Patent Grant
9617605
A sequence listing containing the file named “46_21_54534.txt” which is 19,851 bytes (measured in MS-Windows®) and created on Nov. 6, 2014, comprises 32 nucleotide sequences, is provided herewith via the USPTO's EFS system and is herein incorporated by reference in its entirety. Also incorporated herein by reference are the 32 nucleotide sequences and pertinent identifying information in the Sequence Listing containing the file named “46_21_54534.txt” which is 18477 bytes (measured in MS-Windows®), created on Aug. 30, 2010, and filed with U.S. Provisional Application Ser. No. 61/380,024 on Sep. 3, 2010.
A listing of various soybean linkage group L (chromosome 19) markers is provided herewith in the Specification as Table 2. Also incorporated herein by reference is the listing of various soybean linkage group L (chromosome 19) markers that was provided in the document “Appendix to the Specification as Table 2” that was 4541064 bytes (as measured in MS-Windows®), filed with U.S. Provisional Application Ser. No. 61/380,024 on Sep. 3, 2010. Table 2 appears in the final pages of the specification.
“Yellow Flash” is an undesirable phenotype observed in certain soybean varieties that comprise a transgene that confers tolerance to the broad-spectrum herbicide glyphosate. After application of glyphosate, or applications of glyphosate under certain environmental conditions such as high temperature, the leaves of certain soybean plant varieties comprising the transgene that confers glyphosate tolerance can exhibit a yellowish color (hence, the term “Yellow Flash”). The Yellow Flash phenotype can be observed approximately a week after herbicide application in certain soybean varieties comprising the transgene that confers glyphosate tolerance. The yellow flash phenotype is undesirable as it leads to a visually displeasing off-type yellow leaf color in soybean plants exposed to glyphosate.
Although the Yellow Flash phenotype can be observed approximately a week after herbicide application in certain soybean varieties comprising the transgene that confers glyphosate tolerance, distinct soybean varieties that comprise the same glyphosate tolerant transgene integrated at the same chromosomal locus (i.e. the same transgenic event) can show various degrees of yellow flash upon exposure to high doses of glyphosate. Some varieties comprising the glyphosate tolerant transgene insertion are highly resistance to high dosages of glyphosate, showing no yellow flash phenotype (i.e. a “no flash phenotype”), while other varieties comprising the same glyphosate tolerant transgene insertion are highly susceptible to high dosages of glyphosate, showing a severe yellow flash phenotype. Provided herein are soybean plants comprising an introgressed genomic region associated with a no flash phenotype. Also provided herein are markers that reside outside of a genomic region associated with a no flash 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 no flash phenotype are also provided. Methods of obtaining a soybean plant that exhibits a no flash phenotype and methods of obtaining a soybean plant comprising in its genome at least one no flash locus are also provided. Methods that provide for the introgression of a genomic region associated with a no flash phenotype into soybean germplasm that has a genomic region associated with a yellow flash phenotype. Identification of molecular markers associated with loci that confer the no flash phenotype has significant economic value. By using markers associated with the no flash trait, breeders can select soybean varieties with the favorable alleles (i.e. alleles that are not associated with the yellow flash 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 yellow flash trait) in soybean unfavorable allele. This invention thus provides for commercially desirable transgenic soybean lines that carry a genomic region that is associated with a “no flash” phenotype and tolerate high dosages of glyphosate.
Methods of identifying a soybean plant that comprises a genotype associated with a no flash phenotype are thus provided. In certain embodiments, methods of identifying a soybean plant that comprises a genotype associated with a no flash phenotype, comprising: detecting in the soybean plant an allele in at least one yellow flash marker locus associated with the no flash phenotype wherein the yellow flash marker locus is in a linkage group L genomic region flanked by loci BG406195 (SEQ ID NO: 13) and BU082700 (SEQ ID NO:14), and denoting that the plant comprises a genotype associated with a no flash phenotype. In certain embodiments, the methods further comprise the step of selecting the denoted plant from a population of plants. In certain embodiments of any of the aforementioned methods, the denoted plant comprises a transgene that confers tolerance to glyphosate are provided. In certain embodiments where the denoted plant comprises a transgene that confers tolerance to glyphosate, the soybean plant or progeny thereof is exposed to a dosage of glyphosate sufficient to cause yellow flash in a susceptible variety. In certain embodiments of the aforementioned methods, a plant that exhibits a no flash phenotype is selected. In certain embodiments of the aforementioned methods, the genotype associated with a no flash phenotype 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 BG406195 (SEQ ID NO: 13) and BU551345 (SEQ ID NO: 16) 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 TA14086_34305 (SEQ ID NO: 15) and BU082700 (SEQ ID NO: 14). In certain embodiments of the aforementioned methods, the genotype associated with a no flash phenotype comprises at least one polymorphic allele of at least one marker in the linkage group L region selected from the group consisting of M0129138 (SEQ ID NO:4), M0101742 (SEQ ID NO:5), M0093116 (SEQ ID NO:6), and M0129925 (SEQ ID NO:7) that is associated with a no flash phenotype.
Also provided are methods for obtaining a soybean plant comprising in its genome at least one no flash locus. In certain embodiments, a method for obtaining a soybean plant comprising in its genome at least one no flash locus, compromising the steps of: a. genotyping a plurality of soybean plants with respect to at least one yellow flash locus in a first linkage group L genomic region flanked by loci BG406195 (SEQ ID NO: 13) and BU082700 (SEQ ID NO:14); and, b. selecting a soybean plant comprising in its genome at least one no flash locus comprising a genotype associated with no flash phenotype are provided. In certain embodiments of these methods, the genotype associated with a no flash phenotype 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 BG406195 (SEQ ID NO: 13) and BU551345 (SEQ ID NO: 16); 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 TA14086_34305 (SEQ ID NO: 15) and BU082700 (SEQ ID NO: 14). In certain embodiments of the aforementioned methods, the genotype associated with a no flash phenotype 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 M0129138 (SEQ ID NO:4), M0101742 (SEQ ID NO:5), M0093116 (SEQ ID NO:6), and M0129925 (SEQ ID NO:7). In certain embodiments, the plurality of soybean plants comprises a population that is obtained by: i) crossing a parent plant comprising at least one no flash locus with a parent plant comprising at least one yellow flash locus; or, ii) obtaining seed or progeny from a parental plant segregating for at least one no flash locus. In certain embodiments, the population contains plants that comprise a transgene that confers tolerance to glyphosate. In certain embodiments, the aforementioned methods can 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 the aforementioned methods, the plurality of soybean plants, the soybean plant, and/or progeny thereof are exposed to a dosage of glyphosate sufficient to cause yellow flash in a susceptible variety. In certain embodiments of the aforementioned methods, a plant that exhibits a no flash phenotype is selected.
Also provided herewith are methods for producing a soybean plant comprising in its genome at least one introgressed no flash locus. In certain embodiments, a method for producing a soybean plant comprising in its genome at least one introgressed no flash locus comprising the steps of: a. crossing a first no flash soybean plant with a second soybean plant comprising: a yellow flash locus in a first linkage group L genomic region flanked by loci BG406195 (SEQ ID NO: 13) and BU082700 (SEQ ID NO: 14), and at least one linked polymorphic locus not present in the first no flash soybean plant to obtain a population segregating for the no flash loci and the linked polymorphic locus; b. 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 wherein at least one of the polymorphic amino acids is a linked polymorphic locus not present in the first no flash soybean plant; and c. selecting a soybean plant comprising a genotype associated with a no flash phenotype and at least one linked marker found in the second soybean plant comprising a yellow flash locus but not in the first no flash soybean plant, thereby obtaining a soybean plant comprising in its genome at least one introgressed no flash locus are provided. In certain embodiments of the methods, at least one of the first or the second soybean plants comprises a transgene that confers tolerance to glyphosate. 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 glyphosate sufficient to cause yellow flash in a susceptible variety. In certain embodiments, the yellow flash locus 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 BG406195 (SEQ ID NO: 13) and BU551345 (SEQ ID NO: 16); 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 TA14086_34305 (SEQ ID NO: 15) and BU082700 (SEQ ID NO: 14). In certain embodiments of the aforementioned methods, the polymorphic nucleic acid detected in step (b) is detected with at least one marker selected from the group consisting of M0129138 (SEQ ID NO:4), M0101742 (SEQ ID NO:5), M0093116 (SEQ ID NO:6), and M0129925 (SEQ ID NO:7). In certain embodiments of the aforementioned methods, the polymorphic nucleic acid detected in step (b) is detected with at least one marker selected from the group consisting of M0101742 (SEQ ID NO:5) and M0129925 (SEQ ID NO:7). In certain embodiments of the aforementioned methods, the polymorphic nucleic acids are detected with marker M0101742 (SEQ ID NO:5) and with marker M0129925 (SEQ ID NO:7). In certain embodiments of the aforementioned methods, the linked polymorphic locus is detected with a genotypic marker, a phenotypic marker, or both. In certain embodiments of the 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 no flash locus. In certain embodiments of the methods, the linked polymorphic locus is detected with at least one marker selected from the group consisting of M0205928 (SEQ ID NO:3), M0205537 (SEQ ID NO:8), M0202715 (SEQ ID NO:9), M0206286 (SEQ ID NO:10), M0206054 (SEQ ID NO:11) and M0205375 (SEQ ID NO:12). Also provided herewith are soybean plants comprising an introgressed no flash locus made by the aforementioned methods. In certain embodiments, a soybean plant comprising an introgressed no flash locus and one or more polymorphic loci comprising alleles or combinations of alleles that are not found in a no flash soybean variety and that are linked to the introgressed no flash locus, wherein the plant is produced by the aforementioned methods are provided.
Also provided are soybean plants comprising an introgressed no flash locus and one or more polymorphic loci comprising alleles or combinations of alleles that are not found in a no flash soybean variety and that are linked to the introgressed no flash locus.
Also provided are substantially purified nucleic acid molecules comprising a nucleic acid molecule selected from the group consisting of M0129138 (SEQ ID NO:4), M0101742 (SEQ ID NO:5), M0093116 (SEQ ID NO:6), and M0129925 (SEQ ID NO:7), or a fragment thereof that contains a specific allelic variant of M0129138 (SEQ ID NO:4), M0101742 (SEQ ID NO:5), M0093116 (SEQ ID NO:6), or M0129925 (SEQ ID NO:7). In certain embodiments, the fragment that contains the allelic variant is at least 15, at least 18, at least 20, at least 22, at least 25, or at least 30 nucleotides in length.
In certain embodiments, methods for obtaining a soybean plant that exhibits a no flash phenotype comprising the steps of: a) crossing a soybean plant that exhibits a no flash phenotype with a soybean plant that exhibits a yellow flash phenotype, wherein at least one of the soybean plants comprises a transgene that confers tolerance to glyphosate, and b) selecting a progeny plant from the cross, wherein the progeny plant comprises the transgene that confers glyphosate tolerance and wherein the progeny plant exhibits a no flash phenotype are provided. In certain embodiments of the methods, the selection in step b can comprise: i) genotyping the progeny plant with respect to a yellow flash locus in a linkage group L genomic region flanked by loci BG406195 (SEQ ID NO: 13) and BU082700 (SEQ ID NO: 14); and/or ii) exposing the progeny plant to glyphosate and scoring the plant for a no flash phenotype. In certain embodiments of the methods, a soybean plant that exhibits a yellow flash phenotype comprises at least one linked or unlinked marker not present in the first no flash soybean plant. In certain embodiments, the progeny plant is further selected for the presence of the linked or unlinked marker.
Also provided are methods of breeding soybean plants comprising the steps of:
a. selecting a first soybean plant comprising a genotype in the linkage group L genomic region flanked by loci BG406195 (SEQ ID NO: 13) and BU082700 (SEQ ID NO: 14) that is associated with a no flash phenotype from a population of soybean plants that is segregating for the genotype; and, b) crossing the selected soybean plant with a second soybean plant. In certain embodiments of these methods, one or both of the soybean plants comprises a transgene that confers glyphosate tolerance.
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. Intro gression 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 yellow flash locus with a corresponding no flash locus or by conversion of a locus from a yellow flash genotype to a no flash 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 “Single Plant Selection” (or the acronym “SPS”) refers to a method that is often used instead of bulk method (see immediately above) to advance segregating germplasm in early generations. SPS is always used to advance germplasm to “Progeny Row” (Prow) and “Progeny Row Yield Trial” (PRYT) analyses.
As used herein, the phrase “Progeny Row” (Prow) refers to a plant breeding and analysis method where a row of progeny plants from SPS is grown for observation, further selection, and/or bulking
As used herein, the phrase “Progeny Row Yield Trial” (PRYT) refers to a plant breeding and analysis method where a row of plants from a SPS is grown in a small yield trial with other SPS material. In most instances, the PRYT is usually from the same population and usually consists of one rep at one location.
As used herein, the term “comprising” means “including but not limited to”.
In accordance with the present invention, Applicants have discovered genomic regions, associated markers, and associated methods for identifying and associating genotypes that effect a transgene-mediated glyphosate tolerance trait. 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 glyphosate tolerance 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 transgene mediated glyphosate 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 transgene mediated glyphosate 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 transgene-mediated glyphosate 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 “no flash” or “yellow flash” phenotype can be supplanted by genotypic assays that provide for identification of a plant or plants that contain the desired genomic region.
Provided herewith is a soybean genomic region that is shown herein to be associated with a desirable no flash phenotype when present in certain allelic forms and when combined with certain transgenic loci that confer glyphosate tolerance.
A soybean genomic region provided that can be associated with a desirable no flash 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.
1 The relative positions of the 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.)
2 Both the maternal and paternal alleles of the single nucleotide polymorphisms that can be associated with a non flash phenotype are shown.
3The identified polymorphic allele of marker M0129138 is located at nucleotide 218 of SEQ ID NO: 4.
4The identified polymorphic allele of marker M0101742 is located at nucleotide 1206 of SEQ ID NO: 5.
5The identified polymorphic allele of marker M0093116 is located at nucleotide 183 of SEQ ID NO: 6.
6The identified polymorphic allele of marker M0129925 is located at nucleotide 328 of SEQ ID NO: 7.
7The identified polymorphic allele of marker M0129138 “GG” can be associated with a no flash phenotype when the identified polymorphic alleles of the other markers are: “TT” for M0101742, “AA” for marker M0093116, and either “GG” or “CC” for marker M0129925.
8The identified polymorphic allele of marker M0129138 “AA” can be associated with a no flash phenotype when the identified polymorphic alleles of the other markers are: “CC” for M0101742, “TT” for marker M0093116, and “GG” for marker M0129925.
Also provided herein are sub-regions of the linkage group L region that is flanked by loci BG406195 (SEQ ID NO: 13) and BU082700 (SEQ ID: 14) that are associated with a no flash phenotype. A first sub-region of the linkage group L region associated with a no flash phenotype is flanked by loci BG406195 (SEQ ID NO: 13) and BU551345 (SEQ ID NO: 16). These loci flank a first sub-region that spans telomere proximal nucleotide 107039 to centromere proximal nucleotide 115956 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 no flash phenotype can be detected with markers that include, but are not limited to, M0129138 (SEQ ID NO: 4) and M0101742 (SEQ ID NO: 5). A second sub-region of the linkage group L region associated with a no flash phenotype is flanked by loci TA14086_34305 (SEQ ID NO: 15) and BU082700 (SEQ ID NO: 14). These loci flank the second sub-region that spans telomere proximal nucleotide 800932 to centromere proximal nucleotide 864449 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 no flash phenotype can be detected with markers that include, but are not limited to, M0093116 (SEQ ID NO: 6), and M0129925 (SEQ ID NO: 7). In certain embodiments of invention, a polymorphism associated with a no-flash phenotype is detected in only one of these sub-regions. In other embodiments of the invention, at least one polymorphism associated with a no-flash phenotype is detected in both of these sub-regions. Thus, one or more markers selected from the group consisting of M0129138 (SEQ ID NO: 4), M0101742 (SEQ ID NO: 5), and markers located between loci BG406195 (SEQ ID NO: 13) and BU551345 (SEQ ID NO: 16) can be used either independently of, or in combination with, one or more markers selected from the group consisting of M0093116 (SEQ ID NO: 6), and M0129925 (SEQ ID NO: 7), and markers located between loci TA14086_34305 (SEQ ID NO: 15) and BU082700 (SEQ ID NO: 14) to detect polymorphisms associated with a no flash phenotype. Conversely, one or more markers selected from the group consisting of M0093116 (SEQ ID NO:6), and M0129925 (SEQ ID NO:7), and markers located between loci TA14086_34305 (SEQ ID NO: 15) and BU082700 (SEQ ID NO: 14) can also be used independently of, or in combination with, any markers located in the first sub-region to detect polymorphisms associated with a no flash 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 marker M0129925 (SEQ ID NO: 7).
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 BG406195 (SEQ ID NO: 13) of Table 2 and the centromere proximal locus BU082700 (SEQ ID NO: 14) of Table 2 as well as an assortment of soybean germplasms exhibiting either a “yellow-flash” or “no flash” 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 BG406195 (SEQ ID NO: 13) of Table 2 and the centromere proximal locus BU082700 (SEQ ID NO: 14) 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.
To observe the presence or absence of the “Yellow Flash” or no flash phenotypes, transgenic soybean plants comprising a transgene that confers glyphosate tolerance are typically exposed in early to mid-vegetative growth stages to one or more high doses of glyphosate. Typical doses of glyphosate that can elicit a yellow flash phenotype can range from about a 2-fold label application rate of a commercially available glyphosate formulation to about a 3-fold label application rate of a commercially available glyphosate formulation. In terms of acid equivalents of glyphosate acid applied, typical doses of glyphosate that can elicit a yellow flash phenotype can range from an application rate of about 1.5 pounds of acid equivalent per acre (about 1.68 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 yellow flash 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 Yellow Flash phenotype can be observed approximately a week after herbicide application in certain soybean varieties comprising the transgene that confers glyphosate tolerance. Glyphosate is typically applied during vegetative growth stages. 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 yellow flash phenotype. In certain embodiments, soybean plants at about the V3-V4 vegetative development stage are exposed to an initial glyphosate spray followed by three subsequent sprays at weekly intervals. The first spray can be based on stage of growth and remaining sprays were scheduled at 7 day intervals following initial 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). A description of the soybean vegetative 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 yellow flash trait can also be influenced by temperature, where the trait in varieties that display the yellow flash phenotype is more pronounced following treatment at temperatures of about 32 degrees Celsius or more.
A rating scale that evaluates the degree of yellow flash can also be employed to identify “flash” and “no flash” plants. An exemplary and non limiting scale for evaluating the yellow flash phenotype is as follows, where the low numbers correspond to a “no flash” phenotype and the high numbers correlate to a “flash” phenotype:
1: Green—No Yellowing
2: Mostly green, very slight yellowing <5% (1-2 plants with some yellowing)
3: 5-10% plants with yellowing
4: 11-20% plants with yellowing
5: 21-35% plants with yellowing
6: 36-50% plants with yellowing
7: 51-65% plants with yellowing
8: 66-80% plants with yellowing, some necrosis
9: 81-100% plants with yellowing and or necrosis
Also provided herewith are unique soybean germplasm comprising an introgressed genomic region that is associated with a no flash 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 no flash germplasm) and both linked and unlinked markers characteristic of the desired genetic background of a second germplasm (i.e. a yellow flash germplasm). In addition to the markers provided herewith that identify alleles of genomic region that is associated with a no flash 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. 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 no flash 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 “yellow flash” phenotype. Telomere proximal flanking markers that can be used in these methods include, but are not limited to, M0205928 (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 107039 of locus BG406195. Such polymorphisms can be identified by sequencing loci from flash and no flash germplasms. Centromere proximal flanking markers that can be used in these methods include, but are not limited to, M0202715 (SEQ ID NO: 9), M0206286 (SEQ ID NO: 10), M0206054 (SEQ ID NO: 11), and M0205375 (SEQ ID NO: 12). 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 no flash 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 yellow flash phenotype are thus provided.
A non-limiting and exemplary list of soybean plants that comprise genomic regions associated with either a flash or a no flash 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.
Also provided herewith are additional soybean plants that comprising a genomic region associated with a flash or no flash 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 no flash locus, obtaining a soybean plant that exhibits a no flash phenotype, or obtaining a soybean plant comprising in its genome a genetic region associated with a no flash phenotype.
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. No. 6,803,501, RE39,247, U.S. Pat. No. 6,225,114, U.S. Pat. No. 5,188,642, and U.S. Pat. No. 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, 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.
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/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 no flash loci, regions flanking no flash loci, regions linked to no flash loci, and/or regions that are unlinked to no flash 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 no flash loci, regions flanking no flash loci, regions linked to no flash loci, and/or regions that are unlinked to no flash loci can be used in certain embodiments of the instant invention.
Herein, nucleic acid analysis methods 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. No. 4,683,202; U.S. Pat. No. 4,582,788; and U.S. Pat. No. 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.
In yield trials being conducted at Thomasboro, Ill. in August, 2003, it was observed that some F3:5 soybean lines of the pedigree A3525/DKB31-51 (that contained a transgene that confers glyphosate tolerance and exhibited yellow flash when sprayed with glyphosate (i.e. RoundUp WeatherMax®) while other lines of the same pedigree showed glyphosate tolerance and no yellow flash symptoms. Repeat experiments conducted in 2004 produced similar results in 130 F3:6 lines in yield trials conducted at Hopedale, Ill. A3525 is a commercially available Asgrow® soybean variety and is also known under the variety name SN70025. DKB31-51 is described in U.S. Pat. No. 6,346,658 and samples of this seed have been deposited previously under ATCC accession number PTA-3871.
Fifty-three lines were categorized as either “Flash” or “No-Flash” with the remainder of the material falling between the extremes.
The fifty-three lines categorized in 2004 were planted at Covell, Ill. in 2005 and were sprayed with Roundup WeatherMax® at a rates of about 42 oz/acre on a weekly basis beginning at about the V3 to V4 stage for up to four weeks, thus in certain instances attaining a total rate of glyphosate exposure of a 4 week span of about 168 oz/acre. Tissue samples were collected from one individual plant from each of fifteen lines that demonstrated the most severe yellow flash and fifteen that showed no symptoms. Table 4 shows the breeding and testing history of the materials used in this study.
To identify marker associated with yellow flash symptom, we used a “Genome Scan” approach. Fifteen samples from lines showing no flash and 15 samples from lines with severe flash were collected from a segregating population (F3:6). DNA were extracted from these 30 samples and used as templates to screen a set of 2746 SNP markers that provide a high-density coverage of the entire soybean genome with approximate two markers per CentiMorgan Unit (cM). Allelic scores at each marker were collected and compared across 30 lines. Of the 2746 markers tested, 776 were segregating and 226 were heterozygous. Two markers, M0102027 and M0101742, clearly showed one allele in no flash and another allele in flash samples (Table 5), indicating a linkage between these two markers and the phenotypes. Having 15 plants showing the same allele and the same phenotype by chance is very low (1/215). Therefore, this could not be explained by a random event. A third marker, M0129138, show one allele on the flash lines and another allele on 14 out of 15 no flash lines, indicating a linkage of this marker to the flash phenotypes. M0101742 and M0129138 turned out to be mapped on the same chromosomal location with no recombination on the mapping population. Other data supporting the linkage is that all seven commercial varieties with known flash phenotypes matched perfectly with the allelic data on the two markers.
Approximately 1,700 soybean varieties (lines) were assayed or typed for the presence or absence of allelic variants of the markers M0101742 (SEQ ID NO: 5), M0129138 (SEQ ID NO: 4), M0093116 (SEQ ID NO: 6), and M0129925 (SEQ ID NO: 7). Of these lines, complete scores for all four markers were obtained in 844 lines. A summary of the genotypes observed in these 844 lines is provided in Table 6.
1The genotype of a line is shown as N1N2N3N4N5N6N7N8, where N can be A, T, G, or C depending on the position and the line and where N1 is nucleotide 1, N2 is nucleotide 2, N3 is nucleotide 3, N4 is nucleotide 4, N5 is nucleotide 5, N6 is nucleotide 6, N7 is nucleotide 7, and N8 is nucleotide 8. The genotype represents the both the paternal and maternal allelic forms of the markers M0101742 (SEQ ID NO: 5; position 1206; nucleotides 1 and 2 in genotype shown), M0129138 (SEQ ID NO: 4; position 218; nucleotides 3 and 4 in genotype shown), M0093116 (SEQ ID NO: 6; position 183; nucleotides 5 and 6 in genotype shown), and M0129925 (SEQ ID NO: 7; position 328; nucleotides 3 and 4 in genotype shown). Thus the genotype “TTGGAACC” means that the line is “TT” for both the paternal and maternal contributions to the M0101742 polymorphism, “GG” for both the paternal and maternal contributions to the M0129138 polymorphism, “AA” for both the paternal and maternal contributions to the M0129116 polymorphism, and “CC” for both the paternal and maternal contributions to the M0129925 polymorphism.
Sixty 63 soybean varieties were subsequently selected from various genotype groups and tested for yellow flash essentially as described in Example 1 (i.e. were sprayed with Roundup WeatherMax® at a rates of about 42 oz/acre on a weekly basis beginning at about the V3 to V4 stage for up to four weeks, thus in certain instances attaining a total rate of glyphosate exposure of a 4 week span of about 168 oz/acre). The results of those analyses are provided in Table 7.
For optimal prediction of yellow flash phenotype, all four markers (M0101742, M0129138, M0093116, and M0129925) can be used. However, one can achieve high predictability with only two markers, M0101742 and M0129925. Based on fingerprint information, these two markers would identify two genotypes: CCAAAACC and CCAATTCC. CCAAAACC is the predicted genotype for plants which exhibit yellow flash (193/844 varieties screened based on fingerprint analysis). CCAATTCC is an additional genotype which only represented 9/844 varieties based on fingerprint data. All 9 varieties comprising the CCAATTCC genotype were Plant Introductions (PIs). As used herein in reference to soybean, Plant Introductions (PIs) refer to mostly typical germplasms that have not been subjects of breeding improvements. For example, PIs are include lines that have not been obtained by intercrossing followed by selection. PIs can also include lines obtained from seeds or vegetative propagules of plants that have been introduced from another country. Therefore, the two markers M0101742 and M0129925 could distinguish between the three most common genotypes observed: CCAAAACC (associated with a yellow flash phenotype), TTGGAACC (associated with a non-flash phenotype) and CCAATTGG (associated with a non-flash phenotype). More specifically, where M0101742 is CC and M0129925 is CC, one would predict that most soybean lines thus identified, and that were obtained from, or related to, the varieties analyzed in this Example, would have a Yellow Flash phenotype.
In field tests conducted in 2006, a number of commercial varieties were genotyped with the markers M0101742 (SEQ ID NO: 5), M0129138 (SEQ ID NO: 4), M0093116 (SEQ ID NO: 6), and M0129925 (SEQ ID NO: 7) and exposed to glyphosate to test for the presence or absence of the yellow flash phenotype essentially as described in Example 1 (i.e. were sprayed with Roundup WeatherMax® at a rates of about 42 oz/acre on a weekly basis beginning at about the V3 to V4 stage for up to four weeks, thus in certain instances attaining a total rate of glyphosate exposure of a 4 week span of about 168 oz/acre). The results of this analysis is provided in Table 8.
1 The genotype represents the both the paternal and maternal allelic forms of the markers M0101742 (SEQ ID NO: 5 position 1206), M0129138 (SEQ ID NO: 4; position 218), M0093116 (SEQ ID NO: 6; position 183), and M0129925 (SEQ ID NO: 7; position 328)
2 Deposit numbers of seed available through the American Type Culture Collection (ATCC), 10801 University Blvd., Manassas, Va., USA, 20110-2209.
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 no flash phenotype are given in Table 9.
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 10. 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 PCR primers that can be used to type polymorphisms disclosed in this invention are provided in Table 4 in the columns labeled “Forward Primer SEQ ID” and “Reverse Primer SEQ ID”. 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 10.
Polymorphic nucleotide bases are designated in Table 11 and 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.)
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.
Vigna_unguiculata
Phaseolus_coccineus_release_2
unguiculata (Cowpea)]
Glycine_max_release_2
Arabidopsis thaliana|Rep: ADP-
Arabidopsis thaliana (Mouse-
Glycine_max_release_2
Glycine_max_release_2
Arachis_stenosperma_release_5
thaliana|Rep: ADP-ribosylation
Glycine_max_release_2
Daucus carota (Carrot) =
Glycine_max_release_2
mays (Maize)]
Glycine_max_release_2
Arachis_stenosperma_release_5
Medicago truncatula (Barrel
Medicago|Rep: ADP-
Medicago truncatula (Barrel
Arachis_stenosperma_release_5
Medicago truncatula (Barrel
Phaseolus_coccineus_release_2
Glycine_max_release_2
Lotus_japonicus_release_1
Glycine_soja_release_2
Daucus carota (Carrot) =
Daucus carota (Carrot) =
Arachis_hypogaea_release_5
Medicago truncatula (Barrel
Glycine_max_release_2
Glycine_max_release_2
Glycine_max_release_2
Glycine_max_release_2
Glycine_max_release_2
Glycine_max_release_2
Glycine_max_release_2
Lotus_japonicus_release_1
Glycine_max_release_2
Glycine_max_release_2
Glycine_max_release_2
Glycine_max_release_2
Glycine_max_release_2
Glycine_max_release_2
Glycine_max_release_2
vinifera|Rep: Chromosome
Vitis vinifera (Grape) = partial
Glycine_max_release_2
tabacum|Rep: NPK2 -
Nicotiana tabacum (Common
vinifera|Rep: Chromosome
Vitis vinifera (Grape), partial
Phaseolus_vulgaris
Glycine_max_release_2
truncatula (Barrel medic)]
tabacum|Rep: NPK2 -
Nicotiana tabacum (Common
Glycine_max_release_2
Glycine_max_release_2
truncatula (Barrel medic)]
Lotus_corniculatus_release_1
Lotus_japonicus_release_1
Phaseolus_vulgaris
Phaseolus_vulgaris_release_2
Vigna_unguiculata
Glycine_max_release_2
vinifera|Rep: Chromosome
Vitis vinifera (Grape) = partial
Glycine_max_release_2
vinifera|Rep: Chromosome
Vitis vinifera (Grape), partial
Lotus_japonicus_release_1
Nicotiana tabacum|Rep: DNA
tabacum (Common tobacco),
Lotus_japonicus_release_1
Lotus_corniculatus_release_1
Lotus_japonicus_release_1
truncatula (Barrel medic)]
Phaseolus_vulgaris
japonica (Rice) = partial (5%)
Glycine_max_release_2
vinifera|Rep: Chromosome
Vitis vinifera (Grape) = partial
sativum|Rep: P72 DEAD box
Glycine_max_release_2
sylvestris (Wood tobacco)]
vinifera|Rep: Chromosome
Vitis vinifera (Grape), partial
Lotus_japonicus_release_1
sativum|Rep: P72 DEAD box
Vigna_unguiculata
Glycine_max_release_2
sativum (Garden pea)]
sativum|Rep: P72 DEAD box
vinifera|Rep: Chromosome
Vitis vinifera (Grape) = partial
Glycine_max_release_2
thaliana (Mouse-ear cress)]
Glycine_max_release_2
tabacum (Common tobacco)]
sativum|Rep: Chloroplast
Phaseolus_vulgaris
Phaseolus_vulgaris_release_2
tabacum (Common tobacco)]
Glycine_max_release_2
crystallinum (Common ice
vinifera|Rep: Chromosome
Vitis vinifera (Grape) = partial
Glycine_max_release_2
vinifera|Rep: Chromosome
Vitis vinifera (Grape) = partial
Glycine_max_release_2
Phaseolus_vulgaris
Glycine_max_release_2
Glycine_max_release_2
Glycine_max_release_2
Glycine_max_release_2
truncatula (Barrel medic)]
Arabidopsis thaliana|Rep:
Arabidopsis thaliana (Mouse-
vinifera|Rep: Chromosome
Vitis vinifera (Grape) = partial
Glycine_max_release_2
truncatula (Barrel medic)]
Phaseolus_vulgaris
Glycine_max_release_2
Lotus_japonicus_release_1
Lupinus albus|Rep: NADP-
Glycine_soja_release_2
Glycine_max_release_2
truncatula (Barrel medic)]
Lotus_japonicus_release_1
truncatula (Barrel medic)]
vinifera|Rep: Chromosome
Vitis vinifera (Grape), partial
Glycine_max_release_2
Glycine max|Rep: P24 oleosin
Glycine_max_release_2
Phaseolus_vulgaris
Phaseolus_coccineus_release_2
Arabidopsis thaliana genomic
Glycine_soja_release_2
nigroviridis|Rep: Chromosome
nigroviridis (Green puffer) =
Glycine_max_release_2
Glycine_max_release_2
vinifera|Rep: Chromosome
Vitis vinifera (Grape), partial
Phaseolus_vulgaris
vinifera|Rep: Chromosome
Vitis vinifera (Grape) = partial
Glycine_max_release_2
Phaseolus_vulgaris
Glycine_max_release_2
Lotus_corniculatus_release_1
Lotus_japonicus_release_1
vinifera|Rep: Chromosome
Vitis vinifera (Grape), partial
vinifera|Rep: Chromosome
Vitis vinifera (Grape) = partial
Glycine_max_release_2
vinifera|Rep: Peptidyl-prolyl
vinifera (Grape), partial (18%)
Arabidopsis thaliana|Rep:
thaliana (Mouse-ear cress),
Glycine_soja_release_2
vinifera|Rep: = partial (2%)
Glycine_max_release_2
Lotus_japonicus_release_1
vinifera|Rep: Chromosome
Vitis vinifera (Grape), partial
vinifera|Rep: Chromosome
Vitis vinifera (Grape) = partial
Glycine_max_release_2
Phaseolus_vulgaris
Glycine_max_release_2
Arabidopsis thaliana genomic
thaliana (Mouse-ear cress)]
vinifera|Rep: Chromosome
Vitis vinifera (Grape) = partial
vinifera|Rep: Chromosome
Vitis vinifera (Grape) = partial
Glycine_max_release_2
Glycine_max_release_2
Oryza sativa Japonica
japonica (Rice) = partial (3%)
Lotus_japonicus_release_1
Phaseolus_coccineus_release_2
Phaseolus_coccineus_release_2
Glycine_max_release_2
vinifera|Rep: Chromosome
Vitis vinifera (Grape) = partial
Glycine_max_release_2
Vigna_unguiculata
Glycine_max_release_2
vinifera|Rep: Chromosome
Vitis vinifera (Grape), partial
Glycine_max_release_2
Glycine_max_release_2
Vigna_unguiculata
Phaseolus vulgaris (Kidney
Phaseolus_vulgaris_release_2
Lotus_japonicus_release_1
Phaseolus vulgaris|Rep: Protein
vulgaris (Kidney bean) (French
Phaseolus_vulgaris
Phaseolus_coccineus_release_2
Phaseolus_coccineus_release_2
vinifera|Rep: Chromosome
Vitis vinifera (Grape) = partial
Phaseolus_vulgaris_release_2
Vigna_unguiculata
Glycine_max_release_2
vinifera|Rep: Chromosome chr7
vinifera (Grape) = partial (17%)
Glycine_max_release_2
Lotus_japonicus_release_1
Phaseolus_vulgaris
vinifera
Vigna_unguiculata
Glycine_max_release_2
vinifera|Rep: Chromosome
Vitis vinifera (Grape) = partial
Glycine_max_release_2
thaliana (Mouse-ear cress)]
Glycine_max_release_2
thaliana (Mouse-ear cress)]
Vigna_unguiculata
Arabidopsis thaliana|Rep:
Arabidopsis thaliana (Mouse-
Pisum_sativum_release_2
thaliana (Mouse-ear cress)]
Arabidopsis thaliana|Rep:
Arabidopsis thaliana (Mouse-
Arabidopsis thaliana (Mouse-
Phaseolus_vulgaris
Arabidopsis thaliana (Mouse-
vinifera|Rep: Chromosome
Vitis vinifera (Grape) = partial
Glycine_max_release_2
thaliana (Mouse-ear cress)]
vinifera|Rep: Chromosome chr1
vinifera (Grape), partial (19%)
Arachis_hypogaea_release_5
thaliana|Rep: Hypothetical
Arabidopsis thaliana (Mouse-
Phaseolus_vulgaris
vinifera
vinifera|Rep: Chromosome
Vitis vinifera (Grape) = partial
Glycine_max_release_2
vinifera|Rep: Chromosome
Vitis vinifera (Grape) = partial
Glycine_soja_release_2
Phaseolus_vulgaris
Arabidopsis thaliana (Mouse-
Glycine_max_release_2
Glycine_max_release_2
vinifera|Rep: Chromosome
Vitis vinifera (Grape) = partial
vinifera|Rep: Chromosome
Vitis vinifera (Grape) = partial
Glycine_max_release_2
thaliana (Mouse-ear cress)]
Glycine_max_release_2
Glycine_max_release_2
Arabidopsis thaliana (Mouse-
armeniaca|Rep: Non-specific
armeniaca (Apricot) = partial
Glycine_max_release_2
unguiculata|Rep: Probable non-
unguiculata (Cowpea) = partial
Glycine_max_release_2
androssowii]
Glycine_max_release_2
Phaseolus_vulgaris
Vigna_unguiculata
Glycine_max_release_2
Vitis vinifera (Grape) = partial
Phaseolus_vulgaris
Glycine_soja_release_2
Glycine_max_release_2
Arachis_hypogaea_release_5
Arabidopsis thaliana|Rep:
Arabidopsis thaliana (Mouse-
Vitis vinifera (Grape), partial
Glycine_max_release_2
Phaseolus_vulgaris
Glycine_max_release_2
Glycine_max_release_2
vinifera|Rep: Chromosome
Vitis vinifera (Grape) = partial
Glycine_max_release_2
vinifera|Rep: Chromosome chr3
vinifera (Grape) = partial (20%)
Glycine_max_release_2
truncatula (Barrel medic)]
Glycine_max_release_2
truncatula (Barrel medic)]
Glycine_max_release_2
truncatula (Barrel medic)]
vinifera|Rep: Chromosome
Glycine_max_release_2
truncatula (Barrel medic)]
Glycine_max_release_2
truncatula (Barrel medic)]
vinifera|Rep: Chromosome chr1
vinifera (Grape) = partial (20%)
Glycine_max_release_2
truncatula (Barrel medic)]
Glycine_max_release_2
truncatula (Barrel medic)]
vinifera|Rep: Chromosome
Vigna_unguiculata
Glycine_max_release_2
truncatula (Barrel medic)]
vinifera|Rep: Chromosome chr7
vinifera (Grape), partial (11%)
vinifera|Rep: Chromosome chr7
vinifera (Grape), partial (14%)
vinifera|Rep: = partial (1%)
Glycine_max_release_2
vinifera|Rep: Chromosome chr7
vinifera (Grape), partial (10%)
Glycine_max_release_2
thaliana (Mouse-ear cress)]
Phaseolus_vulgaris
Glycine_max_release_2
Glycine_max_release_2
thaliana (Mouse-ear cress)]
Phaseolus_vulgaris
Arabidopsis thaliana (Mouse-
Glycine_max_release_2
Solanum lycopersicum|Rep:
Solanum lycopersicum
esculentum) = partial (74%)
Glycine_max_release_2
Glycine_max_release_2
thaliana (Mouse-ear cress)]
Phaseolus_vulgaris_release_2
Glycine_max_release_2
Glycine max|Rep: Ribosomal
Vigna_unguiculata
Glycine_max_release_2
Glycine max|Rep: Ribosomal
Glycine_max_release_2
vinifera|Rep: Chromosome
Vitis vinifera (Grape), partial
vinifera|Rep: Chromosome
Vitis vinifera (Grape), partial
Lotus_japonicus_release_1
campestris (Field mustard)]
Phaseolus_vulgaris
Glycine_max_release_2
vinifera|Rep: Chromosome chr1
vinifera (Grape) = partial (56%)
Lotus_corniculatus_release_1
Lotus_japonicus_release_1
tuberosum|Rep: Actin-58 -
Solanum tuberosum (Potato),
Glycine_max_release_2
Glycine_max_release_2
Vigna_unguiculata
tabacum|Rep: Actin-66-
Nicotiana tabacum (Common
Lotus_japonicus_release_1
Arachis_hypogaea_release_5
sativa subsp. indica (Rice)
Glycine_max_release_2
Glycine_max_release_2
Lotus_japonicus_release_1
corniculatum|Rep: Actin -
Aegiceras corniculatum, partial
Glycine_max_release_2
Lotus_japonicus_release_1
truncatula (Barrel medic)]
Glycine_max_release_2
Glycine_max_release_2
Glycine_max_release_2
Prunus mume|Rep: Pm52
mume|Rep: Pm52 protein -
Prunus mume (Japanese
Glycine_max_release_2
Prunus mume|Rep: Pm52
max|Rep: Pyruvate kinase -
Glycine max (Soybean) = partial
Lotus_japonicus_release_1
max|Rep: Pyruvate kinase,
max (Soybean), partial (26%)
Phaseolus_vulgaris_release_2
Vigna_unguiculata
max|Rep: Pyruvate kinase -
Glycine max (Soybean), partial
Glycine_max_release_2
Phaseolus_vulgaris
max|Rep: Pyruvate kinase,
max (Soybean), partial (64%)
Glycine_soja_release_2
Glycine_soja_release_2
max|Rep: Pyruvate kinase -
Glycine max (Soybean) = partial
Glycine_max_release_2
vinifera|Rep: Chromosome
Phaseolus_vulgaris
Glycine_max_release_2
vinifera|Rep: Chromosome
vinifera|Rep: Chromosome
Caranx ignobilis = partial (37%)
Glycine_max_release_2
vinifera|Rep: Chromosome
Glycine_max_release_2
Lotus_japonicus_release_1
japonicus]
japonicus|Rep: Sphingosine
japonicus|Rep: Sphingosine
Glycine_max_release_2
japonicus]
Phaseolus_vulgaris
Glycine_max_release_2
japonicus]
japonicus|Rep: Sphingosine
Glycine_max_release_2
japonicus]
Arachis_stenosperma_release_5
japonicus
Glycine_max_release_2
Glycine_max_release_2
Glycine_max_release_2
vinifera|Rep: Chromosome
Glycine_max_release_2
vinifera|Rep: Chromosome
Glycine_max_release_2
Glycine_max_release_2
Glycine_max_release_2
vinifera|Rep: Chromosome chr5
vinifera (Grape) = partial (15%)
vinifera|Rep: Chromosome chr8
vinifera (Grape), partial (24%)
Glycine_max_release_2
Bacillus cereus subsp.cytotoxis
Glycine_max_release_2
Phaseolus_coccineus_release_2
Arabidopsis thalianagenomic
thaliana (Mouse-ear cress)]
Glycine_max_release_2
Glycine_max_release_2
vinifera|Rep: Chromosome
Vitis vinifera (Grape) = partial
vinifera|Rep: Chromosome
Vitis vinifera (Grape) = partial
Glycine_max_release_2
vinifera|Rep: Chromosome
Vitis vinifera (Grape) = partial
Phaseolus_coccineus_release_2
Phaseolus_vulgaris
vinifera|Rep: Chromosome
Vitis vinifera (Grape) = partial
Glycine_max_release_2
Lotus_corniculatus_release_1
vinifera|Rep: Chromosome
Vitis vinifera (Grape) = partial
Glycine_max_release_2
vinifera|Rep: Chromosome
Vitis vinifera (Grape) = partial
Glycine_max_release_2
Glycine_max_release_2
Glycine_soja_release_2
Glycine_max_release_2
papaya|Rep: Protein kinase -
Carica papaya (Papaya) = partial
papaya|Rep: Protein kinase -
Carica papaya (Papaya) = partial
Vigna_unguiculata
Glycine_max_release_2
Phaseolus_vulgaris
Glycine_max_release_2
Lotus_corniculatus_release_1
Lotus_japonicus_release_1
thaliana (Mouse-ear cress)]
Carica papaya|Rep: Protein
vinifera|Rep: Chromosome
Vitis vinifera (Grape) = partial
vinifera|Rep: Chromosome
Vitis vinifera (Grape), partial
Phaseolus_vulgaris
Glycine_max_release_2
truncatula (Barrel medic)]
Phaseolus_vulgaris
vinifera|Rep: Chromosome
Vitis vinifera (Grape) = partial
Glycine_max_release_2
vinifera|Rep: Chromosome
Vitis vinifera (Grape) = partial
Glycine_max_release_2
Vigna_unguiculata
radiata|Rep: Carbonic
Glycine_max_release_2
aureus (Mung bean) (Vigna
radiata)]
Vigna radiata var. radiata|Rep:
aureus (Mung bean) (Vigna
radiata) = partial (34%)
Glycine_max_release_2
Glycine_soja_release_2
Glycine_max_release_2
aureus (Mung bean) (Vigna
radiata)]
Glycine_max_release_2
Glycine_max_release_2
Glycine_max_release_2
radiata|Rep: Carbonic
Phaseolus_vulgaris_release_2
Vigna radiata var. radiata|Rep:
aureus (Mung bean) (Vigna
radiata) = complete
Lotus_japonicus_release_1
Phaseolus_vulgaris
radiata var. radiata
Phaseolus_vulgaris_release_2
Glycine_soja_release_2
aureus (Mung bean) (Vigna
radiata)]
Glycine_max_release_2
Glycine_max_release_2
sativum (Garden pea)]
sativum|Rep: Carbonic
sativum (Garden pea), partial
Glycine_soja_release_2
aureus (Mung bean) (Vigna
radiata)]
Phaseolus_vulgaris
radiata var. radiata
Glycine_max_release_2
Vigna radiata var. radiata|Rep:
aureus (Mung bean) (Vigna
radiata) = partial (62%)
Glycine_soja_release_2
sativum (Garden pea)]
Phaseolus_vulgaris
radiata var. radiata
sativum|Rep: Carbonic
sativum (Garden pea), partial
Phaseolus_vulgaris_release_2
aureus (Mung bean) (Vigna
radiata)]
Phaseolus_vulgaris_release_2
sativum|Rep: Carbonic
sativum (Garden pea), partial
Vigna_unguiculata
Vigna_unguiculata
Vigna_unguiculata
Glycine_max_release_2
aureus (Mung bean) (Vigna
radiata)]
Vigna radiata var. radiata|Rep:
aureus (Mung bean) (Vigna
radiata) = partial (38%)
Pisum_sativum_release_2
Vigna radiata var. radiata|Rep:
aureus (Mung bean) (Vigna
radiata) = partial (38%)
Glycine_max_release_2
sativum (Garden pea)]
Glycine_max_release_2
aureus (Mung bean) (Vigna
radiata)]
Glycine_max_release_2
aureus (Mung bean) (Vigna
radiata)]
Phaseolus_vulgaris_release_2
aureus (Mung bean) (Vigna
radiata)]
Vigna_unguiculata
Glycine_soja_release_2
aureus (Mung bean) (Vigna
radiata)]
radiata|Rep: Carbonic
Lotus_corniculatus_release_1
Lotus_japonicus_release_1
radiata|Rep: Carbonic
Phaseolus_vulgaris_release_2
aureus (Mung bean) (Vigna
radiata)]
Arabidopsis thaliana|Rep:
Lotus_japonicus_release_1
Pisum_sativum_release_2
Glycine_max_release_2
Glycine_max_release_2
Vigna_unguiculata
Phaseolus_vulgaris
Glycine_max_release_2
truncatula (Barrel medic)]
vinifera|Rep: Chromosome
Vitis vinifera (Grape) = partial
Glycine_max_release_2
sativa (japonica cultivar-
vinifera|Rep: Chromosome
Vitis vinifera (Grape) = partial
thaliana|Rep: Protease Do-like
Arabidopsis thaliana (Mouse-
Glycine_max_release_2
truncatula (Barrel medic)]
Glycine_max_release_2
truncatula (Barrel medic)]
vinifera|Rep: Chromosome
Vitis vinifera (Grape) = partial
Glycine_max_release_2
truncatula (Barrel medic)]
vinifera|Rep: Chromosome
Vitis vinifera (Grape) = partial
vinifera|Rep: Chromosome
Vitis vinifera (Grape) = partial
vinifera|Rep: Chromosome
Vitis vinifera (Grape), partial
Glycine_max_release_2
truncatula (Barrel medic)]
vinifera|Rep: Chromosome
Vitis vinifera (Grape) = partial
Glycine_max_release_2
truncatula (Barrel medic)]
vinifera|Rep: Chromosome
Vitis vinifera (Grape) = partial
Medicago sativa|Rep: Coil
truncatula|Rep: Lipolytic
truncatula (Barrel medic),
vinifera|Rep: Chromosome
Vitis vinifera (Grape) = partial
Glycine_max_release_2
truncatula|Rep: Lipolytic
truncatula (Barrel medic) =
Glycine_max_release_2
Glycine_max_release_2
vinifera|Rep: Chromosome
Vitis vinifera (Grape) = partial
Glycine_max_release_2
Vigna_unguiculata
vinifera|Rep: Chromosome
Vitis vinifera (Grape), partial
Glycine_max_release_2
vinifera|Rep: Chromosome
Vitis vinifera (Grape) = partial
Glycine_max_release_2
Glycine_max_release_2
radiata]
Phaseolus_vulgaris_release_2
vesca|Rep: Ripening-induced
Glycine_soja_release_2
Glycine_soja_release_2
Glycine_max_release_2
Glycine_soja_release_2
Phaseolus_coccineus_release_2
Lotus_japonicus_release_1
radiata = partial (54%)
Glycine_max_release_2
Glycine_max_release_2
radiata = partial (30%)
Glycine_max_release_2
radiata]
Phaseolus_vulgaris
radiata
Vigna_unguiculata
Glycine_max_release_2
Glycine_max_release_2
robur|Rep: Formate
Glycine_max_release_2
Glycine_max_release_2
Glycine_soja_release_2
Vigna_unguiculata
Glycine_soja_release_2
vulgare|Rep: Formate
Glycine_max_release_2
sativa (Rice)]
Glycine_max_release_2
sativa (Rice)]
Glycine_max_release_2
sativa (Rice)]
Glycine_soja_release_2
sativa (Rice)]
Glycine_max_release_2
Glycine_max_release_2
Glycine_max_release_2
Glycine_soja_release_2
vinifera|Rep: Chromosome
Vitis vinifera (Grape) = partial
Glycine_max_release_2
Glycine_max_release_2
Glycine_max_release_2
Glycine_max_release_2
robur|Rep: Formate
robur|Rep: Formate
Glycine_max_release_2
Vigna_unguiculata
robur|Rep: Formate
Glycine_max_release_2
Glycine_max_release_2
sativa (Rice)]
Phaseolus_vulgaris_release_2
Phaseolus_vulgaris
Phaseolus_coccineus_release_2
Glycine_max_release_2
Vigna_unguiculata
Glycine_soja_release_2
Lotus_japonicus_release_1
tuberosum|Rep: Calcium
Solanum tuberosum (Potato),
Phaseolus_vulgaris
vinifera|Rep: Chromosome
Vitis vinifera (Grape) = partial
Phaseolus_vulgaris
Glycine_max_release_2
trifida (Morning glory)]
vinifera|Rep: Chromosome
Vitis vinifera (Grape) = partial
Glycine_soja_release_2
trifida (Morning glory)]
Glycine_max_release_2
vinifera|Rep: Chromosome
Vitis vinifera (Grape) = partial
Glycine_max_release_2
vinifera|Rep: Chromosome
Vitis vinifera (Grape) = partial
vinifera|Rep: Chromosome
Vitis vinifera (Grape) = partial
Glycine_max_release_2
Glycine_max_release_2
vinifera|Rep: Chromosome
Vitis vinifera (Grape), partial
vinifera|Rep: Chromosome
Vitis vinifera (Grape) = partial
Vigna_unguiculata
vinifera|Rep: Chromosome
Vitis vinifera (Grape) = partial
Glycine_max_release_2
Arabidopsis thaliana|Rep:
thaliana (Mouse-ear cress),
Lotus_japonicus_release_1
thaliana (Mouse-ear cress)]
Glycine_max_release_2
Glycine_max_release_2
sativa|Rep: Peptidyl-prolyl cis-
Phaseolus_vulgaris_release_2
Glycine_max_release_2
sativum (Garden pea)]
Phaseolus_vulgaris_release_2
sativum (Garden pea)]
Phaseolus_vulgaris
sativum RepID = O22636_PEA
Lotus_japonicus_release_1
sativum (Garden pea)]
Lotus_corniculatus_release_1
sativum (Garden pea), partial
Glycine_max_release_2
sativum (Garden pea)]
sativum (Garden pea) = partial
Phaseolus_coccineus_release_2
sativum (Garden pea)]
Vigna_unguiculata
Glycine_max_release_2
vinifera|Rep: Chromosome
Vitis vinifera (Grape) = partial
Glycine_max_release_2
Glycine_max_release_2
vulgaris (Kidney bean) (French
vulgaris|Rep: Homeodomain
Phaseolus vulgaris (Kidney
Phaseolus_vulgaris
Phaseolus_vulgaris_release_2
vulgaris (Kidney bean) (French
Vigna_unguiculata
Glycine_soja_release_2
thaliana (Mouse-ear cress)]
Phaseolus vulgaris|Rep:
vulgaris (Kidney bean) (French
Phaseolus vulgaris|Rep:
vulgaris (Kidney bean) (French
Glycine_max_release_2
vulgaris (Kidney bean) (French
Glycine_max_release_2
vulgaris (Kidney bean) (French
Glycine_soja_release_2
Phaseolus_vulgaris
Japonica Group|Rep: PHD
sativa subsp. japonica (Rice),
vinifera|Rep: Chromosome
Vitis vinifera (Grape), partial
Lotus_japonicus_release_1
sativa (japonica cultivar-
Phaseolus_coccineus_release_2
Glycine_max_release_2
Glycine_max_release_2
vinifera|Rep: Chromosome chr8
vinifera (Grape) = partial (21%)
Glycine_max_release_2
Glycine_max_release_2
Glycine_max_release_2
vinifera|Rep: Chromosome chr8
vinifera (Grape) = partial (45%)
Glycine_max_release_2
Glycine_max_release_2
Glycine_max_release_2
Glycine_max_release_2
vinifera|Rep: Chromosome chr1
vinifera (Grape) = partial (63%)
Glycine_max_release_2
Glycine_max_release_2
vinifera|Rep: Chromosome chr1
vinifera (Grape) = partial (54%)
Glycine_max_release_2
sativa (japonica cultivar-
Glycine_max_release_2
truncatula|Rep:
Medicago truncatula (Barrel
Phaseolus_coccineus_release_2
Phaseolus_vulgaris
tabacum|Rep: Aldehyde
tabacum (Common tobacco) =
Glycine_max_release_2
Phaseolus_vulgaris
vinifera|Rep: Chromosome
Vitis vinifera (Grape), partial
vinifera|Rep: Chromosome
Vitis vinifera (Grape) = partial
Glycine_max_release_2
tabacum (Common tobacco)]
Phaseolus_vulgaris
vinifera|Rep: Chromosome
Vitis vinifera (Grape) = partial
vinifera|Rep: Chromosome
Vitis vinifera (Grape), partial
Vigna_unguiculata
Glycine_max_release_2
esculenta (Food pokeberry)]
Phaseolus_vulgaris
vinifera|Rep: Chromosome
Vitis vinifera (Grape) = partial
Glycine_max_release_2
vinifera|Rep: Chromosome
Vitis vinifera (Grape) = partial
Glycine_max_release_2
crystallinum (Common ice
Glycine_max_release_2
thaliana|Rep: NHL repeat-
Arabidopsis thaliana = partial
Glycine_max_release_2
Glycine_max_release_2
vinifera|Rep: Chromosome
Vitis vinifera (Grape) = partial
Vigna_unguiculata
Glycine_max_release_2
vinifera|Rep: Chromosome chr1
vinifera (Grape) = partial (17%)
Phaseolus_vulgaris
Glycine_max_release_2
scrofa|Rep: Skinkine - Sus
scrofa (Pig) = partial (12%)
Glycine_max_release_2
sativa Japonica Group|Rep:
sativa = partial (2%)
Glycine_max_release_2
Gossypium hirsutum (Upland
mexicanum) = partial (22%)
max|Rep: Hsp22.3 - Glycine
max (Soybean) = complete
Glycine_soja_release_2
vinifera|Rep: Chromosome
Vitis vinifera (Grape) = partial
Glycine_max_release_2
truncatula (Barrel medic)]
vinifera|Rep: Chromosome chr1
vinifera (Grape), partial (7%)
Phaseolus_vulgaris
vinifera|Rep: Chromosome
Vitis vinifera (Grape) = partial
Glycine_max_release_2
Glycine_soja_release_2
Glycine_max_release_2
thaliana|Rep: Nodulin MtN21-
thaliana (Mouse-ear cress) =
Phaseolus_vulgaris_release_2
Arachis_stenosperma_release_5
Medicago truncatula (Barrel
Vigna_unguiculata
Glycine_max_release_2
Glycine_soja_release_2
Phaseolus_vulgaris
Glycine_max_release_2
vinifera|Rep: Chromosome
Vitis vinifera (Grape) = partial
Glycine_soja_release_2
vinifera|Rep: Chromosome
Vitis vinifera (Grape) = partial
Glycine_max_release_2
sativum (Garden pea)]
Vigna_unguiculata
Lupinus_albus_release_2
sativum (Garden pea)]
Phaseolus_vulgaris
sativum RepID = Q5GQ66_PEA
sativum (Garden pea) = partial
Vigna_unguiculata
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:
a) “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);
b) “phytozome.net” or “phytozome.net/cgi-bin/gbrowse/soybean/?name=Gm09”;
c) “www.plantgdb.org” or “plantgdb.org/GmGDB/(Assembly version Glyrnal.170 (April 2009)”; and,
d) “ncbi.nlm.nih.gov/sites/entrez” and subsites “ncbi.nlm.nih.gov/nucest”, “ncbi.nlm.nih.gov/dbEST”, “ncbi.nlm.nih.gov/genbank/”, “.ncbi.nlm.nih.gov/sites/genome”, “ncbi.nlm.nih.gov/unigene”, and “ncbi.nlm.nih.gov/UniGene/UGOrg.cgi?TAXID=3847”.
This application is a continuation of U.S. application Ser. No. 13/224,036, now U.S. Pat. No. 8,921,647, filed Sep. 1, 2011 and incorporated herein by reference in its entirety, which claims the benefit of U.S. Provisional Application Ser. No. 61/380,024, filed Sep. 3, 2010 and incorporated herein by reference in its entirety.
| 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 |
| 7067723 | Narvel | Jun 2006 | B2 |
| 7071388 | Narvel | Jul 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 |
| 7504565 | Jenkinson et al. | Mar 2009 | B2 |
| 7554014 | Moots et al. | Jun 2009 | B2 |
| 7569750 | Behm | Aug 2009 | B2 |
| 7728197 | Bowers et al. | Jun 2010 | B1 |
| 20060288444 | McCarroll et al. | Dec 2006 | A1 |
| 20090036308 | Guida, Jr. et al. | Feb 2009 | A1 |
| 20090064354 | Narvel | Mar 2009 | A1 |
| 20090105077 | Bhatti et al. | Apr 2009 | A1 |
| 20090165166 | Feng et al. | 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 |
| Entry |
|---|
| GenBank BG406195.1, “sac36g03.y1 Gm-c1051 Glycine Max cDNA CloneGenome Systems Clone ID: Gm-c1051-4518 5-, mRNA Sequence”, Jul. 22, 2004 (online), retrieved Dec. 27, 2011, Available on the Internet: <URL: http://www.ncbi.nlm.nih.gov/nucest/BG406195>. |
| Song et al., “A New Integrated Genetic Linkage Map of the Soybean”, Theoretical and Applied Genetics, Jun. 2004, pp. 122-128, vol. 109, No. 1. |
| Wang et al., “Association Mapping of Iron Deficiency Chlorosis Loci in Soybean (Glycine max L. Merr.) Advance Breeding Lines”, Theoretical and Applied Genetics, Apr. 2008, pp. 777-787, vol. 116, No. 6. |
| GenBank BU082700.1, “saq36h09.y.1 Gm-c1045 Glycine Max cDNA Clone Soybean Clone ID: Gm-c1045-6906 5—Similar to TR:Q9SSA4 Hypothetical 46.0 KD Protein, mRNA Sequence”, Jul. 2, 2004 (online), retrieved Dec. 27, 2011, Available on the Internet: <URL: http://www.ncbi.nlm.nih.gov/nucest/BU082700>. |
| Padgette et al., “Development, Identification, and Characterization of a Glyphosate-Tolerant Soybean Line”, Crop Science, 1995, pp. 1451-1461, vol. 35. |
| Delannay et al., “Yield Evaluation of a Glyphosate-Tolerant Soybean Line after Treatment with Glyphosate”, Crop Science, 1995, pp. 1461-1467, vol. 35. |
| Meyer et al., “Genetic Factors Influencing Adverse Effects of Mesotrione and Nicosulfuron on Sweet Corn Yield”, Agronomy Journal, 2010, pp. 1138-1144, vol. 102, Issue 4. |
| Choi et al., “A Soybean Transcript Map: Gene Distribution, Haplotype and Single-Nucleotide Polymorphism Analysis”, Genetics, May 2007, pp. 685-696, vol. 176. |
| Grant et al., “SoyBase, the USDA-ARS Soybean Genetics and Genomics Database”, Nucleic Acids Research, 2010, pp. D843-D846, vol. 38. |
| Hyten et al., “A High Density Integrated Genetic Linkage Map of Soybean and the Development of a 1536 Universal Soy Linkage Panel for Quantitative Trait Locus Mapping”, Crop Science, May-Jun. 2010, pp. 960-968, vol. 50. |
| Hyten et al., “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, 2010, pp. 1-8, vol. 11 No. 38. |
| Yoon et al., “BARCSoySNP23: A Panel of 23 Selected SNPs for Soybean Cultivar Identification”, Theoretical and Applied Genetics, 2007, pp. 885-899, vol. 114. |
| Shoemaker et al., “Public Soybean EST Project”, Genbank [database online], 1999 [retrieved on Dec. 6, 2013], retrieved from the internet: <URL: http://www.ncbi.nlm.nih.gov/nucest/23735360?report=genbank> Acession: BU765955. |
| Que et al., “Trait Stacking in Transgenic Crops: Challenges and Opportunities”, GM Crops, Jul.-Aug. 2010, pp. 220-229, vol. 1 No. 4. |
| Heffner, Elliot L. et al., “Genomic Selection for Crop Improvement”, Crop Science, Jan.-Feb. 2009, pp. 1-12, vol. 49. |
| Number | Date | Country | |
|---|---|---|---|
| 20150067912 A1 | Mar 2015 | US |
| Number | Date | Country | |
|---|---|---|---|
| 61380024 | Sep 2010 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 13224036 | Sep 2011 | US |
| Child | 14535106 | US |
