, or groups thereof. Thus, for example, a kit comprising one pair of oligonucleotide primers may have two or more pairs of oligonucleotide primers. Additionally, the term
"comprising" is intended to include examples encompassed by the terms "consisting essentially of and "consisting of." Similarly, the term "consisting essentially of is intended to include examples encompassed by the term "consisting of."
Certain definitions used in the specification and claims are provided below. In order to provide a clear and consistent understanding of the specification and claims, including the scope to be given such terms, the following definitions are provided:
Agronomics," "agronomic traits," and "agronomic performance" refer to the traits (and underlying genetic elements) of a given plant variety that contribute to yield over the course of a growing season. Individual agronomic traits include emergence vigor, vegetative vigor, stress tolerance, disease resistance or tolerance, insect resistance or tolerance, herbicide resistance or tolerance, branching, flowering, seed set, seed size, seed density, standability, threshability, and the like.
"Allele" means any of one or more alternative forms of a genetic sequence. In a diploid cell or organism, the two alleles of a given sequence typically occupy
corresponding loci on a pair of homologous chromosomes. With regard to a SNP marker, allele refers to the specific nucleotide base present at that SNP locus in that individual plant.
The term "amplifying" in the context of nucleic acid amplification is any process whereby additional copies of a selected nucleic acid (or a transcribed form thereof) are produced. Typical amplification methods include various polymerase based replication methods, including the polymerase chain reaction (PCR), ligase mediated methods, such as the ligase chain reaction (LCR), and RNA polymerase based amplification (e.g., by transcription) methods. An "amplicon" is an amplified nucleic acid, e.g., a nucleic acid that is produced by amplifying a template nucleic acid by any available amplification method (e.g., PCR, LCR, transcription, or the like).
An "ancestral line" is a parent line used as a source of genes, e.g., for the development of elite lines.
An "ancestral population" is a group of ancestors that have contributed the bulk of the genetic variation that was used to develop elite lines.
"Backcrossing" is a process in which a breeder crosses a progeny variety back to one of the parental genotypes one or more times.
"Breeding" means the genetic manipulation of living organisms.
The term "chromosome segment" designates a contiguous linear span of genomic DNA that resides in planta on a single chromosome.
The term "crossed" or "cross" means the fusion of gametes via pollination to produce progeny (e.g., cells, seeds or plants). The term encompasses both sexual crosses (the pollination of one plant by another) and selfing (self-pollination, e.g., when the pollen and ovule are from the same plant).
"Cultivar" and "variety" are used synonymously and mean a group of plants within a species (e.g. , Glycine max) that share certain genetic traits that separate them from other possible varieties within that species. Soybean cultivars are inbred lines produced after several generations of self-pollinations. Individuals within a soybean cultivar are homogeneous, nearly genetically identical, with most loci in the homozygous state.
An "elite line" is an agronomically superior line that has resulted from many cycles of breeding and selection for superior agronomic performance. Numerous elite lines are available and known to those of skill in the art of soybean breeding. An "elite population" is an assortment of elite individuals or lines that can be used to represent the state of the art in terms of agronomically superior genotypes of a given crop species, such as soybean.
An "equivalent position" in a polynucleotide and/or polypeptide sequence is a position that correlates to a position in the reference sequence when the sequences are aligned for a maximum correspondence. In some examples, the sequences are aligned across their whole length using a global alignment program. In other examples, a portion of the sequence or sequences may be aligned using a local alignment program or a global alignment program, for example a sequence may comprise exons and introns, conserved motifs or domains, or functional motifs or domains which may be aligned to the reference sequence(s) to identify equivalent positions. Equivalent positions in polynucleotides encoding a polypeptide can be determined using the encoded amino acid, and/or using a FrameAlign program to align the polynucleotide and polypeptide for maximal
correspondence.
As used herein, the terms "exogenous" or "heterologous," as applied to
polynucleotides or polypeptides, refer to molecules that have been artificially supplied to a biological system (e.g., a plant cell, a plant gene, a particular plant species or a plant chromosome under study) and are not native to that particular biological system. The terms indicate that the relevant material originated from a source other than the naturally occurring source, or refers to molecules having a non-natural configuration, genetic location or arrangement of parts. For example, exogenous polynucleotides include polynucleotides from another organism or from the same organism which have been modified by linkage to a distinct non-endogenous polynucleotide and/or inserted to a distinct non-endogenous locus. In contrast, for example, a "native" or "endogenous" gene is a gene that does not contain nucleic acid elements encoded by sources other than the chromosome or other genetic element on which it is normally found in nature. An endogenous gene, transcript or polypeptide is encoded by its natural chromosomal locus, and not artificially supplied to the cell. The term "introduced," when referring to a heterologous or exogenous nucleic acids, refers to the incorporation of a nucleic acid into a eukaryotic or prokaryotic cell using any type of suitable vector (e.g., naked linear DNA, plasmid, plastid, or virion), converted into an autonomous replicon, or transiently expressed (e.g., transfected mRNA). The term includes such nucleic acid introduction means as "transfection," 'transformation," and "transduction." The term "host cell" means a cell that contains an exogenous nucleic acid, such as a vector, and supports the replication and/or expression of the nucleic acid. Host celfs may be prokaryotic cells such as E. colt, or eukaryotic cells such as yeast, insect, amphibian or mammalian cells. In some examples, host cells are plant cells, including, but not limited to, dicot and monocot cells.
An "exotic soybean strain" or an "exotic soybean germplasm" is a strain or germplasm derived from a soybean not belonging to an available elite soybean line or strain of germplasm. In the context of a cross between two soybean plants or strains of germplasm, an exotic germplasm is not closely related by descent to the elite germplasm with which it is crossed. Most commonly, the exotic germplasm is not derived from any known elite line of soybean, but rather is selected to introduce novel genetic elements (typically novel alleles) into a breeding program.
A "genetic map" is a description of genetic linkage relationships among loci on one or more chromosomes (or linkage groups) within a given species, generally depicted in a diagrammatic or tabular form.
"Genotype" refers to the genetic constitution of a cell or organism.
"Germplasm" means the genetic material that comprises the physical foundation of the hereditary qualities of an organism. As used herein, germplasm includes seeds and living tissue from which new plants may be grown; or, another plant part, such as leaf, stem, pollen, or cells, that may be cultured into a whole plant. Germplasm resources provide sources of genetic traits used by plant breeders to improve commercial cultivars.
An individual is "homozygous" if the individual has only one type of allele at a given locus (e.g., a diploid individual has a copy of the same allele at a locus for each of two homologous chromosomes). An individual is "heterozygous" if more than one allele type is present at a given locus (e.g., a diploid individual with one copy each of two different alleles). The term "homogeneity" indicates that members of a group have the same genotype at one or more specific loci. In contrast, the term "heterogeneity" is used to indicate that individuals within the group differ in genotype at one or more specific loci.
"Haplotype" means a combination of sequence polymorphisms that are located closely together on the same chromosome and that can discriminate between different genotypes. The combination represented by the haplotype tends to be inherited together, and this combination may represent sequence differences or alleles within a region. The region may contain one gene, or more than one gene.
The term "homologous" refers to nucleic acid sequences that are derived from a common ancestral gene through natural or artificial processes (e.g., are members of the same gene family), and thus, typically share sequence similarity. Typically, homologous nucleic acids have sufficient sequence identity that one of the sequences or a subsequence thereof or its complement is able to selectively hybridize to the other under selective (e.g. , stringent) hybridization conditions. The term "selectively hybridizes" includes reference to hybridization, under stringent hybridization conditions, of a nucleic acid sequence to a specified nucleic acid target sequence to a detectably greater degree (e.g., at least 2-fold over background) than its hybridization to non-target nucleic acid sequences and to the substantial exclusion of non-target nucleic acids. Selectively hybridizing nucleic acid sequences typically have about at least 70% sequence identity, at least 80% sequence identity, or about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.75%, or 100% sequence identity with each other. A nucleic acid that exhibits at least some degree of homology to a reference nucleic acid can be unique or identical to the reference nucleic acid or its complementary sequence.
The term "introgression" refers to the transmission of a desired allele of a genetic locus from one genetic background to another by sexual crossing, transgenic means, or any other means known in the art. For example, introgression of a desired allele at a specified locus can be transmitted to at least one progeny plant via a sexual cross between two parent plants, at least one of the parent plants having the desired allele within its genome.
Alternatively, for example, transmission of an allele can occur by recombination between two donor genomes, e.g., in a fused protoplast, where at least one of the donor protoplasts has the desired allele in its genome. The desired allele can be, e.g. , a transgene or a gene allele that imparts resistance to a plant pathogen.
The term "isolated" refers to material, such as polynucleotides or polypeptides, which are identified and separated from at least one contaminant with which it is ordinarily associated in its natural or original source. Furthermore, an isolated polynucleotide or polypeptide is typically present in a form or setting that is different from the form or setting that is normally found in nature. In some examples, the isolated molecule is substantially free from components that normally accompany or interact with it in its naturally occurring environment. In some embodiments, the isolated material optionally comprises material not found with the material in its natural environment, e.g., in a cell.
A "line" or "strain" is a group of individuals of identical parentage that are generally inbred to some degree and that are generally homozygous and homogeneous at most loci (isogenic or near isogenic). A "subline" refers to an inbred subset of descendents that are genetically distinct from other similarly inbred subsets descended from the same progenitor. Traditionally, a subline has been derived by inbreeding the seed from an individual soybean plant selected at the F3 to F5 generation until the residual segregating loci are "fixed" or homozygous across most or all loci. Commercial soybean varieties (or lines) are typically produced by aggregating ("bulking") the self-pollinated progeny of a single F3 to F5 plant from a controlled cross between two genetically different parents. While the variety typically appears uniform, the self-pollinating variety derived from the selected plant eventually (e.g., F8) becomes a mixture of homozygous plants that can vary in genotype at any locus that was heterozygous in the originally selected F3 to F5 plant. Marker-based sublines that differ from each other based on qualitative polymorphism at the DNA level at one or more specific marker loci are derived by genotyping a sample of seed derived from individual self-pollinated progeny derived from a selected F3-F5 plant. The seed sample can be genotyped directly as seed, or as plant tissue grown from such a seed sample. Optionally, seed sharing a common genotype at the specified locus (or loci) are bulked providing a subline that is genetically homogenous at identified loci important for a trait of interest (e.g., yield, tolerance, etc.).
"Linkage" refers to a phenomenon wherein alleles on the same chromosome tend to segregate together more often than expected by chance if their transmission was independent. Genetic recombination occurs with an assumed random frequency over the entire genome. Genetic maps are constructed by measuring the frequency of recombination between pairs of traits or markers. The closer the traits or markers lie to each other on the chromosome, the lower the frequency of recombination, and the greater the degree of linkage. Traits or markers are considered herein to be linked if they generally co-segregate. A 1/100 probability of recombination per generation is defined as a map distance of 1.0 centiMorgan (1.0 cM). For example, in soybean, 1 cM correlates, on average, to about 400,000 base pairs (400 Kb). The genetic elements or genes located on a single chromosome segment are physically linked. Advantageously, the two loci are located in close proximity such that recombination between homologous chromosome pairs does not occur between the two loci during meiosis with high frequency, e.g., such that linked loci co-segregate at least about 90% of the time, e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.75%, or more of the time. The genetic elements located within a chromosome segment are also genetically linked, typically within a genetic recombination distance of less than or equal to 50 centimorgans (cM), e.g., about 49, 40, 30, 20, 10, 5, 4, 3, 2, 1, 0.75, 0.5, or 0.25 cM or less. That is, two genetic elements within a single chromosome segment undergo recombination during meiosis with each other at a frequency of less than or equal to about 50%, e.g., about 49%, 40%, 30%, 20%, 10%, 5%, 4%, 3%, 2%, 1%, 0.75%, 0.5%, or 0.25% or less. Closely linked markers display a cross over frequency with a given marker of about 10% or less (the given marker is within about lOcM of a closely linked marker). Put another way, closely linked loci co-segregate at least about 90% of the time.
When referring to the relationship between two genetic elements, such as a genetic element contributing to resistance and a proximal marker, "coupling" phase linkage indicates the state where the "favorable" allele at the resistance locus is physically associated on the same chromosome strand as the "favorable" allele of the respective linked marker locus. In coupling phase, both favorable alleles are inherited together by progeny that inherit that chromosome strand. In "repulsion" phase linkage, the "favorable" allele at the locus of interest (e.g., a QTL for resistance) is physically linked with an "unfavorable" allele at the proximal marker locus, and the two "favorable" alleles are not inherited together (i.e., the two loci are "out of phase" with each other).
"Linkage disequilibrium" refers to a phenomenon wherein alleles tend to remain together in linkage groups when segregating from parents to offspring, with a greater frequency than expected from their individual frequencies.
"Linkage group" refers to traits or markers that generally co-segregate. A linkage group generally corresponds to a chromosomal region containing genetic material that encodes the traits or markers.
"Locus" is a defined segment of DNA.
A "map location" is an assigned location on a genetic map relative to linked genetic markers where a specified marker can be found within a given species. Markers are frequently described as being "above" or "below" other markers on the same linkage group; a marker is "above" another marker if it appears earlier on the linkage group, whereas a marker is "below" another marker if it appears later on the linkage group.
"Mapping" is the process of defining the linkage relationships of loci through the use of genetic markers, populations segregating for the markers, and standard genetic principles of recombination frequency.
"Marker" or "molecular marker" is a term used to denote a nucleic acid or amino acid sequence that is sufficiently unique to characterize a specific locus on the genome. Examples include Restriction Fragment Length Polymorphisms (RFLPs), Single Sequence Repeats (SSRs), Target Region Amplification Polymorphisms (TRAPs), Isozyme
Electrophoresis, Randomly Amplified Polymorphic DNAs (RAPDs), Arbitrarily Primed Polymerase Chain Reaction (AP-PCR), DNA Amplification Fingerprinting (DAF), Sequence Characterized Amplified Regions (SCARs), Amplified Fragment Length Polymorphisms (AFLPs), and Single Nucleotide Polymorphisms (SNPs). Additionally, other types of molecular markers are known to the art, and phenotypic traits may also be used as markers. All markers are used to define a specific locus on the soybean genome. Large numbers of these markers have been mapped. Each marker is therefore an indicator of a specific segment of DNA, having a unique nucleotide sequence. The map positions provide a measure of the relative positions of particular markers with respect to one another. When a trait is stated to be linked to a given marker it will be understood that the actual DNA segment whose sequence affects the trait generally co-segregates with the marker. More precise and definite localization of a trait can be obtained if markers are identified on both sides of the trait. By measuring the appearance of the marker(s) in progeny of crosses, the existence of the trait can be detected by relatively simple molecular tests without actually evaluating the appearance of the trait itself, which can be difficult and time-consuming because the actual evaluation of the trait requires growing plants to a stage and/or under specific conditions where the trait can be expressed. Molecular markers have been widely used to determine genetic composition in soybeans. Shoemaker and Olsen, ((1993) Molecular Linkage Map of Soybean (Glycine max L. Merr.). p. 6.131- 6.138. In S.J. O'Brien (ed.) Genetic Maps: Locus Maps of Complex Genomes. Cold
Spring Harbor Laboratory Press. Cold Spring Harbor, New York.), developed a molecular genetic linkage map that consisted of 25 linkage groups with about 365 RFLP, 11 RAPD, three classical markers, and four isozyme loci. See also Shoemaker R.C. (1994) RFLP Map of Soybean, pp. 299-309 in R.L. Phillips and I. K. Vasil (ed.) DNA-based markers in plants. Kluwer Academic Press Dordrecht, the Netherlands.
"Marker assisted selection" refers to the process of selecting a desired trait or desired traits in a plant or plants by detecting one or more molecular markers from the plant, where the molecular marker is linked to the desired trait.
The term "plant" includes reference to an immature or mature whole plant, including a plant from which seed or grain or anthers have been removed. Seed or embryo that will produce the plant is also considered to be the plant.
As used herein, the term "plant cell" includes, without limitation, cells within or derived from, for example and without limitation, plant seeds, plant tissue suspension cultures, plant tissue, plant tissue explants, plant embryos, meristematic tissue, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen and microspores.
"Plant parts" means any portion or piece of a plant, including leaves, stems, buds, roots, root tips, anthers, seed, grain, embryo, pollen, ovules, flowers, cotyledons, hypocotyls, pods, flowers, shoots, stalks, tissues, tissue cultures, cells and the like.
"Polymorphism" means a change or difference between two related nucleic acids. A "nucleotide polymorphism" refers to a nucleic acid comprising at least one nucleotide difference when compared to a related sequence when the two nucleic acids are aligned for maximal correspondence. A "genetic nucleotide polymorphism" refers to a nucleic acid comprising at least one nucleotide difference when compared to a related sequence when the two nucleic acids are aligned for maximal correspondence, where the two nucleic acids are genetically related, i.e., homologous, for example, where the nucleic acids are isolated from different strains of a soybean plant, or from different alleles of a single strain, or the like.
"Polynucleotide," "polynucleotide sequence," "nucleic acid sequence," "nucleic acid fragment," and "oligonucleotide" are used interchangeably herein. These terms encompass nucleotide sequences and the like. A polynucleotide may be a polymer of RNA or DNA. that is single- or double-stranded, that optionally contains synthetic, non-natural, or altered nucleotide bases. A polynucleotide in the form of a polymer of DNA may be comprised of one or more strands of cDNA, genomic DNA, synthetic DNA, or mixtures thereof: "Positional cloning" is a cloning procedure in which a target nucleic acid is identified and isolated by its genomic proximity to marker nucleic acid. For example, a genomic nucleic acid clone can include part or all of two more chromosomal regions that are proximal to one another. If a marker can be used to identify the genomic nucleic acid clone from a genomic library, standard methods such as sub-cloning or sequencing can be used to identify and or isolate subsequences of the clone that are located near the marker.
"Primer" refers to an oligonucleotide (synthetic or occurring naturally), which is capable of acting as a point of initiation of nucleic acid synthesis or replication along a complementary strand when placed under conditions in which synthesis of a
complementary strand is catalyzed by a polymerase. Typically, primers are
oligonucleotides from 10 to 30 nucleic acids in length, but longer or shorter sequences can be employed. Primers may be provided in double-stranded form, though the single- stranded form is preferred. A primer can further contain a detectable label, for example a 5' end label.
"Probe" refers to an oligonucleotide (synthetic or occurring naturally) that is complementary (though not necessarily fully complementary) to a polynucleotide of interest and forms a duplexed structure by hybridization with at least one strand of the polynucleotide of interest. Typically, probes are oligonucleotides from 10 to 50 nucleic acids in length, but longer or shorter sequences can be employed. A probe can further contain a detectable label. The terms "label" and "detectable label" refer to a molecule capable of detection, including, but not limited to, radioactive isotopes, fluorescers, chemiluminescers, enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors, chromophores, dyes, metal ions, metal sols, semiconductor nanocrystals, ligands (e.g., biotin, avidin, streptavidin, or haptens), and the like. A detectable label can also include a combination of a reporter and a quencher, such as are employed in FRET probes or
TaqMan™ probes. The term "reporter" refers to a substance or a portion thereof which is capable of exhibiting a detectable signal, which signal can be suppressed by a quencher. The detectable signal of the reporter is, e.g., fluorescence in the detectable range. The term "quencher" refers to a substance or portion thereof which is capable of suppressing, reducing, inhibiting, etc., the detectable signal produced by the reporter. As used herein, the terms "quenching" and "fluorescence energy transfer" refer to the process whereby, when a reporter and a quencher are in close proximity, and the reporter is excited by an energy source, a substantial portion of the energy of the excited state nonradiatively transfers to the quencher where it either dissipates nonradiatively or is emitted at a different emission wavelength than that of the reporter.
"RAPD marker" means random amplified polymorphic DNA marker. Chance pairs of sites complementary to single octa- or decanucleotides may exist in the correct orientation and close enough to one another for PCR amplification. With some randomly chosen decanucleotides no sequences are amplified. With others, the same length products are generated from DNAs of different individuals. With still others, patterns of bands are not the same for every individual in a population. The variable bands are commonly called random amplified polymorphic DNA (RAPD) bands.
The term "recombinant" indicates that the material (e.g., a recombinant nucleic acid, gene, polynucleotide or polypeptide) has been altered by human intervention.
Generally, the arrangement of parts of a recombinant molecule is not a native
configuration, or the primary sequence of the recombinant polynucleotide or polypeptide has in some way been manipulated. The alteration to yield the recombinant material can be performed on the material within or removed from its natural environment or state. For example, a naturally occurring nucleic acid becomes a recombinant nucleic acid if it is altered, or if it is transcribed from DNA which has been altered, by means of human intervention performed within the cell from which it originates. A gene sequence open reading frame is recombinant if that nucleotide sequence has been removed from it natural text and cloned into any type of artificial nucleic acid vector. Protocols and reagents to produce recombinant molecules, especially recombinant nucleic acids, are common and routine in the art (see, e.g., aniatis et al. (eds.), Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY, [1982]; Sambrook et al. (eds.), Molecular Cloning: A Laboratory Manual, Second Edition, Volumes 1-3, Cold Spring Harbor Laboratory Press, NY, [1989]; and Ausubel et al. (eds.), Current Protocols in Molecular Biology, Vol. 1-4, John Wiley & Sons, Inc., New York [1994]). The term recombinant can also refer to an organism that harbors a recombinant material, e.g., a plant that comprises a recombinant nucleic acid is considered a recombinant plant. In some embodiments, a recombinant organism is a transgenic organism.
"Recombination frequency" is the frequency of a crossing over event
(recombination) between two genetic loci. Recombination frequency can be observed by following the segregation of markers and/or traits during meiosis. A marker locus is "associated with" another marker locus or some other locus (for example, a tolerance locus), when the relevant loci are part of the same linkage group and are in linkage disequilibrium. This occurs when the marker locus and a linked locus are found together in progeny plants more frequently than if the two loci segregated randomly. Similarly, a marker locus can also be associated with a trait, e.g., a marker locus can be "associated with tolerance or improved tolerance," when the marker locus is in linkage disequilibrium with the trait.
"RFLP" means restriction fragment length polymorphism. Any sequence change in DNA, including a single base substitution, insertion, deletion or inversion, can result in loss or gain of a restriction endonuclease recognition site. The size and number of fragments generated by one such enzyme is therefore altered. A probe that hybridizes specifically to DNA in the region of such an alteration can be used to rapidly and specifically identify a region of DNA that displays allelic variation between two plant varieties. Isozyme Electrophoresis and RFLPs have been widely used to determine genetic composition
"Self crossing" or "self pollination" or "selfing" a process through which a breeder crosses progeny with itself; for example, a second generation hybrid F2 with itself to yield progeny designated F2:3.
"SNP" or "single nucleotide polymorphism" means a sequence variation that occurs when a single nucleotide (A, T, C, or G) in the genome sequence is altered or variable. "SNP markers" exist when SNPs are mapped to sites on the soybean genome. Many techniques for detecting SNPs are known in the art, including allele specific hybridization, primer extension, direct sequencing, and real-time PCR, such as the TaqMan™ assay.
"SSR" means short sequence repeats. "SSR markers" are genetic markers based on polymorphisms in repeated nucleotide sequences, such as microsatellites. A marker system based on SSRs can be highly informative in linkage analysis relative to other marker systems in that multiple alleles may be present. The PCR detection is done by use of two oligonucleotide primers flanking the polymorphic segment of repetitive DNA. Repeated cycles of heat denaturation of the DNA followed by annealing of the primers to their complementary sequences at low temperatures, and extension of the annealed primers with DNA polymerase, comprise the major part of the methodology. "Tolerance" and "improved tolerance" are used interchangeably herein and refer to plants in which higher doses of an herbicide are required to produce effects similar to those seen in non-tolerant plants. Tolerant plants typically exhibit fewer necrotic, lytic, chlorotic, or other lesions when subjected to the herbicide at concentrations and rates typically employed by the agricultural community. A "tolerant plant" or "tolerant plant variety" need not possess absolute or complete tolerance such that no detrimental effect to the plant or plant variety is observed when the given herbicide is applied. Instead, a "tolerant plant," "tolerant plant variety," or a plant or plant variety with "improved tolerance" will simply be less affected by the given herbicide than a comparable susceptible plant or variety.
"Transgenic plant" refers to a plant that comprises within its cells an exogenous polynucleotide, e.g., a polynucleotide from another organism (including a polynucleotide from another soybean plant). Generally, the exogenous polynucleotide is stably integrated within a genome such that the polynucleotide is passed on to successive generations. The exogenous polynucleotide may be integrated into the genome alone or as part of a recombinant expression cassette. "Transgenic" is used herein to refer to any cell, cell line, callus, tissue, plant part, or plant, the genotype of which has been altered by the presence of exogenous nucleic acid including those transgenic organisms or cells initially so altered, as well as those created by crosses or asexual propagation from the initial transgenic organism or cell. The term "transgenic" as used herein does not encompass the alteration of the genome (chromosomal or extra-chromosomal) by conventional plant breeding methods (e.g., crosses) or by naturally occurring events such as random cross-fertilization, non- recombinant viral infection, non-recombinant bacterial transformation, non-recombinant transposition, or spontaneous mutation.
"TRAP marker" means target region amplification polymorphism marker. The
TRAP technique employs one fixed primer of known sequence in combination with a random primer to amplify genomic fragments. The differences in fragments between alleles can be detected by gel electrophoresis.
The term "vector" is used in reference to polynucleotide or other molecules that transfer nucleic acid segment(s) into a cell. A vector optionally comprises parts which mediate vector maintenance and enable its intended use (e.g., sequences necessary for replication, genes imparting drug or antibiotic resistance, a multiple cloning site, operably linked promoter/enhancer elements which enable the expression of a cloned gene, etc.). Vectors are often derived from plasmids, bacteriophages, or plant or animal viruses.
The term "yield" refers to the productivity per unit area of a particular plant product of commercial value. For example, yield of soybean is commonly measured in bushels of seed per acre or metric tons of seed per hectare per season. Yield is affected by both genetic and environmental factors. Yield is the final culmination of all agronomic traits.
MESOTRIONE AND ISOXAZQLE
Mesotrione and isoxazole are two herbicide classes from different chemical families, but both can act as hydroxyphenyl pyruvate dioxygenase (HPPD) inhibitors.
Isoxazole is used as a pre-plant herbicide while mesotrione is used as either a pre-plant or post-emergent herbicide. Isoxazole is member of the isoxazole chemical family.
Following either foliar or root uptake, isoxazole is rapidly converted to a diketonitrile derivative (2-cyclopropyI-3 -(2-mesyl-4-trifluoromethylphenyl)-3 -oxopropanenitrile) by opening of the isoxazole ring. This diketonitrile undergoes degradation to a benzoic acid derivative (2-mesyl-4-trifiuoromethyl benzoic acid) in treated plants and the extent of this degradation is correlated to the degree of susceptibility, being most rapid in tolerant plants and slowest in susceptible plants.
Mesotrione belongs to the triketone family of herbicides, which are chemically derived from a natural phytotoxin produced by the bottlebrush plant Callistemon citrinus. Mesotrione works by inhibiting HPPD (p-hydroxyphenylpyruvate dioxygenase), an essential enzyme in the biosynthesis of carotenoids. Carotenoids protect chlorophyll from excess light energy. MOLECULAR MARKERS AND GENETIC LINKAGE
In traditional linkage analysis, no direct knowledge of the physical relationship of genes on a chromosome is required. Mendel's first law is that factors of pairs of characteristics are segregated, meaning that alleles of a diploid trait separate into two gametes and then into different offspring. Classical linkage analysis can be thought of as a statistical description of the relative frequencies of cosegregation of different traits.
Linkage analysis, as described previously, is the well-characterized descriptive framework of how traits are grouped together based upon the frequency with which they segregate together. Because chromosomal distance is approximately proportional to the frequency of crossing over events between traits, there is an approximate physical distance that correlates with recombination frequency.
Marker loci are traits, and can be assessed according to standard linkage analysis by tracking the marker loci during segregation. Thus, one cM is equal to a 1% chance that a marker locus will be separated from another locus (which can be any other trait, e.g., another marker locus, or another trait locus that encodes a QTL), due to crossing over in a single generation. Any detectible polymorphic trait can be used as a marker so long as it is inherited differentially and exhibits linkage disequilibrium with a phenotypic trait of interest. A number of soybean markers have been mapped and linkage groups created, as described in Cregan, P.B. et ah, "An Integrated Genetic Linkage Map of the Soybean Genome" (1999) Crop Science 39: 1464-90, and more recently in Choi et i, "A Soybean Transcript Map: Gene Distribution, Haplotype and Single-Nucleotide Polymorphism Analysis" (2007) Genetics 176:685-96. Many soybean markers are publicly available at the USDA affiliated soybase website.
Most plant traits of agronomic importance are polygenic, otherwise known as quantitative traits. A quantitative trait is controlled by two or more genes located at various locations, or loci, in the plant's genome. The multiple genes have a cumulative effect which contributes to the continuous range of phenotypes observed in many plant traits. These genes are referred to as quantitative trait loci (QTL). Recombination frequency measures the extent to which a molecular marker is linked with a QTL. Lower recombination frequencies, typically measured in centiMorgans (cM), indicate greater linkage between the QTL and the molecular marker. The extent to which two features are linked is often referred to as the genetic distance. The genetic distance is also typically related to the physical distance between the marker and the QTL; however, certain biological phenomenon (including recombinational "hot spots") can affect the relationship between physical distance and genetic distance. Generally, the usefulness of a molecular marker is determined by the genetic and physical distance between the marker and the selectable trait of interest.
The method for determining the presence or absence of a QTL associated with tolerance or sensitivity to mesotrione and/or isoxazole herbicides in soybean germplasm, comprises analyzing genomic DNA from a soybean germplasm for the presence of at least one molecular marker, wherein at least one molecular marker is linked to the QTL, and wherein the QTL maps to soybean major linkage group L and is associated with tolerance or sensitivity to mesotrione and/or isoxazole herbicides. The term "is associated with" in this context means that the QTL associated with tolerance or sensitivity to mesotrione and/or isoxazole herbicides has been found to be present in soybean plants showing tolerance or sensitivity to mesotrione and/or isoxazole herbicides as described herein.
Any marker that is linked to a trait of interest {e.g., in the present case, a tolerance or improved tolerance trait) can be used as a marker for that trait. Thus, in addition to the markers described herein, markers linked to the markers itemized herein can also be used to predict the tolerance, improved tolerance, or susceptibility/sensitivity trait. Such linked markers are particularly useful when they are sufficiently proximal to a given marker so that they display a low recombination frequency with the given marker. Markers linked and/or closely linked to the given markers are provided, for example, in Figure 1. These include, for example, SATT495, SATT723, Sat_408, A169_l, EV2_1, Sle3_4s,
BLT010_2, BLT007J, SATT232, S04867-1-A, S08102-1-Q1, S08103-1-Q1, S08104-1- Ql, S08106-1-Q1, S08107-1-Q1, S08109-1-Q1, S08110-1-Q1, S08111-1-Q1, S08115-2- Ql, S08117-1-Q1, S08119-1-Q1, S08116-1-Q1, S08112-1-Q1, S08108-1-Q1, S08101-4- Ql, S08101-1-Q1, S08101-2-Q1, S08101-3-Q1, S08118-1-Q1, S08114-1-Q1, S08113-1- Ql, S03859-1-A, Sat_301, SATT446, P10649C-3, SATT232, SO8105-1-Q1, SALT 182, S08010-1-Q1 , S08010-2-Q1, R176_l, JUBC090, SATT238, Sat_071, BLT039 1,
Bng071_l, SATT388, A264_l, RGA 7, RGA7, SATT523, Sat_134, S00224-1, S01659-1, LbA, i8_2, A450 2, A106_l, Sat_405, SATT143, B124_2, A459_l, SATT398, SATT694, Sat_195, Sat_388, SATT652, SATT711, Sat_187, SATT418, SATT278, Sat_397, Sat_191, Sat_320, O109_l, A204 2, SATT497, G214_17, SATT313, B164_l, G214 16, SATT613, A023_l, SATT284, AW508247, SATT462, L050J7, E014_l, A071_5, B046_l, LI, and B162_2.
Marker loci are especially useful when they are closely linked to target loci {e.g., QTL for tolerance, or, alternatively, simply other marker loci, such as those identified herein, that are linked to such QTL) for which they are being used as markers. A marker more closely linked to a target locus is a better indicator for the target locus (due to the reduced cross-over frequency between the target locus and the marker). Thus, in one example, closely linked loci such as a marker locus and a second locus {e.g., a given marker or a QTL) display an inter-locus cross-over frequency of about 10% or less, about 9% or less, about 8% or less, about 7% or less, about 6% or less, about 5% or less, about 4% or less, about 3% or less, or about 2% or less. In some examples, the relevant loci (e.g. , a marker locus and a target locus such as a QTL) display a recombination a frequency of about 1% or less, e.g., about 0.75% or less, about 0.5% or less, or about 0.25% or less. Thus, the loci are about 10 cM, 9 cM, 8 cM, 7 cM, 6 cM, 5 cM, 4 cM, 3 cM, 2cM, lcM, 0.75 cM, 0.5 cM or 0.25 cM or less apart. Put another way, two loci that are localized to the same chromosome, and at such a distance that recombination between the two loci occurs at a frequency of no more than 10% (e.g., about 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.75%, 0.5%, 0.25%, or less) are said to be proximal to each other.
Many marker alleles can be detected or selected for or against. Optionally, one, two, three, or more marker allele(s) can be identified in or introgressed into the plant. Plants or germplasm frequently are identified that have at least one favorable allele that positively correlates with tolerance or improved tolerance. However, it is useful for exclusionary purposes during breeding to also identify alleles that negatively correlate with tolerance, to eliminate such plants or germplasm from subsequent rounds of breeding.
The identification of favorable marker alleles may be germplasm-specific. The determination of which marker alleles correlate with tolerance (or non-tolerance) is determined for the particular germplasm under study. One of skill recognizes that methods for identifying favorable alleles are routine and well known, and, furthermore, that the identification and use of such favorable alleles is well within the scope of the invention.
Numerous markers disclosed herein have been found to be associated with or to correlate with tolerance, improved tolerance, or susceptibility/sensitivity to mesotrione and/or isoxazole herbicides in soybean. Generally, markers that map closer to the QTL mapped to linkage group L and associated with tolerance or sensitivity to mesotrione and/or isoxazole herbicides are superior to markers that map farther from the QTL. In some examples, a marker used to determine the presence or absence of a QTL mapping to soybean linkage group L and associated with tolerance or sensitivity to mesotrione and/or isoxazole herbicides includes one or more of SATT495, P10649C-3, S ATT 182, S03859-1, S00224-1, SATT388, SATT313, and SATT613, or other markers above marker SATT613 on LG-L. Additional useful and/or relevant markers include S03859-1-A, S08103-1-Q1, S08104-1-Q1, S08106-1-Q1, S08110-1-Q1, S08111-1-Q1, S08U5-2-Q1, S08117-1-Q1, S081 19-1-Ql, S08118-1-Q1, S08116-1-Q1, S08114-1-Q1, S08113-1-Q1, S08112-1-Q1, S08108-1-Q1, S08101-2-Q1, S08101-3-Q1, S08101-4-Q1, S08105-1-Q1, S08102-1-Q1, S08107-1-Q1, S08109-1-Q1, and S08101-1-Q1. Any marker assigned to soybean linkage group L and linked or closely linked to a marker disclosed herein as associated with tolerance or sensitivity to mesotrione and/or isoxazole herbicides may be used. Generally, a linked marker is within 50 cM of the referenced marker or trait, and a closely linked marker is within 10 cM of the referenced marker or trait. Updated information regarding markers assigned to soybean linkage group L may be found on the USDA's Soybase website. Further, linkage group L is now formally referred to as chromosome #19.
Intervals defined by markers flanking the QTL associated with tolerance or sensitivity to mesotrione and/or isoxazole herbicides are useful, as well. For interval determination, the genomic DNA of soybean germplasm is typically tested for the presence of at least two of the foregoing molecular markers, one marker on each side of the QTL. Examples of such intervals include the interval flanked by and including SATT613 and above on LG-L, the interval flanked by and including markers SATT495 and SATT613, the interval flanked by and including SATT313 and above on LG-L, the interval flanked by and including markers SATT495 and SATT313, the interval flanked by and including markers SATT495 and SATT388, the interval flanked by and including markers P10649C- 3 and S ATT 182, the interval flanked by and including markers S04867-1-A and S03859-1- A, the interval flanked by and including markers S08110-1-Ql and S08010-1-Q1, the interval flanked by and including markers S08117-1-Ql and S08010-1-Q1, the interval flanked by and including markers S08110-1-Q1 and S08105-1-Q1, the interval flanked by and including markers S08117-1-Ql and S08105-1-Q1, and the interval flanked by and including markers S08113-1-Q1 and S08105-1-Q1.
Initial fine mapping isolated the location of the QTL associated with herbicide tolerance/sensitivity to a -56 kb interval between marker S08117-1-Ql and S08105-1-Q1 on linkage group L. Further fine mapping refined the location of the QTL to a -44 kb interval between marker S08113-1-Q1 and S08105-1-Q1 on linkage group L.
Accordingly, markers that map within the interval defined by and including these markers are particularly useful for selecting for this QTL. These markers include S08117-1-Ql, S08119-1-Q1, S08118-1-Q1, S08U6-1-Q1, S08101-1-Q1, S08112-1-Q1, S08108-1-Q1, S08101-1-Q1, S08101-2-Q1, S08101-3-Q1, S08101-4-Q1, and S08105-1-Q1. In some examples, the markers are S08112-1-Q1, S08108-1-Q1, S08101-2-Q1, S08101-3-Q1, and S08101-4-Q1.
Methods of introgressing tolerance to mesotrione and/or isoxazole herbicides into non-tolerant or less-tolerant soybean germplasm are provided. Any method for introgressing QTLs into soybean plants can be used. In some examples, a first soybean germplasm that contains tolerance or sensitivity to mesotrione and/or isoxazole herbicides derived from the QTL mapped to linkage group L which is associated with tolerance or sensitivity to mesotrione and/or isoxazole herbicides and a second soybean germplasm that lacks tolerance or sensitivity to mesotrione and/or isoxazole herbicides derived from the QTL mapped to linkage group L are provided. The first soybean plant may be crossed with the second soybean plant to provide progeny soybeans. Phenotypic and/or marker screening is performed on the progeny plants to determine the presence of tolerance or sensitivity to mesotrione and/or isoxazole herbicides derived from the QTL mapped to linkage group L. Progeny that test positive for the presence of tolerance or sensitivity to mesotrione and/or isoxazole herbicides derived from the QTL mapped to linkage group L can be selected.
In some examples, the screening and selection are performed by using marker- assisted selection using any marker or combination of markers on major linkage group L provided. Any method of identifying the presence or absence of these markers may be used, including for example single-strand conformation polymorphism (SSCP) analysis, base excision sequence scanning (BESS), RFLP analysis, heteroduplex analysis, denaturing gradient gel electrophoresis, temperature gradient electrophoresis, allelic PCR, ligase chain reaction direct sequencing, mini sequencing, nucleic acid hybridization, or micro-array-type detection.
Amplification primers for amplifying marker loci and suitable marker probes to detect marker loci or to genotype SNP alleles are provided, for example, in Figure 2 and the related sequence listing (SEQ ID NOs: 1-121). Optionally, other sequences to either side of the given primers can be used in place of the given primers, so long as the primers can amplify a region that includes the allele to be detected. Further, it will be appreciated that the precise probe to be used for detection can vary, e.g., any probe that can identify the region of a marker amplicon to be detected can be substituted for those examples provided herein. The configuration of the amplification primers and detection probes can, of course, vary. Thus, the invention is not limited to the primers and probes specifically recited herein.
Systems, including automated systems for selecting plants that comprise a marker of interest and/or for correlating presence of the marker with tolerance are also provided. These systems can include probes relevant to marker locus detection, detectors for detecting labels on the probes, appropriate fluid handling elements and temperature controllers that mix probes and templates and/or amplify templates, and systems and/or instructions that correlate label detection to the presence of a particular marker locus or allele.
Kits are also provided. For example, a kit can include appropriate primers or probes for detecting tolerance associated marker loci and instructions for using the primers or probes for detecting the marker loci and correlating the loci with predicted tolerance to mesotrione and/or isoxazole herbicides. The kits can further include packaging materials for packaging the probes, primers, or instructions; controls, such as control amplification reactions that include probes, primers, or template nucleic acids for amplifications;
molecular size markers; or the like.
Isolated nucleic acid fragments comprising a nucleic acid sequence coding for soybean tolerance or sensitivity to mesotrione and/or isoxazole herbicides, are provided. The nucleic acid fragment comprises at least a portion of a nucleic acid belonging to linkage group L. The nucleic acid fragment is capable of hybridizing under stringent conditions to a nucleic acid of a soybean cultivar tolerant to mesotrione and/or isoxazole. herbicides containing a QTL associated with mesotrione and/or isoxazole herbicide tolerance that is located on major linkage group L.
Vectors comprising such nucleic acid fragments, expression products of such vectors expressed in a host compatible therewith, antibodies to the expression product
(both polyclonal and monoclonal), and antisense nucleic acid to the nucleic acid fragment are also provided.
Seed of a soybean produced by crossing a soybean variety having mesotrione and/or isoxazole herbicide tolerance QTL located on major linkage group L in its genome with another soybean variety, and progeny thereof, are provided.
DETECTION METHODS Any suitable detection method known in the art can be used to detect the markers, QTL, or traits discussed herein. Γη some examples, the presence of marker loci is directly detected in unamplified genomic DNA by performing a Southern blot on a sample of genomic DNA using probes to the marker loci. In other examples, amplification based techniques are employed. PCR, RT-PCR, and LCR are in particularly broad use as amplification and amplification-detection methods for amplifying nucleic acids of interest, thus facilitating detection of markers. Procedures for performing Southern blotting, amplification (PCR, LCR, or the like), and many other nucleic acid detection methods are well established and are taught, e.g., in Sambrook et al, Molecular Cloning - A Laboratory Manual (3d ed.), Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 2000 ("Sambrook"); Current Protocols in Molecular Biology, F.M. Ausubel et al, eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (supplemented through 2002) ("Ausubel")) and PCR Protocols A Guide to Methods and Applications (Innis et al. eds) Academic Press Inc. San Diego, CA (1990) (Innis). Additional details regarding detection of nucleic acids in plants can also be found, e.g., in Plant Molecular Biology (1993) Croy (ed.) BIOS Scientific Publishers, Inc.
Typically, molecular markers are detected by any established method available, including, without limitation, allele specific hybridization (ASH), real-time PCR assays for detecting single nucleotide polymorphisms (SNP), amplified fragment length
polymorphism (AFLP) detection, amplified variable sequence detection, randomly amplified polymorphic DNA (RAPD) detection, restriction fragment length polymorphism (RFLP) detection, self-sustained sequence replication detection, simple sequence repeat (SSR) detection, single-strand conformation polymorphisms (SSCP) detection, isozyme markers detection, or the like. While the exemplary markers provided in the tables herein are either SSR or SNP markers, any of the aforementioned marker types can be employed to identify chromosome segments encompassing genetic element that contribute to superior agronomic performance (e.g., tolerance or improved tolerance).
In another example, the presence or absence of a molecular marker is determined by nucleotide sequencing of the polymorphic marker region. This method is readily adapted to high throughput analysis, as are the other methods noted above, e.g. , using available high throughput sequencing methods such as sequencing by hybridization. In general, the majority of genetic markers rely on one or more properties of nucleic acids for their detection. For exam le, some techniques for detecting genetic markers utilize hybridization of a probe nucleic acid to nucleic acids corresponding to the genetic marker (e.g., amplified nucleic acids produced using genomic soybean DNA as a template). Hybridization formats, including but not limited to solution phase, solid phase, mixed phase, or in situ hybridization assays are useful for allele detection. An extensive guide to the hybridization of nucleic acids is found in Tijssen (1993) Laboratory
Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes Elsevier, New York, as well as in Sambrook and Ausubel.
For example, markers that comprise restriction fragment length polymorphisms
(RFLP) are detected, e.g., by hybridizing a probe which is typically a sub-fragment (or a synthetic oligonucleotide corresponding to a sub-fragment) of the nucleic acid to be detected to restriction-digested genomic DNA. The restriction enzyme is selected to provide restriction fragments of at least two alternative (or polymorphic) lengths in different individuals or populations. Determining one or more restriction enzymes that produce informative fragments for each cross is a simple procedure. After separation by length in an appropriate matrix (e.g., agarose, polyacrylamide, etc.) and transfer to a membrane (e.g., nitrocellulose, nylon, etc.), the labeled probe is hybridized under conditions which result in equilibrium binding of the probe to the target followed by removal of excess probe by washing.
In some examples, molecular markers are detected using a suitable PCR-based detection method. This includes methods where the size or sequence of the PCR amplicon is indicative of the absence or presence of the marker (e.g., a particular marker allele), as well as methods where a labeled allele-specific probe is used for detection (e.g., a TaqMan® assay). In these types of methods, PCR primers and, optionally, probes are hybridized to the conserved regions flanking the polymorphic marker region. Suitable primers can be designed using any suitable method. It is not intended that the invention be limited to any particular primer or primer pair. For example, primers can be designed using any suitable software program, such as LASERGENE®.
In some examples, primers are labeled by any suitable means (e.g., using a nonradioactive fluorescent tag) to allow for rapid visualization of the different size amplicons following an amplification reaction without any additional labeling step or visualization step. In some examples, the primers are not labeled, and the amplicons are visualized following their size resolution, e.g., following agarose gel electrophoresis. In some examples, ethidium bromide staining of the PCR amplicons following size resolution allows visualization of the different size amplicons.
The primers used to amplify the marker loci and alleles herein are not limited to amplifying the entire region of the relevant locus. In some examples, marker amplification produces an amplicon at least 20 nucleotides in length, or alternatively, at least 50 nucleotides in length, or alternatively, at least 100 nucleotides in length, or alternatively, at least 200 nucleotides in length, or up to and including the full length of the amplicon.
Nucleic acid probes to the marker loci can also be cloned and/or synthesized. Any suitable label can be used with a probe. Detectable labels suitable for use with nucleic acid probes include, for example, any composition detectable by spectroscopic, radioisotopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Useful labels include biotin for staining with labeled streptavidin conjugate, magnetic beads, fluorescent dyes, radiolabels, enzymes, and colorimetric labels. Other labels include ligands, which bind to antibodies labeled with fluorophores, chemiluminescent agents, and enzymes. A probe can also constitute radiolabelled PCR primers that are used to generate a radiolabelled amplicon. Methods and reagents for labeling nucleic acids and
corresponding detection strategies can be found, e.g., in Haugland (1996) Handbook of Fluorescent Probes and Research Chemicals Sixth Edition by Molecular Probes, Inc. (Eugene OR); or Haugland (2001) Handbook of Fluorescent Probes and Research
Chemicals Eighth Edition by Molecular Probes, Inc. (Eugene OR).
Separate detection probes can also be omitted in amplification/detection methods, e.g., by performing a real time amplification reaction that detects product formation by modification of the relevant amplification primer upon incorporation into a product, incorporation of labeled nucleotides into an amplicon, or by monitoring changes in molecular rotation properties of amplicons as compared to unamplified precursors (e.g., by fluorescence polarization).
In alternative embodiments, in silico methods can be used to detect the marker loci of interest. For example, the sequence of a nucleic acid comprising the marker locus of interest can be stored in a computer. The desired marker locus sequence or its homolog can be identified using an appropriate nucleic acid search algorithm as provided by, for example, in such readily available programs as BLAST, or even simple word processors.
Real Time Amplification/ Detection Methods:
In one aspect, real time PCR or LCR is performed on the amplification mixtures described herein, e.g., using molecular beacons or TaqMan™ probes. A molecular beacon (MB) is an oligonucleotide or peptide nucleic acid (PNA) which, under appropriate hybridization conditions, self-hybridizes to form a stem and loop structure. The MB has a label and a quencher at the termini of the oligonucleotide or PNA; thus, under conditions that permit intra-molecular hybridization, the label is typically quenched (or at least altered in its fluorescence) by the quencher. Under conditions where the MB does not display intra-molecular hybridization (e.g., when bound to a target nucleic acid, e.g., to a region of an amplicon during amplification), the MB label is unquenched and signal is detected. Standard methods of making and using MBs are known and MBs and reagents are commercially available. See also, e.g., Leone et al. (1995) "Molecular beacon probes combined with amplification by NASBA enable homogenous real-time detection of RNA." Nucleic Acids Res. 26:2150-2155; Tyagi and Kramer (1996) "Molecular beacons: probes that fluoresce upon hybridization" Nature Biotechnology 14:303-308; Blok and Kramer (1 97) "Amplifiable hybridization probes containing a molecular switch" Mol Cell Probes 11:187-194; Hsuih et al. (1997) "Novel, ligation-dependent PCR assay for detection of hepatitis C in serum" J Clin Microbiol 34:501-507; Kostrikis et al. (1998) "Molecular beacons: spectral genotyping of human alleles" Science 279:1228-1229; Sokol et al. (1998) "Real time detection of DNA:RNA hybridization in living cells" Proc. Natl. Acad. Sci. U.S.A. 95:11538-11543; Tyagi et al. (1998) "Multicolor molecular beacons for allele discrimination" Nature Biotechnology 16:49-53; Bonnet et al. (1999) "Thermodynamic basis of the chemical specificity of structured DNA probes" Proc. Natl. Acad. Sci. U.S.A. 96:6171-6176; Fang et al. (1999) "Designing a novel molecular beacon for surface- immobilized DNA hybridization studies" J. Am. Chem. Soc. 121:2921-2922; Marras et al. (1999) "Multiplex detection of single-nucleotide variation using molecular beacons" Genet. Anal. Biomol. Eng. 14:151-156; and Vet et al. (1999) "Multiplex detection of four pathogenic retroviruses using molecular beacons" Proc. Natl. Acad. Sci. U.S.A. 96:6394- 6399. See also, e.g., US Patent 5,925,517 (July 20, 1999) to Tyagi et al. entitled "Detectably labeled dual conformation oligonucleotide probes, assays and kits;" US Patent 6,1 0,097 to Tyagi et al (November 21, 2000) entitled "Nucleic acid detection probes having non-FRET fluorescence quenching and kits and assays including such probes" and US Patent 6,037,130 to Tyagi et al (March 14, 2000), entitled "Wavelength-shifting probes and primers and their use in assays and kits."
PCR detection and quantification using dual-labeled fluorogenic oligonucleotide probes can be done, using, for example, TaqMan® probes. These probes are composed of short (e.g., 10-40 bases) oligodeoxynucleotides that are labeled with two different fluorescent dyes. On the 5' terminus of each probe is a reporter dye, and on the 3' terminus of each probe a quenching dye is found. The oligonucleotide probe sequence is complementary to an internal target sequence present in a PGR amplicon. When the probe is intact, energy transfer occurs between the two fluorophores and emission from the reporter is quenched by the quencher via FRET. During the extension phase of PCR, the probe is cleaved by 5' nuclease activity of the polymerase used in the reaction, thereby releasing the reporter from the oligonucleotide-quencher and producing an increase in reporter emission intensity. Accordingly, TaqMan® probes are oligonucleotides that have a label and a quencher, where the label is released during amplification by the exonuclease action of the polymerase used in amplification. This provides a real time measure of amplification during synthesis. A variety of TaqMan® reagents are commercially available, e.g., from Applied Biosystems (Division Headquarters in Foster City, CA) as well as from a variety of specialty vendors such as Biosearch Technologies (e.g., black hole quencher probes).
In general, synthetic methods for making oligonucleotides, including probes, primers, molecular beacons, PNAs, LNAs (locked nucleic acids), etc., are well known. For example, oligonucleotides can be synthesized chemically according to the solid phase phosphoramidite triester method described by Beaucage and Caruthers (1981), Tetrahedron Letts 22: 1 859-1862, e.g., using a commercially available automated synthesizer.
Oligonucleotides, including modified oligonucleotides and PNAs, can also be ordered from a variety of commercial sources known to persons of skill.
Additional Details Regarding Amplified Variable Sequences, SSR, AFLP ASH, SNPs, and Isozyme Markers Amplified variable sequences refer to amplified sequences of the plant genome, which exhibit high nucleic acid residue variability between members of the same species. All organisms have variable genomic sequences and each organism (with the exception of a clone) has a different set of variable sequences. Once identified, the presence of specific variable sequence can be used to predict phenotypic traits. Typically, DNA from the plant serves as a template for amplification with primers that flank a variable sequence of DNA. The variable sequence is amplified and then sequenced.
Alternatively, self-sustained sequence replication can be used to identify genetic markers. Self-sustained sequence replication refers to a method of nucleic acid
amplification using target nucleic acid sequences which are replicated exponentially in vitro under substantially isothermal conditions by using three enzymatic activities involved in retroviral replication: (1) reverse transcriptase, (2) RNase H, and (3) a DNA-dependent RNA polymerase (Guatelli et al. (1990) Proc Natl Acad Sci USA 87:1874). By mimicking the retroviral strategy of RNA replication by means of cDNA intermediates, this reaction accumulates cDNA and RNA copies of the original target.
Amplified fragment length polymorphisms (AFLP), which are amplified before or after cleavage by a restriction endonuclease, can also be used as genetic markers (V os et al. (1995) Nucl Acids Res 23:4407). The amplification step allows easier detection of specific restriction fragments. AFLP allows the detection large numbers of polymorphic markers and has been used for genetic mapping of plants (Becker et al. (1995) Mol Gen Genet 249:65; and Meksem et al (1995) Mol Gen Genet 249:74).
Allele-specific hybridization (ASH) can be used to identify the genetic markers. ASH technology is based on the stable annealing of a short, single-stranded,
oligonucleotide probe to a completely complementary single-strand target nucleic acid. Detection is via an isotopic or non-isotopic label attached to the probe.
For each polymorphism, two or more different ASH probes are designed to have identical DNA sequences except at the polymorphic nucleotides. Each probe will have exact homology with one allele sequence so that the range of probes can distinguish all the known alternative allele sequences. Each probe is hybridized to the target DNA. With appropriate probe design and hybridization conditions, a single-base mismatch between the probe and target DNA will prevent hybridization. In this manner, only one of the alternative probes will hybridize to a target sample that is homozygous or homogenous for an allele. Samples that are heterozygous or heterogeneous for two alleles will hybridize to both of two alternative probes.
ASH markers are used as dominant markers where the presence or absence of only one allele is determined from hybridization or lack of hybridization by only one probe. The alternative allele may be inferred from the lack of hybridization. ASH probe and target molecules are optionally RNA or DNA; the target molecules are any length of nucleotides beyond the sequence that is complementary to the probe; the probe is designed to hybridize with either strand of a DNA target; the probe ranges in size to conform to variously stringent hybridization conditions, etc.
PCR allows the target sequence for ASH to be amplified from low concentrations of nucleic acid in relatively small volumes. Otherwise, the target sequence from genomic DNA is digested with a restriction endonuclease and size separated by gel electrophoresis. Hybridizations typically occur with the target sequence bound to the surface of a membrane or, as described in U.S. Patent 5,468,613, the ASH probe sequence may be bound to a membrane. In one example, ASH data are typically obtained by amplifying nucleic acid fragments (amplicons) from genomic DNA using PCR, transferring the amplicon target DNA to a membrane in a dot-blot format, hybridizing a labeled oligonucleotide probe to the amplicon target, and observing the hybridization dots by autoradiography.
Single nucleotide polymorphi sms (SNP) are markers that consist of a shared sequence differentiated on the basis of a single nucleotide. Typically, this distinction is detected by differential migration patterns of an amplicon comprising the SNP on, e.g., an acrylamide gel. However, alternative modes of detection, such as hybridization, e.g., ASH, or RFLP analysis are also appropriate.
Isozyme markers can be employed as genetic markers, e.g. , to track markers other than the tolerance markers herein, or to track isozyme markers linked to the markers herein. Isozymes are multiple forms of enzymes that differ from one another in their amino acid sequence, and therefore their nucleic acid sequences. Some isozymes are multimeric enzymes containing slightly different subunits. Other isozymes are either multimeric or monomeric but have been cleaved from the proenzyme at different sites in the amino acid sequence. Isozymes can be characterized and analyzed at the protein level, or alternatively, isozymes, which differ at the nucleic acid level, can be determined. In such cases any of the nucleic acid based methods described herein can be used to analyze isozyme markers.
MARKER ASSISTED SELECTION AND BREEDING OF PLANTS
The identification of markers associated with a particular phenotypic trait can allow for selection of plants possessing that trait, for example, via marker assisted selection (MAS). In general, the application of MAS uses the identification of a population of tolerant plants and genetic mapping of the tolerance trait. Polymorphic loci in the vicinity of the mapped tolerance trait are chosen as potential tolerance markers. Typically, a marker locus closest to the tolerance locus is a preferred marker. Linkage analysis is then used to determine which polymorphic marker allele sequence demonstrates a statistical likelihood of co-segregation with the tolerant phenotype (thus, a "tolerance marker allele").
Following identification of a marker allele for co-segregation with the tolerance allele, it is possible to use this marker for rapid, accurate screening of plant lines for the tolerance allele without the need to grow the plants through their life cycle and await phenotypic evaluations, and furthermore, permits genetic selection for the particular tolerance allele even when the molecular identity of the actual tolerance QTL is anonymous. Tissue samples can be taken, for example, from the first leaf of the plant and screened with the appropriate molecular marker, and within days it is determined which progeny will advance. Linked markers also remove the impact of environmental factors that can often influence phenotypic expression.
After a desired phenotype (e.g., tolerance or sensitivity to mesotrione and/or isoxazole herbicides) and a polymorphic chromosomal marker locus are determined to cosegregate, the polymorphic marker locus can be used to select for marker alleles that segregate with the desired tolerance phenotype. This general process is typically called marker-assisted selection (MAS). In brief, a nucleic acid corresponding to the marker nucleic acid is detected in a biological sample from a plant to be selected. This detection can take the form of hybridization of a probe nucleic acid to a marker allele or amplicon thereof, e.g., using allele-specific hybridization, Southern analysis, northern analysis, in situ hybridization, hybridization of primers followed by PCR amplification of a region of the marker, or the like. After the presence (or absence) of a particular marker in the biological sample is verified, the plant is selected, e.g., used to make progeny plants by selective breeding.
Soybean plant breeders desire combinations of tolerance loci with genes for high yield and other desirable traits to develop improved soybean varieties. Screening large numbers of samples by non-molecular methods (e.g., trait evaluation in soybean plants) can be expensive, time consuming, and unreliable. Use of the polymorphic markers described herein genetically linked to tolerance loci provide effective methods for selecting tolerant varieties in breeding programs. For example, one advantage of marker-assisted selection over field evaluations for tolerance is that MAS can be done at any time of year, regardless of the growing season. Moreover, environmental effects are largely irrelevant to marker-assisted selection.
When a population is segregating for multiple loci affecting one or multiple traits, e.g., multiple loci involved in tolerance, or multiple loci each involved in tolerance or tolerance to different herbicides, the efficiency of MAS compared to phenotypic screening becomes even greater, because all of the loci can be evaluated in the lab together from a single sample of DNA. In the present instance, for linkage group L, relevant markers include: SATT495, P10649C-3, S ATT 182, S03859-1, S00224-1, SATT388, SATT313, and SATT613 (or other markers above SATT613). Additional relevant markers on linkage group L include S03859-1-A, S08103-1-Q1, S08104-1-Q1, S08106-1-Q1, S08110-1-Q1, S081 1 1-1-Q1, S08115-2-Q1, S08117-1-Q1, S08119-1-Q1, S08118-1-Q1, S08116-1-Q1, S081 14-1-Q1, S08113-1-Q1, S08112-1-Q1, S08108-1-Q1, S08101-2-Q1, S08101-3-Q1, S08101-4-Q1, S08105-1-Q1, S08102-1-Q1, S08107-1-Q1, S08109-1-Q1, and S08101-1- Ql, , and markers for other traits, transgenes, and/or loci can be assayed simultaneously or sequentially in a single sample or population of samples. Markers for other traits, transgenes, and/or loci can be assayed simultaneously or sequentially in a single sample or population of samples.
Another use of MAS in plant breeding is to assist the recovery of the recurrent parent genotype by backcross breeding. Backcross breeding is the process of crossing a progeny back to one of its parents or parent lines. Backcrossing is usually done for the purpose of introgressing one or a few loci from a donor parent (e.g. , a parent comprising desirable tolerance marker loci) into an otherwise desirable genetic background from the recurrent parent (e.g., an otherwise high yielding soybean line). The more cycles of backcrossing that are done, the greater the genetic contribution of the recurrent parent to the resulting introgressed variety. This is often necessary, because tolerant plants may be otherwise undesirable, e.g., due to low yield, low fecundity, or the like. In contrast, strains which are the result of intensive breeding programs may have excellent yield, fecundity or the like, merely being deficient in one desired trait, such as tolerance or sensitivity to mesotrione and/or isoxazole herbicides.
The determination of the presence and/or absence of a particular genetic marker or allele, e.g., SATT495, P10649C-3, S ATT 182, S03859-1, S00224-1, SATT388, SATO 13, SATT613 (including markers above SATT613), S03859-1-A, S08103-1-Q1, S08104-1-Q1, S08106-1-Q1, S08110-1-Q.1, S08111-1-Q1, S08115-2-Q1, S08117-1-Q1, S08119-1-Q1, S08118-1-Q1, S08116-1-Q1, S08114-1 -Q1, S08113-1-Q1, S08112-1-Q1, S08108-1-Q1, S08101-2-Q1, S08101-3-Q1, S08101-4-Q1, S08105-1-Q1, S08102-1-Q1, S08107-1-Q1, S08109-1-Q1, or S08101-1-Q1, in the genome of a plant exhibiting a preferred phenotypic trait can be made by any method noted herein. If the nucleic acids from the plant are positive for a desired genetic marker, the plant can be self fertilized to create a true breeding line with the same genotype, or it can be crossed with a plant with the same marker or with other desired characteristics to create a sexually crossed hybrid generation.
Introgression of Favorable Alleles— Efficient Crossing: of Tolerance Markers into Other Lines
One application of MAS is to use the tolerance or improved tolerance markers to increase the efficiency of an introgression or backcrossing effort aimed at introducing a tolerance QTL into a desired (typically high yielding) background. In marker assisted backcrossing of specific markers (and associated QTL) from a donor source, e.g., to an elite genetic background, one selects among progeny or backcross progeny for the donor trait.
Thus, the markers and methods can be utilized to guide marker assisted selection or breeding of soybean varieties with the desired complement (set) of allelic forms of chromosome segments associated with herbicide tolerance as well as markers associated with superior agronomic performance (tolerance, along with any other available markers for yield, disease tolerance, etc.). Any of the disclosed marker alleles can be introduced into a soybean line via, for example, introgression, traditional breeding, or transformation, or a combination thereof, to yield a soybean plant with superior agronomic performance. The number of alleles associated with mesotrione and/or isoxazole tolerance that can be introduced or be present in a soybean plant ranges from 1 to the number of alleles disclosed herein, each integer of which is incorporated herein as if explicitly recited.
Methods of making a progeny soybean plant, and these progeny soybean plants having tolerance or susceptibility to mesotrione and/or isoxazole, are provided. These methods comprise crossing a first parent soybean plant with a second soybean plant and growing the female soybean plant under plant growth conditions to yield soybean plant progeny. Such soybean plant progeny can be assayed for alleles associated with tolerance and, thereby, the desired progeny selected. Such progeny plants or seed can be sold commercially for soybean production, used for food, processed to obtain a desired constituent of the soybean, or further utilized in subsequent rounds of breeding. At least one of the first or second soybean plants is a soybean plant comprising at least one of the allelic forms of the markers provided, such that the progeny are capable of inheriting the allele.
Inheritance of the desired tolerance allele can be traced, such as from progenitor or descendant lines in the subject soybean plants pedigree such that the number of generations separating the soybean plants being subject to the methods will generally be from 1 to 20, commonly 1 to 5, and typically 1, 2, or 3 generations of separation, and quite often a direct descendant or parent of the soybean plant will be subject to the method (i.e., 1 generation of separation).
POSITIONAL CLONING
The molecular marker loci and alleles associated with tolerance or susceptibility to mesotrione and/or isoxazole, e.g., SATT495, P10649C-3, SATT182, S03859-1, S00224-1, SATT388, SATT313, SATT613 (including markers above SATT613), S03859-1-A, S08103-1-Q1, S08104-1-Q1, S08106-1-Q1, S081 10-I-Q1, S08111-1-Q1, S08115-2-Q1, S08117-1-Q1, S08119-1-Q1, S08118-1-Q1, S08116-1-Q1, S08114-1-Q1, S08113-1-Q1, S08112-1-QL S08108-1-Q1, S08101-2-Q1, S08101-3-Q1, S08101-4-Q1, S08105-1-Q1, S08102-1-Q1 , S08107-1-Q1, S08109-1-Q1, and S08101-1 -Ql, can be used, as indicated previously, to identify a tolerance QTL, which can be cloned by well-established procedures, e.g., as described in detail in Ausubel, Berger and Sambrook. These tolerance clones are first identified by their genetic linkage to markers provided herein. Isolation of a nucleic acid of interest is achieved by any number of methods as discussed in detail in such references as Ausubel, Berger and Sambrook, herein, and Clark, ed. (1997) Plant Molecular Biology: A Laboratory Manual Springer- Verlag, Berlin.
For example, "positional gene cloning" uses the proximity of a tolerance marker to physically define an isolated chromosomal fragment containing a tolerance QTL gene. The isolated chromosomal fragment can be produced by such well known methods as digesting chromosomal DNA with one or more restriction enzymes, or by amplifying a chromosomal region in a polymerase chain reaction (PCR), or any suitable alternative amplification reaction. The digested or amplified fragment is typically ligated into a vector suitable for replication, and, e.g., expression, of the inserted fragment. Markers that are adjacent to an open reading frame (ORF) associated with a phenotypic trait can hybridize to a DNA clone (e.g., a clone from a genomic DNA library), thereby identifying a clone on which an ORF (or a fragment of an ORF) is located. If the marker is more distant, a fragment containing the open reading frame is identified by successive rounds of screening and isolation of clones which together comprise a contiguous sequence of DNA, a process termed
"chromosome walking", resulting in a "contig" or "contig map." Protocols sufficient to guide one of skill through the i solation of clones associated with linked markers are found in, e.g. Berger, Sambrook and Ausubel, all herein.
Variant sequences have a high degree of sequence similarity. For polynucleotides, conservative variants include those sequences that, because of the degeneracy of the genetic code, encode the amino acid sequence of one of the native recombinase polypeptides. Variants such as these can be identified with the use of well-known molecular biology techniques, as, for example, with polymerase chain reaction (PCR) and hybridization techniques. Variant polynucleotides also include synthetically derived nucleotide sequences, such as those generated, for example, by using site-directed mutagenesis but which still encode a recombinase protein. Generally, variants of a particular polynucleotide will have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to that particular polynucleotide as determined by known sequence alignment programs and parameters. Variants of a particular polynucleotide (the reference nucleotide sequence) can also be evaluated by comparison of the percent sequence identity between the polypeptide encoded by a variant polynucleotide and the polypeptide encoded by the reference polynucleotide. Percent sequence identity between any two polypeptides can be calculated using sequence alignment programs and parameters described. Where any given pair of polynucleotides is evaluated by comparison of the percent sequence identity shared by the two polypeptides they encode, the percent sequence identity between the two encoded polypeptides is at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity.
Variant proteins include proteins derived from the native protein by deletion, addition, and/or substitution of one or more amino acids to the N-terminal, internal region(s), and/or C-terminal end of the native protein. Variant proteins can be biologically active, that is they continue to possess the desired biological activity of the native protein, for example a variant recombinase can implement a recombination event between appropriate recombination sites. Such variants may result from, for example, genetic polymorphism or from human manipulation. A biologically active variant of a protein may differ from that protein by as few as 1-15 amino acid residues, as few as 1-10, such as 6- 10, as few as 5, as few as 4, 3, 2, or even 1 amino acid residue.
Sequence relationships can be analyzed and described using computer-implemented algorithms. The sequence relationship between two or more polynucleotides or two or more polypeptides can be determined by generating the best alignment of the sequences, and scoring the matches and the gaps in the alignment, which yields the percent sequence identity, and the percent sequence similarity. Polynucleotide relationships can also be described based on a comparison of the polypeptide each encodes. Many programs and algorithms for the comparison and analysis of sequences are available.
Unless otherwise stated, sequence identity/similarity values provided herein refer to the value obtained using GAP Version 10 (GCG, Accelrys, San Diego, CA) using the following parameters: % identity and % similarity for a nucleotide sequence using a gap creation penalty weight of 50 and a gap length extension penalty weight of 3, and the nwsgapdnaxmp scoring matrix; % identity and % similarity for an amino acid sequence using a GAP creation penalty weight of 8 and a gap length extension penalty of 2, and the BLOSUM62 scoring matrix (Henikoff & Henikoff (1989) Proc Natl Acad Sci USA 89:10915).
GAP uses the algorithm of Needleman & Wunsch (1970) J Mol Biol 48:443-453, to find an alignment of two complete sequences that maximizes the number of matches and minimizes the number of gaps. GAP considers all possible alignments and gap positions and creates the alignment with the largest number of matched bases and the fewest gaps. It allows for the provision of a gap creation penalty and a gap extension penalty in units of matched bases. GAP must make a profit of gap creation penalty number of matches for each gap it inserts. If a gap extension penalty greater than zero is chosen, GAP must, in addition, make a profit for each gap inserted of the length of the gap times the gap extension penalty. GAP presents one member of the family of best alignments.
Sequence identity, or identity, is a measure of the residues in the two sequences that are the same when aligned for maximum correspondence. Sequences, particularly polypeptides, that differ by conservative substitutions are said to have sequence similarity or similarity. Means for making this adjustment are known, and typically involve scoring a conservative substitution as a partial rather than a full mismatch. For example, where an identical amino acid is given a score of 1 and a non-conser ative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated using the selected scoring matrix (BLOSUM62 by default for GAP).
Equivalent positions between two or more polynucleotides, and/or polypeptides can be identified using any searching, sequence assembly, and/or alignment tool including, but not limited to, BLAST, GAP, PILEUP, FrameAIign, Sequencher, or similar tools. In some examples, GAP alignment can be used to identify equivalent positions, using the following parameters: for a nucleotide sequence using a gap creation penalty weight of 50 and a gap length extension penalty weight of 3, and the nwsgapdna.cmp scoring matrix; for an amino acid sequence using a gap creation penalty weight of 8 and a gap length extension penalty of 2, and the BLOSUM62 scoring matrix (Henikoff & Henikoff (1989) Proc Natl Acad Sci USA 89:10915). In some examples, PILEUP can be used to identify equivalent positions, using the following parameters for a nucleotide sequence: a gap weight of 5 and a gap length weight of 1, and the pileupdna.cmp scoring matrix; for an amino acid sequence using a gap weight of 8 and a gap length weight of 2, and the BLOSUM62 scoring matrix (Henikoff & Henikoff (1989) Proc Natl Acad Sci USA 89:10915).
Proteins may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions. Methods for such manipulations are generally known. Methods for mutagenesis and nucleotide sequence alterations are described, for example, in Kunkel (1 85) Proc Natl Acad Sci USA 82:488-492; Kunkel et al. (1987) Methods in Enzymol 154:367-382; U.S. Patent 4,873,192; Walker & Gaastra, eds. (1983) Techniques in Molecular Biology (MacMillan Publishing Company, New York) and the references cited therein. Guidance as to appropriate amino acid substitutions that do not affect biological activity of the protein of interest may be found in the model of Dayhoff et al. (1978) Atlas of Protein Sequence and Structure (Natl Biomed Res Found, Washington, D.C.). Conservative substitutions, such as exchanging one amino acid with another having similar properties, may be preferable. GENERATION OF TRANSGENIC CELLS AND PLANTS
The present invention also relates to host cells and organisms which are transformed with nucleic acids corresponding to the tolerance, improved tolerance, or susceptibility/sensitivity markers, traits, or QTLs identified herein. For example, such nucleic acids include chromosome intervals (e.g., genomic fragments), ORFs, and/or cDNAs that encode a tolerance or improved tolerance trait. Additionally, production of polypeptides that provide tolerance or improved tolerance by recombinant techniques are provided.
General texts which describe molecular biological techniques for the cloning and manipulation of nucleic acids and production of encoded polypeptides include Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology volume 152 Academic Press, Inc., San Diego, Calif. (Berger); Sambrook et al, Molecular Cloning— A Laboratory Manual (3rd Ed.), Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 2001 ("Sambrook") and Current Protocols in Molecular Biology, F. M. Ausubel et al, eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (supplemented through 2004 or later)
("Ausubel")). These texts describe mutagenesis, the use of vectors, promoters and many other relevant topics related to, e.g., the generation of clones that comprise nucleic acids of interest, e.g., marker loci, marker probes, QTL that segregate with marker loci, etc.
Host cells are genetically engineered {e.g., transduced, transfected, transformed, etc.) with the vectors {e.g., vectors, such as expression vectors which comprise an ORF derived from or related to a tolerance QTL) which can be, for example, a cloning vector, a shuttle vector, or an expression vector. Such vectors are, for example, in the form of a plasmid, a phagemid, an agrobacterium, a virus, a naked polynucleotide (linear or circular), or a conjugated polynucleotide. Vectors can be introduced into bacteria, especially for the purpose of propagation and expansion. The vectors are also introduced into plant tissues, cultured plant cells, or plant protoplasts by a variety of standard methods known in the art, including but not limited to electroporation (From et al. (1985) Proc. Natl. Acad. Sci. USA 82; 5824), infection by viral vectors such as cauliflower mosaic virus (CaMV) (Hohn et al. (1982) Molecular Biology of Plant Tumors (Academic Press, New York, pp. 549-560; Howell U.S. Pat. No. 4,407,956), high velocity ballistic penetration by small particles with the nucleic acid either within the matrix of small beads or particles or on the surface (Klein et al. (1987) Nature 327;70), use of pollen as vector (WO 85/01856), or use of
Agrobacterium tumefaciens or A. rhizogenes carrying a T-DNA plasmid in which DNA fragments are cloned. The T-DNA plasmid is transmitted to plant cells upon infection by Agrobacterium tumefaciens, and a portion is stably integrated into the plant genome (Horsch et al. (1984) Science 233:496; Fraley et al. (1983) Proc. Natl. Acad. Sci. USA 80:4803). Additional details regarding nucleic acid introduction methods are found in Sambrook, Berger and Ausubel. The method of introducing a nucleic acid into a host cell is not critical, and therefore should not be limited to any particular method for introducing exogenous genetic material into a host cell. Thus, any suitable method that provides for effective introduction of a nucleic acid into a cell or protoplast can be employed.
The engineered host cells can be cultured in conventional nutrient media modified as appropriate for such activities as, for example, activating promoters or selecting transformants. These cells can optionally be cultured into transgenic plants. In addition to Sambrook, Berger and Ausubel, Plant regeneration from cultured protoplasts is described in Evans et al. (1983) "Protoplast Isolation and Culture," Handbook of Plant Cell Cultures 1, 124-176 (MacMillan Publishing Co., New York; Davey (1983) "Recent Developments in the Culture and Regeneration of Plant Protoplasts," Protoplasts, pp. 12-29, (Birkhauser, Basel); Dale (1983) "Protoplast Culture and Plant Regeneration of Cereals and Other Recalcitrant Crops," Protoplasts pp. 31-41, (Birkhauser, Basel); Binding (1985)
"Regeneration of Plants," Plant Protoplasts, pp. 21-73, (CRC Press, Boca Raton, Fla.). Additional details regarding plant cell culture and regeneration include Payne et al. (1 92) Plant Cell and Tissue Culture in Liquid Systems John Wiley & Sons, Inc. New York, N.Y.; Gamborg and Phillips (eds) (1995) Plant Cell, Tissue and Organ Culture; Fundamental Methods Springer Lab Manual, Springer- Verlag (Berlin Heidelberg New York) and Plant Molecular Biology (1993) R. R. D. Croy, Ed. Bios Scientific Publishers, Oxford, U.K. ISBN 0 12 198370 6. Cell culture media in general are also set forth in Atlas and Parks (eds), The Handbook of Microbiological Media (1993) CRC Press, Boca Raton, Fla.
Additional information for cell culture is found in available commercial literature such as the Life Science Research Cell Culture Catalogue (1998) from Sigma-Aldrich, Inc (St Louis, Mo.) ("Sigma-LSRCCC") and the Plant Culture Catalogue and supplement (e.g., 1997 or later), also from Sigma-Aldrich, Inc (St Louis, Mo.) ("Sigma-PCCS").
The production of transgenic organisms is provided, which may be bacteria, yeast, fungi, animals or plants, transduced with the nucleic acids (e.g., nucleic acids comprising the marker loci and/or QTL noted herein). A thorough discussion of techniques relevant to bacteria, unicellular eukaryotes, and cell culture is found in references enumerated herein. Several well-known methods of introducing target nucleic acids into bacterial cells are available, any of which may be used. These include: fusion of the recipient cells with bacterial protoplasts containing the DNA, treatment of the cells with liposomes containing the DNA, electroporation, microinjection, cell fusions, projectile bombardment (biolistics), carbon fiber delivery, and infection with viral vectors (discussed further, below). Bacterial cells can be used to amplify the number of plasmids containing DNA constructs. The bacteria are grown to log phase and the plasmids within the bacteria can be isolated by a variety of methods known in the art (see, for instance, Sambrook). In addition, a plethora of kits are commercially available for the purification of plasmids from bacteria. For their proper use, follow the manufacturer's instructions (see, for example, EasyPrep.TM., FlexiPrep.TM., both from Pharmacia Biotech; StrataClean.TM., from Stratagene; and, QIAprep.TM. from Qiagen). The isolated and purified plasmids are then further manipulated to produce other plasmids, used to transfect plant cells or incorporated into Agrobacterium tumefaciens related vectors to infect plants. Typical vectors contain transcription and translation terminators, transcription and translation initiation sequences, and promoters useful for regulation of the expression of the particular target nucleic acid. The vectors optionally comprise generic expression cassettes containing at least one independent terminator sequence, sequences permitting replication of the cassette in eukaryotes, or prokaryotes, or both, (e.g., shuttle vectors), and selection markers for both prokaryotic and eukaryotic systems. Vectors are suitable for replication and integration in prokaryotes, eukaryotes, or both. See, Giliman & Smith (1979) Gene 8:81; Roberts et al. (1987) Nature 328:731 ; Schneider et al. (1995) Protein Expr. Purif. 6435:10; Ausubel, Sambrook, Berger (all infra). A catalogue of bacteria and bacteriophages useful for cloning are well known in the art, e.g., The ATCC Catalogue of Bacteria and Bacteriophage (1992) Gheraa et al. (eds), published by the ATCC.
Polynucleotide Constructs:
In specific embodiments, one or more of the herbicide-tolerant polynucleotides employed in the methods and compositions can be provided in an expression cassette for expression in the plant or other organism of interest The cassette will include 5' and 3' regulatory sequences operably linked to an herbicide-tolerance polynucleotide. "Operably linked" is intended to mean a functional linkage between two or more elements. For example, an operable linkage between a polynucleotide of interest and a regulatory sequence (e.g., a promoter) is functional link that allows for expression of the
polynucleotide of interest. Operably linked elements may be contiguous or noncontiguous. When used to refer to the joining of two protein coding regions, by "operably linked" is intended that the coding regions are in the same reading frame. When used to refer to the effect of an enhancer, "operably linked" indicates that the enhancer increases the expression of a particular polynucleotide or polynucleotides of interest. Where the polynucleotide or polynucleotides of interest encode a polypeptide, the encoded polypeptide is produced at a higher level.
The cassette may additionally contain at least one additional gene to be co- transformed into the organism. Alternatively, the additional gene(s) can be provided on multiple expression cassettes. Such an expression cassette is provided with a plurality of restriction sites and/or recombination sites for insertion of the herbicide-tolerance polynucleotide to be under the transcriptional regulation of the regulatory regions. The expression cassette may additionally contain other genes, including other selectable marker genes. Where a cassette contains more than one polynucleotide, the polynucleotides in the cassette may be transcribed in the same direction or in different directions (also called "divergent" transcription).
An expression cassette comprising an herbicide-tolerance polynucleotide will include, in the 5'-3' direction of transcription, a transcriptional and translational initiation region (i.e., a promoter), an herbicide-tolerance polynucleotide, and a transcriptional and translational termination region (i.e., termination region) functional in plants or the other organism of interest. Accordingly, plants having such expression cassettes are also provided. The regulatory regions (i.e., promoters, transcriptional regulatory regions, and translational termination regions) and/or the herbicide-tolerance polynucleotide may be native (i.e., analogous) to the host cell or to each other. Alternatively, the regulatory regions and/or the herbicide-tolerance polynucleotide may be heterologous to the host cell or to each other.
While it may be optimal to express polynucleotides using heterologous promoters, native promoter sequences may be used. Such constructs can change expression levels and/or expression patterns of the encoded polypeptide in the plant or plant cell. Expression levels and/or expression patterns of the encoded polypeptide may also be changed as a result of an additional regulatory element that is part of the construct, such as, for example, an enhancer. Thus, the phenotype of the plant or cell can be altered even though a native promoter is used.
The termination region may be native with the transcriptional initiation region, may be native with the operably linked herbicide-tolerance polynucleotide of interest, may be native with the plant host, or may be derived from another source (i.e., foreign or heterologous) to the promoter, the herbicide-tolerance polynucleotide of interest, the plant host, or any combination thereof. Convenient termination regions are available from the Ti-plasmid of A. tumefaciens, such as the octopine synthase and nopaline synthase termination regions, or can be obtained from plant genes such as the Solanum tuberosum proteinase inhibitor II gene. See Guerineau et al. (1991) Mol. Gen. Genet. 262: 141-144; Proudfoot (1991) Cell 64: 671-674; Sanfacon et al. (1991) Genes Dev. 5: 141-149; Mogen et al. (1990) Plant Cell 2: 1261-1272; Munroe et al. (1990) Gene 91: 151-158; Ballas et al. (1989) Nucleic Acids Res. 17: 7891-7903; and Joshi et al. (1987) Nucleic Acids Res. 15: 9627-9639.
A number of promoters can be used, including the native promoter of the polynucleotide sequence of interest. The promoters can be selected based on the desired outcome. The polynucleotides of interest can be combined with constitutive, tissue- preferred, or other promoters for expression in plants.
Such constitutive promoters include, for example, the core promoter of the Rsyn7 promoter and other constitutive promoters disclosed in WO 99/43838 and U.S. Pat. No. 6,072,050; the core CaMV 35S promoter (Odell et al (1985) Nature 313:810-812); rice actin (McElroy et al. (1990) Plant Cell 2:163-171); the maize actin promoter; the ubiquitin promoter (see, e.g., Christensen et al. (1989) Plant Mol. Biol. 12:619-632; Christensen et al. (1992) Plant Mol. Biol. 18:675-689; Callis et al. (1995) Genetics 139:921-39); pEMU (Last et al (1991) Theor. Appl. Genet. 81 :581-588); MAS (Velten et al. (1984) EMBO J. 3: 2723-2730); ALS promoter (U.S. Pat. No. 5,659,026), and the like. Other constitutive promoters include, for example, those described in U.S. Pat. Nos. 5,608,149; 5,608, 144; 5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463; 5,608,142; and 6, 177,611. Some promoters show improved expression when they are used in conjunction with a native 5' untranslated region and/or other elements such as, for example, an intron. For example, the maize ubiquitin promoter is often placed upstream of a polynucleotide of interest along with at least a portion of the 5' untranslated region of the ubiquitin gene, including the first intron of the maize ubiquitin gene.
Chemical-regulated promoters can be used to modulate the expression of a gene in a plant through the application of an exogenous chemical regulator. Depending upon the objective, the promoter may be a chemical-inducible promoter for which application of the chemical induces gene expression or the promoter may be a chemical-repressible promoter for which application of the chemical represses gene expression. Chemical-inducible promoters are known in the art and include, but are not limited to, the maize In2-2 promoter, which is activated by benzenesulfonamide herbicide safeners, the maize GST promoter, which is activated by hydrophobic electrophilic compounds that are used as pre- emergent herbicides, and the tobacco PR-la promoter, which is activated by salicylic acid. Other chemical-regulated promoters of interest include steroid-responsive promoters (see, e.g., the glucocorticoid-inducible promoter in Schena et al. (1991) Proc. Natl. Acad. Sci. USA 88:10421-10425 and McNellis et al. (1998) Plant J. 14:247-257) and tetracycline- inducible and tetracycline-repressible promoters {see, e.g., Gatz et al. (1991) Mol. Gen. Genet. 227:229-237, and U.S. Pat. Nos. 5,814,618 and 5,789,156), herein incorporated by reference.
Tissue-preferred promoters can be utilized to target enhanced herbicide-tolerance polypeptide expression within a particular plant tissue. Tissue-preferred promoters include Yamamoto et al. (1997) Plant J. 12:255-265; Kawamata et al. (1997) Plant Cell Physiol. 38:792-803; Hansen et al. (1997) Mol. Gen Genet. 254:337-343; Russell et al. (1997) Transgenic Res. 6:157-168; Rinehart et al. (1996) Plant Physiol. 112:1331-1341 ; Van Camp et al. (1996) Plant Physiol. 112:525-535; Canevascini et al. (1996) Plant Physiol. 112:513-524; Yamamoto et al. (1994) Plant Cell Physiol. 35:773-778; Lam (1994) Results Probl. Cell Differ. 20:181-196; Orozco et al. (1993) Plant Mol Biol. 23: 1129-1138;
Matsuoka et al. (1993) Proc Natl. Acad. Sci. USA 90:9586-9590; and Guevara- Garcia et al. (1993) Plant J. 4:495-505. Such promoters can be modified, if necessary, for weak expression.
Leaf-preferred promoters are known in the art. See, e.g., Yamamoto et al. (1997) Plant J. 12:255-265; Kwon et al. (1994) Plant Physiol. 105:357-67; Yamamoto et al (1994) Plant Cell Physiol. 35:773-778; Gotor et al. (1993) Plant J. 3:509-18; Orozco et al. (1993) Plant Mol. Biol. 23:1129-1138; and Matsuoka et al. (1993) Proc. Natl. Acad. Sci. USA 90(20):9586-9590.
Root-preferred promoters are known and can be selected from the many available from the literature or isolated de novo from various compatible species. See, e.g., Hire et al. (1992) Plant Mol. Biol. 20:207-218 (soybean root-specific glutamine synthetase gene); Keller and Baumgartner (1991) Plant Cell 3:1051-1061 (root-specific control element in the GRP 1.8 gene of French bean); Sanger et al. (1990) Plant Mol. Biol. 14(3):433-443 (root-specific promoter of the mannopine synthase (MAS) gene of Agrobacterium tumefaciens); and Miao et al. (1991) Plant Cell 3:11-22 (full-length cDNA clone encoding cytosolic glutamine synthetase (GS), which is expressed in roots and root nodules of soybean). See also Bogusz et al. (1990) Plant Cell 2(7): 633-641, where two root-specific promoters are described. Leach and Aoyagi (1991) describe their analysis of the promoters of the highly expressed rolC and roID root- inducing genes of Agrobacterium rhizogenes {see Plant Science (Limerick) 79:69-76). They concluded that enhancer and tissue- preferred DNA determinants are dissociated in those promoters. Teeri et al. (1989) used gene fusion to lacZ to show that the Agrobacterium T-DNA gene encoding octopine synthase is especially active in the epidermis of the root tip and that the TR2' gene is root specific in the intact plant and stimulated by wounding in leaf tissue, an especially desirable combination of characteristics for use with an insecticidal or larvicidal gene {see EMBO J. 8:343-350). The TRT gene, fused to nptll (neomycin phosphotransferase II) showed similar characteristics. Additional root-preferred promoters include the VfENOD- GRP3 gene promoter (Kuster et al (1995) Plant Mol. Biol. 29:759-772); and rolB promoter (Capana et al. (1994) Plant Mol. Biol. 25:681-691. See also U.S. Pat. Nos. 5,837,876; 5,750,386; 5,633,363; 5,459,252; 5,401,836; 5,110,732; and 5,023,179.
Seed-preferred promoters include both seed-specific promoters (those promoters active during seed development such as promoters of seed storage proteins) as well as seed-germinating promoters (those promoters active during seed germination). See Thompson et al. (1989) BioEssays 10: 108, herein incorporated by reference. Such seed- preferred promoters include, but are not limited to, Ciml (cytokinin-induced message); cZ19Bl (maize 19 kDa zein); milps (myo-inositol-1 -phosphate synthase) (see WO 00/11177 and U.S. Pat. No. 6,225,529; herein incorporated by reference). Gamma-zein is an endosperm-specific promoter. Globulin 1 (GIb-1) is a representative embryo-specific promoter. For dicots, seed-specific promoters include, but are not limited to, bean β- phaseolin, napin, β-conglycinin, soybean lectin, cruciferin, and the like. For monocots, seed-specific promoters include, but are not limited to, maize 1 kDa zein, 22 kDa zein, 27 kDa zein, gamma-zein, waxy, shrunken 1, shrunken 2, globulin 1, etc. See also WO 00/12733, where seed-preferred promoters from endl and end2 genes are disclosed; herein incorporated by reference.
Additional promoters of interest include the SCP1 promoter (U.S. Pat. No.
6,072,050), the HB2 promoter (U.S. Pat. No. 6,177,611) and the SAMS promoter
(US20030226166 and SEQ ID NO: 87 and biologically active variants and fragments thereof); each of which is herein incorporated by reference. In addition, as discussed elsewhere herein, various enhancers can be used with these promoters including, for example, the ubiquitin intron {i.e., the maize ubiquitin intron 1 {see, e.g., NCBI sequence S94464), the omega enhancer or the omega prime enhancer (Gallie et al. (1989) Molecular Biology of RNA ed. Cech (Liss, N.Y.) 237-256 and Gallie et al. Gene (1987) 60:217-25), or the 35S enhancer; each of which is incorporated by reference.
The expression cassette can also comprise a selectable marker gene for the selection of transformed cells. Selectable marker genes are utilized for the selection of transformed cells or tissues. Marker genes include genes encoding antibiotic resistance, such as those encoding neomycin phosphotransferase II (NEO) and hygromycin phosphotransferase (HPT), as well as genes conferring resistance to herbicidal compounds, such as glufosinate ammonium, bromoxynil, imidazolinones, and 2,4-dichlorophenoxyacetate (2,4-D).
Additional selectable markers include phenotypic markers such as beta-galactosidase and fluorescent proteins such as green fluorescent protein (GFP) (Su et al. (2004) Biotechnol Bioeng 85:610-9 and Fetter et al. (2004) Plant Cell 16:215-28), cyan florescent protein (CYP) (Bolte et al. (2004) J. Cell Science 117:943-54 and Kato et al. (2002) Plant Physiol 129:913-42), and yellow fluorescent protein (PhiYFP from Evrogen, see, Bolte et al (2004) J. Cell Science 117:943-54). For additional selectable markers, see generally Yarranton (1992) Curr. Opin. Biotech. 3:506-511; Christopherson et al. (1992) Proc. Natl. Acad. Sci. USA 89:6314-6318; Yao et al. (1992) Cell 71 :63-72; Reznikoff (1992) Mol. Microbiol. 6:2419-2422; Barkley et al (1980) in The Operon, pp. 177-220; Hu et al.
(1987) Cell 48:555-566; Brown et al. (1987) Cell 49:603-612; Figge et al. (1988) Cell 52:713-722; Deuschle et al (1989) Proc. Natl. Acad. Aci. USA 86:5400-5404; Fuerst et al. (1989) Proc. Natl. Acad. Sci. USA 86:2549-2553; Deuschle et al (1990) Science 248:480- 483; Gossen (1993) Ph.D. Thesis, University of Heidelberg; Reines et al. (1993) Proc. Natl. Acad. Sci. USA 90: 1917-1921; Labow et al. (1990) Mol. Cell. Biol. 10:3343-3356; Zambretti et al. (1992) Proc. Natl. Acad. Sci. USA 89:3952-3956; Bairn et al. (1991) Proc. Natl. Acad. Sci. USA 88:5072-5076; Wyborski et al. (1991) Nucleic Acids Res. 19:4647- 4653; Hillenand-Wissman (1989) Topics Mol. Struc. Biol. 10: 143-162; Degenkolb et al. (1991) Antimicrob. Agents Chemother. 35: 1591-1595; Kleinschnidt et al (1988)
Biochemistry 27:1094-1104; Bonin (1993) Ph.D. Thesis, University of Heidelberg; Gossen et al. (1992) Proc. Natl. Acad. Sci. USA 89:5547-5551; Oliva et al. (1992) Antimicrob. Agents Chemother. 36:913-919; Hlavka et al. (1985) Handbook of Experimental
Pharmacology, Vol. 78 (Springer-Verlag, Berlin); Gill et al. (1988) Nature 334:721-724. Such disclosures are herein incorporated by reference. The above list of selectable marker genes is not meant to be limiting. Any selectable marker gene can be used, including the GAT gene and or H A gene.
Methods are known in the art of increasing the expression level of a polypeptide in a plant or plant cell, for example, by inserting into the polypeptide coding sequence one or two G/C-rich codons (such as GCG or GCT) immediately adjacent to and downstream of the initiating methionine ATG codon. Where appropriate, the polynucleotides may be modified for increased expression in the transformed plant. That is, the polynucleotides can be synthesized substituting in the polypeptide coding sequence one or more codons which are less frequently utilized in plants for codons encoding the same amino acid(s) which are more frequently utilized in plants, and introducing the modified coding sequence into a plant or plant cell and expressing the modified coding sequence. See, e.g., Campbell and Gowri (1990) Plant Physiol. 92:1-11 for a discussion of host-preferred codon usage. Methods are available in the art for synthesizing plant-preferred genes. See, e.g., U.S. Pat. Nos. 5,380,831, and 5,436,391, and Murray et al. (1989) Nucleic Acids Res. 17:477-498, herein incorporated by reference. Embodiments comprising such modifications are also a feature disclosed.
Additional sequence modifications are known to enhance gene expression in a cellular host. These include elimination of sequences encoding spurious polyadenylation signals, exon-intron splice site signals, transposon-like repeats, and other such well- characterized sequences that may be deleterious to gene expression. The G-C content of the sequence may be adjusted to levels average for a given cellular host, as calculated by reference to known genes expressed in the host cell. When possible, the sequence is modified to avoid predicted hairpin secondary mRNA structures. Enhancers such as the CaMV 35S enhancer may also be used (see, e.g., Benfey et al. (1990) EMBO J. 9:1685- 96), or other enhancers may be used. For example, the sequence set forth in SEQ ID NO: 1, 72, 79, 84, 85, 88, or 89 or a biologically active variant or fragment thereof can be used. See also published application US2007/0061917. As used herein, an enhancer, when operably linked to an appropriate promoter, will modulate the level of transcription of an operably linked polynucleotide of interest. Biologically active fragments and variants of the enhancer domain may retain the biological activity of modulating (increase or decrease) the level of transcription when operab y linked to an appropriate promoter. Generally, variants of a particular polynucleotides will have at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to another polynucleotides as determined by sequence alignment programs and parameters. Variants of a particular polynucleotides also include those encoding a polypeptide having at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference polypeptide as determined by sequence alignment programs and parameters. Polypeptide variants include those encoded by variant polynucleotides, and those having at least about 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference polypeptide as determined by sequence alignment programs and parameters.
It is also recognized that the level and/or activity of a polypeptide of interest may be modulated by employing a polynucleotide that is not capable of directing, in a transformed plant, the expression of a protein or an RNA. For example, the
polynucleotides may be used to design polynucleotide constructs that can be employed in methods for altering or mutating a genomic nucleotide sequence in an organism. Such polynucleotide constructs include, but are not limited to, RNA:DNA vectors, RNArDNA mutational vectors, RNA:DNA repair vectors, mixed-duplex oligonucleotides, self- complementary RNA:DNA oligonucleotides, and recombinogenic oligonucleobases. Such nucleotide constructs and methods of use are known in the art. See, U.S. Pat. Nos.
5,565,350; 5,731,181; 5,756,325; 5,760,012; 5,795,972; and 5,871,984; all of which are herein incorporated by reference. See also, WO 98/49350, WO 99/07865, WO 99/25821, and Beetham et al. (1999) Proc. Natl. Acad. Sci. USA 96: 8774-8778; herein incorporated by reference.
The expression cassette may additionally contain 5' leader sequences. Such leader sequences can act to enhance translation. Translation leaders are known in the art and include: picornavirus leaders, for example, EMCV leader (Encephalomyocarditis 5' noncoding region) (Elroy-Stein et al. (1989) Proc. Natl. Acad. Sci. USA 86: 6126-61 0); potyvirus leaders, for example, TEV leader (Tobacco Etch Virus) (Gallie et al. (1995) Gene 165(2): 233-238), MDMV leader (Maize Dwarf Mosaic Virus) (Kong et al. (1988) Arch Virol 143:1791-1799), and human immunoglobulin heavy-chain binding protein (BiP) (Macejak et al. (1991) Nature 353: 90-94); untranslated leader from the coat protein mRNA of alfalfa mosaic virus (AMV RNA 4) (Jobling et al. (1987) Nature 325: 622-625); tobacco mosaic virus leader (TMV) (Gallie et al. (1989) in Molecular Biology of RNA, ed. Cech (Liss, N.Y.), pp. 237-256); and maize chlorotic mottle virus leader (MCMV) (Lommel et al. (1991) Virology 81 : 382-385). See also, Della-Cioppa et al. (1987) Plant Physiol. 84: 965-968.
In preparing the expression cassette, the various polynucleotide fragments may be manipulated, so as to provide for sequences to be in the proper orientation and, as appropriate, in the proper reading frame. Toward this end, adapters or linkers may be employed to join the fragments or other manipulations may be involved to provide for convenient restriction sites, removal of superfluous material such as the removal of restriction sites, or the like. For this purpose, in vitro mutagenesis, primer repair, restriction, annealing, resubstitutions, e.g., transitions and transversions, may be involved. Standard recombinant DNA and molecular cloning techniques used herein are well known in the art and are described more fully, for example, in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor) (also known as "Maniatis").
In some embodiments, the polynucleotide of interest is targeted to the chloroplast for expression. In this manner, where the polynucleotide of interest is not directly inserted into the chloroplast, the expression cassette will additionally contain a nucleic acid encoding a transit peptide to direct the gene product of interest to the chloroplasts. Such transit peptides are known in the art. See, e.g., Von Heijne et al. (1991) Plant Mol. Biol. Rep. 9: 104-126; Clark et al. (1989) J. Biol. Chem. 264: 17544-17550; Della-Cioppa et al. (1987) Plant Physiol. 84: 965-968; Romer et al. (1993) Biochem. Biophys. Res. Comm. 196: 1414-1421; and Shah et al. (1986) Science 233: 478-481.
Chloroplast targeting sequences are known in the art and include the chloroplast small subunit of ribulose-l,5-bisphosphate carboxylase (Rubisco) (de Castro Silva Filho et al. (1996) Plant Mol. Biol. 30: 769-780; Schnell et al. (1991) J. Biol. Chem. 266(5): 3335- 3342); 5-(enolpyruvyl)shikimate-3-phosphate synthase (EPSPS) (Archer et al. (1990) J. Bioenerg Biomemb. 22(6): 789-810); tryptophan synthase (Zhao et al. (1995) J. Biol. Chem. 270(11): 6081-6087); plastocyanin (Lawrence et al (1997) J. Biol. Chem. 272(33): 20357-20363); chorismate synthase (Schmidt et al (1993) J. Biol. Chem. 268(36): 27447- 27457); and the light harvesting chlorophyll a/b binding protein (LHBP) (Lamppa et al. (1988) J. Biol. Chem. 263: 14996-14999). See also Von Heijne et al. (1991) Plant Mol. Biol. Rep. 9: 104-126; Clark et al. (1989) J. Biol. Chem. 264: 17544-17550; Della-Cioppa et al. (1987) Plant Physiol. 84: 965-968; Romer et al. (1993) Biochem. Biophys. Res. Comm. 196: 1414-1421; and Shah et al. (1986) Science 233: 478-481.
Methods for transformation of chloroplasts are known in the art. See, e.g., Svab et al. (1990) Proc. Natl. Acad. Sci. USA 87: 8526-8530; Svab and Maliga (1993) Proc. Natl. Acad. Sci. USA 90: 913-917; Svab and Maliga (1993) EMBO J. 12: 601-606. The method relies on particle gun delivery of DNA containing a selectable marker and targeting of the DNA to the plastid genome through homologous recombination. Additionally, plastid transformation can be accomplished by transactivation of a silent plastid-borne transgene by tissue-preferred expression of a nuclear-encoded and plastid-directed RNA polymerase. Such a system has been reported in McBride et al. (1994) Proc. Natl. Acad. Sci. USA 91: 7301-7305.
The polynucleotides of interest to be targeted to the chloroplast may be optimized for expression in the chloroplast to account for differences in codon usage between the plant nucleus and this organelle. In this manner, the polynucleotide of interest may be synthesized using chloroplast-preferred codons. See, e.g., U.S. Pat. No. 5,380,831, herein incorporated by reference. Introducing Nucleic Acids into Plants:
Methods for the production of transgenic plants comprising the cloned nucleic acids, e.g., isolated ORFs and cDNAs encoding tolerance genes, are provided. Techniques for transforming plant cells with nucleic acids are widely available and can be readily adapted. In addition to the Berger, Ausubel, and Sambrook references, useful general references for plant cell cloning, culture and regeneration include Jones (ed) (1995) Plant Gene Transfer and Expression Protocols—Methods in Molecular Biology, Volume 49 Humana Press Towata N. J.; Payne et al. (1992) Plant Cell and Tissue Culture in Liquid Systems John Wiley & Sons, Inc. New York, N.Y. (Payne); and Gamborg and Phillips (eds) (1995) Plant Cell, Tissue and Organ Culture; Fundamental Methods Springer Lab Manual, Springer-Verlag (Berlin Heidelberg N.Y.) (Gamborg). A variety of cell culture media are described in Atlas and Parks (eds) The Handbook of Microbiological Media (1993) CRC Press, Boca Raton, Fla. (Atlas). Additional information for plant cell culture is found in available commercial literature such as the Life Science Research Cell Culture Catalogue (1998) from Sigma-Aldrich, Inc (St Louis, Mo.) (Sigma-LSRCCC) and, e.g., the Plant Culture Catalogue and supplement (1997) also from Sigma-Aldrich, Inc (St Louis, Mo.) (Sigma-PCCS). Additional details regarding plant cell culture are found in Croy, (ed.) (1 93) Plant Molecular Biology, Bios Scientific Publishers, Oxford, U.K.
The nucleic acid constructs, e.g., DNA molecules plasmids, cosmids, artificial chromosomes, DNA, and RNA polynucleotides, are introduced into plant cells, either in culture or in the organs of a plant by a variety of conventional techniques. Where the sequence is expressed, the sequence is optionally combined with transcriptional and translational initiation regulatory sequences which direct the transcription or translation of the sequence from the exogenous DNA in the intended tissues of the transformed plant.
Isolated nucleic acid acids can be introduced into plants according to any of a variety of techniques known in the art. Techniques for transforming a wide variety of higher plant species are also well known and described in widely available technical, scientific, and patent literature. See, e.g., Weising et al. (1988) Ann. Rev. Genet. 22:421- 477.
Such methods for introducing polynucleotide or polypeptides into plants include stable transformation methods, transient transformation methods, virus-mediated methods, and breeding. "Stable transformation" is intended to mean that the nucleotide construct introduced into a plant integrates into the genome of the plant and is capable of being inherited by the progeny thereof. "Transient transformation" is intended to mean that a polynucleotide is introduced into the plant and does not integrate into the genome of the plant or a polypeptide is introduced into a plant.
The DNA constructs, for example DNA fragments, plasmids, phagemids, cosmids, phage, naked or variously conjugated-DNA polynucleotides, (e.g., polylysine-conjugated DNA, peptide-conjugated DNA, lipo some-conjugated DNA, etc.), or artificial
chromosomes, can be introduced directly into the genomic DNA of the plant cell using techniques such as electroporation and microinjection of plant cell protoplasts, or the DN A constructs can be introduced directly to plant cells using ballistic methods, such as DNA particle bombardment.
Transformation protocols as well as protocols for introducing polypeptides or polynucleotide sequences into plants may vary depending on the type of plant or plant cell (i.e., monocot or dicot) targeted for transformation. Suitable methods of introducing polypeptides and polynucleotides into plant cells include microinjection (Crossway et al. (1986) Biotechniques 4:320-334), electroporation (Riggs et al. (1986) Proc. Natl. Acad. Sci. USA 83: 5602-5606, Agrobacterium-mediated transformation (U.S. Pat. No.
5,563,055 and U.S. Pat. No. 5,981,840), direct gene transfer (Paszko ski et al. (1984) EMBO J. 3: 2717-2722), and ballistic particle acceleration (see, for example, U.S. Pat. No. 4,945,050; U.S. Pat. No. 5,879,918; U.S. Pat. Nos. 5,886,244; and, 5,932,782; Tomes et al. (1995) in Plant Cell, Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg and Phillips (Springer-Verlag, Berlin); McCabe et al (1988) Biotechnology 6: 923-926); and Lecl transformation (WO 00/28058). Also see Weissinger et al. (1988) Ann. Rev. Genet. 22: 421-477; Sanford et al. (1987) Particulate Science and Technology 5: 27-37 (onion); Christou et al. (1988) Plant Physiol. 87: 671-674 (soybean); McCabe et al. (1988)
Bio/Technology 6: 923-926 (soybean); Finer and McMullen (1991) In Vitro Cell Dev. Biol. 27P: 175-182 (soybean); Singh et al. (1998) Theor. Appl. Genet. 96:319-324
(soybean); Datta et al. (1990) Biotechnology 8: 736-740 (rice); Klein et al. (1988) Proc. Natl. Acad. Sci. USA 85:4305-4309 (maize); Klein et al. (1988) Biotechnology 6:559-563 (maize); U.S. Pat. Nos. 5,240,855; 5,322,783; and, 5,324,646; Klein et al. (1988) Plant Physiol. 91 : 440-444 (maize); Fromm et al. (1990) Biotechnology 8: 833-839 (maize); protocols published electronically by "IP.com" under the permanent publication identifiers IPCOM000033402D, IPCOM000033402D, and IPCOM000033402D and available at the "IP.com" website (cotton); Hooykaas-Van Slogteren et al. (1984) Nature (London) 311: 763-764; U.S. Pat. No. 5,736,369 (cereals); Bytebier et al. (1987) Proc. Natl. Acad. Sci. USA 84: 5345-5349 (Liliaceae); De Wet et al (1985) in The Experimental Manipulation of Ovule Tissues, ed. Chapman et al. (Longman, N.Y.), pp. 197-209 (pollen); Kaeppler et al. (1990) Plant Cell Reports 9: 415-418 and Kaeppler et al. (1992) Theor. Appl. Genet. 84: 560-566 (whisker-mediated transformation); D'Halluin et al. (1992) Plant Cell 4: 1495- 1505 (electroporation); Li et al. (1993) Plant Cell Reports 12: 250-255 and Christou and Ford (1995) Annals of Botany 75: 407-413 (rice); Osjoda et al. (1996) Nature
Biotechnology 14; 745-750 (maize via Agrobacterium tumefaciens); al] of which are herein incorporated by reference.
Microinjection techniques for injecting plant, e.g., cells, embryos, callus, and protoplasts, are known in the art and well described in the scientific and patent literature. For example, a number of methods are described in Jones (ed) (1995) Plant Gene Transfer and Expression Protocols—Methods in Molecular Biology, Volume 49 Humana Press, Towata, N.J., as well as in the other references noted herein and available in the literature.
For example, the introduction of DNA constructs using polyethylene glycol precipitation is described in Paszkowski et al, EMBO J. 3:2717 (1984). Electroporation techniques are described in Fromm et al, Proc. Natl. Acad. Sci. USA 82:5824 (1985). Ballistic transformation techniques are described in Klein et al, Nature 327:70-73 (1987). Additional details are found in Jones (1995) and Gamborg and Phillips (1995), supra, and in U.S. Pat. No. 5,990,387.
Alternatively, Agrobacterium-mediated transformation is employed to generate transgenic plants. Agrobacterium-mediated transformation techniques, including disarming and use of binary vectors, are also well described in the scientific literature. See, e.g., Horsch, et al (1984) Science 233:496; and Fraley et al. (1984) Proc. Natl. Acad. Sci. USA 80:4803 and recently reviewed in Hansen and Chilton (1998) Current Topics in
Microbiology 240:22 and Das (1998) Subcellular Biochemistry 29: Plant Microbe Interactions, pp 343-363.
DNA constructs are optionally combined with suitable T-DNA flanking regions and introduced into a conventional Agrobacterium tumefaciens host vector. The virulence functions of the Agrobacterium tumefaciens host will direct the insertion of the construct and adjacent marker into the plant cell DNA when the cell is infected by the bacteria. See, U.S. Pat. No. 5,591,616. Although Agrobacterium is useful primarily in dicots, certain monocots can be transformed by Agrobacterium. For instance, Agrobacterium
transformation of maize is described in U.S. Pat. No. 5,550,318.
Other methods of transfection or transformation include (1) Agrobacterium rhizogenes-mediated transformation (see, e.g., Lichtenstein and Fuller (1987) In: Genetic Engineering, vol. 6, P W J Rigby, Ed., London, Academic Press; and Lichtenstein; C. P., and Draper (1985) In: DNA Cloning, Vol. II, D. M. Glover, Ed., Oxford, IRI Press; WO 88/02405, published Apr. 7, 1988, describes the use of A. rhizogenes strain A4 and its Ri plasmid along with v4. tumefaciens vectors pARC8 or pARC16 (2) liposome-mediated DNA uptake (see, e.g., Freeman et al (1984) Plant Cell Physiol. 25:1353), (3) the vortexing method (see, e.g., Kindle (1990) Proc. Natl. Acad. Sci., (USA) 87:1228. DNA can also be introduced into plants by direct DNA transfer into pollen as described by Zhou et al. (1983) Methods in Enzymology, 101 :433; D. Hess (1987) Intern Rev. Cytol. 107:367; Luo et al. (1988) Plant Mol. Biol. Reporter 6: 165. Expression of polypeptide coding genes can be obtained by injection of the DNA into reproductive organs of a plant as described by Pena et al. (1987) Nature 325:274. DNA can also be injected directly into the cells of immature embryos and the desiccated embryos rehydrated as described by Neuhaus et al. (1987) Theor. Appl. Genet. 75:30; and Benbrook et al. (1986) in Proceedings Bio Expo Butterworth, Stoneham, Mass., pp. 27-54. A variety of plant viruses that can be employed as vectors are known in the art and include cauliflower mosaic virus (CaMV), geminivirus, brome mosaic virus, and tobacco mosaic virus.
Methods are known in the art for the targeted insertion of a polynucleotide at a specific location in the plant genome. In one embodiment, the insertion of the
polynucleotide at a desired genomic location is achieved using a site-specific
recombination system. See, e.g., W099/25821, W099/25854, WO99/25840,
W099/25855, and 099/25853, all of which are herein incorporated by reference.
Briefly, a polynucleotide can be contained in transfer cassette flanked by two non- recombinogenic recombination sites. The transfer cassette is introduced into a plant having stably incorporated into its genome a target site which is flanked by two non- recombinogenic recombination sites that correspond to the sites of the transfer cassette. An appropriate recombinase is provided and the transfer cassette is integrated at the target site. The polynucleotide of interest is thereby integrated at a specific chromosomal position in the plant genome.
Generation/Regeneration of Transgenic Plants:
Transformed plant cells which are derived by any of the above transformation techniques can be cultured to regenerate a whole plant that possesses the transformed genotype and thus the desired phenotype. Such regeneration techniques rely on
manipulation of certain phytohormones in a tissue culture growth medium, typically relying on a biocide and/or herbicide marker which has been introduced together with the desired nucleotide sequences. Plant regeneration from cultured protoplasts is described in Payne et al. (1992) Plant Cell and Tissue Culture in Liquid Systems John Wiley & Sons, Inc. New York, N.Y.; Gamborg and Phillips (eds) (1995) Plant Cell, Tissue and Organ Culture; Fundamental Methods Springer Lab Manual, Springer- Verlag (Berlin Heidelberg New York); Evans et al. (1983) Protoplasts Isolation and Culture, Handbook of Plant Cell Culture pp. 124-176, Macmillian Publishing Company, New York; and Binding (1985) Regeneration of Plants, Plant Protoplasts pp. 21-73, CRC Press, Boca Raton. Regeneration can also be obtained from plant callus, explants, somatic embryos (Dandekar et al. (1989) J. Tissue Cult. Meth. 12:145; McGranahan, et al. (1990) Plant Cell Rep. 8:512) organs, or parts thereof. Such regeneration techniques are described generally in Klee et al. (1987)., Ann. Rev. of Plant Phys. 38:467-486. Additional details are found in Payne (1992) and Jones (1995), both supra, and Weissbach and eissbach, eds.(1988) Methods for Plant Molecular Biology Academic Press, Inc., San Diego, Calif. This regeneration and growth process includes the steps of selection of transformant cells and shoots, rooting the transformant shoots and growth of the plantlets in soil. These methods are adapted to produce transgenic plants bearing QTLs and other genes isolated according to the methods.
In addition, the regeneration of plants containing the polynucleotides and introduced by Agrobacterium into cells of leaf explants can be achieved as described by Horsch et al. (1985) Science 227:1229-1231. In this procedure, transformants are grown in the presence of a selection agent and in a medium that induces the regeneration of shoots in the plant species being transformed as described by Fraley et al (1983) Proc. Natl. Acad. Sci. (U.S.A.) 80:4803. This procedure typically produces shoots within two to four weeks and these transformant shoots are then transferred to an appropriate root-inducing medium containing the selective agent and an antibiotic to prevent bacterial growth. Transgenic plants may be fertile or sterile.
It is not intended that plant transformation and expression of polypeptides that provide herbicide tolerance be limited to soybean species. Indeed, it is contemplated that the polypeptides that provide tolerance in soybean can also provide a similar phenotype when transformed and expressed in other plants. Examples of plant genuses and species of interest include, but are not limited to, monocots and dicots such as corn (Tea mays), Brassica sp. (e.g., B. napus, B. rapa, B.junced), particularly those Brassica species useful as sources of seed oil, alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secale cereale), sorghum
(Sorghum bicolor, Sorghum vulgare), millet (e.g., pearl millet (Pennisetum ghucum), proso millet (Panicum miliaceum), foxtail millet (Setaria italica), finger millet (Eleusine coracana)), sunflower (Helianthus annuus), safflower (Carthamus tinctorius), wheat (Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanuts (Arachis hypogaea), cotton (Gossypium barbadense, Gossypium hirsutum), sweet potato (Ipomoea batatus), cassava (Manihot escuienta), coffee (Coffea spp.), coconut (Cocos nucifera), pineapple (Ananas comosus), citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musa spp.), avocado (Per ea americana), fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica), olive (plea europaea), papaya (Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamia inlegrifolid), almond (Primus amygdalus), sugar beets (Beta vulgaris), sugarcane (Saccharum spp.), oats (Avena), barley (Hordeum), palm, legumes including beans and peas such as guar, locust bean, fenugreek, garden beans, cowpea, mungbean, lima bean, fava bean, lentils, chickpea, and castor, Arabidopsis, vegetables, ornamentals, grasses, conifers, crop and grain plants that provide seeds of interest, oil-seed plants, and other leguminous plants. Vegetables include tomatoes (Lycopersicon esculentum), lettuce (e.g., Lactuca sativa), green beans (Phaseolus vulgaris), lima beans (Phaseolus limensis), peas (Lathyrus spp.), and members of the genus Cucumis such as cucumber (C. sativus), cantaloupe (C. cantalupensis), and musk melon (C. melo). Ornamentals include azalea (Rhododendron spp.), hydrangea (Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosa spp.), tulips (Tulipa spp.), daffodils (Narcissus spp.), petunias (Petunia hybrida), carnation (Dianthus caryophyllus), poinsettia (Euphorbia pulch rrimd), and chrysanthemum. Conifers include, for example, pines such as loblolly pine (Pinus taeda), slash pine (Pinus elliotii), ponderosa pine (Pinus ponderosd), lodgepole pine (Pinus contorta), and Monterey pine (Pinus radiata); Douglas-fir (Pseudotsuga menziesii) Western hemlock (Tsuga canadensis); Sitka spruce (Picea glauca); redwood (Sequoia sempervirens); true firs such as silver fir (Abies amabilis) and balsam fir (Abies balsamea); and cedars such as Western red cedar (Thuja plicata) and Alaska yellow-cedar (Chamaecyparis nootkatensis).
Promoters of bacterial origin that operate in plants include the octopine synthase promoter, the nopaline synthase promoter and other promoters derived from native Ti plasmids. See, Herrara-Estrella et al. (1983), Nature, 303:209. Viral promoters include the 35S and 19S RNA promoters of cauliflower mosaic virus. See, Odell et al (1985) Nature, 313:810. Other plant promoters include Kunitz trypsin inhibitor promoter (ΚΊΊ), SCP1, SUP, UCD3, the ribulose-l,3-bisphosphate carboxylase small subunit promoter and the phaseolin promoter. The promoter sequence from the E8 gene and other genes may also be used. The isolation and sequence of the E8 promoter is described in detail in Deikman and Fischer (1988) EMBO J. 7:3315. Many other promoters are in current use and can be coupled to an exogenous DNA sequence to direct expression of the nucleic acid.
If expression of a polypeptide from a cDNA is desired, a polyadenylation region at the 3 '-end of the coding region is typically included. The polyadenylation region can be derived from the natural gene, from a variety of other plant genes, or from, e.g., T-DNA.
The vector comprising the sequences (e.g., promoters or coding regions) from genes encoding expression products and transgenes will typically include a nucleic acid subsequence, a marker gene which confers a selectable, or alternatively, a screenable, phenotype on plant cells. For example, the marker can encode biocide tolerance, particularly antibiotic tolerance, such as tolerance to kanamycin, G418, bleomycin, hygromycin, or herbicide tolerance, such as tolerance to chlorsulforon, or phosphinothricin (the active ingredient in the herbicides bialaphos or Basta). See, e.g., Padgette et at. (1996) In: Herbicide-Resistant Crops (Duke, ed.), pp 53-84, CRC Lewis Publishers, Boca Raton ("Padgette, 1996"). For example, crop selectivity to specific herbicides can be conferred by engineering genes into crops that encode appropriate herbicide metabolizing enzymes from other organisms, such as microbes. See Vasif (1996) In: Herbicide-Resistant Crops (Duke, ed.), pp 85-91, CRC Lewis Publishers, Boca Raton) ("Vasil", 1996).
One of skill will recognize that after the recombinant expression cassette is stably incorporated in transgenic plants and confirmed to be operable, it can be introduced into other plants by sexual crossing. Any of a number of standard breeding techniques can be used, depending upon the species to be crossed. In vegetatively propagated crops, mature transgenic plants can be propagated by the taking of cuttings or by tissue culture techniques to produce multiple identical plants. Selection of desirable transgenics is made and new varieties are obtained and propagated vegetatively for commercial use. In seed propagated crops, mature transgenic plants can be self crossed to produce a homozygous inbred plant. The inbred plant produces seed containing the newly introduced heterologous nucleic acid. These seeds can be grown to produce plants that would produce the selected phenotype. Parts obtained from the regenerated plant, such as flowers, seeds, leaves, branches, fruit, and the like are included, provided that these parts comprise cells comprising the isolated nucleic acid. Progeny and variants, and mutants of the regenerated plants are also included, provided that these parts comprise the introduced nucleic acid sequences. Transgenic or introgressed plants expressing a polynucleotide can be screened for transmission of the nucleic acid by, for example, standard nucleic acid detection methods or by immunoblot protocols. Expression at the RNA level can be determined to identify and quantitate expression-positive plants. Standard techniques for RNA analysis can be employed and include RT-PCR amplification assays using oligonucleotide primers designed to amplify only heterologous or introgressed RNA templates and solution hybridization assays using marker or linked QTL specific probes. Plants can also be analyzed for protein expression, e.g., by Western immunoblot analysis using antibodies that recognize the encoded polypeptides. In addition, in situ hybridization and
immunocytochemistry according to standard protocols can be done using heterologous nucleic acid specific polynucleotide probes and antibodies, respectively, to localize sites of expression within transgenic tissue. Generally, a number of transgenic lines are usually screened for the incorporated nucleic acid to identify and select plants with the most appropriate expression profiles.
In one example a transgenic plant that is homozygous for the added heterologous nucleic acid; e.g. , a transgenic plant that contains two added nucleic acid sequence copies, such as a gene at the same locus on each chromosome of a homologous chromosome pair, is provided. A homozygous transgenic plant can be obtained by sexually mating (self- fertilizing) a heterozygous transgenic plant that contains a single added heterologous nucleic acid, germ inating some of the seed produced and analyzing the resulting plants produced for altered expression of a polynucleotide relative to a control plant (e.g., a native, non-transgenic plant). Back-crossing to a parental plant and out-crossing with a non-trans genie plant can be used to introgress the heterologous nucleic acid into a selected background (e.g., an elite or exotic soybean line).
Plants may be produced by any suitable method, including breeding. Plant breeding can be used to introduce desired characteristics (e.g., a stably incorporated transgene or a genetic variant or genetic alteration of interest) into a particular plant line of interest, and can be performed in any of several different ways. Pedigree breeding starts with the crossing of two genotypes, such as an elite line of interest and one other elite inbred line having one or more desirable characteristics (i.e., having stably incorporated a polynucleotide of interest, having a modulated activity and/or level of the polypeptide of interest, etc.) which complements the elite plant line of interest. If the two original parents do not provide all the desired characteristics, other sources can be included in the breeding population. In the pedigree method, superior plants are selfed and selected in successive filial generations. After a sufficient amount of inbreeding, successive filial generations will serve to increase seed of the developed inbred. In specific embodiments, the inbred line comprises homozygous alleles at about 95% or more of its loci. Various techniques known in the art can be used to facilitate and accelerate the breeding (e.g., backcrossing) process, including, for example, the use of a greenhouse or growth chamber with accelerated day/night cycles, the analysis of molecular markers to identify desirable progeny, and the like.
In addition to being used to create a backcross conversion, backcrossing can also be used in combination with pedigree breeding to modify an elite line of interest and a hybrid that is made using the modified elite line. As discussed previously, backcrossing can be used to transfer one or more specifically desirable traits from one line, the donor parent, to an inbred called the recurrent parent, which has overall good agronomic characteristics yet lacks that desirable trait or traits. However, the same procedure can be used to move the progeny toward the genotype of the recurrent parent but at the same time retain many components of the non-recurrent parent by stopping the backcrossing at an early stage and proceeding with selfing and selection. For example, an Fl, such as a commercial hybrid, is created. This commercial hybrid may be backcrossed to one of its parent lines to create a BCl or BC2. Progeny are selfed and selected so that the newly developed inbred has many of the attributes of the recurrent parent and yet several of the desired attributes of the nonrecurrent parent. This approach leverages the value and strengths of the recurrent parent for use in new hybrids and breeding.
Therefore, a method of making a backcross conversion of an inbred line of interest comprising the steps of crossing a plant from the inbred line of interest with a donor plant comprising at least one mutant gene or transgene conferring a desired trait (e.g., herbicide tolerance), selecting an Fl progeny plant comprising the mutant gene or transgene conferring the desired trait, and backcrossing the selected Fl progeny plant to a plant of the inbred line of interest is provided. This method may further comprise the step of obtaining a molecular marker profile of the inbred line of interest and using the molecular marker profile to select for a progeny plant with the desired trait and the molecular marker profile of the inbred line of interest. In the same manner, this method may be used to produce an Fl hybrid seed by adding a final step of crossing the desired trait conversion of the inbred line of interest with a different plant to make Fl hybrid seed comprising a mutant gene or transgene conferring the desired trait.
Recurrent selection is a method used in a plant breeding program to improve a population of plants. The method entails individual plants cross pollinating with each other to form progeny. The progeny are grown and the superior progeny selected by any number of selection methods, which include individual plant, half-sib progeny, full-sib progeny, selfed progeny and topcrossing. The selected progeny are cross-pollinated with each other to form progeny for another population. This population is planted and again superior plants are selected to cross pollinate with each other. Recurrent selection is a cyclical process and therefore can be repeated as many times as desired. The objective of recurrent selection is to improve the traits of a population. The improved population can then be used as a source of breeding material to obtain inbred lines to be used in hybrids or used as parents for a synthetic cultivar. A synthetic cultivar is the resultant progeny formed by the intercrossing of several selected inbreds.
Mass selection is a useful technique when used in conjunction with molecular marker enhanced selection. In mass selection seeds from individuals are selected based on phenotype and/or genotype. These selected seeds are then bulked and used to grow the next generation. Bulk selection requires growing a population of plants in a bulk plot, allowing the plants to self-pollinate, harvesting the seed in bulk and then using a sample of the seed harvested in bulk to plant the next generation. Instead of self pollination, directed pollination could be used as part of the breeding program.
Mutation breeding is one of many methods that could be used to introduce new traits into an elite line. Mutations that occur spontaneously or are artificially induced can be useful sources of variability for a plant breeder. The goal of artificial mutagenesis is to increase the rate of mutation for a desired characteristic. Mutation rates can be increased by many different means including temperature, long-term seed storage, tissue culture conditions, radiation such as X-rays, gamma rays (e.g., cobalt 60 or cesium 137), neutrons, (product of nuclear fission of uranium 235 in an atomic reactor), Beta radiation (emitted from radioisotopes such as phosphorus 32 or carbon 14), or ultraviolet radiation (typically from 2500 to 2900 ran), or chemical mutagens (such as base analogues (5-bromo-uracil), related compounds (8-ethoxy caffeine), antibiotics (streptonigrin), alkylating agents (sulfur mustards, nitrogen mustards, epoxides, ethylenamines, sulfates, sulfonates, sulfones, lactones), azide, hydroxylamine, nitrous acid, or acridines. Once a desired trait is observed through mutagenesis the trait may then be incorporated into existing germplasm by traditional breeding techniques, such as backcrossing. Details of mutation breeding can be found in "Principals of Cultivar Development" Fehr, 1993 Macmillan Publishing Company the disclosure of which is incorporated herein by reference. In addition, mutations created in other lines may be used to produce a backcross conversion of elite lines that comprises such mutations. Methods of Modulating Expression:
In some embodiments, the activity and/or level of the polypeptide is modulated (i.e., increased or decreased). An increase in the level and/or activity of the polypeptide can be achieved by providing the polypeptide to the plant. As discussed elsewhere herein, many methods are known the art for providing a polypeptide to a plant including, but not limited to, direct introduction of the polypeptide into the plant, introducing into the plant (transiently or stably) a polynucleotide construct encoding a polypeptide having the desired activity.
METHODS FOR IDENTIFYING MESOTRIONE OR ISOXAZOLE TOLERANT OR SUSCEPTIBLE SOYBEAN PLANTS
Experienced plant breeders can recognize tolerant soybean plants in the field, and can select the tolerant individuals or populations for breeding purposes or for propagation. In this context, the plant breeder recognizes tolerant, and non-tolerant soybean plants. In some examples, the tolerance is observed in the context of herbicide carryover from the previous crop season.
The screening and selection may also be performed by exposing plants containing said progeny germplasm to mesotrione and/or isoxazole in an assay and selecting those plants showing tolerance or sensitivity to mesotrione and/or isoxazole herbicides as containing soybean germplasm into which germplasm having tolerance or sensitivity to mesotrione and/or isoxazole herbicides derived from the QTL mapped to linkage group L has been introgressed. The live assay may be any such assay known to the art, e.g., Taylor- Lovell et al. (2001) Weed Tech 15:95-102. However, plant tolerance is a phenotypic spectrum consisting of extremes of high tolerance to non-tolerance with a continuum of intermediate tolerance phenotypes.
Evaluation of these intermediate phenotypes using reproducible assays are of value to scientists who seek to identify genetic loci that impart tolerance, conduct marker assisted selection for tolerant population, and for introgression techniques to breed a tolerance trait into an elite soybean line, for example. Describing the continuum of tolerance can be done using any known scoring system or derivative thereof, including the scoring systems described in the Examples. AUTOMATED DETECTION/ CORRELATION SYSTEMS
In some examples, the methods include an automated system for detecting markers and or correlating the markers with a desired phenotype (e.g. , tolerance or susceptibility). Thus, a typical system can include a set of marker probes or primers configured to detect at least one favorable allele of one or more marker locus associated with tolerance or improved tolerance or sensitivity to mesotrione and/or isoxazole herbicides. These probes or primers are configured to detect the marker alleles noted in the tables and examples herein, e.g., using any available allele detection format, e.g., solid or liquid phase array based detection, microfluidic-based sample detection, etc.
In some examples markers involving linkage group L are used. In some examples a marker closely linked to the marker locus of SATT495, P10649C-3, SATT182, S03859-1, S00224-1, SATT388, SATT313, SATT613 (or another marker above SATT613), S03859- l-A, S08103-1-Q1, S08104-1-Q1, S08106-1-Q1, S081 10-1-Q1, S08111-1-Q1, S08115-2- Ql, S081 17-1-Q1, S08119-1-Q1, S08118-1-Q1, S08116-1-Q1, S08114-1-Q1, S081 13-1- Ql, S081 12-1-Q1, S08108-1-Q1, S08101-2-Q1, S08101-3-Q1, S08101-4-Q1, S08105-1- Ql, S08102-1-Q1, S08107-1-Q1, S08109-1-Q1, and S08101-1 -Q1 is used, and the probe set is configured to detect the closely linked marker(s). In some examples, the marker locus is SATT495, P10649C-3, S ATT 182, S03859-1, S00224-1, SATT388, SATT313, SATT613 (or another marker above SATT613), S03859-1-A, S08103-1-Q1, S08104-1-Q1 , S08106-1-Q1, S08110-1-Q1, S08111-1-Q1, S08115-2-Q1, S08117-1-Q1, S08119-1 -Q1, S08118-1-Q1, S08116-1-Q1, S08114-1-Q1, S08113-1-Q1, S08112-1-Q1, S08108-1-Q1, S08101-2-Q1, S08101-3-Q1, S08101-4-Q1, S08105-1-Q1, S08102-1-Q1, S08107-1-Q1, S08109-1 -Ql, and S08101-1-Q1, and the probe set is configured to detect the locus. Similarly, alleles of SATT495, P10649C-3, S ATT 182, S03859-1, S00224-1, SATT388, SATT313, SATT613 (or another marker above SATT613), S03859-1-A, S081O3-1-Q1, S08104-1-Q1, S08106-1-Q1, S08110-1-Ql, S08111-1-Ql, S08115-2-Q1, S081 17-1-Ql, S081 19-1-Q1, S08118-1-Q1, S08116-1-Q1, S08114-1-Q1, S08113-1-Q1, S08112-1 -Q1, S081O8-1-Q1, S08101-2-Q1, S08101-3-Q1, S08101-4-Q1, S08105-1-Q1, S08102-1-Q1, S08107-1-Q1, S08109-1-Q1, and S08101-1 -Ql can be detected.
The typical system includes a detector that is configured to detect one or more signal outputs from the set of marker probes or primers, or amplicon thereof, thereby identifying the presence or absence of the allele. A wide variety of signal detection apparatus are available, including photo multiplier tubes, spectrophotometers, CCD arrays, arrays and array scanners, scanning detectors, phototubes and photodiodes, microscope stations, galvo-scans, microfluidic nucleic acid amplification detection appliances and the like. The precise configuration of the detector will depend, in part, on the type of label used to detect the marker allele, as well as the instrumentation that is most conveniently obtained for the user. Detectors that detect fluorescence, phosphorescence, radioactivity, pH, charge, absorbance, luminescence, temperature, magnetism or the like can be used. Typical detector examples include light (e.g., fluorescence) detectors or radioactivity detectors. For example, detection of a light emission (e.g., a fluorescence emission) or other probe label is indicative of the presence or absence of a marker allele. Fluorescent detection is generally used for detection of amplified nucleic acids (however, upstream and/or downstream operations can also be performed on amplicons, which can involve other detection methods). In general, the detector detects one or more label (e.g., light) emission from a probe label, which is indicative of the presence or absence of a marker allele. The detector(s) optionally monitors one or a plurality of signals from an amplification reaction. For example, the detector can monitor optical signals which correspond to "real time" amplification assay results.
System instructions that correlate the presence or absence of the favorable allele with the predicted tolerance are also provided. For example, the instructions can include at least one look-up table that includes a correlation between the presence or absence of the favorable alleles and the predicted tolerance or improved tolerance. The precise form of the instructions can vary depending on the components of the system, e.g., they can be present as system software in one or more integrated unit of the system (e.g., a microprocessor, computer or computer readable medium), or can be present in one or more units {e.g., computers or computer readable media) operably coupled to the detector. As noted, in one typical example, the system instructions include at least one look-up table that includes a correlation between the presence or absence of the favorable alleles and predicted tolerance or improved tolerance. The instructions also typically include instructions providing a user interface with the system, e.g., to permit a user to view results of a sample analysis and to input parameters into the system.
The system typically includes components for storing or transmitting computer readable data representing or designating the alleles detected, e.g., in an automated system. The computer readable media can include cache, main, and storage memory and/or other electronic data storage components (hard drives, floppy drives, storage drives, etc.) for storage of computer code. Data representing alleles detected can also be electronically, optically, magnetically o transmitted in a computer data signal embodied in a transmission medium over a network such as an intranet or internet or combinations thereof. The system can also or alternatively transmit data via wireless, I , or other available transmission alternatives.
During operation, the system typically comprises a sample that is to be analyzed, such as a plant tissue, or material isolated from the tissue such as genomic DNA, amplified genomic DNA, cDNA, amplified cDNA, RNA, amplified RNA, or the like.
The phrase "allele detection/correlation system" refers to a system in which data entering a computer corresponds to physical objects or processes external to the computer, e.g., a. marker allele, and a process that, within a computer, causes a physical
transformation of the input signals to different output signals. In other words, the input data, e.g., amplification of a particular marker allele is transformed to output data, e.g., the identification of the allelic form of a chromosome segment. The process within the computer is a set of instructions, or "program," by which positive amplification or hybridization signals are recognized by the integrated system and attributed to individual samples as a genotype. Additional programs correlate the identity of individual samples with phenotypic values or marker alleles, e.g., statistical methods. In addition there are numerous e.g., C/C++ programs for computing, Delphi and/or Java programs for GUI interfaces, and productivity tools (e.g., Microsoft Excel and/or SigmaPlot) for charting or creating look up tables of relevant allele-trait correlations. Other useful software tools in the context of the integrated systems of the invention include statistical packages such as SAS, Genstat, Matlab, Mathematica, and S-Plus and genetic modeling packages such as QU-GENE. Furthermore, additional programming languages such as visual basic are also suitably employed in the integrated systems.
For example, tolerance marker allele values assigned to a population of progeny descending from crosses between elite lines are recorded in a computer readable medium, thereby establishing a database corresponding tolerance alleles with unique identifiers for members of the population of progeny. Any file or folder, whether custom-made or commercially available (e.g., from Oracle or Sybase) suitable for recording data in a computer readable medium is acceptable as a database. Data regarding genotype for one or more molecular markers, e.g., ASH, SSR, RFLP, RAPD, AFLP, SNP, isozyme markers or other markers as described herein, are similarly recorded in a computer accessible database. Optionally, marker data is obtained using an integrated system that automates one or more aspects of the assay (or assays) used to determine marker(s) genotype. In such a system, input data corresponding to genotypes for molecular markers are relayed from a detector, e.g., an array, a scanner, a CCD, or other detection device directly to files in a computer readable medium accessible to the central processing unit. A set of system instructions (typically embodied in one or more programs) encoding the correlations between tolerance and the alleles of the invention is then executed by the computational device to identify correlations between marker alleles and predicted trait phenotypes.
Typically, the system also includes a user input device, such as a keyboard, a mouse, a touchscreen, or the like, for, e.g., selecting files, retrieving data, reviewing tables of maker information, etc., and an output device (e.g., a monitor, a printer, etc.) for viewing or recovering the product of the statistical analysis.
Integrated systems comprising a computer or computer readable medium comprising set of files and/or a database with at least one data set that corresponds to the marker alleles herein are provided. The systems optionally also includes a user interface allowing a user to selectively view one or more of these databases. In addition, standard text manipulation software such as word processing software (e.g., Microsoft Word™ or Corel Wordperfect™) and database or spreadsheet software (e.g., spreadsheet software such as Microsoft Excel™, Corel Quattro Pro™, or database programs such as Microsoft Access™ or Paradox™) can be used in conjunction with a user interface (e.g., a GUI in a standard operating system such as a Windows, Macintosh, Unix or Linux system) to manipulate strings of characters corresponding to the alleles or other features of the database.
The systems optionally include components for sample manipulation, e.g., incorporating robotic devices. For example, a robotic liquid control armature for transferring solutions (e.g., plant cell extracts) from a source to a destination, e.g., from a microtiter plate to an array substrate, is optionally operably linked to the digital computer (or to an additional computer in the integrated system). An input device for entering data to the digital computer to control high throughput liquid transfer by the robotic liquid control armature and, optionally, to control transfer by the armature to the solid support is commonly a feature of the integrated system. Many such automated robotic fluid handling systems are commercially available. For example, a variety of automated systems are available from Caliper Technologies (Hopkinton, MA), which utilize various Zymate systems, which typically include, e.g., robotics and fluid handling modules. Similarly, the common O CA® robot, which is used in a variety of laboratory systems, e.g. , for microtiter tray manipulation, is also commercially available, e.g., from Beckman Coulter, Inc. (Fullerton, CA). As an alternative to conventional robotics, microfluidic systems for performing fluid handling and detection are now widely available, e.g. , from Caliper Technologies Corp. (Hopkinton, MA) and Agilent technologies (Palo Alto, CA).
Systems for molecular marker analysis can include a digital computer with one or more of high-throughput liquid control software, image analysis software for analyzing data from marker labels, data interpretation software, a robotic liquid control armature for transferring solutions from a source to a destination operably linked to the digital computer, an input device (e.g., a computer keyboard) for entering data to the digital computer to control high throughput liquid transfer by the robotic liquid control armature and, optionally, an image scanner for digitizing label signals from labeled probes hybridized, e.g., to markers on a solid support operably linked to the digital computer. The image scanner interfaces with the image analysis software to provide a measurement of, e.g., nucleic acid probe label intensity upon hybridization to an arrayed sample nucleic acid population (e.g., comprising one or more markers), where the probe label intensity measurement is interpreted by the data interpretation software to show whether, and to what degree, the labeled probe hybridizes to a marker nucleic acid (e.g., an amplified marker allele). The data so derived is then correlated with sample identity, to determine the identity of a plant with a particular genotype(s) for particular markers or alleles, e.g., to facilitate marker assisted selection of soybean plants with favorable allelic forms of chromosome segments involved in agronomic performance (e.g., tolerance or improved tolerance).
Optical images, e.g., hybridization patterns viewed (and, optionally, recorded) by a camera or other recording device (e.g., a photodiode and data storage device) are optionally further processed in any of the embodiments herein, e.g., by digitizing the image and/or storing and analyzing the image on a computer. A variety of commercially available peripheral equipment and software is available for digitizing, storing and analyzing a digitized video or digitized optical image.
STACKING OF TRAITS AND ADDITIONAL TRAITS OF INTEREST
In some embodiments, the polynucleotide conferring the tolerance in the plants are engineered into a molecular stack with at least one additional polynucleotide. The additional polynucleotide may confer any additional trait of interest, such as tolerance to an additional herbicide, insects, disease, or any other desirable trait. A trait, as used herein, refers to the phenotype derived from a particular sequence or groups of sequences. For example, herbicide-tolerance polynucleotides may be stacked with any other
polynucleotides encoding polypeptides having pesticidal and/or insecticidal activity, such as Bacillus thuringiensis toxic proteins (described in U.S. Pat. Nos. 5,366,892; 5,747,450; 5,737,514; 5,723,756; 5,593,881; Geiser et al. (1986) Gene 48:109; Lee et al. (2003) Appl. Environ. Microbiol. 69:4648-4657 (Vip3A); Galitzky et al. (2001) Acta Crystallogr. D. Biol. Crystallogr. 57:1101-1109 (Cry3Bbl); and Herman et al. (2004) J. Agric. Food Chem. 52:2726-2734 (CrylF)), lectins (Van Damme et al. (1994) Plant Mol. Biol. 24: 825, pentin (described in U.S. Pat. No. 5,981,722), and the like. The combinations generated can also include multiple copies of any one of the polynucleotides of interest.
In some embodiments, an herbicide-tolerance polynucleotide described herein may be stacked with other herbicide-tolerance traits to create a transgenic plant with further improved properties. Other herbicide-tolerance polynucleotides that could be used in such embodiments include those conferring tolerance to the same herbicide by other modes of action, or a different herbicide. Other traits that could be combined with herbicide- tolerance polynucleotides include those derived from polynucleotides that confer on the plant the capacity to produce a higher level of 5-enolpyravylshikimate-3-phosphate synthase (EPSPS), for example, as more fully described in U.S. Pat. Nos. 6,248,876 Bl; 5,627,061; 5,804,425; 5,633,435; 5, 145,783; 4,971,908; 5,312,910; 5,188,642; 4,940,835; 5,866,775; 6,225,114 Bl ; 6,130,366; 5,310,667; 4,535,060; 4,769,061 ; 5,633,448;
5,510,471 ; U.S. Pat. No. Re. 36,449; U.S. Pat. Nos. RE 37,287 E; and 5,491,288; and WO 97/04103; WO 00/66746; WO 01/66704; and WO 00/66747. Other traits that could be combined with herbicide-tolerance polynucleotides include those conferring tolerance to sulfonylurea and/or imidazolinone, for example, as described more fully in U.S. Pat. Nos. 5,605,011 ; 5,013,659; 5, 141,870; 5,767,361; 5,731,180; 5,304,732; 4,761,373; 5,331,107; 5,928,937; and 5,378,824; and international publication WO 96/33270.
In some embodiments, herbicide-tolerance polynucleotides of the plants may be stacked with, for example, hydroxyphenylpyruvatedioxygenases which are enzymes that catalyze the reaction in which para-hydroxyphenylpyruvate (HPP) is transformed into homogentisate. Molecules which inhibit this enzyme and which bind to the enzyme in order to inhibit transformation of the HPP into homogentisate are useful as herbicides. Traits conferring tolerance to such herbicides in plants are described in U.S. Pat. Nos. 6,245,968 Bl; 6,268,549; and 6,069,115; and WO 99/23886. Other examples of suitable herbicide-tolerance traits that could be stacked with herbicide-tolerance polynucleotides include aryloxyalkanoate dioxygenase polynucleotides (which reportedly confer tolerance to 2,4-D and other phenoxy auxin herbicides as well as to aryloxyphenoxypropionate herbicides as described, for example, in WO2005/107437) and dicamba-tolerance polynucleotides as described, for example, in Herman et al (2005) J. Biol. Chem. 280: 24759-24767.
Other examples of herbicide-tolerance traits that could be combined with herbicide- tolerance polynucleotides include those conferred by polynucleotides encoding an exogenous phosphinothricin acetyltransferase, as described in U.S. Pat. Nos. 5,969,213; 5,489,520; 5,550,318; 5,874,265; 5,919,675; 5,561,236; 5,648,477; 5,646,024; 6,177,616; and 5,879,903. Plants containing an exogenous phosphinothricin acetyltransferase can exhibit improved tolerance to glufosinate herbicides, which inhibit the enzyme glutamine synthase. Other examples of herbicide-tolerance traits that could be combined with the herbicide-tolerance polynucleotides include those conferred by polynucleotides conferring altered protoporphyrinogen oxidase (protox) activity, as described in U.S. Pat. Nos.
6,288,306 Bl; 6,282,837 Bl; and 5,767,373; and WO 01/12825. Plants containing such polynucleotides can exhibit improved tolerance to any of a variety of herbicides which target the protox enzyme (also referred to as protox inhibitors).
Other examples of herbicide-tolerance traits that could be combined with herbicide- tolerance polynucleotides include those conferring tolerance to at least one herbicide in a plant such as, for example, a maize plant or horseweed. Herbicide-tolerant weeds are known in the art, as are plants that vary in their tolerance to particular herbicides. See, e.g. , Green and Williams (2004) "Correlation of Corn (Zea mays) Inbred Response to
Nicosulfuron and Mesotrione," poster presented at the WSSA Annual Meeting in Kansas City, Mo., Feb. 9-12, 2004; Green (1998) Weed Technology 12:474-477; Green and Ulrich (1993) Weed Science 41:508-516. The trait(s) responsible for these tolerances can be combined by breeding or via other methods with herbicide-tolerance polynucleotides to provide a plant as well as methods of use thereof.
In this manner, plants that are more tolerant to multiple herbicides are disclosed.
Accordingly, methods for growing a crop {i.e., for selectively controlling weeds in an area of cultivation) that comprise treating an area of interest {e.g., a field or area of cultivation) with at least one herbicide to which the plant is tolerant are likewise disclosed. In some embodiments, methods further comprise treatment with additional herbicides to which the plant is tolerant. In such embodiments, generally the methods permit selective control of weeds without significantly damaging the crop. As used herein, an "area of cultivation" comprises any region in which one desires to grow a plant. Such areas of cultivations include, but are not limited to, a field in which a plant is cultivated (such as a crop field, a sod field, a tree field, a managed forest, a field for culturing fruits and vegetables, etc), a greenhouse, a growth chamber, etc.
Herbicide-tolerant traits can also be combined with at least one other trait to produce plants that further comprise a variety of desired trait combinations including, but not limited to, traits desirable for animal feed such as high oil content {e.g., U.S. Pat. No. 6,232,529); increased digestibility {e.g., modified storage proteins (U.S. application Ser. No. 10/053,410, filed Nov. 7, 2001); and thioredoxins (U.S. application Ser. No.
10/005,429, filed Dec. 3, 2001)); the disclosures of which are herein incorporated by reference. Desired trait combinations also include LLNC (low linolenic acid content; see, e.g., Dyer et al. (2002) Appl. Microbiol. Biotechnol. 59:224-230) and OLCH (high oleic acid content; see, e.g., Fernandez-Moya et al. (2005) J. Agric. Food Chem. 53:5326-5330).
Herbicide-tolerant traits of interest can also be combined with other desirable traits such as, for example, fumonisim detoxification genes (U.S. Pat. No. 5,792,931), avirulence and disease resistance genes (Jones et al. (1994) Science 266:789; Martin et al. (1993) Science 262: 1432; Mindrinos et al. (1994) Cell 78: 1089), and traits desirable for processing or process products such as modified oils {e.g., fatty acid desaturase genes (U.S. Pat. No. 5,952,544; WO 94/11 16)); modified starches {e.g., ADPG yrophosphorylases (AGPase), starch synthases (SS), starch branching enzymes (SBE), and starch debranching enzymes (SDBE)); and polymers or bioplastics (e.g., U.S. Pat. No. 5,602,321; beta- ketothiolase, polyhydroxybutyrate synthase, and acetoacetyl-CoA reductase (Schubert et al. (1988) J. Bacteriol. 170:5837-5847) facilitate expression of polyhydroxyalkanoates (PHAs)); the disclosures of which are herein incorporated by reference. One could also combine herbicide-tolerant polynucleotides with polynucleotides providing agronomic traits such as male sterility (e.g. , see U.S. Pat. No. 5,583,210), stalk strength, flowering time, or transformation technology traits such as cell cycle regulation or gene targeting (e.g., WO 99/61619, WO 00/17364, and WO 99/25821); the disclosures of which are herein incorporated by reference.
In another embodiment, the herbicide-tolerant traits of interest can also be combined with the Rcgl sequence or biologically active variant or fragment thereof. The Rcgl sequence is an anthracnose stalk rot resistance gene in com. See, e.g., U.S. patent application Ser. No. 11 /397, 153, 11/397,275, and 11/397,247, each of which is herein incorporated by reference.
These stacked combinations can be created by any method including, but not limited to, breeding plants by any conventional or TopCross methodology, or genetic transformation. If the sequences are stacked by genetically transforming the plants, the polynucleotide sequences of interest can be combined at any time and in any order. For example, a transgenic plant comprising one or more desired traits can be used as the target to introduce further traits by subsequent transformation. The traits can be introduced simultaneously in a co-transformation protocol with the polynucleotides of interest provided by any combination of transformation cassettes. For example, if two sequences will be introduced, the two sequences can be contained in separate transformation cassettes (trans) or contained on the same transformation cassette (cis). Expression of the sequences can be driven by the same promoter or by different promoters. In certain cases, it may be desirable to introduce a transformation cassette that will suppress the expression of the polynucleotide of interest. This may be combined with any combination of other suppression cassettes or overexpression cassettes to generate the desired combination of traits in the plant. It is further recognized that polynucleotide sequences can be stacked at a desired genomic location using a site-specific recombination system. See, e.g.,
W099/25821, W099/25854, WO99/25840, W099/25855, and W099/25853, all of which are herein incorporated by reference.
Insect resistance genes may encode resistance to pests that have great yield drag such as rootworm, cutworm, European Corn Borer, and the like. Such genes include, for example, Bacillus thuringiensis toxic protein genes (U.S. Pat. Nos. 5,366,892; 5,747,450; 5,736,514; 5,723,756; 5,593,881; and Geiser et al. (1986) Gene 48: 109); and the like.
Genes encoding disease resistance traits include detoxification genes, such as against fumonosin (U.S. Pat. No. 5,792,931); avirulence (avr) and disease resistance (R) genes (Jones et al. (1994) Science 266: 789; Martin et al. (1993) Science 262: 1432; and Mindrinos et al. (1994) Cell 78: 1089); and the like.
Sterility genes can also be encoded in an expression cassette and provide an alternative to physical detasseling. Examples of genes used in such ways include male tissue-preferred genes and genes with male sterility phenotypes such as QM, described in U.S. Pat. No. 5,583,210. Other genes include kinases and those encoding compounds toxic to either male or female gametophytic development.
METHODS OF CONTROLLING WEEDS
Methods are provided for controlling weeds in an area of cultivation, preventing the development or the appearance of herbicide resistant weeds in an area of cultivation, producing a crop, and increasing crop safety. The term "controlling," and derivations thereof, for example, as in "controlling weeds" refers to one or more of inhibiting the growth, germination, reproduction, and/or proliferation of; and/or killing, removing, destroying, or otherwise diminishing the occurrence and/or activity of a weed.
The mesotrione and/or isoxazole tolerant plants display a modified tolerance to herbicides and therefore allow for the application of one or more herbicides at rates that would significantly damage control plants and further allow for the application of combinations of herbicides at lower concentrations than normally applied which still continue to selectively control weeds. The mesotrione and/or isoxazole tolerant plants may also display tolerance to HPPD-inhibitor herbicide carryover from a previous crop growing season. In addition, the mesotrione and/or isoxazole tolerant plants can be used in combination with herbicide blends technology and thereby make the application of chemical pesticides more convenient, economical, and effective for the producer.
The methods comprise planting the area of cultivation with mesotrione and/or isoxazole tolerant crop seeds or plants, and applying to any crop, crop part, weed or area of cultivation thereof an effective amount of a mesotrione and/or isoxazole herbicide of interest. It is recognized that the herbicide can be applied before or after the crop is planted in the area of cultivation. Such herbicide applications can include an application of a mesotrione and/or an isoxazole chemistry, or any combination thereof.
In certain examples, the combination of herbicides comprises a glyphosate, a glufosinate, a dicamba, a bialaphos, a phosphinothricin, a protox inhibitor, a sulfonylurea, an imidazolinone, a chlorsulfuron, an imazapyr, a chlorimuron-etbyl, a quizalofop, an HPPD, a PPO, and/or a fomesafen, or combinations thereof, wherein said effective amount is tolerated by the crop and controls weeds. Any effective amount of these herbicides can be applied, wherein the effective amount is any amount that differentiates between plant cells, plants, and/or seed comprising a mesotrione and/or isoxazole tolerance allele, a mesotrione and/or isoxazole tolerance polynucleotide, and/or a polynucleotide encoding an ABC transporter protein that confers tolerance to herbicide formulations comprising a mesotrione and/or isoxazole. In some examples the herbicides are applied simultaneously, in some examples the herbicides are applied sequentially, in some examples the herbicides are applied as pre-emergent treatments, in some examples the herbicides are applied as post-emergent treatments, in some examples the herbicides are applied as a combination of pre- and post-emergent treatments.
In some examples, the method of controlling weeds comprises planting the area with mesotrione and/or isoxazole tolerant crop seeds or plants and applying to the crop, crop part, seed of said crop or the area under cultivation, an effective amount of a herbicide, wherein said effective amount comprises an amount that is not tolerated by a control crop when applied to the control crop, crop part, seed or the area of cultivation, wherein the control crop does not express a polynucleotide that encodes an herbicide- tolerance polypeptide. In specific embodiments, combinations of herbicides may be used, such as when an additional tolerance trait is incorporated into the plant.
In another embodiment, the method of controlling weeds comprises planting the area with a mesotrione and/or isoxazole tolerant crop seeds or plant and applying to the crop, crop part, seed of said crop or the area under cultivation, an effective amount of a herbicide, wherein said effective amount comprises a level that is above the recommended label use rate for the crop, wherein said effective amount is tolerated when applied to the mesotrione and/or isoxazole tolerant crop, crop part, seed, or the area of cultivation thereof.
Any herbicide can be applied to the tolerant crop, crop part, or the area of cultivation containing said crop plant. Classifications of herbicides (i.e., the grouping of herbicides into classes and subclasses) is well-known in the art and includes classifications by HRAC (Herbicide Resistance Action Committee) and WSSA (the Weed Science Society of America) (see also, etzinger and Mallory-Smith (1997) Weed Technology 1 1 : 384-393). An abbreviated version of the HRAC classification (with notes regarding the corresponding WSSA group) is set forth in Table 1.
Table 1. Abbreviated HRAC classification table.
Figure imgf000079_0001
HRAC WSSA
Mode of Action Chemical Family Active Ingredient
Group Group
amidosulfuron
azimsulfuron
bensulfuron-methyl chlorimuron-ethyl chlorsulfuron
cinosulfuron
cyclosulfamuron
ethametsulfuron- methyl
ethoxysulfuron
flazasulfuron
flupyrsulfuron- methyl-Na
foramsulf ron
halosulfuron- methyl
imazosulfuron
Inhibition of iodosulfuron
acetolactate mesosulfuron
B synthase ALS Sulfonylurea metsulfuron-methyl 2
(acetohydroxyacid nicosulfuron
synthase AHAS) oxasulfuron
primisulfuron- methyl
prosulfuron
pyrazosulfuron- ethyl
rimsulfuron
sulfometuron- methyl
sulfosulfuron
thifensulfuron- methyl
triasulfuron
tribenuron-methyl trifloxysulfuron
triflusulfuron- methyl
tritosulfuron
imazapic
imazamethabenz- methyl
Imidazolinone imazamox
imazapyr
imazaquin
imazethapyr HRAC WSSA
Mode of Action Chemical Family Active Ingredient
Group Group
cloransulam-methyl diclosulam
florasulam
Triazolopyrimidine
fiumetsulam
metosulam
penoxsulam
bispyribac-Na
pyribenzoxim
pyriftalid
Pyrimidinyl(thio)benzoate
pyrithiobac-Na
pyriminobac- methyl
flucarbazone-Na
Sulfonylaminocarbonyl- propoxycarbazone- triazolinone
Na
ametryne
atrazine
cyanazine
desmetryne
dimethametryne
prometon
Inhibition of
prometryne
CI photosynthesis at Triazine 5 propazine
photosystem II
simazine
simetryne
terbumeton
terbuthylazine
terbutryne
trietazine
hexazinone
Triazinone metamitron
metribuzin
Triazolinone amicarbazone
bromacil
Uracil lenacil
terbacil
pyrazon =
Pyridazinone
chloridazon
desmedipham
Phenyl-carbamate
phenmedipham HRAC WSSA
Mode of Action Chemical Family Active Ingredient
Group Group
chlorobromuron
chlorotoluron
chloroxuron
dimefuron
diuron
ethidimuron
fenuron
fluometuron (see
Inhibition of F3)
C2 photosynthesis at Urea isoproturon 7 photosystem II isouron
linuron
methabenzthiazuron metobromuron
metoxuron
monolinuron
neburon
siduron
tebuthiuron
propanil
Amide
pentanochlor
Inhibition of bromofenoxim
C3 photosynthesis at Nitrile bromoxynil 6 photosystem II ioxynil
Benzothiadiazinone bentazon
pyridate
Phenyl-pyridazine
pyridafol
Photosystem-I- diquat
D Bipyridylium 22 electron diversion paraquat
acifluorfen-Na
bifenox
chlomethoxyfen
Inhibition of fluoroglycofen-
E protoporphyrinogen Diphenylether ethyl 14 oxidase (PPO) fomesafen
halosafen
lactofen
oxyfluorfen
fluazolate
Phenylpyrazole
pyraflufen-ethyl
cinidon-ethyl
N-phenylphthalimide flumioxazin
flumiclorac-pentyl fluthiacet-methyl
Thiadiazole
thidiazimin
Figure imgf000083_0001
Diphenylether aclonifen HRAC WSSA
Mode of Action Chemical Family Active Ingredient
Group Group
Inhibition of EPSP glyphosate
G Glycine 9 synthase sulfosate
glufosinate-
Inhibition of
ammonium
H glutamine Phosphinic acid 10 bialaphos =
synthetase
bilanaphos
Inhibition of DHP
I (dihydropteroate) Carbamate asulam 18 synthase
benefin =
benfluralin
butralin
Microtubule dinitramine
Kl Dinitroaniline 1 ί assembly inhibition ethalfluralin
oryzalin
pendimethalin
trifluralin
amiprophos-methyl
Phosphoroamidate
butamiphos
dithiopyr
Pyridine
thiazopyr
propyzamide =
Benzamide pronamide
tebutam
DCPA = chlorthal-
Benzoic acid
dimethyl
Inhibition of
chlorpropham
mitosis /
K2 Carbamate propham 23 microtubule
carbetamide
organisation
Inhibition of
acetochlor
VLCFAs
K3 Chloroacetamide alachlor 15 (Inhibition of cell
butachlor
division)
dimethachlor
dimethanamid
metazachlor
metolachlor
pethoxamid
pretilachlor
propachlor
propisochlor
thenylchlor
diphenamid
Acetamide napropamide
naproanilide HRAC WSSA
Mode of Action Chemical Family Active Ingredient
Group Group
flufenacet
Oxyacetamide
mefenacet
Tetrazolinone fentrazamide
anilofos
Other cafenstrole
piperophos
Inhibition of cell
dichlobenil
L wall (cellulose) Nitrile 20 chlorthiamid
synthesis
Benzamide isoxaben 21
Triazolocarboxamide flupoxam
quinclorac (for
Quinoline carboxylic acid monocots) 26
(also group 0)
Uncoupling DNOC
M (Membrane Dinitrophenol dinoseb 24 disruption) dinoterb
butylate
eye lo ate
dimepiperate
EPTC
esprocarb
molinate
Inhibition of lipid
orbencarb
N synthesis - not Thiocarbamate 8 pebulate
ACCase inhibition
prosulfocarb
thiobencarb =
benthiocarb
tiocarbazil
triallate
vernolate
Phosphorodithioate bensulide
benfuresate
Benzofuran
ethofumesate
TCA
Chloro-Carbonic-acid dalapon 26 flupropanate
clomeprop
2,4-D
2,4-DB
Action like indole dichlorprop = 2,4-
0 acetic acid Phenoxy-carboxylic-acid DP 4 (synthetic auxins) MCPA
MCPB
mecoprop = MCPP
= CMPP HRAC WSSA
Mode of Action Chemical Family Active Ingredient
Group Group
chloramben
Benzoic acid dicamba
TBA
clopyralid
fluroxypyr
Pyridine carboxylic acid
picloram
triclopyr
quinclorac
Quinoline carboxylic acid (also group L)
quinmerac
Other benazolin-ethyl
p Inhibition of auxin Phthalamate naptalam
Γ 19 transport Semicarbazone diflufenzopyr-Na
Unknown (actual
mode of action
unknown, but likely
that they differ in Flamprop-M- z Arylaminoprop ionic acid 25 mode of action methyl /-isopropyl between themselves
and from other
groups)
Pyrazolium difenzoquat 26
DSMA
Organoarsenical 17
MSMA
bromobutide
Other 27
(chloro)-flurenol cinmethylin
cumyluron
dazomet
dymron = daimuron methyl-dimuron= methyl-dymron
etobenzanid
fosamine
indanofan
metam
oxaziclomefone
oleic acid
pelargonic acid
pyributicarb
Herbicides can be classified by their mode of action and/or site of action and can also be classified by the time at which they are applied (e.g., pre-emergent or post- emergent), by the method of application (e.g., foliar application or soil application), or by how they are taken up by or affect the plant. Mode of action generally refers to the metabolic or physiological process within the plant that the herbicide inhibits or otherwise impairs, whereas site of action generally refers to the physical location or biochemical site within the plant where the herbicide acts or directly interacts. Herbicides can be classified in various ways, including by mode of action and/or site of action. Often, an herbicide- tolerance gene that confers tolerance to a particular herbicide or other chemical on a plant expressing it will also confer tolerance to other herbicides or chemicals in the same class or subclass, for example, a class or subclass set forth in the table above. Thus, in some examples, a transgenic plant is tolerant to more than one herbicide or chemical in the same class or subclass, such as, for example, an inhibitor of PPO, a sulfonylurea, a glyphosate, or a synthetic auxin. In some examples the plant is transgenic for one or more of the herbicide tolerance traits, non-transgenic for one of more of the tolerance traits, or any combination thereof
Typically, the plants provided can tolerate treatment with different types of herbicides (i.e., herbicides having different modes of action and/or different sites of action) as well as with higher amounts of herbicides than previously known plants, thereby permitting improved weed management strategies that are recommended in order to reduce the incidence and prevalence of herbicide-tolerant weeds. Specific herbicide combinations can be employed to effectively control weeds.
A transgenic crop plant which can be selected for use in crop production based on the prevalence of herbicide-tolerant weed species in the area where the transgenic crop is to be grown is provided. Methods are known in the art for assessing the herbicide tolerance of various weed species. Weed management techniques are also known in the art, such as for example, crop rotation using a crop that is tolerant to an herbicide to which the local weed species are not tolerant. A number of entities monitor and publicly report the incidence and characteristics of herbicide-tolerant weeds, including the Herbicide
Resistance Action Committee (HRAC), the Weed Science Society of America, and various state agencies (see, e.g., herbicide tolerance scores for various broadleaf weeds from the 2004 Illinois Agricultural Pest Management Handbook), and one of skill in the art would be able to use this information to determine which crop and herbicide combinations should be used in a particular location. These entities also publish advice and guidelines for preventing the development and/or appearance of and controlling the spread of herbicide tolerant weeds (see, e.g., Owen and Hartzler (2004), 2005 Herbicide Manual for Agricultural Professionals, Pub. WC 92 Revised (Iowa State University Extension, Iowa State University of Science and Technology, Ames, Iowa); Weed Control for Cora, Soybeans, and Sorghum, Chapter 2 of "2004 Illinois Agricultural Pest Management Handbook" (University of Illinois Extension, University of Illinois at Urbana-Champaign, 111.); Weed Control Guide for Field Crops, MSU Extension Bulletin E434 (Michigan State University, East Lansing, Mich.)).
Also included are plant cells, plants, and/or seeds produced by any of the foregoing methods.
The present invention is illustrated by the following examples. The foregoing and following description of the present invention and the various embodiments are not intended to be limiting of the invention but rather are illustrative thereof. Hence, it will be understood that the invention is not limited to the specific details of these examples.
EXAMPLES
Example 1: Identification of Isoxafutole Tolerant and Sensitive Soybean Lines - herbicide screening bioassay and intergroup association marker based diagnostic
Two soybean mapping populations were used to confirm significant QTLs related to tolerance and susceptibility to mesotrione and/or isoxazole herbicides, to identify any potential QTLs associated with the tolerance or susceptibility to these herbicides, and to identify any varietal variation due to differences between the two herbicide chemistries used in the study. The mesotrione herbicide used in this study was Callisto® (referred to as Herbicide B); the isoxazole herbicide used in this study was Balance Pro® (referred to as Herbicide A).
Part 1 :
Studies were conducted using herbicides A and B and were performed at two locations, Princeton, IL and Johnston, IA. Herbicide screening protocols developed in the summer of 2008 determined the optimum herbicide rate, application timing and the best time to evaluate soybean injury following application. Herbicide A and B were applied as a pre-plant incorporated herbicide. The application rate for Herbicide A was based on soil organic matter and was applied at half the recommended labeled rate. Herbicide B was applied at half the pre-plant incorporated label rate. Both herbicides were applied using an ATV sprayer outfitted with a 10-foot boom, GPS and a Raven control system. The herbicides were applied at a rate of 30 gallons of water per acre and a spray pressure of 35-40 psi. An agitation system was used to maintain herbicide suspension in the water spray solution. Since both herbicides A and B were used in the same field, the sprayer was cleaned out between applications and a 10- foot buffer strip was used to help separate the two herbicides in the field to ensure no spray overlap.
The herbicides were incorporated into the soil to a depth of 1-2 inches using a field cultivator with rolling baskets 2-5 days following application (Table 2). Incorporation was performed in two directions to ensure even distribution of the herbicide in the soil.
The soybeans were planted into the soil to a depth of 1-1.5 inches using an Almaco 4-row index planter set on 30-inch row spacing. All plots were planted as single row plots with 25 seeds for 4.5 feet of planted row with a three-foot alleyway. The planted population was approximately 90,000 seeds per acre. Both Princeton and Johnston locations were planted on June 4. The herbicide application, planting, and rating dates for both Princeton and Johnston locations are presented in Table 2.
Table 2. Herbicide A lication, tilla e, lantin and ratin dates
Figure imgf000089_0001
Soybean varietal herbicide reactions were evaluated using visual scores for plant growth reduction (STNT) and crop injury rating (HERSC) using descriptions defined below. Two ratings were conducted at both locations; the initial rating (V3) was based off of the clearest distinction of symptoms across the experiments. A second rating (V5 or V6) was conducted to ensure accuracy and note any varietal variation over time. An untreated check was used as a guide for the expected plant growth and development over time. Plant Growth Reduction Rating (STNT)
1-9 herbicide reaction scale for plant growth reduction:
9 = no plant growth reduction from the herbicide
8 = <5% plant growth reduction
7 = >5% and <20% plant growth reduction
6 = >20% and <35% plant growth reduction
5 = >35% and <50% plant growth reduction
4 = >50 and <65% plant growth reduction
3 = >65 and <80% plant growth reduction
2 = >80 and <95% plant growth reduction
1 = >95% plant growth reduction
Crop Injury Rating (HERSC)
1-9 herbicide reaction scale for crop injury (both chlorotic and necrotic tissue):
• 9 = no crop injury
8 = <5% crop injury
7 = >5% and <20% crop injury
6 = >20% and <35% crop injury
5 = >35% and <50% crop injury
• 4 = >50 and <65% crop injury
3 = >65 and <80% crop injury
2 = >80 and <95% crop injury
1 = >95% crop injury Two mapping populations were used that contained known susceptible and tolerant parents that were fixed and carried different alleles for two QTLs identified on linkage group L. The mapping populations were screened using herbicide A and the herbicide screening protocol described above. Two populations (Pop A and Pop B) of 90 randomly selected F3:F5 lines were used in the study. Four replications of the populations were placed in a row by column design to help adjust means due to field variation. The parents of each population were replicated 3 times per replication for a total of 12 times per location. An analysis of variance (ANOVA) was conducted to identify significant differences between the varieties within the populations.
A variety trial was conducted using 144 varieties with herbicides A and B and the herbicide screening protocol described above. Four replications of the varieties were placed in a row by column design to help adjust means due to field variation for both herbicides. The 144 lines included 52 susceptible and 61 tolerant lines identified previously as well as lines identified as moderate but containing the susceptible or tolerant allele for the QTLs.
An ANOVA was run on the STNT and HERSC data to determine any significant differences between soybean varieties. The herbicide response and the varieties were classified into tolerant and susceptible groups to be analyzed using available SSR and SNP markers for identification of other potential QTLs associated with the trait. The tolerant and susceptible classes were analyzed to observe marker trait associations by comparing the allelic frequencies of tolerant and susceptible varieties. This analysis used all available genome wide data produced for the markers and the varieties to run the analysis.
Significant markers were then identified and potential QTL regions were recognized for candidates causing tolerant reactions. This data was used to help identify additional polymorphic markers within the mapping populations. Table 3 indicates the results of the various varieties tested.
Table 3: Mapping population analysis
Figure imgf000091_0001
sus 3 3.252 sus 3 3.3115
SEG 4 3.427
SEG? 4 3.5305
SEG 4 3.544
SUS 4 3.5845
SEG 4 3.589
SUS 4 3.6135
SUS 4 3.6575
SEG 4 3.662
SEG 5 3.729
SEG 5 3.73
SUS 5 3.7435
SEG 5 3.843
TOL 5 3.89
SEG 5 3.9255
SEG 5 3.9925
SEG 5 4.0415
SEG 5 4.0755
SEG 5 4.1025
TOL 5 4.1315
TOL 5 4.171
SEG 6 4.273
SEG 6 4.276
SEG 6 4.325
SEG? 4 4.331
SEG 4 4.375
TOL 6 4.4225
SEG 6 4.436
SEG 6 4.456
SEG 6 4.4955
TOL 6 4.5525
TOL 6 4.5905
TOL 6 4.6135
SEG 6 4.627
TOL 6 4.652
SEG 6 4.652
SEG 6 4.7305
TOL 6 4.7785
TOL 6 4.7805
TOL 6 4.814
SEG? 6 4.815
TOL 6 4.827
TOL 6 4.883
SEG 6 4.955
SEG 7 5.005 SEG 7 5.018
TOL 7 5.0185
SEG 7 5.048
TOL 7 5.053
TOL 7 5.0635
TOL 7 5.0895
TOL 7 5.151
TOL 6 5.239
SEG 7 5.2525
TOL 7 5.258
SEG 7 5.263
TOL 5 5.312
TOL 7 5.357
TOL 7 5.358
SEG? 8 5.4165
TOL 8 5.4475
SEG? 8 5.512
TOL 8 5.5645
TOL 8 5.5975
TOL 8 5.6185
TOL 8 5.707
TOL 8 5.713
TOL 9 5.8935
TOL 9 5.904
TOL 9 6.022
TOL 7 6.0235
SEG? 9 6.1865
TOL 9 6.591
TOL 9 6.842 sus 1 2.049
TOL 9 5.9145
SUS 2 2.733 sus 2 2.748 sus 2 2.801 sus 2 2.883 sus 2 2.938 sus 2 2.9515 sus 2 2.9955
SEG? 2 3.0555 sus 1 2.4295 sus 1 2.5185 sus 1 2.6205 sus 1 2.6395 sus 3 3.1315
SEG? 3 3.1925
SUS 3 3.193 SEG 3 3.216
SEG 3 3.267
SUS 3 3.2875
SUS 3 3.2885
SUS 3 3.3375
SUS 4 3.3435
SUS 4 3.3555
SUS 4 3.361
SUS 4 3.428
SUS 4 3.449
SUS 4 3.539
SUS 4 3.5505
SUS 4 3.6335
SUS 5 3.681
SUS 5 3.793
SUS 5 3.8215
SUS 4 3.8395
SUS 5 4.01
SEG? 5 4.0635
SUS 5 4.072
SEG 5 4.1245
SUS 5 4.1535
TOL 5 4.239
SEG 6 4.3385
SEG 6 4.489
TOL 6 4.5315
SEG 6 4.5915
SEG 6 4.721
TOL 6 4.7345
TOL 6 4.8215
TOL 6 4.8495
TOL 6 4.8625
TOL 6 4.867
TOL 6 4.944
TOL 4 4.973
TOL 7 5.0845
TOL 7 5.0885
TOL 5 5.0955
TOL 7 5.1245
TOL 7 5.1505
TOL 7 5.1695
TOL 4 5.1885
TOL 7 5.221
TOL 7 5.228
SEG 7 5.2285
SEG 7 5.2805 TOL 7 5.3305
SEG 7 5.3345
TOL 8 5.381
TOL 8 5.3835
TOL 8 5.414
TOL 8 5.5165
TOL 8 5.535
TOL 8 5.55
TOL 8 5.5925
TOL 8 5.6125
TOL 8 5.617
TOL 8 5.6275
TOL 8 5.6435
TOL 8 5.67
TOL 8 5.7255
TOL 8 5.7645
TOL 8 5.8155
TOL 8 5.8245
TOL 8 5.8835
TOL 9 5.885
TOL 9 5.8885
TOL 9 5.9165
TOL 9 5.964
TOL 9 6.0095
TOL 9 6.2345
TOL 9 6.2655
TOL 9 6.286
TOL 7 6.3875
TOL 9 6.599 sus 3 3.069
TOL 7 5.359
The experimental means for Herbicide A across both locations for HERSC2 was 4.695 with a standard deviation of 1.94. The coefficient of variation across the locations was 26.7.
Example 2: Determination of QTL and marker associations / intergroup analysis
There was significant (PO.001) difference across varieties for Herbicide A. The LSD was 1.316 across all varieties. Predicted means by location were calculated using a linear model for the locations. The varieties were looked at individually by the adjusted means by location, the LSD value, the average score by location, and the 2008 data for each variety. This gave an overall view of each variety and allowed for a simple classification across all varieties. Any variety that showed a high rate of variability across the data was automatically placed in the segregating group.
The results of the ANOVA for both Herbicides A and B are reported in Table 4. The model used for the analysis was the incomplete block design and the affect of the model is described through the relative efficiency and the Czekanowski Coefficient (Czek Coeff). The relative efficiency is comparing the error terms of the more complex block (incomplete block, ICB) to the less complex model (randomized complete block, RCB). The relative efficiency for the variety trial using herbicide A was 111% and herbicide B wasl23%. The Czek Coeff which reports the top 10 and 20 percent of the entries that were the same for both the RCB and ICB designs was 73 and 83% for the top 10% of entries and 86 and 90% for the top 20% of the entries for the variety trials using herbicides A and B, respectfully.
Table 4. Analysis of variance for the variety trial
Figure imgf000096_0001
Using this method of classification the varieties were grouped according to their reactions to herbicides A and B. For herbicide A there were 32 tolerant, 67 moderate, 37 susceptible and 8 segregating lines. For herbicide B there were 29 tolerant, 75 moderate, 32 susceptible and 8 segregating lines.
An Intergroup Allele Frequency Distribution analysis was conducted using
GeneFlow™ version 7.0 software. An intergroup allele frequency distribution analysis provides a method for finding non-random distributions of alleles between two phenotypic groups.
During processing, a contingency table of allele frequencies was constructed and from this a G-statistic and probability were calculated. The G-statistic was adjusted by using the William's correction factor. The probability value was adjusted to take into account the fact that multiple tests are being done (thus, there is some expected rate of false positives). The adjusted probability is proportional to the probability that the observed allele distribution differences between the two classes would occur by chance alone. The lower that probability value, the greater the likelihood that the tolerance phenotype and the marker will co-segregate. A more complete discussion of the derivation of the probability values can be found in the GeneFlow™ version 7.0 software documentation. See also Sokal and Rolf (1981), Biometry: The Principles and Practices of Statistics in Biological Research, 2nd ed., San Francisco, W. H. Freeman and Co.
The underlying logic is that markers with significantly different allele distributions between the tolerant and non-tolerant groups (z. e. , non-random distributions) might be associated with the trait and can be used to separate them for purposes of marker assisted selection of soybean lines with previously uncharacterized tolerance or non-tolerance or sensitivity to mesotrione and/or isoxazole herbicides. The present analysis examined one marker locus at a time and determined if the allele distribution within the tolerant group is significantly different from the allele distribution within the non-tolerant group. A statistically different allele distribution is an indication that the marker is linked to a locus that is associated with tolerance or non-tolerance or sensitivity to mesotrione and/or isoxazole herbicides. In this analysis, unadjusted probabilities less than one are considered significant (the marker and the phenotype show linkage disequilibrium), and adjusted probabilities less than approximately 0.05 are considered highly significant. Allele classes represented by less than 5 observations across both groups were not included in the statistical analysis. In this analysis, 1043 marker loci had enough observations for analysis.
This analysis compares the plants' phenotypic score with the genotypes at the various loci. This type of intergroup analysis neither generates nor requires any map data. Subsequently, map data (for example, a composite soybean genetic map) is relevant in that multiple significant markers that are also genetically linked can be considered as collaborating evidence that a given chromosomal region is associated with the trait of interest.
For the herbicide A variety trial, the analysis identified 275 markers (PO.05), of these 275 markers identified, 243 of the markers had been previously mapped to a particular genomic position. This allowed for further analysis to identify potential genomic regions for genes and to eliminate marker regions that are likely not associated with the native tolerance to the herbicide. There were a total of 6 regions identified where multiple markers were pointing to a particular genomic region (Table 5). The regions identified included regions on B2, D2, E, G, L and 7 unmapped (UM) markers. Of the original 275 markers identified, 1 12 markers were used to identify the 6 genomic regions.
Table 5. Potential genomic regions for Herbicide A
Figure imgf000098_0001
A similar analysis was conducted for the tolerant and susceptible classes to Herbicide B where 274 markers were identified (PO.05). Of the 274 markers identified 239 markers had been previously mapped to a particular genomic position. Similar regions to Herbicide A were identified that included B2, E, G, L, and 10 UM markers (Table 6). A potential region on N was added and the region on D2 was taken off for Herbicide B tolerance. Of the original 274 markers identified 1 16 markers were used to help identify the 6 regions.
Table 6. Potential genomic regions for Herbicide B
Figure imgf000098_0002
N 37.11 -53.27 21
UM 10 Highly significant <.008
Additional observations were made through looking at the tolerant, moderate, susceptible and segregating classes to each herbicide as assigned through the variety trial. Of the 144 varieties in the herbicide trial, 21 were tolerant to both herbicides, 54 displayed moderate tolerance to both herbicides, and 28 displayed susceptible reactions to both herbicides. There were a total of 31 varieties that were classified one class higher or lower to herbicide A or B. For example 11 varieties were tolerant to herbicide A and moderate to herbicide B, where they were lowered 1 class from herbicide A classification to herbicide B classification. There were zero lines that were tolerant to one herbicide and susceptible to the other. This is summarized in Table 7.
Table 7. Variety reactions to both herbicides
Figure imgf000099_0001
The significant markers were observed for both variety classes to each of the chemistries. Of the 275 and 274 markers that were significant for the Herbicide A and Herbicide B reactions, 144 markers were significant for both variety reactions. As observed in the variety trial analysis, the regions of potential QTLs were observed for both classes on B2, E, G, and L; with the most significant markers on L from 10.1-14.31 cM (Tables 5 and 6).
Table 8 shows the allele distribution for marker S03859, which is closely linked to this region, among the 144 lines analyzed; 32 tolerant lines, 37 non-tolerant (susceptible) lines, 67 moderate, and 8 segregating lines analyzed. Marker calls for the S03859 locus were available for 1 11 of the 144 lines.
Table 8. Allele distribution
Figure imgf000099_0002
S03859 allele (LG-L) Phenotype Adjusted mean
1,1 Susceptible 1.7085
1,1 Susceptible 1.8675
Susceptible 1.9175
1,1 Susceptible 2.006
1,1 Susceptible 2.212
Susceptible 2.272
Susceptible 2.283
1,1 Susceptible 2.2845
1,1 Susceptible 2.327
1,1 Susceptible 2.4255
1,1 Susceptible 2.643
Susceptible 2.6815
1,1 Susceptible 2.781
1,1 Susceptible 2.8325
1,1 Susceptible 2.838
1,1 Susceptible 2.881
1,1 Susceptible 2.883
1,1 Susceptible 2.9005
1,1 Susceptible 2.927
1,1 Susceptible 2.992
1,1 Susceptible 2.994
Susceptible 3.056
1,1 Susceptible 3.1475
3,3 Susceptible 3.215
1,1 Susceptible 3.2835
1,3 Susceptible 3.3085
Susceptible 3.3385
1,1 Susceptible 3.3875
1,1 Susceptible 3.3885
1,1 Susceptible 3.4085
1,1 Segregating 3.5095
1,1 Susceptible 3.6345
1,1 Susceptible 3.711
1,1 Susceptible 3.7595
1,3 Moderate 3.851
Moderate 3.919
Susceptible 3.9385
1,1 Moderate 3.967
3,3 Moderate 4.067
Moderate 4.107
3,3 Susceptible 4.1605
1,1 Susceptible 4.1645
1,3 Moderate 4.17
3,3 Moderate 4.179
3,3 Moderate 4.3415 S03859 allele (LG-L) Phenotype Adjusted mean
3,3 Moderate 4.35
1,1 Moderate 4.357
3,3 Moderate 4.4295
3,3 Moderate 4.4495
Moderate 4.4765
3,3 Moderate 4.524
1,1 Moderate 4.555
Moderate 4.6375
3,3 Moderate 4.6615
3,3 Moderate 4.676
3,3 Moderate 4.678
Segregating 4.723
Moderate 4.7575
3,3 Segregating 4.7645
3,3 Moderate 4.7675
Moderate 4.771
Moderate 4.7875
3,3 Moderate 4.797
1,3 Moderate 4.846
Tolerant 4.85
Moderate 4.891
1,1 Moderate 4.9085
3,3 Moderate 4.9095
3,3 Moderate 4.9395
3,3 Tolerant 4.943
1,1 Moderate 4.961
1,1 Moderate 5.015
3,3 Moderate 5.0195
3,3 Moderate 5.051
3,3 Moderate 5.1305
Tolerant 5.1475
3,3 Moderate 5.1495
Moderate 5.2105
3,3 Tolerant 5.2125
3,3 Moderate 5.214
3,3 Tolerant 5.215
3,3 Moderate 5.2565
3,3 Moderate 5.2795
3,3 Moderate 5.2945
3,3 Moderate 5.3125
3,3 Moderate 5.3145
3,3 Moderate 5.3345
3,3 Moderate 5.335
3,3 Tolerant 5.3365
Tolerant 5.377 S03859 allele (LG-L) Phenotype Adjusted mean
3,3 Moderate 5.39
3,3 Segregating 5.3905
3,3 Segregating 5.4675
3,3 Moderate 5.4835
3,3 Segregating 5.4925
3,3 Moderate 5.4975
3,3 Tolerant 5.518
3,3 Moderate 5.524
Segregating 5.541
Tolerant 5.574
1,1 Moderate 5.5895
3,3 Moderate 5.6025
Moderate 5.616
3,3 Tolerant 5.6685
3,3 Tolerant 5.6925
3,3 Moderate 5.7005
3,3 Moderate 5.7095
Tolerant 5.7165
3,3 Moderate 5.7315
Moderate 5.741
Moderate 5.75
3,3 Tolerant 5.7545
3,3 Moderate 5.7625
3,3 Moderate 5.7705
3,3 Moderate 5.7715
3,3 Moderate 5.7835
3,3 Segregating 5.7875
3,3 Moderate 5.798
3,3 Moderate 5.851
3,3 Moderate 5.852
3,3 Tolerant 5.857
3,3 Tolerant 5.87
3,3 Moderate 5.8705
Tolerant 5.914
Tolerant 5.948
3,3 Tolerant 5.9525
3,3 Tolerant 5.9675
Tolerant 6.0245
3,3 Moderate 6.026
3,3 Moderate 6.0275
3,3 Tolerant 6.034
3,3 Tolerant 6.042
3,3 Tolerant 6.054
Tolerant 6.092
Tolerant 6.1295 S03859 allele (LG-L) Phenotype Adjusted mean
3,3 Moderate 6.2395
3,3 Tolerant 6.2475
3,3 Tolerant 6.2695
Tolerant 6.275
3,3 Tolerant 6.387
3,3 Tolerant 6.431
3,3 Tolerant 6.6535
Tolerant 6.674
The non-random distribution of alleles between the tolerant and non-tolerant plant groups at the marker loci in Table 8 is good evidence that a QTL influencing tolerance or sensitivity to mesotrione and/or isoxazole herbicides is linked to these marker loci.
QTLs related to tolerance and susceptibility to mesotrione and/or isoxazole herbicides were found to essentially co-localize with QTLs related to tolerance to PPO inhibitor herbicides to linkage group L, as shown in the Examples below. Thus, PPO tolerance could be used to fine map the QTL and to identify putative candidate genes as shown below.
Example 3: Identification of Sulfentrazone Tolerant and Sensitive Soybean Lines - herbicide screening bioassay and intergroup association marker based diagnostic
Sulfentrazone is a PPO inhibitor and is the active ingredient in Authority® herbicide. Authority® 75DF (FMC Corp., Philadelphia, PA, USA) is a 75% active ingredient formulation of sulfentrazone containing no other active ingredients.
Part 1 : Herbicide Bioassay
One hundred sixteen (1 16) elite soybean lines were screened for sulfentrazone tolerance using the following protocol. Seed of soybean varieties with adequate seed quality, having greater than 85% warm germination were used.
DESIGN AND REPLICATION: After planting, entries were set up in a randomized complete block design, blocked by replication. Three replications per experiment were used. One or more of well established check variety were included in the experiment, such as available public sector check lines.
Non-tolerant check: Pioneer 9692, Asgrow A4715 Tolerant check: Pioneer 9584, Syngenta S5960
Growing conditions were as follows (greenhouse/ growth chamber): 16 hr photoperiod @ 85° F (w/ 75° nighttime set back). Lighting is critical to the success of the screening as stated below.
METHOD OF SCREENING: Four inch plastic pots were filled with a high quality universal potting soil. Entries were planted 1 inch deep at the rate of 5 seeds/pot. A bar- coded plastic stake was used to identify each entry. After planting the pots were allowed to sit in greenhouse overnight to acclimate to soil and improve germination. The following day a sulfentrazone herbicide solution was slowly poured over each pot and allowed to evenly soak through entire soil profile. This ensured that each seed was exposed to an equal amount of sulfentrazone. Pots were placed on aluminum trays and placed in a greenhouse or growth chamber under high intensity light conditions with photo synthetic photon flux density (PPFD) of at least 500 ΓηοΙ/ητ . Proper lighting conditions were necessary for this screening due to the nature of the PPO inhibitor used. Pots were lightly watered so that herbicide was not leached from the soil profile. After soybean emergence the pots were watered by keeping aluminum trays filled with ¾" of water under each pot.
HERBICIDE SOLUTION:
A) Mix a stock solution of 0.926 g Authority® 75DF (FMC Corp.), thoroughly dissolved in 1000 ml of water.
B) Mix 10 ml of STOCK SOLUTION in 1000 ml of water to create final solution.
C) Pour 100 ml of FINAL SOLUTION over each pot. RECORDING DATA: 10-14 days after treatment, plants were ready to be scored. All scores were based on a comparison to the checks and evaluated as follows:
9 = Equivalent or better when compared to the tolerant check
7 = Very little damage or response noted.
5 = Intermediate response or damage
3 = Major damage, including stunting and foliar necrosis
1 = Severe damage, including severe stunting and necrosis; equivalent or worse when compared to the non-tolerant check Of the 116 soybean lines screened, 102 showed at least some tolerance to sulfentrazone based herbicides and 11 showed high sensitivity. A reference relevant to this protocol would be: Dayan et al, (1997) 'Soybean {Glycine max) cultivar differences in response to sulfentrazone' Weed Science 45:634-641.
Part 2: Inter group analysis
An "Intergroup Allele Frequency Distribution" analysis was conducted using GeneFlow™ version 7.0 software as described above. An intergroup allele frequency distribution analysis provides a method for finding non-random distributions of alleles between two phenotypic groups.
During processing, a contingency table of allele frequencies was constructed and from this a G-statistic and probability were calculated. The G statistic was adjusted by using the William's correction factor. The probability value was adjusted to take into account the fact that multiple tests are being done (thus, there is some expected rate of false positives). The adjusted probability is proportional to the probability that the observed allele distribution differences between the two classes would occur by chance alone. The lower that probability value, the greater the likelihood that the tolerance phenotype and the marker will co-segregate. A more complete discussion of the derivation of the probability values can be found in the GeneFlow™ version 7.0 software documentation. See also Sokal and Rolf (1981), Biometry: The Principles and Practices of Statistics in Biological Research, 2nd ed., San Francisco, W. H. Freeman and Co.
The underlying logic is that markers with significantly different allele distributions between the tolerant and non-tolerant groups (i.e., non-random distributions) might be associated with the trait and can be used to separate them for purposes of marker assisted selection of soybean lines with previously uncharacterized tolerance or non-tolerance to protoporphyrinogen oxidase inhibitors. The present analysis examined one marker locus at a time and determined if the allele distribution within the tolerant group is significantly different from the allele distribution within the non-tolerant group. A statistically different allele distribution is an indication that the marker is linked to a locus that is associated with tolerance or non-tolerance to protoporphyrinogen oxidase inhibitors. In this analysis, unadjusted probabilities less than one are considered significant (the marker and the phenotype show linkage disequilibrium), and adjusted probabilities less than approximately 0.05 are considered highly significant. Allele classes represented by less than 5 observations across both groups were not included in the statistical analysis. In this analysis, 1043 marker loci had enough observations for analysis.
This analysis compares the plants' phenotypic score with the genotypes at the various loci. This type of intergroup analysis neither generates nor requires any map data. Subsequently, map data (for example, a composite soybean genetic map) is relevant in that multiple significant markers that are also genetically linked can be considered as collaborating evidence that a given chromosomal region is associated with the trait of interest.
RESULTS: Table 9 lists the soybean markers that demonstrated linkage disequilibrium with the tolerance to protoporphyrinogen oxidase inhibitor phenotype. There were 1043 markers used in this analysis. Also indicated in that table are the chromosomes on which the markers are located and their approximate map position relative to other known markers, given in cM, with position zero being the first (most distal) marker known at the beginning of the chromosome. These map positions are not absolute, and represent an estimate of map position. The statistical probabilities that the marker allele and tolerance phenotype are segregating independently are reflected in the Adjusted Probability values. Out of 584 loci studied in 38 sensitive and 160 tolerant soybean lines, QTLs on Lg-L were highly significant, as shown in the table below.
Table 9. Inter rou anal sis results for L -L markers
Figure imgf000106_0001
Table 10 shows the allele distribution between 101 tolerant lines and 32/33 non-tolerant lines analyzed. Lines exhibiting tolerance are indicated in the first column as "TOL," and lines exhibiting non-tolerance are indicated in the first column as " ON. " Marker calls for the P 10649C-3 locus and the S60167-TB locus were available for 132 and 63 of the lines respectively. Table 10. Allele distribution
Figure imgf000107_0001
TOL 1
TOL 1
TOL 1
TOL 1
TOL 1
TOL 1
TOL 1
TOL 1
TOL 1
TOL 1
TOL 1
TOL 1
TOL 1
TOL 1
TOL 1
TOL 1
TOL 1
TOL 1
TOL 1
TOL 1
TOL 1
TOL 1
TOL 1
TOL 1
TOL 1
TOL 1
TOL 1
TOL 1
TOL 1
TOL 1
TOL 1
TOL 1
TOL 1
TOL 1
TOL 1
TOL 1
TOL 1
TOL 1
TOL 1
TOL 1
TOL 1
TOL 1
TOL 1
TOL 1
TOL 1
TOL 1 TOL 1
TOL 1
TOL 1
TOL 1
TOL 1
TOL 1
TOL
TOL 1
TOL 1
TOL 2
TOL 1
TOL 1
TOL 1
NON 3
NON 3
NON 1
NON 1 2
NON 3
NON 3
NON 1
NON 3
NON 2
NON 3
NON 2
NON 2
NON 2
NON 1
NON 2 3
NON 3
NON 3
NON 2 3
NON 3
NON 3
NON 1
NON 2
NON 3
NON 3
NON 3
NON 2
NON 3
NON 1 3
NON 3
NON 2
NON 1
NON 3 The non-random distribution of alleles between the tolerant and non-tolerant plant groups at the marker loci in Table 10 is good evidence that a QTL influencing tolerance to protoporphyrinogen oxidase inhibitors is linked to these marker loci. Example 4: Predication and confirmation of marker based selection for response to PPO chemistries in a set of diverse public soybean lines.
Marker haplotype data for a set of 17 diverse public soybean lines was determined for two QTL identified in Example 3 for Linkage Group L molecular markers P10649C-3 (approximate position 3.6) and S00224-1 (approximate position 12.0). The response of these lines to sulfentrazone herbicide was published by Hulting et al. (Soybean (Glycine max (L.) Merr.) cultivar tolerance to sulfentrazone. 2001 Science Direct, Vol. 20(8): 679- 683). The phenotypic response was reported as a growth reduction index: plant height and visual injury as expressed as a percentage of check plot of each cultivar. Data for the marker haplotype on Linkage Group L and the herbicide bioassay results are presented in Table 11. Use of the molecular diagnostic P10649C-3 (linked QTL on Linkage Group L, approximate position 3.6) for this set of phentoyped soybean lines is 92% predictive of tolerance to sulfentrazone when injury is set at 39% or less GRI and is 100% predictive of non-tolerance to sulfentrazone when injury is set at 40% or higher GRI. Use of the S00224-1 marker (approximate position 12.0) for this set of soybean lines is 88% predictive of tolerance to sulfentrazone when injury is set at 39% or less GRI and is 100% predictive of non- tolerance to sulfentrazone when injury is set at 40% or more GRI.
Table 11. Marker haplotype at/near QTL on Linkage Group L for PPO herbicide (sulfentrazone) response and phenotypic measure of crop response, expressed in terms of Growth Reduction Index, for soybean cultivars (italicized items indicate deviations from expected)
Figure imgf000110_0001
Illini 8 1,1 3,3
S100 8 1,1 3,3
Mukden 8 1,1 3,3
Arksoy 10 1,1 3,3
Capital 10 1,1 3,3
Haberlandt 10 3,3 2,2
Ralsoy 13 1,1 2,3
Dunfield 16 1,1 3,3
Peking 22 1,1 3,3
Roanoke 40 3,3 2,2
Ogden 42 3,3 2,2
Hutcheson 46 3,3 2,2
Ransom 52 3,3 2,2 allele call load percent
accuracy
(alleles 1) 24/26 = (allele 3) 23/26 = correct tolerant 92% 88%
(allele 2) = 8/8 = correct non-tolerant (allele 3) 8/8 = 100% 100%
*growth reduction index (plant height and visual injury as expressed as
a percentage of check plot of each cultivar); Pre-emergence
sulfentrazone application of 0.28 kg ai/ha, from Hulting, et al. (supra)
Example 5: Predication and confirmation of marker based selection for response to PPO chemistries in a set of soybean commercial lines
Haplotype data for a set of 7 commercial soybean lines was determined for two QTL identified in the previous example for Linkage Group L molecular markers P 10649C- 3 (position 3.6) and S00224-1 (position 12.0). The response of these lines to sulfentrazone herbicide was determined by method used in Example 3. In addition, the same scale was used for scoring such that:
9 = Equivalent or better when compared to the tolerant check
7 = Very little damage or response noted.
5 = Intermediate response or damage
3 = Major damage, including stunting and foliar necrosis 1 = Severe damage, including severe stunting and necrosis; equivalent or worse when compared to the non-tolerant check
Data for the marker haplotype on Linkage Group L and the herbicide bioassay results are presented in Table 12. Use of either/both of these markers for this set of phentoyped soybean lines is 100% predictive of both tolerance (score of a 7 or 9) and non-tolerance (score of a 1 for the non-tolerant check).
Table 12. Prediction and confirmation of marker based selection at QTL for linkage group L for response to PPO chemistry (sulfentrazone) in a set of commercial soybean varieties.
Figure imgf000112_0001
Example 6: Predication and confirmation of marker based selection for response to PPO chemistries (sulfentrazone) in ten lines from a set of soybean lines phenotyped at the University of Illinois
A comparison for the marker predictiveness of PPO response was conducted. The herbicide bioassay experiment used is described in Phytoxic Response and Yield of Soybean (Glycine max) Varieties Treated with Sulfentrazone or Flumioxazin (Taylor- Lovell et at, 2001 Weed Technology 15:96-102). Phenotypic data was taken from Table 2 of the publication for those varieties for which in-house marker data was available.
Phenotypic score and haplotype data for a set of 10 soybean lines (1 public and 9 commercial) in the chromosomal regions around the QTL for Linkage group L is presented in Table 13. The phenotypic score was determined as percent injury which is defined as visible injury ratings including stunting, chlorosis, and bronzing symptomology (0 = no injury; 100 = complete death) with 448 g ai/ha field application. Ratings were taken 12 days after treatment. Use of marker PI 0649 C (linked QTL on Linkage Group L, approximate position 3.6, allele call 1) for this set of phentoyped soybean lines is 100% predictive of tolerance (allele call 1) to sulfentrazone when injury is 21% or less and is 100% predictive of non-tolerance (allele call 2 or 3) to sulfentrazone when injury is 43% or greater. The predictiveness of marker S00224-1 is also 100% accurate for tolerance (allele 3) and non-tolerance (allele 2) for this set of material.
Table 13. Marker haplotype at/near QTL on Linkage Group L for PPO herbicide
(sulfentrazone) response and phenotypic measure of crop injury
Figure imgf000113_0001
Example 7: Fine mapping of the LG-L herbicide tolerance QTL
The herbicide tolerance QTL on LG-L was initially mapped in two different soybean mapping populations: GEID1653063 x GEID3495695 (F4-derived F6) and GEID4520632 x GEID7589905 (F3-derived F5). From these populations, 184 and 180 lines respectively were genotyped and scored for PPO herbicide tolerance as described above. This data was used to map the herbicide tolerance QTL to chromosome GM19 near the closely linked marker S03859-1-A, which explains 80% of the phenotypic variation. From these two populations, lines with recombination breakpoints near S03859-1-A were identified to define the borders of the QTL and to facilitate fine-mapping. Subsequent analysis of the recombinants indicated that the closely linked marker S03859-1-A was actually the left flanking marker. The GEID1653063 x GEID3495695 population had 37 recombinants that set the flanking markers for the herbicide tolerance QTL as S04867-1-A (GM19: 841543-841958) and S03859-1-A (GM19: 1634882- 1635399) (Table 17). The GEID4520632 x GEID7589905 population had 42
recombinants that delimit the QTL to the same interval (Table 18).
Because S03859-1-A was determined to be closely linked to the herbicide tolerance QTL, annotated loci in the vicinity of this marker were targeted for SNP discovery and marker development. Primers were designed from target loci using Primer3 (open source software available from SourceForge.net) and checked for uniqueness using bioinformatics software. A panel composed of 20 PPO tolerant and 8 PPO susceptible lines, including the four mapping parents from the mapping population, was re-sequenced at the target loci to identify informative SNPs. DNA was extracted using the urea extraction protocol below and PCR amplified using standard lab protocols (see Tables 14-1 ). The PCR was then cleaned up using the ExoSAP-IT® protocol (USB -Cleveland, OH, USA) (Table 15) before being sequenced by Sanger sequencing.
In total, 104 loci were re-sequenced and 235 informative SNPs were identified. From these SNPs, 22 Taqman® probe markers were designed to distinguish between tolerant versus susceptible alleles in the mapping populations. Taqman® assays were designed generally following ABI suggested parameters. These markers were then run on 86 select recombinants combined from the two mapping populations to facilitate fine- mapping and to further delimit the herbicide tolerance QTL interval (Table 19).
Urea extraction protocol
1. Grind 2g fresh tissue or .5g lyophilized tissue and add it to 6mL Urea
Extraction Buffer and mix well.
2. Add RNase A ( 1 OOmg/mL) and incubate @ 37°C for 20min.
a. 3uL - Leaf
b. 12uL - Seed
3. Add 4-5 mL Phenol:Chloroform:Isoamyl 25:24:1. Mix well. (Sigma P3803)
4. Put on rocker inside hood.
a. Fresh - 15 min
b . Lyophilized - 30 min
. Centrifuge @ 8000 rpm at 10°C for 10 min.
6. Transfer supernatant to clean tube.
7. Add 700 uL of 3M NaOAC (pH 5.0) and 5mL cold isopropanol. Mix well. 8. Hook DNA and wash in 70% EtOH.
9. Repeat 70% wash.
10. Transfer pellet to 1.5mL tube and allow to dry.
11. Dissolve pellet in lmL lOmM Tris.
7 M Urea Extraction Buffer
Water 350mL
Urea 336g
5M NaCl 50mL (14.61g)
1M Tris 40mL (pH 8.0)
.5M EDTA 32mL (pH 8.0)
20% Sarcosine Sol. 40mL (8g)
Adjust volume to 800 mL with ddH20
Table 14. PCR Reaction Mix for SNP Discovery
Figure imgf000115_0001
Table 15. PCR Setup for SNP Discovery
Dipper Setup
PCR conditions Temp Time #Cycles initial denature 94 C 3 min IX denature 94 C 45 sec
anneal 65 C 60 sec 35X extension 72 C 75 sec
final extension 72 C 5 min IX end
Table 16. Protocol for PCR clean up
Figure imgf000115_0002
Table 17. Initial recombinants identified from GEID1653063 x GEID3495695 mapping population that delimited herbicide tolerance QTL to interval between S04867-1-A and S03859-1-A
Figure imgf000116_0001
SJ22186074 H A SUS 1 L border
SJ22186906 H A SUS 1 L border
SJ22185984 H A SUS 1 L border
Table 18. Initial recombinants identified from GEID4520632 x GEID7589905 mapping population that delimited herbicide tolerance QTL to interval between
S04867-1-A and S03859-1-A
Figure imgf000117_0001
SP21669584 A H SEG 5 L border
SP21669694 A H SEG 5.666667 L border
SP21669763 A H SEG 5 L border
SP21669533 A H SEG 5 L border
SP21669417 A H SEG 6.333333 L border
SP21669647 A H SEG? 7.666667 L border
SP216696 1 A H SEG? 7.666667 L border
SP21669541 H B SEG? 7.666667 border
SP21669749 H A SEG 5 R border
SP21669356 H A SEG 5 R border
SP21669674 H A SEG? 3.666667 R border
In an initial analysis of the GEID1653063 x GEID3495695 mapping population, four key recombinants were identified which served to further fine-map the herbicide tolerance QTL interval (Table 20). A recombination breakpoint at S08110-1-Ql in line SJ22186052 set the left border, while breakpoints at S08105-1 -Q1 in SJ22186019, SJ22186894, and SJ22185941 set the right border. These recombinants delimit the herbicide tolerance QTL to an -70 kb interval. Initial analysis of the GEID4520632 x GEID7589905 mapping population identified eight key recombinants (Table 13). A recombination breakpoint in line SP21669503 at S08117-1-Ql set the left border, while breakpoints in SP21669249, SP21669332, SP21669615, SP21669616, SP21669670, SP21669458, and SP21669760 set the right border at S08010-1-Q1. These recombinants delimit the herbicide tolerance QTL to a -526 kb interval. However, when the data from these two mapping populations are combined into a single set, the herbicide tolerance QTL interval was delimited to a -56 kb interval between S08117-1-Ql and S08105-1 -Q1 (Table 19 and Table 20).
To facilitate higher resolution mapping of the herbicide tolerance QTL interval, lines from the initial set of 86 recombinants were re-scored for herbicide tolerance to confirm their phenotype. Moreover, new markers were developed and used to genotype these recombinants. Consequently, this further analysis resulted in the identification of a key recombinant (SP21669417) from the GEID4520632 x GEID7589905 mapping population which set the left border of the herbicide tolerance QTL interval at S08113-1 - Ql (Table 13). In summary, key recombinants from the two mapping populations, scored in two fine-mapping experiments, define the herbicide tolerance QTL to a -44 kb interval between S081 13-1-Ql and S08105-1-Q1 (Table 19 and Table 20). Table 19. Summary of SNP markers used for initial QTL mapping and fine-mapping of herbicide tolerance QTL. Combined data from the two o ulations delimits the TL to a -44 kb interval between S08113-1- 1 and S8105-1- 1
Figure imgf000119_0001
Figure imgf000120_0001
* Population A = GEID1653063/GEID3495695; Population B = GEID4520632/GEID7589905
Tables 20A - 20H. Fine-mapping of the herbicide tolerance QTL interval with recombinants from the GEID1653063 x GEID3495695 population. Key recombinants delimit the QTL to the -70 kb interval between S08110-1-Q1 and S08105-1-Q1.
Table 20A.
Figure imgf000121_0001
Table 20B.
Figure imgf000122_0001
Table 20C.
Figure imgf000123_0001
Table 20D.
Figure imgf000124_0001
Table 20E.
Figure imgf000125_0001
Table 20F.
Figure imgf000126_0001
Table 20G.
Figure imgf000127_0001
Table 20H.
Figure imgf000128_0001
Tables 201— 20L. Fine-mapping of the herbicide tolerance QTL interval with recombinants from the GEID4520632 x GEID7589905 population
Table 201.
Figure imgf000129_0001
Table 20J.
Figure imgf000130_0001
Table 20K.
Figure imgf000131_0001
Figure imgf000132_0001
Example 8: SNP haplotype association analysis
Association analysis of SNP haplotypes across the herbicide tolerance QTL region provides an independent method of validating the herbicide tolerance interval. From the panel of susceptible and tolerant lines used to identify SNPs for Taqman® probe development, 235 SNPs from 49 amplicons were identified in the vicinity of the closely linked marker S03859-1 -A. The resulting SNP haplotype data was analyzed to identify an interval in which all of the haplotypes from the susceptible and tolerant lines co-segregated with each other (Table 21). Table 21. SNP haplotype association analysis of the herbicide tolerance QTL interval. Perfect association between haplotype and phenotype between amplicons 1563kl and
1618k2 defines the QTL interval
Figure imgf000133_0001
Although it is difficult to definitively define the co-segregating region, it can conservatively be estimated to reside between amplicons PPO Gml9_1563kl and PPO Gml9_1618k2-1. Within the borders defined by these loci, there are 38 SNP differences that are shared between all of the susceptible lines as compared to all the tolerant lines. This interval overlaps with the ~44 kb QTL interval identified by fine- mapping.
Example 9: QTL Analysis
The F2 population derived from GEID1653063 x GEID6461257 consisting of 251 progeny and segregating for the herbicide tolerance trait was used for mapping. The trait has been previously mapped to LG-L. The population was screened with a total of 15 polymorphic markers from LG-L (Ch 19). Five of these 19 markers showed severe segregation distortion and were excluded in the mapping analysis. A significant QTL for herbicide tolerance was detected on the LG-L (LRS=364) which was closely linked with the PPO production marker S08101-2-Q1 and flanked by markers S04867-1-A (7.81 cM) and S03859-1-A (10.00 cM). The QTL explained around 76% of phenotypic variation.
Material and Methods
Population: An F2 mapping population GErD1653063/GEID6461257 consisting of 251 F2 progeny was used. DNA extraction of the tissue was prepared using a citrate extraction protocol and quantified using the GW DNA quantification protocol.
Phenotype: The herbicide tolerance phenotypes were for each line were evaluated using chi-square analysis to establish a goodness to fit to the expected 1:2:1 genetic segregation ratio. The goodness of fit test indicated that the phenotypic data for the 2 1 progeny follows the expected 1:2:1 genetic ratio (p-value = 0.769).
Genotype: PolyM was used to identify polymorphic markers between the two parents. A total of 15 polymorphic markers from LG-L were assayed. Allele nomenclature used were maternal alleles were assigned "A" and paternal alleles "B", and heterozygous "H". The 10 of 15 markers were linked together on LG-L with 5 markers showing severe segregation distortion in the population. The 5 markers showing severe segregation distortion were excluded for mapping analysis.
Linkage Analysis: Map Manager QTX.b20 (Manly et al. (2001) Mammalian Genome 12:930-932) was used to construct the linkage map and perform the QTL analysis. A 1000 permutation test was used to establish the threshold for statistical significance (LOD ratio statistic - LRS) to declare a putative QTL.
Map Manager parameters were set to:
1) Linkage Evaluation: Intercross
2) Search Criteria: P = le-5
3) Map Function: Kosambi
4) Cross Type: Line Cross
QTL Analysis
Permutation Test: The thresholds at, 0.01 and 0.05 level based on a 1000 permutation test for herbicide tolerance trait are 7.0 and 17.3, respectively. The marker regression analysis showed that the QTL associated with herbicide tolerance could locate on the LG- L.
Interval mapping showed a highly significant region on LG L (LRS =346). The QTL was closely linked with marker S08101 -2-Q1 and flanked by markers S04867-1-A (7.81cM) and S03859-1-A (lO.OOcM). This region was estimated to explain -76% of the phenotypic variation.
It will be apparent to those of skill in the art that it is not intended that the invention be limited by such illustrative embodiments or mechanisms, and that modifications can be made without departing from the scope or spirit of the invention, as defined by the appended claims. It is intended that all such obvious modifications and variations be included within the scope of the present invention as defined in the appended claims. The claims are meant to cover the claimed components and steps in any sequence which is effective to meet the objectives there intended, unless the context specifically indicates to the contrary.
All publications referred to herein are incorporated by reference herein for the purpose cited to the same extent as if each was specifically and individually indicated to be incorporated by reference herein.

Claims

What is claimed is:
1. A method of selecting a plant or germplasm with tolerance, improved tolerance, susceptibility, or increased susceptibility to one or more herbicides selected from the group consisting of a mesotrione herbicide and an isoxazole herbicide, the method comprising: a) detecting in a plant or germplasm at least one allele of one or more marker loci that are associated with the tolerance, improved tolerance, susceptibility, or increased susceptibility to the one or more herbicides, wherein the one or more marker loci localize within a chromosome interval flanked by and including SATT613 and above on linkage group L; and
b) selecting the plant or germplasm comprising the at least one allele of the one or more marker loci, thereby selecting a plant with tolerance, improved tolerance, susceptibility, or increased susceptibility to the one or more herbicides.
2. The method of claim 1, wherein the one or more marker loci localize within a chromosomal interval flanked by and including markers SATT495 and SATT613 on linkage group L.
3. The method of claim 1, wherein the one or more marker loci localize within a chromosomal interval flanked by and including SATT313 and above on linkage group L.
4. The method of claim 1, wherein the one or more marker loci localize within a chromosomal interval flanked by and including markers SATT495 and SATT3 I3 on linkage group L.
5. The method of claim 1, wherein the one or more marker loci localize within a chromosomal interval flanked by and including markers SATT495 and SATT388 on linkage group L.
6. The method of claim 1, wherein the one or more marker loci localize within a chromosomal interval flanked by and including markers P10649C-3 and SATT182 on linkage group L.
7. The method of claim 1, wherein the one or more marker loci localize within a chromosomal interval flanked by and including markers S04867-1-A and S03859-1-A on linkage group L.
8. The method of claim 1, wherein the one or more marker loci localize within a chromosomal interval flanked by and including markers S08110-1-Ql and S08010-1-Q1 on linkage group L.
9. The method of claim 1, wherein the one or more marker loci localize within a chromosomal interval flanked by and including markers S08117-1-Q1 and S08010-1-Q1 on linkage group L.
10. The method of claim 1, wherein the one or more marker loci localize within a chromosomal interval flanked by and including markers S08110-1-Q1 and S08105-1-Q1 on linkage group L.
11. The method of claim 1, wherein the one or more marker loci localize within a chromosomal interval flanked by and including markers S08117-1-Q1 and S08105-1-Q1 on linkage group L.
12. The method of claim 1, wherein the one or more marker loci localize within a chromosomal interval flanked by and including markers S08113-1-Ql and S08105-1-Q1 on linkage group L.
13. The method of claim 1, wherein the one or more marker loci are selected from the group consisting of SATT495, SATT723, Sat_408, A169_l, EV2_1, Sle3 4s, BLT010_2, BLT007^1, SATT232, S04867-1-A, S08102-1-Q1, S08103-1-Q1, S08104-1-Q1, S08106- 1-Ql, S08107-1-Q1, S08109-1-Q1, S08110-1-Q1, S08111-1-Q1, S08115-2-Q1, S08117-1- Ql, S08119-1-Q1, S08116-1-Q1, S08112-1-Q1, S08108-1-Q1, S08101-4-Q1, S0810M- Ql, S08101-2-Q1, S08101-3-Q1, S08U8-1-Q1, S08114-1-Q1, S08113-1-Q1, S03859-1-A, Sat_301, SATT446, P10649C-3, SATT232, S08105-1-Q1, S ATT 182, S08010-1-Q1, S08010-2-Q1, R176 1, JUBC090, SATT238, Sat_071, BLT039 1, Bng071_l, SATT388, A264_l, RGA 7, RGA7, SATT523, Sat_134, S00224-1, S01659-1, LbA, i8_2, A450_2, A106_l, Sat_405, SATT143, B124_2, A459_l, SATT398, SATT694, Sat_195, Sat_388, SATT652, SATT711, Sat 187, SATT418, SATT278, Sat_397, Sat 191, Sat 320, O109_l, A204_2, SATT497, G214 17, SATT313, B164_l, G214_16, SATT613, A023_l, SATT284, AW508247, SATT462, L050_7, E014_l, A071_5, B046_l, LI, and B162_2.
14. The method of claim 1, wherein the selection occurs as part of further breeding to improve a soybean variety's tolerance to one or more mesotrione herbicides or one or more isoxazole herbicides.
15. The method of claim 14, wherein the further breeding comprises crosses with other lines, crosses with hybrids, backcrossing, self-crossing, or combinations thereof.
16. A plant selected by the method of claim 1.
17. A kit for selecting at least one soybean plant by marker assisted selection of one or more quantitative trait loci associated with tolerance, improved tolerance, susceptibility, or increased susceptibility to one or more herbicides selected from the group consisting of a mesotrione herbicide and an isoxazole herbicide, the kit comprising:
a) primers or probes for detecting one or more marker loci associated with the one or more quantitative trait loci, wherein the one or more marker loci are selected from the group consisting of SATT495, SATT723, Sat_408, A169J , EV2_1, Sle3 4s, BLT010_2, BLT007_1, SATT232, S04867-1-A, S08102-1-Q1, S08103-1-Q1, S08104-1-Q1, S08106-1-Q1, S08107-1-Q1, S08109-1-Q1, S08110-1-Q1, S08111-1-Q1, S08115-2-Q1, S08117-1-Q1, S08119-1-Q1, S08116-1-Q1, S08112-1-Q1, S08108-1-Q1, S08101-4-Q1, S08101-1-Q1, S08101-2-Q1, S08101-3-Q1, S08118-1-Q1, S081 14-1-Ql, S08113-1-Q1, S03859-1-A, SatJOl, SATT446, P10649C-3, SATT232, S081O5-1-Q1, SATT182, S08010-1-Q1, S08010-2-Q1, R176_l, JUBC090, SATT238, Sat_071, BLT039_1, Bng071 1, SATT388, A264_l, RGA_7, RGA7, SATT523, Sat_134, S00224-1, S01659-1, LbA, i8_2, A450_2, A106J, Sat 405, SATT143, B124_2, A459_l, SATT3 8, SATT694, Sat_195, Sat_388, SATT652, SATT711, Sat_187, SATT418, SATT278, Sat_397, Sat_1913 Sat_320, O109_l, A204_2, SATT497, G214 17, SATT313, B164_l, G214J6, SATT613, A023 1, SATT284, AW508247, SATT462, L050_7, E014_l, A071_5, B046 1, LI, and B162_2; and
b) instructions for using the primers or probes for detecting the one or more
marker loci and correlating the detected marker loci with predicted tolerance, improved tolerance, susceptibility, or increased susceptibility to the one or more herbicides.
18. A method of screening a soybean to determine the presence or absence of a quantitative trait locus associated with tolerance to one or more herbicides selected from the group consisting of a mesotrione herbicide and an isoxazole herbicide, the method comprising:
a) screening the soybean for a nucleic acid that localizes within an interval flanked and including a marker pair selected from the group consisting of:
i) SATT495 and SATT613,
ϋ) SATT495 and SATT313,
iii) SATT495 and SATT388, and
iv) P10649C-3 and S ATT 182,
v) S04867-1-A and S03859-1-A,
vi) S08110-1-Q1 and S08010-1-Q1,
vii) S08117-1-Q1 and S08010-1-Q1,
viii) S08110-1-Q1 and S08105-1-Q1,
ix) S08117-1-Q1 and S08105-1-Q1, and
S08113-1-Q1 and S08105-1-Q1; and
b) determining whether the soybean comprises said nucleic acid, thereby
determining the presence or absence of a quantitative trait locus associated with tolerance to the one or more herbicides.
1 . A method for selectively controlling weeds in a field containing a soybean crop, the method comprising: (a) planting a field with crop seeds or plants which are tolerant to one or more herbicides selected from the group consisting of a mesotrione herbicide and an isoxazole herbicide, said tolerance resulting from the presence of a tolerance allele in a marker locus within a chromosomal interval flanked by and including SATT613 and above on linkage group L; and
(b) applying a sufficient amount of the one or more herbicides to control the weeds without significantly affecting the crop.
20. The method of claim 19, wherein the marker locus is within a chromosomal interval flanked by and including markers SATT495 and SATT613 on linkage group L.
21. The method of claim 19, wherein the marker locus is within a chromosomal interval flanked by and including SATT313 and above on linkage group L.
22. The method of claim 19, wherein the marker locus is within a chromosomal interval flanked by and including markers SATT495 and SATT313 on linkage group L.
23. The method of claim 19, wherein the marker locus is within a chromosomal interval flanked by and including markers SATT495 and SATT388 on linkage group L.
24. The method of claim 19, wherein the marker locus is within a chromosomal interval flanked by and including markers P10649C-3 and SATT182 on linkage group L.
25. The method of claim 19, wherein the marker locus is within a chromosomal interval flanked by and including markers S04867-1-A and S03859-1-A on linkage group L.
26. The method of claim 19, wherein the marker locus is within a chromosomal interval flanked by and including markers S08110-1-Q1 and S08010-1-Q1 on linkage group L.
27. The method of claim 19, wherein the marker locus is within a chromosomal interval flanked by and including markers S08U7-1-Q1 and S08010-1-Q1 on linkage group L.
28. The method of claim 19, wherein the marker locus is within a chromosomal interval flanked by and including markers S08110-1-Ql and S08105-1-Q1 on linkage group L.
29. The method of claim 19, wherein the marker locus is within a chromosomal interval flanked by and including markers S08117-1-Ql and S081O5-1-Q1 on linkage group L.
30. The method of claim 19, wherein the marker locus is within a chromosomal interval flanked by and including markers S08113-1-Ql and S08105-1-Q1 on linkage group L.
31. The method of claim 19, wherein the one or more herbicide is applied as a pre- emergent herbicide.
32. The method of claim 19, further comprising applying to the crop and weeds in the field a simultaneous or chronologically staggered application of the one or more herbicides and an additional herbicide formulation.
33. The method of claim 32, wherein the additional herbicide formulation comprises an active ingredient selected from the group consisting of a
hydroxyphenylpyruvatedioxygenase inhibitor, a glyphosate, a sulfonylurea, a sulfonamide, an imidazolinone, a bialaphos, a phosphinothricm, a mesotrione, an isoxazole, an azafenidin, a butafenacil, a sulfosate, a glufosinate, a dicamba, and a protox inhibitor.
34. The method of claim 32, wherein the crop seeds or plants further comprise tolerance to the active ingredient of the additional herbicide formulation.
35. The method of claim 34, wherein the tolerance to the active ingredient of the additional herbicide formulation is provided by insertion of a transgene which confers the tolerance.
36. A method for selectively screening soybean plants for tolerance to one or more herbicides selected from the group consisting of a mesotrione herbicide and an isoxazole herbicide, the method comprising: (a) planting soybean seeds or plants comprising a marker locus within a chromosomal interval flanked by and including SATT613 and above on linkage group L;
(b) treating the plants by applying a sufficient amount of the one or more herbicides to differentiate between susceptible and tolerant plants; and
(c) scoring the treated plants for tolerance to the herbicide.
37. The method of claim 36, wherein the marker locus is within a chromosome interval flanked by and including markers SATT495 and SATT613 on linkage group L.
38. The method of claim 36, wherein the marker locus is within a chromosome interval flanked by and including SATT313 and above on linkage group L.
39. The method of claim 36, wherein the marker locus is within a chromosome interval flanked by and including markers SATT495 and SATT313 on linkage group L.
40. The method of claim 36, wherein the marker locus is within a chromosome interval flanked by and including markers SATT495 and SATT388 on linkage group L.
41. The method of claim 36, wherein the marker locus is within a chromosome interval flanked by and including markers P10649C-3 and SATT182 on linkage group L.
42. The method of claim 36, wherein the marker locus is within a chromosomal interval flanked by and including markers S04867-1-A and S03859-1-A on linkage group L.
43. The method of claim 36, wherein the marker locus is within a chromosomal interval flanked by and including markers S08110-1-Ql and S08010-1-Q1 on linkage group L.
44. The method of claim 36, wherein the marker locus is within a chromosomal interval flanked by and including markers S08117-1-Q1 and S08010-1-Q1 on linkage group L.
45. The method of claim 36, wherein the marker locus is within a chromosomal interval flanked by and including markers S08110-1-Q1 and SO8105-1-Q1 on linkage group L.
46. The method of claim 36, wherein the marker locus is within a chromosomal interval flanked by and including markers S08117-1-Ql and S08105-1-Q1 on linkage group L.
47. The method of claim 36, wherein the marker locus is within a chromosomal interval flanked by and including markers S08113-1-Ql and S08105-1-Q1 on linkage group L.
PCT/US2011/022395 2010-01-26 2011-01-25 Hppd-inhibitor herbicide tolerance WO2011094205A1 (en)

Priority Applications (3)

Application Number Priority Date Filing Date Title
CA2788198A CA2788198C (en) 2010-01-26 2011-01-25 Hppd-inhibitor herbicide tolerance
BR112012018618A BR112012018618A2 (en) 2010-01-26 2011-01-25 method for selecting a plant or germplasm having tolerance, increased tolerance, susceptibility, or increased susceptibility to one or more herbicides, plant, kit for selecting at least one soybean plant, screening method for a soybean, method for controlling so selective weed, method for selectively screening soybean plants for tolerance to one or more herbicides
EP11701602A EP2529027A1 (en) 2010-01-26 2011-01-25 Hppd-inhibitor herbicide tolerance

Applications Claiming Priority (8)

Application Number Priority Date Filing Date Title
US29852310P 2010-01-26 2010-01-26
US29852810P 2010-01-26 2010-01-26
US61/298,528 2010-01-26
US61/298,523 2010-01-26
US37145410P 2010-08-06 2010-08-06
US37139210P 2010-08-06 2010-08-06
US61/371,392 2010-08-06
US61/371,454 2010-08-06

Publications (1)

Publication Number Publication Date
WO2011094205A1 true WO2011094205A1 (en) 2011-08-04

Family

ID=43629267

Family Applications (2)

Application Number Title Priority Date Filing Date
PCT/US2011/022378 WO2011094199A1 (en) 2010-01-26 2011-01-25 Polynucleotide and polypeptide sequences associated with herbicide tolerance
PCT/US2011/022395 WO2011094205A1 (en) 2010-01-26 2011-01-25 Hppd-inhibitor herbicide tolerance

Family Applications Before (1)

Application Number Title Priority Date Filing Date
PCT/US2011/022378 WO2011094199A1 (en) 2010-01-26 2011-01-25 Polynucleotide and polypeptide sequences associated with herbicide tolerance

Country Status (6)

Country Link
US (4) US9970021B2 (en)
EP (2) EP2529028A1 (en)
AR (2) AR080021A1 (en)
BR (2) BR112012018618A2 (en)
CA (2) CA2788198C (en)
WO (2) WO2011094199A1 (en)

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014138339A2 (en) 2013-03-07 2014-09-12 Athenix Corp. Toxin genes and methods for their use
WO2018098214A1 (en) 2016-11-23 2018-05-31 Bayer Cropscience Lp Axmi669 and axmi991 toxin genes and methods for their use
WO2018119336A1 (en) 2016-12-22 2018-06-28 Athenix Corp. Use of cry14 for the control of nematode pests
WO2018136611A1 (en) 2017-01-18 2018-07-26 Bayer Cropscience Lp Use of bp005 for the control of plant pathogens
WO2018136604A1 (en) 2017-01-18 2018-07-26 Bayer Cropscience Lp Bp005 toxin gene and methods for its use
WO2023218475A1 (en) * 2022-05-11 2023-11-16 Rallis India Limited Maize snp markers for hppd-inhibitor resistance
WO2024137438A2 (en) 2022-12-19 2024-06-27 BASF Agricultural Solutions Seed US LLC Insect toxin genes and methods for their use

Families Citing this family (57)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8097712B2 (en) 2007-11-07 2012-01-17 Beelogics Inc. Compositions for conferring tolerance to viral disease in social insects, and the use thereof
US8697941B2 (en) * 2008-07-23 2014-04-15 Pioneer Hi-Bred International, Inc. Molecular markers linked to PPO inhibitor tolerance in soybeans
US8962584B2 (en) 2009-10-14 2015-02-24 Yissum Research Development Company Of The Hebrew University Of Jerusalem, Ltd. Compositions for controlling Varroa mites in bees
WO2011112570A1 (en) 2010-03-08 2011-09-15 Monsanto Technology Llc Polynucleotide molecules for gene regulation in plants
US10829828B2 (en) 2011-09-13 2020-11-10 Monsanto Technology Llc Methods and compositions for weed control
EP3434780A1 (en) 2011-09-13 2019-01-30 Monsanto Technology LLC Methods and compositions for weed control
UA116089C2 (en) 2011-09-13 2018-02-12 Монсанто Текнолоджи Ллс Methods and compositios for weed control
CN104160028A (en) 2011-09-13 2014-11-19 孟山都技术公司 Methods and compositions for weed control
US10806146B2 (en) 2011-09-13 2020-10-20 Monsanto Technology Llc Methods and compositions for weed control
US10760086B2 (en) 2011-09-13 2020-09-01 Monsanto Technology Llc Methods and compositions for weed control
EP2755467B1 (en) 2011-09-13 2017-07-19 Monsanto Technology LLC Methods and compositions for weed control
US9840715B1 (en) 2011-09-13 2017-12-12 Monsanto Technology Llc Methods and compositions for delaying senescence and improving disease tolerance and yield in plants
US9920326B1 (en) 2011-09-14 2018-03-20 Monsanto Technology Llc Methods and compositions for increasing invertase activity in plants
US20130254944A1 (en) * 2012-03-21 2013-09-26 E.I. Du Pont De Nemours And Company Herbicide Tolerant Soybeans and Methods of Use
MX360866B (en) 2012-05-24 2018-11-09 A B Seeds Ltd Compositions and methods for silencing gene expression.
WO2014036299A2 (en) * 2012-08-30 2014-03-06 Monsanto Technology Llc Synergistic herbicidal combinations and methods of use
AR092564A1 (en) 2012-09-14 2015-04-22 Bayer Cropscience Lp VARIANTS OF THE ENZYME 4-HYDROXYPHENYL PIRUVATO DEOXIGENASA (HPPD) AND METHODS OF USE TO CONFER TOLERANCE TO HERBICIDES IN PLANTS
CN104870647A (en) 2012-10-18 2015-08-26 孟山都技术公司 Methods and compositions for plant pest control
US10683505B2 (en) 2013-01-01 2020-06-16 Monsanto Technology Llc Methods of introducing dsRNA to plant seeds for modulating gene expression
UY35251A (en) 2013-01-01 2014-07-31 Seeds Ltd Ab ISOLATED DsRNA MOLECULES AND METHODS TO USE THEM TO SILENCE DIANA OF INTEREST MOLECULES
US10000767B2 (en) 2013-01-28 2018-06-19 Monsanto Technology Llc Methods and compositions for plant pest control
WO2014164797A2 (en) 2013-03-13 2014-10-09 Monsanto Technology Llc Methods and compositions for weed control
UY35379A (en) 2013-03-13 2014-09-30 Monsanto Technology Llc ? METHODS AND COMPOSITIONS FOR WEED CONTROL ?.
US20140283211A1 (en) 2013-03-14 2014-09-18 Monsanto Technology Llc Methods and Compositions for Plant Pest Control
US10568328B2 (en) 2013-03-15 2020-02-25 Monsanto Technology Llc Methods and compositions for weed control
US9850496B2 (en) 2013-07-19 2017-12-26 Monsanto Technology Llc Compositions and methods for controlling Leptinotarsa
MX359191B (en) 2013-07-19 2018-09-18 Monsanto Technology Llc Compositions and methods for controlling leptinotarsa.
US20160272997A1 (en) 2013-10-25 2016-09-22 Pioneer Hi-Bred International, Inc. Stem canker tolerant soybeans and methods of use
CA2928855A1 (en) 2013-11-04 2015-05-07 Dow Agrosciences Llc Optimal maize loci
UA120426C2 (en) 2013-11-04 2019-12-10 Монсанто Текнолоджі Елелсі Compositions and methods for controlling arthropod parasite and pest infestations
KR102269769B1 (en) 2013-11-04 2021-06-28 코르테바 애그리사이언스 엘엘씨 Optimal maize loci
NZ746567A (en) 2013-11-04 2019-09-27 Dow Agrosciences Llc Optimal soybean loci
UA119253C2 (en) 2013-12-10 2019-05-27 Біолоджикс, Інк. Compositions and methods for virus control in varroa mite and bees
EP3116303B1 (en) 2014-01-15 2020-07-22 Monsanto Technology LLC Methods and compositions for weed control using epsps polynucleotides
BR112016020889B1 (en) 2014-03-11 2022-10-04 BASF Agricultural Solutions Seed US LLC RECOMBINANT NUCLEIC ACID MOLECULE, BACTERIAL HOST CELL, RECOMBINANT HPPD PROTEIN, RECOMBINANT NUCLEIC ACID USE AND BASE PRODUCT
MX360999B (en) 2014-03-11 2018-11-23 Bayer Cropscience Ag Hppd variants and methods of use.
WO2015153339A2 (en) 2014-04-01 2015-10-08 Monsanto Technology Llc Compositions and methods for controlling insect pests
CA2953347A1 (en) 2014-06-23 2015-12-30 Monsanto Technology Llc Compositions and methods for regulating gene expression via rna interference
EP3161138A4 (en) 2014-06-25 2017-12-06 Monsanto Technology LLC Methods and compositions for delivering nucleic acids to plant cells and regulating gene expression
CN114009454A (en) 2014-07-29 2022-02-08 孟山都技术公司 Compositions and methods for controlling insect pests
PL3256589T3 (en) 2015-01-22 2022-02-21 Monsanto Technology Llc Compositions and methods for controlling leptinotarsa
AU2016270870A1 (en) 2015-06-02 2018-01-04 Monsanto Technology Llc Compositions and methods for delivery of a polynucleotide into a plant
AU2016270913A1 (en) 2015-06-03 2018-01-04 Monsanto Technology Llc Methods and compositions for introducing nucleic acids into plants
KR20180043838A (en) 2015-09-11 2018-04-30 바이엘 크롭사이언스 악티엔게젤샤프트 HPPD variants and methods of use
US11096344B2 (en) 2016-02-05 2021-08-24 Pioneer Hi-Bred International, Inc. Genetic loci associated with brown stem rot resistance in soybean and methods of use
US11624076B2 (en) 2016-04-21 2023-04-11 BASF Agricultural Solutions Seed US LLC TAL-effector mediated herbicide tolerance
US10667478B1 (en) 2017-02-16 2020-06-02 Syngenta Participations Ag Metribuzin tolerance alleles in soybean
BR112019018059A2 (en) 2017-03-07 2020-08-04 BASF Agricultural Solutions Seed US LLC recombinant nucleic acid molecule, host cell, plants, transgenic seeds, recombinant polypeptide, method for producing a polypeptide, weed control method, use of nucleic acid and utility product
CN107130029A (en) * 2017-05-17 2017-09-05 江苏农林职业技术学院 A kind of fig germ plasm resource relationship authentication method based on SSR marker
WO2019083808A1 (en) 2017-10-24 2019-05-02 Basf Se Improvement of herbicide tolerance to hppd inhibitors by down-regulation of putative 4-hydroxyphenylpyruvate reductases in soybean
BR112020008092A2 (en) 2017-10-24 2020-09-15 BASF Agricultural Solutions Seed US LLC method for checking tolerance to a GM herbicide and soy plant
CN107988424B (en) * 2018-01-29 2021-03-09 吉林省农业科学院 Molecular marker, interval, primer and application related to methionine content of soybean seeds
CN108060260B (en) * 2018-01-29 2021-03-09 吉林省农业科学院 SNP (Single nucleotide polymorphism) marker related to methionine content of soybean seeds, interval, primer and application
CN114480697B (en) * 2020-10-23 2023-10-03 广西壮族自治区亚热带作物研究所（广西亚热带农产品加工研究所） Tea tree glutamine synthetase gene SSR molecular marker primer and application
US11818977B2 (en) * 2020-11-30 2023-11-21 Deere & Company System and method for intercrop planting
CN113430299B (en) * 2021-08-13 2022-08-26 山西农业大学经济作物研究所 SNP molecular marker associated with castor stem color and application thereof
IL292199B2 (en) * 2022-04-12 2024-02-01 Plantarc Bio Ltd Method for optimizing gene expression levels in plants

Citations (112)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US542901A (en) 1895-07-16 Cabinet
US4407956A (en) 1981-03-13 1983-10-04 The Regents Of The University Of California Cloned cauliflower mosaic virus DNA as a plant vehicle
WO1985001856A1 (en) 1983-11-03 1985-05-09 Johannes Martenis Jacob De Wet Method for the transfer of exogenous genes in plants using pollen as a vector
US4535060A (en) 1983-01-05 1985-08-13 Calgene, Inc. Inhibition resistant 5-enolpyruvyl-3-phosphoshikimate synthetase, production and use
WO1988002405A1 (en) 1986-10-01 1988-04-07 The Plant Cell Research Institute, Inc. Genetic transformation and controlled regeneration of cucumis sp. plants in vitro
US4761373A (en) 1984-03-06 1988-08-02 Molecular Genetics, Inc. Herbicide resistance in plants
US4873192A (en) 1987-02-17 1989-10-10 The United States Of America As Represented By The Department Of Health And Human Services Process for site specific mutagenesis without phenotypic selection
US4940835A (en) 1985-10-29 1990-07-10 Monsanto Company Glyphosate-resistant plants
US4945050A (en) 1984-11-13 1990-07-31 Cornell Research Foundation, Inc. Method for transporting substances into living cells and tissues and apparatus therefor
US4971908A (en) 1987-05-26 1990-11-20 Monsanto Company Glyphosate-tolerant 5-enolpyruvyl-3-phosphoshikimate synthase
US5013659A (en) 1987-07-27 1991-05-07 E. I. Du Pont De Nemours And Company Nucleic acid fragment encoding herbicide resistant plant acetolactate synthase
US5023179A (en) 1988-11-14 1991-06-11 Eric Lam Promoter enhancer element for gene expression in plant roots
US5110732A (en) 1989-03-14 1992-05-05 The Rockefeller University Selective gene expression in plants
US5145783A (en) 1987-05-26 1992-09-08 Monsanto Company Glyphosate-tolerant 5-endolpyruvyl-3-phosphoshikimate synthase
US5188642A (en) 1985-08-07 1993-02-23 Monsanto Company Glyphosate-resistant plants
US5240855A (en) 1989-05-12 1993-08-31 Pioneer Hi-Bred International, Inc. Particle gun
US5268463A (en) 1986-11-11 1993-12-07 Jefferson Richard A Plant promoter α-glucuronidase gene construct
US5304732A (en) 1984-03-06 1994-04-19 Mgi Pharma, Inc. Herbicide resistance in plants
US5310667A (en) 1989-07-17 1994-05-10 Monsanto Company Glyphosate-tolerant 5-enolpyruvyl-3-phosphoshikimate synthases
US5312910A (en) 1987-05-26 1994-05-17 Monsanto Company Glyphosate-tolerant 5-enolpyruvyl-3-phosphoshikimate synthase
WO1994011516A1 (en) 1992-11-17 1994-05-26 E.I. Du Pont De Nemours And Company Genes for microsomal delta-12 fatty acid desaturases and related enzymes from plants
US5322783A (en) 1989-10-17 1994-06-21 Pioneer Hi-Bred International, Inc. Soybean transformation by microparticle bombardment
US5324646A (en) 1992-01-06 1994-06-28 Pioneer Hi-Bred International, Inc. Methods of regeneration of Medicago sativa and expressing foreign DNA in same
US5331107A (en) 1984-03-06 1994-07-19 Mgi Pharma, Inc. Herbicide resistance in plants
US5341001A (en) 1992-02-13 1994-08-23 Matsushita Electric Industrial Co., Ltd. Sulfide-selenide manganese-zinc mixed crystal photo semiconductor and laser diode
US5366892A (en) 1991-01-16 1994-11-22 Mycogen Corporation Gene encoding a coleopteran-active toxin
US5378824A (en) 1986-08-26 1995-01-03 E. I. Du Pont De Nemours And Company Nucleic acid fragment encoding herbicide resistant plant acetolactate synthase
US5380831A (en) 1986-04-04 1995-01-10 Mycogen Plant Science, Inc. Synthetic insecticidal crystal protein gene
US5399680A (en) 1991-05-22 1995-03-21 The Salk Institute For Biological Studies Rice chitinase promoter
US5401836A (en) 1992-07-16 1995-03-28 Pioneer Hi-Bre International, Inc. Brassica regulatory sequence for root-specific or root-abundant gene expression
US5436391A (en) 1991-11-29 1995-07-25 Mitsubishi Corporation Synthetic insecticidal gene, plants of the genus oryza transformed with the gene, and production thereof
US5459252A (en) 1991-01-31 1995-10-17 North Carolina State University Root specific gene promoter
US5466785A (en) 1990-04-12 1995-11-14 Ciba-Geigy Corporation Tissue-preferential promoters
US5468613A (en) 1986-03-13 1995-11-21 Hoffmann-La Roche Inc. Process for detecting specific nucleotide variations and genetic polymorphisms present in nucleic acids
US5489520A (en) 1990-04-17 1996-02-06 Dekalb Genetics Corporation Process of producing fertile transgenic zea mays plants and progeny comprising a gene encoding phosphinothricin acetyl transferase
US5491288A (en) 1991-03-05 1996-02-13 Rhone Poulenc Agrochimie Chimeric gene comprising the arabidopsis histone H4 promoter for the transformation of plants
US5510471A (en) 1991-03-05 1996-04-23 Rhone-Poulenc Agrochimie Chimeric gene for the transformation of plants
US5561236A (en) 1986-03-11 1996-10-01 Plant Genetic Systems Genetically engineered plant cells and plants exhibiting resistance to glutamine synthetase inhibitors, DNA fragments and recombinants for use in the production of said cells and plants
US5563055A (en) 1992-07-27 1996-10-08 Pioneer Hi-Bred International, Inc. Method of Agrobacterium-mediated transformation of cultured soybean cells
US5565350A (en) 1993-12-09 1996-10-15 Thomas Jefferson University Compounds and methods for site directed mutations in eukaryotic cells
WO1996033270A1 (en) 1995-04-20 1996-10-24 American Cyanamid Company Structure-based designed herbicide resistant products
US5569597A (en) 1985-05-13 1996-10-29 Ciba Geigy Corp. Methods of inserting viral DNA into plant material
US5583210A (en) 1993-03-18 1996-12-10 Pioneer Hi-Bred International, Inc. Methods and compositions for controlling plant development
US5591616A (en) 1992-07-07 1997-01-07 Japan Tobacco, Inc. Method for transforming monocotyledons
US5593881A (en) 1994-05-06 1997-01-14 Mycogen Corporation Bacillus thuringiensis delta-endotoxin
WO1997004103A2 (en) 1995-07-19 1997-02-06 Rhone-Poulenc Agrochimie Mutated 5-enol pyruvylshikimate-3-phosphate synthase, gene coding for said protein and transformed plants containing said gene
US5602321A (en) 1992-11-20 1997-02-11 Monsanto Company Transgenic cotton plants producing heterologous polyhydroxy(e) butyrate bioplastic
US5604121A (en) 1991-08-27 1997-02-18 Agricultural Genetics Company Limited Proteins with insecticidal properties against homopteran insects and their use in plant protection
US5605011A (en) 1986-08-26 1997-02-25 E. I. Du Pont De Nemours And Company Nucleic acid fragment encoding herbicide resistant plant acetolactate synthase
US5608144A (en) 1994-08-12 1997-03-04 Dna Plant Technology Corp. Plant group 2 promoters and uses thereof
US5608142A (en) 1986-12-03 1997-03-04 Agracetus, Inc. Insecticidal cotton plants
US5608149A (en) 1990-06-18 1997-03-04 Monsanto Company Enhanced starch biosynthesis in tomatoes
US5627061A (en) 1990-08-31 1997-05-06 Monsanto Company Glyphosate-tolerant 5-enolpyruvylshikimate-3-phosphate synthases
US5633363A (en) 1994-06-03 1997-05-27 Iowa State University, Research Foundation In Root preferential promoter
US5659026A (en) 1995-03-24 1997-08-19 Pioneer Hi-Bred International ALS3 promoter
US5723756A (en) 1990-04-26 1998-03-03 Plant Genetic Systems, N.V. Bacillus thuringiensis strains and their genes encoding insecticidal toxins
US5731181A (en) 1996-06-17 1998-03-24 Thomas Jefferson University Chimeric mutational vectors having non-natural nucleotides
US5731180A (en) 1991-07-31 1998-03-24 American Cyanamid Company Imidazolinone resistant AHAS mutants
US5737514A (en) 1995-11-29 1998-04-07 Texas Micro, Inc. Remote checkpoint memory system and protocol for fault-tolerant computer system
US5736514A (en) 1994-10-14 1998-04-07 Nissan Chemical Industries, Ltd. Bacillus strain and harmful organism controlling agents
US5736369A (en) 1994-07-29 1998-04-07 Pioneer Hi-Bred International, Inc. Method for producing transgenic cereal plants
US5747450A (en) 1991-08-02 1998-05-05 Kubota Corporation Microorganism and insecticide
US5750386A (en) 1991-10-04 1998-05-12 North Carolina State University Pathogen-resistant transgenic plants
US5760012A (en) 1996-05-01 1998-06-02 Thomas Jefferson University Methods and compounds for curing diseases caused by mutations
US5767373A (en) 1994-06-16 1998-06-16 Novartis Finance Corporation Manipulation of protoporphyrinogen oxidase enzyme activity in eukaryotic organisms
US5789156A (en) 1993-06-14 1998-08-04 Basf Ag Tetracycline-regulated transcriptional inhibitors
US5792931A (en) 1994-08-12 1998-08-11 Pioneer Hi-Bred International, Inc. Fumonisin detoxification compositions and methods
US5814618A (en) 1993-06-14 1998-09-29 Basf Aktiengesellschaft Methods for regulating gene expression
WO1998049350A1 (en) 1997-04-30 1998-11-05 Regents Of The University Of Minnesota In vivo use of recombinagenic oligonucleobases to correct genetic lesions in hepatocytes
US5837876A (en) 1995-07-28 1998-11-17 North Carolina State University Root cortex specific gene promoter
US5866775A (en) 1990-09-28 1999-02-02 Monsanto Company Glyphosate-tolerant 5-enolpyruvyl-3-phosphoshikimate synthases
WO1999007865A1 (en) 1997-08-05 1999-02-18 Kimeragen, Inc. The use of mixed duplex oligonucleotides to effect localized genetic changes in plants
US5879903A (en) 1986-08-23 1999-03-09 Hoechst Aktiengesellschaft Phosphinothricin-resistance gene, and its use
US5879918A (en) 1989-05-12 1999-03-09 Pioneer Hi-Bred International, Inc. Pretreatment of microprojectiles prior to using in a particle gun
US5886244A (en) 1988-06-10 1999-03-23 Pioneer Hi-Bred International, Inc. Stable transformation of plant cells
WO1999023886A1 (en) 1997-11-12 1999-05-20 Aventis Cropscience S.A. Method of controlling weeds in transgenic crops
WO1999025853A1 (en) 1997-11-18 1999-05-27 Pioneer Hi-Bred International, Inc. Targeted manipulation of herbicide-resistance genes in plants
WO1999025855A1 (en) 1997-11-18 1999-05-27 Pioneer Hi-Bred International, Inc. Mobilization of viral genomes from t-dna using site-specific recombination systems
WO1999025821A1 (en) 1997-11-18 1999-05-27 Pioneer Hi-Bred International, Inc. Compositions and methods for genetic modification of plants
WO1999025840A1 (en) 1997-11-18 1999-05-27 Pioneer Hi-Bred International, Inc. A novel method for the integration of foreign dna into eukaryoticgenomes
US5925517A (en) 1993-11-12 1999-07-20 The Public Health Research Institute Of The City Of New York, Inc. Detectably labeled dual conformation oligonucleotide probes, assays and kits
US5928937A (en) 1995-04-20 1999-07-27 American Cyanamid Company Structure-based designed herbicide resistant products
US5932782A (en) 1990-11-14 1999-08-03 Pioneer Hi-Bred International, Inc. Plant transformation method using agrobacterium species adhered to microprojectiles
WO1999043838A1 (en) 1998-02-24 1999-09-02 Pioneer Hi-Bred International, Inc. Synthetic promoters
US5952544A (en) 1991-12-04 1999-09-14 E. I. Du Pont De Nemours And Company Fatty acid desaturase genes from plants
US5981840A (en) 1997-01-24 1999-11-09 Pioneer Hi-Bred International, Inc. Methods for agrobacterium-mediated transformation
US5981722A (en) 1995-11-20 1999-11-09 Board Of Regents For The University Of Oklahoma Trypsin inhibitors with insecticidal properties obtained from PENTACLETHRA MACROLOBA
WO1999061619A2 (en) 1998-05-22 1999-12-02 Pioneer Hi-Bred International, Inc. Cell cycle genes, proteins and uses thereof
USRE36449E (en) 1991-03-05 1999-12-14 Rhone-Poulenc Agro Chimeric gene for the transformation of plants
WO2000011177A1 (en) 1998-08-20 2000-03-02 Pioneer Hi-Bred International, Inc. Seed-preferred promoters
WO2000012733A1 (en) 1998-08-28 2000-03-09 Pioneer Hi-Bred International, Inc. Seed-preferred promoters from end genes
US6037130A (en) 1998-07-28 2000-03-14 The Public Health Institute Of The City Of New York, Inc. Wavelength-shifting probes and primers and their use in assays and kits
WO2000017364A2 (en) 1998-09-23 2000-03-30 Pioneer Hi-Bred International, Inc. Cyclin d polynucleotides, polypeptides and uses thereof
WO2000028058A2 (en) 1998-11-09 2000-05-18 Pioneer Hi-Bred International, Inc. Transcriptional activator lec1 nucleic acids, polypeptides and their uses
US6130366A (en) 1984-12-28 2000-10-10 Plant Genetic Systems Chimaeric gene coding for a transit peptide and a heterologous polypeptide
WO2000066746A1 (en) 1999-04-29 2000-11-09 Syngenta Limited Herbicide resistant plants
WO2000066747A1 (en) 1999-04-29 2000-11-09 Syngenta Limited Herbicide resistant plants
US6150097A (en) 1996-04-12 2000-11-21 The Public Health Research Institute Of The City Of New York, Inc. Nucleic acid detection probes having non-FRET fluorescence quenching and kits and assays including such probes
US6177611B1 (en) 1998-02-26 2001-01-23 Pioneer Hi-Bred International, Inc. Maize promoters
US6177616B1 (en) 1996-12-16 2001-01-23 Hoecsht Schering Agrevo Gmbh Genes coding for amino acid deacetylases with specificity for N-acetyl-L-phosphinothricin, their isolation and their use
WO2001012825A1 (en) 1999-08-13 2001-02-22 Syngenta Participations Ag Herbicide-tolerant protoporphyrinogen oxidase
US6232529B1 (en) 1996-11-20 2001-05-15 Pioneer Hi-Bred International, Inc. Methods of producing high-oil seed by modification of starch levels
US6245968B1 (en) 1997-11-07 2001-06-12 Aventis Cropscience S.A. Mutated hydroxyphenylpyruvate dioxygenase, DNA sequence and isolation of plants which contain such a gene and which are tolerant to herbicides
US6268549B1 (en) 1995-06-02 2001-07-31 Aventis Cropscience S.A. DNA sequence of a gene of hydroxy-phenyl pyruvate dioxygenase and production of plants containing a gene of hydroxy-phenyl pyruvate dioxygenase and which are tolerant to certain herbicides
WO2001066704A2 (en) 2000-03-09 2001-09-13 Monsanto Technology Llc Methods for making plants tolerant to glyphosate and compositions thereof
US20030226166A1 (en) 1998-12-21 2003-12-04 Falco Saverio Carl S-adenosyl-L-methionine synthetase promoter and its use in expression of transgenic genes in plants
WO2005012576A2 (en) * 2003-08-01 2005-02-10 Pioneer Hi-Bred International, Inc. Molecular markers for the selection of soybean plants having superior agronomic performance
WO2005107437A2 (en) 2004-04-30 2005-11-17 Dow Agrosciences Llc Novel herbicide resistance genes
WO2006017840A2 (en) * 2004-08-06 2006-02-16 Pioneer Hi-Bred International Inc. Genetic loci associated with iron deficiency tolerance in soybean
US20070061917A1 (en) 2005-08-24 2007-03-15 Pioneer Hi-Bred International, Inc. Methods and compositions for the expression of a polynucleotide of interest
WO2009144079A1 (en) * 2008-04-14 2009-12-03 Bayer Bioscience N.V. New mutated hydroxyphenylpyruvate dioxygenase, dna sequence and isolation of plants which are tolerant to hppd inhibitor herbicides
US20100024080A1 (en) * 2008-07-23 2010-01-28 Pioneer Hi-Bred International, Inc. Molecular markers linked to ppo inhibitor tolerance in soybeans

Family Cites Families (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US476137A (en) * 1892-05-31 John p
JPH05242636A (en) 1992-02-28 1993-09-21 Sony Corp Tape cassette
US5464024A (en) * 1994-07-05 1995-11-07 Standard Textile Co., Inc. Reusable surgical drape with fluid-retaining trough
US5750853A (en) * 1997-01-14 1998-05-12 Fuller; Peter Armstrong Soybean variety 93B41
US5852226A (en) * 1997-01-14 1998-12-22 Pioneer Hi-Bred International, Inc. Soybean variety 93B82
US6448472B1 (en) * 1999-02-05 2002-09-10 Board Of Regents, The University Of Texas System Genetic and epigenetic manipulation of ABC transporters and ectophosphatases for the conference of hormone and herbicide resistance
US20040031072A1 (en) * 1999-05-06 2004-02-12 La Rosa Thomas J. Soy nucleic acid molecules and other molecules associated with transcription plants and uses thereof for plant improvement
GB0118928D0 (en) * 2001-08-02 2001-09-26 Syngenta Participations Ag DNA molecules conferring tolerance to herbicidal compounds
WO2003031937A2 (en) * 2001-10-12 2003-04-17 Morphotek, Inc. Genetic hypermutability of plants for gene discovery and diagnosis
EP1394267A1 (en) * 2002-08-19 2004-03-03 Bayer HealthCare AG Single nucleotide polymorphisms predictive for cardiovascular disease, adverse drug reactions, and drug efficacy
AU2007299219A1 (en) * 2006-04-05 2008-03-27 Metanomics Gmbh Process for the production of a fine chemical
US9029636B2 (en) * 2008-02-05 2015-05-12 Monsanto Technology Llc Isolated novel nucleic acid and protein molecules from soy and methods of using those molecules to generate transgenic plants with enhanced agronomic traits
US8748695B2 (en) * 2008-07-23 2014-06-10 Pioneer Hi-Bred International, Inc. Molecular markers linked to PPO inhibitor tolerance in soybeans

Patent Citations (137)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US542901A (en) 1895-07-16 Cabinet
US4407956A (en) 1981-03-13 1983-10-04 The Regents Of The University Of California Cloned cauliflower mosaic virus DNA as a plant vehicle
US4535060A (en) 1983-01-05 1985-08-13 Calgene, Inc. Inhibition resistant 5-enolpyruvyl-3-phosphoshikimate synthetase, production and use
US4769061A (en) 1983-01-05 1988-09-06 Calgene Inc. Inhibition resistant 5-enolpyruvyl-3-phosphoshikimate synthase, production and use
WO1985001856A1 (en) 1983-11-03 1985-05-09 Johannes Martenis Jacob De Wet Method for the transfer of exogenous genes in plants using pollen as a vector
US4761373A (en) 1984-03-06 1988-08-02 Molecular Genetics, Inc. Herbicide resistance in plants
US5331107A (en) 1984-03-06 1994-07-19 Mgi Pharma, Inc. Herbicide resistance in plants
US5304732A (en) 1984-03-06 1994-04-19 Mgi Pharma, Inc. Herbicide resistance in plants
US4945050A (en) 1984-11-13 1990-07-31 Cornell Research Foundation, Inc. Method for transporting substances into living cells and tissues and apparatus therefor
US6130366A (en) 1984-12-28 2000-10-10 Plant Genetic Systems Chimaeric gene coding for a transit peptide and a heterologous polypeptide
US5569597A (en) 1985-05-13 1996-10-29 Ciba Geigy Corp. Methods of inserting viral DNA into plant material
US5188642A (en) 1985-08-07 1993-02-23 Monsanto Company Glyphosate-resistant plants
US4940835A (en) 1985-10-29 1990-07-10 Monsanto Company Glyphosate-resistant plants
US5646024A (en) 1986-03-11 1997-07-08 Plant Genetic Systems, N.V. Genetically engineered plant cells and plants exhibiting resistance to glutamine synthetase inhibitors, DNA fragments and recombinants for use in the production of said cells and plants
US5561236A (en) 1986-03-11 1996-10-01 Plant Genetic Systems Genetically engineered plant cells and plants exhibiting resistance to glutamine synthetase inhibitors, DNA fragments and recombinants for use in the production of said cells and plants
US5648477A (en) 1986-03-11 1997-07-15 Plant Genetic Systems, N.V. Genetically engineered plant cells and plants exhibiting resistance to glutamine synthetase inhibitors, DNA fragments and recombinants for use in the production of said cells and plants
US5468613A (en) 1986-03-13 1995-11-21 Hoffmann-La Roche Inc. Process for detecting specific nucleotide variations and genetic polymorphisms present in nucleic acids
US5380831A (en) 1986-04-04 1995-01-10 Mycogen Plant Science, Inc. Synthetic insecticidal crystal protein gene
US5879903A (en) 1986-08-23 1999-03-09 Hoechst Aktiengesellschaft Phosphinothricin-resistance gene, and its use
US5378824A (en) 1986-08-26 1995-01-03 E. I. Du Pont De Nemours And Company Nucleic acid fragment encoding herbicide resistant plant acetolactate synthase
US5605011A (en) 1986-08-26 1997-02-25 E. I. Du Pont De Nemours And Company Nucleic acid fragment encoding herbicide resistant plant acetolactate synthase
WO1988002405A1 (en) 1986-10-01 1988-04-07 The Plant Cell Research Institute, Inc. Genetic transformation and controlled regeneration of cucumis sp. plants in vitro
US5268463A (en) 1986-11-11 1993-12-07 Jefferson Richard A Plant promoter α-glucuronidase gene construct
US5608142A (en) 1986-12-03 1997-03-04 Agracetus, Inc. Insecticidal cotton plants
US4873192A (en) 1987-02-17 1989-10-10 The United States Of America As Represented By The Department Of Health And Human Services Process for site specific mutagenesis without phenotypic selection
US5312910A (en) 1987-05-26 1994-05-17 Monsanto Company Glyphosate-tolerant 5-enolpyruvyl-3-phosphoshikimate synthase
US4971908A (en) 1987-05-26 1990-11-20 Monsanto Company Glyphosate-tolerant 5-enolpyruvyl-3-phosphoshikimate synthase
US5145783A (en) 1987-05-26 1992-09-08 Monsanto Company Glyphosate-tolerant 5-endolpyruvyl-3-phosphoshikimate synthase
US5013659A (en) 1987-07-27 1991-05-07 E. I. Du Pont De Nemours And Company Nucleic acid fragment encoding herbicide resistant plant acetolactate synthase
US5141870A (en) 1987-07-27 1992-08-25 E. I. Du Pont De Nemours And Company Nucleic acid fragment encoding herbicide resistant plant acetolactate synthase
US5886244A (en) 1988-06-10 1999-03-23 Pioneer Hi-Bred International, Inc. Stable transformation of plant cells
US5990387A (en) 1988-06-10 1999-11-23 Pioneer Hi-Bred International, Inc. Stable transformation of plant cells
US5023179A (en) 1988-11-14 1991-06-11 Eric Lam Promoter enhancer element for gene expression in plant roots
US5110732A (en) 1989-03-14 1992-05-05 The Rockefeller University Selective gene expression in plants
US5879918A (en) 1989-05-12 1999-03-09 Pioneer Hi-Bred International, Inc. Pretreatment of microprojectiles prior to using in a particle gun
US5240855A (en) 1989-05-12 1993-08-31 Pioneer Hi-Bred International, Inc. Particle gun
US5310667A (en) 1989-07-17 1994-05-10 Monsanto Company Glyphosate-tolerant 5-enolpyruvyl-3-phosphoshikimate synthases
US5322783A (en) 1989-10-17 1994-06-21 Pioneer Hi-Bred International, Inc. Soybean transformation by microparticle bombardment
US5466785A (en) 1990-04-12 1995-11-14 Ciba-Geigy Corporation Tissue-preferential promoters
US5874265A (en) 1990-04-17 1999-02-23 Dekalb Genetics Corporation Methods and compositions for the production of stably transformed fertile monocot plants and cells thereof
US5489520A (en) 1990-04-17 1996-02-06 Dekalb Genetics Corporation Process of producing fertile transgenic zea mays plants and progeny comprising a gene encoding phosphinothricin acetyl transferase
US5969213A (en) 1990-04-17 1999-10-19 Dekalb Genetics Corporation Methods and compositions for the production of stably transformed fertile monocot plants and cells thereof
US5919675A (en) 1990-04-17 1999-07-06 Dekalb Genetics Corporation Methods and compositions for the production of stably transformed, fertile monocot plants and cells thereof
US5550318A (en) 1990-04-17 1996-08-27 Dekalb Genetics Corporation Methods and compositions for the production of stably transformed, fertile monocot plants and cells thereof
US5723756A (en) 1990-04-26 1998-03-03 Plant Genetic Systems, N.V. Bacillus thuringiensis strains and their genes encoding insecticidal toxins
US5608149A (en) 1990-06-18 1997-03-04 Monsanto Company Enhanced starch biosynthesis in tomatoes
US6248876B1 (en) 1990-08-31 2001-06-19 Monsanto Company Glyphosate-tolerant 5-enolpyruvylshikimate-3-phosphate synthases
US5804425A (en) 1990-08-31 1998-09-08 Monsanto Company Glyphosate-tolerant 5-enolpyruvylshikimate-3-phosphate synthases
US5633435A (en) 1990-08-31 1997-05-27 Monsanto Company Glyphosate-tolerant 5-enolpyruvylshikimate-3-phosphate synthases
US5627061A (en) 1990-08-31 1997-05-06 Monsanto Company Glyphosate-tolerant 5-enolpyruvylshikimate-3-phosphate synthases
US5866775A (en) 1990-09-28 1999-02-02 Monsanto Company Glyphosate-tolerant 5-enolpyruvyl-3-phosphoshikimate synthases
US6225114B1 (en) 1990-09-28 2001-05-01 Monsanto Company Modified gene encoding glyphosate-tolerant 5-enolpruvyl-3-phosphoshikimate synthase
US5932782A (en) 1990-11-14 1999-08-03 Pioneer Hi-Bred International, Inc. Plant transformation method using agrobacterium species adhered to microprojectiles
US5366892A (en) 1991-01-16 1994-11-22 Mycogen Corporation Gene encoding a coleopteran-active toxin
US5459252A (en) 1991-01-31 1995-10-17 North Carolina State University Root specific gene promoter
US5633448A (en) 1991-03-05 1997-05-27 Rhone-Poulenc Agrochimie Chimeric gene for the transformation of plants
USRE37287E1 (en) 1991-03-05 2001-07-17 Aventis Cropscience S.A. Chimeric gene for the transformation of plants
US5491288A (en) 1991-03-05 1996-02-13 Rhone Poulenc Agrochimie Chimeric gene comprising the arabidopsis histone H4 promoter for the transformation of plants
US5510471A (en) 1991-03-05 1996-04-23 Rhone-Poulenc Agrochimie Chimeric gene for the transformation of plants
USRE36449E (en) 1991-03-05 1999-12-14 Rhone-Poulenc Agro Chimeric gene for the transformation of plants
US5399680A (en) 1991-05-22 1995-03-21 The Salk Institute For Biological Studies Rice chitinase promoter
US5767361A (en) 1991-07-31 1998-06-16 American Cyanamid Company Imidazolinone resistant AHAS mutants
US5731180A (en) 1991-07-31 1998-03-24 American Cyanamid Company Imidazolinone resistant AHAS mutants
US5747450A (en) 1991-08-02 1998-05-05 Kubota Corporation Microorganism and insecticide
US5604121A (en) 1991-08-27 1997-02-18 Agricultural Genetics Company Limited Proteins with insecticidal properties against homopteran insects and their use in plant protection
US5750386A (en) 1991-10-04 1998-05-12 North Carolina State University Pathogen-resistant transgenic plants
US5436391A (en) 1991-11-29 1995-07-25 Mitsubishi Corporation Synthetic insecticidal gene, plants of the genus oryza transformed with the gene, and production thereof
US5952544A (en) 1991-12-04 1999-09-14 E. I. Du Pont De Nemours And Company Fatty acid desaturase genes from plants
US5324646A (en) 1992-01-06 1994-06-28 Pioneer Hi-Bred International, Inc. Methods of regeneration of Medicago sativa and expressing foreign DNA in same
US5341001A (en) 1992-02-13 1994-08-23 Matsushita Electric Industrial Co., Ltd. Sulfide-selenide manganese-zinc mixed crystal photo semiconductor and laser diode
US5591616A (en) 1992-07-07 1997-01-07 Japan Tobacco, Inc. Method for transforming monocotyledons
US5401836A (en) 1992-07-16 1995-03-28 Pioneer Hi-Bre International, Inc. Brassica regulatory sequence for root-specific or root-abundant gene expression
US5563055A (en) 1992-07-27 1996-10-08 Pioneer Hi-Bred International, Inc. Method of Agrobacterium-mediated transformation of cultured soybean cells
WO1994011516A1 (en) 1992-11-17 1994-05-26 E.I. Du Pont De Nemours And Company Genes for microsomal delta-12 fatty acid desaturases and related enzymes from plants
US5602321A (en) 1992-11-20 1997-02-11 Monsanto Company Transgenic cotton plants producing heterologous polyhydroxy(e) butyrate bioplastic
US5583210A (en) 1993-03-18 1996-12-10 Pioneer Hi-Bred International, Inc. Methods and compositions for controlling plant development
US5789156A (en) 1993-06-14 1998-08-04 Basf Ag Tetracycline-regulated transcriptional inhibitors
US5814618A (en) 1993-06-14 1998-09-29 Basf Aktiengesellschaft Methods for regulating gene expression
US5925517A (en) 1993-11-12 1999-07-20 The Public Health Research Institute Of The City Of New York, Inc. Detectably labeled dual conformation oligonucleotide probes, assays and kits
US5565350A (en) 1993-12-09 1996-10-15 Thomas Jefferson University Compounds and methods for site directed mutations in eukaryotic cells
US5871984A (en) 1993-12-09 1999-02-16 Thomas Jefferson University Compounds and methods for site directed mutations in eukaryotic cells
US5756325A (en) 1993-12-09 1998-05-26 Thomas Jefferson University Compounds and methods for site directed mutations in eukaryotic cells
US5593881A (en) 1994-05-06 1997-01-14 Mycogen Corporation Bacillus thuringiensis delta-endotoxin
US5633363A (en) 1994-06-03 1997-05-27 Iowa State University, Research Foundation In Root preferential promoter
US6288306B1 (en) 1994-06-16 2001-09-11 Syngenta Investment Corporation Methods of selecting plants, plant tissue or plant cells resistant to a protoporphyrinogen oxidase inhibitor
US6282837B1 (en) 1994-06-16 2001-09-04 Novartis Finance Corporation Methods of controlling the growth of undesired vegetation with herbicide tolerant plants or plant seeds having altered protoporphyrinogen oxidase activity
US5767373A (en) 1994-06-16 1998-06-16 Novartis Finance Corporation Manipulation of protoporphyrinogen oxidase enzyme activity in eukaryotic organisms
US5736369A (en) 1994-07-29 1998-04-07 Pioneer Hi-Bred International, Inc. Method for producing transgenic cereal plants
US5608144A (en) 1994-08-12 1997-03-04 Dna Plant Technology Corp. Plant group 2 promoters and uses thereof
US5792931A (en) 1994-08-12 1998-08-11 Pioneer Hi-Bred International, Inc. Fumonisin detoxification compositions and methods
US5736514A (en) 1994-10-14 1998-04-07 Nissan Chemical Industries, Ltd. Bacillus strain and harmful organism controlling agents
US5659026A (en) 1995-03-24 1997-08-19 Pioneer Hi-Bred International ALS3 promoter
US5928937A (en) 1995-04-20 1999-07-27 American Cyanamid Company Structure-based designed herbicide resistant products
WO1996033270A1 (en) 1995-04-20 1996-10-24 American Cyanamid Company Structure-based designed herbicide resistant products
US6268549B1 (en) 1995-06-02 2001-07-31 Aventis Cropscience S.A. DNA sequence of a gene of hydroxy-phenyl pyruvate dioxygenase and production of plants containing a gene of hydroxy-phenyl pyruvate dioxygenase and which are tolerant to certain herbicides
WO1997004103A2 (en) 1995-07-19 1997-02-06 Rhone-Poulenc Agrochimie Mutated 5-enol pyruvylshikimate-3-phosphate synthase, gene coding for said protein and transformed plants containing said gene
US5837876A (en) 1995-07-28 1998-11-17 North Carolina State University Root cortex specific gene promoter
US5981722A (en) 1995-11-20 1999-11-09 Board Of Regents For The University Of Oklahoma Trypsin inhibitors with insecticidal properties obtained from PENTACLETHRA MACROLOBA
US5737514A (en) 1995-11-29 1998-04-07 Texas Micro, Inc. Remote checkpoint memory system and protocol for fault-tolerant computer system
US6150097A (en) 1996-04-12 2000-11-21 The Public Health Research Institute Of The City Of New York, Inc. Nucleic acid detection probes having non-FRET fluorescence quenching and kits and assays including such probes
US5760012A (en) 1996-05-01 1998-06-02 Thomas Jefferson University Methods and compounds for curing diseases caused by mutations
US6072050A (en) 1996-06-11 2000-06-06 Pioneer Hi-Bred International, Inc. Synthetic promoters
US5795972A (en) 1996-06-17 1998-08-18 Thomas Jefferson University Chimeric mutational vectors having non-natural nucleotides
US5731181A (en) 1996-06-17 1998-03-24 Thomas Jefferson University Chimeric mutational vectors having non-natural nucleotides
US6232529B1 (en) 1996-11-20 2001-05-15 Pioneer Hi-Bred International, Inc. Methods of producing high-oil seed by modification of starch levels
US6177616B1 (en) 1996-12-16 2001-01-23 Hoecsht Schering Agrevo Gmbh Genes coding for amino acid deacetylases with specificity for N-acetyl-L-phosphinothricin, their isolation and their use
US5981840A (en) 1997-01-24 1999-11-09 Pioneer Hi-Bred International, Inc. Methods for agrobacterium-mediated transformation
WO1998049350A1 (en) 1997-04-30 1998-11-05 Regents Of The University Of Minnesota In vivo use of recombinagenic oligonucleobases to correct genetic lesions in hepatocytes
WO1999007865A1 (en) 1997-08-05 1999-02-18 Kimeragen, Inc. The use of mixed duplex oligonucleotides to effect localized genetic changes in plants
US6245968B1 (en) 1997-11-07 2001-06-12 Aventis Cropscience S.A. Mutated hydroxyphenylpyruvate dioxygenase, DNA sequence and isolation of plants which contain such a gene and which are tolerant to herbicides
WO1999023886A1 (en) 1997-11-12 1999-05-20 Aventis Cropscience S.A. Method of controlling weeds in transgenic crops
US6069115A (en) 1997-11-12 2000-05-30 Rhone-Poulenc Agrochimie Method of controlling weeds in transgenic crops
WO1999025853A1 (en) 1997-11-18 1999-05-27 Pioneer Hi-Bred International, Inc. Targeted manipulation of herbicide-resistance genes in plants
WO1999025855A1 (en) 1997-11-18 1999-05-27 Pioneer Hi-Bred International, Inc. Mobilization of viral genomes from t-dna using site-specific recombination systems
WO1999025821A1 (en) 1997-11-18 1999-05-27 Pioneer Hi-Bred International, Inc. Compositions and methods for genetic modification of plants
WO1999025854A1 (en) 1997-11-18 1999-05-27 Pioneer Hi-Bred International, Inc. A method for directional stable transformation of eukaryotic cells
WO1999025840A1 (en) 1997-11-18 1999-05-27 Pioneer Hi-Bred International, Inc. A novel method for the integration of foreign dna into eukaryoticgenomes
WO1999043838A1 (en) 1998-02-24 1999-09-02 Pioneer Hi-Bred International, Inc. Synthetic promoters
US6177611B1 (en) 1998-02-26 2001-01-23 Pioneer Hi-Bred International, Inc. Maize promoters
WO1999061619A2 (en) 1998-05-22 1999-12-02 Pioneer Hi-Bred International, Inc. Cell cycle genes, proteins and uses thereof
US6037130A (en) 1998-07-28 2000-03-14 The Public Health Institute Of The City Of New York, Inc. Wavelength-shifting probes and primers and their use in assays and kits
US6225529B1 (en) 1998-08-20 2001-05-01 Pioneer Hi-Bred International, Inc. Seed-preferred promoters
WO2000011177A1 (en) 1998-08-20 2000-03-02 Pioneer Hi-Bred International, Inc. Seed-preferred promoters
WO2000012733A1 (en) 1998-08-28 2000-03-09 Pioneer Hi-Bred International, Inc. Seed-preferred promoters from end genes
WO2000017364A2 (en) 1998-09-23 2000-03-30 Pioneer Hi-Bred International, Inc. Cyclin d polynucleotides, polypeptides and uses thereof
WO2000028058A2 (en) 1998-11-09 2000-05-18 Pioneer Hi-Bred International, Inc. Transcriptional activator lec1 nucleic acids, polypeptides and their uses
US20030226166A1 (en) 1998-12-21 2003-12-04 Falco Saverio Carl S-adenosyl-L-methionine synthetase promoter and its use in expression of transgenic genes in plants
WO2000066746A1 (en) 1999-04-29 2000-11-09 Syngenta Limited Herbicide resistant plants
WO2000066747A1 (en) 1999-04-29 2000-11-09 Syngenta Limited Herbicide resistant plants
WO2001012825A1 (en) 1999-08-13 2001-02-22 Syngenta Participations Ag Herbicide-tolerant protoporphyrinogen oxidase
WO2001066704A2 (en) 2000-03-09 2001-09-13 Monsanto Technology Llc Methods for making plants tolerant to glyphosate and compositions thereof
WO2005012576A2 (en) * 2003-08-01 2005-02-10 Pioneer Hi-Bred International, Inc. Molecular markers for the selection of soybean plants having superior agronomic performance
WO2005107437A2 (en) 2004-04-30 2005-11-17 Dow Agrosciences Llc Novel herbicide resistance genes
WO2006017840A2 (en) * 2004-08-06 2006-02-16 Pioneer Hi-Bred International Inc. Genetic loci associated with iron deficiency tolerance in soybean
US20070061917A1 (en) 2005-08-24 2007-03-15 Pioneer Hi-Bred International, Inc. Methods and compositions for the expression of a polynucleotide of interest
WO2009144079A1 (en) * 2008-04-14 2009-12-03 Bayer Bioscience N.V. New mutated hydroxyphenylpyruvate dioxygenase, dna sequence and isolation of plants which are tolerant to hppd inhibitor herbicides
US20100024080A1 (en) * 2008-07-23 2010-01-28 Pioneer Hi-Bred International, Inc. Molecular markers linked to ppo inhibitor tolerance in soybeans

Non-Patent Citations (245)

* Cited by examiner, † Cited by third party
Title
"2004 Illinois Agricultural Pest Management Handbook", UNIVERSITY OF ILLINOIS EXTENSION, ILLINOIS, article "Weed Control for Corn, Soybeans, and Sorghum"
"Current Protocols in Molecular Biology", vol. 1-4, 1994, JOHN WILEY & SONS, INC.
"Current Protocols", 2002, GREENE PUBLISHING ASSOCIATES, INC. AND JOHN WILEY & SONS, INC., article "Current Protocols in Molecular Biology"
"Current Protocols", 2004, JOHN WILEY & SONS, INC., article "Current Protocols in Molecular Biology"
"Fundamental Methods Springer Lab Manual", 1995, SPRINGER-VERLAG, article "Plant Cell, Tissue and Organ Culture"
"Genetic Engineering", vol. 6, 1987, ACADEMIC PRESS
"Methods for Plant Molecular Biology", 1988, ACADEMIC PRESS, INC., SAN DIEGO, CALIF.
"Molecular Cloning: A Laboratory Manual", 1982, COLD SPRING HARBOR LABORATORY PRESS
"Molecular Cloning: A Laboratory Manual", vol. 1-3, 1989, COLD SPRING HARBOR LABORATORY PRESS
"MSU Extension Bulletin E434", MICHIGAN STATE UNIVERSITY, MICHIGAN, article "Weed Control Guide for Field Crops"
"PCR Protocols A Guide to Methods and Applications", 1990, ACADEMIC PRESS INC.
"Plant Cell, Tissue and Organ Culture; Fundamental Methods", 1995, SPRINGER LAB MANUAL
"Plant Cell, Tissue and Organ Culture; Fundamental Methods", 1995, SPRINGER-VERLAG
"Plant Gene Transfer and Expression Protocols-Methods in Molecular Biology", vol. 49, 1995, HUMANA PRESS
"Plant Gene Transfer and Expression Protocols--Methods in Molecular Biology", vol. 49, 1995, HUMANA PRESS
"Plant Molecular Biology", 1993, BIOS SCIENTIFIC PUBLISHERS
"Plant Molecular Biology", 1993, BIOS SCIENTIFIC PUBLISHERS, INC
"Plant Molecular Biology", 1993, SCIENTIFIC PUBLISHERS
"Plant Molecular Biology: A Laboratory Manual", 1997, SPRINGER-VERLAG
"Techniques in Molecular Biology", 1983, MACMILLAN PUBLISHING COMPANY
"The ATCC Catalogue of Bacteria and Bacteriophage", 1992, ATCC
"The Handbook of Microbiological Media", 1993, CRC PRESS
ARCHER ET AL., J. BIOENERG BIOMEMB., vol. 22, no. 6, 1990, pages 789 - 810
BAIM ET AL., PROC. NATL. ACAD. SCI. USA, vol. 88, 1991, pages 5072 - 5076
BALLAS ET AL., NUCLEIC ACIDS RES., vol. 17, 1989, pages 7891 - 7903
BARKLEY ET AL., THE OPERON, 1980, pages 177 - 220
BEAUCAGE; CARUTHERS, TETRAHEDRON LETTS, vol. 22, 1981, pages 1859 - 1862
BECKER ET AL., MOL GEN GENET, vol. 249, 1995, pages 65
BEETHAM ET AL., PROC. NATL. ACAD. SCI. USA, vol. 96, 1999, pages 8774 - 8778
BENBROOK ET AL.: "Proceedings Bio Expo Butterworth", 1986, STONEHAM, MASS., pages: 27 - 54
BENFEY ET AL., EMBO J., vol. 9, 1990, pages 1685 - 96
BERGER; KIMMEL: "Methods in Enzymology", vol. 152, ACADEMIC PRESS, INC., article "Guide to Molecular Cloning Techniques"
BINDING: "Regeneration of Plants, Plant Protoplasts", 1985, CRC PRESS, pages: 21 - 73
BLOK; KRAMER: "Amplifiable hybridization probes containing a molecular switch", MOL CELL PROBES, vol. 11, 1997, pages 187 - 194, XP004457808, DOI: doi:10.1006/mcpr.1997.0103
BOGUSZ ET AL., PLANT CELL, vol. 2, no. 7, 1990, pages 633 - 641
BOLTE ET AL., J. CELL SCIENCE, vol. 117, 2004, pages 943 - 54
BONIN, PH.D. THESIS, 1993
BONNET ET AL.: "Thermodynamic basis of the chemical specificity of structured DNA probes", PROC. NATL. ACAD. SCI. U.S.A., vol. 96, 1999, pages 6171 - 6176, XP002244958, DOI: doi:10.1073/pnas.96.11.6171
BROWN ET AL., CELL, vol. 49, 1987, pages 603 - 612
BYTEBIER ET AL., PROC. NATL. ACAD. SCI. USA, vol. 84, 1987, pages 5345 - 5349
CALLIS ET AL., GENETICS, vol. 139, 1995, pages 921 - 39
CAMPBELL; GOWRI, PLANT PHYSIOL., vol. 92, 1990, pages 1 - 11
CANEVASCINI ET AL., PLANT PHYSIOL., vol. 112, 1996, pages 513 - 524
CAPANA ET AL., PLANT MOL. BIOL., vol. 25, 1994, pages 681 - 691
CHOI ET AL.: "A Soybean Transcript Map: Gene Distribution, Haplotype and Single-Nucleotide Polymorphism Analysis", GENETICS, vol. 176, 2007, pages 685 - 96, XP002703639, DOI: doi:10.1534/genetics.107.070821
CHRISTENSEN ET AL., PLANT MOL. BIOL., vol. 12, 1989, pages 619 - 632
CHRISTENSEN ET AL., PLANT MOL. BIOL., vol. 18, 1992, pages 675 - 689
CHRISTOPHERSON ET AL., PROC. NATL. ACAD. SCI. USA, vol. 89, 1992, pages 6314 - 6318
CHRISTOU AND FORD, ANNALS OF BOTANY, vol. 75, - 1995, pages 407 - 413
CHRISTOU ET AL., PLANT PHYSIOL., vol. 87, 1988, pages 671 - 674
CLARK ET AL., J. BIOL. CHEM., vol. 264, 1989, pages 17544 - 17550
CREGAN, P.B. ET AL.: "An Integrated Genetic Linkage Map of the Soybean Genome", CROP SCIENCE, vol. 39, 1999, pages 1464 - 90, XP002309701
CROSSWAY ET AL., BIOTECHNIQUES, vol. 4, 1986, pages 320 - 334
D. HESS, INTERN REV. CYTOL., vol. 107, 1987, pages 367
DALE: "Protoplasts", 1983, BIRKHAUSER, article "Protoplast Culture and Plant Regeneration of Cereals and Other Recalcitrant Crops", pages: 31 - 41
DANDEKAR ET AL., J. TISSUE CULT. METH., vol. 12, 1989, pages 145
DAS: "Subcellular Biochemistry", PLANT MICROBE INTERACTIONS, vol. 29, 1998, pages 343 - 363
DATTA ET AL., BIOTECHNOLOGY, vol. 8, 1990, pages 736 - 740
DAVEY: "Protoplasts", 1983, article "Recent Developments in the Culture and Regeneration of Plant Protoplasts", pages: 12 - 29
DAYAN ET AL.: "Soybean (Glycine max) cultivar differences in response to sulfentrazone", WEED SCIENCE, vol. 45, 1997, pages 634 - B41
DAYHOFF ET AL.: "Atlas of Protein Sequence and Structure", 1978, NATL BIOMED RES FOUND
DE CASTRO SILVA FILHO ET AL., PLANT MOL. BIOL., vol. 30, 1996, pages 769 - 780
DE WET ET AL.: "The Experimental Manipulation of Ovule Tissues", 1985, LONGMAN, pages: 197 - 209
DEGENKOLB ET AL.: "Antimicrob", AGENTS CHEMOTHER., vol. 35, 1991, pages 1591 - 1595
DEIKMAN; FISCHER, EMBO J., vol. 7, 1988, pages 3315
DELLA-CIOPPA ET AL., PLANT PHYSIOL., vol. 84, 1987, pages 965 - 968
DEUSCHLE ET AL., PROC. NATL. ACAD. ACI. USA, vol. 86, 1989, pages 5400 - 5404
DEUSCHLE ET AL., SCIENCE, vol. 248, 1990, pages 480 - 483
D'HALLUIN ET AL., PLANT CELL, vol. 4, 1992, pages 1495 - 1505
DYER ET AL., APPL. MICROBIOL. BIOTECHNOL., vol. 59, 2002, pages 224 - 230
ELROY-STEIN ET AL., PROC. NATL. ACAD. SCI. USA, vol. 86, 1989, pages 6126 - 6130
EMBO J., vol. 8, pages 343 - 350
EVANS ET AL.: "Handbook of Plant Cell Cultures", vol. 1, 1983, MACMILLAN PUBLISHING CO., article "Protoplast Isolation and Culture", pages: 124 - 176
EVANS ET AL.: "Protoplasts Isolation and Culture, Handbook of Plant Cell Culture", 1983, MACMILLIAN PUBLISHING COMPANY, pages: 124 - 176
FANG ET AL.: "Designing a novel molecular beacon for surface- immobilized DNA hybridization studies", J. AM. CHEM. SOC., vol. 121, 1999, pages 2921 - 2922, XP002315845, DOI: doi:10.1021/ja9837809
FEHR: "Principals of Cultivar Development", 1993, MACMILLAN PUBLISHING COMPANY
FEMANDEZ-MOYA ET AL., J. AGRIC. FOOD CHEM., vol. 53, 2005, pages 5326 - 5330
FETTER ET AL., PLANT CELL, vol. 16, 2004, pages 215 - 28
FIGGE ET AL., CELL, vol. 52, 1988, pages 713 - 722
FINER; MCMULLEN, IN VITRO CELL DEV. BIOL., vol. 27P, 1991, pages 175 - 182
FRALEY ET AL., PROC. NATL. ACAD. SCI. (U.S.A.), vol. 80, 1983, pages 4803
FRALEY ET AL., PROC. NATL. ACAD. SCI. USA, vol. 80, 1983, pages 4803
FRALEY ET AL., PROC. NATL. ACAD. SCI. USA, vol. 80, 1984, pages 4803
FREEMAN ET AL., PLANT CELL PHYSIOL., vol. 25, 1984, pages 1353
FROM ET AL., PROC. NATL. ACAD. SCI. USA, vol. 82, 1985, pages 5824
FROMM ET AL., BIOTECHNOLOGY, vol. 8, 1990, pages 833 - 839
FROMM ET AL., PROC. NATL. ACAD. SCI. USA, vol. 82, 1985, pages 5824
FUERST ET AL., PROC. NATL. ACAD. SCI. USA, vol. 86, 1989, pages 2549 - 2553
GALITZKY ET AL., ACTA CRYSTALLOGR. D. BIOL. CRYSTALLOGR., vol. 57, 2001, pages 1101 - 1109
GALLIE ET AL., GENE, vol. 165, no. 2, 1995, pages 233 - 238
GALLIE ET AL., GENE, vol. 60, 1987, pages 217 - 25
GALLIE ET AL.: "Molecular Biology of RNA", 1989, LISS, pages: 237 - 256
GALLIE ET AL.: "Molecular Biology ofRNA", 1989, LISS, pages: 237 - 256
GATZ ET AL., MOL. GEN. GENET., vol. 227, 1991, pages 229 - 237
GEISER ET AL., GENE, vol. 48, 1986, pages 109
GILIMAN; SMITH, GENE, vol. 8, 1979, pages 81
GILL ET AL., NATURE, vol. 334, 1988, pages 721 - 724
GOSSEN ET AL., PROC. NATL. ACAD. SCI. USA, vol. 89, 1992, pages 5547 - 5551
GOSSEN, PH.D. THESIS, 1993
GOTOR ET AL., PLANT J., vol. 3, 1993, pages 509 - 18
GREEN, WEED TECHNOLOGY, vol. 12, 1998, pages 474 - 477
GREEN; ULRICH, WEED SCIENCE, vol. 41, 1993, pages 508 - 516
GREEN; WILLIAMS: "WSSA Annual Meeting in Kansas City, Mo., Feb. 9-12, 2004", 2004, article "Correlation of Corn (Zea mays) Inbred Response to Nicosulfuron and Mesotrione"
GUATELLI ET AL., PROC NATL ACAD SCI USA, vol. 87, 1990, pages 1874
GUERINEAU ET AL., MOL. GEN. GENET., vol. 262, 1991, pages 141 - 144
GUEVARA-GARCIA, PLANT J., vol. 4, 1993, pages 495 - 505
HANSEN ET AL., MOL. GEN GENET., vol. 254, 1997, pages 337 - 343
HANSEN; CHILTON, CURRENT TOPICS IN MICROBIOLOGY, vol. 240, 1998, pages 22
HAUGLAND: "Handbook of Fluorescent Probes and Research Chemicals", 1996, MOLECULAR PROBES, INC.
HAUGLAND: "Handbook of Fluorescent Probes and Research Chemicals", 2001, MOLECULAR PROBES, INC.
HENIKOFF; HENIKOFF, PROC NATL ACAD SCI USA, vol. 89, 1989, pages 10915
HENIKÖFF; HENIKOFF, PROC NATL ACAD SCI USA, vol. 89, 1989, pages 10915
HERMAN ET AL., J. AGRIC. FOOD CHEM., vol. 52, 2004, pages 2726 - 2734
HERMAN ET AL., J. BIOL. CHEM., vol. 280, 2005, pages 24759 - 24767
HERRARA-ESTRELLA ET AL., NATURE, vol. 303, 1983, pages 209
HILLENAND-WISSMAN, TOPICS MOL. STRUC. BIOL., vol. 10, 1989, pages 143 - 162
HIRE ET AL., PLANT MOL. BIOL., vol. 20, 1992, pages 207 - 218
HLAVKA ET AL.: "Handbook of Experimental Pharmacology", vol. 78, 1985, SPRINGER-VERLAG
HOHN ET AL.: "Molecular Biology of Plant Tumors", 1982, ACADEMIC PRESS, pages: 549 - 560
HOOYKAAS-VAN SLOGTEREN ET AL., NATURE (LONDON), vol. 311, 1984, pages 763 - 764
HORSCH ET AL., SCIENCE, vol. 227, 1985, pages 1229 - 1231
HORSCH ET AL., SCIENCE, vol. 233, 1984, pages 496
HSUIH ET AL.: "Novel, ligation-dependent PCR assay for detection of hepatitis C in serum", J CLIN MICROBIOL, vol. 34, 1997, pages 501 - 507
HU ET AL., CELL, vol. 48, 1987, pages 555 - 566
HULTING ET AL.: "Soybean (Glycine max (L.) Merr.) cultivar tolerance to sulfentrazone", SCIENCE DIRECT, vol. 20, no. 8, 2001, pages 679 - 683, XP002547823, DOI: doi:10.1016/S0261-2194(01)00035-7
JOBLING ET AL., NATURE, vol. 325, 1987, pages 622 - 625
JONES ET AL., SCIENCE, vol. 266, 1994, pages 789
JOSHI ET AL., NUCLEIC ACIDS RES., vol. 15, 1987, pages 9627 - 9639
KAEPPLER ET AL., PLANT CELL REPORTS, vol. 9, 1990, pages 415 - 418
KAEPPLER ET AL., THEOR. APPL. GENET., vol. 84, 1992, pages 560 - 566
KATO ET AL., PLANT PHYSIOL, vol. 129, 2002, pages 913 - 42
KAWAMATA ET AL., PLANT CELL PHYSIOL., vol. 38, 1997, pages 792 - 803
KELLER; BAUMGARTNER, PLANT CELL, vol. 3, 1991, pages 1051 - 1061
KINDLE, PROC. NATL. ACAD. SCI., vol. 87, 1990, pages 1228
KLEE ET AL., ANN. REV. OF PLANT PHYS., vol. 38, 1987, pages 467 - 486
KLEIN ET AL., BIOTECHNOLOGY, vol. 6, 1988, pages 559 - 563
KLEIN ET AL., NATURE, vol. 327, 1987, pages 70
KLEIN ET AL., NATURE, vol. 327, 1987, pages 70 - 73
KLEIN ET AL., PLANT PHYSIOL., vol. 91, 1988, pages 440 - 444
KLEIN ET AL., PROC. NATL. ACAD. SCI. USA, vol. 85, 1988, pages 4305 - 4309
KLEINSCHNIDT ET AL., BIOCHEMISTRY, vol. 27, 1988, pages 1094 - 1104
KONG ET AL., ARCH VIROL, vol. 143, 1988, pages 1791 - 1799
KOSTRIKIS ET AL.: "Molecular beacons: spectral genotyping of human alleles", SCIENCE, vol. 279, 1998, pages 1228 - 1229, XP002942660, DOI: doi:10.1126/science.279.5354.1228
KUNKEL ET AL., METHODS IN ENZYMOL, vol. 154, 1987, pages 367 - 382
KUNKEL, PROC NATL ACAD SCI USA, vol. 82, 1985, pages 488 - 492
KUSTER ET AL., PLANT MOL. BIOL., vol. 29, 1995, pages 759 - 772
KWON ET AL., PLANT PHYSIOL., vol. 105, 1994, pages 357 - 67
LABOW ET AL., MOL. CELL. BIOL., vol. 10, 1990, pages 3343 - 3356
LAM, RESULTS PROBL. CELL DIFFER., vol. 20, 1994, pages 181 - 196
LAMPPA ET AL., J. BIOL. CHEM., vol. 263, 1988, pages 14996 - 14999
LAST ET AL., THEOR. APPL. GENET., vol. 81, 1991, pages 581 - 588
LAWRENCE ET AL., J. BIOL. CHEM., vol. 272, no. 33, 1997, pages 20357 - 20363
LEE ET AL., APPL. ENVIRON. MICROBIOL, vol. 69, 2003, pages 4648 - 4657
LEONE ET AL.: "Molecular beacon probes combined with amplification by NASBA enable homogenous real-time detection of RNA", NUCLEIC ACIDS RES., vol. 26, 1995, pages 2150 - 2155
LI ET AL., PLANT CELL REPORTS, vol. 12, 1993, pages 250 - 255
LICHTENSTEIN; C. P.; DRAPER: "DNA Cloning", vol. II, 1985, IRI PRESS
LOMMEL ET AL., VIROLOGY, vol. 81, 1991, pages 3 82 - 3 85
LUO ET AL., PLANT MOL. BIOL. REPORTER, vol. 6, 1988, pages 165
MACEJAK ET AL., NATURE, vol. 353, 1991, pages 90 - 94
MANLY ET AL., MAMMALIAN GENOME, vol. 12, 2001, pages 930 - 932
MARRAS ET AL.: "Multiplex detection of single-nucleotide variation using molecular beacons", GENET. ANAL. BIOMOL. ENG., vol. 14, 1999, pages 151 - 156
MARTIN ET AL., SCIENCE, vol. 262, 1993, pages 1432
MATSUOKA ET AL., PROC NATL. ACAD. SCI. USA, vol. 90, 1993, pages 9586 - 9590
MATSUOKA ET AL., PROC. NATL. ACAD. SCI. USA, vol. 90, no. 20, 1993, pages 9586 - 9590
MCBRIDE ET AL., PROC. NATL. ACAD. SCI. USA, vol. 91, 1994, pages 7301 - 7305
MCCABE ET AL., BIO/TECHNOLOGY, vol. 6, 1988, pages 923 - 926
MCCABE ET AL., BIOTECHNOLOGY, vol. 6, 1988, pages 923 - 926
MCELROY ET AL., PLANT CELL, vol. 2, 1990, pages 163 - 171
MCGRANAHAN ET AL., PLANT CELL REP., vol. 8, 1990, pages 512
MCNELLIS ET AL., PLANT J., vol. 14, 1998, pages 247 - 257
MEKSEM ET AL., MOL GEN GENET, vol. 249, 1995, pages 74
MIAO ET AL., PLANT CELL, vol. 3, 1991, pages 11 - 22
MINDRINOS ET AL., CELL, vol. 78, 1994, pages 1089
MOGEN ET AL., PLANT CELL, vol. 2, 1990, pages 1261 - 1272
MUNROE ET AL., GENE, vol. 91, 1990, pages 151 - 158
MURRAY ET AL., NUCLEIC ACIDS RES., vol. 17, 1989, pages 477 - 498
NEEDLEMAN; WUNSCH, J MOL BIOL, vol. 48, 1970, pages 443 - 453
NEUHAUS ET AL., THEOR. APPL. GENET., vol. 75, 1987, pages 30
ODELL ET AL., NATURE, vol. 313, 1985, pages 810
ODELL ET AL., NATURE, vol. 313, 1985, pages 810 - 812
OLIVA ET AL., ANTÙNICROB. AGENTS CHEMOTHER., vol. 36, 1992, pages 913 - 919
OROZCO ET AL., PLANT MOL BIOL., vol. 23, 1993, pages 1129 - 1138
OROZCO ET AL., PLANT MOL. BIOL., vol. 23, 1993, pages 1129 - 1138
OSJODA ET AL., NATURE BIOTECHNOLOGY, vol. 14, 1996, pages 745 - 750
OWEN; HARTZLER: "Herbicide Manual for Agricultural Professionals,", 2004, IOWA STATE UNIVERSITY EXTENSION, IOWA
PADGETTE ET AL.: "Herbicide-Resistant Crops", 1996, CRC LEWIS PUBLISHERS, pages: 53 - 84
PASZKOWSKI ET AL., EMBO J., vol. 3, 1984, pages 2717
PASZKOWSKI ET AL., EMBO J., vol. 3, 1984, pages 2717 - 2722
PAYNE ET AL.: "Plant Cell and Tissue Culture in Liquid Systems", 1992, JOHN WILEY & SONS, INC.
PENA ET AL., NATURE, vol. 325, 1987, pages 274
PLANT SCIENCE (LIMERICK), vol. 79, pages 69 - 76
PROUDFOOT, CELL, vol. 64, 1991, pages 671 - 674
REINES ET AL., PROC. NATL. ACAD. SCI. USA, vol. 90, 1993, pages 1917 - 1921
RETZINGER; MALLORY-SMITH, WEED TECHNOLOGY, vol. 11, 1997, pages 384 - 393
REZNIKOFF, MOL. MICROBIOL., vol. 6, 1992, pages 2419 - 2422
RIGGS ET AL., PROC. NATL. ACAD. SCI. USA, vol. 83, 1986, pages 5602 - 5606
RINEHART ET AL., PLANT PHYSIOL., vol. 112, 1996, pages 1331 - 1341
ROBERTS ET AL., NATURE, vol. 328, 1987, pages 731
ROMER ET AL., BIOCHEM. BIOPHYS. RES. COMM., vol. 196, 1993, pages 1414 - 1421
RUSSELL ET AL., TRANSGENIC RES., vol. 6, 1997, pages 157 - 168
SAMBROOK ET AL.: "Molecular Cloning - A Laboratory Manual", vol. 1-3, 2000, COLD SPRING HARBOR LABORATORY
SAMBROOK ET AL.: "Molecular Cloning: A Laboratory Manual", 1989, COLD SPRING HARBOR LABORATORY PRESS
SAMBROOK ET AL.: "Molecular Cloning--A Laboratory Manual", vol. 1-3, 2001, COLD SPRING HARBOR LABORATORY
SANFACON ET AL., GENES DEV., vol. 5, 1991, pages 141 - 149
SANFORD ET AL., PARTICULATE SCIENCE AND TECHNOLOGY, vol. 5, 1987, pages 27 - 37
SANGER ET AL., PLANT MOL. BIOL., vol. 14, no. 3, 1990, pages 433 - 443
SCHENA ET AL., PROC. NATL. ACAD. SCI. USA, vol. 88, 1991, pages 10421 - 10425
SCHMIDT ET AL., J. BIOL. CHEM., vol. 268, no. 36, 1993, pages 27447 - 27457
SCHNEIDER ET AL., PROTEIN EXPR. PURIF., vol. 6435, 1995, pages 10
SCHNELL ET AL., J. BIOL. CHEM., vol. 266, no. 5, 1991, pages 3335 - 3342
SCHUBERT ET AL., J. BACTERIOL., vol. 170, 1988, pages 5837 - 5847
SHAH ET AL., SCIENCE, vol. 233, 1986, pages 478 - 481
SHOEMAKER R.C.: "DNA-based markers in plants", 1994, KLUWER ACADEMIC PRESS, article "RFLP Map of Soybean", pages: 299 - 309
SHOEMAKER; OLSEN: "Genetic Maps: Locus Maps of Complex Genomes", 1993, COLD SPRING HARBOR LABORATORY PRESS, article "Molecular Linkage Map of Soybean (Glycine max L. Merr.)", pages: 6.131 - 6.138
SINGH ET AL., THEOR. APPL. GENET., vol. 96, 1998, pages 319 - 324
SOKAL; ROLF: "Biometry: The Principles and Practices of Statistics in Biological Research", 1981, W. H. FREEMAN AND CO, SAN FRANSCISCO
SOKAL; ROLF; 1981: "Biometry: The Principles and Practices of Statistics in Biological Research", W. H. FREEMAN AND CO, SAN FRANCISCO
SOKOL: "Real time detection of DNA:RNA hybridization in living cells", PROC. NATL. ACAD. SCI. U.S.A., vol. 95, 1998, pages 11538 - 11543, XP002123471, DOI: doi:10.1073/pnas.95.20.11538
SU ET AL., BIOTECHNOL BIOENG, vol. 85, 2004, pages 610 - 9
SVAB ET AL., PROC. NATL. ACAD. SCI. USA, vol. 87, 1990, pages 8526 - 8530
SVAB; MALIGA, EMBO J., vol. 12, 1993, pages 601 - 606
SVAB; MALIGA, PROC. NATL. ACAD. SCI. USA, vol. 90, 1993, pages 913 - 917
TAYLOR-LOVELL ET AL., WEED TECH, vol. 15, 2001, pages 95 - 102
TAYLOR-LOVELL ET AL., WEED TECHNOLOGY, vol. 15, 2001, pages 96 - 102
TIJSSEN: "Laboratory Techniques in Biochemistry and Molecular Biology--Hybridization with Nucleic Acid Probes", 1993, ELSEVIER
TOMES ET AL.: "Plant Cell, Tissue, and Organ Culture: Fundamental Methods", 1995, SPRINGER-VERLAG
TYAGI ET AL.: "Multicolor molecular beacons for allele discrimination", NATURE BIOTECHNOLOGY, vol. 16, 1998, pages 49 - 53, XP002949994, DOI: doi:10.1038/nbt0198-49
TYAGI; KRAMER: "Molecular beacons: probes that fluoresce upon hybridization", NATURE BIOTECHNOLOGY, vol. 14, 1996, pages 303 - 308, XP002914999, DOI: doi:10.1038/nbt0396-303
VAN CAMP ET AL., PLANT PHYSIOL., vol. 112, 1996, pages 525 - 535
VAN DAMME ET AL., PLANT MOL. BIOL., vol. 24, 1994, pages 825
VASIL: "Herbicide-Resistant Crops", 1996, CRC LEWIS PUBLISHERS, pages: 85 - 91
VELTEN ET AL., EMBO J., vol. 3, 1984, pages 2723 - 2730
VET ET AL.: "Multiplex detection of four pathogenic retroviruses using molecular beacons", PROC. NATL. ACAD. SCI. U.S.A., vol. 96, 1999, pages 6394 - 6399, XP002145609, DOI: doi:10.1073/pnas.96.11.6394
VON HEIJNE ET AL., PLANT MOL. BIOL. REP., vol. 9, 1991, pages 104 - 126
WEISING ET AL., ANN. REV. GENET., vol. 22, 1988, pages 421 - 477
WEISSINGER ET AL., ANN. REV. GENET., vol. 22, 1988, pages 421 - 477
WYBORSKI ET AL., NUCLEIC ACIDS RES., vol. 19, 1991, pages 4647 - 4653
YAMAMOTO ET AL., PLANT CELL PHYSIOL., vol. 35, 1994, pages 773 - 778
YAMAMOTO ET AL., PLANT CELLPHYSIOL., vol. 35, 1994, pages 773 - 778
YAMAMOTO ET AL., PLANT J., vol. 12, 1997, pages 255 - 265
YAO ET AL., CELL, vol. 71, 1992, pages 63 - 72
YARRANTON, CURR. OPIN. BIOTECH., vol. 3, 1992, pages 506 - 511
ZAMBRETTI ET AL., PROC. NATL. ACAD. SCI. USA, vol. 89, 1992, pages 3952 - 3956
ZHAO ET AL., J. BIOL. CHEM., vol. 270, no. 11, 1995, pages 6081 - 6087
ZHOU, METHODS IN ENZYMOLOGY, vol. 101, 1983, pages 433

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014138339A2 (en) 2013-03-07 2014-09-12 Athenix Corp. Toxin genes and methods for their use
EP3626828A2 (en) 2013-03-07 2020-03-25 BASF Agricultural Solutions Seed US LLC Toxin genes and methods for their use
WO2018098214A1 (en) 2016-11-23 2018-05-31 Bayer Cropscience Lp Axmi669 and axmi991 toxin genes and methods for their use
WO2018119336A1 (en) 2016-12-22 2018-06-28 Athenix Corp. Use of cry14 for the control of nematode pests
WO2018136611A1 (en) 2017-01-18 2018-07-26 Bayer Cropscience Lp Use of bp005 for the control of plant pathogens
WO2018136604A1 (en) 2017-01-18 2018-07-26 Bayer Cropscience Lp Bp005 toxin gene and methods for its use
WO2023218475A1 (en) * 2022-05-11 2023-11-16 Rallis India Limited Maize snp markers for hppd-inhibitor resistance
WO2024137438A2 (en) 2022-12-19 2024-06-27 BASF Agricultural Solutions Seed US LLC Insect toxin genes and methods for their use

Also Published As

Publication number Publication date
CA2788198C (en) 2021-01-19
CA2818599A1 (en) 2011-08-04
US10968492B2 (en) 2021-04-06
EP2529028A1 (en) 2012-12-05
US9970021B2 (en) 2018-05-15
US20110185444A1 (en) 2011-07-28
CA2788198A1 (en) 2011-08-04
WO2011094199A1 (en) 2011-08-04
AR080022A1 (en) 2012-03-07
BR112012018618A2 (en) 2017-01-10
BR112012018616A2 (en) 2017-01-10
AR080021A1 (en) 2012-03-07
US20170233757A1 (en) 2017-08-17
EP2529027A1 (en) 2012-12-05
US9611485B2 (en) 2017-04-04
US20180201949A1 (en) 2018-07-19
US20110185445A1 (en) 2011-07-28

Similar Documents

Publication Publication Date Title
US10968492B2 (en) HPPD-inhibitor herbicide tolerance
US20150218658A1 (en) Molecular markers linked to ppo inhibitor tolerance in soybeans
US20080096218A1 (en) Polynucleotide Encoding a Maize Herbicide Resistance Gene and Methods for Use
US10000820B2 (en) Genetic loci associated with resistance of soybean to cyst nematode and methods of use
AU2014318041B2 (en) Molecular markers for blackleg resistance gene Rlm2 in Brassica napus and methods of using the same
US20220195537A1 (en) Methods and compositions to select and/or predict cotton plants resistant to fusarium race-4 resistance in cotton
US11136630B2 (en) Markers linked to reniform nematode resistance
CA3228149A1 (en) Methods of identifying, selecting, and producing anthracnose stalk rot resistant crops
WO2015038470A1 (en) Molecular markers for blackleg resistance gene rlm4 in brassica napus and methods of using the same
AU2014386227A1 (en) Markers linked to reniform nematode resistance

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 11701602

Country of ref document: EP

Kind code of ref document: A1

REEP Request for entry into the european phase

Ref document number: 2011701602

Country of ref document: EP

WWE Wipo information: entry into national phase

Ref document number: 2011701602

Country of ref document: EP

ENP Entry into the national phase

Ref document number: 2788198

Country of ref document: CA

WWE Wipo information: entry into national phase

Ref document number: 2012002072

Country of ref document: CL

NENP Non-entry into the national phase

Ref country code: DE

REG Reference to national code

Ref country code: BR

Ref legal event code: B01A

Ref document number: 112012018618

Country of ref document: BR

ENP Entry into the national phase

Ref document number: 112012018618

Country of ref document: BR

Kind code of ref document: A2

Effective date: 20120726