Patent Grant
10604767
A sequence listing containing the file named “46_21_59246_A_PCT.txt” which is 33,292 bytes (measured in MS-Windows) and created on May 20, 2013, comprises 56 nucleotide sequences, is provided herewith via the USPTO's EFS system and is herein incorporated by reference in its entirety.
A listing of various soybean linkage group L (chromosome 19) markers is provided herewith in the Specification as Table 2.
International Patent Application Publication WO 2012/031097 describes genetic regions of soybean linkage group L that contain polymorphic loci that are associated with an undesirable “yellow flash” phenotype that is observed in the foliage of certain soybean varieties that comprise a transgene that confers resistance to glyphosate that are exposed to glyphosate.
“Dicamba intolerance” is an undesirable phenotype observed in certain soybean varieties that comprise a transgene that can confer resistance to the broad-spectrum herbicide dicamba. After application of dicamba, it has been discovered that the leaves of certain soybean plant varieties comprising the transgene that confers resistance to dicamba can exhibit a “dicamba intolerance phenotype” comprising malformation (epinasty) of the main stem and petioles upon exposure to dicamba. The epinastic growth habit of such “dicamba intolerant” transgenic plants is manifest in pronounced bending/twisting of the main stem and petioles. In dicamba intolerant transgenic soybean plants exposed to dicamba, the upper nodes and petioles may die, but lower portion of the plant may remain vegetative and new growth can be limited. However, other soybean plant varieties containing the same transgene that confers resistance to dicamba do not exhibit the dicamba intolerance phenotype when co-exposed to the same dosage of dicamba. The dicamba intolerance phenotype can be observed within approximately 2 to 10 days after herbicide application in certain soybean varieties comprising the transgene that confers resistance to dicamba. The dicamba intolerance phenotype is undesirable as it can lead to reduced yield in certain transgenic soybean plant varieties exposed to dicamba.
Although the dicamba intolerance phenotype can be observed within approximately 2 to 10 days after dicamba application in certain soybean varieties comprising the transgene that confers dicamba resistance, distinct soybean varieties that comprise the same dicamba resistance transgene integrated at the same chromosomal locus (i.e. the same transgenic event) can show various degrees of dicamba intolerance upon exposure to high doses of dicamba. Some varieties comprising the dicamba resistance transgene insertion are highly tolerant to high dosages of dicamba, showing no dicamba intolerance phenotype (i.e. a “dicamba tolerance phenotype”), while other varieties comprising the same dicamba resistance transgene insertion are highly susceptible to high dosages of dicamba, showing a severe dicamba intolerance phenotype. Provided herein are soybean plants comprising an introgressed genomic region associated with a dicamba tolerance phenotype. Also provided herein are markers that reside outside of a genomic region associated with a dicamba tolerance phenotype and that facilitate breeding activities that include, but are not limited to, introgression of this genomic region. Markers and specific alleles thereof that are associated with a dicamba tolerance phenotype are also provided. Methods of obtaining a soybean plant that exhibits a dicamba tolerance phenotype and methods of obtaining a soybean plant comprising in its genome at least one dicamba tolerance locus are also provided. Methods that provide for the introgression of a genomic region associated with a dicamba tolerance phenotype into soybean germplasm that has a genomic region associated with a dicamba intolerance phenotype are also provided. Identification of molecular markers associated with loci that confer the dicamba tolerance phenotype has significant economic value. By using markers associated with the dicamba tolerance trait, breeders can select soybean varieties with the favorable alleles (i.e. alleles that are not associated with the dicamba intolerance trait) for use in trait integration. They can also use the markers to help them eliminate unfavorable alleles (i.e. alleles that are associated with the dicamba intolerance trait) in soybeans. In certain embodiments, commercially desirable transgenic soybean lines that carry a genomic region that is associated with a “dicamba tolerance” phenotype and tolerate high dosages of dicamba are thus provided.
It has also been surprisingly observed that soybean plants comprising the dicamba tolerance loci, a transgene conferring resistance to dicamba, and a transgene conferring resistance to glyphosate also exhibit improved reproductive tolerance to glyphosate application relative to plants with the same two transgenes that lack the dicamba tolerance loci. Although the glyphosate reproductive intolerance phenotype can be observed after late stage (i.e. V6/R1) glyphosate application in certain soybean varieties comprising the transgenes that confer dicamba and glyphosate resistance, distinct soybean varieties that comprise the same dicamba and glyphosate resistance transgene integrated at the same chromosomal loci (i.e. the same transgenic events) can show various degrees of glyphosate reproductive intolerance (i.e. varying degrees of sterility) upon such exposure to glyphosate. Some varieties comprising the dicamba and glyphosate resistance transgene insertions are highly tolerant to late stage glyphosate application, showing no sterility phenotype (i.e. a “glyphosate reproductive intolerance phenotype”), while other varieties comprising the same dicamba and glyphosate resistance transgene insertions are highly susceptible to late stage glyphosate application, showing varying levels of sterility. Provided herein are soybean plants comprising an introgressed genomic region associated with a dicamba tolerance phenotype that also provide for reproductive tolerance to glyphosate. Also provided herein are markers that reside outside of a genomic region associated with a dicamba tolerance/reproductive tolerance to glyphosate phenotype and that facilitate breeding activities that include, but are not limited to, introgression of this genomic region. Markers and specific alleles thereof that are associated with a dicamba tolerance/reproductive tolerance to glyphosate are also provided. Methods of obtaining a soybean plant that exhibits reproductive tolerance to glyphosate and methods of obtaining a soybean plant comprising in its genome at least one dicamba tolerance/reproductive tolerance to glyphosate locus are also provided. Methods that provide for the introgression of a genomic region associated with reproductive tolerance to glyphosate into soybean germplasm that has a genomic region associated with a reproductive tolerance to glyphosate are also provided. Identification of molecular markers associated with loci that confer reproductive tolerance to glyphosate has significant economic value. By using markers associated with the reproductive tolerance to glyphosate trait, breeders can select soybean varieties with the favorable alleles (i.e. alleles that are not associated with the glyphosate reproductive intolerance trait) for use in trait integration. They can also use the markers to help them eliminate unfavorable alleles (i.e. alleles that are associated with the glyphosate reproductive intolerance trait) in soybeans. In certain embodiments, commercially desirable transgenic soybean lines that carry a genomic region that is associated with a “glyphosate reproductive tolerance” phenotype and tolerate late stage (i.e. V6/R1) application of glyphosate are thus provided.
Methods of identifying a soybean plant that comprises a genotype associated with a dicamba tolerance phenotype and/or a glyphosate reproductive tolerance phenotype are thus provided.
In certain embodiments, the plurality of soybean plants comprises a population that is obtained by: i) crossing a parent plant comprising at least one dicamba tolerance locus with a parent plant comprising at least one dicamba intolerance locus; or, ii) obtaining seed or progeny from a parental plant segregating for at least one dicamba tolerance locus. In certain embodiments, the population contains plants that comprise a transgene that confers resistance to dicamba. In certain embodiments, the aforementioned methods can further comprise the step of assaying for the presence of at least one additional marker, where the additional marker is either linked or unlinked to the linkage group L genomic region. In certain embodiments of the aforementioned methods, the plurality of soybean plants, the soybean plant, and/or progeny thereof are exposed to a dosage of dicamba sufficient to cause dicamba intolerance in a susceptible variety. In certain embodiments of the aforementioned methods, a plant that exhibits a dicamba tolerance phenotype is selected.
Also provided herewith are methods for producing a soybean plant comprising in its genome at least one introgressed dicamba tolerance locus. Also provided herewith are soybean plants comprising an introgressed dicamba tolerance locus made by the aforementioned methods. In certain embodiments, a soybean plant comprising an introgressed dicamba tolerance locus and one or more polymorphic loci comprising alleles or combinations of alleles that are not found in a dicamba tolerant soybean variety and that are linked to the introgressed dicamba tolerance locus, where the plant is produced by the aforementioned methods are provided.
Also provided are soybean plants comprising an introgressed dicamba tolerance locus and one or more polymorphic loci comprising alleles or combinations of alleles that are not found in a dicamba tolerant soybean variety and that are linked to the introgressed dicamba tolerance locus.
Methods of identifying a soybean plant that comprises a genotype associated with dicamba tolerance and/or reproductive tolerance to glyphosate are thus provided. In certain embodiments, the methods can comprise detecting in a soybean plant an allele in at least one genetic locus associated with dicamba tolerance and/or reproductive tolerance to glyphosate, where the genetic locus is in a linkage group L genomic region flanked by loci M0205928 (SEQ ID NO: 4) and BU765955 (SEQ ID NO: 12), and denoting that the plant comprises a genotype associated with dicamba tolerance. In certain embodiments, the methods can further comprise the step of selecting the denoted plant from a population of plants. In certain embodiments, the plant comprises a transgene that confers resistance to dicamba and/or a transgene that confers resistance to glyphosate. In certain embodiments, the soybean plant or progeny thereof is exposed to a dosage of dicamba sufficient to cause a deleterious effect in a susceptible variety comprising the transgene and/or is exposed to a dosage of glyphosate sufficient to cause sterility in a susceptible variety comprising the transgene(s). In certain embodiments of any of the aforementioned methods, a plant that exhibits dicamba tolerance and/or reproductive tolerance to glyphosate is selected. In certain embodiments of any of the aforementioned methods, a genotype associated with a dicamba tolerance comprises at least one polymorphic allele of at least one marker in a first sub-region of the linkage group L region that is flanked by loci M0205928 (SEQ ID NO: 4) and M0129138 (SEQ ID NO: 6) and/or at least one polymorphic allele of at least one marker in a second sub-region of the linkage group L region that is flanked by loci BU551363 (SEQ ID NO: 9) and BU765955 (SEQ ID NO: 12) and/or at least one polymorphic allele of at least one marker in a third sub-region of the linkage group L region that is flanked by loci BU55345 (SEQ ID NO:7) and M0114388 (SEQ ID NO:8) is provided. In certain embodiments of any of the aforementioned methods, the genotype associated with dicamba tolerance comprises at least one polymorphic allele of at least one marker in the linkage group L region selected from the group consisting of a TT allele M0205350 (SEQ ID NO: 10), a TT allele of M0101742 (SEQ ID NO: 5), a CC allele of M0102027 (SEQ ID NO: 11), and an AA allele of NGMAX008197032 (SEQ ID NO:52).
Methods for obtaining a soybean plant comprising in its genome at least one dicamba tolerance locus are also provided. In certain embodiments, these methods can compromise the steps of: (a) genotyping a plurality of soybean plants with respect to at least one genetic locus in a linkage group L genomic region flanked by loci M0205928 (SEQ ID NO: 4) and BU765955 (SEQ ID NO: 12); and, (b) selecting a soybean plant comprising in its genome at least one genetic locus comprising a genotype associated with dicamba tolerance. In certain embodiments of these methods, the genotype associated with dicamba tolerance comprises at least one polymorphic allele of at least one marker in a first sub-region of the linkage group L region flanked by loci M0205928 (SEQ ID NO: 4) and M0129138 (SEQ ID NO: 6) and/or at least one polymorphic allele of at least one marker in a second sub-region of the linkage group L region that is flanked by loci BU551363 (SEQ ID NO: 9) and BU765955 (SEQ ID NO: 12) and/or at least one polymorphic allele of at least one marker in a third sub-region of the linkage group L region that is flanked by loci BU55345 (SEQ ID NO:7) and M0114388 (SEQ ID NO:8). In certain embodiments of any of these aforementioned methods, the genotype associated with dicamba tolerance comprises at least one polymorphic allele of at least one marker in the first linkage group L region, the first sub-region, the second sub-region, or the third sub-region, where the marker is selected from the group consisting of a TT allele M0205350 (SEQ ID NO: 10), a TT allele of M0101742 (SEQ ID NO: 5), a CC allele of M0102027 (SEQ ID NO: 11), and an AA allele of NGMAX008197032 (SEQ ID NO:52). In certain embodiments of these methods, the plurality of soybean plants comprises a population that is obtained by: i) crossing a parent plant comprising at least one dicamba tolerance locus with a parent plant comprising at least one dicamba sensitivity locus; or, ii) obtaining seed or progeny from a parental plant segregating for at least one dicamba tolerance locus. In certain embodiments of these methods, the population contains plants that comprise at least one transgene that confers resistance to dicamba and/or a transgene that confers resistance to glyphosate. In certain embodiments of any of the aforementioned methods, the methods can further comprise the step of assaying for the presence of at least one additional marker, where the additional marker is either linked or unlinked to the linkage group L genomic region. In certain embodiments of any of the aforementioned methods, the plurality of soybean plants, the soybean plant, and/or progeny thereof are exposed to a dosage of dicamba sufficient to cause a deleterious effect in a susceptible variety comprising the transgene and/or is exposed to a dosage of glyphosate sufficient to cause sterility in a susceptible variety comprising the transgene. In certain embodiments of any of the aforementioned methods, a plant that exhibits dicamba tolerance and/or reproductive tolerance to glyphosate is selected.
Methods for producing a soybean plant comprising in its genome at least one introgressed dicamba tolerance locus are also provided. In certain embodiments, these methods comprise the steps of: (a) crossing a first soybean plant with a dicamba tolerance locus with a second soybean plant comprising: a dicamba sensitivity locus in a first linkage group L genomic region flanked by loci M0205928 (SEQ ID NO: 4) and M0129138 (SEQ ID NO: 6) and/or at least one polymorphic allele of at least one marker in a second sub-region of the linkage group L region that is flanked by loci BU551363 (SEQ ID NO: 9) and BU765955 (SEQ ID NO: 12) and/or at least one polymorphic allele of at least one marker in a third sub-region of the linkage group L region that is flanked by loci BU55345 (SEQ ID NO:7) and M0114388 (SEQ ID NO:8) and at least one linked polymorphic locus not present in the first soybean plant to obtain a population segregating for the dicamba tolerance loci and the linked polymorphic locus; (b) detecting at least two polymorphic nucleic acids in at least one soybean plant from the population, where at least one of the polymorphic nucleic acids is located in the first linkage group L region and/or the second linkage group L region and where at least one of the polymorphic amino acids is a linked polymorphic locus not present in the first soybean plant; and (c) selecting a soybean plant comprising a genotype associated with dicamba tolerance and at least one linked marker found in the second soybean plant comprising a dicamba sensitivity locus but not in the first soybean plant, thereby obtaining a soybean plant comprising in its genome at least one introgressed dicamba tolerance locus. In certain embodiments of these methods, at least one of the first or the second soybean plants comprises a transgene that confers resistance to dicamba and/or a transgene that confers resistance to glyphosate. In certain embodiments of these methods, the population, the selected soybean plant, and/or progeny of selected soybean plant is exposed to a dosage of dicamba sufficient to cause a deleterious effect in a susceptible variety comprising the transgene and/or is exposed to a dosage of glyphosate sufficient to cause sterility in a susceptible variety comprising the transgene. In certain embodiments of these methods, the polymorphic nucleic acid detected in step (b) is detected with at least one marker selected from the group consisting of M0205350 (SEQ ID NO: 10), M0101742 (SEQ ID NO: 5), M0102027 (SEQ ID NO: 11), and NGMAX008197032 (SEQ ID NO:52). In certain embodiments of these methods, the polymorphic nucleic acid detected in step (b) comprises a TT allele of M0205350 (SEQ ID NO: 10), a TT allele of M0101742 (SEQ ID NO: 5), a CC allele of M0102027 (SEQ ID NO: 11), and an AA allele of NGMAX008197032 (SEQ ID NO:52). In certain embodiments of these methods, the polymorphic nucleic acid detected in step (b) is detected with marker M0205350 (SEQ ID NO: 10) or M0102027 (SEQ ID NO: 11). In certain embodiments of these methods, the polymorphic nucleic acids are detected with marker M0101742 (SEQ ID NO: 5). In certain embodiments of these methods, the polymorphic nucleic acids are detected with marker NGMAX008197032 (SEQ ID NO:52). In certain embodiments of any of the aforementioned methods, the linked polymorphic locus is detected with a genotypic marker, a phenotypic marker, or both. In certain embodiments of these methods, the linked polymorphic locus is detected with a marker that is located within about 1000, 500, 100, 40, 20, 10, or 5 kilobases (Kb) of the dicamba tolerance locus. In certain embodiments of these methods, the linked polymorphic locus is detected with at least one marker selected from the group consisting of asmbl_11856 (SEQ ID NO: 1), TC122822 (SEQ ID NO: 2), BI967232 (SEQ ID NO: 3), M0205537 (SEQ ID NO: 15), M0202715 (SEQ ID NO: 16), M0206286 (SEQ ID NO: 17), M0206054 (SEQ ID NO: 18), and M0205375 (SEQ ID NO: 19).
Transgenic soybean plants comprising introgressed linkage group L regions comprising at least one polymorphic allele of at least one marker in a first sub-region of the linkage group L region that flanked by loci M0205928 (SEQ ID NO: 4) and M0129138 (SEQ ID NO: 6) and/or at least one polymorphic allele of at least one marker in a second sub-region of the linkage group L region that is flanked by loci BU551363 (SEQ ID NO: 9) and BU765955 (SEQ ID NO: 12) and/or at least one polymorphic allele of at least one marker in a third sub-region of the linkage group L region that is flanked by loci BU55345 (SEQ ID NO:7) and M0114388 (SEQ ID NO:8), where the polymorphic alleles are associated with dicamba tolerance and/or reproductive tolerance to glyphosate, and where the plant comprises a transgene that confers resistance to dicamba are also provided. In certain embodiments, the polymorphic alleles comprise a TT allele of M0205350 (SEQ ID NO: 10), a TT allele of M0101742 (SEQ ID NO: 5), a CC allele of M0102027 (SEQ ID NO: 11), and an AA allele of NGMAX008197032 (SEQ ID NO:52). In certain embodiments, the transgenic plant exhibits dicamba tolerance. In certain embodiments, the transgenic plant further comprises a transgene that confers resistance to glyphosate and exhibits reproductive tolerance to glyphosate. In certain embodiments of any one of the aforementioned methods, the plant further comprises at least one of a 2,4-D, glufosinate, bromoxynil, acetolactate synthase (ALS), acetyl CoA carboxylase (ACCase), hydroxyphenyl pyruvate dioxygenase (HPPD), or sulfonylurea herbicide resistance transgenes and/or at least one transgene selected from the group of transgenes conferring insect resistance, nematode resistance, fungal resistance, an improvement in seed oil quantity, an improvement in seed oil quality, abiotic stress resistance, and intrinsic yield increases. In certain embodiments, the insect resistance conferring transgene is a transgene that expresses an insecticidal Bacillus thuringiensis protein.
Also provided herein are soybean plants comprising a dicamba tolerance locus, a transgene conferring resistance to glyphosate, a transgene conferring resistance to dicamba, where the plants exhibit both improved dicamba tolerance and improved reproductive tolerance to glyphosate relative to soybean plants comprising the same two transgenes but lacking the dicamba tolerance locus. Such improved reproductive tolerance to glyphosate is reflected in reduced sterility when the plants are exposed to glyphosate.
In certain embodiments, the dicamba tolerance locus provided herein can provide for improved performance of additional combinations of transgenic traits (i.e. “stacked transgenic traits”) in soybean plants. In such embodiments, the dicamba tolerance locus provided herein can be alternatively referred to and considered a “stacked transgenic trait improvement” locus. Allele(s) of the dicamba tolerance locus or “stacked transgenic trait improvement” locus that do not confer such dicamba tolerance or such stacked transgenic trait improvements are referred to herein as dicamba sensitivity or “stacked transgenic trait sensitivity” loci. Transgenic plants comprising the stacked transgenic trait improvement locus provided herein exhibit improved performance of both transgenes present in the transgenic plant relative to plants comprising the same two transgenes that lack the stacked transgenic trait improvement locus. Such improved performance can manifest in any of enhanced transgenic trait performance, increased transgene efficacy, and/or increased transgene expression. Transgenic plants comprising the stacked trait improvement locus and two transgenes are thus provided herein. Thus, in certain embodiments the two independent and distinct transgenes that exhibit improved performance in the presence of the stacked transgenic trait improvement locus both contribute to the same trait. In certain embodiments, this same trait is selected from the group consisting of resistance to a single herbicide, resistance to an insect, resistance to a nematode, resistance to a fungal disease, resistance to an abiotic stress, an improvement in seed oil quantity, an improvement in seed oil quality, and intrinsic yield increases. In certain embodiments, the two transgenes can contribute to the same herbicide resistance trait where the herbicide resistance is selected from the group consisting of glyphosate, dicamba, 2,4-D, glufosinate, bromoxynil, synthetic auxins, and inhibitors of acetolactate synthase (ALS), acetyl CoA carboxylase (ACCase) and hydroxyphenyl pyruvate dioxygenase (HPPD) resistance. In certain embodiments, the two transgenes can contribute to the same insect, fungal, or nematode resistance trait where the resistance to the same insect, fungal, or nematode pest is by a different mode of action to provide for improved pest resistance management. In other embodiments, the two transgenes can be two independent and distinct transgenes that encode different genes but contribute to a different trait. In certain embodiments, this different trait is independently selected from the group consisting of resistance to one or more herbicide(s), resistance to one or more insect(s), resistance to one or more nematode(s), resistance to one or more fungal disease(s), resistance to one or more abiotic stress(es), one or more improvement(s) in seed oil quantity or quantities, one or more improvement(s) in seed oil quality or qualities, intrinsic yield increases, and combinations thereof. In certain embodiments the stacked trait improvement locus is an herbicide tolerance locus that provides for improved tolerance to at least two distinct herbicides in plants comprising at least two transgenes that respectively confer resistance to those two herbicides. In certain embodiments, the herbicide tolerance locus provides for improved tolerance to at least two herbicides selected from the group consisting of glyphosate, dicamba, 2,4-D, glufosinate, bromoxynil, synthetic auxins, and inhibitors of acetolactate synthase (ALS), acetyl CoA carboxylase (ACCase) and hydroxyphenyl pyruvate dioxygenase (HPPD) resistance in plants comprising at least two transgenes that confer resistance to those two herbicides. In certain embodiments, the herbicide tolerance locus confers improved tolerance to dicamba, improved reproductive tolerance to glyphosate, and improved tolerance to a synthetic auxin that includes, but is not limited to 2,4-D, in a plant comprising transgenes that confer resistance to dicamba, glyphosate, and the synthetic auxin that includes, but is not limited to 2,4-D. In certain embodiments, the two transgenes can confer a distinct herbicide resistance trait where the herbicide resistance is selected from the group consisting of glyphosate, dicamba, 2,4-D, glufosinate, bromoxynil, synthetic auxins other than 2,4-D, acetolactate synthase (ALS), acetyl CoA carboxylase (ACCase) and hydroxyphenyl pyruvate dioxygenase (HPPD) resistance. Provided herein are soybean plants comprising any combination of a stacked trait improvement locus and at least two transgenes conferring herbicide tolerance selected from the group consisting of glyphosate, dicamba, 2,4-D, glufosinate, bromoxynil, synthetic auxins, and inhibitors of acetolactate synthase (ALS), acetyl CoA carboxylase (ACCase) and hydroxyphenyl pyruvate dioxygenase (HPPD). In certain embodiments, soybean plants comprising an introgressed stacked trait improvement locus, at least one transgene selected from the group consisting of glyphosate, dicamba, 2,4-D, glufosinate, bromoxynil, synthetic auxins other than 2,4-D, acetolactate synthase (ALS), acetyl CoA carboxylase (ACCase), hydroxyphenyl pyruvate dioxygenase (HPPD), and sulfonylurea herbicide resistance transgenes, and at least one transgene selected from the group of transgenes conferring insect resistance, nematode resistance, fungal resistance, an improvement in seed oil quantity, an improvement in seed oil quality, abiotic stress resistance, and intrinsic yield increases are provided. In still other embodiments, soybean plants comprising an introgressed stacked trait improvement locus, at least one transgene selected from the group consisting of glyphosate, dicamba, 2,4-D, and at least one transgene conferring resistance to an insect are provided. In still other embodiments, soybean plants comprising an introgressed stacked trait improvement locus, at least one transgene selected from the group consisting of glyphosate, dicamba, glufosinate, and 2,4-D resistance conferring transgenes, and at least one transgene conferring resistance to an insect that encodes a Bacillus thuringiensis toxin are provided. In certain embodiments, soybean plants comprising an introgressed stacked trait improvement locus, a glyphosate and a dicamba resistance conferring transgene, and a cry1Ac insect resistance transgene are provided. In still other embodiments, soybean plants comprising at least one herbicide resistance transgene selected from the group consisting of a dicamba resistance conferring transgene. a glyphosate resistance conferring transgene, a 2,4-D resistance conferring transgene, and a glufosinate resistance conferring transgene and/or at least one transgene encoding a product that confers insect resistance selected from the group consisting of a dsRNA that inhibits a target gene of an insect pest, a patatin, a Bacillus thuringiensis insecticidal protein, a Xenorhabdus insecticidal protein, a Photorhabdus insecticidal protein, a Bacillus laterosporous insecticidal protein, a Bacillus sphaericus insecticidal protein, and a lignin are provided.
Methods of identifying a soybean plant that comprises a genotype associated with stacked transgenic trait improvement are thus provided. In certain embodiments, the methods can comprise detecting in a soybean plant an allele in at least one genetic locus associated with stacked transgenic trait improvement, where the genetic locus is in a linkage group L genomic region flanked by loci M0205928 (SEQ ID NO: 4) and BU765955 (SEQ ID NO: 12), and denoting that the plant comprises a genotype associated with stacked transgenic trait improvement. In certain embodiments, the methods can further comprise the step of selecting the denoted plant from a population of plants. In certain embodiments, the plant comprises at least two transgenes that contribute to the same trait. In certain embodiments, this same trait is selected from the group consisting of resistance to a single herbicide, resistance to an insect, resistance to a nematode, resistance to a fungal disease, resistance to an abiotic stress, an improvement in seed oil quantity, an improvement in seed oil quality, and intrinsic yield increases. In certain embodiments, the plant comprises at least two transgenes that contribute to different traits. In certain embodiments, this different trait is independently selected from the group consisting of resistance to one or more herbicide(s), resistance to one or more insect(s), resistance to one or more nematode(s), resistance to one or more fungal disease(s), resistance to one or more abiotic stress(es), one or more improvement(s) in seed oil quantity or quantities, one or more improvement(s) in seed oil quality or qualities, intrinsic yield increases, and combinations thereof. In certain embodiments, the soybean plant or progeny thereof is exposed to a dosage of an herbicide sufficient to cause a deleterious effect in a susceptible variety comprising the transgene conferring resistance to that herbicide but lacking the stacked transgenic trait improvement locus. In certain embodiments of any of the aforementioned methods, a plant that exhibits stacked transgenic trait improvement is selected. In certain embodiments of any of the aforementioned methods, a genotype associated with stacked transgenic trait improvement comprises at least one polymorphic allele of at least one marker in a first sub-region of the linkage group L region that is flanked by loci M0205928 (SEQ ID NO: 4) and M0129138 (SEQ ID NO: 6) and/or at least one polymorphic allele of at least one marker in a second sub-region of the linkage group L region that is flanked by loci BU551363 (SEQ ID NO: 9) and BU765955 (SEQ ID NO: 12) and/or at least one polymorphic allele of at least one marker in a third sub-region of the linkage group L region that is flanked by loci BU55345 (SEQ ID NO:7) and M0114388 (SEQ ID NO:8) is provided. In certain embodiments of any of the aforementioned methods, the genotype associated with stacked transgenic trait improvement comprises at least one polymorphic allele of at least one marker in the linkage group L region selected from the group consisting of a TT allele M0205350 (SEQ ID NO: 10), a TT allele of M0101742 (SEQ ID NO: 5), a CC allele of M0102027 (SEQ ID NO: 11), and an AA allele of NGMAX008197032 (SEQ ID NO:52).
Methods for obtaining a soybean plant comprising in its genome at least one stacked transgenic trait improvement locus are also provided. In certain embodiments, these methods can compromise the steps of: (a) genotyping a plurality of soybean plants with respect to at least one genetic locus in a linkage group L genomic region flanked by loci M0205928 (SEQ ID NO: 4) and BU765955 (SEQ ID NO: 12); and, (b) selecting a soybean plant comprising in its genome at least one genetic locus comprising a genotype associated with stacked transgenic trait improvement. In certain embodiments, the plant comprises at least two transgenes that contribute to the same trait. In certain embodiments, this same trait is selected from the group consisting of resistance to a single herbicide, resistance to an insect, resistance to a nematode, resistance to a fungal disease, resistance to an abiotic stress, an improvement in seed oil quantity, an improvement in seed oil quality, and intrinsic yield increases. In certain embodiments, the plant comprises at least two transgenes that contribute to different traits. In certain embodiments, this different trait is independently selected from the group consisting of resistance to one or more herbicide(s), resistance to one or more insect(s), resistance to one or more nematode(s), resistance to one or more fungal disease(s), resistance to one or more abiotic stress(es), one or more improvement(s) in seed oil quantity or quantities, one or more improvement(s) in seed oil quality or qualities, intrinsic yield increases, and combinations thereof. In certain embodiments of these methods, the genotype associated with stacked transgenic trait improvement comprises at least one polymorphic allele of at least one marker in a first sub-region of the linkage group L region flanked by loci M0205928 (SEQ ID NO: 4) and M0129138 (SEQ ID NO: 6) and/or at least one polymorphic allele of at least one marker in a second sub-region of the linkage group L region that is flanked by loci BU551363 (SEQ ID NO: 9) and BU765955 (SEQ ID NO: 12) and/or at least one polymorphic allele of at least one marker in a third sub-region of the linkage group L region that is flanked by loci BU55345 (SEQ ID NO:7) and M0114388 (SEQ ID NO:8). In certain embodiments of any of these aforementioned methods, the genotype associated with stacked transgenic trait improvement comprises at least one polymorphic allele of at least one marker in the first linkage group L region, the first sub-region, the second sub-region, the third sub-region, where the marker is selected from the group consisting of a TT allele M0205350 (SEQ ID NO: 10), a TT allele of M0101742 (SEQ ID NO: 5), a CC allele of M0102027 (SEQ ID NO: 11), and an AA allele of NGMAX008197032 (SEQ ID NO:52). In certain embodiments of these methods, the plurality of soybean plants comprises a population that is obtained by: i) crossing a parent plant comprising at least one stacked transgenic trait improvement locus with a parent plant lacking a stacked transgenic trait improvement locus; or, ii) obtaining seed or progeny from a parental plant segregating for at least one stacked transgenic trait improvement locus. In certain embodiments of any of the aforementioned methods, the methods can further comprise the step of assaying for the presence of at least one additional marker, where the additional marker is either linked or unlinked to the linkage group L genomic region. In certain embodiments of any of the aforementioned methods, the plurality of soybean plants, the soybean plant, and/or progeny thereof are exposed to a dosage of an herbicide sufficient to cause a deleterious effect in a susceptible variety comprising the transgene conferring resistance to that herbicide but lacking the stacked transgenic trait improvement locus. In certain embodiments of any of the aforementioned methods, a plant that exhibits stacked transgenic trait improvement is selected.
Methods for producing a soybean plant comprising in its genome at least one introgressed stacked transgenic trait improvement locus are also provided. In certain embodiments, these methods comprise the steps of (a) crossing a first soybean plant with a stacked transgenic trait improvement locus with a second soybean plant lacking a stacked transgenic trait improvement locus in a first linkage group L genomic region flanked by loci M0205928 (SEQ ID NO: 4) and M0129138 (SEQ ID NO: 6) and/or at least one polymorphic allele of at least one marker in a second sub-region of the linkage group L region that is flanked by loci BU551363 (SEQ ID NO: 9) and BU765955 (SEQ ID NO: 12) and/or at least one polymorphic allele of at least one marker in a third sub-region of the linkage group L region that is flanked by loci BU55345 (SEQ ID NO:7) and M0114388 (SEQ ID NO:8) and at least one linked polymorphic locus not present in the first soybean plant to obtain a population segregating for the stacked transgenic trait improvement loci and the linked polymorphic locus; (b) detecting at least two polymorphic nucleic acids in at least one soybean plant from the population, where at least one of the polymorphic nucleic acids is located in the first linkage group L region and/or the second linkage group L region and where at least one of the polymorphic amino acids is a linked polymorphic locus not present in the first soybean plant; and (c) selecting a soybean plant comprising a genotype associated with stacked transgenic trait improvement and at least one linked marker found in the second soybean plant lacking the stacked transgenic trait improvement locus but not found in the first soybean plant, thereby obtaining a soybean plant comprising in its genome at least one introgressed stacked transgenic trait improvement locus. In certain embodiments, the first and/or second plant comprises at least two transgenes that contribute to the same trait. In certain embodiments, this same trait is selected from the group consisting of resistance to a single herbicide, resistance to an insect, resistance to a nematode, resistance to a fungal disease, resistance to an abiotic stress, an improvement in seed oil quantity, an improvement in seed oil quality, and intrinsic yield increases. In certain embodiments, the plant comprises at least two transgenes that contribute to different traits. In certain embodiments, this different trait is independently selected from the group consisting of resistance to one or more herbicide(s), resistance to one or more insect(s), resistance to one or more nematode(s), resistance to one or more fungal disease(s), resistance to one or more abiotic stress(es), one or more improvement(s) in seed oil quantity or quantities, one or more improvement(s) in seed oil quality or qualities, intrinsic yield increases, and combinations thereof. In certain embodiments of these methods, the population, the selected soybean plant, and/or progeny of selected soybean plant is exposed to a dosage of an herbicide sufficient to cause a deleterious effect in a susceptible variety comprising the transgene that confers resistance to the herbicide but lacking the stacked transgenic trait improvement locus. In certain embodiments of these methods, the polymorphic nucleic acid detected in step (b) is detected with at least one marker selected from the group consisting of M0205350 (SEQ ID NO: 10), M0101742 (SEQ ID NO: 5), M0102027 (SEQ ID NO: 11), and an AA allele of NGMAX008197032 (SEQ ID NO:52). In certain embodiments of these methods, the polymorphic nucleic acid detected in step (b) comprises a TT allele of M0205350 (SEQ ID NO: 10), a TT allele of M0101742 (SEQ ID NO: 5), a CC allele of M0102027 (SEQ ID NO: 11) and an AA allele of NGMAX008197032 (SEQ ID NO:52). In certain embodiments of these methods, the polymorphic nucleic acid detected in step (b) is detected with marker M0205350 (SEQ ID NO: 10), M0102027 (SEQ ID NO: 11) or NGMAX008197032 (SEQ ID NO: 52). In certain embodiments of these methods, the polymorphic nucleic acids are detected with marker M0101742 (SEQ ID NO: 5). In certain embodiments of these methods, the polymorphic nucleic acids are detected with marker NGMAX008197032 (SEQ ID NO:52). In certain embodiments of any of the aforementioned methods, the linked polymorphic locus is detected with a genotypic marker, a phenotypic marker, or both. In certain embodiments of these methods, the linked polymorphic locus is detected with a marker that is located within about 1000, 500, 100, 40, 20, 10, or 5 kilobases (Kb) of the stacked transgenic trait improvement locus. In certain embodiments of these methods, the linked polymorphic locus is detected with at least one marker selected from the group consisting of asmbl_11856 (SEQ ID NO: 1), TC122822 (SEQ ID NO: 2), BI967232 (SEQ ID NO: 3), M0205537 (SEQ ID NO: 15), M0202715 (SEQ ID NO: 16), M0206286 (SEQ ID NO: 17), M0206054 (SEQ ID NO: 18), and M0205375 (SEQ ID NO: 19).
Transgenic soybean plants comprising introgressed linkage group L regions comprising at least one polymorphic allele of at least one marker in a first sub-region of the linkage group L region that flanked by loci M0205928 (SEQ ID NO: 4) and M0129138 (SEQ ID NO: 6) and/or at least one polymorphic allele of at least one marker in a second sub-region of the linkage group L region that is flanked by loci BU551363 (SEQ ID NO: 9) and BU765955 (SEQ ID NO: 12) and/or at least one polymorphic allele of at least one marker in a third sub-region of the linkage group L region that is flanked by loci BU55345 (SEQ ID NO:7) and M0114388 (SEQ ID NO:8), where the polymorphic alleles are associated with a stacked transgenic trait improvement locus, and where the plant comprises: (a) at least two transgenes that contribute to the same trait; or, (b) at least two transgenes that contribute different traits. In certain embodiments, this same trait is selected from the group consisting of resistance to a single herbicide, resistance to an insect, resistance to a nematode, resistance to a fungal disease, resistance to an abiotic stress, an improvement in seed oil quantity, an improvement in seed oil quality, and intrinsic yield increases. In certain embodiments, this different trait is independently selected from the group consisting of resistance to one or more herbicide(s), resistance to one or more insect(s), resistance to one or more nematode(s), resistance to one or more fungal disease(s), resistance to one or more abiotic stress(es), one or more improvement(s) in seed oil quantity or quantities, one or more improvement(s) in seed oil quality or qualities, intrinsic yield increases, and combinations thereof. In certain embodiments, the polymorphic alleles comprise a TT allele of M0205350 (SEQ ID NO: 10), a TT allele of M0101742 (SEQ ID NO: 5), a CC allele of M0102027 (SEQ ID NO: 11), and an AA allele of NGMAX008197032 (SEQ ID NO:52). In certain embodiments, the transgenic plant exhibits a stacked transgenic trait improvement. In certain embodiments, the transgenic plant further comprises a transgene that confers resistance to glyphosate and exhibits reproductive tolerance to glyphosate. In certain embodiments of any one of the aforementioned methods, the plant further comprises at least one of a 2,4-D, glufosinate, bromoxynil, synthetic auxins other than 2,4-D, acetolactate synthase (ALS), acetyl CoA carboxylase (ACCase), hydroxyphenyl pyruvate dioxygenase (HPPD), or sulfonylurea herbicide resistance transgenes and/or at least one transgene selected from the group of transgenes conferring insect resistance, nematode resistance, fungal resistance, an improvement in seed oil quantity, an improvement in seed oil quality, abiotic stress resistance, and intrinsic yield increases.
Also provided herein are methods of identifying a soybean plant that comprises a genotype associated with stacked transgenic trait improvement, comprising: detecting in a soybean plant an allele in at least one genetic locus associated with stacked transgenic trait improvement, wherein the genetic locus is in a linkage group L genomic region flanked by loci M0205928 (SEQ ID NO: 4) and BU765955 (SEQ ID NO: 12), and denoting that the plant comprises a genotype associated with stacked transgenic trait improvement. In certain embodiments, the method further comprises the step of selecting the denoted plant from a population of plants and wherein the detection is performed either before or after the selection. In certain embodiments, the denoted plant comprises at least one transgene that confer resistance to an herbicide and is selected for improved tolerance to that herbicide. In certain embodiments, the selection comprises exposing the population of plants to a dosage of herbicide sufficient to cause a deleterious effect in a susceptible variety comprising the transgene that confers resistance to the herbicide. In certain embodiments: (i) the plants comprise an herbicide resistance transgene selected from the group consisting of a dicamba resistance conferring transgene, a glyphosate resistance conferring transgene, a 2,4-D resistance conferring transgene, and a glufosinate resistance conferring transgene; and (ii) the plants comprising the herbicide resistance transgene are exposed to a dosage of a corresponding herbicide selected from the group consisting of dicamba, glyphosate, glufosinate, and 2,4-D that is sufficient to cause a deleterious effect in a susceptible variety comprising the herbicide resistant transgene that confers resistance to the corresponding herbicide. In certain embodiments, the stacked transgenic trait improvement is independently selected from the group consisting of an improvement in transgene-mediated resistance to one or more herbicide(s), an improvement in transgene-mediated resistance to one or more insect(s), an improvement in transgene-mediated resistance to one or more nematode(s), an improvement in transgene-mediated resistance to one or more fungal disease(s), an improvement in transgene-mediated resistance to one or more abiotic stress(es), in one or more improvement(s) in transgene-mediated seed oil quantity trait(s), one or more improvement(s) in seed oil quality trait(s), an improvement in transgene-mediated intrinsic yield increases, and combinations thereof. In certain embodiments, the denoted plant comprises at least one herbicide resistance transgene and/or at least one insect resistance conferring transgene that encodes a Bacillus thuringiensis toxin. In certain embodiments, the denoted plant comprises at least one herbicide resistance transgene selected from the group consisting of a dicamba resistance conferring transgene. a glyphosate resistance conferring transgene, a 2,4-D resistance conferring transgene, and a glufosinate resistance conferring transgene and/or at least one transgene encoding a product that confers insect resistance selected from the group consisting of a dsRNA that inhibits a target gene of an insect pest, a patatin, a Bacillus thuringiensis insecticidal protein, a Xenorhabdus insecticidal protein, a Photorhabdus insecticidal protein, a Bacillus laterosporous insecticidal protein, a Bacillus sphaericus insecticidal protein, and a lignin. In certain embodiments of any of the preceding methods, the genotype associated with stacked transgenic trait improvement comprises at least one polymorphic allele of at least one marker in a first sub-region of the linkage group L region that is flanked by loci M0205928 (SEQ ID NO: 4) and M0129138 (SEQ ID NO: 6) and/or at least one polymorphic allele of at least one marker in a second sub-region of the linkage group L region that is flanked by loci BU551363 (SEQ ID NO: 9) and BU765955 (SEQ ID NO: 12) and/or at least one polymorphic allele of at least one marker in a third sub-region of the linkage group L region that is flanked by loci BU55345 (SEQ ID NO:7) and M0114388 (SEQ ID NO:8). In certain embodiments of any of the preceding methods, the genotype associated with stacked transgenic trait improvement comprises at least one polymorphic allele of at least one marker in the linkage group L region selected from the group consisting of a TT allele M0205350 (SEQ ID NO: 10), a TT allele of M0101742 (SEQ ID NO: 5), a CC allele of M0102027 (SEQ ID NO: 11), and an AA allele of NGMAX008197032 (SEQ ID NO:52).
Also provided are methods for obtaining a soybean plant comprising in its genome at least one stacked transgenic trait improvement locus, compromising the steps of genotyping a plurality of soybean plants with respect to at least one genetic locus in a linkage group L genomic region flanked by loci M0205928 (SEQ ID NO: 4) and BU765955 (SEQ ID NO: 12); and selecting a soybean plant comprising in its genome at least one genetic locus comprising a genotype associated with stacked transgenic trait improvement. In certain embodiments of the methods, the genotype associated with stacked transgenic trait improvement comprises at least one polymorphic allele of at least one marker in a first sub-region of the linkage group L region flanked by loci M0205928 (SEQ ID NO: 4) and M0129138 (SEQ ID NO: 6) and/or at least one polymorphic allele of at least one marker in a second sub-region of the linkage group L region that is flanked by loci BU551363 (SEQ ID NO: 9) and BU765955 (SEQ ID NO: 12) and/or at least one polymorphic allele of at least one marker in a third sub-region of the linkage group L region that is flanked by loci BU55345 (SEQ ID NO:7) and M0114388 (SEQ ID NO:8). In certain embodiments of any of the preceding methods, the genotype associated with stacked transgenic trait improvement comprises at least one polymorphic allele of at least one marker in the first linkage group L region, the first sub-region, or the second sub-region, wherein the marker is selected from the group consisting of a TT allele M0205350 (SEQ ID NO: 10), a TT allele of M0101742 (SEQ ID NO: 5), a CC allele of M0102027 (SEQ ID NO: 11), and an AA allele of NGMAX008197032 (SEQ ID NO:52). In certain embodiments of the methods, the plurality of soybean plants comprises a population that is obtained by: i) crossing a parent plant comprising at least one stacked transgenic trait improvement locus with a parent plant comprising at least one stacked transgenic trait sensitivity locus; or, ii) obtaining seed or progeny from a parental plant segregating for at least one stacked transgenic trait improvement locus. In certain embodiments of the methods, the population contains plants that comprise at least one transgene that confers resistance to an herbicide and the stacked transgenic trait improvement comprises improved tolerance to a corresponding herbicide. In certain embodiments of any of the preceding methods, the methods further comprise the step of assaying for the presence of at least one additional marker, wherein the additional marker is either linked or unlinked to the linkage group L genomic region. In certain embodiments of any of the preceding methods, the plurality of soybean plants, the soybean plant, and/or progeny thereof are exposed to a dosage of herbicide sufficient to cause a deleterious effect in a susceptible variety comprising the transgene that confers resistance to the herbicide. In certain embodiments of any of the preceding methods, a plant that exhibits dicamba tolerance and/or reproductive tolerance to glyphosate and/or glufosinate tolerance and/or 2,4-D tolerance is selected. In certain embodiments of any of the preceding methods, the stacked transgenic trait improvement is selected from the group consisting of an improvement in transgene-mediated resistance to one or more herbicide(s), an improvement in transgene-mediated resistance to one or more insect(s), an improvement in transgene-mediated resistance to one or more nematode(s), an improvement in transgene-mediated resistance to one or more fungal disease(s), an improvement in transgene-mediated resistance to one or more abiotic stress(es), in one or more improvement(s) in transgene-mediated seed oil quantity trait(s), one or more improvement(s) in seed oil quality trait(s), an improvement in transgene-mediated intrinsic yield increases, and combinations thereof.
Also provided herein are methods for producing a soybean plant comprising in its genome at least one introgressed stacked transgenic trait improvement locus comprising the steps of: crossing a first soybean plant with a stacked transgenic trait improvement locus with a second soybean plant comprising: a stacked transgenic trait sensitivity locus in a first linkage group L genomic region flanked by loci M0205928 (SEQ ID NO: 4) and M0129138 (SEQ ID NO: 6) and/or at least one polymorphic allele of at least one marker in a second sub-region of the linkage group L region that is flanked by loci BU551363 (SEQ ID NO: 9) and BU765955 (SEQ ID NO: 12) and/or at least one polymorphic allele of at least one marker in a third sub-region of the linkage group L region that is flanked by loci BU55345 (SEQ ID NO:7) and M0114388 (SEQ ID NO:8) and at least one linked polymorphic locus not present in the first soybean plant to obtain a population segregating for the stacked transgenic trait improvement loci and the linked polymorphic locus; detecting at least two polymorphic nucleic acids in at least one soybean plant from the population, wherein at least one of the polymorphic nucleic acids is located in the first linkage group L region and/or the second linkage group L region and at least one of the polymorphic amino acids is a linked polymorphic locus not present in the first soybean plant; and selecting a soybean plant comprising a genotype associated with stacked transgenic trait improvement and at least one linked marker found in the second soybean plant comprising a stacked transgenic trait sensitivity locus but not in the first soybean plant, thereby obtaining a soybean plant comprising in its genome at least one introgressed stacked transgenic trait improvement locus. In certain embodiments of the methods, at least one of the first or the second soybean plants comprises a transgene that confers resistance to an herbicide. In certain embodiments of the methods, the population, the selected soybean plant, and/or progeny of selected soybean plant is exposed to a dosage of herbicide sufficient to cause a deleterious effect in a susceptible variety comprising the transgene that confers resistance to a corresponding herbicide. In certain embodiments of the methods, the polymorphic nucleic acid detected in step (b) is detected with at least one marker selected from the group consisting of M0205350 (SEQ ID NO: 10), M0101742 (SEQ ID NO: 5), M0102027 (SEQ ID NO: 11), and NGMAX008197032 (SEQ ID NO:52). In certain embodiments of the methods, the polymorphic nucleic acid detected in step (b) comprises a TT allele of M0205350 (SEQ ID NO: 10), a TT allele of M0101742 (SEQ ID NO: 5), a CC allele of M0102027 (SEQ ID NO: 11), and an AA allele of NGMAX008197032 (SEQ ID NO:52). In certain embodiments of the methods, the polymorphic nucleic acid detected in step (b) is detected with marker M0205350 (SEQ ID NO: 10), M0102027 (SEQ ID NO: 11), or marker NGMAX008197032 (SEQ ID NO:52). In certain embodiments of the methods, the polymorphic nucleic acids are detected with marker M0101742 (SEQ ID NO: 5). In certain embodiments of any of the preceding methods, the linked polymorphic locus is detected with a genotypic marker, a phenotypic marker, or both. In certain embodiments, the linked polymorphic locus is detected with a marker that is located within about 1000, 500, 100, 40, 20, 10, or 5 kilobases (Kb) of the stacked transgenic trait improvement locus. In certain embodiments, the linked polymorphic locus is detected with at least one marker selected from the group consisting of asmbl_11856 (SEQ ID NO: 1), TC122822 (SEQ ID NO: 2), BI967232 (SEQ ID NO: 3), M0205537 (SEQ ID NO: 15), M0202715 (SEQ ID NO: 16), M0206286 (SEQ ID NO: 17), M0206054 (SEQ ID NO:18), and M0205375 (SEQ ID NO: 19).
Also provided are transgenic soybean plants comprising introgressed linkage group L regions comprising at least one polymorphic allele of at least one marker in a first sub-region of the linkage group L region that flanked by loci M0205928 (SEQ ID NO: 4) and M0129138 (SEQ ID NO: 6) and/or at least one polymorphic allele of at least one marker in a second sub-region of the linkage group L region that is flanked by loci BU551363 (SEQ ID NO: 9) and BU765955 (SEQ ID NO: 12) and/or at least one polymorphic allele of at least one marker in a third sub-region of the linkage group L region that is flanked by loci BU55345 (SEQ ID NO:7) and M0114388 (SEQ ID NO:8), wherein the polymorphic alleles are associated with stacked transgenic trait improvement and wherein the plant comprises at least one transgene. In certain embodiments, the transgene confers resistance to an herbicide. In certain embodiments, the polymorphic alleles comprise a TT allele of M0205350 (SEQ ID NO: 10), a TT allele of M0101742 (SEQ ID NO: 5), a CC allele of M0102027 (SEQ ID NO: 11), and an AA allele of NGMAX008197032 (SEQ ID NO:52). In certain embodiments of any of the preceding methods, the plant exhibits tolerance to at least one herbicide. In certain embodiments, the plant comprises: i) a transgene that confers resistance to glyphosate and exhibits reproductive tolerance to glyphosate; and/or (ii) a dicamba resistance conferring transgene and exhibits dicamba tolerance; and/or (iii) a glufosinate resistance conferring transgene and exhibits glufosinate tolerance; and/or (iv) a 2,4-D resistance conferring transgene and exhibits 2,4-D tolerance. In certain embodiments, the plant comprises at least one transgene conferring resistance to a herbicide selected from the group consisting of dicamba, 2,4-D, glufosinate, bromoxynil, synthetic auxins other than 2,4-D, acetolactate synthase (ALS), acetyl CoA carboxylase (ACCase), hydroxyphenyl pyruvate dioxygenase (HPPD), and a sulfonylurea herbicide and/or at least one transgene selected from the group of transgenes conferring insect resistance, nematode resistance, fungal resistance, an improvement in seed oil quantity, an improvement in seed oil quality, abiotic stress resistance, and intrinsic yield increases. In certain embodiments, the plant comprises at least one herbicide resistance transgene and/or at least one insect resistance conferring transgene that encodes a Bacillus thuringiensis toxin. In certain embodiments, the plant comprises at least one herbicide resistance transgene selected from the group consisting of a dicamba resistance conferring transgene a glyphosate resistance conferring transgene, a 2,4-D resistance conferring transgene, and a glufosinate resistance conferring transgene and/or at least one transgene encoding a product that confers insect resistance selected from the group consisting of a dsRNA that inhibits a target gene of an insect pest, a patatin, a Bacillus thuringiensis insecticidal protein, a Xenorhabdus insecticidal protein, a Photorhabdus insecticidal protein, a Bacillus laterosporous insecticidal protein, a Bacillus sphaericus insecticidal protein, and a lignin.
Also provided herein are methods of identifying a transgenic soybean plant that comprises a genotype associated with stacked transgenic trait improvement, the method comprising: (a) scoring at least one transgenic plant in a population of transgenic soybean plants that had been exposed to dicamba for dicamba tolerance, the plants having a transgene that confers resistance to dicamba; and, (b) selecting a transgenic plant that exhibits dicamba tolerance, thereby identifying a transgenic soybean plant that comprises a genotype associated with stacked transgenic trait improvement. In certain embodiments, the population is segregating for to at least one genetic locus in a linkage group L genomic region flanked by loci M0205928 (SEQ ID NO: 4) and BU765955 (SEQ ID NO: 12) that is associated with stacked transgenic trait improvement. In certain embodiments, the method further comprises genotyping the selected soybean plant with respect to at least one genetic locus in a linkage group L genomic region flanked by loci M0205928 (SEQ ID NO: 4) and BU765955 (SEQ ID NO: 12). In certain embodiments, the selected transgenic plant further comprises a transgene that confers resistance to glyphosate and the selected transgenic plant or progeny thereof is scored for reproductive tolerance to glyphosate following exposure to glyphosate. In certain embodiments of any of the preceding methods, the methods further comprise exposing the population of transgenic soybean plants to dicamba. In certain embodiments of any of the preceding methods, dicamba tolerance is scored by determining a reduction in malformation when compared to a dicamba sensitive transgenic plant that comprises the transgene that confers resistance to dicamba. In certain embodiments of any of the preceding methods, the stacked transgenic trait improvement is selected from the group consisting of an improvement in transgene-mediated resistance to one or more herbicide(s), an improvement in transgene-mediated resistance to one or more insect(s), an improvement in transgene-mediated resistance to one or more nematode(s), an improvement in transgene-mediated resistance to one or more fungal disease(s), an improvement in transgene-mediated resistance to one or more abiotic stress(es), in one or more improvement(s) in transgene-mediated seed oil quantity trait(s), one or more improvement(s) in seed oil quality trait(s), an improvement in transgene-mediated intrinsic yield increases, and combinations thereof. In certain embodiments of any of the preceding methods, the selected plant comprises at least one additional herbicide resistance transgene selected from the group consisting of a glyphosate resistance conferring transgene, a 2,4-D resistance conferring transgene, and a glufosinate resistance conferring transgene and/or at least transgene encoding a product that confers insect resistance selected from the group consisting of a dsRNA that inhibits a target gene of an insect pest, a patatin, a Bacillus thuringiensis insecticidal protein, a Xenorhabdus insecticidal protein, a Photorhabdus insecticidal protein, a Bacillus laterosporous insecticidal protein, a Bacillus sphaericus insecticidal protein, and a lignin.
Also provided herein are methods of identifying a transgenic soybean plant that comprises a genotype associated with stacked transgenic trait improvement, comprising: (a) scoring at least one plant in a population of transgenic soybean plants that had been exposed to glyphosate for reproductive tolerance to glyphosate, wherein the plants comprise a transgene that confers resistance to glyphosate; and, (b) selecting a transgenic plant that exhibits reproductive tolerance to glyphosate, thereby identifying a transgenic soybean plant that comprises a genotype associated with stacked transgenic trait improvement. In certain embodiments, the population is segregating for to at least one genetic locus in a linkage group L genomic region flanked by loci M0205928 (SEQ ID NO: 4) and BU765955 (SEQ ID NO: 12) that is associated with stacked transgenic trait improvement. In certain embodiments, the method further comprises genotyping the selected soybean plant with respect to at least one genetic locus in a linkage group L genomic region flanked by loci M0205928 (SEQ ID NO: 4) and BU765955 (SEQ ID NO: 12). In certain embodiments of any of the preceding methods, the selected transgenic plant further comprises a transgene that confers resistance to dicamba and the selected transgenic plant or progeny thereof is scored for tolerance to dicamba following exposure to dicamba. In certain embodiments of any of the preceding methods, the methods further comprise exposing the population of transgenic soybean plants to glyphosate. In certain embodiments of any of the preceding methods, glyphosate reproductive tolerance is scored by determining a reduction in sterility when compared to a transgenic plant that exhibits glyphosate reproductive sensitivity and comprises the transgene that confers resistance to glyphosate. In certain embodiments of any of the preceding methods, the stacked transgenic trait improvement is selected from the group consisting of an improvement in transgene-mediated resistance to one or more herbicide(s), an improvement in transgene-mediated resistance to one or more insect(s), an improvement in transgene-mediated resistance to one or more nematode(s), an improvement in transgene-mediated resistance to one or more fungal disease(s), an improvement in transgene-mediated resistance to one or more abiotic stress(es), in one or more improvement(s) in transgene-mediated seed oil quantity trait(s), one or more improvement(s) in seed oil quality trait(s), an improvement in transgene-mediated intrinsic yield increases, and combinations thereof. In certain embodiments of any of the preceding methods, the selected plant comprises at least one additional herbicide resistance transgene selected from the group consisting of a dicamba resistance conferring transgene, a 2,4-D resistance conferring transgene, and a glufosinate resistance conferring transgene and/or at least one transgene encoding a product that confers insect resistance selected from the group consisting of a dsRNA that inhibits a target gene of an insect pest, a patatin, a Bacillus thuringiensis insecticidal protein, a Xenorhabdus insecticidal protein, a Photorhabdus insecticidal protein, a Bacillus laterosporous insecticidal protein, a Bacillus sphaericus insecticidal protein, and a lignin.
Also provided are methods of obtaining a transgenic soybean plant that comprises a genotype associated with stacked transgenic trait improvement, the methods comprising: exposing a population of transgenic soybean plants to an herbicide, wherein the plants have a transgene that confers resistance to the herbicide; observing herbicide tolerance exhibited by one or more soybean plants following exposure to the herbicide; and, (c) selecting a transgenic plant that exhibits herbicidetolerance, thereby obtaining a transgenic soybean plant that comprises a genotype associated with stacked transgenic trait improvement. In certain embodiments, the population is segregating for to at least one genetic locus in a linkage group L genomic region flanked by loci M0205928 (SEQ ID NO: 4) and BU765955 (SEQ ID NO: 12) that is associated with stacked transgenic trait improvement. In certain embodiments, the method further comprises genotyping the selected soybean plant with respect to at least one genetic locus in a linkage group L genomic region flanked by loci M0205928 (SEQ ID NO: 4) and BU765955 (SEQ ID NO: 12). In certain embodiments of any of the preceding methods, the transgene that confers resistance to the herbicide is selected from the group consisting of a dicamba resistance conferring transgene, a glyphosate resistance conferring transgene, a 2,4-D resistance conferring transgene, and a glufosinate resistance conferring transgene and the plants are exposed to the corresponding herbicide. In certain embodiments of any of the preceding methods, the transgene confers resistance to glyphosate and the selected transgenic plant or progeny thereof is scored for reproductive tolerance to glyphosate following exposure to glyphosate. In certain embodiments of any of the preceeding methods, the transgene confers resistance to dicamba and the selected transgenic plant or progeny thereof are scored for dicamba tolerance.
Also provided herein are methods of identifying a transgenic soybean plant that comprises a genotype associated with reproductive tolerance to glyphosate, the method comprising: (a) scoring at least one transgenic plant in a population of transgenic soybean plants that had been exposed to dicamba for dicamba tolerance, the plants having a transgene that confers resistance to dicamba; and, (b) selecting a transgenic plant that exhibits dicamba tolerance, thereby identifying a transgenic soybean plant that comprises a genotype associated with reproductive tolerance to glyphosate. In certain embodiments of the methods, the population is segregating for to at least one genetic locus in a linkage group L genomic region flanked by loci M0205928 (SEQ ID NO: 4) and BU765955 (SEQ ID NO: 12) that is associated with dicamba tolerance. In certain embodiments of the methods, the methods further comprise genotyping the selected soybean plant with respect to at least one genetic locus in a linkage group L genomic region flanked by loci M0205928 (SEQ ID NO: 4) and BU765955 (SEQ ID NO: 12). In certain embodiments of any of the aforementioned methods, the selected transgenic plant further comprises a transgene that confers resistance to glyphosate and wherein the selected transgenic plant or progeny thereof is scored for reproductive tolerance to glyphosate following exposure to glyphosate. In certain embodiments of any of the aforementioned methods, the methods further comprise exposing the population of transgenic soybean plants to dicamba. In certain embodiments of any of the aforementioned methods, the dicamba tolerance is scored by determining a reduction in malformation when compared to a dicamba sensitive transgenic plant that comprises the transgene that confers resistance to dicamba.
Also provided herein are methods of identifying a transgenic soybean plant that comprises a genotype associated with tolerance to dicamba, comprising: (a) scoring at least one plant in a population of transgenic soybean plants that had been exposed to glyphosate for reproductive tolerance to glyphosate, wherein the plants comprise a transgene that confers resistance to glyphosate; and, (b) selecting a transgenic plant that exhibits reproductive tolerance to glyphosate, thereby identifying a transgenic soybean plant that comprises a genotype associated with dicamba tolerance. In certain embodiments of the methods, the population is segregating for to at least one genetic locus in a linkage group L genomic region flanked by loci M0205928 (SEQ ID NO: 4) and BU765955 (SEQ ID NO: 12) that is associated with dicamba tolerance. In certain embodiments of the methods, the method further comprises genotyping the selected soybean plant with respect to at least one genetic locus in a linkage group L genomic region flanked by loci M0205928 (SEQ ID NO: 4) and BU765955 (SEQ ID NO: 12). In certain embodiments of any of the aforementioned methods, the selected transgenic plant further comprises a transgene that confers resistance to dicamba and wherein the selected transgenic plant or progeny thereof is scored for tolerance to dicamba following exposure to dicamba. In certain embodiments of any of the aforementioned methods, the methods further comprise exposing the population of transgenic soybean plants to glyphosate. In certain embodiments of any of the aforementioned methods, the glyphosate reproductive tolerance is scored by determining a reduction in sterility when compared to a transgenic plant that exhibits glyphosate reproductive sensitivity and comprises the transgene that confers resistance to glyphosate.
Also provided herein are methods of obtaining a transgenic soybean plant that comprises a genotype associated with reproductive tolerance to glyphosate, the methods comprising: (a) exposing a population of transgenic soybean plants to dicamba, wherein the plants have a transgene that confers resistance to dicamba; (b) observing dicamba tolerance exhibited by one or more soybean plants following exposure to dicamba; and, (c) selecting a transgenic plant that exhibits dicamba tolerance, thereby obtaining a transgenic soybean plant that comprises a genotype associated with reproductive tolerance to glyphosate. In certain embodiments of the methods, the population is segregating for to at least one genetic locus in a linkage group L genomic region flanked by loci M0205928 (SEQ ID NO: 4) and BU765955 (SEQ ID NO: 12) that is associated with dicamba tolerance. In certain embodiments of the methods, the methods further comprise genotyping the selected soybean plant with respect to at least one genetic locus in a linkage group L genomic region flanked by loci M0205928 (SEQ ID NO: 4) and BU765955 (SEQ ID NO: 12). In certain embodiments of any of the aforementioned methods, the selected transgenic plant further comprises a transgene that confers resistance to glyphosate and the selected transgenic plant or progeny thereof is scored for reproductive tolerance to glyphosate following exposure to glyphosate. In certain embodiments of any of the aforementioned methods, the dicamba tolerance is scored by determining a reduction in malformation when compared to a dicamba sensitive transgenic plant that comprises the transgene that confers resistance to dicamba. Further areas of applicability will become apparent from the description provided herein. It should be understood that the description and specific examples are intended for purposes of illustration only and are not intended to limit the scope of the present disclosure.
As used herein, an “allele” refers to one of two or more alternative forms of a genomic sequence at a given locus on a chromosome. When all the alleles present at a given locus on a chromosome are the same, that plant is homozygous at that locus. If the alleles present at a given locus on a chromosome differ, that plant is heterozygous at that locus.
As used herein, the term “denoting” when used in reference to a plant genotype refers to any method whereby a plant is indicated to have a certain genotype. Such indications of a certain genotype include, but are not limited to, any method where a plant is physically marked or tagged. Physical markings or tags that can be used include, but not limited to, a barcode, a radio-frequency identification (RFID), a label or the like. Indications of a certain genotype also include, but are not limited to, any entry into any type of written or electronic database whereby the plant's genotype is provided.
A “locus” is a position on a genomic sequence that is usually found by a point of reference; e.g., a short DNA sequence that is a gene, or part of a gene or intergenic region. A locus may refer to a nucleotide position at a reference point on a chromosome, such as a position from the end of the chromosome.
As used herein, “linkage group L” corresponds to the soybean linkage group L described in Choi, et al., Genetics. 2007 May; 176(1): 685-696. Linkage group L, as used herein, also corresponds to soybean chromosome 19 (as described on the World Wide Web at soybase.org/LG2Xsome.php). As used herein, “polymorphism” means the presence of one or more variations of a nucleic acid sequence at one or more loci in a population of at least two members. The variation can comprise but is not limited to one or more nucleotide base substitutions, the insertion of one or more nucleotides, a nucleotide sequence inversion, and/or the deletion of one or more nucleotides.
As used herein, the term “single nucleotide polymorphism,” also referred to by the abbreviation “SNP,” means a polymorphism at a single site wherein the polymorphism constitutes any or all of a single base pair change, an insertion of one or more base pairs, and/or a deletion of one or more base pairs.
As used herein, “marker” means a detectable characteristic that can be used to discriminate between organisms. Examples of such characteristics include, but are not limited to, genetic markers, biochemical markers, fermentation yield, fermentation efficiency, energy yield, secondary compounds, metabolites, morphological characteristics, and agronomic characteristics.
As used herein, “marker assay” means a method for detecting a polymorphism at a particular locus using a particular method. Marker assays thus include, but are not limited to, measurement of at least one phenotype (such as seed color, flower color, or other visually detectable trait as well as any biochemical trait), restriction fragment length polymorphism (RFLP), single base extension, electrophoresis, sequence alignment, allelic specific oligonucleotide hybridization (ASO), random amplified polymorphic DNA (RAPD), microarray-based polymorphism detection technologies, and the like.
As used herein, “genotype” means the genetic component of the phenotype and it can be indirectly characterized using markers or directly characterized by nucleic acid sequencing.
As used herein, the term “introgressed”, when used in reference to a genetic locus, refers to a genetic locus that has been introduced into a new genetic background. Introgression of a genetic locus can thus be achieved through both plant breeding methods or by molecular genetic methods. Such molecular genetic methods include, but are not limited to, various plant transformation techniques and/or methods that provide for homologous recombination, non-homologous recombination, site-specific recombination, and/or genomic modifications that provide for locus substitution or locus conversion. In certain embodiments, introgression could thus be achieved by substitution of a dicamba intolerance locus with a corresponding dicamba tolerance locus or by conversion of a locus from a dicamba intolerance genotype to a dicamba tolerance genotype.
As used herein, “phenotype” means the detectable characteristics of a cell or organism which can be influenced by gene expression.
As used herein, “linkage” refers to relative frequency at which types of gametes are produced in a cross. For example, if locus A has genes “A” or “a” and locus B has genes “B” or “b” and a cross between parent I with AABB and parent B with aabb will produce four possible gametes where the genes are segregated into AB, Ab, aB and ab. The null expectation is that there will be independent equal segregation into each of the four possible genotypes, i.e. with no linkage ¼ of the gametes will of each genotype. Segregation of gametes into a genotypes differing from ¼ are attributed to linkage.
As used herein, the termed “linked”, when used in the context of markers and/or genomic regions, means that the markers and/or genomic regions are located on the same linkage group or chromosome.
As used herein, a “nucleic acid molecule,” be it a naturally occurring molecule or otherwise may be “substantially purified”, if desired, referring to a molecule separated from substantially all other molecules normally associated with it in its native state. More preferably, a substantially purified molecule is the predominant species present in a preparation. A substantially purified molecule may be at least about 60% free, preferably at least about 75% free, more preferably at least about 90% free, and most preferably at least about 95% free from the other molecules (exclusive of solvent) present in the natural mixture. The term “substantially purified” is not intended to encompass molecules present in their native state.
As used herein, “quantitative trait locus (QTL)” means a locus that controls to some degree numerically representable traits that are usually continuously distributed. As used herein, the term “transgene” means nucleic acid molecules in the form of DNA, such as cDNA or genomic DNA, and RNA, such as mRNA or microRNA, which may be single or double stranded.
As used herein, the term “event”, when used in the context of describing a transgenic plant, refers to a particular transformed plant line. In a typical transgenic breeding program, a transformation construct responsible for a trait is introduced into the genome via a transformation method. Numerous independent transformants (events) are usually generated for each construct. These events are evaluated to select those with superior performance.
As used herein, the term “soybean” means Glycine max and includes all plant varieties that can be bred with soybean, including wild soybean species. In certain embodiments, soybean plants from the species Glycine max and the subspecies Glycine max L. ssp. max or Glycine max ssp. formosana can be genotyped using the compositions and methods of the present invention. In an additional aspect, the soybean plant is from the species Glycine soja, otherwise known as wild soybean, can be genotyped using these compositions and methods. Alternatively, soybean germplasm derived from any of Glycine max, Glycine max L. ssp. max, Glycine max ssp. Formosana, and/or Glycine soja can be genotyped using compositions and methods provided herein.
As used herein, the term “bulk” refers to a method of managing a segregating population during inbreeding that involves growing the population in a bulk plot, harvesting the self-pollinated seed of plants in bulk, and using a sample of the bulk to plant the next generation.
As used herein, the phrase “transgene that confers tolerance to dicamba” refers to the ability of a transgene to provide a soybean plant capable of surviving exposure to dicamba at a rate of about 0.5 pounds of acid equivalent per acre of dicamba acid to about 1.5 pounds of acid equivalent per acre of dicamba acid applied at either pre-emergence and/or postemergence. Transgenic plants comprising a transgene that confers tolerance to dicamba can exhibit, either a “dicamba tolerant” phenotype in certain soybean germplasms or a “dicamba sensitive” phenotype in other distinct soybean germplasms when exposed to dicamba.
As used herein, the phrase “dicamba intolerant” refers to undesirable phenotypic traits observed in certain soybean germplasms that comprise a transgene that confers resistance to dicamba after exposure to dicamba at a rate of about 0.5 pounds of acid equivalent per acre of dicamba acid to about 1.5 pounds of acid equivalent per acre of dicamba acid. Such undesirable phenotypic traits include, but are not limited to, pronounced bending/twisting of the main stem and petioles, necrosis of the upper nodes and petioles, and/or limitation of new growth.
As used herein, the phrase “dicamba tolerant” refers to either the absence or reduction of undesirable phenotypic traits observed after exposure to dicamba in “dicamba intolerant” soybean germplasms that comprise a transgene that confers resistance to dicamba.
As used herein, the term “comprising” means “including but not limited to”.
As used herein, the terms “scoring” or “score”, refer to any qualitive, semi-quantitive, or quantitive method for determining the presence, absence, and/or the partial presence or absence, of a phenotypic trait.
As used herein, the phrase “susceptible variety”, when used in reference to herbicide tolerance in a soybean plant comprising a transgene that confers resistance to that herbicide, refers to a soybean variety that allele(s) of the stacked transgenic trait improvement locus that do not confer such stacked transgenic trait improvements. “Susceptible varieties’ are also referred to herein as “sensitive varieties” in the context of herbicide tolerance in a soybean plant comprising a transgene that confers resistance to that herbicide.
As used herein, the phrase “corresponding herbicide”, when used in reference to a transgene that confers herbicide resistance, refers to the herbicide that the transgene confers resistance to. Thus, a corresponding herbicide for a transgene that confers resistance to glyphosate, dicamba, 2,4-D, or glufosinate is respectively glyphosate, dicamba, 2,4-D, or glufosinate.
In accordance with the present invention, Applicants have discovered genomic regions, associated markers, and associated methods for identifying and associating genotypes that effect the levels of dicamba tolerance observed in soybean plants comprising a transgene that confers resistance to dicamba. Dicamba (3,6-dichloro-o-anisic acid) is a useful broad spectrum herbicide for controlling weeds. For example, in one embodiment, a method of the invention comprises screening a plurality of transgenic germplasm entries displaying a heritable variation for at least one transgene mediated dicamba resistance trait wherein the heritable variation is linked to at least one genotype; and associating at least one genotype from the transgenic germplasm entries to at least one dicamba tolerance trait. In another embodiment, a method of the invention comprises crossing at least two germplasm entries with a test germplasm entry for the evaluation of performance of at least one dicamba tolerance trait in order to determine preferred crossing schemes. The methods of the present invention can be used with traditional breeding techniques as described below to more efficiently screen and identify genotypes affecting a dicamba tolerance trait.
The use of markers to infer a phenotype of interest results in the economization of a breeding program by substituting costly, time-intensive phenotyping assays with genotyping assays. Further, breeding programs can be designed to explicitly drive the frequency of specific, favorable phenotypes by targeting particular genotypes (U.S. Pat. No. 6,399,855). Fidelity of these associations may be monitored continuously to ensure maintained predictive ability and, thus, informed breeding decisions (US Patent Application 2005/0015827). In this case, costly, time-intensive phenotyping assays required for determining if a plant or plants contains a genomic region associated with a “dicamba tolerance” or “Dicamba intolerance” phenotype can be supplanted by genotypic assays that provide for identification of a plant or plants that contain the desired genomic region that confers dicamba tolerance.
A Genomic Region Associated with a Dicamba Tolerance Phenotype
Provided herewith is a soybean genomic region that is shown herein to be associated with a desirable dicamba tolerance phenotype when present in certain allelic forms and when combined with certain transgenic loci that confer dicamba tolerance.
A soybean genomic region provided that can be associated with a desirable dicamba tolerance phenotype when present in certain allelic forms is located on the telomere proximal end of the short arm of soybean linkage group L (chromosome 19). A series of markers useful in practicing the methods of this invention are provided herewith in Table 1. Additional markers useful in the practice of the invention are provided herewith in Table 2 of the Specification, which is incorporated herewith by reference in its entirety. Table 2 provides the Table 1 markers, additional nucleic acid markers or loci that have been disclosed in various databases, the relative positions of the markers on a physical map of linkage group L (soybean chromosome 19), and sources for the markers.
1The relative positions of the approximate middle position of the listed markers or loci based on nucleotide positions on a physical map of soybean linkage group L (chromosome 19) of Table 2 are provided where nucleotide position 0 (zero) is telomere proximal and nucleotide position 2009800 is centromere proximal. Polymorphic nucleotide bases are designated in the sequence listing provided herewith according to the WIPO Standard ST.25 (1998), Table 1, as follows: r = g or a (purine); y = t/u or c (pyrimidine); m = a or c; (amino); k = g or t/u (keto); s = g or c (strong interactions 3 H-bonds); w = a or t/u (weak interactions 2H-bonds); b = g or c or t/u (not a); d = a or g or t/u (not c); h = a or c or t/u (not g); v = a or g or c (not t, not u); and n = a or g or c or t/u (unknown, or other; any.)
2Both the maternal and paternal alleles of the single nucleotide polymorphisms that can be associated with a dicamba tolerance phenotype are shown.
3The identified polymorphic allele of marker M0101742 is located at nucleotide 1206 of SEQ ID NO: 5.
4The identified polymorphic allele of marker M0205350 is located at nucleotide 148 of SEQ ID NO: 10.
5The identified polymorphic allele of marker M0102027is located at nucleotide 349 of SEQ ID NO: 11.
6The identified polymorphic allele of marker M0101742 “TT” can be associated with a dicamba tolerance phenotype when the identified polymorphic alleles of the other markers are: “TT” for M0205350 and, in certain embodiments, “CC” for M0102027.
7The identified polymorphic allele of marker M020350 “TT” can be associated with a dicamba tolerance phenotype when the identified polymorphic alleles of the other markers are: “TT” for M0101742 and, in certain embodiments, “CC” for M0102027.
8In certain embodiments, the identified polymorphic allele “CC” for marker M0102027 can be associated with a dicamba tolerance phenotype when the identified polymorphic alleles of the other markers are: “TT” for M0101742 and “TT” for M020350.
9The identified polymorphic allele of marker NGMAX008197032 is located at nucleotide 201 of SEQ ID NO: 52.
10In certain embodiments, the identified polymorphic allele of marker NGMAX008197032 “AA” can be associated with a dicamba tolerance phenotype when the identified polymorphic alleles of the other markers are: “TT” for M0205350 and, in certain embodiments, “CC” for M0102027, and “TT” for M0101742.
Also provided herein are sub-regions of the linkage group L region that is flanked by loci M0205928 (SEQ ID NO: 4) and BU765995 (SEQ ID NO: 12) that are associated with a dicamba tolerance phenotype. A first sub-region of the linkage group L region associated with a dicamba tolerance phenotype is flanked by loci M0205928 (SEQ ID NO: 4) and M0129138 (SEQ ID NO: 6). These loci flank a first sub-region that spans telomere proximal nucleotide 92334 to centromere proximal nucleotide 113494 in the physical map of linkage group L provided in Table 2 of the specification. Polymorphisms located in this first sub-region that are associated with a dicamba tolerance phenotype can be detected with markers that include, but are not limited to, M0101742 (SEQ ID NO: 5). A second sub-region of the linkage group L region associated with a dicamba tolerance phenotype is flanked by loci BU551363 (SEQ ID NO: 9) and BU765955 (SEQ ID NO: 12). These loci flank the second sub-region that spans telomere proximal nucleotide 422447 to centromere proximal nucleotide 474316 in the physical map of linkage group L provided in Table 2 of the specification. Polymorphisms located in this second sub-region that are associated with a dicamba tolerance phenotype can be detected with markers that include, but are not limited to, M0205350 (SEQ ID NO: 10) or M0102027 (SEQ ID NO: 11). A third sub-region of the linkage group L region associated with a dicamba tolerance phenotype is flanked by loci BU55345 (SEQ ID NO:7) and M0114388 (SEQ ID NO:8). These loci flank the second sub-region that spans telomere proximal nucleotide 115,956 to centromere proximal nucleotide 380,486 in the physical map of linkage group L provided in Table 2 of the specification. Polymorphisms located in this third sub-region that are associated with a dicamba tolerance phenotype can be detected with markers that include, but are not limited to, NGMAX008197032 (SEQ ID NO:52). In certain embodiments of invention, a polymorphism associated with a dicamba tolerant phenotype is detected in only one of these sub-regions. In other embodiments of the invention, at least one polymorphism associated with a dicamba tolerant phenotype is detected in any two of these sub-regions. Thus, a marker including, but not limited to, M0101742 (SEQ ID NO: 5) can be used either independently of, or in combination with, one or more markers selected from the group consisting of M0205350 (SEQ ID NO: 10) and/or M0102027 (SEQ ID NO: 11) to detect polymorphisms associated with a dicamba tolerance phenotype. In certain embodiments, a marker including, but not limited to, M0101742 (SEQ ID NO: 5) can be used either independently of, or in combination with, marker NGMAX008197032 (SEQ ID NO:52) to detect polymorphisms associated with a dicamba tolerance phenotype. In certain embodiments, a marker including, but not limited to, marker NGMAX008197032 (SEQ ID NO:52) can be used either independently of, or in combination with, one or more markers selected from the group consisting of M0205350 (SEQ ID NO: 10) and/or M0102027 (SEQ ID NO: 11) to detect polymorphisms associated with a dicamba tolerance phenotype. In certain embodiments, a polymorphism in the first sub-region is detected with marker M0101742 (SEQ ID NO: 5) and a polymorphism in the second sub-region is detected with markers M0205350 (SEQ ID NO: 10) and/or M0102027 (SEQ ID NO: 11). In certain embodiments, a polymorphism in the first sub-region is detected with marker M0101742 (SEQ ID NO: 5) and a polymorphism in the third sub-region is detected with marker NGMAX008197032 (SEQ ID NO: 52). In certain embodiments, the alleles of these markers associated with dicamba tolerance are a TT allele M0101742 (SEQ ID NO: 5), a TT allele of M0205350 (SEQ ID NO: 10), and, in certain embodiments, a CC allele of M0102027 (SEQ ID NO: 11), and an AA allele of NGMAX008197032 (SEQ ID NO:52).
Additional genetic markers can be used either in conjunction with the markers provided in Table 1 and/or Table 2 or independently of the markers provided in Table 1 and/or Table 2 to practice the methods of the instant invention. Publicly available marker databases from which useful markers can be obtained include, but are not limited to, the soybase.org website on the internet (World Wide Web) that is administered by the United States Agricultural Research Service, the United States Department of Agriculture, and Iowa State University. Additional soybean markers that can be used and that have been described in the literature include, but are not limited to, Hyten et al., BMC Genomics. 11:38, 2010; Choi et al., Genetics. 176(1):685-96, 2007; Yoon et al., Theor Appl Genet. 2007 March; 114(5):885-99; and Hyten et al. Crop Sci. 2010 50: 960-968. Given the provision herein of a genomic region on linkage group L (chromosome 19) delimited or flanked by the telomere proximal locus M0205928 (SEQ ID NO: 4) of Table 2 and the centromere proximal locus BU765955 (SEQ ID NO: 12) of Table 2 as well as an assortment of soybean germplasms exhibiting either a “dicamba intolerant” or “dicamba tolerant” phenotype, additional markers located either within or near this genomic region that are associated with these phenotypes can be obtained by merely typing the new markers in the various germplasms provided herewith. The genomic region on linkage group L (chromosome 19) delimited or flanked by the telomere proximal locus M0205928 (SEQ ID NO: 5) of Table 2 and the centromere proximal locus BU765955 (SEQ ID NO: 12) of Table 2 can also be mapped relative to markers provided in any publicly available or other soybean physical or genetic map to place this genetic locus on that map.
Identification of Plants Exhibiting the “Dicamba Intolerance” or “Dicamba Tolerance” Phenotype
To observe the presence or absence of the “dicamba intolerance” or dicamba tolerance phenotypes, transgenic soybean plants comprising a transgene that confers resistance to dicamba are typically exposed in early to mid-vegetative growth stages to one or more high doses of dicamba. Typical doses of dicamba that can elicit a dicamba intolerance phenotype can range from about a 2-fold label application rate of a commercially available dicamba formulation to about a 3-fold label application rate of a commercially available dicamba formulation. In terms of acid equivalents of dicamba acid applied, typical doses of dicamba that can elicit a dicamba intolerance phenotype can range from an application rate of about 1.0 pounds of acid equivalent per acre of dicamba acid to about 1.5 pounds of acid equivalent per acre of dicamba acid when the indicated amounts of dicamba acid are provided in either a commercially available dicamba formulation or when the indicated amounts of dicamba acid is provided in a similar formulation suitable for application to dicamba-tolerant crops. Commercially available dicamba formulations that can be used include, but are not limited to, Clarity® (BASF, NC, USA); Banvel®, Banvel M®, Banvel II®, Banvel SGF®, or Vanquish® (Syngenta, Wilmington, Del., USA); or Rifle® (Loveland Products, Inc., Loveland, Colo., USA). In certain embodiments, the commercially available dicamba formulation used is Clarity®. In certain embodiments, doses of dicamba that can elicit a dicamba intolerance phenotype can range from about a 2 fold application rate of about 0.25 gallons per acre Clarity® to about a three fold application rate of about 0.375 gallons per acre per acre Clarity®.
The dicamba intolerance phenotype can be observed approximately a week after herbicide application in certain soybean varieties comprising the transgene that confers resistance to dicamba. Dicamba is typically applied during pre and post-emergent vegetative growth stages. In certain embodiments of these methods, dicamba can be applied in weekly intervals (i.e. once a week) for any of 2, 3, 4 or more successive weeks to score for the presence of the dicamba intolerance phenotype. In certain embodiments, soybean plants at about the V3 vegetative development stage are exposed to an initial dicamba spray followed by a subsequent spray at V6/R1. Genotypes provided herein are especially useful for providing dicamba tolerance to plants sprayed at the V6 stage. As discussed herein, the vegetative stages of soybean are as follows: VE (emergence), VC (cotyledon stage), V1 (first trifoliate leaf), V2 (second trifoliate leaf), V3 (third trifoliate leaf), V(n) (nth trifoliate leaf), and V6 (flowering will soon start). As discussed herein, the reproductive stages of soybean are as follows: R1 (beginning bloom), R2 (full bloom), R3 (beginning pod), R4 (full pod), R5 (beginning seed), R6 (full seed), R7 (beginning maturity) and R8 (full maturity). A description of the soybean vegetative and reproductive stages can be found on the world wide web (interne) at ag.ndsu.edu/pubs/plantsci/rowcrops/a1174/a1174w.htm (North Dakota State University publication A-1174, June 1999, Reviewed and Reprinted August 2004).
A rating scale that evaluates the degree of dicamba intolerance can also be employed to identify “dicamba intolerant” and “dicamba tolerant” plants. An exemplary and non-limiting scale for evaluating the Dicamba intolerance phenotype is as follows, where a low number corresponds to a “dicamba tolerance” phenotype and the a high number correlates to a “dicamba intolerance” phenotype:
A rating of 1: Less than 10% of plants show malformation
A rating of 2: 10-50% of plants show malformation
A rating of 3: Greater than 50% of plants show malformation
Identification of Plants Exhibiting Reproductive Tolerance to Glyphosate Phenotype
To observe the presence or absence of reproductive tolerance to glyphosate phenotypes, transgenic soybean plants comprising a transgene that confers glyphosate resistance are typically exposed in mid- to late-vegetative growth stages to one or more high doses of glyphosate. Doses of glyphosate that can elicit a reproductive sensitivity phenotype are usually at least about twice the typical application rates of commercial glyphosate formulations that are used to provide weed control in transgenic, glyphosate resistant soybean plants. In terms of acid equivalents of glyphosate acid applied, typical doses of glyphosate that can elicit a reproductive sensitivity phenotype can range from an application rate of about 1.0 pounds of acid equivalent per acre (about 1.12 kilograms per hectare) of glyphosate acid to about 2.25 pounds of acid equivalent per acre (i.e. about 2.52 kilograms per hectare) of glyphosate acid when the indicated amounts of glyphosate acid are provided in either a commercially available glyphosate formulation or when the indicated amounts of glyphosate acid is provided in a similar formulation suitable for application to glyphosate-tolerant crops. Commercially available glyphosate formulations that can be used include, but are not limited to, Roundup Original MAX®, Roundup PowerMAX®, Roundup UltraMax®, or RoundUp WeatherMAX® (Monsanto Co., St. Louis, Mo., USA); Touchdown IQ® or Touchdown Total® (Syngenta, Wilmington, Del., USA); Glyphomax®, Glyphomax Plus®, or Glyphomax XRT® (Dow Agrosciences LLC, Indianapolis, Ind., USA). In certain embodiments, the commercially available glyphosate formulation used is RoundUp WeatherMAX®. In certain embodiments, doses of glyphosate that can elicit a reproductive sensitivity phenotype can range from about a 2 fold application rate of about 42.6 ounces per acre RoundUp WeatherMax® (1.68 kilograms per hectare) to about a three fold application rate of about 63.9 ounces per acre RoundUp WeatherMax® (i.e. about 2.52 kilograms per hectare).
The reproductive sensitivity phenotype can be observed at an appropriate stage of reproductive development after herbicide application in certain soybean varieties comprising the transgene that confers glyphosate resistance. Glyphosate is typically applied during vegetative growth stages, where applications in later vegetative growth stages can typically elicit reproductive sensitivity at lower application rates. In certain embodiments of these methods, glyphosate can be applied in weekly intervals (i.e. once a week) for any of 2, 3, 4 or more successive weeks to score for the presence of the reproductive sensitivity phenotype. In certain embodiments, soybean plants at about the V3 vegetative development stage are exposed to an initial glyphosate spray followed by a subsequent spray at the V6 vegetative stage. In certain embodiments, soybean plants at about the V6 vegetative development stage are exposed to a glyphosate spray. As discussed herein, the vegetative stages of soybean are as follows: VE (emergence), VC (cotyledon stage), V1 (first trifoliolate leaf), V2 (second trifoliolate leaf), V3 (third trifoliolate leaf), V(n) (nth trifoliolate leaf), and V6 (flowering will soon start). As discussed herein, the reproductive stages of soybean are as follows R1 (beginning bloom, first flower); R2 (full bloom, flower in top 2 nodes); R3 (beginning pod, 3/16″ pod in top 4 nodes); R4 (full pod, ¾″ pod in top 4 nodes); R5 (⅛″ seed in top 4 nodes); R6 (full size seed in top 4 nodes); R7 (beginning maturity, one mature pod); and, R8 (full maturity, 95% of pods on the plant are mature). A description of the soybean vegetative and reproductive stages can be found on the world wide web (internet) at ag.ndsu.edu/pubs/plantsci/rowcrops/a1174/a1174w.htm (North Dakota State University publication A-1174, June 1999, Reviewed and Reprinted August 2004). Expression of the reproductive sensitivity trait can also be influenced by temperature, where the trait in varieties that display the reproductive sensitivity phenotype is more pronounced following treatment at temperatures of about 32 degrees Celsius or more.
A rating scale that evaluates the degree of reproductive sensitivity can also be employed to identify “tolerant” and “sensitive” plants. An exemplary and non limiting scale for evaluating the reproductive sensitivity phenotype is as follows, where the low numbers correspond to a “glyphosate reproductive tolerance” phenotype and the high numbers correlate to a “glyphosate reproductive sensitivity” phenotype where sterility is monitored as follows:
Also provided herewith is unique soybean germplasm comprising an introgressed genomic region that is associated with a dicamba tolerance phenotype and methods of obtaining the same. Marker-assisted introgression involves the transfer of a chromosomal region, defined by one or more markers, from one germplasm to a second germplasm. Offspring of a cross that contain the introgressed genomic region can be identified by the combination of markers characteristic of the desired introgressed genomic region from a first germplasm (i.e. such as a dicamba tolerance germplasm) and both linked and unlinked markers characteristic of the desired genetic background of a second germplasm (i.e. a dicamba intolerance germplasm). In addition to the markers provided herewith that identify alleles of genomic region that is associated with a dicamba tolerance phenotype, flanking markers that fall on both the telomere proximal end of the genomic region on linkage group L (chromosome 19) and the centromere proximal end of the linkage group L (chromosome 19) genomic region are also provided in Tables 1 and 2. Table 2 is provided at the end of the specification immediately before the claims. Such flanking markers are useful in a variety of breeding efforts that include, but are not limited to, introgression of the genomic region associated with a dicamba tolerance phenotype into a genetic background comprising markers associated with germplasm that ordinarily contains the allelic forms of the genomic region that is associated with a “Dicamba intolerance” phenotype. Telomere proximal flanking markers that can be used in these methods include, but are not limited to, asmbl_11856 (SEQ ID NO: 1), TC122822 (SEQ ID NO: 2), BI967232 (SEQ ID NO: 3), and/or polymorphisms in any of the loci listed in Table 2 of the Specification located between starting base 16426 (the telomere proximal base) of locus asmbl_11856 and starting base 92334 of locus M0205928 (SEQ ID NO: 4). Centromere proximal flanking markers that can be used in these methods include, but are not limited to, M0205537 (SEQ ID NO: 15), M0202715 (SEQ ID NO: 16), M0206286 (SEQ ID NO: 17), M0206054 (SEQ ID NO: 18) and M0205375 (SEQ ID NO: 19) and/or polymorphisms in any of the other loci listed in Table 2 that are centromere proximal to BU765955 (SEQ ID NO: 12). Soybean plants wherein the two subregions that are respectively flanked by loci M0205928 (SEQ ID NO: 4) and M0129138 (SEQ ID NO: 6 and by loci BU551363 (SEQ ID NO: 9) and BU765955 (SEQ ID NO: 12) are selectively introgressed can be obtained by using the BU551345 (SEQ ID NO: 7), SATT723, and/or M0114388 (SEQ ID NO: 8) markers, or by using any of the markers located between these two subregions that are provided in Table 2. Any of the aforementioned polymorphisms can be identified by sequencing loci from dicamba intolerant and dicamba tolerance germplasms. Additional markers located on linkage group L (chromosome 19) and other chromosomes are disclosed in US Patent Application Publication 20090208964. Publicly available marker databases from which additional useful markers located on linkage group L (chromosome 19) and other chromosomes can be obtained include, but are not limited to, the soybase.org website on the internet that is administered by the United States Agricultural Research Service, the United States Department of Agriculture, and Iowa State University. Soybean plants or germplasm comprising an introgressed genomic region that is associated with a dicamba tolerance phenotype wherein at least 10%, 25%, 50%, 75%, 90%, or 99% of the remain genomic sequences carry markers characteristic of soybean plants or germplasm that are otherwise or ordinarily comprise a genomic region associated with the Dicamba intolerance phenotype are thus provided.
Soybean Plants Comprising Genomic Region Associated with the Dicamba Intolerance and Dicamba Tolerance Phenotypes and Transgenes that Confer Resistance to Dicamba
A non-limiting and exemplary list of soybean plants that comprise genomic regions associated with either a dicamba-intolerance or a dicamba tolerance phenotype are provided herewith in Table 3.
1 Branded names of Asgrow ® (designated “AG”) and DEKALB ® soybean varieties from Monsanto Co. 800 N. Lindbergh Blvd., St. Louis, MO, USA.
2 Deposit numbers of seed available through the American Type Culture Collection (ATCC), 10801 University Blvd., Manassas, Va., USA, 20110-2209.
3 Dicamba phenotype is the phenotype observed in the indicated germplasm containing a transgene that confers resistance to dicamba when exposed to dicamba.
Also provided herewith are additional soybean plants that comprising a genomic region associated with a dicamba intolerant or dicamba tolerance phenotype that are identified by use of the markers provided in Table 1 and/or Table 2 and/or methods provided herein. Any of the soybean plants identified in Table 3 or other soybean plants that are otherwise identified using the markers or methods provided herein can be used in methods that include, but are not limited to, methods of obtaining soybean plants with an introgressed dicamba tolerance locus, obtaining a soybean plant that exhibits a dicamba tolerance phenotype, or obtaining a soybean plant comprising in its genome a genetic region associated with a dicamba tolerance phenotype.
In certain embodiments, the soybean plants provided herein or used in the methods provided herein can comprise a transgene that confers resistance to dicamba. In certain embodiments, the dicamba tolerant soybean plants can comprise a transgene encoding a dicamba-degrading dicamba monoxygenase (DMO) enzyme that catalyzes the conversion of herbicidal dicamba (3,6-dichloro-o-anisic acid) to a non-toxic 3,6-dichlorosalicylic acid. In certain embodiments, the dicamba-degrading dicamba monoxygenase (DMOw) comprise a DMO enzyme disclosed in U.S. Pat. Nos. 7,022,896, 7,105,724, and 7,812,224, each incorporated herein by reference in their entireties. Exemplary and non-limiting DMOw dicamba monooxygenase encoding nucleic acid and protein sequences are provided herewith as SEQ ID NO: 20 and SEQ ID NO: 21. In certain embodiments, the dicamba tolerant soybean plants can comprise a dicamba monoxygenase variant which exhibits improved catalytic parameters such as increased turnover number and/or a lower km for the substrate, improved catalysis at lower pH values, and/or improved catalysis at higher temperatures relative to an unaltered dicamba monooxygenase. In certain embodiments, the dicamba monoxygenase variant comprises a DMOc variant enzyme disclosed in U.S. Pat. No. 7,884,262, incorporated herein by reference in its entirety. Exemplary and non-limiting DMOc dicamba monooxygenase variant encoding nucleic acid and protein sequences are provided herewith as SEQ ID NO: 22 and SEQ ID NO: 23. In certain embodiments, a dicamba monooxygenase is operably linked to a chloroplast transit peptide (CTP). Operable linkage of certain CTPs to DMO is disclosed in U.S. Pat. No. 8,084,666, which is incorporated herein by reference in its entirety. In certain embodiments, it is contemplated that the soybean plants used herein can comprise one or more specific genomic insertion(s) of a dicamba tolerant transgene including, but not limited to, as those found in MON87708 soybean (deposited under ATCC accession number PTA-9670 and described in US Patent Application Publication Number 20110067134).
In certain embodiments, the soybean plants provided herein or used in the methods provided herein can comprise a transgene that confers tolerance to glyphosate. Transgenes that can confer tolerance to glyphosate include, but are not limited to, transgenes that encode glyphosate tolerant Class I EPSPS (5-enolpyruvylshikimate-3-phosphate synthases) enzymes or glyphosate tolerant Class II EPSPS (5-enolpyruvylshikimate-3-phosphate synthases) enzymes. Useful glyphosate tolerant EPSPS enzymes provided herein are disclosed in U.S. Pat. Nos. 6,803,501, RE39,247, 6,225,114, 5,188,642, and 4,971,908. In certain embodiments, the glyphosate tolerant soybean plants can comprise a transgene encoding a glyphosate oxidoreductase or other enzyme which degrades glyphosate. Glyphosate oxidoreductase enzymes had been described in U.S. Pat. No. 5,776,760 and U.S. Reissue Pat. RE38,825. In certain embodiments the soybean plant can comprise a transgene encoding a glyphosate N-acetyltransferase gene that confers tolerance to glyphosate. In certain embodiments, the soybean plant can comprise a glyphosate n-acetyltransferase encoding transgene such as those described in U.S. Pat. No. 7,666,644. In still other embodiments, soybean plants comprising combinations of transgenes that confer glyphosate tolerance are provided. Soybean plants comprising both a glyphosate resistant EPSPS and a glyphosate N-acetyltransferase are also provided herewith. In certain embodiments, it is contemplated that the soybean plants used herein can comprise one or more specific genomic insertion(s) of a glyphosate tolerant transgene including, but not limited to, as those found in: i) MON89788 soybean (deposited under ATCC accession number PTA-6708 and described in US Patent Application Publication Number 20100099859), ii) GTS 40-3-2 soybean (Padgette et al., Crop Sci. 35: 1451-1461, 1995), iii) event 3560.4.3.5 soybean (seed deposited under ATCC accession number PTA-8287 and described in US Patent Publication 20090036308), or any combination of i (MON89788 soybean), ii (GTS 40-3-2 soybean), and iii (event 3560.4.3.5 soybean).
In certain embodiments, the gene that confers resistance to dicamba is a gene encoding a Dicamba monooxygenase (DMO). The DMO gene is a microbial gene that has been transformed into soybean and cotton to confer tolerance to the dicamba herbicide. The DMO protein expressed in the plants transformed with the DMO gene actively metabolizes dicamba to 3,6-dichloro salicylic acid (DCSA), which lacks herbicidal activity. In certain embodiments, Dicamba resistant (DR) soybeans can be crossed with “RoundUp Ready 2 Yield™” (RR2Y) soybeans to generate a stack (RR2YxDR) which can confer resistance to both dicamba and glyphosate. It has been observed in certain germplasms that a herbicide traits (i.e. transgene conferred glyphosate and dicamba resistance)×germplasm interaction can result in increased sensitivity to dicamba (i.e. “dicamba intolerance”) that may be commercially undesirable. In certain embodiments, favorable haplotypes are provided herein which are associated with robust tolerance to dicamba and glyphosate, and which are useful for selection of RR2YxDR soybeans that do not exhibit dicamba intolerance.
In certain embodiments, it is contemplated that genotypic assays that provide for non-destructive identification of the plant or plants can be performed either in seed, the emergence stage, the “VC” stage (i.e. cotyledons unfolded), the V1 stage (appearance of first node and unifoliate leaves), the V2 stage (appearance of the first trifoliate leaf), and thereafter. In certain embodiments, non-destructive genotypic assays are performed in seed using apparati and associated methods as described in U.S. Pat. Nos. 6,959,617; 7,134,351; 7,454,989; 7,502,113; 7,591,101; 7,611,842; and 7,685,768, which are incorporated herein by reference in their entireties. In certain embodiments, non-destructive genotypic assays are performed in seed using apparati and associated methods as described in US Patent Application Publications 20100086963, 20090215060, and 20090025288, which are incorporated herein by reference in their entireties. Published U.S. Patent Applications US 2006/0042527, US 2006/0046244, US 2006/0046264, US 2006/0048247, US 2006/0048248, US 2007/0204366, and US 2007/0207485, which are incorporated herein by reference in their entirety, also disclose apparatus and systems for the automated sampling of seeds as well as methods of sampling, testing and bulking seeds. Thus, in a certain embodiments, any of the methods provided herein can comprise screening for markers in individual seeds of a population wherein only seed with at least one genotype of interest is advanced.
Soybean Plants Comprising a Genomic Region Associated with Stacked Transgenic Trait Improvement and Transgenes that Confer Resistance to Other Herbicides and/or Insects
In certain embodiments, soybean plants comprising a genomic region associated with stacked transgenic trait improvement (or the dicamba tolerance phenotype) and at least one additional herbicide resistance transgene selected from the group consisting of a dicamba resistance conferring transgene, a 2,4-D resistance conferring transgene, and a glufosinate resistance conferring transgene and/or at least one transgene encoding a product that confers insect resistance selected from the group consisting of a dsRNA that inhibits a target gene of an insect pest, a patatin, a Bacillus thuringiensis insecticidal protein, a Xenorhabdus insecticidal protein, a Photorhabdus insecticidal protein, a Bacillus laterosporous insecticidal protein, a Bacillus sphaericus insecticidal protein, and a lignin are provided herein. Such transgenic trait improvements that can occur in plants comprising the genomic regions provided herein can be ascertained by comparing transgenic trait performance in varieties containing the genomic regions to the transgenic trait performance in other varieties lacking the genomic region. Such transgenic herbicide resistance trait improvements that can occur in plants comprising the genomic regions provided herein can include, but are not limited to, decreased phytotoxicity upon herbicide exposure in varieties containing the genomic regions conferring the improved transgenic trait performance and the corresponding herbicide resistance conferring transgene in comparison to other varieties lacking the genomic region and the corresponding herbicide resistance conferring transgene upon herbicide exposure. Various dsRNAs that inhibit a target gene of an insect pest are described in US Patent Application Publication Number 20120137387, which is specifically incorporated herein by reference in its entirety. A Bacillus thuringiensis insecticidal protein can be any of a number of insecticidal proteins including but not limited to a Cry1, a Cry3, a TIC851, a CryET70, a Cry22, a TIC901, a TIC1201, a TIC407, a TIC417, a binary insecticidal protein CryET33 and CryET34, a binary insecticidal protein CryET80 and CryET76, a binary insecticidal protein TIC100 and TIC101, a binary insecticidal protein PS149B1, a VIP insecticidal protein, a TIC900 or related protein, or combinations of the insecticidal proteins ET29 or ET37 with insecticidal proteins TIC810 or TIC812, and insecticidal chimeras of any of the preceding insecticidal proteins. A Bacillus thuringiensis insecticidal protein can be any of a number of insecticidal proteins including but not limited to a Cry1Aa, Cry1Ab, Cry1Ac, Cry1Ad, Cry1Ae, Cry1Ba, Cry1Bb, Cry1Ca, Cry1Cb, Cry1Da, Cry1Db, Cry1Ea, Cry1Eb, Cry1Fa, Cry1Fb, Cry1Ga, Cry1Ha, Cry2Aa, Cry2Ab, Cry1Ja, Cry1 Ka, Cry11 Aa, Cry11Ab, Cry12Aa, Cry3Ba, Cry3Bb, Cry3C, Cry4a, Cry4Ba, Cry5a, Cry5Ab, Cry6Aa, Cry6Ba, Cry7Aa, Cry7Ab, Cry8Aa, Cry8Ba, Cry8Ca, Cry9Aa, Cry9Ba, Cry9Ca, Cry10Aa, Cry11Aa, Cry12Aa, Cry13Aa, Cry14Aa, Cry15Aa, Cyt1Aa, and Cyt2Aa protein or an insecticidal chimeras thereof. Insecticidal chimeras of certain Bacillus thuringiensis insecticidal proteins include, but are not limited to, Cry1A/F and other chimeras disclosed in US Patent Application Publication No. 20090143298. Such transgenic insect resistance trait improvements that can occur in plants comprising the genomic regions provided herein can include, but are not limited to, decreased insect-mediated plant damage, or increased insect death, inhibition, stunting, or cessation of insect feeding in varieties containing the genomic regions that confer the transgenic trait performance in comparison to other varieties lacking the genomic region.
Molecular Assisted Breeding Techniques
Genetic markers that can be used in the practice of the instant invention include, but are not limited to, are Restriction Fragment Length Polymorphisms (RFLP), Amplified Fragment Length Polymorphisms (AFLP), Simple Sequence Repeats (SSR), Single Nucleotide Polymorphisms (SNP), Insertion or Deletion Polymorphisms (Indels), Variable Number Tandem Repeats (VNTR), and Random Amplified Polymorphic DNA (RAPD), and others known to those skilled in the art. Marker discovery and development in crops provides the initial framework for applications to marker-assisted breeding activities (US Patent Applications 2005/0204780, 2005/0216545, 2005/0218305, and 2006/00504538). The resulting “genetic map” is the representation of the relative position of characterized loci (DNA markers or any other locus for which alleles can be identified) along the chromosomes. The measure of distance on this map is relative to the frequency of crossover events between sister chromatids at meiosis.
As a set, polymorphic markers serve as a useful tool for fingerprinting plants to inform the degree of identity of lines or varieties (U.S. Pat. No. 6,207,367). These markers form the basis for determining associations with phenotype and can be used to drive genetic gain. The implementation of marker-assisted selection is dependent on the ability to detect underlying genetic differences between individuals.
Certain genetic markers for use in the present invention include “dominant” or “codominant” markers. “Codominant markers” reveal the presence of two or more alleles (two per diploid individual). “Dominant markers” reveal the presence of only a single allele. The presence of the dominant marker phenotype (e.g., a band of DNA) is an indication that one allele is present in either the homozygous or heterozygous condition. The absence of the dominant marker phenotype (e.g., absence of a DNA band) is merely evidence that “some other” undefined allele is present. In the case of populations where individuals are predominantly homozygous and loci are predominantly dimorphic, dominant and codominant markers can be equally valuable. As populations become more heterozygous and multiallelic, codominant markers often become more informative of the genotype than dominant markers.
In another embodiment, markers that include, but are not limited to, single sequence repeat markers (SSR), AFLP markers, RFLP markers, RAPD markers, phenotypic markers, isozyme markers, single nucleotide polymorphisms (SNPs), insertions or deletions (Indels), single feature polymorphisms (SFPs, for example, as described in Borevitz et al. 2003 Gen. Res. 13:513-523), microarray transcription profiles, DNA-derived sequences, and RNA-derived sequences that are genetically linked to or correlated with dicamba tolerance loci, regions flanking dicamba tolerance loci, regions linked to dicamba tolerance loci, and/or regions that are unlinked to dicamba tolerance loci can be used in certain embodiments of the instant invention.
In one embodiment, nucleic acid-based analyses for determining the presence or absence of the genetic polymorphism (i.e. for genotyping) can be used for the selection of seeds in a breeding population. A wide variety of genetic markers for the analysis of genetic polymorphisms are available and known to those of skill in the art. The analysis may be used to select for genes, portions of genes, QTL, alleles, or genomic regions (Genotypes) that comprise or are linked to a genetic marker that is linked to or correlated with dicamba tolerance loci, regions flanking dicamba tolerance loci, regions linked to dicamba tolerance loci, and/or regions that are unlinked to dicamba tolerance loci can be used in certain embodiments of the instant invention.
Nucleic acid analysis methods provided herein include, but are not limited to, PCR-based detection methods (for example, TaqMan™ assays), microarray methods, mass spectrometry-based methods and/or nucleic acid sequencing methods. In one embodiment, the detection of polymorphic sites in a sample of DNA, RNA, or cDNA may be facilitated through the use of nucleic acid amplification methods. Such methods specifically increase the concentration of polynucleotides that span the polymorphic site, or include that site and sequences located either distal or proximal to it. Such amplified molecules can be readily detected by gel electrophoresis, fluorescence detection methods, or other means.
A method of achieving such amplification employs the polymerase chain reaction (PCR) (Mullis et al. 1986 Cold Spring Harbor Symp. Quant. Biol. 51:263-273; European Patent 50,424; European Patent 84,796; European Patent 258,017; European Patent 237,362; European Patent 201,184; U.S. Pat. Nos. 4,683,202; 4,582,788; and 4,683,194), using primer pairs that are capable of hybridizing to the proximal sequences that define a polymorphism in its double-stranded form.
Methods for typing DNA based on mass spectrometry can also be used. Such methods are disclosed in U.S. Pat. Nos. 6,613,509 and 6,503,710, and references found therein. Polymorphisms in DNA sequences can be detected or typed by a variety of effective methods well known in the art including, but not limited to, those disclosed in U.S. Pat. Nos. 5,468,613, 5,217,863; 5,210,015; 5,876,930; 6,030,787; 6,004,744; 6,013,431; 5,595,890; 5,762,876; 5,945,283; 5,468,613; 6,090,558; 5,800,944; 5,616,464; 7,312,039; 7,238,476; 7,297,485; 7,282,355; 7,270,981 and 7,250,252 all of which are incorporated herein by reference in their entireties. However, the compositions and methods of the present invention can be used in conjunction with any polymorphism typing method to type polymorphisms in genomic DNA samples. These genomic DNA samples used include but are not limited to genomic DNA isolated directly from a plant, cloned genomic DNA, or amplified genomic DNA.
For instance, polymorphisms in DNA sequences can be detected by hybridization to allele-specific oligonucleotide (ASO) probes as disclosed in U.S. Pat. Nos. 5,468,613 and 5,217,863. U.S. Pat. No. 5,468,613 discloses allele specific oligonucleotide hybridizations where single or multiple nucleotide variations in nucleic acid sequence can be detected in nucleic acids by a process in which the sequence containing the nucleotide variation is amplified, spotted on a membrane and treated with a labeled sequence-specific oligonucleotide probe.
Target nucleic acid sequence can also be detected by probe ligation methods as disclosed in U.S. Pat. No. 5,800,944 where sequence of interest is amplified and hybridized to probes followed by ligation to detect a labeled part of the probe.
Microarrays can also be used for polymorphism detection, wherein oligonucleotide probe sets are assembled in an overlapping fashion to represent a single sequence such that a difference in the target sequence at one point would result in partial probe hybridization (Borevitz et al., Genome Res. 13:513-523 (2003); Cui et al., Bioinformatics 21:3852-3858 (2005). On any one microarray, it is expected there will be a plurality of target sequences, which may represent genes and/or noncoding regions wherein each target sequence is represented by a series of overlapping oligonucleotides, rather than by a single probe. This platform provides for high throughput screening a plurality of polymorphisms. A single-feature polymorphism (SFP) is a polymorphism detected by a single probe in an oligonucleotide array, wherein a feature is a probe in the array. Typing of target sequences by microarray-based methods is disclosed in U.S. Pat. Nos. 6,799,122; 6,913,879; and 6,996,476.
Target nucleic acid sequence can also be detected by probe linking methods as disclosed in U.S. Pat. No. 5,616,464, employing at least one pair of probes having sequences homologous to adjacent portions of the target nucleic acid sequence and having side chains which non-covalently bind to form a stem upon base pairing of the probes to the target nucleic acid sequence. At least one of the side chains has a photoactivatable group which can form a covalent cross-link with the other side chain member of the stem.
Other methods for detecting SNPs and Indels include single base extension (SBE) methods. Examples of SBE methods include, but are not limited, to those disclosed in U.S. Pat. Nos. 6,004,744; 6,013,431; 5,595,890; 5,762,876; and 5,945,283. SBE methods are based on extension of a nucleotide primer that is adjacent to a polymorphism to incorporate a detectable nucleotide residue upon extension of the primer. In certain embodiments, the SBE method uses three synthetic oligonucleotides. Two of the oligonucleotides serve as PCR primers and are complementary to sequence of the locus of genomic DNA which flanks a region containing the polymorphism to be assayed. Following amplification of the region of the genome containing the polymorphism, the PCR product is mixed with the third oligonucleotide (called an extension primer) which is designed to hybridize to the amplified DNA adjacent to the polymorphism in the presence of DNA polymerase and two differentially labeled dideoxynucleosidetriphosphates. If the polymorphism is present on the template, one of the labeled dideoxynucleosidetriphosphates can be added to the primer in a single base chain extension. The allele present is then inferred by determining which of the two differential labels was added to the extension primer. Homozygous samples will result in only one of the two labeled bases being incorporated and thus only one of the two labels will be detected. Heterozygous samples have both alleles present, and will thus direct incorporation of both labels (into different molecules of the extension primer) and thus both labels will be detected.
In another method for detecting polymorphisms, SNPs and Indels can be detected by methods disclosed in U.S. Pat. Nos. 5,210,015; 5,876,930; and 6,030,787 in which an oligonucleotide probe having a 5′ fluorescent reporter dye and a 3′ quencher dye covalently linked to the 5′ and 3′ ends of the probe. When the probe is intact, the proximity of the reporter dye to the quencher dye results in the suppression of the reporter dye fluorescence, e.g. by Forster-type energy transfer. During PCR forward and reverse primers hybridize to a specific sequence of the target DNA flanking a polymorphism while the hybridization probe hybridizes to polymorphism-containing sequence within the amplified PCR product. In the subsequent PCR cycle DNA polymerase with 5′→3′ exonuclease activity cleaves the probe and separates the reporter dye from the quencher dye resulting in increased fluorescence of the reporter.
In another embodiment, the locus or loci of interest can be directly sequenced using nucleic acid sequencing technologies. Methods for nucleic acid sequencing are known in the art and include technologies provided by 454 Life Sciences (Branford, Conn.), Agencourt Bioscience (Beverly, Mass.), Applied Biosystems (Foster City, Calif.), LI-COR Biosciences (Lincoln, Nebr.), NimbleGen Systems (Madison, Wis.), Illumina (San Diego, Calif.), and VisiGen Biotechnologies (Houston, Tex.). Such nucleic acid sequencing technologies comprise formats such as parallel bead arrays, sequencing by ligation, capillary electrophoresis, electronic microchips, “biochips,” microarrays, parallel microchips, and single-molecule arrays, as reviewed by R.F. Service Science 2006 311:1544-1546.
The markers to be used in the methods of the present invention should preferably be diagnostic of origin in order for inferences to be made about subsequent populations. Experience to date suggests that SNP markers may be ideal for mapping because the likelihood that a particular SNP allele is derived from independent origins in the extant populations of a particular species is very low. As such, SNP markers appear to be useful for tracking and assisting introgression of QTLs, particularly in the case of Genotypes.
The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.
Marker-assisted backcrossing (MABC) is a common breeding methodology to transfer a gene of interest into a desired recurrent parent. MABC was used to transfer both the dicamba resistance (DMO) transgene (U.S. Patent Appl. US20110067134) and the glyphosate resistant RoundUp Ready 2 Yield™ (RR2Y) CP4 genes (U.S. Pat. No. 7,632,985) into several recurrent parents. The process involved making three backcrosses to the recurrent parent and using genome wide markers to target the recovery of 95% or greater of the recurrent parent genome. In this process, markers were used to confirm the presence of the DMO and RR2Y CP4 genes and the absence of the RR1 CP4 gene. The haplotype at the dicamba tolerance locus for each recurrent parent was known.
Observations on MABC lines whose recurrent parents contained different haplotypes were made. For each MABC, there were several variants, with each variant tracing to a unique BC3F1 plant. The MABC lines were grown in spray trials at two locations throughout the United States with two replications at each location. Each line was planted in a paired, twelve foot plot. Each plot was sprayed at V3 with 0.75 lb a.e./acre of glyphosate and 1.0 lb a.e./acre dicamba followed by the same treatment at V6. Malformation was measured as the percentage of plants within a plot that were malformed. For each MABC, the data were averaged across variants, replications, and locations to place into one the following categories:
1. No malformation: No severe malformation detected
2. Malformation: >20% severe malformation
The data are presented in Table 4. The 6 MABC lines with a CCAATT or TTAATT haplotype all showed malformation whereas the 25 lines with the TTTTCC haplotype showed no malformation. These observations indicated that the presence of certain haplotypes at the dicamba tolerance locus in lines containing the DMO and RR2Y CP4 transgenes leads to malformation following dicamba treatment.
The effect of different haplotypes on response to dicamba and glyphosate were evaluated by comparing MG6-7 BC2F3:4 lines that contained a CP4 transgene that confers tolerance to glyphosate and a DMO transgene that confers tolerance to dicamba. The recurrent parent of the population was RP1 that had the CCAACC haplotype for markers NS0101742, NS0205350, and NS0102027 markers, respectively. The donor parent of the population was DP1 that had a TTTTCC haplotype. During the breeding process, the haplotype for each line at the dicamba tolerance locus was not known. Markers were used to select for the absence of the RR1 CP4 gene, and for the presence of the RR2Y CP4 gene and the Dicamba resistance DMO gene. Table 5 describes the breeding history for this material.
A total of 360 BC2F3:4 lines were grown in Mount Olive, N.C. in 2011. The lines were grown in a single four foot rows with one replication. The lines were sprayed with 0.75 lb a.e./acre of glyphosate at V3 plant stage followed by the same rate of glyphosate at V6 plant stage plus 0.5 lb a.e./acre of dicamba.
Rating Scales:
Malformation:
A rating of 1: Less than 10% of plants show malformation
A rating of 2: 10-50% of plants show malformation
A rating of 3: Greater than 50% of plants show malformation
Sterility:
A rating of 1: Less than 10% of plants show sterility
A rating of 2: 10-50% of plants show sterility
A rating of 3: Greater than 50% of plants show sterility
Table 6 shows the distribution of lines across the different rating classes.
The 51 lines that were rated “1” for malformation and “1” for sterility and the 62 lines that were rated “3” for malformation and “2” or “3” for sterility were genotyped for the three dicamba tolerance markers as shown in Table 7.
The 34 lines with the TTTTCC haplotype had a rating of “1” for malformation and “1” for sterility as summarized in Table 8.
Out of the 66 lines with the CCAACC haplotype, 59 had a rating of “3” for malformation and a rating of “2” or “3” for sterility. The 13 lines heterozygous for the markers had a range of ratings for malformation and sterility.
These results support the observations that certain haplotypes at the dicamba tolerance locus in lines containing the RR2Y CP4 and DMO transgenes causes sterility from glyphosate and malformation from dicamba applications made at the V6 plant stage
The effect of different haplotypes on response to glyphosate were evaluated by measuring observing sterility in MG3 to MG4 lines in glyphosate spray trials in Fontezuela, Argentina in 2012. The lines were from populations known to segregate for markers at the dicamba tolerance locus based on parental haplotypes. During the breeding process, the haplotype for each line at the dicamba tolerance locus was not known. Table 9 describes the breeding history for this material.
Markers were used to select for the absence of the RR1 CP4 gene, and for the presence of the RR2Y CP4 gene and the Dicamba resistance (DMO) gene. A total of 1,083 F3:4 lines across six populations were planted in 4 foot single row plots. Remnant seed from each line was used for genotyping the lines across two markers at the dicamba tolerance locus. The lines were sprayed with 1.125 lb a.e./acre of glyphosate at V3 plant stage followed by the same rate applied at V6. Sterility ratings were taken at maturity.
A rating of 1: Less than 10% of plants show sterility
A rating of 2: 10-50% of plants show sterility
A rating of 3: Greater than 50% of plants show sterility
The sterility ratings by haplotype class are shown in Table 10.
Lines with the TTTT haplotype for markers M0101742 and M0205350 did not show sterility across all populations. Nearly all lines with a CĆAA or TTAA haplotype had at least 10% of plants that showed sterility. It is not unexpected that some plants in these lines did no show sterility as some variation in the spray application or other environmental variations can influence the expression of sterility. Lines genotyped as CTAT or TTAT were segregating at the dicamba tolerance locus and progeny from these lines showed a range of response for sterility.
These results support the observations that certain haplotypes at the dicamba tolerance locus in lines containing the RR2Y CP4 and DMO transgenes causes sterility from glyphosate applications made at the V6 plant stage.
In one embodiment, the detection of polymorphic sites in a sample of DNA, RNA, or cDNA may be facilitated through the use of nucleic acid amplification methods. Such methods specifically increase the concentration of polynucleotides that span the polymorphic site, or include that site and sequences located either distal or proximal to it. Such amplified molecules can be readily detected by gel electrophoresis, fluorescence detection methods, or other means. Exemplary primers and probes for amplifying and detecting genomic regions associated with a dicamba tolerance phenotype are given in Table 11.
Oligonucleotides can also be used to detect or type the polymorphisms disclosed herein by single base extension (SBE)-based SNP detection methods. Exemplary oligonucleotides for use in SBE-based SNP detection are provided in Table 12. SBE methods are based on extension of a nucleotide primer that is hybridized to sequences adjacent to a polymorphism to incorporate a detectable nucleotide residue upon extension of the primer. It is also anticipated that the SBE method can use three synthetic oligonucleotides. Two of the oligonucleotides serve as PCR primers and are complementary to the sequence of the locus which flanks a region containing the polymorphism to be assayed. Exemplary extension primers that can be used to type polymorphisms disclosed in this invention are provided in Table 12 in the column labeled “Probe (SBE)”. Following amplification of the region containing the polymorphism, the PCR product is hybridized with an extension primer which anneals to the amplified DNA adjacent to the polymorphism. DNA polymerase and two differentially labeled dideoxynucleoside triphosphates are then provided. If the polymorphism is present on the template, one of the labeled dideoxynucleoside triphosphates can be added to the primer in a single base chain extension. The allele present is then inferred by determining which of the two differential labels was added to the extension primer. Homozygous samples will result in only one of the two labeled bases being incorporated and thus only one of the two labels will be detected. Heterozygous samples have both alleles present, and will thus direct incorporation of both labels (into different molecules of the extension primer) and thus both labels will be detected. Exemplary forward and reverse SBE probes are provided in Table 12.
The effect of different haplotypes on response to dicamba and glyphosate were evaluated by comparing genetically similar lines that contained a CP4 transgene that confers tolerance to glyphosate and a DMO transgene that confers tolerance to dicamba. In 2010, two plants from each of fourteen BC1F2:4 lines, or families, across five backcross populations were harvested individually to develop pairs of BC1F4:6 lines from each family. The haplotype of each recurrent parent for each backcross population is shown in Table 13.
The donor parent for each population was A3244-RR2Y/A3525-DT that had a TTTTCC haplotype for markers M0101742, M0205350, and M0102027 markers, respectively. During the breeding process, the haplotype for each line at the dicamba tolerance locus was not known. Markers were used to select for the absence of the RR1 CP4 gene, and for the presence of the RR2Y CP4 gene and the Dicamba DMO gene. The BC1F4:5 rows were sprayed with glyphosate at the V6 plant growth stage in Quillota Chile where a pair of lines per family were rated for sterility to glyphosate. Table 14 describes the breeding history for this plant material.
DNA was extracted from each line to generate haplotypes across three markers at the dicamba tolerance locus. The lines were evaluated across four locations in the United States (Stuttgart, Ark.; Stonington Ill.; Evansville, Ind.; and Galena, Md.) in 2011. At each location the lines were grown in four to five foot single-row plots replicated two times and one of seven different herbicide treatments were applied at different plant growth stages (V3 or V6) as described in Table 15.
Rating Scale:
Malformation to dicamba was rated by the percentage of plants showing malformation Sterility to glyphosate was rated as:
A rating scale of 1: Less than 10% of plants show sterility
A rating scale of 2: Less than 10-50% of plants show sterility
A rating scale 3: Greater than 50% of plants show sterility
Data were averaged across replications and locations to place into following classes as described in Table 16.
As described in Example 3, marker haplotypes corresponded to reaction to glyphosate application. There were several populations grown in Fontezuela, Argentina where plants sprayed with glyphosate were selected for reproductive tolerance to glyphosate in the absence of haplotype information on each line. Table 17 describes four populations that segregated for the haplotype based on parental haplotypes. Table 18 describes the number of lines grown and the number of lines selected.
The 46 selected lines were subsequently genotyped and found to possess the favorable AATT haplotype. In addition, the lines were grown at Stonington, Ill. in 2012 and evaluated for herbicide response. The lines were sprayed with 1.0 lb a.e/acre dicamba and 1.5 lb a.e/acre glyphosate at the V6 plant stage and did not show malformation to dicamba or sterility to glyphosate. These results further support the ability to use glyphosate selection as a means to recover the favorable haplotype and tolerance to both glyphosate and dicamba.
The effect of different haplotypes on response to dicamba was evaluated by comparing F2 families that contained the DMO transgene for dicamba resistance, but that lacked the CP4 transgene for glyphosate resistance. F2 plants across six different populations that were growing in Kunia, Hi. in 2012 were tissue sampled and genotyped for the CP4 and DMO transgenes and for markers NGMAX008197032 and M0205350. F2 plants that were fixed homozygous for the presence of DMO and absence of CP4 and that were fixed homozygous for a haplotype class were selected and harvested individually to create families. Table 19 shows the number of F2 families per haplotype class. The F2 families were evaluated for tolerance to dicamba in a greenhouse environment.
1An “AA” allele for NGMAX-008197032 (SEQ ID NO: 52) and a “TT” allele for
2A “GG” allele for NGMAX-008197032 (SEQ ID NO: 52) and an “AA” allele for
A comparison of dicamba tolerance in the plants from segregating populations of Table 19 having various “favorable” or “unfavorable” haplotypes of the indicated parental germplasm is provided in
A comparison of selections of favorable and unfavorable dicamba tolerance haplotypes based on either one or two spray treatments was made. Transgenic soybean plants containing a dicamba resistance conferring transgene and various favorable or unfavorable haplotypes were treated with dicamba at a rate of 1 pound/acre at either: (a) the V3 and V6 stages; or (b) the V6 stage only. The results of this comparison are shown in
Having illustrated and described the principles of the present invention, it should be apparent to persons skilled in the art that the invention can be modified in arrangement and detail without departing from such principles.
Although the materials and methods of this invention have been described in terms of various embodiments and illustrative examples, it will be apparent to those of skill in the art that variations can be applied to the materials and methods described herein without departing from the concept, spirit and scope of the invention. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined by the appended claims.
thaliana|Rep: ADP-
Arabidopsis thaliana
thaliana|Rep: ADP-
Arabidopsis thaliana
Daucus carota (Carrot) =
Medicago|Rep: ADP-
Medicago truncatula
Medicago|Rep: ADP-
Medicago truncatula
Medicago|Rep: ADP-
Medicago truncatula
tuberosum (Potato)]
Daucus carota (Carrot) =
Daucus carota (Carrot) =
Medicago|Rep: ADP-
Medicago truncatula
Vitis vinifera (Grape) =
Nicotiana tabacum|Rep:
vinifera (Grape), partial
Nicotiana tabacum|Rep:
truncatula (Barrel medic)]
truncatula (Barrel medic)]
truncatula (Barrel medic)]
Vitis vinifera (Grape) =
truncatula (Barrel
vinifera (Grape), partial
truncatula (Barrel medic)]
Nicotiana tabacum|Rep:
Nicotiana tabacum
truncatula (Barrel medic)]
sativa Japonica Group|Rep:
Oryza sativa subsp.
japonica (Rice) = partial
Vitis vinifera (Grape) =
sativum|Rep: P72 DEAD
sativum (Garden pea) =
vinifera (Grape), partial
truncatula (Barrel medic)]
sativum|Rep: P72 DEAD
sativum (Garden pea),
sativum|Rep: P72 DEAD
sativum (Garden pea) =
Vitis vinifera (Grape) =
sativum (Garden pea) =
trichocarpa
crystallinum (Common ice
Vitis vinifera (Grape) =
Vitis vinifera (Grape) =
Arabidopsis thaliana|Rep:
thaliana (Mouse-ear cress) =
Vitis vinifera (Grape) =
truncatula (Barrel medic)]
albus (White lupin)]
albus (White lupin), partial
albus (White lupin)]
vinifera (Grape), partial
max|Rep: P24 oleosin
Arabidopsis thaliana
thaliana (Mouse-ear cress)]
nigroviridis|Rep:
Tetraodon nigroviridis
vinifera (Grape), partial
vinifera
Vitis vinifera (Grape) =
vinifera (Grape), partial
Vitis vinifera (Grape) =
vinifera|Rep: Peptidyl-
Vitis vinifera (Grape),
Arabidopsis thaliana|Rep:
thaliana (Mouse-ear cress),
vinifera (Grape), partial
Vitis vinifera (Grape) =
Arabidopsis thaliana
thaliana (Mouse-ear cress)]
Vitis vinifera (Grape) =
Vitis vinifera (Grape) =
Oryza sativa subsp.
japonica (Rice) = partial
thaliana (Mouse-ear cress)]
thaliana (Mouse-ear cress)]
Vitis vinifera (Grape) =
thaliana (Mouse-ear cress)]
vinifera (Grape), partial
vulgaris|Rep: Protein kinase
vulgaris (Kidney bean)
vulgaris|Rep: Protein kinase
vulgaris (Kidney bean)
Vitis vinifera (Grape) =
Vitis vinifera (Grape) =
japonicus]
Vitis vinifera (Grape) =
thaliana (Mouse-ear cress)]
thaliana (Mouse-ear cress)]
thaliana|Rep:
Arabidopsis thaliana
thaliana|Rep:
Arabidopsis thaliana
thaliana|Rep:
Arabidopsis thaliana
thaliana|Rep:
Arabidopsis thaliana
Vitis vinifera (Grape) =
Vitis vinifera|Rep:
vinifera (Grape), partial
Arabidopsis thaliana|Rep:
thaliana (Mouse-ear cress)
Vitis vinifera (Grape) =
Vitis vinifera (Grape) =
thaliana|Rep:
Arabidopsis thaliana
Vitis vinifera (Grape) =
Vitis vinifera (Grape) =
thaliana (Mouse-ear cress)]
Arabidopsis thaliana|Rep:
thaliana (Mouse-ear cress) =
Prunus armeniaca|Rep:
armeniaca (Apricot) =
armeniaca (Apricot)]
unguiculata|Rep: Probable
Vigna unguiculata
vinifera
vinifera|Rep: Methionine
vinifera (Grape) = partial
Arabidopsis thaliana|Rep:
vinifera|Rep: Methionine
vinifera (Grape), partial
Vitis vinifera (Grape) =
thaliana (Mouse-ear cress)]
Vitis vinifera|Rep:
vinifera (Grape) = partial
vinifera|Rep: Chromosome
Vitis vinifera|Rep:
vinifera (Grape) = partial
Vitis vinifera (Grape) =
Vitis vinifera|Rep:
vinifera (Grape), partial
Vitis vinifera|Rep:
vinifera (Grape), partial
Vitis vinifera|Rep: = partial
Vitis vinifera|Rep:
vinifera (Grape), partial
thaliana (Mouse-ear cress)]
trichocarpa
thaliana|Rep: Elongation
thaliana (Mouse-ear cress) =
esculentum (Tomato)]
lycopersicum|Rep:
lycopersicum (Tomato)
esculentum) = partial (74%)
esculentum (Tomato)]
thaliana (Mouse-ear cress)]
max|Rep: Ribosomal
max|Rep: Ribosomal
vinifera (Grape), partial
vinifera (Grape), partial
campestris (Field mustard)]
Vitis vinifera|Rep:
vinifera (Grape) = partial
Solanum tuberosum|Rep:
tuberosum (Potato), partial
thaliana (Mouse-ear cress)]
Nicotiana tabacum|Rep:
tabacum (Common
thaliana (Mouse-ear cress)]
Oryza sativa subsp. indica
Glycine max|Rep: Actin-1 -
Glycine max (Soybean) =
vulgare)]
Aegiceras
corniculatum|Rep: Actin -
Aegiceras corniculatum,
truncatula (Barrel medic)]
Prunus mume|Rep: Pm52
Prunus mume|Rep: Pm52
Prunus mume|Rep: Pm52
max (Soybean) = partial
max (Soybean)]
Glycine max|Rep: Pyruvate
Glycine max (Soybean),
max (Soybean), partial
max (Soybean)]
max
Glycine max|Rep: Pyruvate
Glycine max (Soybean),
sinensis (Sweet orange)]
sinensis (Sweet orange)]
max (Soybean) = partial
Vitis vinifera (Grape) =
Vitis vinifera (Grape) =
Vitis vinifera (Grape) =
ignobilis|Rep: ATP
ignobilis = partial (37%)
Bos taurus|Rep: Lysosomal
Vitis vinifera (Grape) =
japonicus]
japonicus|Rep: Sphingosine
japonicus|Rep: Sphingosine
japonicus]
japonicus]
japonicus|Rep: Sphingosine
japonicus]
japonicus|Rep: Sphingosine
Vitis vinifera (Grape) =
Vitis vinifera (Grape) =
Vitis vinifera|Rep:
vinifera (Grape) = partial
Vitis vinifera|Rep:
vinifera (Grape), partial
Bacillus cereus subsp.
Arabidopsis thaliana
thaliana (Mouse-ear cress)]
sativa (japonica cultivar-
Vitis vinifera (Grape) =
Vitis vinifera (Grape) =
Vitis vinifera (Grape) =
Vitis vinifera (Grape) =
Vitis vinifera (Grape) =
thaliana (Mouse-ear cress)]
Vitis vinifera (Grape) =
papaya (Papaya) = partial
papaya (Papaya) = partial
thaliana (Mouse-ear cress)]
papaya (Papaya), partial
Vitis vinifera (Grape) =
vinifera (Grape), partial
truncatula (Barrel medic)]
vulgaris
Vitis vinifera (Grape) =
Vitis vinifera (Grape) =
radiata var. radiata|Rep:
Phaseolus aureus (Mung
radiata var. radiata|Rep:
Phaseolus aureus (Mung
mays (Maize)]
mays (Maize)]
mays (Maize)]
mays (Maize)]
mays (Maize)]
radiata var. radiata|Rep:
Phaseolus aureus (Mung
mays (Maize)]
radiata var. radiata|Rep:
Phaseolus aureus (Mung
mays (Maize)]
mays (Maize)]
mays (Maize)]
sativum|Rep: Carbonic
Pisum sativum (Garden
mays (Maize)]
radiata var. radiata|Rep:
Phaseolus aureus (Mung
sativum|Rep: Carbonic
Pisum sativum (Garden
mays (Maize)]
sativum|Rep: Carbonic
Pisum sativum (Garden
radiata var. radiata|Rep:
Phaseolus aureus (Mung
radiata var. radiata|Rep:
Phaseolus aureus (Mung
radiata var. radiata|Rep:
Phaseolus aureus (Mung
radiata var. radiata|Rep:
Phaseolus aureus (Mung
thaliana|Rep: Carbonic
thaliana (Mouse-ear cress),
Vitis vinifera (Grape) =
Vitis vinifera (Grape) =
Arabidopsis thaliana|Rep:
Arabidopsis thaliana
Vitis vinifera (Grape) =
Vitis vinifera (Grape) =
Vitis vinifera (Grape) =
vinifera (Grape), partial
Vitis vinifera (Grape) =
Vitis vinifera (Grape) =
Medicago sativa|Rep: Coil
truncatula|Rep: Lipolytic
Medicago truncatula
Vitis vinifera (Grape) =
truncatula|Rep: Lipolytic
Medicago truncatula
Vitis vinifera (Grape) =
vinifera (Grape), partial
Vitis vinifera (Grape) =
truncatula (Barrel medic)]
annuus (Common
vesca|Rep: Ripening-
vesca (Woodland
annuus (Common
annuus (Common
truncatula (Barrel medic)]
truncatula (Barrel medic)]
radiata|Rep: Auxin-induced
radiata|Rep: Auxin-induced
annuus (Common
truncatula (Barrel medic)]
radiata|Rep: Auxin-induced
sativum (Garden pea)]
sativum|Rep: Protein
Quercus robur|Rep:
Quercus robur (English
Hordeum vulgare (Barley) =
Vitis vinifera (Grape) =
Quercus robur|Rep:
Quercus robur (English
Quercus robur|Rep:
Quercus robur (English
Quercus robur|Rep:
Quercus robur (English
sativa Indica Group
sativa (japonica cultivar-
tuberosum (Potato)]
tuberosum|Rep: Calcium
tuberosum (Potato), partial
Vitis vinifera (Grape) =
Vitis vinifera (Grape) =
thaliana (Mouse-ear cress)]
Vitis vinifera (Grape) =
thaliana (Mouse-ear cress)]
Vitis vinifera (Grape) =
Vitis vinifera (Grape) =
sativa (japonica cultivar-
thaliana (Mouse-ear cress)]
vinifera (Grape), partial
Vitis vinifera (Grape) =
Vitis vinifera (Grape) =
thaliana (Mouse-ear cress)]
thaliana|Rep: Protein kinase -
Arabidopsis thaliana
thaliana (Mouse-ear cress)]
sativa|Rep: Peptidyl-prolyl
sativa subsp. indica (Rice) =
oleracea (Spinach)]
sativum (Garden pea)]
sativum (Garden pea)]
sativum (Garden pea)]
sativum|Rep: Poly(A)
sativum (Garden pea),
sativum (Garden pea)]
sativum|Rep: Poly(A)
sativum (Garden pea) =
sativum (Garden pea)]
Vitis vinifera (Grape) =
borgpetersenii serovar
vulgaris|Rep:
Phaseolus vulgaris (Kidney
thaliana (Mouse-ear cress)]
vulgaris|Rep:
Phaseolus vulgaris (Kidney
vulgaris|Rep:
Phaseolus vulgaris (Kidney
sativa subsp. japonica
vinifera (Grape), partial
Vitis vinifera|Rep:
Vitis vinifera (Grape) =
Vitis vinifera (Grape) =
Vitis vinifera|Rep:
vinifera (Grape) = partial
Vitis vinifera|Rep:
vinifera (Grape) = partial
truncatula|Rep:
truncatula (Barrel medic) =
corniculatus (Bird's-foot
Nicotiana tabacum|Rep:
Nicotiana tabacum
vinifera (Grape), partial
Vitis vinifera (Grape) =
tabacum (Common
Vitis vinifera (Grape) =
vinifera (Grape), partial
esculenta (Food
Vitis vinifera (Grape) =
Vitis vinifera (Grape) =
crystallinum (Common ice
Arabidopsis thaliana|Rep:
thaliana = partial (4%)
Vitis vinifera (Grape) =
Vitis vinifera (Grape) =
vinifera
scrofa|Rep: Skinkine - Sus
scrofa (Pig) = partial (12%)
hirsutum|Rep: Nodulin-like
hirsutum (Upland cotton)
Glycine max|Rep: Hsp22.3 -
Glycine max (Soybean) =
Vitis vinifera (Grape) =
Vitis vinifera|Rep:
vinifera (Grape), partial
Vitis vinifera (Grape) =
Arabidopsis thaliana|Rep:
thaliana (Mouse-ear cress) =
truncatula|Rep:
Medicago truncatula
Vitis vinifera (Grape) =
Vitis vinifera (Grape) =
sativum (Garden pea)]
sativum (Garden pea)
sativum|Rep: Alpha-
sativum (Garden pea) =
Sequences for the genes provided above can be obtained from the World Wide Web (or Internet) using the identifiers provided in Column 1 (Locus/Display Name) or Column 5 (ADDITIONAL LOCUS INFORMATION) from the following internet locations: “soybase.org” (described in Grant et al., Nucleic Acids Research, 2010, Vol. 38, Database issue D843-D846) or soybase.org/gbrowse/cgi-bin/gbrowse/gmax1.01/ (see Hyten D L, Choi I-Y, Song Q, Specht J E, Carter T E et al. (2010) A high density integrated genetic linkage map of soybean and the development of a 1,536 Universal Soy Linkage Panel for QTL mapping. Crop Science 50:960-968. (Crop Science); and Hyten D L, Cannon S B, Song Q, Weeks N, Fickus E W et al. (2010) High-throughput SNP discovery through deep resequencing of a reduced representation library to anchor and orient scaffolds in the soybean whole genome sequence. BMC Genomics 11(1): 38);
This application is a 35 U.S.C. § 371 National Phase of International Patent Application No. PCT/US2013/042349, filed May 23, 2013 and incorporated herein by reference in its entirety, which claims the benefit of U.S. Patent Application No. 61/779,739, filed Mar. 13, 2013; U.S. Patent Application No. 61/753,725, filed Jan. 17, 2013; U.S. Patent Application No. 61/753,693, filed Jan. 17, 2013; U.S. Patent Application No. 61/650,869, filed May 23, 2012; and U.S. Patent Application No. 61/650,852, filed May 23, 2012, each of which is incorporated herein by reference in its entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2013/042349 | 5/23/2013 | WO | 00 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2013/177356 | 11/28/2013 | WO | A |
| Number | Name | Date | Kind |
|---|---|---|---|
| 5659116 | Rhodes | Aug 1997 | A |
| 5750857 | Rhodes | May 1998 | A |
| 5973235 | Holmes | Oct 1999 | A |
| 6005170 | Lussenden | Dec 1999 | A |
| 6080917 | Lussenden | Jun 2000 | A |
| 6143953 | Buettner | Nov 2000 | A |
| 6184442 | Nickell | Feb 2001 | B1 |
| 6346658 | Moots | Feb 2002 | B1 |
| 6348644 | Rhodes | Feb 2002 | B1 |
| 6632982 | Floyd | Oct 2003 | B1 |
| 6660912 | Owen | Dec 2003 | B1 |
| 6683233 | Owen | Jan 2004 | B1 |
| 6858783 | Eby et al. | Feb 2005 | B2 |
| 6881879 | Floyd | Apr 2005 | B2 |
| 6884927 | Eby | Apr 2005 | B1 |
| 6900372 | Hicks | May 2005 | B2 |
| 6933423 | Narvel | Aug 2005 | B2 |
| 7022896 | Weeks et al. | Apr 2006 | B1 |
| 7067723 | Narvel | Jun 2006 | B2 |
| 7071388 | Narvel | Jul 2006 | B2 |
| 7105724 | Weeks et al. | Sep 2006 | B2 |
| 7135626 | Davis | Nov 2006 | B2 |
| 7294764 | Leitz | Nov 2007 | B2 |
| 7378578 | Narvel | May 2008 | B2 |
| 7388131 | Hicks | Jun 2008 | B1 |
| 7479582 | Moots et al. | Jan 2009 | B2 |
| 7482516 | Hicks | Jan 2009 | B2 |
| 7498489 | Jenkinson et al. | Mar 2009 | B2 |
| 7502113 | Deppermann et al. | Mar 2009 | B2 |
| 7504565 | Jenkinson et al. | Mar 2009 | B2 |
| 7554014 | Moots et al. | Jun 2009 | B2 |
| 7569750 | Behm | Aug 2009 | B2 |
| 7592516 | Floyd et al. | Sep 2009 | B2 |
| 7728197 | Bowers et al. | Jun 2010 | B1 |
| 7812224 | Weeks et al. | Oct 2010 | B2 |
| 7964777 | Eby | Jun 2011 | B2 |
| 8921647 | Cerny et al. | Dec 2014 | B2 |
| 9617605 | Cerny et al. | Apr 2017 | B2 |
| 20060288444 | McCarroll et al. | Dec 2006 | A1 |
| 20090036308 | Guida, Jr. et al. | Feb 2009 | A1 |
| 20090064354 | Marvel et al. | Mar 2009 | A1 |
| 20090105077 | Bhatti | Apr 2009 | A1 |
| 20090165166 | Feng | Jun 2009 | A1 |
| 20090208964 | McCarroll et al. | Aug 2009 | A1 |
| 20100099859 | Malven et al. | Apr 2010 | A1 |
| 20100122372 | Sebastian et al. | May 2010 | A1 |
| 20120084879 | Cerny et al. | Apr 2012 | A1 |
| 20150216135 | Gilsinger et al. | Aug 2015 | A1 |
| Number | Date | Country |
|---|---|---|
| 2012031097 | Mar 2012 | WO |
| WO 2012031097 | Mar 2012 | WO |
| Entry |
|---|
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| 20150143577 A1 | May 2015 | US |
| Number | Date | Country | |
|---|---|---|---|
| 61650869 | May 2012 | US | |
| 61650852 | May 2012 | US | |
| 61753725 | Jan 2013 | US | |
| 61753693 | Jan 2013 | US | |
| 61779739 | Mar 2013 | US |
