Brassica juncea line NUBJ1207

Abstract

To provide a land-based source of DPA.SOLUTION: Provided is a seed of Brassica juncea line designated as NUBJ1207, wherein a representative sample of the seed has been deposited under ATCC Accession No. PTA-125954.SELECTED DRAWING: None

Description

[Technical field]

[0001] Related Applications This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 891,908, filed August 26, 2019, which is hereby incorporated by reference in its entirety for all purposes.

[0002] Sequence Listing This application contains a Sequence Listing (created on June 20, 2019 and first submitted via EFS-Web in ASCII format on August 26, 2019 as a 2,914 byte file titled 87376_0015PRO_ST25.txt), which is filed contemporaneously herewith and is hereby fully incorporated by reference in its entirety.

[0003] The present embodiment relates to a novel and unique, DPA-producing Brassica juncea strain designated NUBJ1207. [Background technology]

[0004] Long-chain omega-3 polyunsaturated fatty acids (ω3 LC PUFAs) are widely recognized for their critical role in the human diet. The ω3 LC PUFA Δ7,10,13,14,17-docosapentaenoic acid (DPA) contributes to various aspects of human healthy living, from cardiovascular health to brain development and brain function throughout life. Land-based sources of DPA are needed. Summary of the Invention

[0005] The present embodiments provide a plant, derivative or progeny plant, plant part, or seed of DPA Brassica juncea line NUBJ1207 ("DPA juncea"), a representative sample of which has been deposited under ATCC Accession No. PTA-125954 (see Appendix). One aspect of the embodiments provides a method for producing DPA juncea seed, the method comprising crossing two Brassica plants and harvesting the resulting seed, where at least one of the two Brassica plants is DPA juncea line NUBJ1207 or a progeny thereof. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0006] It should be noted that the invention is not limited to, and may vary from, the particular methodology, protocols, and reagents described herein. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the invention, which is defined solely by the claims.

[0007] All patents or other publications are incorporated herein by reference for the purpose of, for example, describing and disclosing the methods described in such publications that may be used in connection with the present invention, but are not intended to provide definitions of terms that are inconsistent with those provided herein. Such publications are provided solely for their disclosure prior to the filing date of this application. Nothing in this regard should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention or for any other reason. All statements as to the date or representation as to the contents of such documents are based on the information available to the applicant and do not constitute any admission as to the accuracy of the dates or contents of such documents.

[0008] As used in this specification and claims, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Throughout this specification, unless otherwise specified, "comprise," "comprises," and "comprising" are used inclusively rather than exclusively, such that a recited integer or group of integers may include one or more other unrecited integers or groups of integers. The term "or" is inclusive unless modified, for example, by "either." Thus, unless the context dictates otherwise, the word "or" means any one member of a particular list and also includes any combination of members of that list.

[0009] All values ​​are approximate because there is some variation in fatty acid composition due to environmental conditions. Values ​​are generally expressed as area percent, which is the approximate weight percent of total fatty acids or weight percent of total seeds. Therefore, except in the operating examples or where otherwise indicated, all numbers expressing amounts or reaction conditions used herein should be understood to be modified in all instances by the term "about."

[0010] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. The terms used herein are intended to describe specific embodiments only and are not intended to limit the scope of the present invention, which is defined only by claims. In order to better understand this disclosure, certain terms are defined first. Further definitions are set forth throughout the detailed description.

[0011] The present embodiments provide Brassica lines that produce LC ω3 PUFA DPA in their seed oil, and in particular lines that embody the attributes of B. juncea line NUBJ 1207. LC ω3 PUFAs are known in the art with appropriate discussion of these and other fatty acids, see, for example, U.S. Patent No. 10,570,405.

[0012] Brassica juncea belongs to the Cruciferae (Brassicaceae) plant family, commonly known as the mustard family. Generally, B. juncea has pale green leaves with few hairs on the primary leaf and the leaf blade attached to the petiole, the plant reaches 1-2 meters in height, the lower leaves are deeply lobed, the upper leaves are narrow and open all over and do not close, the flowers are pale yellow and open gradually from the base to the top of the elongated raceme, the siliques (seed pods) are slightly close together and 2.5-5 cm long except for the 0.5-1 cm long beak, the seeds are round and may be yellow or brown. B. juncea is an amphidiploid (AABB genome, 2n = 36) of B. rapa (AA genome, 2n = 20) by B. nigra (BB genome, 2n = 16).

[0013] Brassica juncea ssp. juncea is primarily cultivated for its seeds. More specifically, oilseed B. juncea is grown as a spice crop (mustard seeds) in North America, but is also used as a source of cooking oil in Asia and other regions. Nearly all oilseed B. juncea grown (mostly in the Indian subcontinent) are high in both erucic acid and glucosinolates, although some low-glucosinolate and low-erucic acid subspecies do exist that provide canola-quality edible oils. More specifically, "canola oil" is defined as having less than 2% erucic acid, and canola meal must contain less than 30 μmoles of 3-butenyl glucosinolate, 4-pentenyl glucosinolate, 2-hydroxy-3-butenyl glucosinolate, 2-hydroxy-4-pentenyl glucosinolate, or mixtures thereof per gram of air-dried, non-oil meal. See, e.g., CODEX ALIMENTARIUS: FATS, OILS & RELATED PRODUCTS, Vol. 8 (2nd ed., Food & Agriculture Org. United Nations, Rome, Italy, 2001). In particular, the seed oil of B. juncea line NUBJ1207 contains less than 1% erucic acid.

[0014] Compared to the more widely grown canola species B. napus and B. rapa, B. juncea is more tolerant to heat and drought stress. Typically, B. juncea may be combined in either bands or straight lines, as it is more tolerant to shattering than some B. napus. Further information on the biology, hybridization, and diseases of B. juncea is readily available. See, for example, Canadian Food Inspection Agency, Biology Document BIO2007-01, Biology of Brassica juncea (Canola / Mustard) (2012). Neither wild type B. juncea, B. napus, nor B. rapa produce DPA in seed oil.

[0015] A "line" is a group of plants that exhibit little overall variation among individuals sharing that designation. A "line" also refers to a homogenous population of plants with substantially the same genetic material, which exhibits little or no genetic variation among individuals for at least one trait, particularly in the transgenes present in line NUBJ1207 and the resulting DPA product. A "subspecies" or "cultivar" may be used interchangeably with "line," but generally the former two terms refer to a line suitable for commercial production. For example, "genetically derived," as used in the phrase "genetically derived from parent line," means that the characteristic in question is dictated in whole or in part by some aspect of the genetic makeup of the plant in question.

[0016] "Progeny" means all descendants, including descendants and derivatives, of a plant, and includes first, second, third, and subsequent generations, and may be produced by self-pollination of the plant, or crossing with the same plant or plants having different genotypes, and may be modified by various suitable genetic engineering techniques. A vegetative species usually refers to a plant that has been intentionally altered and selected by humans. "T0" refers to the first generation of transformed plant material, "T1" refers to the seed produced on the T0 plant, the T1 seed gives rise to a plant that produces a T2 seed, and so on to the Tx progeny.

[0017] "Part of a plant" includes intact plant cells in a plant or part thereof, such as plant cells, plant organs, plant protoplasts, plant cell tissue cultures from which plants can regenerate, plant callus, plant clumps, and embryos, pollen, ovules, seeds, pods, leaves, flowers, branches, fruits, stalks, roots, root tips, anthers, cotyledons, hypocotyls, radicles, single cells, gametes, cell cultures, tissue cultures, etc. Cotyledons are a type of seed leaf, a small leaf contained on a plant embryo. Cotyledons comprise the food storage tissue of a seed. An embryo is a small plant contained within a mature seed. "Plant cell" also encompasses non-regenerative plant cells. Progeny, derivatives, variants and mutants of the regenerated plants are also included within the scope of the present embodiments if any of them contain one, two or three of the nucleic acid molecules as exemplified by SEQ ID NOs: 1 and 2, SEQ ID NOs: 3 and 4, and SEQ ID NOs: 5 and 6, which molecules have the characteristics of DPA juncea NUBJ1207. The present embodiments also relate to the use of elite event NUBJ1207 transgenes in plant cell and tissue cultures. The present embodiments include plants and plant parts from elite event NUBJ1207 lines, as well as other plants produced by the methods described.

[0018] At least one embodiment described herein provides a novel B. juncea line, designated NUBJ1207, which produces DPA in its seed oil. Other aspects of this embodiment provide seeds, plants, and plant parts of DPA juncea NUBJ1207, methods for producing canola plants produced by crossing DPA juncea NUBJ1207 with itself or with other canola genotypes, and the production of mutants by mutagenesis or transformation of DPA juncea NUBJ1207. Thus, this embodiment includes any method of using line NUBJ1207, examples of which include selfing, backcrossing, hybridization, and crossing into populations. All plants produced using line NUBJ1207 as a parent are within the scope of this embodiment. In at least one embodiment, DPA juncea line NUBJ1207 is used in crosses with other different canola plants to generate first generation (F1) hybrid seeds and plants with superior characteristics. Thus, examples of members of the genus Brassica useful in practicing the present embodiment include, in addition to B. juncea, B. napus, B. napobrassica, B. oleracea, B. carinata, B. rapa, and B. campesteris, as well as any other plant belonging to the genus Brassica that allows for breeding between Brassica species. Typically, "oilseed plant" refers to any one of the species B. napus, B. rapa (or campesteris), or B. juncea.

[0019] Phenotypic characterization of the DPA juncea line NUBJ1207, in addition to production of DPA in seed oil, includes the following:

[0020] [Table 1]

[0021] The DPA Juncea described herein may be an A line, a B line, or an R line. The A line is a male sterile line, the B line is similar to "A" in all characteristics but is a male fertile line, and the R line, when crossed with the B line, restores fertility in the F1 hybrid. The B line is sometimes called a maintainer line because it is used to maintain fertility. The restorer line is a pollinating subspecies that is used to pollinate the male sterile line to generate F1 progeny that are male fertile and therefore self-produce seed. An inbred line may allow for the restoration of fertility to the progeny of the male sterile line to which it is crossed. Thus, a hybrid line may be grown from F1 seeds generated by crossing two inbred (pure) lines, one of which may be male sterile.

[0022] For pure-bred line selection, the progeny (self-pollination) of a single plant selection are kept separate and are not combined into bulk. A set of genetically homogeneous lines is the expected outcome. Thus, a pure-bred line may be the progeny of a homozygous plant from a single self-fertilization.

[0023] Thus, the present embodiments provide a method of introducing a desirable trait into DPA Juncea line NUBJ1207, the method comprising the steps of crossing a NUBJ1207 plant with a plant of another Brassica genotype that contains a desirable trait to generate a progeny plant, where the desirable trait is selected from the group consisting of male sterility, herbicide resistance, insect resistance, modified fatty acid metabolism, modified carbohydrate metabolism, modified seed yield, modified oil percent, modified protein percent, modified lodging resistance, and resistance to bacterial, fungal, or viral diseases; and crossing the NUBJ1207 plant with a plant of another Brassica genotype that contains a desirable trait to generate a progeny plant, where the desirable trait is selected from the group consisting of male sterility, herbicide resistance, insect resistance, modified fatty acid metabolism, modified carbohydrate metabolism, modified seed yield, modified oil percent, modified protein percent, modified lodging resistance, and resistance to bacterial, fungal, or viral diseases to generate the selected progeny plant. selecting one or more progeny plants having the desired traits, crossing the selected progeny plants with a NUBJ1207 plant to generate a backcross progeny plant, selecting a backcross progeny plant having substantially all of the desired traits, and physiological and morphological characteristics of the NUBJ1207 line to generate a selected backcross progeny plant, and repeating these steps three or more times to generate a selected fourth or subsequent progeny plant by backcrossing that includes substantially all of the desired traits, and physiological and morphological characteristics of the line NUBJ1207 as described herein. Included in this aspect of the embodiment are plants generated by the method, where the plant has substantially all of the desired traits (i.e., DPA production), and physiological and morphological characteristics of the NUBJ1207 line.

[0024] "Breeding" includes all methods of developing or propagating plants, including both inter- and intra-specific and inter- and intra-line crosses, as well as all appropriate conventional and artificial breeding techniques. Desired traits (e.g., the DPA trait of NUBJ1207) may be transferred to other canola or B. juncea lines, cultivars, or species, or may be transferred through conventional breeding methods, and may also be transferred to other Brassica species, such as B. napus and B. rapa, through interspecific crosses. Both conventional breeding methods and interspecific cross methods, as well as other methods of transferring genetic material between plants, are well known in the art.

[0025] B. juncea strain NUBJ1207 was developed by Agrobacterium tumefaciens-mediated transformation with the gene construct pJP3416_GA7-modB ("modB") and contains seven genes capable of promoting the accumulation of omega-3 fatty acids in seeds. Briefly, the modB construct contains genes encoding a Δ6 desaturase cloned from the microalga Micromonas pusilla, a Δ6-elongase and a Δ5 elongase cloned from the Microalga Pyramimonas cordata, a Δ5 desaturase cloned from the Microalga Pavlova salina, a Δ15 / ω3-desaturase cloned from the yeast Pichia pastoris, a Δ12 desaturase cloned from the yeast Lachancea kluyveri, and a Δ4 desaturase cloned from Pavlova salina, each with an appropriate transcription promoter and terminator. See, e.g., U.S. Patent Nos. 10,570,405 (FIG. 1), 10,563,218, 10,125,084, 9,718,759, and 9,932,541. However, upon insertion into B. juncea (a low erucic acid, low glucosinolate strain) from which NUBJ1207 was derived, the Δ4-desaturase gene of modB was truncated and rendered inoperable.

[0026] Genetic analysis revealed that NUBJ1207 contains three recombinant transgene loci, each with a separate transgene insert containing an expression cassette, with each insert located in a different chromosome. In particular, the transgene insert in NUBJ1207 chromosome A06 contains a truncated MAR sequence (640 bp Rb7 matrix attachment region from Nicotiana tabacum), the Arabidopsis thaliana FA elongase 1 promoter (Pro_Arath-FAE1), the P. cordata Δ6-elongase gene, the Glycine max lectin terminator (Ter_Glyma-Lectin), the cauliflower mosaic virus 35S RNA promoter, the coding sequence for the phosphinothricin acetyltransferase (PAT) enzyme, the terminator from A. tumefaciens nopaline synthase (TER_Agrtu-NOS), and the A. tumefaciens left border (LB) used for transcription of the T-DNA. The transgene insert in NUBJ1207 chromosome B07 contains the A. tumefaciens right border (RB) used for transcription of the T-DNA, the Linum usitatissimum conlinin2 terminator (TER_Linus-Cnl2), the M. pusilla Δ6 desaturase, and the truncated L. usitatissimum conlinin2 promoter (PRO_Linus-Cnl2) (minus 5' 228 bp).The transgene inserts in NUBJ1207 chromosome A08 contained RB, TER_Linus-Cnl2, M. pusilla Δ6 desaturase, PRO_Linus-Cnl2 and Pro_Arath-FAE1, P. cordata Δ5 elongase, Ter_Glyma-lectin and the promoter of B. napus napin (PRO_Brana-Fp1), P. salina Δ5 desaturase, and the promoter of β-lactamase (PRO_Brana-Fp2). The expression cassettes contain the P. pastoris Δ15 / ω3-desaturase, TER_Agrtu-NOS, the terminator of L. oustatizimum conlinin 1 (TER_Linus-Cnl1), the promoter of L. oustatizimum conlinin 1 (PRO_Linus-Cnl1), the promoter of L. oustatizimum conlinin 1 (PRO_Linus-Cnl2), and 21 base pairs of P. salina Δ4-desaturase. (Note that the expression cassettes described above reflect a vector design in which the genes are not arranged in the same 5' to 3' orientation.) Furthermore, for the transgene insert in A08, the truncated Δ4-desaturase sequence only encodes the first seven N-terminal amino acid residues of the enzyme and is not expected to have Δ4-desaturase activity, consistent with the fatty acid content observed for line NUBJ1207 seed oil.

[0027] The three transgene inserts in strain NUBJ1207 provide most of the genes from the binary vector modB, except for the Δ12-desaturase and Δ4-desaturase genes. However, B. juncea contains endogenous Δ12-desaturase and Δ15 / ω3-desaturase genes, such that the Δ12-desaturation activity is complemented by the endogenous genes. Additionally, strain NUBJ1207 contains two copies of the Δ6-desaturase transgene. These transgene inserts convert oleic acid (OA) to DPA, but do not further convert DPA to DHA due to the lack of Δ4 desaturase activity.

[0028] Thus, one embodiment provides seed of Brassica juncea lineage designation NUBJ1207, a representative sample of which has been deposited under ATCC accession number PTA-125954, wherein the seed comprises within its genome a first transgenic locus located on a first chromosome comprising a P. cordata Δ6-elongase and a phosphinothricin acetyltransferase, a second transgenic locus located on a second chromosome comprising a M. pusilla Δ6 desaturase, and a third transgenic locus located on a third chromosome comprising the M. pusilla Δ6 desaturase, the P. cordata Δ5-elongase, the P. salina Δ5 desaturase, the Pichia pastoris Δ15 / ω3 desaturase, and non-functional portions of the P. salina Δ4 desaturase.

[0029] From the initial transformation, the progeny were self-pollinated and a plant breeding pedigree system was then used to develop the line NUBJ1207 of this embodiment. Some of the criteria used for selection at various generations include DPA content (as % of total fatty acids in seeds), vigor, fertility, standability, disease resistance, oil content, and maturity. NUBJ1207 is therefore a genetically stable and uniform transgenic Brassica and no off-type plants were noted during evaluation. The NUBJ1207 line shows genetic stability and uniformity as described in the phenotypic description information. It has been self-pollinated for a sufficient number of generations with careful attention to uniformity in terms of plant type. The line has been propagated with continuous observations for uniformity.

[0030] A major phenotypic feature of NUBJ1207 is the production of significant amounts of DPA in its seeds. As shown herein, bulk seeds of NUBJ1207 generally contain about 10% DPA or more (as a % of total fatty acids in the seed).

[0031] At least one embodiment relates to a method for generating a DPA Brassica plant by crossing a first parent plant with a second parent plant, where the first plant or the second plant is a plant from Brassica juncea line NUBJ1207. In at least one embodiment, both the first parent plant and the second parent plant may be from line NUBJ1207. Any breeding method using line NUBJ1207 is foreseen, examples of which include selfing, backcrossing, hybrid breeding, and crossing into populations. Any plant generated using B. juncea line NUBJ1207 as a parent is within the scope of the present embodiments. For example, B. juncea line NUBJ1207 may be crossed with a canola-quality low glucosinolate B. juncea line to provide a low glucosinolate DPA B. juncea.

[0032] Additional methods of the present embodiment include the introduction of expression vectors introduced into plant tissues using direct gene transfer methods, such as by using microprojectile-mediated delivery, DNA injection, electroporation, or Agrobacterium-mediated transformation. In some embodiments, transgenic variants of NUBJ1207 may contain at least one additional transgene (in addition to those present in NUBJ1207), such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 additional transgenes. Thus, the present invention also relates to super-transformed variants of the claimed line NUBJ1207, or variants obtained by breeding NUBJ1207 with one or more other transgenic Brassicas. Such transgenes are expressed under the control of regulatory sequences (e.g., promoters, enhancers, intervening sequences, terminators) well known in the art. In one embodiment, the desirable trait may be one or more of herbicide resistance, insect resistance, disease resistance, modified seed yield, modified oil percent, modified protein percent, modified lodging resistance, or modified fatty acid or carbohydrate metabolism. The specific gene may be any gene known in the art or enumerated herein, examples of which include, but are not limited to, a marker gene, a polynucleotide (nucleic acid molecule) that confers resistance or tolerance to imidazolinones, sulfonylureas, glyphosate, glufosinate, 2,4-D, dicamba, L-phosphinothricin, triazines, hydroxyphenylpyruvate, dioxygenase inhibitors, protoporphyrinogen oxidase inhibitors, phenoxypropionic acid, cyclohexanone, or benzonitrile, a polynucleotide encoding Bacillus thuringiensis, a polynucleotide encoding a phytase, a polynucleotide encoding 1-acyl-glycerol-3-phosphate acyltransferase (LPAAT), a galactinol synthase, or a raffinose artificial enzyme, or a polynucleotide that confers resistance to blackleg, white rust, or other common canola diseases.Such transgenes and related techniques are well known in the art, see, for example, U.S. Patent Nos. 9,718,759 and 8,143,488. Alternatively, the DPA trait of such transgenic embodiments may be obtained by breeding canola containing at least one transgene with NUBJ1207 as just described. For example, DPA yield may be increased by introgressing NUBJ1207 with a transgenic B. napus A02 chromosome as described in U.S. Patent No. 10,563,218. The trait to produce DHA as well as DPA may be obtained by introgressing at least one complete modB insert (one locus containing two modB constructs), such as, for example, a transgenic A05 chromosome, into NUBJ1207 as described in U.S. Patent No. 10,570,405.

[0033] Thus, transgenes or other phenotypic traits can be introduced into NUBJ1207, or the DPA production trait of NUBJ1207 can be introduced into other Brassica or canola lines using conventional breeding techniques such as introgression or backcrossing, which are well known in the art. Examples of canola plant breeding techniques that can be utilized in producing progeny of NUBJ1207 include, for example, recurrent selection, bulk selection, mass selection, mutation breeding, backcrossing, pedigree breeding, tissue culture, open pollination breeding, restriction fragment length polymorphism enhanced selection, genetic marker enhanced selection, and generation of doubled haploids. A combination of such techniques is often used. Development of Brassica subspecies in plant breeding programs usually requires the development and evaluation of homozygous subspecies. See, e.g., US2018 / 0016591, U.S. Patent No. 8,143,488, Downey et al., Rapeseed & Mustard, at 437 in PRINCIPLES OF CULTIVAR DEVEL. (Fehr (ed.), Macmillan & Co., NY, 1987), Thompson, Breeding winter oilseed rape Brassica napus, 7 Adv. Appl. Biol. 1-104 (1983), Ward et al., Oilseed Rape (Farming Press Ltd., Wharefedale Road, Ipswich, Suffolk, 1985).

[0034] In another aspect of this embodiment, NUBJ1207 and its progeny can be identified not only by its phenotypic trait (i.e., production of DPA), but also by identifying its genotype. As described in Example 3 herein, NUBJ1207 not only has a unique genetic profile provided by its transgenes, but also contains unique DNA sequences at the junctions created upon insertion of the transgenic cassette into the B. juncea genome. More specifically, portions of the genetic construct GA7-modB reside at three loci, each of which resides on a different chromosome (chromosomes A08, A06, and B07), each creating six unique junctions with unique DNA sequences.

[0035] The marker profile of NUBJ1207 can be recognized using techniques such as RFLP, random amplified polymorphic DNA (RAPD), arbitrarily primed polymerase chain reaction (AP-PCR), DNA amplification fingerprinting (DAF), sequence characterized amplified region (SCAR), amplified fragment length polymorphism (AFLP), SSR, also called microsatellite, or SNP. See, for example, Glick et al., METHODS IN PLANT MOLEC. BIOL. & BIOTECHNOL. (CRC Press, Boca Raton, 1993). An exemplary assay for the identification of NUBJ1207 junctions is the Kompetitive Allele Specific PCR (KASP) assay, the method of which is illustrated in US2018 / 0016591.

[0036] Currently, canola is recognized as an increasingly important oilseed crop and source of meal in many parts of the world. DPA juncea line NUBJ1207 or its progeny can be used in the production of oil, meal, or other food or edible supplies, including dietary supplements or medicines, according to known techniques. Advantageously, oil obtained from DPA juncea NUBJ1207 is low in erucic acid (less than 1%). Glucosinolates may be found in the meal portion of the treated seeds and can be minimized by methods known in the art.

[0037] In accordance with the Budapest Treaty, applicant has deposited seeds of DPA B. juncea NUBJ1207 under accession number PTA-125954 with the American Type Culture Collection (ATCCR), located at 0801 University Blvd., Manassas, Va., 20110-2209 USA. Applicant has satisfied the requirements of 37 C.FR §§ 1.801-1.809. Applicant is not authorized to waive any restrictions imposed by statute on the movement of biological material or its transportation in commerce. Applicant does not waive any rights provided under patent law, nor any plant breeders' rights.

[0038] Thus, an aspect of the present embodiments provides seeds of Brassica juncea lineage designation NUBJ1207 (DPA juncea NUBJ1207), a representative sample of which has been deposited under ATCC Accession Number PTA-125954. At least one embodiment provides a Brassica plant or portion thereof, including a seed genome of DPA juncea NUBJ1207, produced by growing the seed of DPA juncea NUBJ1207, such as a seed, pollen, cotyledon, half-cotyledon, tissue culture of regenerative cells, or ovule. The tissue culture or regenerative cells according to one embodiment regenerate a plant capable of expressing all of the morphological and physiological characteristics of DPA juncea NUBJ1207, and may comprise leaves, pollen, embryos, roots, root tips, sheaths, flowers, ovules, and stalks. Another embodiment provides a Brassica plant or part thereof having all of the physiological and morphological characteristics of DPA juncea NUBJ 1207. Another embodiment provides cells of B. juncea line NUBJ 1207, a representative sample of seeds of which has been deposited under ATCC Accession No. PTA-125954.

[0039] Another aspect of this embodiment provides a method for producing Brassica seeds, the method comprising growing and cultivating DPA juncea NUBJ1207 to produce seeds and harvest the seeds, a representative sample of which has been deposited under ATCC Accession No. PTA-125954. The growing or cultivating may be performed in a greenhouse, tent, or field. The growing or cultivating may be performed on a commercial scale. At least one embodiment provides a composition comprising an oil, meal, or meal protein obtained by practicing this method. The oil may be a crude oil, a refined oil, or a concentrated oil of one or more fatty acids.

[0040] Another aspect of this embodiment provides a method of producing an oil or meal, the method comprising obtaining seeds of DPA juncea NUBJ1207, or related progeny thereof, and processing the seeds to obtain an oil or meal. At least one embodiment provides a composition comprising an oil of seeds of DPA juncea NUBJ1207, or related progeny thereof. The oil may be a crude oil, a refined oil, or a concentrated oil of one or more fatty acids. At least one embodiment provides a composition comprising a meal of seeds of DPA juncea NUBJ1207, or related progeny thereof.

[0041] Another aspect of this embodiment provides a Brassica plant or part (e.g., progeny) derived from DPA juncea NUBJ1207, where the plant or part has been transformed such that its genetic material includes one or more transgenes operably linked to one or more regulatory elements in addition to the transgenes present in Brassica juncea line NUBJ1207. Thus, at least one embodiment provides a method of generating a Brassica plant whose genetic material includes at least one transgene in addition to the transgenes of line NUBJ1207, the method comprising crossing a NUBJ1207 plant or a plant derived therefrom with a second plant of another Brassica line, such that the genetic material of the progeny resulting from the cross expresses the additional at least one transgene. Alternatively, at least one embodiment provides a method of generating a Brassica plant whose genetic material includes at least one transgene in addition to the transgenes of line NUBJ1207, the method comprising introducing at least one additional transgene into NUBJ1207. In such methods, the genetic material of the progeny resulting from crossing or transformation expresses additional desirable traits. The additional transgene may confer herbicide resistance, insect resistance, bacterial disease resistance, fungal disease resistance, viral disease resistance, or sterility.

[0042] Another embodiment provides a method for producing a canola plant derived from Brassica line NUBJ1207, the method comprising: (a) crossing B. juncea line NUBJ1207 with a second canola plant to produce a progeny canola seed; and (b) growing the progeny canola seed under plant growth conditions to produce a canola plant derived from Brassica line NUBJ1207. The embodiment further includes (c) crossing the NUBJ1207-derived canola plant with itself or with another canola plant to produce an additional NUBJ1207-derived canola progeny seed, (d) growing the progeny canola seed of step (c) under plant growth conditions to produce an additional NUBJ1207-derived canola plant, and (e) repeating the crossing of (c) and the growing of (d) zero (0) to ten (10) times to produce further NUBJ1207-derived canola plants, wherein the NUBJ1207-derived canola plants express at least two NUBJ1207 traits selected from DPA products and at least one additional agronomically desirable trait. Related embodiments provide NUBJ1207-derived canola plants or portions thereof produced by such methods.

[0043] Another embodiment provides a method for producing Brassica seeds, the method comprising crossing a first parent Brassica plant with a second parent Brassica plant and harvesting the resulting first generation Brassica seeds, wherein the first parent Brassica plant or the second parent Brassica plant is a Brassica line NUBJ1207 plant. Specifically, the first parent Brassica plant is different from the second parent Brassica plant, and the resulting seeds are first generation (F1) hybrid Brassica seeds. Related embodiments provide the further steps of backcrossing the selected F1 hybrid plants to line NUBJ1207 or to a different Brassica plant having the desired trait to generate backcross progeny seeds, growing the resulting backcross progeny seeds and selecting backcross progeny plants having the desired traits, and repeating the backcrossing and growing steps on the selected backcross progeny plants for 1 to 10 generations to generate progeny Brassica plants derived from B. juncea line NUBJ1207. In related embodiments, progeny derived from B. juncea line NUBJ1207 can be obtained by self-pollinating the F1 progeny.

[0044] Another embodiment provides a method for obtaining a Brassica inbred line, the method comprising: (a) planting a seed collection comprising hybrid seeds, one of which is Brassica NUBJ1207, the collection further comprising Brassica NUBJ1207 seeds; (b) growing canola plants from the seed collection; (c) identifying inbred plants from the inbred line; (d) selecting the inbred plants; and (e) controlling pollination in a manner that preserves homozygosity of the inbred plants. Seeds of inbred Brassica line NUBJ1207 have been deposited under ATCC accession number PTA-125954. In some embodiments of this method, the Brassica inbred line may be B. juncea or B. napus.

[0045] At least one embodiment provides a method of generating a doubled haploid subpopulation, the method comprising: (a) isolating a bud of an F1 plant provided herein; (b) dissecting the haploid grains; (c) placing the haploid grains in culture; (d) inducing the grains to differentiate into embryos and then into smaller embryos (plantlets); (e) identifying whether the smaller embryos contain a diploid chromosome number, where the diploid chromosome number arises through chromosome doubling; and continuing to develop the smaller embryos if they contain a diploid chromosome number.

[0046] Further aspects of the present embodiment provide novel nucleic acid molecules (e.g., DNA molecules) embodied by the combination of the transgene insert and the B. juncea genome, and cells or compositions comprising such nucleic acids. Exemplary embodiments provide DNA molecules comprising one, two, or three DNA molecules selected from (a) a DNA molecule comprising a sequence provided as SEQ ID NO:1 and a sequence provided as SEQ ID NO:2, (b) a DNA molecule comprising a sequence provided as SEQ ID NO:3 and a sequence provided as SEQ ID NO:4, or (c) a DNA molecule comprising a sequence provided as SEQ ID NO:5 and a sequence provided as SEQ ID NO:6. An exemplary composition according to this aspect may be a seed meal comprising such a DNA molecule. At least one embodiment provides a plant (such as a Brassica plant), a seed, a plant part, a seed meal, etc., as described herein, comprising such a DNA molecule.

[0047] Related embodiments provide a cell comprising one, two, or three nucleic acid molecules selected from: (a) a DNA molecule comprising a sequence provided as SEQ ID NO:1 and a sequence provided as SEQ ID NO:2; (b) a DNA molecule comprising a sequence provided as SEQ ID NO:3 and a sequence provided as SEQ ID NO:4; (c) a DNA molecule comprising a sequence provided as SEQ ID NO:5 and a sequence provided as SEQ ID NO:6. The cell may be a B. juncea cell. The cell may be present in a plant, seed, or part of a plant as described herein. The cell may be present in a meal (i.e., a seed meal).

[0048] Another aspect provides a method of using the DNA molecules of SEQ ID NO:1-SEQ ID NO:6, or a portion thereof. Such uses include, for example, identifying the presence of at least one of the locus features of NUBJ1207. An embodiment provides a method of detecting a DNA molecule, such as amplifying a DNA molecule that includes a sequence of at least 20 contiguous nucleotides from one of SEQ ID NO:1-SEQ ID NO:6. In certain embodiments, the method may be a KASP assay. A related embodiment provides a single-stranded portion of any one of the nucleic acids of SEQ ID NO:1-SEQ ID NO:6, such as a primer or probe.

[0049] Another aspect of this embodiment provides a method of detecting the presence of event NUBJ1207 in a sample containing plant DNA, the method comprising contacting said sample with at least one primer that binds to at least one transgene flanking junction region of the Brassica genome as set forth in SEQ ID NO:1-SEQ ID NO:6. The method may utilize a KASP™ genotyping assay. Related embodiments provide plants, plant material, or plant-derived material identified by such methods. Another related embodiment includes a kit comprising components for carrying out such methods.

[0050] At least one embodiment provides a method for detecting the presence of event NUBJ1207 in a sample containing plant DNA, the method comprising: (a) contacting the sample with a first primer that binds to a flanking junction region of the Brassica genome of NUBJ1207 and a second primer that binds to a transgene of NUBJ1207; (b) subjecting the sample to polymerase chain reaction; and (c) characterizing an amplicon produced between the primers.

[0051] Another aspect of the present embodiments provides genomic DNA of the inbred B. juncea line NUBJ1207, representative seeds of said inbred line being deposited under ATCC Accession No. PTA-125954. A related embodiment provides a plant cell comprising: (a) DNA having a sequence as set forth in at least one of SEQ ID NO:1-6; (b) DNA having at least 95% identity to at least one of SEQ ID NO:1-6; or (c) the complement of (a) or (b).

[0052] Another aspect of this embodiment provides seeds of B. juncea line NUBJ1207, or plants derived therefrom (i.e., progeny or related progeny), comprising about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, or about 20% DPA (as a % of total fatty acids in the seed oil). Another embodiment provides a collection of seeds comprising at least 95% seeds of B. juncea line NUBJ1207, or plants derived therefrom, wherein the seeds comprise about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, or about 20% DPA (as a % of total fatty acids in the seed oil). Certain embodiments provide seed oils obtained from such seeds or collections of seeds.

[0053] A further embodiment provides a method for obtaining a DPA-containing Brassica grain comprising: (a) introgressing the DPA trait of NUBJ1207 into an elite Brassica line that is male sterile; (b) introgressing the DPA trait of NUBJ1207 into a second elite Brassica line that is fertile; (c) crossing the two lines (a) and (b) to obtain hybrid progeny; (d) cultivating seeds of the hybrid progeny; and (e) harvesting grain produced by the cultivated hybrid progeny. EXAMPLES

[0054] Example 1. Cultivation attempt (North America) Seeds obtained from B lines derived from the elite event of NUBJ1207 had the following fatty acid profile as determined by gas chromatography (Table 2).

[0055] [Table 2]

[0056] Seeds were sown in the spring in North American Zone 7. During cultivation, certain agronomic characteristics were monitored approximately weekly, as shown in Table 3 below.

[0057] [Table 3]

[0058] Example 2. Cultivation attempt (Australia) Four DPA-producing B. juncea accessions, including NUBJ1207, were field-trialed in separate tents in Australia and the fatty acid content of mature seeds (pooled bulk) was analyzed by gas chromatography (alternatively, seed fatty acids can be measured using high resolution NMR). The fatty acid content with fatty acids present greater than 1% (of total fatty acids %) is shown in Table 4 below. The data show consistency between generations derived from bulk plants and single plants.

[0059] [Table 4]

[0060] DPA juncea NUBJ1207 was grown in a field in south-eastern Australia during the winter of 2017. Seeds were sown in May, the resulting plants began to flower 139 days after sowing, and seeds were harvested 218 days after sowing.

[0061] Pooled seed data from single plants from siblings of tent-grown NUBJ1207 are shown below in Table 5. The data show consistency between siblings in the T5 generation.

[0062] [Table 5]

[0063] In further experiments, seed pods were harvested from 10 individual tent-grown plants at various stages of maturity and plant position and the seeds analyzed. The data below, Table 6, shows the consistency between pods at different stages of maturity and positions on the plant.

[0064] [Table 6]

[0065] Example 4. Unique flanking regions The methods and kits herein are useful for identifying the presence of plant material containing transgenes, particularly in NUBJ1207, as well as transgenic Brassica plants, plant material, and seeds containing such events in biological samples. The elite event NUBJ1207 described herein can be identified by genotype, which can be characterized through a genetic marker profile that can identify plants of the same cultivar or related cultivars, or can be used to determine or confirm genealogy. Genetic marker profiles can be obtained by techniques such as restriction fragment length polymorphism (RFLP), random amplified polymorphic DNA (RAPD), arbitrarily primed polymerase chain reaction (AP-PCR), DNA amplification fingerprinting (DAF), sequence-characterized amplified regions (SCAR), amplified fragment length polymorphism (AFLP), simple sequence repeats (SSR) (also called microsatellites), and single nucleotide polymorphism (SNP).

[0066] For example, the elite event NUBJ1207 described herein can be identified by generating a genetic map from a sample of plant material. The genetic map can be generated by conventional RFLP, polymerase chain reaction (PCR) analysis, or SSR to identify the approximate chromosomal location of the integrated DNA molecule encoding the heterologous protein. See Glick & Thompson, METHODS IN PLANT MOLEC. BIOL. & BIOTECHNOL. 269 (CRC Press, Boca Raton, FL, 1993). Map information regarding chromosomal location is useful for protecting the owner of the transgenic plant of interest. For example, a map of the integration region can be compared with a similar map of a suspect plant to determine whether the latter shares a common lineage with the plant of interest. Examples of map comparison include hybridization, RFLP, PCR, SSR, and sequencing, all of which are conventional techniques.

[0067] DPA juncea NUBJ1207 is an inbred line from an elite event that can be characterized and identified by unique DNA sequences at the junctions of the parental plant genome and the inserted transgenic construct. More specifically, chromosomes A08, A06, and B07 each contain 5' and 3' junctions that are unique to DPA juncea.

[0068] For example, the DNA sequence of the 5' junction of chromosome A08 contains 100 nucleotides of B. juncea (italicized) and 100 nucleotides of the transgene (non-italicized).

[0069] [ka]

[0070] For example, the DNA sequence of the 3' junction of chromosome A08 contains 100 nucleotides of the transgene (non-italicized) and 100 nucleotides of B. juncea (italicized).

[0071] [ka]

[0072] For example, the DNA sequence of the 5' junction of chromosome A06 contains 100 nucleotides of B. juncea (italicized) and 100 nucleotides of the transgene (non-italicized).

[0073] [ka]

[0074] For example, the DNA sequence of the 3' junction of chromosome A06 contains 100 nucleotides of the transgene (non-italicized) and 100 nucleotides of B. juncea (italicized).

[0075] [ka]

[0076] For example, the DNA sequence of the 5' junction of chromosome B07 contains 100 nucleotides of B. juncea (italicized) and 100 nucleotides of the transgene (non-italicized).

[0077] [ka]

[0078] For example, the DNA sequence of the 3' junction of chromosome B07 contains 100 nucleotides of the transgene (non-italicized) and 100 nucleotides of B. juncea (italicized).

[0079] [ka]

[0080] For the identification of DPA Juncea NUBJ1207, the primer for identifying the junction can be any suitable length derived from the larger junction sequence. The length of the junction sequence used to identify DPA Juncea NUBJ1207 can be any suitable length that provides unique junction identification, for example, at least 10, or at least 15, or 20 consecutive nucleotides from the junction between the transgene and B. juncea DNA.

[0081] Although the foregoing embodiments have been described in some detail by way of illustration and example, for purposes of clarity and understanding, it will be apparent to those skilled in the art that certain variations and modifications, such as single gene modifications and mutations, somaclonal variants, mutants selected from large populations of plants of this inbred line, and the like, may be practiced within the scope of the invention, which is limited only by the appended claims.

Claims

1. Seeds of Brassica juncea lineage designation NUBJ1207, a representative sample of said seeds being deposited under ATCC Accession No. PTA-125954.

2. 13. A Brassica plant or part thereof comprising the genome of the seed of claim 1 and produced by growing the seed of claim 1.

3. Pollen of the plant according to claim 2.

4. An ovule of the plant according to claim 2.

5. A Brassica plant or part thereof, having all the physiological and morphological characteristics of the plant of claim 2.

6. A tissue culture of regenerative cells produced from the Brassica plant of claim 2.

7. 7. The tissue culture of claim 6, wherein the cells or protoplasts are from a tissue selected from the group consisting of leaves, pollen, embryos, roots, root tips, sheaths, flowers, ovules, and stalks.

8. 3. A tissue culture of regenerative cells of the Brassica plant of claim 2, wherein the tissue regenerates into a plant capable of expressing all of the morphological and physiological characteristics of the plant of claim 2.

9. 10. A method for producing Brassica seeds, the method comprising growing the plant of claim 2 to produce seeds and harvesting the seeds.

10. 10. The method of claim 9, wherein the growing step occurs in a greenhouse.

11. 10. The method of claim 9, wherein the growing step is performed in a tent.

12. 10. The method of claim 9, wherein the growing step occurs in a field.

13. 3. A method for producing oil comprising obtaining seeds of the plant of claim 2 and processing the seeds to obtain oil.

14. 6. The Brassica plant or part thereof of claim 5, wherein the plant or part thereof has been transformed such that its genetic material contains one or more transgenes operably linked to one or more regulatory elements in addition to the transgenes present in Brassica juncea line NUBJ1207.

15. 15. A method for generating a Brassica plant whose genetic material contains at least one transgene in addition to the transgene of the NUBJ1207 lineage, said method comprising the step of crossing the plant of claim 14 with either a second plant of another Brassica lineage or a plant of the NUBJ1207 genus, such that the genetic material of the progeny resulting from said cross expresses the additional at least one transgene.

16. 16. The method of claim 15, wherein the additional transgene confers herbicide resistance, insect resistance, bacterial disease resistance, fungal disease resistance, viral disease resistance, or sterility.

17. 6. The Brassica plant or part thereof of claim 5, wherein the plant or part thereof is the product of conventional breeding techniques and has not been modified by recombinant, mutant, or other genetic modification other than the elite event associated with NUBJ1207.

18. 20. A method for producing a Brassica plant having at least one additional desirable trait in addition to traits of the inbred Brassica line NUBJ1207, said method comprising the step of crossing a plant of claim 17 with a plant of another Brassica line or with a plant of the inbred line NUBJ1207, such that progeny genetic material resulting from said cross expresses the desired additional trait.

19. 20. The method of claim 18, wherein the additional transgene confers herbicide resistance, insect resistance, bacterial disease resistance, fungal disease resistance, viral disease resistance, or sterility.

20. 1. A method for producing a canola plant derived from Brassica line NUBJ1207, the method comprising: (a) crossing a plant of Brassica juncea accession NUBJ1207 with a second canola plant to produce progeny canola seed; (b) growing the progeny canola seed under plant growth conditions to produce a canola plant derived from Brassica line NUBJ1207.

21. (c) crossing a NUBJ1207-derived canola plant with itself or with another canola plant to produce additional NUBJ1207-derived progeny canola seed; (d) growing the progeny canola seed of step (a) under plant growth conditions to produce additional NUBJ1207-derived canola plants; 21. The method of claim 20, comprising: (e) repeating said crossing step of (a) and said growing step of (b) zero (0) to seven (7) times to produce additional NUBJ1207-derived canola plants, wherein said NUBJ1207-derived canola plants express at least two NUBJ1207 traits selected from DPA products and at least one additional agronomically desirable trait.

22. 21. A canola plant or part thereof derived from Brassica line NUBJ1207 produced by the method of claim 20.

23. 22. A canola plant or part thereof derived from canola NUBJ1207 produced by the method of claim 21.

24. 10. A method for producing Brassica seed, the method comprising crossing a first parent Brassica plant with a second parent Brassica plant and harvesting the resulting first generation Brassica seed, wherein the first parent Brassica plant or the second parent Brassica plant is the Brassica plant of claim 2.

25. 25. The method of claim 24, wherein the first parent Brassica plant is different from the second parent Brassica plant and the resulting seed is a first generation (F1) hybrid Brassica seed.

26. 1. A method for obtaining a canola inbred line, the method comprising: (a) planting a collection of seeds comprising hybrid seeds, one of whose parents is the Brassica plant of claim 2, said collection further comprising seeds of said inbred lines; (b) growing canola plants from said collection of seeds; (c) identifying inbred plants from said inbred lines; (d) selecting the inbred plants; (e) controlling pollination in a manner that preserves homozygosity of the inbred plants; A method comprising:

27. 27. The method of claim 26, wherein one parent has all the physiological and morphological characteristics of the inbred Brassica line NUBJ1207, and seeds of said line have been deposited under ATCC Accession No. PTA-125954.

28. 27. The method of claim 26, wherein the canola inbred line is B. juncea or B. napus.

29. A cell of Brassica juncea NUBJ1207, wherein a representative sample of seeds of Brassica juncea NUBJ1207 has been deposited under ATCC Accession No. PTA-125954.

30. (a) a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO:1 and the nucleic acid sequence of SEQ ID NO:2; (b) a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO:3 and the nucleic acid sequence of SEQ ID NO:4; or (c) a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO:5 and the nucleic acid sequence of SEQ ID NO:6; A nucleic acid molecule comprising a DNA molecule, or a combination of two or three DNA molecules, selected from:

31. 31. A plant, seed, plant part, or seed meal comprising the nucleic acid molecule of claim 30.

32. 32. The plant, seed, plant part, or seed meal of claim 31 , wherein the plant, seed, plant part, or seed meal is a Brassica juncea plant, seed, plant part, or seed meal.

33. (a) a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO:1 and the nucleic acid sequence of SEQ ID NO:2; (b) a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO:3 and the nucleic acid sequence of SEQ ID NO:4; (c) a nucleic acid molecule comprising the nucleic acid sequence of SEQ ID NO:5 and the nucleic acid sequence of SEQ ID NO:6; or (d) the complement of (a), (b), or (c); A cell comprising one, two, or three DNA molecules selected from:

34. 34. The cell of claim 33, wherein the cell is a Brassica juncea cell.

35. 34. The cell of claim 33, wherein the cell is present in a plant, a seed, a plant part, or a seed meal.

36. 1. A method for identifying the presence of at least one locus characteristic of event NUBJ1207 in a sample comprising plant DNA, said method comprising: (a) contacting the sample with at least one of the nucleic acid molecules of claim 30, or a portion thereof; (b) subjecting the sample to a polymerase chain reaction; (c) assaying an amplicon comprising a nucleic acid molecule comprised of at least 20 contiguous nucleotides from one of SEQ ID NO:1 through SEQ ID NO:

6.

37. 37. The method of claim 36, wherein the method is a KASP™ genotyping assay.

38. 37. The method of claim 36, wherein a single-stranded portion of any one of the nucleic acid molecules of SEQ ID NO:1 to SEQ ID NO:6 is used as a primer or probe.

39. 1. A method for detecting the presence of event NUBJ1207 in a sample containing plant DNA, the method comprising contacting the sample with at least one primer that binds to at least one transgene flanking junction region of the Brassica genome as set forth in SEQ ID NO:1 to SEQ ID NO:

6.

40. 40. The method of claim 39, wherein the method is a KASP™ genotyping assay.

41. 41. A plant, plant material, or plant-derived material identified by the method of claim 39 or claim 40.

42. A kit comprising components for carrying out the method of claim 39 or claim 40.

43. 1. A method for detecting the presence of event NUBJ1207 in a sample containing plant DNA, the method comprising: (a) contacting the sample with a first primer that binds to a flanking junction region of the Brassica genome of NUBJ1207 and a second primer that binds to a transgene of NUBJ1207; (b) exposing the sample to polymerase chain reaction; and (c) characterizing an amplicon generated between the primers.

44. Genomic DNA of the inbred Brassica line NUBJ1207, seed of which is representative of an inbred canola line, has been deposited under ATCC Accession No. PTA-125954.

45. A plant cell comprising: (a) DNA having a sequence as set forth in at least one of SEQ ID NO:1 to SEQ ID NO:6; (b) DNA having at least 95% identity to at least one of SEQ ID NO:1 to SEQ ID NO:6; or (c) the complement of (a) or (b).

46. Seeds of a Brassica juncea lineage containing about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, or about 20% DPA (as a % of total fatty acids in the seed oil).

47. A collection of seeds comprising at least 95% seeds of Brassica juncea line NUBJ1207, wherein the seeds contain about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, or about 20% DPA (as a % of total fatty acids in the seed oil).

48. 48. A seed oil obtained from the seed of claim 46 or the collection of seeds of claim 47.

49. 48. A meal obtained from the seed of claim 46 or the collection of seeds of claim 47.

50. A meal protein obtained from the seed of claim 46 or the collection of seeds of claim 47.

51. 1. A method for obtaining a DHA-containing Brassica grain, the method comprising: (a) introgressing the DPA trait of B. juncea line NUBJ1207 into an elite Brassica line that is male sterile; (b) introgressing the DPA trait of line NUBJ1207 into a second elite Brassica line that is fertile; (c) crossing the two lines (a) and (b) to obtain a hybrid progeny; (d) cultivating seeds of the hybrid progeny; (e) harvesting the grain produced by the cultivated hybrid progeny.

52. 1. An extracted seed oil comprising fatty acids in an esterified form, said fatty acids comprising palmitic acid (C16), stearic acid (C18), oleic acid (C18:1), cis-vaccenic acid (C18:1n7c), linoleic acid (C18:2n6c), alpha-linolenic acid (C18:3n3), stearidonic acid (C18:4n3), gondoic acid (C20:1n9c), eicosatetraenoic acid (C20:4n3), omega-3 docosatrienoic acid (C22:4n3), and omega-3 docosapentaenoic acid (C22:5n3); Here, in the total fatty acid content of the seed oil, 1. An extracted seed oil having a palmitic acid level of less than 5%, a stearic acid level of less than 4%, an oleic acid level of 35%-48%, a cis-vaccenic acid level of less than 4%, a linoleic acid level of less than 9%, an alpha-linolenic acid level of 15%-20%, a stearidonic acid level of less than 2%, a gondoic acid level of less than 2%, an eicosatetraenoic acid level of less than 4.5%, an omega-3 docosatrienoic acid level of 1%-3%, and an omega-3 docosapentaenoic acid level of 8%-15%.

53. 53. The extracted seed oil of claim 52, wherein the seed oil is from Brassica juncea.

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