OA16515A - Soybean event MON89788 and methods for detection thereof. - Google Patents
Soybean event MON89788 and methods for detection thereof. Download PDFInfo
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- OA16515A OA16515A OA1201300398 OA16515A OA 16515 A OA16515 A OA 16515A OA 1201300398 OA1201300398 OA 1201300398 OA 16515 A OA16515 A OA 16515A
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Abstract
The present invention provides for soybean plant and seed comprising transformation event MON89788 and DNA molecules unique to these events. The invention also provides methods for detecting the presence of these DNA molecules in a sample.
Description
DESCRIPTION
SOYBEAN EVENT MON89788 AND METHODS
FOR DETECTION THEREOF
BACKGROUND OF THE INVENTION
This application claims priority from U.S. Piovisional Application No. 60/685,584, filed May 27, 2005, the entire disclosure of which is herein incorporated by reference.
1. Field of the Invention
The présent invention relates to a new and distinctive transgenic soybean transformation event, designated MON89788, a soybean cultivar derived therefrom, and plant parts, seed, and products thereof. The invention also relates to assays for detecting the presence of a DNA molécule spécifie to MON89788 in a plant part extract or seed extract.
2, Description of Related Art
Soybean (Glycine max) is an important crop in many areas of the world. The methods of biotechnology hâve been applied to soybean for improvement of the agronomie traits and the quality of the product. One such agronomie trait-important in soybean production is herbicide tolérance, in particular, tolérance to glyphosate herbicide. 20 A herbicide tolérant soybean event would be a useful trait for managing weeds.
N-phosphonomethylglycine, also-known as glyphosate, is a well-known herbicide that has activity on a broad spectrum of plant species. Glyphosate is the active ingrédient of Roundup® (Monsanto Co., St Louis, MO), a safe herbicide having a desirably short half-life in the environment. When applied to a plant surface, glyphosate moves 25 systemically through the plant Glyphosate is phytotoxic due to its inhibition of the shikimic acid pathway, which provîdes a precursor for the synthesis of aromatic amino acids. Glyphosate inhibits the eu2yme 5-enolpyruyyl-3-phosphoshikirnate synthase (EPSPS) found in plants.
Glyphosate tolérance can be achieved by the expression of EPSPS variants that hâve lower afSnîty for glyphosate and therefore retain their catalytic activity in the presence of glyphosate (U.S. Patent Nos. 5,633,435; 5,094,945; 4,535,060, and 6,040,497). Enzymes that dégradé glyphosate in plant tissues (U.S. Patent No. 5,463,175) are also capable of conferring cellular tolérance to glyphosate. Such genes are used for the production of transgenic crops that are tolérant to glyphosate, thereby allowing glyphosate to be used for. effective weed control with minimal concem of crop damage. For example, glyphosate tolérance has been genetically engineered into corn (U.S. Patent No. 5,554,798), wheat (U.S. Patent 6,689,880), cotton (U.S. Patent 6,740,488), soybean (WO 9200377) and canola (US Patent Appl. 20040018518). The transgenes for glyphosate tolérance and the transgenes for tolérance to other herbicides, e.g. the bar gene, (Toki ez al., 1992; Thompson et al., 1987; phosphinothricin acetyltransferase (DeBlock et al., 1987), for tolérance to glufosinate herbicide) are also useful as selectable markers or scorable markers and can provide a useful phenotype for sélection of plants linked with other agronomically useful traits.
The expression of foreign genes in plants is known to be influenced by their chromosomal position, perhaps due to chromatin structure (e.g., heterochromatin) or the proxiinity of transcriptional régulation éléments (e.g., enhancers) close to the intégration site (Weising et al., 1988). For fois reason, it is often necessary to screen a large number of events in order to identify an event characterized by optimal expression of an introduced gene of interest. For example, it has been observed in plants and in other organisms that there may be a wide variation in levels of expression of an introduced gene among events.* There may also be différences in spatial or temporal patterns of expression, for example, différences in the relative expression of a transgene in various plant tissues, that may not correspond to the patterns expected from transcriptional regulatory éléments présent ïn the introduced gene construct. For this reason, it is common to produce hundreds to thousands of different events and screen those events for a single event that has desired transgene expression levels and patterns for commercial purposes. An event that has desired levels or patterns of transgene expression is useful for introgressing foe transgene into other genetic backgrounds by sexual outcrossing using conventional breeding methods. Progeny of such crosses maintain foe transgene expression characteristics of the original transformant. This strategy is used to ensure reliable gene expression in a number of varieties that are well adapted to local growing conditions.
It would be advantageous to be able to detect the presence of a particular event in order to détermine whether progeny of a sexual cross contain. a transgene of interest. In addition, a method for detecting a particular event would be helpfol for complying with régulations requiring the pre-market approval and labeling of foods derived from recombinant crop plants, for example. It is possible to detect the presence of a transgene by any well known polynucleic acid détection method such as the polymerase chain reaction (PCR) or DNA hybridization using polynucleic acid probes. These détection methods generally fbcus on frequently used genetic éléments, such as promoters, termînators, marker genes, etc. As a resuit, such methods may not be useful for discrimmating between different events, particularly those produced using the same DNA construct unless the sequence of chromosomal DNA (“flanking DNA”) adjacent to the inserted transgene DNA is known. An event-specific PCR assay is discussed, for example, by Windels et al. (1999), who identified glyphosate tolérant soybean event 40-32 by PCR using a primer set spanning the junction between the insert transgene and flanking DNA, specifically one primer that included sequence from the insert and a second primer that included sequence from flanking DNA. Transgenïc plant event spécifie DNA détection methods hâve also been described in US Patent Nos. 6,893,826; 6,825,400; 6,740,488; 6,733,974 and 6,689,880; 6,900,014 and 6,818,807, herein incorporated by reference in their entirety.
This invention relates to the glyphosate tolérant soybean event MON89788 (also referred to as MON19788 or GM_A19788) and to the DNA molécules contained in these soybean plants that are useful in détection methods for the plant and progeny thereof and plant tissues derived from MON89788.
SUMMARY OF THE INVENTION
The présent invention provides a soybean transgemc event designated MON89788 (also referred to as MON19788) and progeny thereof having représentative seed deposited with American Type Culture Collection (ATCC) with accession No. PTA-6708. Another aspect of the invention is the plant cells or regenerable parts of the plant and seeds of the soybean event MON89788. The invention also includes plant parts of soybean event —
MON89788 that include, but are not limited to a cell, pollen, ovule, fîowers, shoots, roots, leaves, andproducts derived from MON89788, for example soybeanmeal, flour and oil.
One aspect of the invention provides compositions and methods for detecting the presence of a DNA transgene/genomic jonction région from a soybean event MON89788 plant or seed or products derived from plant parts or seed. DNA molécules are provided that comprise at least one transgene/genomic junction DNA molécule selected from the group consisting of SEQ ID NO:1 and SEQ ID NO:2, and compléments thereof, wherein the junction molécule sp ans the insertion site that comprises a heterologous DNA inserted into the genome of the soybean cell and the genoniic DNA from the soybean cell flariking the insertion site soybean event MON89788. Such junction sequences may, in one aspect of the invention, be defined as comprising nucléotides 1093-1113 or 5396-5416 of SEQ ID NO:9, respectively. In other aspects of the invention, the junctions may be defined as including additional portions of the flanking genome and trans gene, for example, and may be defined as comprising one or more sequence as given by nucléotides 1073-1113,10431113,1093-1133, 1093-1163,1043-1163,5376-5416, 5346-5416, 5396-5436, 5396-5416, 5396-5466, or 5346-5466 of SEQ ID NO:9. Such sequences and plants and seeds comprising these sequences therefore form one aspect of the invention.
A novel DNA molécule is provided that is a DNA transgene/genomic région SEQ ID NO:3 or the complément thereof from soybean event MON89788. A soybean plant and seed comprising SEQ ID NO:3 in its genome is an aspect of this invention. SEQ ID NO:3 further comprises SEQ ID NO:1 in its entirety.
According to another aspect of the invention, a DNA molécule is provided that is a DNA transgene/genomic région SEQ ID NO:4, or the complément thereof wherein this DNA molécule is novel in soybean event MON89788. A soybean plant and seed comprising SEQ ID NO:4 in its genome is an aspect of this invention. SEQ ID NO:4 further comprises SEQ ID NO:2 in its entirety.
According to another aspect of the invention, two nucleic acid molécules are provided for use in a DNA détection method, wherein the first nucleic acid molécule comprises at least 11 or more contiguous polynucleotides of any portion of the transgene région of the DNA molécule of SEQ ID NO:3 and the second nucleic acid is a molécule of similar length of any portion of a 5’ flanking soybean genomic DNA région of SEQ ID NO:3, wherein these nucleic acid molécules when used together are useful as primera in a >
DNA amplification method that produces an amplicori. The amplicon produced using these primers in the DNA amplification method is diagnostic for soybean event
MON89788 DNA. The amplicon produced by the described primers that is homologous or complementary to a portion of SEQ JD NO:3 comprising SEQ ID NO:1 is an aspect of the invention.
According to another aspect of the invention, two nucleic acid molécules are provided for use in a DNA détection method, wherein the first nucleic acid molécule comprises at least 11 or more contiguous polynucleotides of any portion of the transgene région of the DNA molécule of SEQ ID NO:4 and a second nucleic acid molécule of similar length of any portion of a 3* flanking soybean genomic DNA of SEQ ID NO:4, wherein these nucleic acid molécules when used together are useful as primers in a DNA amplification method that produces an amplicon. The amplicon produced using these primers in the DNA amplification method is diagnostic for soybean event MON89788 DNA. The amplicon produced by the described primers that is homologous or complementary to a portion of SEQ ID NO:4 comprising SEQ ID NO:2 is an aspect of the invention.
Any nucleic acid primer pair derived from SEQ ID NO:3 or SEQ ID NO:4, or SEQ ID NO:9 or the compléments thereof, that when used in a DNA amplification reaction produces an amplicon diagnostic for soybean event MON89788-derived tissue, such as an amplicon that comprises SEQ ID NO:1 or SEQ ID NO:2 or any portion of SEQ ID NO:9 respectively, is another embodiment of the invention. Ih a particular embodiment, the primer pair may consist of primer A (SEQ ID NO:5) and primer D (SEQ IDNO:8).
Another aspect of the invention is a soybean plant, or seed, or product derived from a plant or seed comprising event MON89788, in which the genomic DNA when isolated from the soybean plant; or seed, or product produces an amplicon in a DNA amplification method that comprises SEQ ID NO:1 or SEQ ID NO:2.
Still another aspect of the invention is a soybean plant, or seed, or product derived from a plant or seed comprising MON89788, in which the genomic DNA when isolated from the soybean plant, or seed, or product produces an amplicon in a DNA amplification method, wherein DNA primer molécules SEQ ID NO:5 and SEQ ID NO:6 are used in the DNA amplification method.
Yet another aspect of the invention is a soybean plant, seed, product, or commodity derived from the plant or seed, comprising MON89788, in which the genomic DNA when isolated from the s.oybean plant, or seed, or product produces an amplicon in a DNA amplification method, wherein DNA primer molécules SEQ ID NO:7 and SEQ ID NO:8 are used in the DNA amplification method. The product or commodity may comprise, without limitation, a food or feed product comprising or derived from one or more of the following products of a soybeanplant comprising event MON89788: lecithin, fatty acids, glycerol, sterol, edible oil, defatted soy fiakes, soy meals including defatted and toasted soy meals, soy milk curd, tofu, soy flour, soy protein concentrate, isolated soy protein, hydrolyzed vegetable protein, textured soy protein, and soy protein fiber.
According to another aspect of the invention, a method of detecting the presence of DNA corresponding specifically to the soybean event MON89788 DNA in a sample is provided. Such method comprising: (a) contacting a sample comprising DNA with a DNA primer pair; (b) performing a nucleic acid amplification réaction, thereby producing the amplicon; and (c) detecting the amplicon, wherein said amplicon comprises SEQ ID ΝΟ.Ί or SEQ ID NO:2. A kit comprising DNA primer molécules that when used in a DNA amplification method produces an amplicon comprising SEQ ID NO:1 or SEQ ID NO:2 is a further aspect of the invention.
According to another aspect of the invention, a method of detecting the presence of DNA corresponding specifically to the soybean event MON89788 DNA in a sample is provided. Such method comprising: (a) contacting a sample comprising DNA with a probe that hÿbridizes under stringent hybridization conditions with genomic DNA from soybean event MON89788 and does not hybridize under the stringent hybridization conditions with a control soybean plant DNA; (b) subjecting the sample and probe to stringent hybridization conditions; and (c) detecting hybridization of the probe to the soybean event MON89788 DNA, wherein said probe comprises SEQ ID NO:1 or SEQ ID NO:2. The sample may comprise a progeny seed, plant, or plant part comprising soybean event MON89788, or any of the following products derived from a plant comprising MON89788: lecithin, fatty acids, glycerol, sterol, edible oil, defatted soy fiakes, soy meals including defatted and toasted soy meals, soy milk curd, tofu, soy flour, soy protein concentrate, isolated soy protein, hydrolyzed vegetable protein, textured soy protein, and soy protein fiber. A kit comprising a DNA probe comprising a DNA molécule that is„ homologous or complementary to SEQ ID NO:1 or SEQ ID NO:2 is an aspect of the invention. A kit comprising a DNA molécule comprising SEQ ID NO: 18, SEQ ID
NO:19, or SEQ ID NO:20, or their compléments, is also an aspect of the invention.
According to another aspect of the invention, a method of producing a soÿbean plant that tolérâtes an application of glyphosate is provided that comprise the steps of: (a) sexually crossing a first parental glyphosate tolérant soÿbean plant comprising event MON89788, and a second parental soybean plant that lacks the glyphosate tolérance, thereby producing a plurality of progeny plants; and (b) selecting a progeny plant that tolérâtes application of glyphosate. Breeding methods may additionally comprise the steps of crossing the parental plant comprising soÿbean event MON89788 to a second parental soybean plant that is also tolérant to glyphosate and selecting for glyphosate tolérant progeny by molecular marker DNA genetically Iinked to the glyphosate tolérant phenotype found in each parent
Another aspect of the invention, is a method to control weeds in a field of soÿbean plants comprising MON89788, wherein said method comprises planting a field with soybean seed comprising event MON89788 said représentative seed deposited as ATCC accession No. PTA-6708, allowing said seed to germinate and treating said plants with an effective dose of glyphosate to control weed growth in said field.
The foregoing and other aspects of the invention will become more apparent from the following detailed description and accompanymg drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG 1. Organization of the transgene insertion in the genome of a soybean plant comprising event MON89788.
FIG. 2A- 2B. Processing of commodity products from soybean.
DETAILED DESCRIPTION
The présent invention relates to a novel soÿbean transformation event designated MON89788 that provides glyphosate tolérance, and the plant parts and seed and products produced from plants, plant parts, seed, and products comprising the event. The invention provides DNA molécules that are novel in the genome of soybean cells comprising MON89788 and DNA molécules that can be used in various DNA détection methods to „ i identify ΜΟΝ89788 DNA in a sample. The invention provides a method. to control weeds in a field of plants containing MON89788 by treating the weeds in the field | comprising plants comprising event MON89788 with a glyphosate herbicide.
| The following définitions and methods are provided to better defîne the présent
Si invention and to guide those of ordinary skill in the art in the practice of the présent | invention. Unless otherwise noted, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art. Définitions of common tenus in molecular biology may also be found in Rieger et al. (1991) and Lewin (1994). The nomenclature for DNA bases as set forth at 37 CFR § 1.822 is used.
As used. herein, the term “soybean” means Glycine max and includes ail plant f varieties that can be bred with soybean.
As used herein, the term “comprising” means “including but not limited to”.
“Glyphosate” refers to N-phosphonomethylglycine and its salts, Glyphosate is the active ingrédient of Roundup® herbicide (Monsanto Co.). Treatments with “glyphosate 15' herbicide” refer to treatments with the Roundup®, Roundup Ultra®, Roundup Pro® I herbicide or any other herbicide formulation containing glyphosate. Examples of commercial formulations of glyphosate include, without restriction, those sold by Monsanto Company as ROUNDUP®, ROUNDUP® ULTRA, ROUNDUP® i ULTRAMAX, ROUNDUP® CT, ROUNDUP® EXTRA, ROUNDUP® BIACTIVE, 2(j| ROUNDUP® BIOFORCE, RODEO®, POLÀRIS®, SPARK® and ACCORD® herbicides, ail of which contain glyphosate as its isopropylammonium sait; ROUNDUP® I WEATHERMAX (glyphosate potassium sait), those sold by Monsanto Company as ROUNDUP® DRY and RIVAL® herbicides, which contain glyphosate as its ammonium sait; that sold by Monsanto Company as ROUNDUP® GEOFORCE, which contains 25f glyphosate as its sodium sait; and that sold by Syngenta Crop Protection as |, TOUCHDOWN® herbicide, which contains glyphosate as its trimethylsulfonium sait. Treatment of a field comprising glyphosate tolérant soybean plants comprising event | MON89788 with any of these glyphosate herbicide formulations will control weed growth in the field and not affect the growth or yield of the soybean plants comprising 30 MON89788.
A transgenic “event” is produced by transformation of plant cells with heterologous DNA, for example, a nucleic acid construct that includes a transgene of interest, régénération of a population of plants resulting from the insertion of the transgene into the genome of the plant, and sélection of a particular plant characterized by insertion into a particular genome location, The term “event” refers to the original transformant and progeny of the transformant that include the heterologous DNA. The tenu “event” also refers to progeny produced by a sexual outcross between the transformant and another variety that include the heterologous transgene DNA and the flanldng genomic DNA. The term “event” also refers to DNA from the original transformant comprising the inserted DNA and flanking genomic sequence immediately adjacent to the inserted DNA that would be expected to be transferred to a progeny that receives inserted DNA including the transgene of interest as the resuit of a sexual cross of one parental line that includes the inserted DNA (for exemple, the original transformant and progeny resulting from the selfing) and a parental line that does not contain the inserted DNA
A glyphosate tolérant soybean plant can be bred by first sexually crossing a first parental soybean plant consisting of a soybean plant grown from a transgenic glyphosate tolérant soybean plant comprising MON89788 or an soybean plant that is a progeny of the cross of such a plant that expresses the glyphosate tolérant phenotype, and a second parental soybean plant that lacks the tolérance to glyphosate, thereby producing a plurality of first progeny plants; and then selecting a progeny plant that is tolérant to application of glyphosate herbicide. These steps can further include the back-crossing of the glyphosate tolérant progeny plant to the second parental soybean plant or a third parental soybean plant, then selecting progeny by application with glyphosate or by identification with molecular markers associated with the trait thereby producing an soybean plant that tolérâtes the application of glyphosate herbicide. Molecular markers may be used that comprise the junction DNA molécules identified at the 5’ and 3’ sites of insertion of the transgene in event MON89788.
It is also to be understood that two different transgenic plants can also be mated to produce ofîspring that contain two independently segregating, exogenous transgenes. Back-crossing to a parental plant and out-crossing with a non-transgenic plant as previously described is also contemplated, as is végétative propagation. Descriptions of other breeding methods that are commonly used for different traits and crops can be found in one of several référencés, e.g., Fehr, (1987). —
A “probe” is an isolated nucleic acid to which is attached a conventional détectable label or reporter molécule, for example, a radioactive isotope, a ligand, a chemiluminescent agent, or an enzyme. Such a prohe is complementary to a strand of a target nucleic acid, in the case of the présent invention, to a strand of genomic DNA from a soybean plant comprising event MON89788 whether from a soybean plant or seed or from a sample or extract of the plant or seed that includes DNA from the event. Probes according to the présent invention include not only deoxyribonucleic or ribonucleic acids, but also polyamides and other probe materials that bind specifically to a target DNA sequence and can be used to detect the presence of that target DNA sequence.
“Primers” are isolated polynucleic acids that are annealed to a complementary target polynucleic acid strand by nucleic acid hybridization to form a hybrid between the primer and the target polynucleic acid strand, then extended along the target polynucleic acid strand by a polymerase, for example, a DNA polymerase. Primer pairs of the présent invention refer to their use for amplification of a target polynucleic acid molécule, for example, by the polymerase chain. reaction (PCR) or other conventional nucleic acid amplification methods.
Probes and primers are generally 11 polynucleotides or more in length, preferably 18 polynucleotides or more, more preferably 24 polynucleotides or 30 polynucleotides or more. Such probes and primers hÿbridize specifically to a target molécule under high stringency hybridization conditions. Preferably, probes and primers according to the présent invention hâve complété sequence identity with the target molécule, although probes differing from the target sequence and that retain the ability to hÿbridize to target sequences under high stringency conditions may be designed by conventional methods.
Methods for preparing and using probes and primers are described, for example, in Sambrook et al. (1989); Ausubel et al. (1992); and ïnnis et al. (1990). PCR-primer pairs (a primer set) can be derived from a known sequence, for example, by using computer programs intended for that propose such as Primer (Version 0.5, © 1991, Whitehead Institute for Biomédical Research, Cambridge, MA).
Primers and probes based on the flanking genomic DNA and insert sequences disclosed herein (SEQ ID NOs: 1-4 and 9) can be used to confirm and, if necessary, to correct the disclosed sequences by conventional methods, for example, by isolatiiig the corresponding DNA molécule from a deposit of seed comprising MON89788, and^y'^ detennining the nucleic acid sequence such molécules. Additional associated DNA molécules may be isolated from the genome of a cell comprising MON89788 that comprise the transgene insert and genomic flanking régions, and fragments of these molécules may be used as primers or probes,
The nucleic acid probes and primers of the présent invention hybridize under stringent conditions to a target DNA sequence. Any conventional nucleic acid hybridization or amplification method can be used to identify the presence of DNA from the MON89788 event in a sample. Nucleic acid molécules or fragments thereof are capable of specifically hybridizing to other nucleic acid molécules under certain circumstances. As used herein, two nucleic acid molécules are said to be capable of specifically hybridizing to one another if the two molécules are capable of fonning an anti-paralleL, double-stranded nucleic acid structure and are of sufficient length to maintain this structure under high stringency conditions. A nucleic acid molécule is said to be the “complément” of another nucleic acid molécule if they exhibit complété complementarity. As used herein, molécules are said to exhibit “complété complementarity” when every nucléotide of one of the molécules is complementary to a nucléotide of the other. Two molécules are said to be “mmimally complementary” if they can hybridize to one another with sufficient stability to permit them to remain annealed to one another under at least conventional “low-stringency” conditions. Similarly, the molécules are said to be “complementary” if they can hybridize to one another with sufficient stability to permit them to remain annealed to one another under conventional “high-stringency” conditions. Conventional stringency conditions are described by Sambrook et al., 1989, and by Ilaymes et aL (1985). Departures from complété complementarity are· therefore permissîble, as long as such departures do not completely preclude the capacity of the molécules to form a double-stranded structure. In order for a nucleic acid molécule to serve as a primer or probe it need only be sufficiently complementary in sequence to be able to form a stable double-stranded structure under the particular solvent and sait concentrations employed.
As used herein, a substantially homologous sequence is a nucleic acid sequence that will specifically hybridize to the complément of the nucleic acid sequence to which it is being compared under high stringency conditions. Appropriate stringency conditions which promote DNA hybridization, for example, 6.0 x sodium chloride/sodium citrate — il (SSC) at about 45°C, followed by a wash of 2.0 x SSC at 50°C, are known to those skilled in the art or can. be found in Carrent Protocole in Molecular Biology, John Wiley & Sons,
N. Y. (1989), 6.3.1-63.6. For example, the sait concentration in the wash step can be selected from a low stringency of about 2.0 x SSC at 50°C to a high stringency of about
O. 2 x SSC at 50°C. In addition, the température in the wash step can be increased from low stringency conditions at room température, about 22°C, to high stringency conditions at about 65°C. Both température and sait may be varied, or either the température or the sait concentration may be held constant while the other variable is changed. In a preferred embodiment, a nucleic acid of the présent invention will specifically hybridize to one or more of the nucleic acid moiecules set forth in SEQ ID NOs:l-4, and 9 compléments thereof or fragments of either under moderately stringent conditions, for example at about 2.0 x SSC and about 65°C. In a particularly preferred embodiment, a nucleic acid of the présent invention will specifically hybridize to one or more of the nucleic acid moiecules set forth in SEQ ID NOs:l-4, and 9 compléments thereof or fragments of either under high stringency conditions. Ih one aspect of the présent invention, a preferred marker nucleic acid moiecule of the présent invention comprises the nucleic acid sequence as set forth in SEQ ID NO:1 or SEQ ID NO:2 or compléments thereof or fragments of either. Ih another aspect of the présent invention, a preferred marker nucleic acid moiecule of the présent invention shares between 80% and 100% or 90% and 100% sequence identity with the nucleic acid sequence set forth in SEQ ID NO;1 or SEQ ID NO:2 or compléments thereof or fragments of either. Molecular marker DNA moiecules that comprise SEQ ID NO:1, or SEQ ID NO:2, or compléments thereof or fragments of either may be used as markers in plant breeding methods to identity the progeny of genetic crosses similar to the methods described for simple sequence repeai DNA marker analysis, in Cregan et al. (1997); ail of which is herein incorporated by reference in its' entirely. The hybridization of the probe to the target DNA moiecule can be detected by any number of methods known to those skilled in the art, these can include, but are not limited to, fluorescent tags, radioactive tags, antibody based tags, and chemilununescent tags.
Regarding the amplification of a target nucleic acid sequence (for example, by PCR) using a particular amplification primer pair, “stringent conditions” are conditions that permit the primer pair to hybridize only to the target nucleic-acid sequence to which a primer having the corresponding wild-type sequence (or its complément) would bind and preferably to produce a unique amplification product, the amplicon, in a DNA thermal amplification reaction.
The tenu “spécifie for (a target sequence)” indicates that a probe or primer hÿbridizes under stringent hybridization conditions only to the target sequence in a sample comprising the target sequence.
As used herein, “amplified DNA” or “amplicon” refers to the product of nucleic acid amplification of a target nucleic acid sequence that is part of a nucleic acid template. For example, to détermine whether the soÿbean plant resulting from a sexual cross eontains transgenic event MON89788 or whether a soybean sample collected from a field comprises MON89788, or a soÿbean extract, such as a meal, flour or oil comprises MON89788. DNA extracted from a soÿbean plant tissue sample or extract may be subjected fô a nucleic acid amplification method using a primer pair that includes a primer derived from the genomie région adjacent to the insertion site of inserted heteiologous transgene DNA, and a second primer derived from the inserted heterologous transgene DNA to produce an amplicon that is diagnostic for the presence of the event DNA. The amplicon is of a length and has a sequence that is also diagnostic for the event The amplicon may range in length from the combined length of the primer pairs plus one nucléotide base pair, or plus about fîfty nucléotide base pairs, or plus about two htmdredfifty nucléotide base pairs, or plus about three hundred-fifly nucléotide base pairs or more.
Alternatively, a primer pair can be derived from flanking genomie sequence on both sides of the inserted DNA so as to produce an amplicon that includes the entire insert nucléotide sequence. A member of a primer pair derived from the plant genomie sequence may be located a distance from the inserted transgene DNA molécule, this distance can range from one nucléotide base pair up to about twenty thousand nucléotide base pairs. The use of the term “amplicon specifically excludes primer dimers that may be formed in the DNA thermal amplification reaction.
Nucleic acid amplification can be accomplished by any of the various nucleic acid amplification reaction methods known in the art, including the polymerase chain reaction (PCR). A variety of amplification methods are known in the art and are described, inter alia, in U.S. Patent Nos. 4,683,195 and 4,683,202 and in Tunis et al. (1990). PCR amplification methods have been developed to amplîfy up to 22 kb of genomie DNA and L up to 42 kb of bactériophage DNA (Cheng et al., 1994). These methods as well as other methods known in the art of DNA amplification may be used in the practice of the present invention. The sequence of the heterologous DNA insert or flanking sequence from soybean event MON89788 and can be verified, and corrected if necessary by amplifying such molécules from the event genome using primers derived from the sequences provided herein followed by standard DNA sequencing methods applied to the PCR ampficon or to isolated cloned transgene/genomic DNA.
The amplicon produced by these methods may be detected by a plurality of techniques. One such method is Genetic Bit Analysis (Nikiforov, et al., 1994) where an DNA oligonucleotide is designed which overlaps both the adjacent flanking genomic DNA sequence and the inserted DNA transgene sequence. The oligonucleotide is immobilized. in wells of a microwell plate. Following PCR of the région of interest (using one primer in the inserted sequence and one in the adjacent flanking genomic sequence), a single-stranded PCR product can be hybridized to the immobilized oligonucleotide and serve as a template for a single base extension reaction using a DNA polymerase and labelled ddNTPs spécifie for the expected next base. Readout may be fluorescent or ELISA-based. A signal indicates presence of the insert/flanking genomic sequence due to successful amplification, hybridization, and single base extension.
Another method is the Pyrosequencing technique as described by Winge (2000). Tn this method an oligonucleotide is designed that overlaps the adjacent genomic DNA and insert DNA junction. The oligonucleotide is hybridized to single-stranded PCR product from the région of interest (one primer in the inserted sequence and one in the flanking genomic sequence) and incubated in the presence of a DNA polymerase, ATP, sulfurylase, Iuciferase, apyrase, adenosine 5’ phosphosulfate and luciferin. DNTPs are added individually and the incorporation results in a light signal which is measured. A light signal indicates the presence of the transgene insert/flanking sequence due to successful amplification, hybridization, and single or multi-base extension.
Fluorescence Polarization as described by Chen et al. (1999) is a method that can be used to detect the amplicon of the present invention. Using this method an oligonucleotide is designed which overlaps the genomic flanking and inserted DNA junction. The oligonucleotide is hybridized to single-stranded PCR product from the région of interest (one primer in the inserted DNA and one in the flanking genomic DNA sequence) and incubated in the presence of a DNA polymerase and a fluorescent-labeled ddNTP. Single base extension results in incorporation of the .ddNTP. Incorporation can be measured as a change in polarization using a fluorometer. A change in polarization indicates the presence of the transgene insert/flanking genomic sequence due to successful amplification, hybridization, and single base extension.
Taqman® (PE Applied Biosystems, Foster City, CA) is described as a method of detecting and quantifying the presence of a DNA sequence and is fully understood in the instructions provided by the manufacturer. Briefly, a FRET oligonucleotide probe is desigued which overlaps the genomic flanking and insert DNA junction. The FRET probe and PCR primera (one primer in the insert DNA sequence and one in the flanking genomic sequence) are cycled in the presence of a thermostable polymerase and dNTPs. Hybridization of the FRET probe results in cleavage and release of the fluorescent moiety away from the quenching moiety on the FRET probe. A fluorescent signal indicates the presence of the flanking genomic /transgene insert sequence due to successful amplification and hybridization.
Molecular Beacons hâve been described for use in sequence détection as described in Tyangi et al. (1996) Briefly, a FRET oligonucleotide probe is desigued that overlaps the flanking genomic and insert DNA junction. The unique structure of the FRET probe results in it containing secondary structure that keeps the fluorescent and quenching moieties in close proximity. The FRET probe and PCR primera (one primer in the insert DNA sequence and one in the flanking genomic sequence) are cycled in the presence of a thermostable polymerase and dNTPs. Following successful PCR amplification, hybridization of the FRET probe to the target sequence results in the removal of the probe secondary structure and spatial séparation of the fluorescent and quenching moieties. A fluorescent signal results. A fluorescent signal indicates the presence of the flanking genomic/transgene insert sequence due to successful amplification and hybridization.
Other described methods, such as, microfluidics (US Patent Pub. 2006068398, US Patent No. 6,544,734) provide methods and devices to separate and amplify DNA samples. Optical dyes used to detect and quantitate spécifie DNA molécules (WO/05017181). Nanotube devices (WO/06024023) that comprise an electronic sensor for the détection of DNA molécules or nanobeads that bind spécifie DNA molécules and can then be detected are useful for deteetmg DNA molécules of the présent invention. ‘
DNA détection kits can be developed using the compositions disclosed herein and the methods described or known in the art of DNA détection. The kits are useful for the identification of soybean event DNA in a sample and can be applied to methods for breeding soybean plants containing DNA. The kits may contain DNA primers or probes that are homologous or complementary to SEQ ID NOs:l-4 and 9 or DNA primers or probes homologous or complementary to DNA container! in the transgene genetic éléments of DNA, these DNA sequences can be used in DNA amplification reactions or as probes in a DNA hybridization method. The structure of the DNA of the transgene genetic éléments contained in the soybean genome and illustrated in Figure 1 comprises a 5’ genomie région of the soybean A3244 genome flanking the transgene insert, the insert comprising a portion of the right border région (RB) from Agrobacterium tumefaciens, the chimeric promoter FMWTsfl and related linked éléments (US Patent 6,660,911; also referred to as FMV/ElFla) is operably connected to an Arabidopsis EPSPS chloroplast transit peptide coding sequence (herein referred to as CTP2 or TS-AtEPSPS CTP2, US Patent 5,633,435, operably connected to a glyphosate résistant EPSPS (herein referred to as CP4 EPSPS or aroA:CP4, isolated from Agrobacterium tumefaciens strain CP4 and coding sequence modified for enhanced expression in plant cells, U.S. Patent 5,633,435), operably connected to the 3’ termination région from pea ribulose 1,5-bisphosphate caiboxylase (herein referred to as E9 3’ or T-Ps.RbcS:E9, Coruzzi et al., (1984), a portion of the left border (LB) région from Agrobacterium tumefaciens, and the 3’ genomie région of the soybean A3244 genome flanking the transgene insert. DNA molécules useful as primers in DNA amplification methods can be derived from the sequences of the genetic éléments of the transgene insert contained in soybean event MON89788. These primer molécules can be used as part of a primer set that also includes a DNA primer molécule derived from the genome of soybean flanking the transgene insert. Soybean event MON89788 was produced by transformation of soybean fine A3244 (US Patent 5,659,114) by an Agrobacterium mediated method, for example, methods described in US Patents 6,384,301 and 7,002,058 (herein incorporated by reference in their entirety).
The inventors of the présent invention hâve discovered that a soybean line comprising the MON89788 T-type genomie région (T-type is combination of a transgene and the associated haplotype région of a plant genome) in its genome has an improved yield relative to a line comprising the previous 40-3-2 T-type genomie région. This was demonstrated in replicated field trials including yield data collected from multiple locations in the United States (US patent application 60/685584).
The following examples are included to demonstrate exemples of certain preferred embodiments of the invention. It should be appreciated by those of skill in the art that the 5 techniques disclosed in the examples that follow represent approaches the inventors hâve found function well in the practice of the invention, and thus can be considered to constitute examples of preferred modes for its practice. However, those of skill in the art should, in light of the présent disclosure, appreciate that many changes can be made in the spécifie embodiments that are disclosed and still obtain a like or similar resuit without 10 departing from the spirit and scope of the invention.
EXAMPLES
EXAMPLE 1
Production of Amplicon Diagnostic for MON89788 Genomic DNA
DNA from transgenic soybean event MON89788 is extracted from tissus comprising soybean seeds, végétative tissus, or meal. The DNA is isolated from the tissue using Qiagen’s DNeasy Plant Miniprep Kit according to the manufacturées instructions (Qiagen Corp. Valencia, CA).
A PCR product is produced that comprises a portion of the genomic DNA flanking the 5’ end of the T-DNA (transfer DNA comprising the transgene) insertion in the genome of a plant comprising MON89788. This DNA product comprises SEQ ID NO.*3. The PCR may be performed using one primer designed to hybridize to the genomic DNA sequences flanking the 5’ end of the transgene insert (DNA primer A, SEQ DD NO:5; see FIG. 1) paired with a second primer (DNA primer B, SEQ ID NO:6) located in the transgene promoter région (US Patent 6,660,911, SEQ ED NO:28, herein incorporated by reference and found within SEQ DD NO: 9).
A PCR product is produced from the 3’ end of the transgene insert that comprises a portion oflhe genomic DNA flanking thé 3’ end of the T-DNA insertion in the genome of a plant comprising MON89788 This DNA product comprises SEQ ID NO:4. PCR may be performed using one primer designed to hybridize to the genomic DNA sequences flanking the 3 ’ end of the insert of each event (DNA primer D, SEQ ID NO:8) and paired '
I with a second primer (DNA primer C, SEQ ID NO:7) located in the T-Ps.RbcS:E9 3’ transcription termination sequence at the 3* end ofthe insert.
The PCR template includes ~50 ng of genomic DNA. As a négative control ~5Û ng of genomic DNA from the non-teansgenic soybean cultivar is utilized. Each PCR réaction contains 5 μΐ 10 X Buffer for REDAccuTaq™ LA DNA Polymerase Mix (Sigma-AIdrich, St Louis, MO), 200 μΜ each dNTP (Sigma-AIdrich), 0.4 μΜ each primer, and 2.5 Units JumpStart™ REDTaq™ DNA Polymerase (Sigma-AIdrich) in a 50 μΐ total volume reaction. The PCR réactions are performed under the following cycling conditions: 1 cycle at 94°C for 3 minutes (min); 32 or 35 cycles at 94°C for 30 seconds (s), 58°C for 30 s, 72°C for 30 s or 1 min; 1 cycle at 72°C for 10 min.
DNA event primer pairs are used to produce an amplicon diagnostic for MON89788 genomic DNA. These event primer pairs include, but are not limited to primers A and B (SEQ ID NO:5 and 6) and event primer pairs C and D (SEQ ID NO: 7 and 8), that are used in the described DNA amplification method. In addition to these primer pairs, any primer pair derived from SEQ ID NOS or SEQ ID NO:4, or the compléments thereof, that when used in a DNA amplification reaction produces an amplicon that comprises SEQ ID NO:1 or SEQ ID NO:2 diagnostic for soybean MON89788 event-derived tissue, respectively, may be utilized. DNA amplification conditions illustrated in Table 1 and Table 2 can be used to produce a diagnostic amplicon for MON89788 using the appropriate event primer pairs. Any modification of these methods used to produce an amplicon diagnostic for MON89788 is within the ordinary slrill of the art. An extract putatively containing DNA of a soybean plant or seed comprising MON89788, or a product derived from a plant comprising MON89788 that when tested in a DNA amplification method produces an amplicon diagnostic for soybean event MON89788 may be utilized as a template for amplification to détermine whether MON89788 is présent.
The amplicon is produced by the use of at least one primer sequence derived from SEQ ID NO:3 or SEQ ID NO:4 that when used in a PCR method produces a diagnostic amplicon for event MON89788. For example, the production of the MON89788 amplicons can be performed using a Stratagene Robocycler, MI Engine, Perkin-Elmer 9700, or Eppendorf Mastercycler Gradient thermocycler as shown in Table 2, or by methods and apparatus known to those skilled in the art ka/''
Table 1. PCR procedure and réaction mixture conditions for the identification of soybean
MON89788 5’ transgeneinserVgenomicjunction région.
Step Reagent Amount Comments
| 1 | Nuclease-free water | add to final volume of 20 pl | - |
| 2 | 10X réaction buffer (withMgCL) | 2.0 pl | IX final concentration of buffer, 1.5 mM final concentration of MgCl2 |
| 3 | 10 mM solution of dATP, dCTP, dGTP, and dTTP | 0.4 μΐ | 200 pM final concentration of each dNTP |
| 4 | Event primer A (SEQ ID NO:5 resuspended in IX TE buffer or nuclease-free water to a concentration of 10 μΜ) | 0.2 pl | 0.1 μΜ final concentration |
| 5 | Event primer B (SEQ ID NO:6 resuspended in IX TE buffer or nuclease-fiee water to a concentration of 10 μΜ) | 0.2 pl | 0.1 pM final concentration |
| 6 | RNase, DNase fiee (500 pg/ml) | 0.1JX1 | 50 ng/reaction |
| 7 | REDTaq DNA polymerase (1 unit/pl) | 1.0 pl (recommended to switch pipets prior to next step) | 1 unit/reaction |
| 8 | Extracted DNA (template): • Samples to be anaiyzed: * indivîdual leaves * pooled leaves (maximum of 10 leaves/pool) | • 10-200 ng of genomic DNA • 200 ng of genomic DNA | |
| • Négative control | • 50 ng of non-transgenic soybean genomic DNA | ||
| • Négative control | • no template DNA (solution in which DNA was resuspended) | ||
| • Positive control | • 50 ng of soybean genomic DNA comprising MON89788 |
Gently mix and, if needed (no hot top on thermocycler), add 1-2 drops of minéral oil on top of each reaction. Proceed with the PCR in a Stratagene Robocycler (Stratagene, La Jolla, CA), MJ Engine (MJR-Biorad, Hercules, CA), Perkin-Elmer 9700 (Perkin Ekner, Boston, MA), or Eppendorf Mastercycler Gradient (Eppendorf, Hamburg, Germany) thennocycler using the following cycling parameters (Table 2). The MJ Engine or EppendorfMastercycler Gradient thermocycler should be nm in the calculated mode.
Run the Perkin-Elmer 9700 thermocycler with the ramp speed set at maximum.
IC
Table 2. Thermocycler conditions
Cycle No.
Settings: Stratagene Robocycler
94°C 3 minutes
94°C 1 minute
64°C 1 minute
72°C 1 minute and 30 seconds
72°C 10 minutes
| Cycle No. | Settings: MJ Engine or Perkin-Elmer 9700 |
| 1 | 94°C 3 minutes |
| 34 | 94°C 30 seconds 64°C 30 seconds 72°C 1 minute |
| 1 | 72°C 10 minutes |
| Cycle No. | Settings: EppendorfMastercycler Gradient |
| 1 | 94°C 3 minutes |
| 34 | 94°C 15 seconds 64°C 15 seconds 72°C 1 minute |
| 1 | 72°C 10 minutes |
EXAMPLE 2
Sequence Détermination ofTransgene/Genomic Région and Sonthem Analysis
DNA sequencing of the PCR products provides for DNA that can be used to design additional DNA molécules as primers and probes for the identification of soybean plants or seed comprising MON89788. PCR products of the expected sizes representmg the 5’ and 3’ transgene/genomic sequences were isolated by séparation of the PCR products on a 2.0% agarose gel by electrophoresis. PCR products are isolated that include the 5’ and 3’ DNA régions that span the insert junction between the transgene insertion into the soybean genome. The 5’ and 3’ PCR products for MON89788 are purified by agarose gel electrophoresis followed by isolation from the agarose matrix using the QIAquick Gel Extraction Kit (catalog # 28704, Qiagen Inc., Valencia, CA). The purified PCR products are then sequenced (e.g. ABI Prism™ 377, PE Biosystems, Foster City, CA) and analyzed (e.g. DNASTAR sequence analysis software, DNASTAR Inc.,
Madison,WI). --20
A DNA sequence was determined for the nucléotide base pair segment representing the transgene/genomic région of event MON89788 as illustrated in FIG 1 and identified as SEQ ID NO:9. The genomic and transgene éléments that are contained in SEQ ID NO:9 are described in Table 3. The 5’ and 3* flanldng régions are included in
SEQ ID NO:9 and given in SEQ ID NOs:21 and 22.
The junction sequences are relatively short polynucleotide molécules that are novel DNA sequences and are diagnostic for MON89788 DNA when detected in a polynucleic acid détection assay. The junction sequences in SEQ ID NO:1 aud SEQ ID NO:2 represent 10 polynucleotides on each side of an insertion site of the transgene fragment and soybean genomic DNA in MON89788. Longer or shorter polynucleotide junction sequences can be selected from SEQ ID NO:3 or SEQ ID NO:4. The junction molécules (5’junction région SEQ ID NO:1, and 3’ junction région SEQ ID NO:2 ) are useful as DNA probes or as DNA primer molécules in methods for DNA détection.
Primera and probes used in a Taqman® method (Roche Molecular Systems, Inc., Pleasanton, CA) for détection of an event spécifie DNA molécule were developed for event MON89788. The primer molécules are reforred to as SQ2824 (SEQ ID NO:10), SQ2826 (SEQ ID NO:11), SQ1141 (SEQ ID NO:12), SQ1142 (SEQ ID NO:13), SQ5543 (SEQ ID NO: 14) and the probe molécules are referred to as PB871-6FAM (SEQ ID NO.T5), PB2191-VIC (SEQ ID NO:16), and PB57-VIC (SEQ ID NO:17). The primera and probes were used in the Taqman® method according to manufacturera instructions to provide a diagnostic amplicon for DNA comprising MON89788. Soybean tissues including processed products, for example meal, are useful sources of DNA for this method. Additional primera used to produce an amplicon from soymeal include SEQ ID NOs:l8~20.
Table 3. Genome and genetic element annotation of the transgene/genomic DNA fragment (SEQ ID NO:9) contained in the genome of soybean comprising MON89788.
| Genetic Elément1 | Location in Sequence2 | Function (Reference) | |
| Sequence flanking 5' end of the insert | 1-1103 | SOYBEAN GENOMIC DNA | |
| 5’Jnnction région | 1093-1113 | DNA région spanning the transgene insertion | |
| B3-Right Border | 1104-1145 | DNA Tegion from Agrobacterium tumefadens containing the right border sequence used for transfer of the T-DNA (Depicker et al., 1982) |
| Genetic Elément1 | Location in Sequence3 | Fnnction (Reference) |
| Intervening Sequence | 1146-1215 | Sequences used in DNA cloning |
| Ÿ-FMWTsfl | 1216-2255 | Chimeric promoter consisting of enhancer sequences from the 35S promoter of the Figwort Mosaic virus (Ricliins et al., 1987) and the promoter from the Tsfl gene of Arabidopsis thaliana (encoding élongation factor EF-lalpha (Axelos, et al., 1989) |
| Ls-Tsfl | 2256-2301 | 5' nontranslated leader (exon 1) from the Tsfl gene of Arabidopsis thaliana encoding élongation factor EF-l alpha (Axelos et al., 1989) |
| t-Tsfl | 2302-2923 | Intron from the Tsfl gene of Arabidopsis thaliana encoding élongation factor EF-l alpha (Axelos et al., 1989) |
| Intervening Sequence | 2924-2932 | SEQUENCES USEE IN DNA CLONING |
| TS’-CZPJ | 2933-3160 | Sequences encoding the chloroplast transit peptide from the ShkG gene of Arabidopsis thaliana encoding EPSPS (KleeeZaÀ, 1987) |
| CS?-cp4 qpsps | 3161-4528 | Codon optimized coding sequence of the aroA gene from the Agrobacterium sp. strain CP4 encoding the CP4 EPSPS protein (Padgette et al., 1996; Barry et al., 1997) |
| Intervening Sequence | 4529-4570 | Sequences used in DNA cloning |
| T’-EÎ | 4571-5213 | 3' nontranslated sequence from the RbcS2 gene of Pisum sativum encoding the Rubisco small subunit (Coruzzi et al., 1984) |
| Intervening Sequence | 5214-5256 | Sequences used in DNA cloning |
| B-Left Border | 5257-5406 | DNA région from Agrobacterium tumefaciens containing the left border sequence used for transfer of the T-DNA (Barker et al., 1983) |
| 3’ junction région | 5396-5416 | DNA région spanning the transgene insertion |
| Sequence flanking 3' end of the insert | 5407-6466 | Soybean genomic DNA |
Southern Blot Analysis
Genomic DNA from a plant comprising MON89788 and control soybean genomic DNA of each) is digested with various restriction enzymes (140U) in a total volume of 150μ1 including 15/zl of the corresponding manufacturées buffer (NEB, Beverly, MA). Restriction endonucléases, e.g., Bglll, BamHl, Ncol, Hindi 11, and Bell, are used in the Southern analysis of MON89788. Endonucléase digests are performed at the appropriate température for at least 6 hours. After incubating, the DNA is precipitated with 3M sodium acetate and 2.5 volumes of éthanol. Subsequently, the DNA is washed with 70% éthanol, dried, and resuspended in 40μ1 of TBE. Loading buffer (0.2X) is added to the samples and then subjected to electrophoresis on agarose ^)/ gels (0.8%) for 16-18 hours at 30 volts. The gels are stained with ethidium-bromide, then treated with a depurination solution (0.125N HCL) for 10 minutes, with a denaturing solution (0.5M sodium hydroxide, 1.5M sodium chloride) for 30 minutes, and fînally with a neutralizing solution (0.5M Trizma base, 1.5M sodium chloride) for 30 minutes, The DNA is transferred to Hybond-N membrane (Amersham Pharmacia Biotech, Buckingamshire, England) using a Turboblotter (Schleicher and Schuell, Dassel, Gennany) for 4-6 hours and then fîxed to the membrane using a UV light
Membranes are prehybridized with 20mls of DIG Easy Hyb solution (Roche Molecular Biochemicals, Indianapolis, IN; cat #1603558) for 2-4 hours at 45°C. Radioactive DNA probes (32P dCTP) homologous or complementary to SEQ ID NO:1, or SEQ ID NO;2, or SEQ ID NO:3, or SEQ ID NO;4, or a portion thereof are made using a Radprime DNA Labeling kit (hivitrogen, Carlsbad, CA; cat. #18428-011). Unincorporated nucléotides are removed using SEPHADEX G-50 columns (hivitrogen). The prehybridization solution is replaced with lOmls of prc-wanned DIG Easy Hyb solution containing the denatured probe to a final concentration of 1 million counts per ml. The blots arehÿbridized at 45°C for 16-18 hours.
Blots are washed with a low stringency solution (5X SSC, 0.1X SDS) at 45°C and then repeatedly washed with a higher stringency solution (0.1X SSC, 0.1% SDS) at 65°C. The blots are exposed to a phosphor screen (Amersham Biosciences, Piscataway, NJ) for >2 hours and the exposure rend using a Data Stoim 860 machine (Amersham Biosciences). These methods and conditions exemplifîed may be modifîed by those skilled in the art of detecting DNA in a sample.
EXAMPLE 3 Weed Control
Controlling the growth of weeds in a field of soybeans comprising MON89788. A field is planted with. soybean seeds comprising MON89788, the seeds are allowed to germinaie into plants and the field of plants is treated with a herbicide formulation containing glyphosate. An effective dose of a glyphosate formulation at treatment rates of from about 0.25 lb ae/A (pounds of glyphosate acid equïvalent/acre) to 3 or more lb ae/A is applied to the field. Rates often applied range from about 0.75 lb ae/A to 1.5 lb ae/A at a frequency of one or more treatments during the growing season as necessary to control
the growth of weeds in a field. Seeds from the plants comprising MON89788 are harvested from the treated plants.
A deposit of the Monsanto Technology LLC, soybean seed représentative of event
MON89788 disclosed above and recited in the claims has been made under the Budapest
Treaty with the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, Va. 20110. The ATCC accession number for the deposit comprising event
MON89788 (also known as MON19788 or GM_A19788) is PTA-6708, deposited May 11, 2005. The deposit will be maintained in the depository for a period of 30 years, or 5 years after the last request, or for the effective life of the patent, whichever is longer, and will be replaced. as necessary during that period.
Having illustrated and described the principles of the présent 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. We claim ail modifications that are
Ail publications and published patent documents cited in this spécification are incorporated herein by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. '
REFERENCES
The following référencés, to the extent that they provide exemplary procédural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.
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n 8 SEPL 2013
Claims (25)
1. A nucleic acid sequence comprising the sequence of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3 or SEQ ID NO:4 and a glyphosate tolérant 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) coding sequence.
2. A soybean plant or part thereof comprising event MON89788, wherein représentative soybean seed comprising event MON89788 have been deposited under ATCC accession number PTA-6708.
3. A seed of the plant of claim 2, wherein the seed comprises event MON89788.
4. A soybean commodity product produced from the seed of claim 3.
5. The soybean commodity product of claim 5, further defined as meal, flour, flakes, or oil.
6. The soybean plant part of claim 2, defined as a cell, pollen, ovule, flower, shoot, root, or leaf.
7. The soybean plant of claim 2, further defmed as a progeny plant of any génération of a soybean plant comprising said event MON89788.
8. The soybean plant of claim 2, wherein the genome of said plant comprises at least one DNA molécule selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:21 and SEQ ID NO:22.
9. The soybean plant of claim 2, wherein the genome of said plant produces an amplicon diagnostic for event MON89788 when tested in a DNA amplification method, said amplicon comprising SEQ ID NO: 1 or SEQ ID NO:2.
10. The seed of claim 3, wherein the DNA of the seed produces an amplicon diagnostic for event MON89788 when tested in a DNA amplification method, said amplicon comprising SEQ ID NO: 1 or SEQ ID NO:2.
11. The meal, flour, flakes, or oil of claim 5, further defined as comprising a nucleic acid that produces an amplicon diagnostic for event MON89788 when tested in a DNA amplification method, said amplicon comprising SEQ ID NO: 1 or SEQ ID NO:2.
12. A DNA polynucleotide primer molécule comprising at least 11 contiguous nucléotides of SEQ ID NO:3, or its complément that is useful in a DNA amplification method to produce an amplicon diagnostic for event MON89788.
13. An isolated DNA polynucleotide primer molécule comprising at least 11 contiguous nucléotides of SEQ ID NO:4, or its complément that is useful in a DNA amplification method to produce an amplicon diagnostic for event MON89788.
14. A DNA détection kit spécifie for event MON89788 comprising at least nucleic acid comprising 11 or more contiguous nucléotides homologous or complementary to SEQ TD NO:3 or SEQ ID NO:4.
15. A method of producing a soybean plant tolérant to glyphosate herbicide comprising introducing into the genome of said plant event MON89788.
16. The method of claim 15, defined as comprising the steps of :
(a) crossing a first soÿbean plant comprising event MON89788 with a second soÿbean plant lacking event MON89788 to produce progeny plants; and (b) selecting at least a first progeny plant that comprises said event MON89788 and is tolérant to glyphosate.
17. The method of claim 16, further comprising selfîng said first progeny plant to produce second génération progeny plants and selecting at least a first plant homozygous for said event MON89788.
18. A method of detecting the presence of DNA corresponding to the soÿbean event MON89788 in a sample, the method comprising:
(a) contacting a sample comprising soÿbean DNA with a primer set, which when used in a nucleic acid amplification reaction with genomie DNA from soÿbean event MON89788, produces a diagnostic amplicon for soÿbean event MON89788; and (b) performing a nucleic acid amplification réaction, thereby producing the diagnostic amplicon; and (c) detecting the diagnostic amplicon.
19. A method of detecting the presence of a nucleic acid corresponding to event MON89788 in a sample, the method comprising:
(a) obtaining a sample of soÿbean DNA; and (b) assaying the sample for the presence of a DNA sequence from event MON89788. m/
20. The method of claim 19, wherein assaying the DNA sample comprises detecting the presence ofthe nucleic acid sequence of at least one of SEQ ED NO:1, SEQ ID NO:2, or compléments thereof.
21. A soybean plant comprising a glyphosate tolérant trait that is genetically linked to a nucleic acid moiecule comprising SEQ ID NO: 1 or SEQ ID NO:2.
22. A method of producing a soybean commodity product comprising, (a) obtaining the soybean plant or part thereof of claim 2; and (b) producing a soybean commodity product from the soybean plant or part thereof.
23. The method of claim 22, wherein the commodity product is defined as meal, flour, flakes, protein isolate, or oil.
24. A method for controlling the growth of weeds in a field comprising soybean plants comprising event MON89788, the method comprising treating the field with an amount of glyphosate effective to control the growth of weeds, wherein the soybean plants exhibit tolérance to the glyphosate.
25. The method of claim 24, wherein treating the field is carried out from VI to R4 stage of growth.
Publications (1)
| Publication Number | Publication Date |
|---|---|
| OA16515A true OA16515A (en) | 2015-10-21 |
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