EA041446B1 - IMBRED TRANSGENIC CANOLA LINE NS-B50027-4 AND ITS SEEDS - Google Patents

Description

Related application

This application claims priority of U.S. Provisional Application No. 62/351,250, filed June 16, 2017, and incorporated herein by reference in its entirety for all purposes.

Sequence listing

This application contains a sequence listing that has been submitted in ASCII format via EFS-Web and is incorporated herein by reference in its entirety.

The technical field to which the invention belongs

Embodiments of the present invention relate to the field of canola breeding and agricultural products, in particular to the inbred seed and plant of an inbred transgenic canola line designated NS-B50027-4 and derivatives of such an inbred plant; and methods for detecting the inbred transgenic canola line NS-B50027-4.

Prerequisites for the creation of the invention

Canola is an important oilseed in many regions of the world. The fatty acid composition of canola oil is rich in monounsaturated and polyunsaturated fatty acids, including short chain omega-3 fatty acids, but does not contain long chain omega-3 fatty acids. Long-chain omega-3 fatty acids (LC-ω3) have proven health benefits, but currently LC-ω3 fatty acids are mainly sourced directly from seaweed or algae-eating marine fish. Recognition of the dietary importance of LC-ω3 fatty acids, especially docosahexaenoic acid (22:6 n-3; DHA), docosapentaenoic acid (22:5 n-3; DPA), and eicosapentaenoic acid (20:5 n-3; EPA) has contributed to a dramatic an increase in the demand for consumed fish oil. Thus, there is a need for alternative direct sources of LC-ω3 fatty acids for human consumption. In addition, since farmed fish, such as Atlantic salmon, accumulate fatty acids in proportion to dietary fatty acids, it is necessary to maintain the amount of LC-polyunsaturated fatty acids (LC-PUFA) in the fish feed and, in turn, ensure the presence of these fatty acids. acids in farmed fish. Accordingly, there is a need for sources rich in LC-PUFA that can be used in aquaculture. For example, there is a need for canola that can produce LC-PUFA, in particular LC-ω3 fatty acid such as DHA, for use in aquaculture as well as for direct human consumption. However, despite advances in plant breeding and manipulation by molecular genetics, there are no commercial sources of canola oil that reach the level of LC-PUFA produced in wild fish. In addition, a canola cultivar (not an F1 hybrid) must be homogeneous, homozygous and reproducible in order to be used for reliable commercial crop production. Thus, there remains a need for a canola line that can be grown as a sustainable crop whose seeds provide commercially viable amounts of ω3 fatty acids, LC-PUFA and LC-ω3 fatty acids such as DHA.

The essence of the invention

The embodiments described herein provide an inbred recombinant canola line, designated NS-B50027-4, whose seeds contain beneficial levels of ω3 and LC-ω3 fatty acid, thus providing a renewable terrestrial system for the production of these valuable oils. A representative seed sample of the inbred canola line NS-B50027-4 was deposited under the Budapest Treaty with the American Type Culture Collection (ATCC®) (Manassas, VA) on June 9, 2016 and assigned accession number PTA-123186 (see Appendix A). ). Also described herein are the cells, tissues, seeds and oil of the inbred canola line NS-B50027-4. The combination of selection and breeding with transgene manipulation provides variability in species where this variability does not exist. For example, the fatty acid profile of the canola line NS-B50027-4 described herein does not exist in native plants such as B. napus; and the features described herein, in particular the advantageous feature of DHA products, have been developed with significant human technical intervention.

In one aspect of the present embodiments, a canola (Brassica napus L.) seed of line NS-B50027-4, a genetically modified canola cultivar AV Jade, is provided that has been selected and bred into a stable, uniform breeding line that accumulates a high proportion of ( mass percent) of ω3 and LC-ω3 fatty acids, in particular LC-ω3 of PUFA, such as DHA, relative to the total fatty acid content. The inbred line NS-B50027-4 was developed to provide canola plants that produce seeds containing LC-ω3 PUFAs, specifically DHA, at levels approaching those found in some wild fish oils. The edible oil derived from NS-850027-4 has a significantly higher DHA content than other B. napus plants. A new uniform breeding line, NS-B50027-4, was developed through initial genetic transformation followed by careful selection and breeding for high DHA content in a stable, high yielding, morphologically fit canola line.

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Thus, at least one embodiment described herein relates to seeds of the inbred canola line NS-B50027-4; plants cultivated from seeds of the inbred canola line NS-B50027-4 and parts thereof such as pollen, ovule or seed; and methods for producing seed from a canola plant by cultivating an inbred canola line NS-B50027-4 or by crossing an inbred canola line NS-B50027-4 with itself or with another canola or Brassica line (such as B. juncea), and obtaining seed from cultured offspring. In a related embodiment, a seed of a canola or Brassica strain derived from NS-B50027-4 by introgression of at least one NS-B5OO27-4 transgenic locus is provided.

In at least one embodiment, the seed of a population of canola plants produced by the method described herein is provided, wherein said population has an average of 10-100% of its alleles derived from the NS-B5OO27-4 canola line. Similarly, the present embodiments provide for the use of the NS-B50027-4 canola line, NS-B50027-4 subline, NS-B50027-4 progeny or subline, or a plant obtained by crossing NS-B50027-4 with a second canola or Brassica plant, for breeding or cultivating a plant to produce seeds, oil, flour or protein.

In at least one embodiment, the seed of an oilseed rape plant, such as a Brassica napus plant, is provided, comprising in its genome at least a portion of the genome of the inbred line NS-B50027-4. In at least one embodiment, a plant is provided, such as a B. napus or B. juncea plant, comprising in its genome at least a portion of the genome of the inbred line NS-B50027-4. In at least one embodiment, there is provided a cell of an oilseed rape plant, such as a B. napus or B. juncea plant, comprising in its genome at least a portion of the genome of the inbred line NS-B50027-4. In another embodiment, the genomic DNA of an oilseed rape plant, such as a B. napus or B. juncea plant, is provided, comprising at least a portion of the genome, eg, at least one transgenic locus of the NS-B50027-4 lineage. At least one embodiment further relates to seeds, cells, tissues, tissue cultures, progeny and progeny of a plant containing at least a portion of the NS-B5OO27-4 genome grown from seed deposited with ATCC® under accession number PTA -123186. In another embodiment, further provided are plants that can be obtained (e.g., by propagating or breeding) from a canola plant containing at least a portion of the NS-B50027-4 genome (such as a plant grown from seed deposited with ATCC ® under accession number PTA-123186).

The reference seed of the inbred line NS-B50027-4 according to the present embodiments has been deposited with ATCC® under accession number PTA-123186. In at least one embodiment, NS-B50027-4 seed, deposited under accession number PTA-123186, is provided that grows into a canola plant whose seed contains at least about 5% DHA, about 6% DHA, about 7% when harvested normally. % DHA, about 8% DHA, about 9% DHA, about 10% DHA, about 11% DHA, about 12% DHA, about 13% DHA, about 14% DHA, about 15% DHA, 16% DHA, including amounts between the specified values and at the specified values, or more DHA, presented in mass percent (wt.%) of the total fatty acids in the seed.

In at least one embodiment, the seed deposited with the ATCC® under accession number PTA-123186 is a seed lot consisting of at least about 95% inbred transgenic seeds containing the NS-B50027-4 elite event transgenes that grow in a canola plant whose seeds contain at least 5% DHA, about 6% DHA, about 7% DHA, about 8% DHA, about 9% DHA, about 10% DHA, about 11% DHA, about 12% DHA, about 13% DHA, about 14% DHA, about 15% DHA, about 16% DHA, inclusive, or more DHA, in wt.% of the total fatty acids in the seed.

Seed deposited with ATCC® under accession number PTA-123186 is a seed lot consisting of at least about 95% or more than 95% transgenic seeds homozygous for the transgenic DNA and containing elite event NS-B50027-4, which grow into a canola plant whose seeds contain at least about 5% LC-PUFA, about 6% LC-PUFA, about 7% LC-PUFA, about 8% LC-PUFA, about 9% LC-PUFA, about 10% LC-PUFA, about 11% LC-PUFA, about 12% LC-PUFA, about 13% LC-PUFA, about 14% LC-PUFA, about 15% LC-PUFA, about 16% LC-PUFA, about 17% LC -PUFA, about 18% LC-PUFA, about 19% LC-PUFA, about 20% LC-PUFA inclusive or more LC-PUFA as the sum of EPA, DPA and DHA in wt.% of the total fatty acids in the seed. The seed or seed oil of this line may contain about 20-35 wt.% LC-PUFA, inclusive.

In at least one embodiment, there is provided a seed batch of hybrid seed obtained by crossing a male sterile canola or Brassica line with a second canola or Brassica line, both of which are homozygous for the NS-B50027-4 elite event, wherein the hybrid seed contains at least about 5% DHA, about 6% DHA, about 7% DHA, about 8% DHA, about 9% DHA, about 10% DHA, about 11% DHA, about 12% DHA, about 13% DHA, about

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14% DHA, about 15% DHA, about 16% DHA, about 17% DHA, about 18% DHA, about 19% DHA, about 20% DHA inclusive or more DHA in wt.% of the total fatty acids in the seed.

In one aspect of the present embodiments, a seed or seed progeny is provided that is obtainable or that is derived from a deposited seed that has been cultured and crossed at least once with another canola or Brassica (e.g., introgression with another canola or a Brassica plant with the same or different genetic background), while the seed can be sown and cultivated, and the seed obtained from such progeny can have essentially the same seed oil phenotype (trait) as NS-B50027-4. In some embodiments, the relative proportions of fatty acids in the seed or seed oil of such seed progeny are similar to those of NS-B50027-4, but with a higher yield than NS-B50027-4. In some embodiments, such progeny seed or seed oil may contain a higher proportion of LC-PUFA, such as EPA, DPA, or DHA, than NS-B50027-4 seed. In some embodiments, such progeny seed may contain ratios of non-LC-PUFA, such as oleic, linoleic, palmitoleic, vaccenic, or linolenic acids, different from their content in the seed or seed oil of NS-B50027-4. In some embodiments, during normal harvesting, the fatty acid content of the progeny seed derived from NS-B5OO27-4 is at least 5% DHA, about 6% DHA, about 7% DHA, about 8% DHA, about 9% DHA, about 10% DHA, about 11% DHA, about 12% DHA, about 13% DHA, about 14% DHA, about 15% DHA, about 16% DHA, about 17% DHA, about 18% DHA, about 19% DHA, about 20% DHA, about 21% DHA, about 22% DHA, about 23% DHA, about 24% DHA, about 25% DHA, about 26%, about 27% DHA, about 28% DHA, about 29% DHA, about 30% DHA inclusive or more DHA, in wt.% of the total fatty acids in the seed. For example, the seed of such progeny may contain 20-35% DHA, inclusive, in wt.% of the total fatty acid content in the seed. In some embodiments, upon normal harvesting, the fatty acid content of such progeny seed derived from NS-B50027-4 is at least about 5% LC-PUFA, about 6% LC-PUFA, about 7% LC-PUFA, about 8 % LC-PUFA, about 9% LC-PUFA, about 10% LC-PUFA, about 11% LC-PUFA, about 12% LC-PUFA, about 13% LC-PUFA, about 14% LC-PUFA, about 15% LC-UFA, about 16% LC-PUFA, about 17% LC-PUFA, about 18% LC-PUFA, about 19% LC-PUFA, about 20% LC-PUFA, about 21% LC-PUFA, about 22% LC - PUFA, about 23% LC-PUFA, about 24% LC-PUFA, about 25% LC-PUFA, inclusive, or more LC-PUFA, as the sum of EPA, DPA and DHA in wt.% of the total fatty acids in seed. For example, the seed of such progeny may contain about 20-40% LC-PUFA (wt.% of total fatty acids), inclusive.

In at least one embodiment, NS-B50027-4 seed contains substantially more ω3 ALA than conventional canola varieties. For example, when harvested normally, the fatty acid content of NS-B50027-4 seed or progeny is at least about 15% ALA, about 15% ALA, about 17% ALA, about 18% ALA, about 19% ALA, about 20% ALA, about 21% ALA, about 22% ALA, about 23% ALA, about 24% ALA, about 25% ALA, about 26% ALA, inclusive, or more ALA in wt.% of the total fatty acids in the seed.

In another aspect of the present embodiments, an oil is provided with advantageous levels of ω3 fatty acid and LC-ω3 fatty acid, wherein the fatty acid composition contains a higher ratio of ω3:ω6 fatty acids compared to conventional commercial canola oil. For example, in one embodiment, the seed oil from NS-B50027-4 has an EPA/DPA/DHA(ω3):LA(ω6) ratio of about 1.25. In comparison, AV Jade seed oil (which does not contain EPA/DPA/DHA) has a ω3:ω6 ratio of about 0.5. In addition, the oil from the AV Jade parent line does not contain DHA, so it does not have a DHA:LA relationship; while exemplary oil from NS-B50027-4 has a DHA:LA ratio of about 1.05; compared to farm-raised salmon oil, which has a DHA:LA ratio of about 0.908. The ω3 fatty acid ratios of NS-B50027-4 are particularly preferred for palmitic acid. In particular, the oil from the AV Jade parent line does not contain DHA and therefore does not have a DHA:palmitate relationship; in contrast, the reference oil from NS-B50027-4 has a DHA:palmitate ratio of about 2.12; in comparison, farmed salmon oil has a DHA:palmitate ratio of about 0.59 and wild salmon oil has a DHA:palmitate ratio of about 1.02. In at least one embodiment, the ω3:ω6 fatty acid ratio in NS-B50027-4 seed oil is about 3-7 inclusive, e.g., the ω3:ω6 fatty acid ratio is about 3, about 3.5, about 4, about 4, 5, about 5, about 5.5, about 6, about 6.5, or about 7. Progeny, hybrid, introgressive hybridization plants, and other such plants derived from NS-B50027-4 have similar or advantageous ω3 ratios :ω6.

In another aspect of the present embodiments, the oil, lipid, ω3-FA, LC-PUFA, or DHA from the seed or progeny of the inbred line NS-B50027-4 is used as a food product or in a food product (food or food material). , including drinks) or as dietary supplements (food additives) for humans or animals. In at least one embodiment, oil, lipid, ω3-FA, LC-PUFA, or DHA

- 3 041446 from event seed NS-B50027-4 is used to supplement food or food additives for aquaculture applications. In at least one embodiment, the oil, lipid, ω3-FA, LC-PUFA or DHA from the seed of the inbred line NS-B50027-4 is used as a pharmaceutical composition or in a pharmaceutical composition. In other embodiments, such an oil, lipid, ω3-FA, LC-PUFA, ω3 LC-PUFA, or DHA is obtained from lines derived from NS-B50027-4.

In another aspect of the present embodiments, the flour or protein obtained from the seed flour obtained from the seed of the inbred line NS-B50027-4 or the progeny derived from it is used as a food product (food or food material) or as food additives ( food additives) for humans or animals. In at least one embodiment, flour produced from the seed of NS-B50027-4 or progeny derived from it is used as a feed additive or feed additives for use in aquaculture. In at least one embodiment, a protein derived from or progeny derived from NS-B5OO27-4 seed is used to supplement feed or feed additives for use in aquaculture.

In one aspect of the present embodiments, a method for increasing LC-PUFA in a plant is provided by providing (eg, by genetic transformation or breeding) to the plant multiple copies of genetic constructs expressing certain front end enzymes of the LC-PUFA biosynthetic pathway. For example, while not all of the enzymes A6-desaturases, A5-desaturases, A5-elongases, and ω3/Δ15-desaturases can be considered solely as the front end of a desaturase or elongase, in specific embodiments, these genes are assembled into an artificial locus that enhances the production of LC-PUFA , for example EPA, DPA or DHA, in a transgenic plant that expresses other genes required for LC-PUFA synthesis. In specific embodiments, the artificial locus containing some front end genes includes at least one of Micromonas pusilla-derived A6-desaturase, Pyramimonas cordata-derived Δ-5-elongase, Pavlova salina-derived Δ5-desaturase, or Δ15 /ω3-desaturase derived from Pichia pastoris. In specific embodiments, an artificial locus containing some front end genes of the LC-PUFA biosynthetic pathway contains Δ6-desaturase derived from Micromonas pusilla, Δ5-elongase derived from Pyramimonas cordata, Δ5-desaturase derived from Pavlova salina, and Δ15/ω3 originating from Pichia pastoris. In a particular embodiment, this transgenic insert of four genes is segregated from NS-B50027-4 (eg, by outcrossing the A02 chromosome) and introduced into a recipient plant line using standard plant breeding techniques.

In one aspect of the described embodiments, a novel canola breeding line, designated NS-B50027-4, and an oilseed rape plant such as Brassica napus L. or B. juncea containing the NS-B50027-4 elite event in its nuclear genome are provided. Canola plants containing the genetic event line NS-B50027-4 are capable of seed-specific production of fatty acids that contain more LC-PUFA than the fatty acids produced in normal canola plants. Canola plants of the inbred line NS-B50027-4 exhibit other agronomic productive traits that are substantially equivalent to non-transgenic isogenic canola plant lines; but such characters are distinguished from other lines to provide an independent line or cultivar designated NS-B50027-4. A representative seed sample of the inbred canola line NS-B50027-4 was deposited with ATCC® and received accession number PTA-123186.

At least one embodiment relates to transgenic canola seeds, plants or plant parts, tissues or cells thereof, having at least one transgenic insert stably integrated into the genome, containing an expression cassette containing sixteen heterologous genes, wherein the transgenes are codon-optimized for plant expression and encode Δ4-desaturase derived from Pavlova salina, Δ5-desaturase derived from Pavlova salina, Δ5-elongase derived from Pyramimonas cordata, Δ6-desaturase derived from Micromonas pusilla, Δ6-elongase derived from Pyramimonas cordata, Δ12 a desaturase derived from Lachancea kluyveri, Δ15/ω3-desaturase derived from Pichia pastoris, and at least one matrix attachment site (MAR) derived from Nicotiana tabacum and a selectable marker gene; and at least one transgene insert containing an expression cassette containing four heterologous genes, wherein the transgenes are codon-optimized for plant expression and encode the transgenes Δ6-desaturase derived from Micromonas pusilla, Δ5-elongase derived from Pyramimonas cordata, Δ5-desaturase derived from Pavlova salina, Δ15/ω3-desaturase derived from Pichia pastoris, and at least one MAR derived from Nicotiana tabacum. The inbred transgenic line NS-B5OO27-4 illustrates this embodiment, and a representative sample of seeds with these heterologous genes is deposited with ATCC® under accession number PTA-123186.

In addition, in one aspect of the present embodiments, methods are provided for identifying a transgenic plant, or cells or tissues thereof, containing a transgenic trait (elite event) of an inbred canola line NS-B50027-4, wherein said method is based on identifying the presence of characteristic DNA molecules that have specific nucleotide sequences or code for specific amino acids. For example, such characteristic DNA molecules contain

- 4 041446 sequences at least 15 bp long. (base pairs), at least 20 bp, at least 30 bp inclusive, which contain the site of embedding or attaching an event, i.e. a fragment of an inserted foreign DNA containing LC-ω3 fatty acid synthesis genes and a fragment of the canola genome (5'- or 3'-flanking regions for each insert) docked to each other, which allows specific identification of NS-B50027-4. As another example of this aspect, the NS-B50027-4 identification method can use a primer set to identify a number of transgenes and flanking regions. Embodiments also apply to plants identified by the methods described herein.

In some embodiments, compositions are provided useful in allele-competitive PCR assays (in which two allele-specific forward primers recognize SNPs), digital droplet PCR (ddPCR) assays, quantitative PCR (qPCR) assays, paralog-specific assays, and in random presence (AR) assays. Particular embodiments of primers useful in performing KASP assays to detect genetic traits of NS-B50027-4, particularly useful in introgression studies and hybrid development, include the primers shown in SEQ ID NOs: 1-90, or complements thereof. At least some of these primers or their complements may be included in a kit to identify NS-B50027-4, progeny of NS-B50027-4, or other plants or plant materials containing at least a partial NS-B50027-4 genome.

In a related aspect, there is provided genomic DNA obtained from or derived from plants containing at least a portion of the genomic DNA of the NS-B50027-4 lineage, in particular regions of the genome comprising transgenes or junction regions thereof. Such genomic DNA can be used, for example, as a reference control material in the identification assays described herein.

Brief description of the figures

In FIG. 1 is a diagram showing enzymes introduced into transgenic plants to provide a biosynthetic pathway for DHA synthesis.

In FIG. 2 shows a plot of grain yield plotted against predicted DHA levels, in kg/ha, across eight cultivation sites. ♦ stands for DHA, kg/ha; - indicates a straight line DHA kg/ha; y=29.296x+2.8315; R ² =0.8567.

In FIG. 3 shows a plot of grain yield versus predicted LC-PUFA (EPA, DPA and DHA) levels in kg/ha for eight plots. ♦ denotes LC-PUFA in kg/ha; - denotes a straight line LC-PUFA in kg/ha; y=34.043x+3.4049; R ² =0.8636.

In FIG. 4 is a block diagram that describes possible processing and product flows from harvested transgenic canola seed that contains ω3 fatty acids. Rounded rectangles represent products or fractions of a process; ovals indicate processing stages; rectangles with sharp corners indicate the possible application of the product.

Detailed description

It should be understood that the present invention is not limited to the specific methodology, protocols, reagents, etc. described herein, and therefore may vary. The terminology given herein is used solely to describe specific embodiments and does not limit the scope of the present invention, which is defined solely by the claims.

The contents of all designated patents and other publications are incorporated herein by reference for the purpose of describing and disclosing, for example, the methodologies described in such publications that may be used in connection with the present invention, but should not define terms inconsistent with those which are presented in this document. These publications are provided solely for their disclosure prior to the filing date of this application. Nothing in this connection should be construed as an admission that the inventors are not entitled to backdate such disclosure by virtue of prior invention or for any other reason. All statements regarding the date of submission or the content of these documents are based on information available to the applicants and do not contain an admission of the truth of the dates or content of these documents.

As used herein and in the claims, the singular forms a, an, and the include the plural unless the context clearly indicates otherwise. Throughout this specification, unless otherwise indicated, contain, contains, and containing are used inclusively, and not exclusively, so that a specified integer or group of integers may include one or more other unspecified integers or groups of integers. The term or is inclusive unless modified, for example, by the word both. Thus, unless the context indicates otherwise, the word or means any one member of a particular list, and also includes any combination of members of that list.

All values are approximate as there is some fluctuation in the composition

- 5 041446 fatty acids due to environmental conditions. The values are generally expressed as weight percent of total fatty acids or weight percent of the whole seed. Accordingly, except in the case of working examples or where otherwise indicated, all numbers expressing amounts of ingredients or reaction conditions used herein are to be understood as modified in all instances by the term about, unless otherwise indicated; refers generally to a deviation of ±1% from the specified value, but may allow a deviation of ±5 or ±10% from the specified value, accepted in the appropriate context by a person skilled in the art.

Recombinant DNA technologies can be applied according to standard protocols known in the art. See, for example, Sambrook et al., Molecular Cloning: Lab. Manual (2nd ed., Cold Spring Harbor Lab. Press, NY (1989); Ausubel et al., Current Protocols Molec. Biol. (1994 and updates); DNA Cloning: Practical Approach, vols. 1-4 (Glover & Hames , Eds., IRL Press 1995, 1996), Croy, Plant Molec. Biol. Labfax (BIOS Sci. Pub. Ltd. & Blackwell Sci. Pub., UK, 1993); WO 2015089587.

The headings are for convenience only and should not be construed as limiting the invention in any way. Unless otherwise indicated, all technical and scientific terms used in this document have the same meanings as they are commonly understood by a person skilled in the art. The terminology used herein is for the purpose of describing specific embodiments only and is not intended to limit the scope of the present invention, which is solely defined by the claims. For easier understanding of the present invention, some terms will be defined first. Additional definitions are set forth throughout the detailed description.

Definitions.

A lineage is a group of plants that show very little overall difference between the individuals to which the designation applies. Also, a line refers to a homogeneous group of plants carrying essentially the same genetic material that exhibit little or no genetic difference between individuals in at least one trait. A variety or cultivar may be used interchangeably with a line, but generally the first two terms refer to a line that is suitable for commercial production. In this application, genetically inherited, as used, for example, in the phrase genetically inherited from parental lines, means that the characteristic in question is wholly or partly due to the properties of the genetic composition of the plant in question.

As used herein, the term Brassica refers to plants in the Brassicaceae family. The Brassica plant may belong to one of the species Brassica napus, B. rapa (or campestris) or B. juncea. Alternatively, the plant may be of a species obtained by crossing these Brassica species, such as B. napocampestris, or by artificially crossing one of these Brassica species with other Cruciferacea species. The term ploidy is used to determine whether the number of chromosomes displayed by a cultivar is diploid or tetraploid. Since Brassica napus is an allotetraploid (amphidiploid) obtained by crossing and retaining both the genomes of Brassica rapa (formerly B. campestris) and B. oleracea, a Brassica napus plant containing the NS-B50027-4 transgenic event can be used by various or conventional methods. dilution to introduce the NS-B50027-4 event, and thus the trait of LC-m3 fatty acid production or increased LC-m3 fatty acid expression, as described herein, into other members of the genus Brassica. Accordingly, examples of members of the genus Brassica useful for practicing the present embodiments include, but are not limited to, B. juncea, B. napobrassica, B. oleracea, B. carinata, B. napus, B. rapa, and B. campestris, and also any other plants belonging to the genus Brassica which permit breeding between Brassica species.

Typically, an oil plant refers to any of the species B. napus, B. rapa (or campestris) or B. juncea.

The Brassica napus plant is commonly known as rapeseed or oilseed rape, and certain cultivars may be referred to as canola. As used herein, the term canola or canola plant refers to the Brassica plant that can be used to produce canola oil (i.e., oil that meets a certain quality requirement of less than 2% erucic acid) and includes B. napus, B. napobrassica, B. rapa, B. juncea and B. campestris. Canola plants are amphidiploids (also called allotetraploids), which refer to an interspecific hybrid having a complete diploid set of chromosomes from each parent, commonly referred to as the AACC genome.

Generally, the terms canola and canola plant refer to Brassica napus, but include all plant varieties that can be crossed with canola. The terms canola and canola plant also include plant parts. Canola oil must contain less than 2% erucic acid (Δ13-22:1) and less than 30 µmol glucosinolates/g air-dried, oil-free hard canola seed (ie flour). See, for example, Codex Alimentarius: Fats, Oils & Related Products, vol. 8 (2nd ed., Food & Agriculture Org. United Nations, Rome, Italy, 2001).

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A plant part includes plant cells, plant organs, plant protoplasts, plant cell tissue culture from which plants can be regenerated, plant calluses, plant masses, and plant cells that are intact in plants or plant parts such as germs, pollen, ovules, seeds. , achenes, leaves, flowers, branches, fruit, stems, roots, root ends, anthers, cotyledons, hypocotyls, primary roots, individual cells, gametes, cell cultures, tissue cultures, etc. The cotyledon is a type of seed leaf; a small leaf found on the embryo of a plant. The cotyledon contains tissues to store the nutrients of the seed. The embryo is a small plant contained in a mature seed. Plant cells also include non-regenerating plant cells. Progeny, derivatives, variants, and mutants of regenerated plants are also included within the scope of the present embodiments, provided that these portions contain at least some of the NS-B50027-4 event nucleic acid molecules, typically one or two of the two NS-B50027 elite event loci -4. The present embodiments also relate to the use of NS-B50027-4 elite event transgenes in cell culture and plant tissue culture. Embodiments include plants and plant parts from the elite event line NS-B50027-4, as well as other plants obtained using the described methods, which are added to the genetic structure of NS-B50027-4, or progeny that contains at least one of the two transgenic loci NS-B50027-4.

An allele is an alternative form of a gene that refers to one trait or characteristic. In a diploid cell or organism, two alleles of a given gene occupy corresponding loci on a pair of homologous chromosomes.

The locus confers one or more traits such as, for example, modified fatty acid metabolism, modified phytic acid metabolism, modified carbohydrate metabolism, male sterility, herbicide resistance, insect resistance, disease resistance, or modified protein metabolism. The trait may, for example, be due to a naturally occurring gene introduced into the line's genome by backcrossing, natural or induced mutation, or a transgene introduced by genetic transformation techniques. A locus may contain one or more alleles integrated into a single chromosomal location. Quantitative trait locus (QTL) refers to a region of DNA (locus) that correlates with phenotype variability (quantitative trait). The QTL is often associated with or contains genes that control this phenotype. QTLs are mapped by identifying which molecular markers (such as SNPs or AFLPs) correlate with the observed trait.

Percent identity refers to the comparison of homozygous alleles of two canola or Brassica varieties. The percentage of identity is determined by comparing the statistically significant number of homozygous alleles of the two developed varieties. For example, a percentage identity of 90% between NS-B50027-4 and a second Brassica means that two Brassica have the same alleles at 90% of their loci. With regard to a certain DNA or protein molecule, percent (%) identity, minimum percent (%) identity of the identified sequence (SEQ ID NO) means a nucleotide sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 76%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99 %, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least and including 99.9% identical to the corresponding sequence shown, indicated by SEQ ID NO.

The degeneracy of the genetic code allows for the possibility of a larger range of percent (%) identity for polynucleotide sequences than would normally be acceptable for proteins due to the many nucleotide combinations that can code for a given protein. In addition, amino acid substitution allows for many minor changes in the protein's primary amino acid structure, such as amino acid substitutions, that do not impair enzymatic function. In addition, polynucleotides may have, when compared to natural molecules, one or more mutations, which are deletions, insertions, or substitutions of nucleotide residues. Polynucleotides that have mutations relative to a reference sequence can either be naturally occurring (ie, isolated from a natural source) or synthetic (eg, by performing site-directed mutagenesis or DNA shuffling).

Typically, an event is an artificial genetic locus that, as a result of genetic manipulation, carries a foreign DNA containing a gene or genes of interest (transgene(s)). Typical allelic event states are the presence or absence of foreign DNA. An event can be characterized phenotypically by the expression of one or more transgenes. At the genetic level, an event is part of the plant's genetic makeup. At the molecular level, an event can be characterized by a restriction map,

- 7 041446

5'-(upstream) or 3'-(downstream) flanking sequences of the transgene(s) or molecular configuration of the transgene(s). Typically, the transformation of plant cells or plant parts with transforming DNA leads to a huge number of events, each of which is unique.

The term gene refers to a DNA molecule, typically containing several operably linked DNA regions, such as a promoter and a 5' untranslated region (5'UTR or 5' non-coding sequences), which together form a promoter region; a coding region (which may or may not encode a protein); and a 3' untranslated region (3'UTR or 3' non-coding sequences) containing a polyadenylation site. Typically in plant cells, the 5'UTR coding region and the 3'UTR are transcribed into an RNA molecule, which in the case of a protein-coding gene is translated into a protein. Thus, a coding sequence refers to a sequence of nucleotides in a DNA molecule that provides codons that translate (via messenger RNA, ribosomal and associated translation machinery) a particular sequence of amino acids. A gene may include additional sections of DNA, such as, for example, introns. Genotype refers to the genetic constitution of a cell or organism. A genetic locus is generally the position of a given gene or set of genes in a plant's genome.

The term transgene refers to a gene of interest that has been introduced into the genome of a plant. Accordingly, a transgenic plant contains at least one transgene in the genome of all its cells. The transgenes of the present embodiments contain at least one copy of the following enzymes expressed during LC-PUFA biosynthesis in line NS-B50027-4: A4 desaturase derived from the marine microalgae Pavlova salina, A5 desaturase derived from P. salina, A5-elongase derived from the microalgae Pyramimonas cordata, A6-desaturase derived from the microalgae Micromonas pusilla, A6-elongase derived from P. cordata, A12-desaturase from the yeast Lachancea kluyveri, and A15/m3-desaturase derived from the yeast Pichia pastoris. Alternatively or additionally, the transgenes of the present embodiments comprise an A5 desaturase derived from P. salina, an A5 elongase derived from P. cordata, an A6 desaturase derived from M. pusilla, and a Δ15/ω3 desaturase derived from P. pastoris. The NS-B50027-4 transgenes are located in a binary manner in expression cassettes that include the respective regulatory regions. The NS-B50027-4 transgenes described above are artificial in the sense that they were designed using a codon optimization strategy and thus the transgenes do not otherwise exist in nature. The transgene expression cassette included at least one matrix attachment site (MAR) from Nicotiana tabacum. The transgenic cassette also included a selectable marker gene. See, for example, WO 2013185184; US 2015/0166928; PCT/US2017/__, filed June 16, 2017, claiming priority of US Provisional Application 62/351246.

The term foreign or heterologous, when referring to a gene or DNA molecule in relation to plant species, means that the gene or DNA molecule, or part of it (for example, a specific region) is not found in nature in these plant species or is not found in nature in this plant species. genetic locus in these plant species. The term foreign DNA also refers to a DNA molecule that will or has been introduced into the plant genome as a result of transformation. In the context of this disclosure, a transgene, transgene cassette or transgene expression cassette contains at least one foreign or heterologous DNA.

The term chimeric, when referring to a gene or DNA molecule, is used to indicate that a gene or DNA molecule contains at least two functionally significant DNA regions (such as a promoter, 5'UTR, coding region, 3'UTR, intron), which are not naturally related to each other and come from different sources, such that at least one DNA region is foreign to another DNA region in the chimeric DNA molecule.

The terms plasmid or vector refer to an extrachromosomal element, often carrying genes that are not part of the cell's central metabolism, and usually in the form of circular double-stranded DNA molecules. Such elements can be autonomously replicating sequences, genome-integrating sequences, phage or nucleotide sequences, linear or circular, single- or double-stranded DNA or RNA, isolated from any source, in which a number of nucleotides have been linked or recombined into a unique construct capable of introducing into a cell, such as an expression cassette. For transgenic plants, such plasmids or vectors may contain T-DNA regions that facilitate the introduction of the transgene(s) into the plant genome.

An expression cassette refers to a genetic construct containing a transgene and having elements in addition to a foreign gene that allow the gene to be expressed in a foreign host (e.g., a 5' promoter (optionally with an enhancer) untranslated region (UTR) of DNA, DNA encoding a selected gene product, and the corresponding 3'-UTR of DNA); and may refer to the cassette before and after insertion into the plant genome. In other words, transgenic

- 8 041446 contains an expression cassette. Accordingly, insert DNA refers to heterologous DNA introduced into plant material by a transformation process and includes DNA (which is different from the original/wild type/native) DNA used for such transformation as described herein. The DNA insert is typically a transgenic expression cassette.

Transforming DNA refers to a recombinant DNA molecule used for transformation, such as an expression vector. The transforming DNA typically contains at least one gene of interest (eg, a chimeric gene) that is capable of conferring one or more specific characteristics to the transformed plant.

Transformation refers to the transfer of a nucleic acid molecule into a host organism, resulting in genetically stable inheritance. The nucleic acid molecule may be, for example, a plasmid that replicates autonomously, or it may integrate into the genome of the host organism. Host organisms containing transformed nucleic acid fragments are referred to as transgenic or recombinant or transformed organisms.

The term recombinant DNA molecule is used for illustrative purposes and thus may include an isolated nucleic acid molecule, which may be DNA and which may be produced by recombinant or other methods such as synthetic DNA synthesis or PCR. PCR (polymerase chain reaction) is a reaction in which identical copies of a target polynucleotide are obtained using primers consisting of an upstream and downstream primer and a polymerization catalyst such as DNA polymerase and a generally thermally stable polymerase enzyme. PCR methods are known in the art. See, for example, PCR (McPherson & Moller, eds., BIOS Sci. Publ. Ltd., Oxford, 2000). PCR can be performed on genomic DNA or cDNA.

Suitable regulatory elements or useful regulatory sequences refer to polynucleotides preceding (eg, 5'UTR), within, or following (3'UTR) a coding region that affect the transcription, processing, or stability of the RNA, or translation of the associated coding region. Regulatory elements may include promoters, enhancer elements, translation leader sequences, introns, polyadenylation recognition sequences, RNA processing sites, effector binding sites, and a stem-loop structure.

A promoter refers to a DNA element capable of controlling the expression of a coding region or functional RNA. In general, the coding region is located 3' to the promoter element. Promoters may be isolated entirely from a native gene, or may be composed of different elements derived from different naturally occurring promoters, or even include synthetic DNA segments. Those of skill in the art will recognize that different promoters may direct gene expression in different tissues or cell types, or at different stages of development, or in response to different environmental or physiological conditions. Promoters that cause gene expression in most cell types are generally referred to as constitutive promoters in most cases. It is generally accepted that, since in most cases the exact boundaries of the regulatory elements have not been fully defined, DNA fragments of different lengths can have identical promoter activity.

The terms 3' non-coding sequences and transcription terminator or 3' untranslated region (UTR) refer to DNA elements located to the right (3') of the coding region of the DNA molecule. These include polyadenylation recognition sequences and other sequences encoding regulatory signals capable of influencing mRNA processing or gene expression. The polyadenylation signal is typically characterized by an influence on the addition of polyadenylic acid tails to the 3' end of the mRNA precursor. The 3' region can affect the transcription, processing or stability of the RNA or the translation of the associated coding region.

Operably linked refers to the association of nucleic acid elements in one nucleic acid molecule or fragment in such a way that one of them affects the function of the other. For example, a promoter is operably linked to a coding region if it is capable of influencing the expression of that coding region (ie, the coding region is under the transcriptional control of the promoter). The coding regions can be operatively linked to regulatory elements in sense or antisense orientation.

Mutagenesis is a process in which an agent, such as ethyl methylsulfonate, is applied to plant material, which is known to induce mutations in the genetic material in order to produce new genetic variability in the species, and is generally carried out with a particular trait in mind. See Swanson et al., 7 Plant Cell Rep. 83 (1988). Other methods include generating mutants in the direction of substitution of specific nucleotides or amino acids (substitutions, deletions or additions). Such methods for performing nucleic acid sequence substitutions are included within the term mutagenesis. Mutagenesis may be useful in knockout experiments for

- 9 041446 violations of gene expression.

Primers are relatively short polynucleotides or oligonucleotides that are complementary to the portion of the polynucleotide to be amplified, eg by polymerase chain reaction. Typically, the primer may be no more than 50 nucleotides in length, such as less than about 30 nucleotides or less than about 24 nucleotides.

As used herein, the term expression refers to the transcription and stable accumulation of sense (mRNA) generated from the nucleic acids of the invention. Expression may also refer to the translation of an mRNA into a polypeptide.

Reference to a cell includes a plant cell, whether isolated, contained in tissue culture, or introduced into a plant or plant part, unless otherwise indicated or clear from the context.

Regeneration includes selection from a plant to be bred or a cell variety capable of regeneration (eg, seeds, microspores, ovules, pollen, vegetative parts). These cells may optionally be subjected to genetic transformation or mutagenesis, after which the plant develops from the cells using regeneration, pollination or cultivation methods based on the type of genetically modified cells. Applicable regeneration techniques are known to those skilled in the art; see, for example, Armstrong & Green, 165 Planta 322 (1985); Close & Ludeman, 52 Planta Sci. 81 (1987).

Progeny means all progeny, including progeny and derivatives of a plant or plants, and includes first, second, third and subsequent generations, and may be formed by self-pollination or crossing with plants of the same or different genotypes, and may be modified by a number of suitable genetic engineering methods. Cultigen usually refers to plants that have been deliberately modified and selected by humans. T0 refers to the first generation of transformed plant material, T1 refers to seeds derived from T0 plants, T1 seeds give rise to plants that produce T2 seeds, etc. to subsequent Tx offspring.

Breeding includes all methods of plant development or propagation and includes both intra- and interspecific and intra- and inter-line crossings, as well as all suitable methods of traditional breeding and artificial breeding. Desired traits (eg, DHA trait NS-B50027-4) can be transferred to other lines, canola or B. napus cultivars or cultigens; or by traditional breeding methods, and can also be transferred to other Brassica species such as B. juncea and B. rapa by interspecific crossing. Conventional breeding methods and interbreeding methods, as well as other methods for transferring genetic material between plants, are well known in the art.

Backcrossing is the process by which a breeder repeatedly crosses hybrid progeny with the parent line. For example, crossing the first generation of F1 hybrids with one of the parent F1 hybrid genotypes is a backcross. Backcrossing can be used to introduce a trait of interest from a donor parent (transgenic parent) into a recurrent parent (elite line to be modified): the donor parent is crossed to the recurrent parent; the offspring of this cross are then backcrossed to the recurrent parent; the offspring of these crosses are selected for the trait of interest and then backcrossed to the recurrent parent; this process is repeated with as many backcrosses as necessary to create a line in which the recurrent parent is homozygous for the trait passed from the donor parent. Backcrossing can also be combined with selfing, which may be advantageous when introducing recessive genes.

Introgression is the stable incorporation of genes from one gene pool into another. Introgressive hybridization refers to the inclusion (usually by hybridization and backcrossing) of alleles from one entity into the gene pool of a second, distinct entity. Lines with introgressions allow the study of quantitative trait loci and also provide a means to incorporate new traits, i.e. traits NS-B50027-4, into other canola or brassica such as B. napus or B. juncea. Recipient lines of interest include freely pollinated (OP) herbicide resistant lines such as triazine resistant (TT); Roundup Ready® (RR); packaged TT and RR; OR lines resistant to imidazolinone (IMI); or IMI (Rf) reducing agent lines; lines with a high percentage of seed oil; optimal background for DHA lines; or lines chosen for regional adaptation. Such lines can be developed as hybrids or elite developments. Because existing isogenic canola lines exhibit a potential seed oil content of 45%, introgression of the NS-B50027-4 loci can generate seed oil containing 20% DHA in total yield in such batched events. Additional breeding event packages can be selected for EPA or DPA products. Indeed, the NS-B5OO27-4 genes (one or both loci) can be packaged with other transgenic Brassica that contain at least one insert of seven or eight genes (i.e. seven enzymes and optionally a marker) by transformation with the same the vector itself or a similar vector used

- 10 041446 th to generate NS-B50027-4, which leads, for example, to obtaining a plant with three loci, providing the production of LC-PUFA. Alternatively, as described hereinafter, the A02 and A05 loci may be segregated from NS-B50027-4 and the A02 locus may be bundled into other LC-PUFA producing lines (e.g., single insert line for EPA production, DPA or DHA), to increase LC-PUFA production.

Disease resistance generally refers to the ability of a plant to limit the activity of a particular pest, such as an insect, fungus, virus, or bacterium. Disease tolerance refers to the ability of plants to tolerate a disturbance caused by a particular pest (such as an insect, fungus, virus, or bacterium) or adverse environmental condition, yet still function and produce despite that disturbance.

The fatty acid composition or fatty acid composition generally refers to the weight percentage of the various fatty acids present in the endogenously formed oil of mature, whole, partially dried seeds. It is common practice in the industry to present the fatty acid composition as a percentage of area (area normalization) rather than absolute weight percent, but the former approximates the latter. Absolute results can be calculated using individual reference standards of known concentration and an internal standard to calculate results on a mg/kg basis. It is also possible to use correction factors to calculate fatty acid mass without using individual fatty acid standards, although an internal standard may still be required. Typically, fatty acid composition is determined by crushing the seeds and extracting the fatty acids as fatty acid methyl esters (FAMEs), which can be analyzed for fatty acid composition by various methods to give data as percentage of area or data from which percentage can be extracted. from the square. Illustrative analytical approaches include gas chromatography (GC), GC-mass spectrometry (GC-MS), liquid chromatography-mass spectrometry (LC-MS), nuclear magnetic resonance (NMR), or reflectance spectroscopy. The total lipid can be separated using methods known in the art to purify fractions such as the TAG fraction, for example. Other ways of characterizing fatty acid compositions are known to those skilled in the art. See, for example, Canola: Chem., Production, Processing & Utilization (Daun et al., eds., AOCS Press, Urbana, IL, 2011) (Daun et al., 2011); US 2015/0166928; US 20160002566.

Similarly, the oil content is the typical weight percentage of oil present in mature, whole, partially dried seeds (typically containing about 6 or 7% moisture). The oil percentage, also referred to as oil content, is calculated as the mass of oil divided by the mass of the seed at a standardized moisture content. The oil content can be a characteristic of different plant varieties. It can be determined using various analytical methods such as nuclear magnetic resonance (NMR), reflectance spectroscopy (NIR) and Soxhlet extraction. For example, oil content in canola can be measured using NMR methods (Rossell & Pritchar, Analysis of Oilseeds, Fats & Fatty Foods, 48-53 (Elsevier Sci. Pub. Ltd, London, 1991) on an NMS 100 Minispec pulse meter, which simultaneously measures the moisture content The oil content of the seed can also be measured by NIR spectroscopy (Li et al., 67 Phytochem. 904 (2006)).

The expressions extracted plant lipid, isolated plant lipid, extracted lipid, and the like. refer to compositions containing lipids that have been extracted, for example, from a crushed plant or plant parts such as seeds. The extracted lipid may be a relatively crude composition obtained, for example, by grinding plant material such as seeds; or a more refined composition in which most if not all of the water, nucleic acids, proteins or carbohydrates derived from plant material has been removed from the oil. Examples of purification methods are known in the art. In some embodiments, the extracted or isolated plant lipid contains at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% (w/w ) lipid by weight of the composition. The extracted lipid may be solid or liquid at room temperature, the latter being referred to herein as an oil. In some embodiments, the extracted lipid has not been mixed with another lipid, such as DHA produced by another source (eg, DHA from fish oil). In some embodiments, after extraction, the ratio of oleic acid to DHA, palmitic acid to DHA, linoleic acid to DHA, or total ω6 fatty acids to total ω3 fatty acids did not change significantly (e.g., no more than 10% or 5% variation) compared to ratio in an intact seed or cell. In other words, the extracted oil has not been fortified with a particular fatty acid, such as DHA. In other embodiments, the extracted plant lipid has not been subjected to a procedure, such as hydrogenation or fractionation, that changes the ratio of oleic acid to DHA, palmitic acid to DHA, linoleic acid to DHA, or total ω6 fatty acids to total ω3 fatty acids.

- 11 041446 compared with the ratio in the intact seed or cell. In other words, the extracted oil has not been fortified with a particular fatty acid, such as DHA. When the extracted plant lipid according to the present invention is an oil, said oil may further contain non-fatty acid molecules such as sterols.

As noted above, the terms extracted vegetable oil and isolated vegetable oil refer to compositions containing an extracted vegetable lipid or an isolated vegetable lipid that is liquid at room temperature. The oil is obtained from a plant or part of it, such as seeds. The extracted or isolated oil may be a relatively crude composition obtained, for example, by grinding the seeds of a plant; or a more refined composition in which most if not all of the water, nucleic acids, proteins or carbohydrates derived from plant material has been removed from the oil. The composition may contain other components, which may or may not be a lipid. In one embodiment, the oil composition contains at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 95% (w/w) of extracted vegetable lipid. In one embodiment, the extracted oil of the invention has not been mixed with another oil, such as DHA, not produced by another source (eg, DHA from fish oil). In one embodiment, after extraction, the ratio of oleic acid to DHA, palmitic acid to DHA, linoleic acid to DHA, or total ω6 fatty acids to total ω3 fatty acids did not change significantly (e.g., no more than 10% or 5% variation) compared to ratio in an intact seed or cell. In another embodiment, the extracted vegetable oil has not been subjected to a procedure, such as hydrogenation or fractionation, that changes the ratio of oleic acid to DHA, palmitic acid to DHA, linoleic acid to DHA, or total fatty acids ω6 to total fatty acids ω3 when compared to the ratio in intact seed or cell. The extracted vegetable oil according to these embodiments may contain non-fatty acid molecules such as sterols.

As used herein, the term oil is a composition containing predominantly lipid and which is liquid at room temperature. For example, the oil according to the invention preferably contains at least 75%, at least 80%, at least 85% or at least 90% by weight of lipids. Typically, refined vegetable oil contains at least 90% triacylglycerols (TAG) by weight of lipid in the oil. Minor oil components such as diacylglycerols (DAG), free fatty acids (FFA), phospholipid, or sterols may be present in the oil. The edible oil obtained from NS-B50027-4 may have one or more of the following characteristics: a DHA content of at least about 7% by weight, a DPA content of at least about 1% by weight, an EPA content of at least about 0, 4% by weight, oleic/cis-vaccenic acid content about 46% by weight, linoleic acid content about 8.2% by weight, ALA content at least about 19%, combined ALA/arachidic acid/SDA content about 21% by weight by weight, the combined content of EPA/DPA/DHA is at least about 9% (in wt.% of the total content of fatty acids). In some embodiments, the combined content of EPA/DPA/DHA is about 16%.

As used herein, the term fatty acid refers to a carboxylic acid, often with a long aliphatic tail, either saturated or unsaturated. Typically, fatty acids have a carbon-carbon chain length of at least eight carbon atoms, such as at least 12 carbon atoms, 16 carbon atoms, 18 carbon atoms, 20 carbon atoms, 22 carbon atoms, or 24 carbon atoms. Most natural fatty acids have an even number of carbons because their biosynthesis involves acetate, which has two carbons. Fatty acids can be in a free state (non-esterified); in an esterified form such as a triglyceride (TAG) moiety, a diacylglyceride (DAG), a monoacylglyceride; be bound by acyl-CoA (thioether) or in another bound form. The fatty acid may be esterified as a phospholipid such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, or diphosphatidylglycerol.

Saturated fatty acids do not contain carbon-carbon double bonds (alkenes) or other functional groups along the chain. Thus, the term saturated refers to the presence of hydrogen at all possible carbon atoms (except the carboxylic acid group [-COOH]). In other words, in an omega saturated fatty acid (ω), the end (also called the n-terminus) of the fatty acid contains three hydrogen atoms (-CH3) and each carbon within the chain contains two hydrogen atoms (-CH2-). Total saturation generally refers to the combined percentage of palmitic (C16:0), stearic (C18:0), arachidic (C20:0), behenic (C22:0), and tetracosanoic (C24:0) fatty acids.

Unsaturated fatty acids have a similar backbone to saturated fatty acids except that they include at least one alkene group (-CH=CH-) on the carbon

- 12 041446 chains. The two flanking carbons (attached to each side of the alkene group) can occur in the cis or trans configuration. Monounsaturated fatty acids refer to fatty acids that have at least twelve carbon atoms but only one alkene group in the carbon chain. Polyunsaturated fatty acids or PUFAs refer to fatty acids that have at least twelve carbon atoms and at least two alkene groups in the carbon chain. Long chain polyunsaturated fatty acids and LC-PUFAs refer to fatty acids that have at least twenty carbon atoms in the carbon chain and contain at least two alkene groups. Very long chain polyunsaturated fatty acids and VLC-PUFAs refer to fatty acids that have at least twenty-two carbon atoms and at least three alkene groups in the carbon chain. Link to LC-PUFA includes VLC-PUFA. Typically, the number of carbon atoms in the carbon chain of fatty acids refers to the straight carbon chain. If the carbon chain is branched, the atoms present in the side groups are excluded from the number of carbon atoms.

In one embodiment, LC-PUFA is a ω3 fatty acid or an omega-3 fatty acid: it belongs to the family of unsaturated fatty acids (alkene group, double bond, or C=C) having a carbon-carbon bond at the third carbon atom from the methyl end fatty acid. In another embodiment, LC-PUFA is a ω6 fatty acid: it belongs to the family of unsaturated fatty acids (alkene group) having a carbon-carbon bond at the sixth carbon atom from the methyl end of the fatty acid. The position of the alkene in the fatty acid chain is also denoted using Δ (or delta), with the alkene position numbered relative to the carboxy terminus of the fatty acid. For example, linoleic acid may also be referred to as cis-Δ9, cis-Δ12 octadecadienoic acid, or ^Δ9,2 octadecadienoic acid. Fatty acids can also be identified by referring to the lipid number C:D, where C is the number of carbon atoms and D is the number of double bonds in the carbon chain. For example, arachidonic acid can be annotated 20:4Δ ^{5, , ,} , which means a chain of twenty carbons with four alkene groups located at carbons 5, 8, 11, and 14 from the carboxy terminus of the fatty acid. This name also indicates that arachidonic acid is a ω6 fatty acid, because if there are twenty carbons and an alkene at C14 from the carboxyl end, the first alkene from the methyl end must be at C6.

In a further embodiment, the LC-PUFA is selected from the group consisting of arachidonic acid (ARA, 20:4Δ ^5.8,11.14 ; ω6), eicosatetraenoic acid (ETA, 20:4Δ ^8.11,14.17 ; ω3) , eicosapentaenoic acid (EPA, 20:5Δ ^5,8,11,14,17 ; ω3), docosapentaenoic acid (DPA, 22:5Δ ^{7,10,13,16,19} ; ω3) or docosahexaenoic acid (DHA, 22: 6Δ ^{4,7,10,13,16,19} ; ω3). LC-PUFA can also be dihomo-γlinoleic acid (DGLA) or eicosatrienoic acid (ETrA, 20:3Δ ^11,14,17 ; ω3). The LC-PUFA made in accordance with the present embodiments may be a mixture of any or all of the above and may include other LC-PUFAs or derivatives of any of these LC-PUFAs. In at least one embodiment, the ω3 fatty acids are at least one of DHA; DPA and DHA or EPA, DPA and DHA.

In addition, as noted above, LC-PUFA and VLC-PUFA may be free fatty acid (non-esterified), esterified, or in another bound form. Thus, the LC-PUFA of the present embodiments of the invention may be present as a mixture of forms in a cell lipid, an extracted lipid, or a purified oil. In at least one embodiment, the oil contains at least 75% or at least 85% triacylglycerols (TAGs), with the remainder present as other lipid forms such as those mentioned, wherein the TAGs contain at least one LC- PUFA. The oil can then be further refined or processed, such as by hydrolysis with a strong base to release free fatty acids, or by distillation or the like.

Thus, expressions for total fatty acids ω3, total fatty acids ω3, and the like are refer to the sum of all ω3 fatty acids, esterified and non-esterified, in an extracted lipid, oil, recombinant cell, plant part, or seed, as the context defines, usually expressed as a percentage of the total fatty acids. These ω3 fatty acids include ALA, SDA, ETrA, ETA, EPA, DPA or DHA and exclude any ω6 fatty acids or monounsaturated fatty acids. Expressions new fatty acids ω3, content of new fatty acids ω3, etc. refer to the sum of all ω3 fatty acids excluding ALA, esterified and non-esterified, in an extracted lipid, oil, recombinant cell, plant part, or seed, as determined by the context, expressed as a percentage of total fatty acids. These new ω3 fatty acids are fatty acids that are produced in the cells, plants, plant parts, and seeds of the present embodiments by expression of elite event transgene constructs and, when present, include SDA, ETrA, ETA, EPA, DPA, or DHA, but exclude ALA, any ω6 fatty acids or monounsaturated

- 13 041446 fatty acids. Exemplary total ω3 fatty acid content and new ω3 fatty acid content can be determined by converting the fatty acids in a sample to fatty acid methyl esters (FAME) and analyzing by gas chromatography (GC) by methods known in the art. See, for example, American Oilseed Chemists' Society (AOCS) method Celd-91.

Similarly, expressions for total fatty acids ω6, total fatty acids ω6, and the like. refer to the sum of all ω6 fatty acids, esterified and non-esterified, in an extracted lipid, oil, recombinant cell, plant part, or seed, as determined by context, expressed as a percentage of total fatty acids. General ω6 fatty acids, if present, may include LA, GLA, DGLA, ARA, EDA, or ω6-DPA and exclude any ω3 fatty acids or monounsaturated fatty acids. Expressions new fatty acids ω6, content of new fatty acids ω6, etc. refer to the sum of all ω6 fatty acids excluding LA, esterified and non-esterified, in an extracted lipid, oil, recombinant cell, plant part, or seed, as determined by the context, expressed as a percentage of total fatty acids. These new ω6 fatty acids are fatty acids that are produced in cells, plants, plant parts, or seeds as described herein through the expression of elite event transgenes and may include GLA, DGLA, ARA, EDA, or ω6-DPA, but exclude LA, any ω3 fatty acids or monounsaturated fatty acids.

Seed halves analysis is a procedure in which fatty acid analysis is performed on one of two cotyledons (seed halves) and the remaining seedling bearing the second cotyledon is used to form a plant if the results of the analysis are positive.

The protein content is the typical weight percent protein in oil-free flour or essentially oil-free flour, for example, with 95 or 99% oil removed from mature whole dried seeds, as determined by methods known in the art. See, for example, Daun et al., 2011; AOCS Official Meth. Ba 4e-93 Combustion Meth. Determination Crude Protein.

Mature seed produced by commercial growers for purposes other than cultivating or propagating a species is sometimes referred to as a grain.

The skilled artisan will appreciate, for example, that the term obtaining a plant part as a step in a method according to the present embodiments may include obtaining one or more plant parts, such as seeds, for use in the method. Obtaining a plant part includes harvesting a plant part from a plant, such as with a mechanical harvester, or purchasing a plant part, or obtaining a plant part from a supplier, or otherwise obtaining a plant part by purchasing a plant part from someone else who has the harvested part. plants. Thus, for example, obtaining NS-B5OO27-4 seed or NS-B50027-4 progeny may involve growing plants, collecting plant seeds, acquiring seeds, obtaining seeds, purchasing seeds, placing seeds in a container or storing seeds, or transporting seeds to another place.

The expression essentially all physiological and morphological characteristics of the parent refers to a plant that has essentially all the physiological and morphological characteristics of the recurrent parent, with the exception of characteristics derived from the transformed trait. Canola inbred line NS-B50027-4.

The NS-B50027-4 canola line is a stable and uniform breeding line as described herein. Line NS-B50027-4 was bred with emphasis on uniformity of plant type, and said line was propagated while constantly monitoring uniformity. The NS-B50027-4 line has been field tested with regulatory approval at ten sites in major canola growing regions in Australia and Canada. Agronomic performance evaluations were carried out in field studies at multiple sites to evaluate characteristics such as germination, seedling vigor, plant height, lodging and yield. All field trials were also conducted against opportunistic diseases or stress-causing insects, as well as normal phenotypic characteristics.

Line NS-B50027-4 is similar to the Australian AV Jade (parental isogenic line) in terms of leaves, flowers and pods, and produces plants with the same height and appearance when mature. Line NS-B50027-4 has acceptable resistance to blackleg (Leptosphaeria) disease and has a similar seed production potential under typical canola growing conditions in Australia and does not show increased seed cracking or plant lodging at maturity compared to AV Jade. No significant differences were observed between DHA canola and AV Jade in terms of plant pest characteristics, and nothing indicated a selective advantage that would result in an increased likelihood of canola NS-B50027-4 weed infestation.

In addition to the LC-PUFA trait, NS-B50027-4 differs from AV Jade in having a slightly longer time to flower and is similar to Australian in this regard.

- 14 041446 ATR Wahoo cultivar. NS-B50027-4 is notable in particular for the production of LC-PUFA in its seeds, in particular LC-ω3 fatty acids and more specifically DHA. The NS-B50027-4 canola line is not the parent of any other canola cultivar commercialized at the time of the patent filing for this NS-B50027-4 line.

The inbred transgenic canola line NS-B50027-4 has the following morphological and physiological characteristics (based primarily on data obtained and averaged from eight different locations in Australia in 2015).

Table 1

Descriptive information: NS-B50027-4 and AV Jade

NS-B50027-4 Comparison grade: AV Jade Kinds Brassica napus Brassica napus Sheet: Green average average Leaf: Petals present present Leaf: Number of petals average average Leaf: Serrated leaf edge average average Leaf: Length average average flowering time medium to late average Flower: Petal color yellow yellow Flower: Petal Width average average Flower: Pollen production present present Plant: Seedling Power medium to high average Plant: Height at full bloom average average Plant: lodging at maturity low low Black leg disease resistance present present Pod: Length medium to long medium to long Pod: Length to "spout" average average Pod: Pedicel length medium to long medium to long splitting seeds low low Seeds: Exit high high Seeds: % oil moderate moderate Seeds: % Erucic acid zero zero Seeds: % EPA C20:5pZ present absent Seeds: % DPA C22:5p3 present absent Seeds: % DHA C22:6p3 present absent

In another aspect of the present embodiments, a method is provided for obtaining a Brassica plant or canola derived from NS-B50027-4, or parts thereof, such as a seed, comprising obtaining the seed of NS-B50027-4 or the seed of the new Brassica napus line described above, and growing the seed into the plant under Brassica or canola growing conditions. In another embodiment, it is proposed to obtain a hybrid seed by obtaining NS-B50027-4 seed or Brassica napus seed as described above, growing the plant, cross-pollinating the plant, and obtaining a seed that matures as a result of cross-pollination. The seed may then be cultivated under Brassica or canola growing conditions to produce a Brassica or canola hybrid, or parts thereof, including the seed. Thus, in another aspect, a method is provided for growing the line Brassica napus NS-B50027-4 (representative seed of said line is deposited with ATCC® under accession number PTA-123186), sub-line NS-B50027-4, progeny NS-B50027-4 or sub-line, or a plant obtained by crossing NS-B50027-4 with a second Brassica plant, the method comprising: obtaining Brassica NS-B50027-4 seed or Brassica napus line seed as described above, and growing the seed under Brassica plant growing conditions. Additional aspects regarding progeny, hybrids, and introgressive hybridization to produce NS-N50027-4 are described herein.

- 15 041446

Elite event.

The phenotypic expression of transgenes in canola is determined by the structure of the transgene cassette itself and the localization of its insertion in the plant genome: the presence of transgenes in specific localizations in the plant genome can affect transgene expression and the overall plant phenotype. The insertion of a recombinant DNA molecule into the plant genome usually occurs as a result of cell or tissue transformation (or other genetic manipulations). The specific embedding site(s) may be random or pre-defined (if a targeted integration process is used). Agronomically or industrially successful introduction of a trait of commercial interest into a plant by genetic manipulation can be a lengthy procedure, depending on various factors. The actual transformation and regeneration of genetically transformed plants is only the first in a series of selection steps that involve extensive description of genetic characteristics, breeding and evaluation during greenhouse or field trials, which ultimately leads to the selection of an elite event.

The NS-B50027-4 line was selected as an elite development in canola development that produces LC-PUFA, in particular LC-w3 fatty acids and more specifically DHA. An elite event is an event selected from a group of events obtained by transformation of the same transforming DNA or by backcrossing with plants resulting from such transformation, based on the expression and stability of transgenic constructs, compatibility with the optimal agronomic characteristics of a plant containing such construction, and realization of the desired phenotypic trait. Thus, the selection criteria for an elite event are at least one, and preferably all of the following list:

(a) the presence of the transgenes does not compromise other desirable characteristics of the plant, such as characteristics relating to agronomic productivity or commercial value;

(b) the event is described by a well-defined molecular configuration that is stably inherited and for which suitable diagnostic tools can be developed to control identity;

(c) the genes of interest in the transgene cassette exhibit correct, adequate and stable spatial and temporal phenotypic expression at a commercially acceptable level under a range of environmental conditions under which normal agronomic exploitation of the plants carrying said event is possible. Transgenes can also be associated with positions in the plant genome that allow for introgression into a subsequent commercially desirable genetic environment.

The elite status of the event is confirmed by introgression of the elite event into various variants of the genetic environment relevant to the issue under consideration, and the observation of compliance with at least one of the above criteria, for example (a), (b) and (c). In addition, the selection of elite events can also be determined by compatibility, in particular, the progeny resulting from crossing between NS-B50027-4 and a plant carrying at least one other event such that said progeny carries both events. Thus, an elite event refers to a genetic locus containing a transgene(s) cassette(s) that(s) meet(s) the criteria described above. A plant, seed, plant material, or progeny may contain one or more elite events within its genome.

The expression of the transgenes confers on the plant one or more phenotypic traits (eg, LC-w3 fatty acid production) that would be conferred by insertion of transforming DNA (based on the structure and function of some or all of the genes of interest). In the present embodiments, several transgenes provide a biosynthetic pathway for the production of LC-w3 fatty acids in the transformed plant.

One aspect of the present embodiments relates to the surprising copy number of expressed transgenes in the genome of NS-B50027-4. Expressed means that the primary structure of the DNA molecule, i.e. the coding sequence of the transgene indicates that the gene encodes an active protein. However, expressed coding sequences may not be expressed because gene silencing occurs due to various mechanisms of inactivation of homologous transgenes in vivo. Inactivation of homologous transgenes has been described in plants where the transgene was introduced in sense orientation, with the surprising result that both gene and transgene activity was suppressed. See Napoli et al., 2 Plant Cell 279 (1990). Possible mechanisms for the inactivation of homologous genetic sequences include transcription inactivation via methylation, where the endogenous mechanisms of gene silencing are signaled by regions of duplicated DNA, and post-transcriptional silencing, in which the combined levels of mRNA from the endogenous gene and transgene trigger threshold-induced degradation of both transcripts. See van Bokland et al., 6 Plant J. 861 (1994). It is surprising, however, that although the NS-B50027-4

- 16 041446 has at least three copies of several transgenes, some of which are located in the same orientation, NS-B50027-4 shows synergistic expression of DHA.

The original transformants cultured from Brassica napus L. (var. AV Jade) showed wide variability in fatty acid production levels, especially in EPA and DHA levels. For the second and third generations, the selection was based mainly on the content of DHA and EPA in transgenic seeds. In some cases, especially in the T2 or T3 generations, segregation patterns (determined by growing twenty to forty individual seeds from one plant to twenty forty offspring and then measuring the DHA and EPA content of the individual seeds of these offspring) also showed results with a large scatter, indicating for the appearance of complex or multicopy inserts. Thus, many of the original generations of T2 or T3 plants have been discarded. It was initially concluded that multiple copies of the transgenic insert would produce unstable transformants as well as exhibit the classic gene silencing seen in homozygous genotypes as discussed above. Therefore, if PCR analysis of transformed plants showed a copy number >1, these transformants were often discarded.

Surprisingly, the NS-B5OO27-4 elite event was found to contain a stable (and functional) multicopy event: a sixteen-gene insert comprising two T-DNA-limited eight-gene cassettes (each insert of eight genes encoding seven enzymes, and marker) located in binary (inverted) form from the left border to the left border (similar to a massive palindrome); and a separate smaller cassette with four genes; and this combination of transgenic inserts acts synergistically for DHA production in the NS-B50027-4 inbred line. More specifically, the combination of crossing, backcrossing and self-crossing segregated a sixteen-gene insertion on the A05 chromosome (also referred to as N05) and a four-gene insertion on the A02 chromosome (also referred to as N02). The contribution of each transgenic chromosome was determined by breeding each segregant to obtain pure homozygous lines for each event. For example, in one experiment, a segregant containing an insert of sixteen genes produced about 4% DHA; and the segregant containing the four-gene insert did not produce DHA; but when the segregants were grown by combining the transgenic A02 chromosome locus and the transgenic A05 chromosome locus, the combination of the two transgene inserts provided a plant that produced at least about 7% DHA to at least about 14% DHA, inclusive, in its seed. This result was unexpected. As already noted, despite the unusual genetic composition of the NS-B50027-4 elite event, this line was found to be stable and reliable in terms of fatty acid production.

As noted herein, the biosynthetic pathway for LC-PUFA included seed-specific promoters to limit the expression and production of LC-PUFA in the developing seed. No expression of any of the seven transgenes was found in whole plants, roots, flowers, or other plant tissues (eg, flower bud, young pod) of NS-B50027-4, other than seeds. In the developing seed, the content of transgenic proteins ranged from the highest to the lowest (ng/mg total protein): Pavsa-Δ4D>Lackl-Δ12D>Picpa-ω3D>Micpu-Δ6D>Pavsa-Δ5D>Pyrco-Δ6E and PyrcoΔ5Ε were not detected. In mature seed, the transgenic protein content ranged from the highest to the lowest (ng/mg total protein): was not found.

In one aspect of the present embodiments, a method for increasing LC-PUFA in a plant is provided by providing (eg, by genetic transformation or breeding) to the plant multiple copies of genetic constructs expressing certain enzymes at the front end of the LC-PUFA biosynthetic pathway. See Napier et al., 330 Biochem. J. 611 (1998) (characterizes the structure as an N-terminal cytochrome b5 domain and a typical fatty acid desaturase domain having three highly conserved histidine boxes). For example, while not all Δ6-desaturase, Δ5-desaturase, Δ5-elongase, and ω3/Δ15-desaturase enzymes (counted as Nos. 2, 3, 5, and 6 in the biosynthetic pathway) can be considered solely as the front end of the desaturase, in specific embodiments, these the genes are assembled into an artificial locus that enhances the production of LC-PUFA, such as DHA. In specific embodiments, the artificial locus containing some front end genes includes Δ6-desaturase derived from Micromonas pusilla, Δ5-elongase derived from Pyramimonas cordata, Δ5-desaturase derived from Pavlova salina, and Δ15/ω3-desaturase derived from Pichia pastoris. Localization of this artificial locus on chromosome A02 NS-B50027-4 provides a means of segregating this locus into a plant line (segregant) and allowing it to introgress into another plant using conventional plant breeding techniques.

Oil and fatty acid ω3.

With the canola plant line NS-B50027-4 according to the present embodiments, ω3 and LC-ω3 fatty acids can be produced commercially from canola seeds NS-B50027-4. Thus, methods for selecting and propagating transformed plants provide a variety of plants with advantageous NS-B50027-4 traits that are harvested

- 17 041446 in the usual way and the fatty acid is extracted from the tissue of interest, for example seeds.

In a further embodiment, the extracted plant lipid may be treated to increase DHA levels as a percentage of total fatty acids. For example, the treatment is hydrolysis of esterified fatty acids to free fatty acids, or transesterification to modify TAG components. For example, oil from the seed of NS-B5OO27-4 or its progeny can be enriched for DHA content or processed to convert the fatty acids in the oil into alkyl esters such as methyl or ethyl esters, which can then be purified or fractionated for enrichment. lipid or DHA oil. In some embodiments, the lipid fatty acid composition after such treatment contains at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least least 95% DHA inclusive.

The present embodiments also include progeny and descendants of these new B. napus lines from the NS-B50027-4 line. Progeny or offspring can be obtained using breeding or tissue culture methods known to those skilled in the art. For example, the progeny or progeny may contain the canola fatty acid profile developed in these lines. Thus, offspring or offspring can have any number of genes from developed lines. Descendants or progeny may include only those genes that provide the canola fatty acid phenotype provided herein, or additional genes. This can be determined using molecular analysis known to those skilled in the art.

In one aspect, a method is provided for obtaining seed from a Brassica plant, such as B. napus or B. juncea, having an NS-B50027-4 phenotype, such as a seed DHA content of at least 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12% DHA, about 13% DHA, about 14% DHA, about 15%, about 16% DHA, about 17% DHA, about 18% DHA, about 19% DHA, about 20% DHA, about 21% DHA, about 22% DHA, about 23% DHA, about 24% DHA, about 25% DHA inclusive, or more DHA (in wt% of total fatty acids); or, for example, an LC-PUFA fatty acid content of at least 5% LC-PUFA, about 6% LC-PUFA, about 7% LC-PUFA, about 8% LC-PUFA, about 9% LC-PUFA, about 10% LC-PUFA, about 11% LC-PUFA, about 12% LC-PUFA, about 13%, about 14% LC-PUFA, about 15% LC-PUFA, about 16% LC-PUFA, about 17% LC- PUFA about 18% LC-PUFA, about 19% LC-PUFA, about 20% LC-PUFA, about 21% LC-PUFA, about 22% LC-PUFA, about 23% LC-PUFA, about 24% LC-PUFA, about 25% LC-PUFA inclusive or more LC-PUFA (sum of EPA, DPA and DHA in wt% of total fatty acids).

In another aspect, a homogeneous population of crushed Brassica napus seeds derived from the plants described herein (i.e., NS-B5OO27-4 seed or progeny seed containing at least one NS-B50027-4 locus) is provided, wherein the crushed B. napus seeds contain at least about 30%, about 35%, or from about 36% to about 40%, inclusive, by weight of the total fatty acid content (wt.% seed). In specific embodiments, for example, the fatty acid content of NS-B50027-4 seed or seed derived from progeny containing at least one locus from NS-B5OO27-4 is at least 5% DHA, about 6% DHA, about 7% DHA about 8% DHA, about 9% DHA, about 10% DHA, about 11% DHA, about 12% DHA, about 13% DHA, about 14% DHA, about 15%, about 16% DHA, about 17 % DHA, about 18% DHA, about 19% DHA, about 20% DHA, about 21% DHA, about 22% DHA, about 23% DHA, about 24% DHA, about 25% DHA inclusive (as wt.% from total fatty acids) or more DHA. In specific embodiments, for example, the fatty acid content of NS-B50027-4 seed or progeny seed containing at least one locus of NS-B5OO27-4 is at least 5% LC-PUFA, about 6% LC-PUFA , about 7% LC-PUFA, about 8% LC-PUFA, about 9% LC-PUFA, about 10% LC-PUFA, about 11% LC-PUFA, about 12% LC-PUFA, about 13%, about 14% LC-PUFA, about 15% LC-PUFA, about 16% LC-PUFA, about 17% LC-PUFA, about 18% LC-PUFA, about 19% LC-PUFA, about 20% LC-PUFA, about 21% LC -PUFA about 22% LC-PUFA, about 23% LC-PUFA, about 24% LC-PUFA, about 25% LC-PUFA, about 26% LC-PUFA, about 27% LC-PUFA, about 28% LC-PUFA , about 29% LC-PUFA, about 30% LC-PUFA, about 31% LC-PUFA, about 32% LC-PUFA, about 33% or more LC-PUFA (LC-PUFA is the sum of EPA, DPA and DHA in wt.% of the total fatty acids). In other embodiments, for example, the fatty acid content of NS-B50027-4 seed is at least about 18% ALA, about 19% ALA, about 20% ALA, about 21% ALA, about 22% ALA, about 23% ALA about 24% ALA, about 25% ALA, about 26% ALA inclusive or more ALA in wt.% of the total fatty acids of the seed.

Also provided is a homogeneous population of crushed Brassica napus NS-B50027-4 seed disclosed herein, or a homogeneous population of crushed B. napus seed from a progeny or progeny of NS-B50027-4, wherein the DHA content of the crushed B. napus seeds is at least at least 5% DHA, about 6% DHA, about 7% DHA, about 8% DHA, about 9% DHA, about 10% DHA, about 11% DHA, about 12% DHA, about 13% DHA, about 14% DHA, about 15%, about

- 18 041446

16% DHA, about 17% DHA, about 18% DHA, about 19% DHA, about 20% DHA, about 21% DHA, about 22% DHA, about 23% DHA, about 24% DHA, about 25% DHA, inclusive , or more DHA (in wt.% of the total fatty acids). Butter, flour and protein are also offered from this crushed seed.

In a further embodiment, an extracted plant lipid (eg, oil) may be treated to increase DHA levels as a percentage of total fatty acids. For example, the treatment includes hydrolysis of esterified fatty acids to free fatty acids or interesterification. For example, canola oil can be processed to convert the fatty acids in the oil to alkyl esters such as methyl or ethyl esters, which can then be fractionated to enrich the lipid or DHA oil. In embodiments, the lipid fatty acid composition after such enrichment contains at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% DHA, or at least least 95% DHA inclusive.

In another embodiment, a method is provided for producing oil or flour from a Brassica napus plant of line NS-B50027-4, representative seeds of which are deposited with ATCC® under accession number PTA-123186, subline NS-B50027-4, progeny NS-B50027-4 or subline , or a plant obtained by crossing NS-B50027-4 with a second Brassica plant, comprising growing the Brassica napus plant described above under Brassica plant growing conditions; seed collection; and extracting the oil, flour or protein from the seeds.

In another embodiment, provided herein is a method for producing oil from a Brassica napus plant of line NS-B50027-4, representative seeds of which are deposited with ATCC® under accession number PTA-123186, subline NS-B50027-4, progeny NS-B50027-4 or subline, or a plant obtained by crossing NS-B50027-4 with a second Brassica plant, including grinding the seeds of the plant Brassica napus line NS-B50027-4, while representative seeds of this line are deposited in ATCC® under accession number PTA-123186, subline NS -850027-4, progeny of NS-B50027-4 or a subline or plant obtained by crossing NS-B50027-4 with a second Brassica plant; and extracting oil from said seeds.

In another aspect, flour and protein and oil are provided from the seed of NS-B50027-4 or from the seed of a progeny derived from NS-B50027-4. Extraction of protein from plant biomass can be carried out by known methods. See, for example, Heney & Orr, 114 Anal. Biochem. 92 (1981). Seed flour NS-B50027-4 may be particularly beneficial as it contains at least some DHA and other ω3 fatty acids. Similarly, the protein fraction obtained from the NS-B5OO27-4 seed contains at least some beneficial DHA and other ω3 fatty acids. Thus, in another embodiment, a seed meal obtained from the seed of NS-B50027-4 or from the seed of a progeny derived from NS-B50027-4 is provided. In one embodiment, the seed meal contains elite event NS-B50027-4. Preferably, the seed meal retains some of the lipid or oil produced in the seed from which the seed meal is derived, albeit at a lower level, after most of the lipid or oil has been extracted from the seed. The seed meal can be used as a food product, such as a component of animal feed or an ingredient in food production. Given the higher ratio of ω3:ω6 fatty acids in this seed meal, NS-850027-4 seed meal can provide superior nutrition compared to other commercially available seed meal.

As noted above, the expression fatty acid content or fatty acid composition usually refers to the weight percent of various fatty acids present in the endogenously formed oil of mature, whole, partially dried seeds (typically containing about 6 or 7% moisture), calculated as the percentage of a particular fatty acid. in the form of a normalized area; or relative to a known standard; or as a weight ratio of fatty acids per gram of seed (eg, mg DHA/g seed).

In common industry practice, the composition of fatty acids is presented as a percentage by area (normalized by area), and not in absolute amounts. For example, chromatography often generates peak data and the area under each peak is integrated and expressed as a percentage of the total area under all fatty acid peaks in the chromatogram. Percentage of area is easy to calculate and compare with results reported by others in the industry who also report percentage of area. Percentage of area is not absolute, but provides a reasonable approximation. Absolute yield in terms of results presented in mg/kg can be calculated, for example, by including reference standards of known concentration and an internal standard. Correction factors can also be used to calculate the mass fractions of fatty acids.

For example, when determining fatty acid content, seeds can be crushed, extracted with oil triacylglycerides (TAG), followed by saponification and methylation with methanol and sodium methoxide, or by reaction with 1.25% 3-(trifluoromethyl)phenyl-trimethyl-ammonium hydroxide in

- 19 041446 methanol (Meth Prep II™, Fischer Scientific Cat #AT18007) to form fatty acid methyl esters. The resulting fatty acid methyl esters (FAME) can be analyzed by gas liquid chromatography (GLC) using a capillary column that separates FAME based on degree of unsaturation and fatty acid chain length. FAME can also be analyzed using, for example, GC, LC-MS, GC-MS, NMR or reflectance spectroscopy. The fatty acid composition can also be determined from whole seeds, for example by breaking the seed coats and subjecting the broken seeds to direct methylation. The total lipid can be separated by methods known in the art to purify fractions such as the TAG fraction. For example, thin layer chromatography (TLC) can be performed on an analytical scale to separate TAG from other lipid fractions such as DAG, acyl-CoA or phospholipid to determine the composition of fatty acids, in particular TAG. You can use a number of other analytical methods known to experts in this field. See, for example, Tinoco et al., 3 Anal. Biochem. 514 (1962); Canola: Chemistry, Production, Processing & Utilization (Daun et al., eds., AOCS Press, Urbana, IL, 2011) (Daun et al., 2011); US 2015/0166928; US 20160002566.

The lipid or oil of the NS-B50027-4 seed can also be purified or enriched to increase the proportion of TAG, for example by removing free fatty acids or phospholipids. In at least one embodiment, the NS-850027-4 seed lipid is in the form of an oil, wherein at least 75%, at least 80%, at least 85%, at least 90%, at least 95% , at least 98% inclusive or in the range from 95 to 98% inclusive by weight of the lipid is in the form of TAG.

In another embodiment, the lipid from the seed of NS-B50027-4 (or progeny thereof) is treated to increase the amount of DHA as a percentage of total fatty acids. For example, the processing may include interesterification. For example, the lipid may be processed to convert the fatty acids contained in the oil into alkyl esters such as methyl or ethyl esters, which may then be fractionated to enrich the lipid or oil with DHA. In embodiments, the lipid fatty acid composition after such enrichment contains at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% DHA. Similarly, since the lipid from NS-B50027-4 seed contains a higher proportion of ALA compared to typical canola, the lipid from NS-B50027-4 seed can be processed to enrich the lipid in terms of ALA content. In addition, the fatty acid may be in a mixture of fatty acids having a fatty acid composition as described herein, or may be enriched such that the fatty acid content is at least 40% or at least 90% of the fatty acid content in mixtures. In one embodiment, the fatty acid is non-esterified. Alternatively, the fatty acid is esterified to, for example, a methyl, ethyl, propyl or butyl group.

Despite having a lower oleic acid content compared to oils that are claimed to confer stability to DHA and other LC-PUFAs due to their high oleic acid content, ω3 LC-PUFA oil from NS-B50027-4 seed exhibits amazing stability. More specifically, ω3 LC-PUFA fatty acids are generally known to be unstable and particularly prone to oxidation. It is understood in the art that encapsulation, blending with other oils, particularly high oleic acid oils, or the addition of antioxidants is required to extend the shelf life of LC-PUFA and food products containing LC-PUFA. However, despite the absence of such treatments, evidence suggests that the oil extracted from the crushed seed of NS-B50027-4 retains freshness for several months at room temperature. In addition, the levels of nutrients such as phytosterols, vitamin E, vitamin K1, β-carotene, and minerals in NS-B50027-4 oil are in the natural range of other commercial canola oils. Undesirable substances such as pesticides, mycotoxins or polyaromatic hydrocarbons found in the oil obtained from NS-B50027-4 were absent at detectable levels. In addition, no plant DNA was found in the oil obtained from NS-B50027-4.

In another aspect of the present embodiments, a source of DHA and LC-PUFA is provided for use in nutritional supplements and foods for humans and non-human animals. In particular, NS-B50027-4 seed oil provides a sustainable source of DHA and LC-PUFA for aquaculture applications. Due to the high global demand for fish and the resulting overfishing in the seas, marine and freshwater aquaculture is becoming increasingly important. See, for example, Betancor et al., 4 Sci. Rep. 8104 (2014). For example, the farming and consumption of salmon fish has increased dramatically over the past 20 years. However, the diet of wild fish is very different from that of other species in aquaculture. In fact, aquaculture is still heavily dependent on marine fisheries for essential food nutrients such as fishmeal and fish oil. Indeed, fish oil is the main source of ω3-LC PUFA in aquaculture. Since marine fish oil is a limiting factor for the fast growing fish farming industry

- 20 041446 (5 to 10% per year), the aquaculture diet contains a wide range of alternative vegetable ingredients such as legume seeds, bagasse, leaf bagasse and an increasing proportion of vegetable oil in addition to animal oil. Replacing fish oils with vegetable oils that traditionally contain low levels of LC-PUFA means that less LC-PUFA will be available in fish diets, even if some oils, such as flaxseed oil, contain some ALA that can be converted, albeit only in limited amounts, into LC metabolites in fish. In general, current vegetable oils in fish feeds can have a detrimental effect on FA distribution in fish and they can change the ω3/ω6 ratio and reduce the total LC-PUFA content in fish meat.

For example, typical vegetable oils contain high amounts of ω6 PUFA, mainly in the form of linoleic acid (C18:2 ω6; LA). The oil from the parent line AV Jade has a DHA:LA ratio of 0.016; the oil from NS-B50027-4 has a DHA:LA ratio of 1.048; compared to farmed salmon oil having a DHA:LA ratio of 0.908. Strobel et al., 11 Lipids Health Dis., 144 (2012). Interestingly, the ω3 ratios of FA from NS-B50027-4 are particularly advantageous for palmitic acid, a saturated fatty acid associated with cardiovascular disease and dyslipidemia. Diet, Nutrition & Prevention of Chronic Dis., WHO Tech. Rep. Series 916, Report of a Joint WHO/FAO Expert Consultation, 88 (World Health Organization, Geneva, 2003). The oil from the parent line AV Jade does not contain DHA; for example, the oil from NS-B50027-4 has a DHA:palmitate ratio of about 2.12; compared to farmed salmon oil having a DHA:palmitate ratio of about 0.59; and wild salmon oil has a DHA:palmitate ratio of about 1.028. Strobel et al., 2012. Food preparation for use in aquaculture, including LC-PUFA, is also known in the art. See Betancor et al., 2014; Petrie et al., 9 PLOS ONE 1, 2014; Tocher, 449 Aquaculture, 94 (2015).

Thus, the scope of the present embodiments covers the use of oil from NS-B50027-4 as a source of ω3 fatty acids for aquaculture feed and an aquaculture feed containing oil derived from NS-B50027-4 and its progeny. Studies conducted using oil derived from NS-B50027-4 have shown that this oil is safe to include in freshwater salmon feed (2 to 25 g fish fry) and that EPA and DHA from feed containing oil from NS-B50027-4.

In another aspect, the present embodiments provide compositions comprising one or more of lipid, oil, fatty acid, seed, seed meal, protein, cell, oil plant, or plant part of NS-B50027-4. In some embodiments, the composition further comprises a carrier suitable for pharmaceutical, food, or agricultural use, a seed treatment compound, a fertilizer, another food or feed ingredient, or an added protein or vitamins. In FIG. 4 shows a diagram of the possible processing steps, products or fractions obtained at different stages of the process, and the proposed use of such fractions or products.

Also provided are food, cosmetics or chemicals containing one or more of lipid or oil derived from NS-B50027-4, fatty acid derived from NS-B50027-4, cell derived from NS-B50027-4, oily plants, seed meal or other composition derived from or derived from NS-850027-4. In another aspect, a method is provided for preparing a food product, the method comprising mixing one or more of NS-B50027-4 lipid or oil or progeny thereof, NS-B50027-4 fatty acid or progeny thereof, cell, plant part, seed, flour from seeds, oil plant NS-B50027-4 or its progeny, or another composition that is any of the compositions derived from or derived from NS-B50027-4 or its progeny, with at least one other food ingredient. The method may include the steps of mixing, cooking, baking, extruding, emulsifying, or otherwise formulating the food product, or packaging the food product, or analyzing the amount of lipid or oil in the food product.

The food product referred to herein contains an oil, lipid, fatty acid ester or fatty acid obtained directly or indirectly using the methods, cells, plants or seeds disclosed herein, i.e. oil from the seed of NS-B50027-4 or from a seed obtained from a progeny derived from NS-B50027-4 containing at least one locus from NS-B50027-4. Foodstuffs include foodstuffs or preparations for human or animal consumption which, when ingested, nourish or form tissues or provide energy; or maintain, restore or maintain an adequate nutritional status for metabolic function. Food products include nutritional compositions for infants or young children, such as, for example, milk formula and seed meal. Food products also include nutritional supplements such as dietary supplements. In addition, food products may include dietary macronutrients, protein, carbohydrates, vitamins or minerals in amounts suitable for a particular application. The amounts of these ingredients will vary depending on whether the composition is intended for use in normal individuals or

- 21 041446 for use in individuals with special needs, such as individuals suffering from metabolic disorders and the like. The food product may be presented in solid, liquid, jelly or emulsion form. The food product may be added to any type of food for any suitable purpose, ie. it need not be added only for special dietary uses.

The food product referred to herein also contains milled canola, canola seed meal, or derived protein (i.e., meal, bagasse, or meal protein) from NS-B50027-4 seed or progeny derived from NS-B5OO27 -4 containing at least one locus from NS-B50027-4. Currently, the use of canola meal in specific high quality commercial feeds is often limited due to a number of issues including anti-nutritional factors, high fiber content, limited digestibility, and lack of h3-FA or LC-PUFA, or a low density protein source. Processing the canola seed cake fraction into a canola protein concentrate can eliminate these problems such that canola protein derived from the seed of NS-B50027-4 or its progeny provides a suitable substitute for fishmeal, canola meal, soybean meal or soy protein concentrate, as well as a number of other widely used feed ingredients. For example, shrimp require ten essential amino acids. Canola has the right balance of amino acids for shrimp. Four fatty acids are important for shrimp: LA, ALA and, more importantly, EPA and DHA. The ω3:ω6 fatty acid ratio is also important in shrimp feed, with the optimum ω3:ω6 ratio being around 2.5. The total inclusion of lipids in the feed is considered optimal, ranging from 4.5 to 7.5%. It would be beneficial to include some oil in the flour-derived protein from the canola lines described herein. In addition, the apparent digestibility of shrimp crude protein is high at over 80%; and earlier studies have shown that protein from plants is less digestible than protein derived from fishmeal. By fractionating the protein and concentrating the protein derived from the canola meal described herein, digestibility is expected to closely match fishmeal and may be superior to currently available canola meal. Such protein products may also provide important alternatives to fishmeal in aquaculture and terrestrial feed.

In another aspect, NS-B50027-4 of the present invention provides a method for treating or preventing a condition that would benefit from a PUFA such as a ω3 fatty acid or DHA, the method comprising administering to a subject one or more of the following: lipid or oil obtained from NS-B50027-4; a fatty acid derived from NS-B500274; a cell derived from NS-B50027-4; oil plant, seed meal or other composition derived from or derived from NS-B50027-4. In another aspect, a method for making a medicament is provided, the method comprising mixing one or more of the following: NS-B50027-4 lipid or oil; fatty acid NS-B50027-4; NS-B50027-4 cells; oil plant NS-B50027-4, plant part NS-B50027-4, seed NS-B50027-4, seed meal NS-B50027-4; or another composition containing any of these compositions derived from or derived from NS-B500274 or its progeny; wherein the drug may be administered in the form of a pharmaceutical composition containing the PUFA ethyl ester. The subject may be a human or non-human animal.

Examples of conditions that can be beneficially affected by ω3 fatty acid include elevated serum triglyceride levels, elevated serum cholesterol levels such as elevated LDL cholesterol, cardiac arrhythmias, high blood pressure, coronary artery disease, restenosis after angioplasty, inflammation, asthma , rheumatoid arthritis, ulcerative colitis, Crohn's disease, psoriasis, platelet aggregation in eczema, gastrointestinal bleeding, endometriosis, premenstrual syndrome, kidney stones, fetal alcohol syndrome, attention deficit hyperactivity disorder, unipolar depression, bipolar depression, aggressive hostility, schizophrenia, adrenoleukodystophia, hypertension, diabetes, obesity, osteoporosis, Alzheimer's disease, chronic obstructive pulmonary disease, multiple sclerosis, cystic fibrosis, phenylketonuria, myalgic encephalomyelitis, acquired immune deficiency, chronic fatigue after a viral infection, cancer or eye disease.

The manufacture of a medicament may include mixing the oil obtained from NS-B50027-4 with a pharmaceutically acceptable carrier for the treatment of the condition exemplified herein. The method may include oil refining or interesterification, or oil fractionation to increase the level of DHA. In a specific embodiment, the method includes treating a lipid or oil to convert the fatty acids contained in the oil into alkyl esters such as methyl or ethyl esters. Further processing such as fractionation or distillation can be used to enrich the lipid or oil with DHA. In an exemplary embodiment, the drug contains DHA ethyl esters. The level of DHA ethyl esters in a drug may be in

- 22 041446 range from about 30% to about 50%, inclusive, or be at least 80%, at least 85%, at least 90%, or at least 95%, inclusive of the drug, or the fatty acid component of the drug . The drug may further contain EPA or DPA ethyl esters, for example in an amount of 30 to 50% inclusive, or at least 90% of the total fatty acid content of the drug. The drug may further contain ethyl esters of ALA, EPA or DPA, wherein the ω3 fatty acid content is in the range of from about 30% to about 50%, inclusive, or is at least 90% of the total fatty acid content in medicine. Such drugs are suitable for administration to humans or animals as determined by the healthcare professional. The formulation of pharmaceutical compositions containing fatty acids is known in the art. In addition, as is known in the art, it is desirable to obtain at least about 300 mg per day of ω3 fatty acid, especially ω3 LC-PUFA, such as EPA, DPA or DHA; but doses of 0.1 mg to 20 g per day or more of a specific fatty acid may be appropriate as directed by a healthcare professional.

In addition, the oil obtained from NS-B50027-4 can be used for cosmetic purposes: it can be added to already existing cosmetic compositions to form a mixture, or the fatty acid obtained as described herein can be used as an active ingredient in cosmetic composition. See, for example, US 2008/0241082.

In addition, techniques commonly practiced in the art can be used to extract, process, and analyze oils derived primarily from the seeds of NS-B50027-4. The oil can be obtained by cold pressing, water treatment, or more typical processing. See, for example, Mansour et al., 6 Nutrients 776 (2014); US Pat. No. 6,599,513. For example, NS-B50027-4 seed may be cold pressed (eg, with a screw press) and the oil filtered; and the remaining cake meal can be subjected to extraction with hexane to obtain additional amount of oil from the crushed/pressed canola seed.

In the production of processed foods, canola seed is boiled, pressed and extracted to produce crude oil, which is then degummed, refined, bleached and deodorized. The various steps may be carried out under a nitrogen atmosphere. In general, seed grinding methods are known in the art. For example, oilseeds can be softened (if necessary) by spraying with water or soaking in water to increase the moisture content of the seeds, for example, to 7-8.5%. The softened seed is rolled, for example, using a smooth roll with a gap setting of 0.23 to 0.27 mm. Heat can be used to deactivate enzymes, destroy cells, drain oil droplets, and form agglomerates of protein particles. Most of the seed oil is extracted by pressing while passing through a screw press, resulting in a processed seed meal (cake) and pressed crude oil. The crude oil resulting from the pressing operation can be clarified by passing through a sump with a slotted drain cover to remove solids that are released with the oil during the pressing operation. This clarified oil can be pumped through a plate and frame filter to remove any remaining fine solids. The cake leaving the screw press often contains a significant amount of oil, which can be extracted with a solvent, such as hexane, using, for example, a heat-controlled column to obtain additional oil from the seed meal. After removing the solvent from the crude oil obtained from the husk, the pressed and extracted oils can be combined and subjected to additional oil processing procedures. For example, the extracted lipid or oil may be subjected to one or more processing steps to increase the purity of the lipid/oil component. For example, the purification steps may include treating the extracted crude oil in at least one of the following: degumming, deodorization, decolorization, drying, fractionation, or enrichment with antioxidants (eg, mixed tocopherols).

Degumming is the first step in oil refining and is primarily intended to remove most of the phospholipids from the oil, which may be present at about 1-2% of the total extracted lipid. The addition of water, typically containing 2% phosphoric acid, to crude oil at 70-80° C. results in the separation of most of the phospholipids, trace metals, pigments and insoluble lecithin. Degumming can also be done by adding concentrated phosphoric acid to the crude seed oil to convert the non-hydratable phosphatides to a hydrated form and to chelate the minor amounts of metals present. The phospholipid emulsion is separated from the seed oil by centrifugation.

Alkaline refining, sometimes also referred to as neutralization, usually follows degumming and precedes deodorization and bleaching. More specifically, after degumming, the seed oil is titrated with sufficient alkaline solution to neutralize the free fatty acid and phosphoric acids and separate the triglyceride fraction. Suitable alkali compounds

- 23 041446 include sodium hydroxide, potassium hydroxide, sodium carbonate, lithium hydroxide, calcium hydroxide, calcium carbonate and ammonium hydroxide. Soap is removed by centrifugation or extraction into soap solvent and the neutralized oil is washed with water. If necessary, any excess alkali in the oil can be neutralized with a suitable acid such as hydrochloric acid or sulfuric acid.

Some vegetable oils, such as soybean oil, are processed through a combination of degumming and neutralization. To condition the non-hydratable phosphatides, a small amount of phosphoric or citric acid is added to the crude, non-degummed oil. After vigorous stirring, dilute caustic soda (7 and 12%) is added to neutralize the free fatty acids. An adequate amount of water will hydrate the phosphatides in the presence of caustic soda. After the reaction time in the holding mixer, the oil is heated and sent directly to the separator to separate the soap stock. The neutral oil is washed in about 3-10% water to reduce residual soap content and the mixture is separated into wash water and oil. In the vacuum dryer, the residual moisture in the oil is reduced.

Deodorization is the treatment of oils and fats at high temperature (200~260°C) and low pressure (0.1-1mmHg), generally achieved by introducing steam into seed oil at a rate of about 0.1 ml/min/100 ml seed oil. After about 30 minutes of this sparging, the seed oil is cooled under vacuum. The seed oil is usually transferred to a glass container and washed with argon before being stored in the refrigerator. This treatment also improves the color of the seed oil and removes most of the volatile or odorous compounds, including any remaining free fatty acids, monoacylglycerols, or oxidation products.

Bleaching is a refining process in which oils are heated at 90-120°C for 10-30 minutes in the presence of (e.g. 0.2-2.0%) bleaching earth (e.g. Trisyl® Silicas, W.R. Grace & Co., Columbia, Md., US) and in the absence of oxygen (under nitrogen or under vacuum). This stage removes unwanted pigments (carotenoids, chlorophyll, gossypol, etc.), as well as oxidation products, trace metals, sulfur compounds and traces of soap. RDB oil refers to oil that has been refined, deodorized and bleached.

Winterization is a process sometimes used in the industrial oil industry to separate oils and fats into solid (stearic) and liquid (oleic) fractions by crystallization at temperatures below ambient temperature. Some vegetable oils, such as sunflower or corn oil, contain waxes (esters of long-chain fatty alcohols and fatty acid esters) that crystallize at low temperatures and cause the oil to become cloudy. Wet winterization in combination with neutralization is suitable for the removal of these waxes. It was originally applied to cottonseed oil to produce a solids-free product and can also be used to reduce saturated fatty acids in oils.

Interesterification is the process of exchanging fatty acids within and between triglyceride (TAG) molecules, or transferring a fatty acid to another alcohol to form an ester. Initially, this may involve the release of fatty acids from TAG as free fatty acids, or direct production of fatty acid esters such as fatty acid methyl or ethyl esters. In the transesterification reaction of TAG with an alcohol such as methanol or ethanol, the alkyl group of the alcohol forms an ester bond with the acyl groups (including DHA) of TAG. In combination with a fractionation process, interesterification can be used to modify the fatty acid composition of lipids. Marangom et al., 6 Trends Food Sci. Technol. 329 (1995). For transesterification, chemical means (e.g. catalyzed by a strong acid or base) or enzymatic means can be used, the latter using lipases which can be positionally specific (sn-1/3- or sn-2 position specific) for the TAG fatty acid or have a preference for some fatty acids over others. Adamczak, 13 Poll. J. Food Nutr. Sc., 3 (2004); Ferreira-Dias Elec., 16 J. Biotechnol. (2013).

Alternatively, the purification steps may avoid transesterification processes or other processes that alter the fatty acid composition of the lipid or oil so as to increase the DHA content as a percentage of total fatty acids. In other words, the fatty acid composition/profile of the purified lipid or oil obtained from the seed of NS-B50027-4 or its progeny may be substantially the same as that of the crude lipid or oil.

Optionally, fractionation of fatty acids to increase the concentration of LC-PUFA in the oil can be achieved by any of the methods known in the art, such as, for example, freeze crystallization, urea complexation, molecular distillation, supercritical fluid extraction, countercurrent chromatography, and ion complexation. silver. Complexation with urea is the preferred method because of its simplicity and effectiveness in reducing the level of saturated and monounsaturated fatty acids in the oil. Gamez et al., 36 Food Res. Int'l 721 (2003). TAG oils are first broken down into their constituent fatty acids, often in the form of fatty acid esters, by the action of lipases or

- 24 041446 by an acid- or base-catalyzed hydrolysis reaction in which one mole of TAG reacts with at least 3 moles of an alcohol (for example, ethanol for ethyl esters or methanol for methyl esters), while excess alcohol allows separation of the resulting alkyl esters and the resulting glycerol. The free fatty acids or fatty acid esters usually do not change in the fatty acid composition upon processing and can then be mixed with the ethanolic urea solution to form the complex. Saturated and monounsaturated fatty acids readily complex with urea and crystallize on cooling and can subsequently be removed by filtration. Thus, the finished urea-free fraction is enriched in LC-PUFA.

As noted, various steps or multiple steps can be practiced under nitrogen, other inert atmosphere or vacuum, and the desired oil or lipid obtained from the seed of NS-B50027-4 or its progeny can be packaged under nitrogen, an inert gas. or vacuum to avoid oxidation.

Also, as noted herein, ground canola, seed meal, or derived protein (i.e. meal, bagasse, or meal protein) from seed derived from NS-B50027-4 or its progeny may be used for products food or feed. High DHA pressed cake can be used as a high oil cake, or it can be hexane defatted to make a flour. Hexane-treated flour is usually desolventized and roasted (DT-flour). Identification of NS-B50027-4 and its progeny

An elite genetic event can be characterized by the location(s) and configuration at the site(s) of insertion of the recombinant DNA molecule(s) into the plant genome. The site of the plant genome where the recombinant DNA cassette is inserted is also referred to as the insertion site or the target site. A flanking region or flanking sequence refers to a stretch of DNA, e.g., at least 20 base pairs, at least 50 base pairs, or up to 5000 base pairs, of the plant genome that is located at or directly upstream of the transcription (5') and adjacent to the transgenic cassette. , or directly downstream (3') and adjacent to the transgenic cassette. The result of transformation procedures leading to the insertion of foreign DNA in a random manner is the formation of transformants containing various flanking regions characteristic and unique for each transformant (elite event).

In another aspect, a method is provided for producing a Brassica napus plant or parts derived from NS-B50027-4, comprising crossing the above described Brassica napus plant or parts thereof with a second plant to produce first generation progeny seed; growing said first generation progeny seed to produce an F2 generation plant; optionally repeating the crossing and growing steps to obtain subsequent child generations of said seed to obtain a Brassica napus seed, plant or parts thereof derived from the NS-B50027-4 cross line. A plant or plant parts (including any hybrid) produced by this process is also provided. In one embodiment, a genetic trait that has been inserted into the genome of a particular canola plant can be transferred to the genome of another cultivar using conventional breeding techniques well known in the plant breeding art. For example, a backcross approach can be used to transfer a transgene from a transformed canola cultivar into an already developed canola cultivar and the resulting backcross plant will then contain the transgene(s).

Accordingly, in another aspect of the present embodiments, compositions, methods, and kits for detecting NS-B50027-4 are provided. It would be useful to be able to detect the presence of a particular event in order to determine if the progeny of a sexual cross contains a transgene of interest. In addition, a specific event detection method would be useful for complying with regulations requiring, for example, premarket approval and labeling of food products derived from recombinant crops. The presence of a transgene can be detected by any known nucleic acid detection method such as polymerase chain reaction (PCR) or DNA hybridization using nucleic acids as probes. These detection methods are usually based on frequently used genetic elements such as promoters, terminators, marker genes, and so on. As a result, such methods may not be suitable for distinguishing between different events, in particular events generated using similar DNA constructs, unless the chromosomal DNA sequence adjacent to the inserted DNA (flanking DNA) is known. Event-specific PCR analysis is described. See, for example, Windels et al., Med. fac. Landbouww, Univ. Gent 64/5b:459 (1999) (where a soybean glyphosate resistance event was identified by PCR using a primer set spanning the junction: a first primer including the sequence from the insert and a second primer including the sequence from the flanking DNA) . In addition, the insertion of sixteen NS-B50027-4 genes disrupted the expression of the Brassica gene encoding a protein interacting with Pto (Pto-interacting protein, PTI), a serine-threonine kinase involved in signal transduction mediated by a hypersensitivity response,

- 25 041446 located on chromosome A05. Although no phenotypic change was observed, this provides another marker to identify NS-B50027-4 or progeny derived from NS-B50027-4.

The methods and kits herein are useful for identifying in biological samples the presence of plant material containing, in particular, the transgenes in NS-B50027-4, as well as transgenic canola plants, plant materials, and seeds containing such an event. Elite event NS-B50027-4 described herein can be identified by genotype, which can be characterized by a genetic marker profile that can identify plants of the same cultivar or related cultivar, or be used to determine or confirm pedigree. Profiles of genetic markers can be obtained by methods such as restriction fragment length polymorphism (RFLP), randomly amplified polymorphic DNA (Randomly Amplified Polymorphic DNAs, RAPD), randomly primed polymerase chain reaction (Arbitrarily Primed Polymerase Chain Reaction, AP- PCR), DNA Amplification Fingerprinting (DAF), Sequence Characterized Amplified Regions (SCAR), Amplified Fragment Length Polymorphisms (AFLP), Simple Sequence Repeats (SSR) (also called microsatellites) and single nucleotide polymorphism (Single Nucleotide Polymorphisms (SNP).

For example, the NS-B50027-4 elite event described herein can be identified by genetic mapping from a sample of plant material. A genetic map can be created using standard RFLP, polymerase chain reaction (PCR), or SSR assays that identify the approximate position of the inserted DNA molecule on the chromosome that codes for the foreign protein. See Glick & Thompson, Methods in Plant Molec. Biol. & Biotechnol., 269 (CRC Press, Boca Raton, FL, 1993). Mapping information with respect to chromosomal localization is useful for protecting ownership of a given transgenic plant. For example, a map of the embedding area can be compared with similar maps of plants being tested to determine if the latter have common parents with a given plant. Map comparisons may include hybridization, RFLP, PCR, SSR, and sequencing, which are standard techniques.

In another aspect of the present embodiments, kits and methods are provided for determining whether a canola plant is or related to the inbred line NS-B50027-4, or is a canola plant that contains at least a portion of the genetic elite event of the line NS-B50027- 4. This document describes compositions and a method for the simple and unambiguous identification of the NS-B50027-4 elite event in biological samples.

For example, the kit may include at least one primer set for identifying one or more NS-B50027-4 genetic markers, such as a set of sense (forward) and antisense (reverse) primers. Specific primer embodiments include the following primers used in KASP assay kits for detecting genetic traits of NS-B5OO27-4, particularly useful in introgression studies and hybrid breeding. See Example 2. These primers may consist of a nucleic acid molecule containing at least ten consecutive nucleic acids of the sequence shown in SEQ ID NOs: 1-90 (see Table 14, Example 3), or their complements.

The present invention also provides methods for identifying a canola plant containing the NS-B50027-4 elite event, comprising (a) obtaining a mixture comprising a biological sample containing canola plant DNA and first and second nucleic acid primers capable of amplifying a nucleic acid molecule specific for event NS-B50027-4;

(b) carrying out the reaction in the mixture under conditions that allow the first and second nucleic acid primers to amplify a nucleic acid molecule specific for the NS-B50027-4 event; and (c) detecting the presence of an amplified fragment of the nucleic acid molecule, wherein the presence of the nucleic acid molecule specific for the canola elite event NS-B50027-4 indicates that the canola plant is a canola plant NS-B50027-4.

In another embodiment, methods are provided for detecting an NS-B50027-4 elite event nucleic acid molecule in a biological sample, comprising (a) providing a mixture comprising a biological sample containing DNA and a nucleic acid probe capable of hybridizing to an event-specific nucleic acid molecule. NS-B50027-4;

(b) carrying out the reaction in the mixture under conditions that allow the probe to hybridize with a nucleic acid molecule specific for NS-B50027-4; and (c) detecting the presence of a hybridized nucleic acid molecule, wherein the presence of a nucleic acid molecule specific for the NS-B50027-4 event indicates that the sample contains the nucleic acid molecule of the NS-B50027-4 event.

- 26 041446

In yet another embodiment, methods are provided for detecting the presence of an NS-B50027-4 event nucleic acid molecule in a biological sample, comprising (a) obtaining a mixture comprising a biological sample containing DNA and a first primer capable of annealing with a region of the inserted event nucleic acid molecule NS-B50027-4, and a second primer capable of annealing to a flanking nucleic acid molecule in the host cell genome;

(b) carrying out the reaction in the mixture under conditions that allow the first and second nucleic acid primers to produce an amplified nucleic acid molecule containing a fragment of the inserted NS-B5OO27-4 event nucleic acid molecule; and (c) detecting the presence of the amplified nucleic acid molecule, wherein the presence of a fragment of the inserted NS-B50027-4 event nucleic acid molecule indicates that the sample contains the inserted NS-B50027-4 event DNA.

Proper testing should identify any major weaknesses and the level of superiority or improvement over existing commercial canola cultivars. In addition to demonstrating improved performance, there must be demand for a new cultivar that is compatible with industry standards or that creates a new market. The introduction of a new cultivar may introduce additional costs to the seed producer, plant grower, processor and consumer due to ad hoc marketing and sales of modified seed and commercial production techniques and use of the new product. Testing prior to the release of a new cultivar should take into account the costs of research and breeding, as well as the technical superiority of the final cultivar. For seed-propagated cultivars, easy and economical seed production should be possible.

Offspring.

The NS-B50027-4 line described in this document can be used as a parent line to breed other lines. For example, as a source, it can be self-pollinated, crossed, backcrossed, used to obtain double haploids used as starting materials for genetic transformation, or subjected to genetic transformation, further mutagenesis, and used for other forms of breeding, as known to those skilled in the art. in this area. Methods and results of using the starting material to breed other lines are also within the scope of these embodiments.

With the advent of molecular biology techniques that have enabled the isolation and characterization of genes encoding specific protein products, plant biologists have become increasingly interested in generating plant genomes that contain and express foreign genes, or additional or modified variants of native or endogenous genes (perhaps under control of various promoters) in order to change the genetic traits of the plant in a certain way. Any DNA sequences, from different species or from the same species, that are introduced into the genome using transformation or different dilution methods are collectively referred to herein as transgenes. Over the past fifteen to twenty years, several methods have been developed for obtaining transgenic plants, and the present invention in specific embodiments, the implementation also relates to transformed versions of the claimed line.

Nucleic acids, oligonucleotides or polynucleotides refer to RNA or DNA molecules that are linear or branched, single or double stranded, or hybrids thereof, including RNA/DNA hybrids. The terms also cover the 3'-UTR and 5'-UTR, typically at least about 1000 nucleotides of sequence upstream of the 5'end of the coding region and at least about 200 nucleotides of sequence downstream of the 3'end of the coding region of the gene. Less common bases such as inosine, 5-methylcytosine, 6-methyladenine, hypoxanthine and others can also be used for antisense, dsRNA and ribozyme pairing. For example, it has been found that polynucleotides containing the C-5 propyne analogs of uridine and cytidine can bind to RNA with a high degree of affinity and are potent antisense inhibitors of gene expression. Other modifications can also be made, such as changing the phosphodiester backbone or the 2'-hydroxyl group in the ribose sugar group of the RNA. Antisense polynucleotides and ribozymes may be composed entirely of ribonucleotides or may contain a mixture of ribonucleotides and deoxyribonucleotides. The polynucleotides of the invention can be prepared by any means, including genomic preparations, cDNA preparations, in vitro synthesis, RT-PCR, and in vitro or in vivo transcription.

Plant transformation involves constructing an expression vector that will function in plant cells. Such a vector includes DNA comprising a gene under the control of a regulatory element or operably linked to a regulatory element (eg, a promoter). The expression vector may contain one or more of these operatively linked gene/regulatory element combinations. The vector(s) may be in the form of a plasmid and may be used alone or in combination with other plasmids to provide transformed canola plants using transformation methods known in the art to introduce transgenes.

- 27 041446 into the genetic material of canola plant(s), including canola plants NS-B50027-4. For example, a transgenic cassette containing the gene(s) encoding glycerol-3-phosphate acyltransferase (GPAT), lysophosphatidic acid acyltransferase (LPAAT), or diacylglycerol acyltransferase (DGAT) can be transformed into NS-B5OO27-4 for fatty acid modification or TAG synthesis. See, for example, Shrestha et al., 7 Front. Plant Sci. 1402 (2016).

A genetic trait that has been inserted into a particular canola plant using transformation techniques can be transferred to another line using conventional breeding techniques well known in the plant breeding art. For example, plants bearing the elite event NS-B50027-4 can, for example, be obtained from seed deposited with ATCC®. Such plants can then be propagated or used in a conventional breeding scheme to introduce the NS-B50027-4 event (ie LC-PUFA biosynthesis) into other cultivars of the same plant or plants of related species. The deposited seeds belong to Brassica napus species. However, methods for introducing alleles or transgenes located on the A genome or C genome from B. napus into B. juncea are well known in the art and include backcrossing. The backcross method can be used to transfer a transgene from a transformed canola plant into an elite inbred line, and the resulting progeny contains the transgene. In addition, if an inbred line is used for transformation, then the transgenic plants can be crossed with another line to produce a transgenic hybrid canola plant. As used herein, the term crossing may refer to a simple crossing of X with Y, or a backcrossing process, depending on the context.

Various genetic elements can also be introduced into the plant genome using transformation. These elements include, but are not limited to, genes; coding sequences; inducible, constitutive and tissue-specific promoters; enhancer sequences; as well as signal and target sequences. The advent of new molecular biological methods has made it possible to isolate and characterize genetic elements with specific functions, such as encoding specific protein products. Scientists in the field of plant biology have shown a high interest in creating plant genomes that contain and express foreign genetic elements, or additional or modified versions of native or endogenous genetic elements, in order to specifically change the traits of the plant. Any DNA molecules, whether from other species or from the same species, inserted into the genome using transformation are collectively referred to herein as transgenes. The transformation process is the insertion of DNA into the genome. Several methods have been developed for producing transgenic plants, and the present invention, in specific embodiments, also relates to transformed versions of the claimed canola line NS-B50027-4.

Many methods have been developed for plant transformation, including protocols for the biological and physical transformation of plants. In addition, expression vectors and in vitro culture methods are available for plant cell or tissue transformation and plant regeneration. See, for example, Miki et al., Procedures for introducing foreign DNA into plants, in Meth. Plant Molec. Biol. & Biotechnol. at 63 (Glick & Thompson, eds., CRC Press, Boca Raton, 1993); Gruber et al., Vectors for plant transformation, id. at R 89; Genetic transformation for the improvement of Canola, Proc. World Conf. Biotechnol. Fats & Oils Indus. at 43-46 (Am. Oil. Chem. Soc., Champaign, IL, 1988).

The most common types of plant transformation involve the construction of an expression vector. Such a vector includes a DNA molecule that contains a coding region under the control of a regulatory region or operably linked to a regulatory region, such as a promoter. An expression vector may contain one or more genes and one or more regulatory elements. At least one of the coding regions and their respective regulatory elements may be located in opposite orientations within the vector, providing a binary vector. Theoretically, arranging genes affected by gene silencing in binary form can minimize gene silencing. The vector(s) may be in the form of a plasmid and may be used alone or in combination with other plasmids to provide transformed canola plants using transformation methods known in the art to introduce transgenes into the genetic plant material NS-B50027-4 or a plant derived from NS-B50027-4.

For example, the original transformation cassette, pJP3416_GA7-modB, included seven genes capable of stimulating the accumulation of omega-3 fatty acids in canola seed and a selectable marker gene to facilitate the selection of putative transgenic plants in vitro. See WO2013185184; US Patent Publication 20150374654; Petrie et al., 6 Plant Meth. 8 (2010). The expressed genes are all synthetic - codon-optimized and synthesized - since transgenic DNA molecules are not found in any natural organism. The original DNA sequences that were used as templates for codon optimization have been described. See Petrie et al., 12 Metab. Eng'g 233 (2010a); Petrie et al., 11 Plant Methods 6 (2010b); Petrie et al., 21 Transgenic Res. 139 (2012).

As is known in the art, functional gene promoters are regions

- 28 041446

DNAs that are essential for gene transcription but do not code for functional products such as peptides. For example, a typical promoter for constitutive expression is derived from the cauliflower mosaic virus. Kay et al., 236 Sci. 1299 (1987); Coutu et al., 16 Transgenic Res. 771 (2007). Individual developmentally controlled promoters include promoters that preferentially initiate transcription in specific tissues such as seeds, leaves, roots, fibers, xylem vessels, tracheids, or sclerenchyma. Promoters of particular importance are seed-preferred promoters that initiate transcription predominantly or only in the seed. Seed-preferred promoters include seed-specific promoters (ie, promoters active during seed development, such as seed storage protein promoters) and seed germination promoters (ie, promoters active during seed germination). See Thompson et al., 10 BioEssays 108 (1989). Such seed-preferred promoters include, but are not limited to, Cim1 (cytokinin-induced signaling); cZ19B1 (19 kDa maize zein); milps (myo-inositol-1-phosphate synthase) (see WO 2000/11177 and US Pat. No. 6,225,529). For dicots, seed-specific promoters include, but are not limited to, bean beta-phaseolin, napin, betaconglycinin, soybean lectin, cruciferin, conlinin, and the like. Seed-specific promoters used in GA7-modB have been described previously: A. thaliana FAE1 (Rossack et al., 46 Plant Molec. Biol. 717 (2001)); L. usitatissimum Cnl1 and Cnl2 (Chaudhary et al., WO 2001016340); and a truncated napin promoter from B. napus (Stalberg et al., 23 Plant Molec. Biol. 671 (1993)). See also WO 2013185184.

An inducible promoter is a promoter that can be regulated by the environment. Examples of environmental conditions under which transcription by inducible promoters can occur include anaerobic conditions or the presence of light. Tissue-specific, tissue-preferred (eg, seed-specific) and inducible promoters represent a class of non-constitutive promoters. See Ward et al., 22 Plant Mol. Biol. 361 (1993); Meft et al., 90 PNAS 4567 (1993) (copper induced); Gatz et al., 243 Mol. Gen. Genet. 32 (1994) (induced by herbicide antidotes); Gatz et al., 227 Mol. Gen. Genet. 229 (1991) (tetracycline-induced); Schena et al., 88 PNAS 10421 (1991) (glucocorticosteroid-induced). See also WO 2001016340 and the promoters discussed therein.

A constitutive promoter is a promoter that can be active under most environmental conditions. Typical constitutive promoters include promoters from plant viruses such as the 35S promoter from cauliflower mosaic virus (CMV) (Odell et al., 313 Nature 810 (1985)) and promoters from genes such as rice actin (McElroy et al., 2 Plant Cell 163 (1990)); ubiquitin (Christensen et al., 12 Plant Mol. Biol. 619 (1989); Christensen et al., 18 Plant Mol. Biol. 6759 (1992)); pEMU (Last et al., 81 Theor. Appl. Genet. 581 (1991)); MAS (Velten et al., 3 EMBO J. 2723 (1984)), and maize histone H3 (Lepetit et al., 231 Mol. Gen. Genet. 276 (1992); Atanassova et al., 2 Plant J. 291 ( 1992)). The ALS promoter, the 5' Xbal/Ncol fragment to the Brassica napus ALS3 structural gene (or a nucleotide sequence similar to said Xbal/Ncol fragment) is another constitutive promoter. See also WO 1996/30530 and the promoters discussed therein. The CMV promoter is also associated with a useful enhancer region. See, for example, WO 1996/30530 and WO 2013185184 and the promoters discussed therein.

Terminator regions, which include polyadenylation signals, are required for the production of complete and stable mRNA molecules. For example, the A. tumefaciens nopaline synthase (NOS) terminator provides a useful terminator. Bevan, 12 Nucl. Acid Res. 8711 (1984); Rogers et al., in Biotechnol. Plant Sci. at 219 (Acad. Press, Inc., New York, NY, 1985); Sanders et al., 15 Nucl. Acids Res. 1543 (1987). A number of regulatory sequences were used in combination to direct and terminate transcription of various expression cassettes.

Transport of a protein produced by transgenes into the subcellular space, e.g., into the chloroplast, vacuole, peroxisome, glyoxysome, cell wall, or mitochondrion, or for secretion into the apoplast, can be accomplished by operably attaching a nucleotide sequence encoding a signal sequence to the 5' or 3 '-regions of the gene encoding the protein in question. Targeting sequences at the 5' or 3' end of a structural gene during protein synthesis and processing can determine where the final compartmentalization of the encoded protein can occur.

The presence of a signal sequence directs the polypeptide either to an intracellular organoid, or to a subcellular space, or for secretion in the apoplast. Many signal sequences are known in the art. See, for example, Becker et al., 20 Plant Mol. Biol. 49 (1992); Knox et al., 9 Plant Mol. Biol. 3 (1987); Lerner et al., 91 Plant Physiol. 124 (1989); Fontes et al., 3 Plant Cell 483 (1991); Matsuoka et al., 88 PNAS 834 (1991); Creissen et al., 2 Plant J. 129 (1991); Kalderon et al., 39 Cell 499 (1984); Steifel et al., 2 Plant Cell 785 (1990).

Expression vectors typically include at least one genetic marker operably linked to a regulatory element (eg, a promoter) that allows for the recovery of marker-bearing transformed cells or by negative selection, i. growth inhibition of cells that do not contain a selectable marker gene, or by positive selection,

- 29 041446

those. screening of the product encoded by the genetic marker. Many selectable marker genes for plant transformation are known in transformation techniques and include, for example, genes encoding enzymes that metabolically detoxify a selective chemical agent, which may be an antibiotic or herbicide, or genes encoding an altered target that may be insensitive to inhibitor. A number of positive selection methods are also known in the art.

One widely used selectable marker gene suitable for plant transformation may include the neomycin phosphotransferase II (nptII) gene isolated from the Tn5 transposon, which, when placed under the control of plant regulatory signals, confers resistance to kanamycin. Fraley et al., 80 PNAS 4803 (1983). Another commonly used selectable marker gene may be the hygromycin phosphotransferase gene, which confers resistance to the antibiotic hygromycin. Vanden Elzen et al., 5 Plant Mol. Biol., 299 (1985). Additional selectable marker genes of bacterial origin that confer antibiotic resistance include gentamicin acetyltransferase, streptomycin phosphotransferase, aminoglycoside 3'-adenyl transferase, and the bleomycin resistance determinant. Hayford et al., 86 Plant Physiol. 1216 (1988); Jones et al., 210 Mol. Gen. Genet., 86 (1987); Svab et al., 14 Plant Mol. Biol. 197 (1990); Hille et al., 7 Plant Mol. Biol. 171 (1986). Other selectable marker genes confer resistance to herbicides such as glyphosate, glufosinate, or bromoxynil. Comai et al., 317 Nature 741 (1985); Gordon-Kamm et al., 2 Plant Cell 603 (1990); Stalker et al., 242 Sci. 419 (1988). Selectable marker genes suitable for plant transformation that are not of bacterial origin include, for example, mouse dihydrofolate reductase, plant 5-enolpyruvylshikimate-3-phosphate synthase, and plant acetolactate synthase. Eichholtz et al., 13 Somatic Cell Mol. Genet. 67 (1987); Shah et al., 233 Sci. 478 (1986); Charest et al., 8 Plant Cell Rep. 643 (1990).

Another class of marker genes suitable for plant transformation requires screening of putatively transformed plant cells rather than direct genetic selection of transformed cells for resistance to a toxic substance, such as an antibiotic. These genes are particularly useful for quantifying or visualizing the spatial pattern of gene expression in specific tissues, and are often referred to as reporter genes because they can be fused to a gene or gene regulatory sequence to study gene expression. Commonly used genes for screening transformed cells include α-glucuronidase (GUS), α-galactosidase, luciferase, and chloramphenicol acetyltransferase. Jefferson, 5 Plant Mol. Biol. 387 (1987); Teeri et al., 8 EMBO J. 343 (1989); Koncz et al., 84 PNAS 131 (1987); DeBlock et al., 3 EMBO J. 1681 (1984). Some methods for visualizing GUS activity in vivo do not require destruction of plant tissue. Molecular Probes, Publication 2908, IMAGENE GREEN, 1-4 (1993); Naleway et al., 115 J. Cell Biol. 151a (1991). However, these in vivo methods for visualizing GUS activity have not proven useful for recovering transformed cells due to low sensitivity, high initial fluorescence levels, and limitations associated with the use of luciferase genes as selectable markers. Green fluorescent protein (GFP) can be used as a marker of gene expression in prokaryotic and eukaryotic cells. Chalfie et al., 263 Sci. 802 (1994). Fluorescent proteins (GFP) and GFP mutations can be used as possible markers for screening.

NS-B50027-4 and the progeny of NS-B50027-4 can also be transformed to confer disease or pest resistance. For example, a plant line can be transformed with a cloned resistance gene to create plants that are resistant to certain pathogenic strains. See, for example, Jones et al., 266 Sci. 789 (1994) (cloning of the tomato Cf-9 gene for resistance to Cladosporium flavum); Martin, et al., 262 Sci. 1432 (1993) (tomato Pto gene for resistance to Pseudomonas syringae pv. tomato encoding protein kinase); Mindrinos et al., 78 Cell 1089 (1994) (Arabidopsis RSP2 gene for P. syringae resistance); Geiser et al. 48 Gene 109 (1986) (Bacillus thuringiensis δendotoxin gene); Van Damme et al., 24 Plant Mol. Biol. 25 (1994) (lectin gene binding mannose from Clivia miniata); Sumitani et al., 57 Biosci. Biotech. Biochem. 1243 (1993) (amylase inhibitor); Abe et al., 262 J. Biol. Chem. 16793 (1987) (rice cysteine proteinase inhibitor); Huub et al., 21 Plant Mol. Biol. 985 (1993) (tobacco proteinase I inhibitor); Regan, 269 J. Biol. Chem. 9 (1994) (insect diuretic hormone receptor); Pratt et al., 163 Biochem. Biophys. Res. Comm. 1243 (1989) (allostatin); Tomalski et al., US Pat. No. 5,266,317 (genes encoding insect-specific paralytic neurotoxins); Scott et al., WO 1993/02197 (callase gene); Kramer et al., 23 Insect Biochem. Mol. Biol. 691 (1993) (tobacco hawk chitinase); Kawalleck et al., 21 Plant Mol. Biol. 673 (1993) (parsley ubi4-2 polyubiquitin gene); WO 1995/16776 (tachyplesin peptide derivatives which inhibit fungal plant pathogens); WO 199518855 (synthetic antibacterial peptides); Jaynes et al., 89 Plant Sci. 43 (1993) (secropin-v-lytic peptide analogue for conferring resistance to Pseudomonas solanacearum in transgenic tobacco plants); Botella et al., 24 Plant Mol. Biol., 24:757 (1994) (mung bean calmodulin); Griess, et al., 104 Plant Physiol. 1467 (1994) (maize calmodulin);

- 30 041446

Taylor, et al., Abstract No. 497, Seventh International Symposium on Molecular Plant-Microorganism Interactions (Edinburgh, Scotland) (1994) (enzymatic inactivation in transgenic tobacco by production of single chain antibody fragments); Tavladorakiet al., 366 Nature 469 (1993) (virus resistance by transgenic antibody); Lamb et al., 10 Biotechnol. 1436 (1992) (fungal endo-α-1,4-D-polygalacturonase fragments promote fungal colonization and plant nutrient release by solubilization of plant cell wall homo-α-1,4-D-galacturonase); Toubart et al., 2 Plant J. 367 (1992) (bean endopolygalacturonase inhibitory protein); Logemann et al., 10 Bio/technology 305 (1992) (transgenic plants that express a gene that inactivates the barley ribosome have increased resistance to fungal diseases).

As noted, herbicide resistance is another useful trait that can be introduced through genetic modification. For example, resistance to herbicides that inhibit the growth point or meristem, such as imidazolinone or sulfonylurea, can be conferred by mutant ALS and AHAS enzymes. See, for example, Lee et al., 7 EMBO J. 1241 (1988); Miki et al., 80 Theor. Appl. Genet. 449 (1990); glyphosate resistance is conferred by mutant 5enolpyrullshikimate-3-phosphate synthase (EPSPS) genes and aroA genes; resistance to glufosinate is conferred by phosphinothricin acetyltransferase genes; and genes encoding an ACCase inhibitor confer resistance to pyridineoxy or phenoxypropionic acids and cyclohexones. See, for example, US Pat. No. 4,940,835 (EPSPS confers glyphosate resistance); mutant aroA gene, accession number ATCC® 39256, see Comai, US patent 4769061; see also Umaballava-Mobapathie, 8 Transgen. Res. 33 (1999) (glufosinate resistant lettuce Lactuca sativa); Kumada et al., EP 0 333 033; Goodman et al., US Pat. No. 4,975,374 (glutamine synthetase genes confer resistance to herbicides such as L-phosphinothricin); Leemans et al., EP 0242246 (phosphinothricin acetyltransferase); DeGreef et al., 7 Bio/technol. 61 (1989) (chimeric bar genes encoding phosphinothricin acetyltransferase activity); Marshall et al., 83 Theor. Appl. Genet. 435 (1992) (genes Accl-S1, Accl-S2 and Accl-S3 confer resistance to phenoxypropionic acids and cyclohexones such as sethoxydim and haloxyfop); Przibilla et al., 3 Plant Cell 169 (1991) (the PsbA and gs ⁺ genes confer triazine resistance); Stalker, US Pat. No. 4,810,648 (nitrilase genes confer resistance to benzonitrile); Hayes et al., 285 Biochem. J. 173 (1992) (glutathione S transferase); Hattori et al., 246 Mol. Gen. Genet. 419 (1995) (acetohydroxy acid synthase confers resistance to a variety of herbicides); Shiota et al., 106 Plant Physiol. 17 (1994) (NADPH-cytochrome P450 yeast oxidoreductase); Aono et al., 36 Plant Cell Physiol. 1687 (1995) (glutathione reductase and superoxide dismutase); Datta et al., 20 Plant Mol. Biol. 619 (1992) (various phosphotransferases); WO 01/12825; US patents 6288306; 6282837; 5,767,373 (protox-altered plants resistant to duct-targeting herbicides).

NS-B50027-4 and progeny derived from NS-B50027-4 can be further modified to confer any number of value-added traits known in the art. See, for example, Goto, et al., 521 Acta Horticul. 101 (2000) (soy ferritin gene); Curtis et al., 18 Plant Cell Rep. 889 (1999) (nitrate reductase); Knultzon et al., 89 PNAS 2625 (1992) (stearyl ACP desaturase); Shiroza et al., 170 J. Bacteriol. 810 (1988) (nucleotide sequence of the mutant Streptococcus fructosyltransferase gene); Steinmetz et al., 20 Mol. Gen. Genet. 220 (1985) (levansucrase gene of Bacillus subtilis); Pen et al., 10 Bio/technol. 292 (1992) (transgenic plants express Bacillus lichenifonnis α-amylase); Elliot et al., 21 Plant Mol. Biol. 515 (1993) (tomato invertase genes); Sogaard et al., 268 J. Biol. Chem. 22480 (1993) (site-directed mutagenesis of the barley α-amylase gene); Fisher et al., 102 Plant Physiol. 1045 (1993) (maize endosperm starch branching enzyme II).

Canola line NS-B50027-4 can also be manipulated to be male sterile using any of a number of methods known in the art, including using mechanical methods, chemical methods, self-incompatibility (SI), cytoplasmic male sterility (CMS, Ogura or other system) or nuclear male sterility (NMS). The expression manipulated to ensure male sterility refers to the use of any available methods to obtain a male sterile version of the NS-B50027-4 canola line. Male sterility can be partial or complete male sterility. See, for example, WO 2001/29237 (introduction of a deacetylase gene under the control of a tapetum-specific promoter and using the chemical N-Ac-PPT); WO 199213956, WO 199213957 (stamen-specific promoters); Paul et al., 19 Plant Mol. Biol. 611 (1992) (introduction of barnase and barstar genes); see also US Pat. Nos. 5,859,341; 6297426; 5478369; 5824524; 5850014; 6265640; Hanson et. al., 16 Plant Cell S154 (2004).

Numerous methods for plant transformation have been developed, including biological and physical protocols for plant transformation. See, for example, WO 2013185184; Miki et al., in Meths. Plant Molec. Biol. Biotechnol. at 67-88 (Glick & Thompson, Eds., CRC Press, Inc., Boca Raton, FL, 1993). In addition, expression vectors and in vitro culture methods are available for plant cell or tissue transformation and plant regeneration. See, for example, WO 2013185184; Gruber et al., Meths. Plant Molec. Biol. Biotechnol. at 89-119 (Glick & Thompson, Eds., CRC Press, Inc., Boca Raton, FL, 1993). One method for introducing an expression vector into plants uses the natural system

- 31 041446 Agrobacterium transformation, see Horsch et al., 227 Sci. 1229 (1985); Curtis et al., 45 J. Exper. Botany 1441 (1994); Torres et al., 34 Plant Cell Tissue Organ Culture 279 (1993); Dinant et al., 3 Molec. Breeding 75 (1997); Kado, 10 Crit. Rev. Plant Sci. 1 (1991) (A. tumefaciens and A. rhizogenes Ti and Ri plasmids, respectively, carry genes responsible for plant genetic transformation); Gruber et al.; Miki et al.; Moloney et al., 8 Plant Cell Rep. 238 (1989) (Agrobacterium vector systems and Agrobacterium mediated gene transfer methods); US patent No. 5591616.

As an alternative to Agrobacterium-mediated transformation, several plant transformation methods have been developed, collectively referred to as direct gene transfer. A widely used method for plant transformation is microparticle-mediated transformation, in which DNA is transferred to the surface of microparticles ranging in size from 1 to 4 μm. The expression vector is introduced into plant tissues using a biolistic device that accelerates microparticles to a speed of 300-600 m/s, sufficient to penetrate the walls and membranes of plant cells. Russell et al., 12 Plant Cell Rep. 165 (1993); Aragao et al., 20 Plant Mol. Biol. 357 (1992); Aragao et al., 12 Plant Cell Rep. 483 (1993); Aragao, 93 Theor. Appl. Genet. 142 (1996); Kim & Minamikawa, 117 Plant Sci. 131 (1996); Sanford et al., 5 Part. sci. Technol. 27 (1987); Sanford, 6 Trends Biotech. 299 (1988); Klein et al., 6 Bio/technol. 559 (1988); Sanford, 7 Physiol. Plant, 206 (1990); Klein et al., 10 Bio/technol. 268 (1992).

Methods for physically delivering DNA to plants are also known in the art. See, for example, Zhang et al., 9 Bio/technol. 996 (1991) (sonication); Deshayes et al., 4 EMBO J. 2731 (1985) (liposomes); Christou et al., 84 PNAS 3962 (1987) (spheroplast NHW11915); Hain et al., 199 Mol. Gen. Genet. 161 (1985) (Precipitation of CaCl ₂ ); Draper et al., 23 Plant Cell Physiol. 451 (1982) (polyvinyl alcohol or polyA-ornithine); Saker et al., 40 Biology Plantarum, 507 (1997/98) (protoplast electroporation). Additional methods include, but are not limited to, expression vectors introduced into plant tissues using a direct gene transfer method such as microparticle-mediated delivery with a biolistic device, DNA injection, electroporation, and the like. Following transformation, expression of the selectable marker genes described above may allow for the preferential selection of transformed cells, tissues, or plants using regeneration and selection methods well known in the art. See, for example, WO 2013185184.

The above methods of transformation are usually used to obtain a transgenic line. The transgenic line can then be crossed with another (non-transgenic, mutated or transformed) line to produce a new transgenic canola line. Alternatively, a genetic trait inserted into a particular hybrid of a Brassica plant such as B. napus or B. juncea using well known transformation techniques can be transferred to another line using standard crossbreeding, backcrossing and selfing techniques which are also well known. in the field of plant breeding. For example, a backcrossing approach can be used to transfer an inserted trait from a public non-elite inbred line to an elite inbred line, or from an inbred line that contains a foreign gene in its genome to an inbred line or lines that do not contain that gene. As used herein, the term crossing may refer to a simple crossing of X with Y, or a backcrossing process, depending on the context.

When the term plant NS-B50027-4 is used in the context of the present embodiments, this also includes any gene conversions of this line. The term gene-converted plant refers to those NS-B50027-4 plants produced by backcrossing, genetic engineering, or mutation, wherein substantially all of the desired morphological and physiological characteristics of the cultivar are obtained in addition to one or more genes transferred to the line, derived from NS-B50027-4 by backcrossing, genetic engineering or mutation. Backcrossing techniques can be used in the present invention to improve or introduce a characteristic into a plant variety. As used herein, the term backcrossing refers to the repeated backcrossing of a hybrid progeny with a recurrent parent, i. backcrossing 1, 2, 3, 4, 5, 6, 7, 8, 9 or more times with a recurrent parent. A parent plant that contributes a desired characteristic to a gene(s) is called a non-recurrent or donor parent. This terminology refers to the fact that the non-recurrent parent occurs once in a backcross protocol and therefore is not repeated. A Brassica or canola parent that carries a gene or genes from a non-recurrent parent is known as a recurrent parent because it is used for multiple cycles in the backcross protocol. Poehlman & Sleper, 1994; Fehr, 1993. In a typical backcrossing protocol, the original variety of interest (the recurrent parent) is crossed with a second variety (the non-recurrent parent) that carries the single transfer gene of interest. The resulting progeny from this cross is then crossed again with the recurrent parent and the process is repeated until a canola or brassica plant is obtained in which substantially all of the desired morphological and physiological characteristics of the recurrent parent are obtained in the converted plant in addition to the transferred gene(s). am) from a non-recurrent parent. So

- 32 041446 Responsibly one or two loci from NS-B50027-4 can be transferred to another canola or Brassica line.

Choosing a suitable recurrent parent is an important step for a successful backcross procedure. The goal of a backcross protocol is to change or replace a trait or characteristic in the original line. To accomplish this, the gene of the recurrent parent is modified or replaced with the desired gene from the non-recurrent parent, while leaving essentially all the rest of the desired genetic and therefore desirable physiological and morphological composition of the original parental line. The choice of a specific non-recurrent parent will depend on the purpose of the backcross. One of the main goals of introgression is to add some commercially desirable, agronomically important plant trait. The exact backcrossing protocol will depend on the characteristic or trait being modified to determine the appropriate testing protocol. Although backcrossing methods are simplified when the trait to be transferred is a dominant allele, a recessive allele can also be transferred. In this case, it may be necessary to introduce a progeny test to determine if the desired trait has been successfully transferred.

Genetic traits can be identified that are not normally chosen when developing a new line, but which can be improved by backcrossing techniques. Such traits may or may not be transgenic. Examples of these traits include, but are not limited to, male sterility, modified carbohydrate metabolism, herbicide resistance, resistance to bacterial, fungal, or viral diseases, insect resistance, nutritional enhancement, industrial applicability, yield stability, and increased yield. These genes are usually inherited through the nucleus. See, for example, US Pat. Nos. 5,969,212; 7164059.

Further reproduction of the inbred line NS-B50027-4 can occur by tissue culture and regeneration. The term tissue culture refers to a mixture of isolated cells of the same or different species, or an aggregation of such cells, organized into parts of a plant. Typical types of tissue cultures are protoplasts, callus, plant pieces and plant cells, which can generate whole tissue culture in plants or plant parts such as leaves, pollen, embryos, roots, root tips, anthers, pistils, flowers, seeds, petioles, stepchildren, etc. Means for preparing and maintaining plant tissue culture are well known in the art. Tissue culture of various canola tissues and regeneration of plants from it is well known. See, for example, Teng et al., 27 HortSci. 1030 (1992); Teng et al., 28 HortSci. 669 (1993); Zhang et al., 46 J. Genet. Breeding 287 (1992); Webb et al., 38 Plant Cell Tissue Organ Cult. 77 (1994); Curtis et al., 45 J. Exp. Bot. 1441 (1994); Nagata et al., 125 J. Am. Soc'y Hort. sci. 669 (2000); Ibrahim, et al., 28 Plant Cell Tissue Organ Cult. 139 (1992); U.S. Patents 5,959,185; 5973234; 5977445; 8816111. Tissue culture as well as microspore culture can be successfully obtained for the regeneration of canola plants. See Chuong et al. 4 Plant Cell Rep. 4 (1985); Barsby et al., 5 Plant Cell Rep. 101 (1986); Kartha et al., 31 Physiol. Plant 217 (1974); Narasimhulu et al., 7 Plant Cell Rep. 104 (1988); Swanson, 6 Meth. Molec. Biol. 159 (1990); Cell Culture Tech. & Canola Improvement, 66 J. Am. oil chem. soc. 455 (1989). From the literature it is clear that the state of the art is such that these methods of obtaining plants are usually used with a high degree of success. Thus, in another aspect of the present embodiments, cells are provided that, after cultivation and differentiation, reproduce canola plants having the physiological and morphological characteristics of the inbred transgenic line NS-B50027-4.

Typically, when a transgene is introduced into a plant by conventional breeding, its insertion site in the plant genome and its flanking regions are not altered. An insertion region refers to a region corresponding to a region of at least 40 bp, such as at least 100 bp or up to more than 10,000 bp, surrounded by upstream (upstream position) and downstream (downstream position) flanking regions of a transgene in the genome ( untransformed) plant and including the site of insertion (and possible deletion of the target site). Considering minor differences due to mutations within species, the insertion region will retain at least 85%, e.g. 90, 95 or 100%, sequence identity with the sequence containing the upstream (upstream position) and downstream (downstream position) flanking regions. foreign DNA in a given plant of these species. However, the insertion of a transgenic cassette into the plant genome can sometimes be associated with a deletion of plant DNA, referred to as a target site deletion. However, additional transgenes or other genetic manipulations can be made in NS-B50027-4 without undue experimentation; and plants derived from NS-850027-4 can be identified as described herein.

The starting material NS-B50027-4 can be used to produce hybrid seed lines, for example if it is backcrossed with a source of cytoplasmic male sterility or some other source to sterilize the inbred line as female. Alternatively, the line may be used directly. For example, B. napus line NSB50027-4 can be crossed with another canola plant to form a population of F1 plants.

- 33 041446 first generation. The first generation F1 plant population obtained by this method is also an embodiment. This first generation F1 plant population contains a substantially complete set of alleles of the NS-B50027-4 canola lineage.

The F1 hybrid is generally considered to have all the alleles of each parent. One of skill in the art can use breeding books or molecular methods to identify a particular F1 plant produced using the NS-B50027-4 canola line, and any such individual plant is also within the scope of this invention. These embodiments also cover the use of these methods using the NS-B50027-4 transgenic line or single gene conversions of the NS-B50027-4 line.

In another embodiment, a method is provided for using the NS-B50027-4 canola line in a backcross with a recurrent parent any number of times. Using the transgenic methods described herein, backcrossing methods, or other breeding methods known to those skilled in the art, individual plants and plant populations can be developed that retain at least 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 99.5% genetic profile of the canola line NS-B50027-4. The percentage of the genetic profile retained in the offspring can be measured by pedigree analysis or by using genetic methods such as molecular markers or electrophoresis. In pedigree analysis, on average 50% of the original germplasm will be passed on to the progeny line after one crossing with another line, 25% after another crossing with another line, and so on. Molecular markers can also be used to confirm and/or determine the lineage of offspring.

The specific method for obtaining a line derived from the canola line NS-B50027-4 is as follows. One of skill in the art crossed the NS-B5OO27-4 canola line with another canola plant such as the elite/isogenic line. The F1 seed resulting from this cross was grown to obtain a homogeneous population. The F1 seed contained 50% alleles from the canola line NS-B50027-4 and 50% alleles from another plant. The F1 seed was grown and subjected to self-pollination, thereby obtaining the F2 seed. On average, an F2 seed received 50% of its alleles from the NS-B50027-4 line and 50% from another canola plant, but different individual plants in the population had a much higher percentage of their alleles derived from the NS-B50027-4 event. Wang et al., 40 Crop Sci. 659 (2000); Bernardo et al., 102 Theor. Appl. Genet. 986 (2001). As used in this context, the term population refers to a statistically representative sample. F2 seed was grown and plant selection was made based on visual observation or trait measurement. The trait used for selection may be a trait of high DHA production in canola seed line NS-B5OO27-4. Progeny derived from event NS-B50027-4 showing the desired trait derived from NS-B50027-4 were selected and each plant was harvested separately. This F3 seed from each plant was grown in separate rows and self-pollinated. Then the selected rows or plants from the rows were collected and threshed individually. Selection was again based on visual observation of plant phenotype or measurement of desirable plant traits, such as the desirable trait derived from NS-B50027-4. The growing and selection process was repeated any number of times until an inbred canola plant derived from NS-B50027-4 was obtained.

The canola plant derived from NS-B50027-4 contains desirable traits derived from the NS-B50027-4 canola line, some of which may not be expressed by the other canola plant to which the NS-B5OO27-4 canola line was crossed, and some of which can be expressed by both canola lines, but are now at levels equal to or greater than those expressed in NS-850027-4.

Canola plants derived from NS-B50027-4 have an average of 50% of their genes derived from NS-B50027-4, but various individual plants in the population have a much higher percentage of their alleles derived from NS-B50027-4. The process of breeding, crossing, selfing and selection is repeated to produce another population of canola plants derived from NS-B50027-4, with an average of 25% of their genes coming from the canola line NS-B50027-4, but different individual plants from the population have much a larger percentage of their alleles originating from NS-B50027-4. Another embodiment of the invention is an inbred canola plant derived from NS-B50027-4 that has acquired the desirable high DHA trait derived from NS-B50027-4.

The previous example can be modified in a variety of ways, for example, selection may or may not occur in each selfed generation, selection may occur before or after the actual selfing process, or individual selection may be made by collecting individual pods, plants, rows, or plots in any moment during the breeding process described. In addition, haploid double breeding methods can be used at any stage of the process. The population of plants produced in each and any generation of self-pollination is also an embodiment of the present embodiments, and each such population will consist of plants containing approximately 50% of their genes from

- 34 041446 canola lines NS-B50027-4, 25% of their genes from the canola line NS-B50027-4 in the second cycle of crossing, selfing and selection, 12.5% of their genes from the canola line NS-B50027-4 in the third cycle of crossing , self-pollination and selection, etc.

Another embodiment is a method of producing a homozygous canola plant derived from NS-B50027-4 by crossing the NS-B50027-4 canola line with another canola plant and applying dual haploid methods to an F1 seed or F1 plant or to any generation of the NS canola line -B5OO27-4 obtained by self-pollination of this cross. Breeding pure lines is usually used to improve self-pollinating crops or inbred lines of cross-pollinating crops. Two parents that have favorable, complementary traits are crossed to produce F1. An F2 population is produced by self-pollination of one or more F1 populations or by crossing two F1 populations (cross between sibs). The selection of the best individuals usually starts on the F2 population. Then, starting with F3, the best individuals in the best families are selected. To improve the efficiency of selection for traits with low inheritance, retesting of families or hybrid combinations including individuals from these families can begin with the F4 generation. At the late stage of inbreeding (namely F6 and F7), the best lines or mixtures of phenotypically similar lines are tested for possible use as new cultivars.

In addition, the present embodiments relate to methods for obtaining canola plants derived from NS-B50027-4 by crossing the NS-850027-4 canola line with a canola plant and growing the progeny seed, and repeating the steps of crossing and growing with a canola plant derived from NS-B50027-4, 1 to 2 times, 1 to 3 times, 1 to 4 times, or 1 to 5 times, and selfing any number of times after the first, second, third, fourth, or fifth cross. Massive and recurrent selection can be used to improve populations of either self- or cross-pollinated crops. A genetically variable population of heterozygous individuals is either identified or created by crossbreeding several different parents. The best plants are selected on the basis of individual excellence, outstanding offspring, or superior combinative ability. The selected plants are cross-crossed to obtain a new population, from which additional selection cycles are continued. This process can be used to combine NS-B50027-4 with other LC-PUFA transgenic lines, effectively increasing the copy number in the homozygous line to increase seed LC-PUFA production. This method is advantageous for introducing transgenes into strong lines that are not amenable to transformation, or when the transgenic loci are known to be located on chromosomes other than those of NS-B50027-4, for example, the transgenic locus at A06 can be combined with the A02 and A05 NS loci -B50027-4. The homozygous line as a result of such a crossing includes six copies of the transgenic inserts. If gene silencing does not suppress expression to an uncontrolled effect, LC-PUFA production can be substantially increased. A truly homozygous F4 experimental line containing six transgenic loci (Full SinglexNS-B50027-4) produced about 25% DHA (wt.% of the total fatty acids in the seed).

Backcross breeding is used to transfer genes for a simple and highly inherited trait into the desired homozygous cultivar or line that is the recurrent parent. The source of the trait to be transferred is called the donor parent. The resulting plant is expected to have the characteristics of the recurrent parent (eg cultivar) and the desired trait carried over from the donor parent. After the initial crossing, individuals with the phenotype of the donor parent are selected and re-crossed (backcrossed) with the recurrent parent. The resulting plant is expected to have the characteristics of the recurrent parent (eg cultivar) and the desired trait carried over from the donor parent.

The single seed generation method in the strict sense refers to planting a splitting population, collecting samples of one seed per plant, and using that sample to plant the next generation. As the population progresses from F2 to the desired level of inbreeding, each of the plants from which the lines are derived can be traced back to different F2 individuals. The number of plants in a population decreases with each generation due to the inability of some seeds to germinate or some plants to produce at least one seed. As a result, not all F2 plants originally present in the population will be progeny at the completion of the generation improvement process.

In the following embodiment, a single gene conversion of NS-B50027-4 is provided. Gene conversion occurs when DNA sequences are introduced using traditional (non-transformation) breeding methods such as backcrossing. DNA sequences, either naturally occurring or transgenes, can be introduced using these traditional breeding methods. Desired traits tolerated by this method include, but are not limited to, fertility modification, fatty acid profile modification, other nutritional enhancements, industrial application enhancements, disease resistance,

- 35 041446 insects, herbicide resistance and yield improvement. The trait of interest is transferred from the donor parent to the recurrent parent in this case the canola plant described here. Monogenic traits can result from the transfer of either a dominant allele or a recessive allele. The selection of offspring with the trait of interest is carried out by direct selection for the trait associated with the dominant allele. Selecting offspring for a trait that is carried by the recessive allele requires growing and self-crossing (self-pollinating) the first backcross to determine which plants carry the recessive alleles. For recessive traits, additional testing of the offspring in generations from subsequent backcrosses may be required to determine the presence of the gene of interest. Along with selection for the trait of interest, offspring are selected based on the phenotype of the recurrent parent. It should be understood that sometimes additional polynucleotide sequences or genes are transferred along with a feature of interest resulting from single gene conversion. Progeny containing at least 90%, 95%, 96%, 97%, 98%, 99%, or 99.5% of the genes from the recurrent parent of the canola plant described here, plus containing the trait resulting from single gene conversion, is considered single gene conversion NS-B50027 -4. When a trait is controlled by two genes (for example, resistance to certain diseases), selection is made for two genes, and so on. A specific aspect of the embodiment described herein involves the use of the four gene segregant (four gene locus inserted on chromosome A02) NS-B50027-4 to increase LC-PUFA production in another transgenic plant.

Mutation breeding is another way to introduce new traits into canola varieties. Mutations that occur spontaneously or are artificially induced can be useful sources of variability for the breeder. The goal of artificial mutagenesis is to increase the frequency of mutations for a desired characteristic. Mutation rates can be increased in a variety of ways, including exposure to temperature, long-term seed storage, tissue culture conditions, radiation (e.g., x-rays, gamma rays, neutrons, beta rays, or ultraviolet rays), chemical mutagens (such as base analogs like 5-bromouracil), antibiotics, alkylating agents (such as sulfur mustards, nitrogen mustards, epoxides, ethyleneamines, sulfates, sulfonates, sulfones or lactones), azide, hydroxylamine, nitrous acid or acridines. Once a desired trait is observed through mutagenesis, that trait can then be incorporated into existing germplasm using conventional breeding methods. See, for example, Fehr, Principles Cultivar Devel. (Macmillan Pub'l Co., 1993). For example, a mutation can be used to increase DPA production in NS-B50027-4 by disrupting the expression of the A4 desaturase transgene.

It should be understood that the canola line according to the present embodiments, through routine manipulation of cytoplasmic genes, nuclear genes or other factors, can be obtained in the form of male sterility, as described in the references discussed earlier. Such embodiments are also within the scope of the present claims. The present embodiments thus provide F1 hybrid seeds and plants produced using the NS-B50027-4 canola line. Accordingly, in another embodiment, a method is provided for obtaining DHA-containing canola seed by introgression of the DHA trait NS-B50027-4 into an elite Brassica line, which is a male sterile line; introgression of the DHA NS-B5OO27-4 trait into the second elite line Bmssica, which is fertile; crossing two lines to obtain hybrid offspring; seed cultivation of hybrid offspring; collection of grain produced by cultivated hybrid offspring. An additional oil extraction step from the hybrid seed progeny provides a DHA-containing canola oil.

There are many laboratory methods available to analyze, compare and characterize plant genotypes; these include isoenzyme electrophoresis, restriction fragment length polymorphisms (RFLP), randomly amplified polymorphic DNA (RAPD), randomly primed polymerase chain reaction (AP-PCR), DNA amplification fingerprinting (DAF), amplified region characterized by nucleotide sequence (SCAR) , amplified fragment length polymorphism (AFLP), simple repeat sequences (SSR) (also called microsatellites), and single nucleotide polymorphism (SNP). Example 3 provided herein provides KASP assays for the analysis of NS-B50027-4 plant and progeny derived therefrom. Primers selected from those shown in SEQ ID NOs: 1-90 can also be adapted to other sequence based techniques.

Isoenzyme electrophoresis and RFLP are widely used to determine the genetic composition. See Shoemaker & Olsen, in Genetic Maps: Locus Maps of Complex Genomes, at 6.131 (O'Brien, ed., Cold Spring Harbor Lab. Press, Cold Spring Harbor, N.Y. (1993) linkage groups with ~365 RFLPs, 11 RAPDs, 3 classical markers and 4 isozyme loci), see also Shoemaker, in DNA-Based Markers in Plants, 299 (Phillips & Vasil, Eds., Kluwer Acad. Press, Dordrecht, Netherlands, 1994 ).

At present, the SSR technology is an effective marker used in practice.

- 36 041446 technology; more marker loci can be applied in daily practice and more alleles per marker locus can be detected using SSR compared to RFLP. See, e.g., Diwan & Cregan, 95 Theor. Appl. Genet. 22 (1997). SNPs can also be used to identify the unique genetic composition of the invention and the progeny species that retain that unique genetic composition. Various methods using molecular markers can be used in combination to improve overall resolution. Molecular markers, including markers identified using methods such as isozyme electrophoresis, restriction fragment length polymorphism (RFLP), randomly amplified polymorphic DNA (RAPD), random primer polymerase chain reaction (AP-PCR), amplified DNA fingerprinting (DAF) ), amplified sequence characterized regions (SCARs), amplified fragment length polymorphisms (AFLPs), simple repeat sequences (SSRs), and single nucleotide polymorphisms (SNPs) can be used in plant breeding methods. One application of molecular markers is to map quantitative trait loci (QTLs). QTL mapping is the use of markers that are known to be closely related to alleles that have measurable effects on a quantitative trait. Selection in the breeding procedure is based on the accumulation of markers associated with alleles with a positive effect, or the elimination of markers associated with alleles with a negative effect, from the plant genome.

Molecular markers can also be used during the breeding procedure to select for qualitative traits. For example, markers closely associated with alleles, or markers containing sequences within the actual alleles of interest, can be used to select plants that contain alleles of interest during a backcross breeding program. The markers can also be used to select for the genome of the recurrent parent and against the markers of the donor parent. Using this method, the amount of the donor parent's genome that remains in the selected plants can be minimized. It can also be used to reduce the number of backcrosses to a recurrent parent required for a backcross program. The use of molecular markers in a selection procedure is often referred to as genetic marker-enhanced selection or marker-assisted selection. Molecular markers can also be used to identify and exclude specific germplasm sources from parental or ancestral varieties of a plant by providing a means of tracking genetic profiles through crossbreeding.

Thus, it is clear from the prior art that these methods of obtaining plants are generally accepted in the sense that their use has become part of everyday practice and they have a high success rate. The usefulness of the NS-B50027-4 canola line also extends to crosses with other species. Usually suitable species belong to the family Brassicacea. Accordingly, any and all methods utilizing canola elite event NS-B50027-4 in breeding are embraced by the present embodiments, including self-pollination, pure breeding, backcrossing, hybridization, and backcrossing with populations. All plants and plant populations produced using the NS-B50027-4 canola line elite event as parent are within the scope of these embodiments, including those developed from cultivars derived from the NSB50027-4 canola line. Those skilled in the art can use unique molecular marker profiles or breeding records to identify progeny lines or progeny populations derived from the NS-B50027-4 canola line. A particular aspect of the present embodiments provides an unexpected advantage in the production of LC-PUFA in transgenic plants that have been introduced with the A02 locus segregated from NS-B50027-4 using conventional breeding methods.

Examples

Example 1 Characterization and selection of the NS-B50027-4 line in experimental field trials.

A difficult task in plant breeding is the identification of individual plants that are genetically superior, since for most traits the true value of a genotype can be masked by other confounding plant traits or environmental factors. One way to identify a superior plant is by visual evaluation of its characteristics in comparison to other experimental plants and one or more commonly grown standard cultivars. If a single visual assessment does not lead to a definitive conclusion, repeated visual assessments will provide a better estimate of genetic value.

Plants originally identified as B0050-027-18 were selected based on the single seed generation method by collecting a single seed sample per plant and using the single seed sample to plant the next generation. As a rule, the number of plants in a population decreases with each generation due to the fact that some seeds do not germinate or some plants do not produce at least one seed. As a result, not all of the originally selected plants in the population are progeny when the progeny improvement is complete. Moreover, original transgenic events exacerbate the complexity of inheritance so that predictions are often inaccurate about the genotype or phenotype of the offspring. Thus, the experimental plants were subjected to

- 37 041446 pollination and selected by type for subsequent generations until a particular line became homozygous, showed selected traits with excellent agronomic properties and produced a homogeneous population of pure-line offspring. In particular, experimental lines were selected following a reselection breeding program at the Nuseed Innovation Center (NIC), Horsham (Victoria, Australia). The selection and improvement of candidate lines was based on the following:

(a) the number of copies of the T-DNA insert;

(b) pattern of DNA expression segregation;

(c) homozygosity (based on fatty acid phenotype and genotype);

(d) LC-ω3-DHA production; and (e) suitable agronomic traits for growing crops based on progeny testing on plots in winter and summer.

In Australia, canola is grown in the southern drylands zone and mainly under winter rainfall conditions. Production in Australia is mainly from spring-type canola cultivars, which have low vernalization requirements. In general, Australian cultivars generally retain a slight delay in onset of flowering and have relatively high plant vigor or biomass production during the winter months. Canola crops in Australia are usually planted from April to May after the first major rain event and are harvested from October to December. Yield is primarily affected by available water during the growing season and the cultivar's water use efficiency. The gene for resistance to the main pathotype of the blackleg pathogen Leptosphaeria maculans may differentiate cultivars in terms of seedling survival and stem rot, but Australian cultivars are generally considered to be highly resistant when grown according to recommended agronomic practices. Seed development is followed by a growth period of five to seven months and occurs in late spring or early summer. Apart from the availability of water, the yield can be significantly affected by large temperature fluctuations (<0 to >35°C) which can cause underdevelopment of seeds and achenes.

As noted herein, canola germplasm transformation was performed with an eight-gene construct (seven enzymes for the DHA biosynthetic pathway and a marker) resulting in seed-specific accumulation of LC-ω3 fatty acids, in particular DHA. More broadly, the phenotype was distinguished by product quality (PQ) (produced by omega-3 fatty acids), although the plants carried a marker gene (MG). Transformed material was reselected for homozygosity of the loci, DHA seed expression, agronomic traits and potential yield suitable for commercial production. The tested lines originated from transformed seedlings (variety AV Jade). The experimental seeds were germinated, allowing the plants to self-pollinate in isolation (ie, insect-proof tents).

Three siblings derived from the T2 generation from the transgenic event were compared with eight other commercial canola cultivars (lines) for a number of important agronomic and seed traits across eight experimental field locations (sites) in Australia (subsequent field trials in Australia and Canada are described further below). ). The eight sites in Australia represented a wide range of potential yield under different environmental conditions, as evidenced by the range of average yield across sites (ie AV Garnet: 0.7-2.4 t/ha). Transgenic siblings were represented by the tested lines: NS-B50027-4 (T3), B0050-027-18-36-13 (T4) and B0050-02718-105-13 (T4). In addition, NS-B50027-4 was grown in experimental plots in Canada and agronomic and seed traits were compared with non-transgenic canola lines. Additional T3 and T5 generations of NS-B50027-4 were also compared with non-transgenic lines and two transgenic segregants derived from NS-B50027-4 by outcrossing, each containing a different transgenic loci (i.e. segregated A05 and A02) (see example 2). The variability of the agronomic traits of the tested lines was comparable to that of commercial cultivars evaluated on all tested media. This conclusion was supported by the finding that the highest yielding grain yield of the test line was statistically comparable, based on site-by-site analysis (MET-REML), to the highest yielding commercial cultivars. In addition, for each site, the highest yielding line tested was significantly higher yielding than at least one cultivar, with the exception of one site where there was no significant difference. The tested lines produced seeds with a slightly lower oil percentage and with a varying fatty acid composition; but this did not affect yield or agronomic performance. Expression of DHA LC ω3 fatty acid was highly stable across all media tested.

Control cultivars (commercial breeding lines) used for comparison provided an agronomically diverse (e.g., land cover, phenology) range of well-adapted (i.e., high but variable yield potential and oil content) cultivars widely grown in the planting area. All of these cultivars are cross-pollinating and are described and extensively evaluated in, for example, the Australian National Variety Testing Program and

- 38 041446 in the Australian National Variety Testing Program and Regional annual crop reports. See nvtonline website. In addition, the variability in plant disease resistance is well documented for blackleg in Australia. Van De Wouw et al., 67 Crop & Pasture Sci. 273 (2015). In Australia, blackleg disease can result in up to 90% yield loss. Marcroft & Bluett, Agricul. Notes, AG1352, Victoria, Dept. Primary Indian. (2008). Genetic variability among commercial cultivars in regard to the fatty acid composition of a given seed and the oil content of the seed has been recorded over time. See Seberry et al., Quality of Australian Canola 2011 (Australian Oilseeds Fed., 2012). Plants from the AV Jade cultivar were transformed to produce transgenic T0 and therefore AV Jade can be considered a non-transformed isoline of the transgenic event described herein.

Phenotypic variability for the tested lines was characterized by plant germination, plant vigor, flowering time, flowering duration, plant height, seed cracking, resistance to lodging, severity of blackleg infection, number of plants at harvest, grain yield, grain moisture, percentage of oil in seeds and the content of fatty acids, in particular the polyunsaturated fatty acid LC-ω3 (LC-PUFA), especially in relation to the yield of EPA, DPA and DHA. For all measured features, limited likelihood analysis was performed using ASREML in the statistical program GenStat. Gilmour et al., ASREML user guide, release 3.0, Biometric Bulletin (3) (VSV Int'l, Waterhouse Stm Hemel Hempstead, UK, 2009). The statistical method of the linear mixed model was used to account for the spatial variation of the fields, as described in detail and used for field plant breeding and genetic studies. Cullis & Gleeson, 47 Biometrics 1449 (1991); Smith et al., 57 Biometrics 1138 (2001); Cullis & Gleeson, 47 Biometrics 1449 (1991); Smith et al., 57 Biometrics 1138 (2001); Welham et al., Analysis of linear mixed models by ASReml-R with Applications in Plant Breeding: Course Notes (VSV Int'l, Waterhouse Stm Hemel Hempstead, UK, 2013). Meta-REML analysis on the plots was then performed in terms of grain yield (t/ha) to determine the best linear unbiased prediction (BLUP) for the tested lines.

More frequent and detailed measurements were taken in all environments. Evaluation of germination and rooting of plants was carried out on two randomly selected quadrants of one square meter in each plot fourteen days after sowing. The number of plants germinated per square meter was analyzed as a trait variable. A plant emergence score based on a visual assessment of average plant density at a plot was also recorded for each plot across all plots and analyzed as a trait variable (1: low emergence=0-5 plants/m ² ; 5: average emergence=25- 30 plants/m ² 9: B=high germination=45-50 plants/m ² ). Plant emergence based on number per square meter and plant emergence scores were significantly different (P<0.05) between lines for all eight plots. Statistical variability in the germination of plants of transgenic lines was significant (P<0.05) within the range of cultivars in all experiments. Plant vigor was based on biomass, which was scored on a scale from 1 (low: <10% leaf area of land) to 9 (high: >90% of leaf area of land) and analyzed. Plant vigor varied significantly (P<0.05) between lines for six of the seven plots. The average plant power score for the plots was 6 (60-70% of the leaf cover of the land plot) in all eight sites; and variability was relatively consistent, indicating low environmental effects for this trait. Statistically, the variability in plant vigor for the tested lines was significant within the range of cultivars across all plots.

Flowering time was recorded as the number of days from sowing until 50% of the plants in the experimental plot had at least one open flower. This was recorded for each site for all trials and analyzed as a trait variable. Flowering varied significantly (P<0.05) between lines for all plots. The mean on-site flowering time varied from 99 to 110 days and reflected environmental differences on the experimental sites for this trait. Statistically, the variability in flowering time for the transgenic lines was significant across the range of cultivars in all experiments.

Flowering time was the estimated difference between flowering time and flowering end time (given as a number of days). The calculation was made for each site in all trials and analyzed as a trait variable:

Bloom Duration=Blossom End Day-Blossom Time (50%)

The average flowering time on the plots varied from 24 to 30 days, which is shorter than the average and reflects the conditions that prevailed due to seed filling. Statistically, the variability in the duration of flowering of transgenic lines was significant within the range of cultivars for all experiments.

The number of plants at the time of collection, based on the number of plants per square meter, varied significantly between lines for all eight plots. Variation in the number of plants

- 39 041446 at the time of collection for transgenic lines was significant (P<0.05) within the range of cultivars across all stands and locations. The number of plants at emergence was significantly correlated with the number of plants counted at harvest. Some of the calculated survival percentages were in excess of 100%, indicating slow emergence in two cultivars (ATR Wahoo and AV Jade): not all seedlings had emerged at the time the number of emerging plants was recorded.

The height of the plant at the time of physiological maturity was measured from the base to the grown top in the center of the plot. The center of the plot was used to avoid distorting effects likely associated with a region of space within the plot (edge effects). This trait was recorded for each site across all trials and analyzed as a trait variable. Plant height at maturity (cm) varied significantly (P<0.05) between lines for all plots. The average plant height on the plots varied from 63 to 105 cm, indicating differences in the environment on the experimental plots for this trait.

Height variability at maturity for the transgenic lines was significant (P<0.05) within the cultivar range in all experiments.

Seed cracking (sometimes referred to as bean cracking) at maturity was analyzed by counting the number of cracked seeds per 1/8 square meter, which was recorded over a two week period. This was done by placing two trays between planted rows and under a canopy for each plot at all locations and analyzed as a trait variable. A seed cracking score (on a scale of 1 [zero] to 9 [high: +40]) was also recorded for one site based on the number of seeds observed on the ground just before harvest and analyzed as a trait variable. Seed cracking, based on the number of seeds on the ground at harvest, varied significantly (P<0.05) between lines for four of the eight plots. The average number of cracked seeds per plot varied from 3 to 15 (per 1/8th square meter) and indicated low levels of cracking in all plots. The seed cracking score at one of the sites also varied significantly between lines and was in close agreement with the average number of cracked seeds at the sites. This indicates that cracking, recorded as a score, was a good predictor of seed cracking. Statistical variability in seed cracking based on seed count and score for transgenic lines was significant across the cultivar range in all experiments.

Lodging resistance was recorded as a score from 1 (lodging resistant) to 9 (lodging prone) as measured by the angle of the plant from the base of the plant at maturity. There was no statistically significant variability in plant lodging. The lack of variability for this trait appears to be related to below-average rainfall at the late pod-filling stage.

The severity of blackleg symptoms characteristic of Leptosphaeria maculans and Leptosphaeria biglobosa was recorded on a score of 1 (low: <5%) to 9 (high: >40%) for one repetition at five sites. Not all sites were evaluated due to the lack of visible deviation. There were no symptoms associated with rotting and breakage of the trunk. Blackleg symptoms were observed at very low levels at all eight sites. One plot was planted using bare seeds (seeds not treated with fungicide). There were no relative differences in plant germination among the tested lines between this site and other sites treated with the seed fungicide. Foliar symptoms do not always predict the degree of stem rot caused by L. maculans (a leading cause of crop loss and a basis for assessing resistance in Australia, see Sosnowski et al., 33 Australian Plant Pathol. 401 (2004)). Several studies have assessed resistance to blackleg based on pathogenic infection of cotyledons, leaves, stem (rot) and plant survival in the field. Given the absence of rot and stem damage, canola lines can be considered resistant to the effects of this disease for the purposes described in this document.

The number of plants at the time of collection was estimated by counting the plants in two quadrants of one square meter in each plot in all eight plots. The mean of both quadrants was then used to estimate the number of plants per square meter and analyzed as a trait variable. Percentage of plant survival (%) was calculated by representing the mean values of the number of plants per plot as % of the average values of plants emerging per plot:

%Plant Survival=(Number of Plants at Harvest 100)/Number of Emerging Plants

Grain was harvested using harvesters for experimental and breeding plots when the experimental seeds were physiologically mature. The collection direction was kept constant (ie, end to end for each row) for each trial to avoid collection direction errors. The weight of dry grain for each plot was determined and converted into units of t/ha per os

- 40 041446 new plot area and analyzed as a feature variable.

Grain moisture at harvest and in the laboratory sample was recorded and analyzed as a trait variable. A hand-held moisture meter was used to analyze bulk samples directly at the collection point in the experimental field. Moisture percentage was also determined using the kiln method based on Australian Oilseed Federation (AOF) method 4-1.5. This method involved oven drying a 5 g sample in open jars at 130° C. for 1 hour. The samples were cooled in a desiccator for 40 minutes and weighed, and the percent moisture was determined as the percent weight loss. Grain moisture at harvest (in percent) varied significantly between line treatments for all eight sites. The average moisture content of grain on the plots during collection varied from 9 to 12%, which indicated that the seeds were collected at the same stage of grain development. Statistically, grain moisture variability at harvest for the transgenic lines was significant across the cultivar range in all experiments. Grain Moisture Percentage at Harvest also correlated with flowering time, such that seeds from late flowering lines (i.e. ATR Wahoo and Monola515TT) had significantly higher grain moisture percentages at harvest time across all plots. Laboratory seed moisture varied significantly between lines for all plots. However, the differences between lines and across sites were insignificant and averaged ~7%. This indicates the absence of distorting effects of seed storage. The seed moisture variability in the laboratory for the transgenic line was significant (P<0.05) within the range represented by the non-transgenic lines.

Seed oil content (%) was analyzed using Spinlock NMR spectrometry on seeds adjusted to 6% moisture. Briefly, 5-10 g seed samples were weighed into an NMR tube and analyzed with an NMR spectrometer. Seed oil content was determined by calibration software originally created using twenty reference samples of known oil percentage determined by gravimetric oil extraction. Seed oil content varied significantly (P<0.05) between lines in all eight experimental plots. The average percentage of seed oil content on the plots varied from 37.0 to 41.5%, which was generally below the typical average for the planting media and possibly a result of below-average and higher-than-average rainfall, temperatures observed during seed filling. Relative differences along the lines were consistent across sites. Variability in oil content in seeds for transgenic lines was slightly lower compared to non-transgenic lines across all plots: on average, by about 2%, which can serve as an object for genetic improvement. The lower oil content may not be genetically related to the transgenic event, but may result from the transformation of a cultivar with a lower oil content, i.e. AV Jade.

A brief description of the agronomic traits of the NS-B50027-4 event compared to non-transgenic cultivars collected during experimental cultivations is shown in Table 1. 2 (REML analysis; F pr<0.001 Sig for all features).

- 41 041446

table 2

Grain yield (t/ha) and agronomic measurement data for non-transgenic canola cultivars and experimental transgenic test lines in eight environments in 2015

i c3 i co s Line name e O y 0 m per m ² Y-P Quantity I r „ g about the plant at ____sbope E | Germination ’ In ¹ Power bj th plant Home ® flowering Finishing ® flowering tl Continue. ^s ___flowering Height rast. in the g state of ____ ripeness Cracked ___seeds 4 Grain output Humidity about? grains at bop ATR Bonito 18.2 16.0 7.3 6.8 103.8 131.2 27.5 90.0 13.0 1.35 10.6 ATR Gem 17.9 16.6 7.1 6.7 105.3 133.6 28.2 91.0 10.9 1.21 13.0 ATR Stingray 17.6 17.3 7.1 5.9 100.9 129.7 28.8 82.7 14.4 1.34 8.2 ATR Wahoo 11.2 11.8 5.9 6.1 108.2 136.0 27.3 92.3 10.7 1.12 18.7 AV Garnet 18.6 16.3 7.4 7.2 104.4 132.8 28.6 102.1 15.0 1.31 10.2 AV Jade 7.8 12.5 5.0 4.8 106.7 134.8 28.3 89.9 9.8 0.96 9.9 A.V. Zircon 19.0 15.7 7.3 7.0 104.4 132.0 27.6 98.7 22.5 1.31 9.5 Monola 515TT 20.3 18.5 7.5 5.8 108.6 136.1 27.3 87.9 12.3 1.24 12.4 NS-B50027-4 18.1 15.7 7.1 5.9 107.8 135.0 27.2 88.2 10.5 1.17 11.0 B0050-027-18-36-13 22.5 20.3 7.2 5.9 106.6 134.4 27.9 76.4 10.3 0.95 10.8 B-050-27-18-105-13 22.6 19.8 7.6 5.4 108.5 135.8 27.3 70.6 8.9 0.92 11.1 Min value for variety 7.8 11.8 5.0 4.8 100.9 129.7 27.3 82.7 9.8 0.96 8.2 NS-B50027-4 18.1 15.7 7.1 5.9 107.8 135.0 27.2 88.2 10.5 1.17 11.0 Max value for variety 20.3 18.5 7.5 7.2 108.6 136.1 28.8 102.1 22.5 1.35 18.7 Average 17.6 16.4 7.0 6.2 104.7 133.2 28.5 90.0 12.0 1.14 11.0 VAR 0.67 0.98 0.02 0.01 0.04 0.07 0.13 1.35 2.74 0.00 0.15 LSD 1.62 1.95 0.28 0.21 0.41 0.54 0.71 2.30 3.30 0.11 0.78 cv% 4.6 6.0 2.0 1.7 0.2 0.2 1.3 1.3 13.8 5.0 3.6

Fatty acids were determined using solvent extraction followed by simultaneous saponification and methylation and analysis by GC-FID. Briefly, this involved grinding seed samples and extracting the oil into solvent from a subsample of crushed seeds. The solvent was evaporated under nitrogen and the oil-containing sub-sample was diluted in new solvent. An aliquot was reacted with Meth Prep II (saponification/methylation reagent). Samples were heated at 40° C. to speed up the reaction and then injected into GC-FID using a BPX-70 fatty acid column. Fatty acids were calculated as the percentage composition of the oil, where the area of each fatty acid peak was determined as a percentage of the sum of all fatty acid peaks in the chromatogram. These scores were analyzed individually as a feature variable. The percentage of a specific fatty acid was determined for: palmitic acid; stearic acid; oleic and cis-vaccenic acid; linoleic acid; alpha-linolenic acid (ALA); arachidonic acid (also known as eicosanoic acid) and stearidonic acid (SDA); polynic, gondoic and gadoleic acids; erucic acid and eicosatetraenoic acid (ETA); eicosapentaenoic acid (EPA); docosapentaenoic acid (DPA); and docosahexaenoic acid (DHA).

In table. 3 shows a site-by-site analysis of seed fatty acid content (all values are in percent; REML analysis; F pr<0.001 Sig for all traits).

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Table 3

Seed analysis by site

Humidity lab. seeds seed oil Palmitic acid Stearic acid Oleic & cis-vaccenic Linoleic acid ALA, arachidonic, &SDA Polynic, gondola, & gadoleic ERA DPA DHA Sum of EPA, DPA and DHA ATR Bonito 6.7 41.9 3.9 1.7 60.5 20.9 10.1 1.1 0.0 0.0 0.0 0.0 ATRGEM 6.8 41.5 3.7 1.7 66.3 14.9 10.2 1.2 0.0 0.0 0.0 0.1 ATR Stingray 6.6 40.8 4.3 1.8 60.6 20.5 9.7 1.0 0.0 0.0 0.1 0.1 ATR Wahoo 6.8 41.7 3.7 1.6 60.7 20.4 10.4 1.2 0.0 0.0 0.1 0.1 AV Garnet 7.1 40.4 3.6 1.7 69.6 11.8 9.7 1.5 0.0 0.0 0.1 0.1 AV Jade 6.7 41.0 4.0 2.2 61.0 18.7 11.2 1.0 0.0 0.0 0.1 0.2 A.V. Zircon 6.8 41.0 3.8 1.6 69.3 11.8 10.4 1.3 0.0 0.0 0.0 0.0 Monola 515TT 6.9 40.9 3.6 2.1 73.3 12.2 5.2 1.4 0.0 0.0 0.1 0.2 NS-B50027-4 6.9 37.0 4.1 2.1 45.9 8.2 20.7 3.4 0.4 1.0 8.6 9.9 B0050-027-18-36-13 7.1 35.5 4.2 2.4 41.8 7.9 22.2 3.8 0.6 1.2 10.5 12.2 В-050-27-18-105-13 7.2 35.3 4.1 2.4 42.0 7.7 22.1 3.9 0.5 1.2 10.3 12.0 Min value by grade 6.6 40.4 3.6 1.6 60.5 11.8 5.2 1.0 0.0 0.0 0.0 0.0 NS-B50027-4 6.9 37.0 4.1 2.1 45.9 8.2 20.7 3.4 0.4 1.0 8.6 9.9 Max value by grade 7.1 41.9 4.3 2.2 73.3 20.9 11.2 1.5 0.0 0.0 0.1 0.2 Average 6.8 39.6 3.9 2 55 14.8 15 2.3 0.1 0.6 3.1 3.8 VAR 0.002 0.05 0.0005 0.0003 0.1912 0.0385 0.0475 0.0032 0.0001 0.0005 0.0248 0.0345 LSD 0.09 0.44 0.04 0.03 0.87 0.39 0.43 0.11 0.02 0.05 0.31 0.37 cv% 0.7 0.6 0.5 0.9 0.8 1.3 1.5 2.5 6.3 4.1 5.1 4.9

The percentage of fatty acid present in the seed as stearic acid, as determined by GC-FID analysis, varied significantly between lines across all eight plots. The average percentage of stearic acid across sites showed very little variability and ranged from 1.6 to 2.4%. The statistical variability in the percentage of stearic acid for the transgenic line was significant within the range represented by the non-transgenic lines across all sites.

The percentage of fatty acids in the seed, such as oleic and cis-vaccenic acid, as measured by GC-FID analysis, varied significantly between lines across all eight plots. The average percentage of oleic and cis-vaccenic acids for the sites varied from 60 to 73%. The variability in the percentage of oleic and cis-vaccenic acids for the transgenic line was significantly (P<0.05) lower than the range represented by non-transgenic lines across all plots. This result is related to the transgenic insertion and does not affect commercial agronomy or grain production. In addition, a specialized high oleic oil cultivar, Monola515 TT, produced significantly more oleic and cis-vaccenic acids compared to other cultivars due to single nucleotide polymorphisms (SNPs) in the Fad gene.

The percentage of fatty acid in the seed present as linoleic acid, as determined by GC-FID analysis, varied significantly between all lines across all eight plots. The average percentage of linoleic acid per site varied from 12 to 21%. The variability in percentage of linoleic acid for transgenic lines of sibs derived from a single T2 event (plant NS-B50027-4) was significantly (P<0.05) lower than the range represented by cultivars across all sites and sibs derived from sibs other events. A significant difference in the % content of linoleic acid, apparently, is associated with the expression of transgenes. The decrease in % linoleic acid appears to be related to the transgenic insertion but does not affect agronomy or industrial scale grain production.

The percentage of fatty acid present as ALA, arachidic acid and SDA varied significantly between all lines across all eight sites. The average percentage of ALA, arachidic acid and SDA varied from 5 to 11% by site. Variability in % content of ALA, arachidic acid and SDA for transgenic lines was significantly (P<0.05) higher compared to non-transgenic cultivars in all experiments. Significant differences observed for this trait at some sites were associated with the expression of transgenes. This result is related to the transgenic insertion and does not affect commercial agronomy or grain production. A specialized high oleic oil cultivar (Monola515 TT) produced a significantly (P<0.05) lower % ALA compared to other cultivars due to SNPs in the Fad genes.

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The percentage of fatty acid present as pollinic, gondoic and gadoleic acids varied significantly between lanes across all eight plots. The average percentage of pollinic, gondoic and gadoleic acids varied from 1.0 to 1.5%. The variability in the percentage of pollinic, gondoic and gadoleic acids for transgenic lines was significantly (P<0.05) higher compared to non-transgenic cultivars in all experiments. This result is related to the transgenic insertion and does not affect commercial agronomy or grain production.

The percentage of fatty acid present as erucic acid and ETA was recorded at five sites and generally approached 0%. The results associated with the transgenic insertion do not commercially affect agronomy or grain production.

Seed content of LC-ω3 polyunsaturated fatty acid (LC-PUFA), specifically EPA, DPA and DHA, was calculated as a percentage for each sample taken from the plot and analyzed as a trait variable, where lc-pufa=epa%+dpa %+dha%

The predicted DHA content in units of kg/ha was calculated for each plot and analyzed as a trait variable:

DHA, kg/ha=(% Oilχ0.01)χ(%DHAχ0.01)χGrain yield (t/ha)х1000

The predicted content of LC-PUFA in units of kg/ha was calculated for each plot and analyzed as a trait variable:

DHA, kg/ha=(% Oilχ0.01)х(LC-PUFA%χ0.01)хGrain yield (t/ha)х1000

The percentage of fatty acid as EPA varied significantly (P<0.05) between lanes across all eight plots. The percentage variability for the transgenic line was significantly (P<0.05) higher compared to the non-transgenic cultivars. This transgenic insertion outcome does not affect agronomy or seed production, but may indeed make seeds more valuable.

The percentage of fatty acid such as DPA varied significantly between lanes across all sites. Percentage variability for transgenic lines was significantly (P<0.05) higher compared to non-transgenic cultivars across all plots. This result is related to the transgenic insert and does not affect agronomy or grain production.

The percentage of fatty acid such as DHA varied significantly (P<0.05) between lanes across all eight sites. Percentage variability for transgenic lines was significantly (P<0.05) higher than for non-transgenic cultivars across all sites. This result is related to the transgenic insertion and does not affect commercial agronomy or grain production. Variation between transgenic sibling lines was used as a basis for selection.

The percentage of DHA by sites and comparison of the elite event NS-B5OO27-4 with non-transgenic cultivars (according to GC-FID analysis) are shown in Table. 4 (REML analysis; F pr<0.001 Sig for all sites).

Table 4

Average % DHA content (С22:6p3) on sites by cultivar/elite event

Line name Site: A IN WITH D E F G H Site average ATR Bonito 0.0 0.0 0.0 0.1 0.1 0.0 0.1 0.0 0 ATR Gem 0.0 0.0 0.0 0.1 0.1 0.1 0.1 0.0 0.1 ATR Stingray 0.0 0.1 0.2 0.3 0.0 0.1 0.1 0.0 0.1 ATR Wahoo 0.1 0.2 0.1 0.2 0.0 0.2 0.1 0.0 0.1 AV Garnet 0.0 0.0 0.0 0.2 0.1 0.2 0.2 0.0 0.1 AV Jade 0.0 0.3 0.5 0.0 0.2 0.1 0.3 0.0 0.2 A.V. Zircon 0.0 0.1 0.1 0.1 0.2 0.1 0.1 0.0 0.1 Monola 515TT 0.1 0.1 0.3 0.2 0.1 0.1 0.2 0.0 0.1 NS-B50027-4 8.1 9.8 7.8 7.5 8.5 8.4 8.8 10.2 8.6 B0050-027-18-36-13 10.0 12.2 9.5 9.7 10.1 10.4 10.8 13.3 10.8 В-050-27-18-105-13 10.5 11.3 9.0 9.6 10.2 9.7 10.9 12.5 10.5 Min value for varieties 0.0 0.0 0.0 0.0 0.0 0.0 0.1 0.0 0 NS-B50027-4 8.1 9.8 7.8 7.5 8.5 8.4 8.8 10.2 8.6 Max value for varieties 0.1 0.3 0.5 0.3 0.2 0.2 0.3 0.0 0.2 Average 3.5 3.6 3 2.49 3.7 3.56 3.88 4.1 VAR 0.15 0.07 0.079 0.29 0.14 0.14 0.11 0.18 SE 0.39 0.27 0.281 0.53 0.37 0.37 0.33 0.42 LSD 0.78 0.54 0.6 1.1 0.75 0.73 0.65 0.84 cv% eleven 7.5 9.4 21.5 10 10.6 8.4 10.4

Predicted DHA content, expressed in kg/ha, calculated from the fat profile

- 44 041446 acids, the percentage of oil in the seed and the yield of grain, varied significantly (P<0.05) between lines across all sites. Percentage variability for transgenic lines was significantly (P<0.05) higher than for non-transgenic lines across all sites. This result is related to the transgenic insertion and does not affect commercial agronomy or grain production. Variation between transgenic sibling lines was used as a basis for selection. High stability of DHA, expressed in units of production per unit area (kg/ha), was noted due to low site variability in seed oil content and percentage of DHA produced in seeds.

The predicted yield of DHA (kg/ha) by site and comparison of the elite event NS-B50027-4 with non-transgenic cultivars are shown in Table 1. 5 (REML analysis, F pr<0.001 Sig for all sites).

Table 5

Average predicted yield of DHA (kg/ha) at sites by lines

Line name Site: A IN WITH D E F G H Site average ATR Bonito 0 1 0 1 0 0 1 0 0 ATR Gem 0 0 0 0 2 2 2 1 1 ATR Stingray 0 0 0 1 0 0 0 0 0 ATR Wahoo 1 0 1 0 0 1 1 0 0 AV Garnet 0 1 2 1 1 4 1 2 1 AV Jade 0 0 1 0 1 0 0 0 0 A.V. Zircon 0 0 0 2 0 4 1 0 1 Monola 515TT 0 0 2 0 2 0 0 0 0 NS-B50027-4 thirty 28 39 36 50 49 24 41 37 B0050-027-18-36-13 25 27 41 41 52 53 24 36 37 В-050-27-18-105-13 24 25 34 50 46 40 26 33 35 Min value for variety 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 NS-B50027-4 30.0 28.0 39.0 36.0 50.0 49.0 24.0 41.0 37.0 Max value for variety 1.0 1.0 2.0 2.0 2.0 4.0 2.0 2.0 1.0 Average 8 8 12 12 16 17 8 12 VAR 3.93 3.08 14.60 38.66 10.77 24.55 7.60 6.94 SE 1.98 1.75 3.81 6.20 3.28 4.79 2.75 2.63 LSD 4.0 3.5 7.6 12.4 6.6 9.6 5 5 5.3 cv% 23.6 22.5 33.1 53.4 20.3 29.8 34.2 22.7

With regard to omega-3 LC-PUFA seed content - EPA, DPA and DHA percentage (measured by HPLC) - LC-PUFA percentage varied significantly (P<0.05) between lines across all eight plots. Variability in % content for transgenic lines was significantly (P<0.05) higher compared to cultivars in all experiments. This result is related to the transgenic insertion and does not affect agronomy or commercial grain production. Variation between transgenic sibling lines was used as a basis for selection. Trace amounts of LC-PUFA observed in non-transgenic lines were likely due to pollen flow, seed movement, or GC-FID error.

In table. 6 shows the percentage values determined by the GC-FID method (REML analysis; F pr<0.001 Sig for all sites).

Table 6

Site by cultivar or transgenic line: average content of LC-PUFA in seeds (% sum of EPA+DPA+DHA)

Line name Site: A V c D E F G H Average no sites ATR Bonito 0.0 0.0 0.0 0.1 0.1 0.0 0.2 0.2 0.1 ATR Gem 0.0 0.0 0.0 0.1 0.2 0.1 0.1 0.1 0.1 ATR Stingray 0.0 0.1 0.2 0.3 0.0 0.1 0.1 0.5 0.2 ATR Wahoo 0.1 0.2 0.1 0.2 0.0 0.2 0.1 0.2 0.1 AV Garnet 0.0 0.0 0.0 0.1 0.1 0.2 0.2 0.0 0.1 AV Jade 0.0 0.4 0.5 0.0 0.2 0.1 0.3 0.0 0.2 A.V. Zircon 0.0 0.2 0.1 0.1 0.2 0.1 0.1 0.3 0.1 Monola 515TT 0.1 0.1 0.3 0.2 0.2 0.1 0.2 0.0 0.2

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NS-B50027-4 9.5 11.4 9.1 8.9 9.8 9.8 10.3 11.8 10.1 В0050-027-18-36-13 11.7 14.2 11.0 11.4 11.8 12.2 12.7 15.3 12.5 В-050-27-18-105-13 12.2 13.2 10.5 11.3 11.9 11.3 12.7 14.5 12.2 Min value for variety 0.0 0.0 0.0 0.0 0.0 0.0 0.1 0.0 0.0 NS-B50027-4 9.5 11.4 9.1 8.9 9.8 9.8 10.3 11.8 10.1 Max value for variety 0.1 0.4 0.5 0.3 0.2 0.2 0.3 0.5 0.2 Average 4.3 4.4 3.6 3.05 4.5 4.34 4.75 4.9 VAR 0.23 0.11 0.112 0.33 0.19 0.2 0.14 0.23 SE 0.48 0.33 0.334 0.58 0.44 0.43 0.38 0.48 LSD 0.95 0.66 0.7 1.2 0.87 0.87 0.76 0.96 cv% eleven 7.6 9.3 18.9 9.7 10.3 8 9.8

The predicted LC-PUFA content, expressed in kg/ha, calculated from the fatty acid profile, % seed oil content, and grain yield, varied significantly (P<0.05) between treated lines across all sites. Variability in % content for transgenic lines was significantly (P<0.05) higher compared to cultivars in all experiments. This result is due to the transgenic insertion and has no commercial impact on agronomy or grain production. Variation between transgenic sibling lines was used as a basis for selection. Trace amounts in cultivar seeds appear to be related to pollen flow, seed movement, or HPLC error. High stability of LC-PUFA, expressed in units of production per unit area (kg/ha), was noted due to low site variability in seed oil content and the percentage of DHA produced in seeds. See also FIG. 2 and 3.

In table. 7 shows the predicted content of LC-PUFU in kg/ha (F ρκΟ,ΟΟί for all sites).

Table 7

Average predicted content of LC-PUFA (kg/ha) in seeds at sites by cultivars (lines)

Line name Site: A IN C D E F G H Average no site ATR Bonito 0 1 0 1 0 0 1 0 0 ATR Gem 1 0 0 0 2 3 3 0 1 ATR Stingray 1 0 0 1 0 0 0 0 0 ATR Wahoo 0 0 2 0 0 1 1 0 1 AV Garnet 0 1 2 1 1 4 1 0 1 AV Jade 0 1 1 0 1 0 0 0 0 A.V. Zircon 0 0 0 2 0 4 2 0 1 Monola 515TT 0 0 2 0 2 0 0 0 1 NS-B50027-4 35 33 46 43 58 58 28 46 43 В0050-027-18-36-13 29 32 48 48 60 61 28 41 43 В-050-27-18-105-13 28 29 40 58 53 46 thirty 40 41 Average 10 10 14 14 20 20 10 14 VAR 5.50 4.34 21.71 54.48 15.98 34.93 11.14 58.10 SE 2.34 2.08 4.65 7.36 3.99 5.71 3.33 4.04 LSD 4.7 4.2 9.3 14.7 8.0 11.4 6.7 8.1 cv% 22.6 21.9 33.1 51.9 20.1 28.9 33.5 54.8

The oil content of the seeds, determined by NMR, was also tabulated for each of the cultivation sites and presented in table. 8 (in percent; REML analysis; F ρκΟ,ΟΟί Sig for all sites).

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Table 8

Average oil content in seeds (%) on sites by cultivars

Line: Area: A IN WITH D E F G H Site average ATR Bonito 43.0 40.6 43.6 43.4 42.6 41.8 39.1 39.6 41.7 ATR Gem 43.7 40.7 43.9 42.7 42.3 40.5 37.8 39.5 41.3 ATR Stingray 41.4 39.7 42.6 42.8 41.1 39.1 38.9 39.9 40.6 ATR Wahoo 43.1 41.0 43.3 43.4 42.5 40.1 39.0 39.9 41.4 AV Garnet 43.9 39.9 43.4 41.8 41.5 39.1 36.5 36.4 40.2 AV Jade 41.7 40.2 42.6 42.2 42.5 38.6 37.5 39.4 40.6 A.V. Zircon 44.0 40.6 43.6 42.1 41.5 39.8 36.9 38.8 40.9 Monola 515TT 42.2 40.1 42.3 42.1 42.2 38.8 38.1 39.3 40.6 NS-B50027-4 38.7 36.1 39.3 38.2 37.3 36.5 34.2 35.1 36.9 B0050-027-18-36-13 36.7 34.3 37.2 37.9 36.8 34.3 33.4 33.2 35.5 B-050-27-18-105-13 36.3 34.4 37.9 37.4 35.8 34.1 32.1 33.0 35.1 Min value for cultivar 41.4 39.7 42.3 41.8 41.1 38.6 36.5 36.4 40.2 NS-B50027-4 38.7 36.1 39.3 38.2 37.3 36.5 34.2 35.1 36.9 Max value for cultivar 44.0 41.0 43.9 43.4 42.6 41.8 39.1 39.9 41.7 Average 40.8 38.7 41.5 41.2 39.8 38.01 37 37.8 VAR 0.13 0.09 0.23 0.13 0.13 0.19 0.18 0.1 SE 0.36 0.3 0.477 0.36 0.36 0.42 0.43 0.31 LSD 0.71 0.59 1 0.7 0.73 0.85 0.85 0.62 cv% 1 0.8 1.2 0.9 0.9 1.1 1.2 0.8

Additional analysis of the content of fatty acids in the seeds of NS-B50027-4 is presented in table. 9.

Table 9

Detailed Fatty Acid Content of Seed NS-B50027-4

NS-B50027-4, Generation T7, Summer 2015-2016 GLA ALA SDA C14:0 С16:0 S16:1p7s С18:0 S18:1p9s S18:1p7s S18:2p6s S18:3p6 S18:ZpZ S20:0 S18:4pZ S20:1p9s 1* 0.05 4.33 0.24 2.16 38.83 4.26 7.81 0.58 21.54 0.64 2.20 1.31 2 0.05 4.28 0.23 2.19 38.32 4.17 7.76 0.59 21.58 0.65 2.21 1.31 3 0.05 4.20 0.22 2.19 38.77 4.06 7.81 0.60 21.73 0.66 2.22 1.28 4 0.05 4.19 0.21 2.16 38.69 4.09 7.79 0.61 21.66 0.63 2.25 1.34 5 0.05 4.26 0.21 2.18 38.35 4.22 7.81 0.59 21.78 0.64 2.25 1.29 0.05 4.25 0.22 2.18 38.69 4.16 7.80 0.60 21.66 0.64 2.23 1.30 NS-B50027-4, Generation Tb, Winter 2015 1 0.0 4.60 0.21 2.22 41.95 3.10 6.35 0.47 21.06 0.69 2.27 1.14 2 0.0 5.00 0.25 2.01 36.02 3.50 6.70 0.66 21.46 0.66 3.21 1.10 3 0.09 4.67 0.24 2.32 34.45 3.41 6.33 0.60 22.53 0.71 3.35 1.01 4 0.0 4.57 0.20 2.01 34.27 3.08 6.47 0.59 23.14 0.65 3.24 1.08 5 0.0 5.08 0.30 2.22 36.51 3.99 6.55 0.57 21.59 0.72 3.41 1.06 0.02 4.78 0.24 2.15 36.64 3.42 6.48 0.58 21.95 0.69 3.10 1.08 NS-B50027-4, Generation T5, Summer 2014-2015 I 0.05 4.51 1 0-21 2.05 | 39.18 I 4.10 | 8.67 | 0.66 | 21.93 0.60 1.86 1.38 NS-B50027-4, Generation T4, Winter 2014 I 0.17 3.93 | 0.16 2.14 44.54 2.65 | 7.02 θ 45 19-4 0.64 2.21 1.26 *Sample number

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NS-B50027-4, Generation T7, Summer 2015-2016 (continued) DGLA ETE ETA ERA DPA6 DPA3 DHA С21:0 S20:Zp6 S20:ZpZ C22:0 S20:4p3 S22:1p9s S20:5pZ С24:0 S22:5p6 S24:1p9s S22:5pZ S22:6pZ 1* 0 0 0.71 0.31 0 0 0.40 0.24 0.10 0.85 9.69 2 0 0 0.70 0.32 0 0 0.39 0.23 0.11 0.88 9.78 3 0 0 0.72 0.32 0 0 0.40 0.23 0.09 0.89 9.83 4 0 0 0.71 0.33 0 0 0.41 0.23 0.08 0.91 9.92 5 0 0 0.72 0.32 0 0.01 0.41 0.23 0.10 0.89 9.80 0 0 0.71 0.32 0 0 0.40 0.23 0.10 0.88 9.80 NS-B50027-4, TB Generation, Winter 2015 (continued) 1 0 0 0.59 0.34 0 0 0.55 0 0.09 0 0.89 10.22 2 0 0 0.47 0.33 0 0 0.68 0 0.11 0.10 1.21 13.34 3 0 0 0.60 0.33 0 0 0.80 0 0 0.09 1.13 14.02 4 0 0 0.63 0.38 0 0 0.71 0.14 0.10 0.10 1.07 13.99 5 0 0 0.52 0.37 0 0 0.60 0.11 0 0.13 1.06 12.10 0 0 0.56 0.35 0 0 0.67 0.05 0.06 0.08 1.07 12.73 NS-B50027-4, Generation T5, Summer 2014-2015 (continued) 1 | 0.14 1 0 0.83 0.33 0 0 0.32 | 0.8 I 0.16 I 0.13 1 0-71 1 8.43 NS-B50027-4 Generation T4 Winter 2014 (continued) 1 1 1 0-07 0.46 | 0.26 1-09 0 θ 41 1 1 1 | 0.85 I 8.89 *Sample number NS-B50027-4, Generation T7, Summer 2015-2016 (continued) NMR oil Amount of EPA, DPA, DHA General Ω3 General Ω6 Ω3/Ω6 total saturated fat total monounsaturated fat General PUFAs 1* 39.3 10.94 35.39 8.39 4.22 7.74 44.74 43.78 2 38.8 11.04 35.54 8.36 4.25 7.41 44.62 43.89 3 39.2 11.12 35.79 8.41 4.26 7.64 44.41 44.20 4 39.5 11.23 35.85 8.40 4.27 7.59 44.41 44.25 5 39.4 11.10 35.85 8.40 4.27 7.68 44.19 44.25 11.09 35.68 8.39 4.25 7.67 44.48 44.07 NS-B50027-4, TB Generation, Winter 2015 (continued) 1 11.66 35.58 6.91 5.15 7.85 46.40 42.48 2 15.23 40.37 7.47 5.41 7.99 40.96 47.84 3 15.95 42.42 6.93 6.12 8.11 39.19 49.35 4 15.76 42.78 7.17 5.97 7.75 38.73 59.94 5 13.75 39.27 7.12 5.52 8.49 41.98 46.38 14.47 40.08 7.12 5.63 8.04 41.45 47.20 NS-B50027-4, Generation T5, Summer 2014-2015 (continued) I 1 9.46 | 34.09 | 9.49 1 3.59 I 7.76 | 45.00 I 43.58 *Sample number

The data in the table. 9 confirm that, in addition to LC-ω3 fatty acids, NS-B50027-4 seeds also contain significantly higher amounts of ALA compared to conventional canola varieties. See also table. 3. Although ALA is not an LC-PUFA, it is a ω3 fatty acid. The ratio of fatty acids ω3:ω6 in NS-B5OO27-4 seed oil in Table. 9 is in the range of about 3.59 to about 6.12; the fatty acid ratio ω3:ω6 in conventional canola oil is about 0.5. See Patterson et al., J. Nutr. Metab, 2012:539426 (2012).

In table. 10 shows data relating to the percentage of DHA and LC-PUFA in seeds from sixteen generations of elite event NS-B50027-4 grown in experimental sites in Australia. Additional field testing in Australia yielded bulk seeds with 9.6% DHA and 10.1% LC-PUFA.

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Table 10

DHA% and LC-PUFA% in seeds from elite event NS-B50027-4 per generation

Generation seed sample Wednesday growing season Year in the field Area DHA% in seeds LC-PUFA% in seeds 1 T1 one plant Glass greenhouse Controlled environment A 5.7 6.0 2 T2 one plant Glass greenhouse Controlled environment A 9.5 10.1 3 TK one plant glass greenhouse Controlled environment A 12.6 13.1 4 TK-x Volumetric Insulating awning Winter spring 2014 IN 8.9 10.2 5 T3-2x Volumetric Open field Summer 2014-15 WITH 8.4 9.5 6 TZ-Zx Volumetric Open field Winter summer 2015 D 9.0 10.6 7 T4 one plant glass greenhouse Controlled environment A 11.9 13.2 8 T5 one plant glass greenhouse Controlled environment A 13.4 14.6 9 T5's Volumetric Insulating awning Winter spring 2015 IN 12.7 14.5 10 T5-2x Volumetric Open field Summer 2015-16 WITH 9.8 11.1 eleven T5-Zx Volumetric Open field Winter spring 2016 D 9.6 10.6 12 Tb one plant glass greenhouse Controlled environment A 12.9 14.4 13 tb-x Volumetric Insulating awning Summer 2015-16 WITH 17.3 18.8 14 T6-2x Volumetric Insulating awning Winter spring 2016 E 10.1 12.1 15 T7 one plant glass greenhouse Controlled environment A 13.8 15.1 16 T7's Volumetric Insulating awning Winter spring 2016 IN 12.5 14.1

In addition, the ability to grow NS-B50027-4 in Canada was tested under controlled experimental conditions at two different sites in 2016. 11 shows a comparison of agronomic and NS-B5OO27-4 yield data from several non-transgenic canola lines.

Table 11

Agronomic measurement data for non-transgenic canola cultivars and experimental transgenic test lines from two experimental sites in Canada in 2016.

Feature: Germination Height rast. in comp. maturity Beginning of flowering End of flowering ί 1 e £ ς o N-G 03 t-g _a Lodging in comp. maturity cracked seeds Number of plants when collecting Symptoms of Alternariosis Set to black leg plant power Grain moisture % Garnet in Zepna Grain output Line name Rust per m ² cm Day Day days Score (1-9) Number Rust per m ² Score (1-9) Score (1-9) Score (1-9) % % t/ha ATR Bonito 23 90 49 75 26 8 3 24 2 6 6 8 76 1.9 ATR Gem 22 98 48 76 27 7 2 23 3 6 6 8 79 2.0 ATR Stingray 21 88 48 75 27 9 6 26 3 7 5 6 61 1.7 ATR Wahoo 22 98 48 75 27 7 4 26 2 7 6 10 87 2.2 AV Garnet 27 BY 48 76 28 6 7 23 3 7 6 8 100 2.6 AV Jade 27 109 48 76 29 8 3 27 2 7 7 7 85 2.1 A.V. Zircon 14 125 50 76 26 8 9 18 1 7 5 8 101 2.6 Monola 515TT 26 79 47 73 27 8 13 25 2 6 6 6 60 1.6 DK 7444 21 112 47 72 25 7 4 23 2 7 7 5 113 2.8 LL 130 18 123 47 73 26 8 4 21 2 7 6 6 114 2.9 NS-B50027-4 T3 AND 109 49 77 28 8 4 16 2 8 4 8 80 2.1 NS-B50027-4 T5 16 111 49 76 27 8 2 18 2 8 6 9 82 2.3 Min value for variety 14 79 47 72 25 6 2 18 1 6 5 5 60 1.7 NS-B50027-4 14 BY 49 76 28 8 3 18 2 8 5 8 81 2.2 Max value for variety 27 125 50 76 29 9 13 26 3 7 7 10 114 2.9

Since the canola line NS-B50027-4 is essentially homogeneous, it can be reproduced by sowing seeds of this line, growing the resulting canola plants under self-pollination conditions, or

- 49 041446 pollination of siblings under conditions of proper isolation, and collection of the resulting seeds using traditional agronomic methods.

Example 2 Segregants NS-B50027-4.

As noted above, NS-B50027-4 contains a sixteen-gene insert (two inverted cassettes of eight genes each) and a four-gene insert; each insert represents a separate locus in the plant genome. The combination of crossing, backcrossing and self-crossing cleaved off a sixteen-gene insertion on the A05 chromosome (A05 segregant locus) and a four-gene insertion on the A02 chromosome (segregant A02 locus). Agronomic data for these segregants were compared with NS-B5OO27-4 and non-transgenic cultivars grown at four different experimental sites in Australia. The summarized data are shown in Table. 12, which shows mean values from four experimental sites (REML analysis; F pr<0.001 Sig for all traits).

Table 12

Agronomic measurement data for non-transgenic canola cultivars and experimental transgenic test lines from four experimental sites in 2016

Feature: Germination Germination Height rast. in comp. maturity Beginning of flowering End of flowering Flowering time Lodging in comp. maturity cracked seeds Number of plants at plant power Line name Rast, per m ² Score (1-9) cm Day Day days Score (1-9) no. Rast, per m ² Score (1-9) ATR Bonito 15.7 7.5 110.7 102.1 146.9 44.8 4.6 30.6 12.8 6.1 ATR Gem 17.7 7.6 115.4 103.0 148.9 46.0 4.5 40.4 14.0 6.5 ATR Stingray 18.9 7.3 100.9 100.7 147.2 46.5 3.4 74.6 15.4 5.4 ATR Wahoo 17.4 7.7 119.3 104.9 149.8 44.9 4.3 34.6 13.0 6.2 AV Garnet 18.8 7.7 126.9 101.7 148.5 46.7 4.8 41.8 13.9 7.1 AV Jade 17.2 7.7 117.6 100.0 147.9 47.9 4.9 50.9 16.1 7.4 A.V. Zircon 14.3 7.4 119.8 102.7 147.3 44.6 4.8 71.4 12.5 7.2 Monola 515TT 21.7 7.9 115.4 105.9 149.5 43.6 3.3 92.1 17.3 5.7 NS-B50027-4 T3 12.8 6.3 120.7 106.5 149.6 43.2 2.8 45.0 10.7 5.2 NS-B50027-4 T5 17.2 7.6 111.3 106.5 149.2 43.1 1.8 40.2 12.9 5.9 Locus A05 segregant 15.7 7.2 94.9 102.3 150.1 47.7 1.9 27.6 15.1 4.8 Locus A02 segregant 16.5 7.7 123.9 103.5 148.8 45.3 3.3 38.3 14.5 6.9 Min value for variety 14.8 7.3 100.9 100.0 146.9 43.6 3.3 30.6 12.5 5.4 NS-B50027-4 15.0 7.0 116.0 106.2 149.4 43.2 2.3 40.4 11.8 5.6 Max value for variety 21.7 7.9 126.9 105.9 150.1 47.7 4.9 92.1 17.3 7.4

In addition to plant vigor and other agronomic characteristics, grain yield (t/ha) was also characterized, as shown in Table 1. 13, in which the percentage of oil was determined by NMR, and the AV Garnet variety used as a comparison line was taken as 100% in terms of seed oil content.

Table 13

Grain yield (t/ha) and seed characterization data for non-transgenic canola cultivars and experimental transgenic test lines from four experimental sites in 2016

Feature: Grain output Grain moisture at harvest seed oil Oil at 6% humidity Line name: t/ha % % o/ /o ATR Bonito 3.46 8.3 86.8 46.8 ATR Gem 3.48 9.7 87.0 46.4 ATR Stingray 3.61 6.7 90.2 46.3 ATR Wahoo 3.76 10.9 93.8 46.6 AV Garnet 4.00 8.0 100.0 45.8 AV Jade 3.90 6.8 97.8 47.5 A.V. Zircon 3.73 6.8 93.1 47.8 Monola 515TT 3.16 9.3 79.2 45.2 NS-B50027-4 T3 2.96 10.0 74.1 42.8 NS-B50027-4 T5 2.83 9.8 70.7 41.4 Locus A05 segregant 3.10 11.6 77.1 42.2 Locus A02 segregant 4.21 10.0 105.2 46.8 Min value for variety 3.26 6.8 79.2 45.2 NS-B50027-4 2.90 9.9 72.4 42.0 Max Max value for variety 4.00 10.9 100.0 47.8

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The site average DHA and LC-PUFA for NS-B50027-4 T3 was about 6.2% for DHA and about 7.1% for LC-PUFA, and the site average DHA and LC-PUFA for NS-B50027- 4 T5 was about 7.4% for DHA and about 8.6% for LC-PUFA, indicating a genetic effect in generations from T3 to T5.

In addition, with respect to the segregant A02 locus, this transgenic line, derived from NS-B50027-4, contains the following four transgenes: A6-desaturase (derived from M. pusilla), A5-elongase (derived from P. cordata), A5- desaturase (originating from P. salina); and A15/m3 desaturase (originating from P. pastoris). Although this line produces seed oil, the A02 locus of the segregant lacks the enzymes necessary for the production of LC-PUFA and DHA (see FIG. 1). In particular, it lacks A12 desaturase, which provides the early substrate, LA, A6 elongase, which converts SDA to ETA, and A4 desaturase, which converts DPA to DHA. As expected, regulatory field trials showed that the segregant's A02 Locus produced less than 1% LC-PUFA (EPA+DPA+DHA). In comparison, the A05 locus of the segregant contains a complete set of enzymes for DHA biosynthesis, and regulatory field tests have shown that it produced about 4% DHA in about 4.5% LC-PUFA. Surprisingly, in the same regulatory field trial, the NS-B50027-4 line produced about 7.4% DHA in about 8.4% LC-PUFA (this trial showed a sub-optimal collection). That the NS-B50027-4 line produced significantly more DHA than the segregant's A05 locus is surprising since, for example, the segregant's A02 locus does not provide the additional A4 desaturase that converts DPA to DHA. The A02 locus of the segregant provides the potential for yield enhancement.

The A02 and A05 loci can be segregated from NS-B50027-4 and used to further generate progeny derived from NS-B50027-4. For example, the segregated A02 locus can be packaged into other LC-PUFA producing lines, such as the Full Single line containing one insert of seven transgenes for DHA biosynthesis to increase LC-PUFA production in that line. Indeed, seeds of homozygous F4 progeny bundled by introgression of the A02 locus into a Full Single recipient showed a 6% increase in DHA compared to the Full Single recipient line. Interestingly, this homozygous progeny containing the A02 locus produced more DHA than the homozygous Full Single F4 line bundled with the segregated A05 locus. Of note, the NS-B50027-4 line produced more DHA than the Full Single line, and Full Single bundled NS-B50027-4 (both loci) also showed an increase in DHA production compared to Full Single.

The A02 NS-B50027-4 locus can be introgressed into recurrent Brassica or canola that produces other LC-PUFAs such as EPA or DPA to increase LC-PUFA production in that Brassica or canola. For example, a transgenic plant, B. juncea, has been described that produces significant amounts of DPA in seeds. WO 2015089587. Introgression of the segregated A02 locus NS-B50027-4 into this B. juncea plant increased the amount of DPA produced in seeds. Similarly, a Brassica plant biosynthetically transformed to produce EPA (eg, transgenic insert(s) containing neither A5 elongase nor A4 desaturase) produces significant amounts of EPA in seeds; and EPA production was increased by introgressive hybridization with the A02 locus of NS-B50027-4.

Example 3 Comparative allele-specific PCR analysis (KASP).

The phenotypic expression of transgenes in canola is determined both by the structure of the transgene cassette itself and by the localization of its insertion in the plant genome: the presence of transgenes in certain localizations in the plant genome can affect transgene expression and the overall plant phenotype. The incorporation of a recombinant DNA molecule into the plant genome usually occurs as a result of cell or tissue transformation (or other genetic manipulation). The specific inclusion site(s) may be random or pre-defined (if a targeted integration process is used). Agronomically or industrially successful introduction of a trait of commercial interest into a plant by genetic manipulation can be a lengthy procedure, depending on various factors. The actual transformation and regeneration of genetically transformed plants is only the first in a series of selection steps that involve extensive genetic research, breeding and evaluation in field trials, which ultimately leads to the selection of an elite event.

The NS-B50027-4 line was developed after extensive breeding and field trials and provides a canola cultivar that produces about 7-15% DHA (wt% of total fatty acids) in the seed oil. Genetic analysis showed that the NS-B50027-4 line has a transgenic insert on the A02 chromosome and another transgenic insert on the A05 chromosome. Insert on A05 contains two complete T-DNA-restricted cassettes of eight genes (Micpu-A6D, Pyrco-A5E, Pavsa-A5D, Picpaω3D, Pavsa-A4D, Lackl-A12D, Pyrco-A6E and PAT marker) in head-to-orientation -head (RB-LB:LB-RB). The insert on chromosome A02 contains a set of four Micpu-A6D, Pyrco-A5E, Pavsa-A5D, and Picpaω3D genes. Surprisingly, cross segregation showed that insertions on chromosome A02 and chromosome A05 were required to achieve a DHA production of about 11%.

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Approximately 1200 introgressively bred progeny from eight different BC and F ₂ populations of DHA-rich canola were used for DNA extraction based on LGC Octopure's standard operating procedure (SOP) developed at the Nuseed Molecular Lab in Woodland. Briefly, two 0.25 inch disks of lyophilized leaves were ground in 300 µl of DNA isolation buffer (100 mM Tris-HCl, pH 8.0; 25 mM EDTA, pH 8.0; 0.5% SDS, 1.5 M NaCl ) at 1400 rpm for 8 min using the GenoGrinder. After incubation in a water bath at 55°C for 45 min and centrifugation at 4500 rpm for 30 min, 50 µl of the supernatant was transferred into 100 µl of LGC binding buffer with sbeadex magnetic particles. After binding and washing, the DNA was eluted in 80 μl of DNA elution buffer from LGC.

The DNA concentration was measured on a NanoDrop 8000 instrument (Thermo Scientific), which ranged from 5.0-20.0 ng/µl with an average value of 10.0 ng/µl. DNA samples were diluted 1x. For each reaction, 2.0 µl (~5.0 ng/µl) of the genomic DNA sample and 2 µl of primer master mix were added to the wells of a 384-well KASP genotyping plate.

In addition to progeny from introgressed DHA-rich canola populations, eight controls were included in genotyping. They included two non-GMO controls (Dwarf and AV Jade), two hemizygous controls (2.5 ng Av Jade or 2.5 ng Dwarf + 2.5 ng B0050-027-18-2012-19); two positive event controls (B0050-027-18-20-12-19) and four non-template controls (NTC). A positive control (plant T5 B0050-027-18-20-12-19) was previously used to characterize a canola DHA event by sequencing.

The KASP assays were designed to provide a simple, cost-effective, high throughput and flexible way to detect and monitor eight transgenes and four NS-B50027-4 specific compounds, and to further facilitate NS-B50027-4 introgression in breeding programs. KASP™ genotyping chemistry, assay design, genotyping and evaluation were performed according to the manufacturer's standard protocol (LGC Ltd., Middlesex, UK) with modifications.

Sequence information was loaded into the LGC Kraken Workflow Manager and KASP assays were designed using its Primer Picker assay design software. A typical KASP assay includes two allele-specific primers (Primer_Allele X for the transgenic allele and Primer_Allele Y for the wild-type non-transgenic allele) and one common locus-specific primer (PrimerCommon). Primer_Allele X is bound to the FAM fluorescent dye, and Primer_Allele Y to the HEX fluorescent dye.

Most of the assays targeting junction sites were this type of assay with three primers (Table 14). Four primer assays have also been developed for the detection of DHA-enriched canola, in addition to the conventional three primer assays mentioned above. Four primer assays included transgenic allele-specific Primer_Allele X, wild-type allele-specific PrimerAllele Y, PrimerCommon, specific for the Omega 3 gene, and wild-type specific PrimerCommon 2 in reaction. To detect the eight genes in the Omega 3 cassette, only two primers, Primer_Allele X and Primer_Common, were used in each assay (two primer assay); both primers were specific for the Omega-3 gene (Table 14).

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Table 14

Primer sequences from 28 KASP assays for marker-assisted detection and selection (MAS) of DHA-enriched canola

ID Target Primer name SEQID NO Primer sequence (5'-3') NBN01 Micpu-A6D Primer_Allele X NO:1 GAAGGTGACCAAGTTCATGCTCCAAGCA CCGTAGTAAGAGAGCA PrimerCommon NO:2 GCTAAGAAGTGGGGACTCAACTACAA NBN02 Rugso-L5E Primer_Allele X NO:3 GAAGGTGACCAAGTTCATGCTGCTCTTGC TGGAACTCTTGG PrimerCommon NO:4 GGGTTAGCCACATTGTAGGTAACGTA NBN03 Pavsa-A5D Primer_Allele X NO:5 GAAGGTGACCAAGTTCATGCTTAAGAGA CACCCTGGTGGAAAGA PrimerCommon NO:6 TAGCATCAGTTCCAACTTGGTAAGCAAT NBN04 Picpa-o3D Primer_Allele X NO:7 GAAGGTGACCAAGTTCATGCTGAACACG TAAGCAGACCAAGCAG PrimerCommon NO:8 CCCTCTTCTCCCTAACGAATTCCT NBN05 Pavsa-A4D Primer_Allele X NO:9 GAAGGTGACCAAGTTCATGCTGAGGAAC CTGTTGCCTGCTGATGA PrimerCommon NO: 10 GCGATCCTAGCACAAAGTTGAAGGTA NBN06 Lackl-A12D Primer_Allele X NO:11 GAAGGTGACCAAGTTCATGCTGGATGGA TCGCTTACCTCTTCGT PrimerCommon NO:12 CAGGGTAAGGTTGTCCTGTAACGTT NBN07 Rugso-L6E Primer_Allele X NO:13 GAAGGTGACCAAGTTCATGCCTCTATTGGA TGGGGACTCAAGC Primer_Common NO:14 GGGAGATTCCTTAGTAGCAGAAGAGAT NBN08 PAT Primer_Allele X NO:15 GAAGGTGACCAAGTTCATGCTCCTGAGA GGCGTCCTGTTGAAAT PrimerCommon NO:16 AACAGCAGCCATATCAGCAGCAGTA NBN57 Connection area upstream insert A02 Primer_Allele X NO:17 GAAGGTGACCAAGTTCATGCTGCCTTCAG TTTAAACTATCAGTGTTTGA PrimerAllele Y NO:18 GAAGGTCGGAGTCAACGGATTGTTCTGTA TACAACTTGTCGTGCTAC PrimerCommon NO:19 GGGTTGTGTGTGAAAACGTGTGAGCAA NBN68 Connection area upstream insert A02 Primer_Allele X NO:20 GAAGGTGACCAAGTTCATGCTAAAACTATC AGTGTTTGAACACCTC Primer Allele Y NO:21 GAAGGTCGGAGTCAACGGATTACAACTT GTCGTGCTACACACCT PrimerCommon NO :22 GACAAGTGAATCTGTTTGGGGTTG NBN58 Connection area upstream insert A02 Primer_Allele X NO:23 GAAGGTGACCAAGTTCATGCTGCCTTCAG TTTAAACTATCAGTGTTTGA Primer Allele Y NO :24 GAAGGTCGGAGTCAACGGATTGTTCTGTA TACAACTTGTCGTGCTAC PrimerCommon NO:25 GAAAACGTGTGAGCAATTGTTGGAGGT NBN85 Connection area upstream insert A02 Primer_Allele X NO:26 GAAGGTGACCAAGTTCATGCTGCCTTCAG TTTAAACTATCAGTGTTTGA Primer Allele Y NO:27 GAAGGTCGGAGTCAACGGATTGTTCTGTA TACAACTTGTCGTGCTAC PrimerCommon NO:28 GACAAGTGAATCTGTTTGGGGTTG NBN14 Connection area upstream insert A02 Primer_Allele X NO:29 GAAGGTGACCAAGTTCATGCTACAACTTG TCGTGCTACACACCT Primer Allele Y NO:30 GAAGGTCGGAGTCAACGGATTAAACTAT CAGTGTTTTGAACACCTCC PrimerCommon NO:31 GGTTGTGTGAAAACGTGTGAGC NBN15 Connection area Primer_Allele X NO:83 GAAGGTGACCAAGTTCATGCTCTTTTAGC TAAATAAGAGGTTCTGTATACT

- 53 041446

upstream inserts A02 PrimerAllele Y NO:84 GAAGGTCGGAGTCAACGGATTCTTTTAGC TAAATAAGAGGTTCTGTATACA PrimerCommon NO:85 GATTGTGATTCCGGGCAGT Primer_Common2 NO:86 GTGTGAAAACGTGTGAGCAAT NBN16 Connection area downstream insert A02 Primer_Allele X NO:32 GAAGGTGACCAAGTTCATGCTTGTGAGCA ATTGTTGGAGGT Primer Allele Y NO:33 GAAGGTCGGAGTCAACGGATTTTGTGATT CCGGGCAGTAG PrimerCommon NO:34 TCTTATCAACATTAAGAACATAATCTTTT AG NBN62 Connection area downstream insert A02 Primer_Allele X NO35 GAAGGTGACCAAGTTCATGCTTTTAGCTA AATAAGAGGTTCTGTATACT Primer Allele Y NO:36 GAAGGTCGGAGTCAACGGATTCTTTTAGC TAAATAAGAGGTTCTGTATACA PrimerCommon NO:37 CAGGGATTGTGATTCCGGGCAGTA Primer_Common2 NO:38 GTGTGAGCAATTGTTGGAGGTGTGTA NBN64 Connection area downstream insert A02 Primer_Allele X NO:39 GAAGGTGACCAAGTTCATGCTCCGGGCA GTAGTAATTAATAATATAGTATTA Primer Allele Y NO:40 GAAGGTCGGAGTCAACGGATTGGAGGTG TGTAGCACGACAAGTT PrimerCommon NO:41 CTCAAACTTCTTATCAACATTAAGAACAT A NBN52 Connection area upstream insert A05 Primer_Allele X NO :42 GAAGGTGACCAAGTTCATGCTGCCTTCAG TTTAAACTATCAGTGTTTG Primer Allele Y NO:43 GAAGGTCGGAGTCAACGGATTCACGGTG GAGGTCACCATGT PrimerCommon NO :44 CACGGAGATAGGCTGCATCTGAAT NBN51 Connection area upstream insert A05 Primer_Allele X NO:45 GAAGGTGACCAAGTTCATGCTGCCTTCAG TTTAAACTATCAGTGTTTG Primer Allele Y NO:46 GAAGGTCGGAGTCAACGGATTCACGGTG GAGGTCACCATGT PrimerCommon NO:47 GCTGCATCTGAATGACTGGTGTGTT NBN09 Connection area upstream insert A05 Primer_Allele X NO:48 GAAGGTGACCAAGTTCATGCTGTGTTTCTT GGGTGGGTCTGTCCTTA Primer Allele Y NO:49 GAAGGTCGGAGTCAACGGATTTGTTCTTG GGTGGGTCTGTCCTTC PrimerCommon NO:50 GTTGGCTAAGGTCACGGTGGAG Primer_Common2 NO:51 ATCCACTAGCAGATTGTCGTTTCCC NBN50 Connection area upstream insert A05 Primer_Allele X NO:52 GAAGGTGACCAAGTTCATGCTTCTTGGGT GGGTCTGTCCTTC Primer Allele Y NO53 GAAGGTCGGAGTCAACGGATTGTTCTTGG GTGGGTCTGTCCTTA PrimerCommon NO:54 GATTGTCGTTTCCCGCCTTCAGTTTT Primer_Common2 NO55 CGTTGGCTAAGGTCACGGTGGA NBN48 Connection area upstream insert A05 Primer_Allele X NO:56 GAAGGTGACCAAGTTCATGCTTCTTGGGT GGGTCTGTCCTTC Primer Allele Y NO57 GAAGGTCGGAGTCAACGGATTGTTCTTGG GTGGGTCTGTCCTTA PrimerCommon NO:58 CCGCCTTCAGTTTAAACTATCAGTGTTTT

- 54 041446

Primer_Common2 NO:59 CGTTGGCTAAGGTCACGGTGGA NBN10 Connection area upstream insert A05 Primer_Allele X NO:60 GAAGGTGACCAAGTTCATGCTGGTCACG GTGGAGGTCACCA Primer Allele Y NO:61 GAAGGTCGGAGTCAACGGATTCCGCCTTC AGTTTAAACTATCAGTGTT PrimerCommon NO :62 GGTGTGTTCTTGGGTGGGTCTG NBN83 Connection area downstream insert A05 Primer_Allele X NO:63 GAAGGTGACCAAGTTCATGCTTCAGTTTA AACTATCAGTGTTACCT Primer Allele Y NO :64 GAAGGTCGGAGTCAACGGATTACATGGT GACCTCCCACCGTG PrimerCommon NO:65 GTACTTTAAGCTTATAACCCTTTGTC NBN82 Connection area downstream insert A05 Primer_Allele X NO:66 GAAGGTGACCAAGTTCATGCTGGAGATC CACTAGCAGATTGTCGTT Primer Allele Y NO:67 GAAGGTCGGAGTCAACGGATTCTTGGGT GGGTCTGTCCTTAC PrimerCommon NO:68 GCAGGAGGTACTTTAAGCTTATA NBN84 Connection area downstream insert A05 Primer_Allele X NO:69 GAAGGTGACCAAGTTCATGCTGATTGTCG TTTCCCGCCTTCAGTTT Primer Allele Y NO:70 GAAGGTCGGAGTCAACGGATTGTCCTTAC ATGGTGACCTCCAC PrimerCommon NO:71 GTACTTTAAGCTTATAACCCTTTGTC NBN66 Connection area downstream insert A05 Primer_Allele X NO :72 GAAGGTGACCAAGTTCATGCTGATTGTCG TTTCCCGCCTTCAGTTT Primer Allele Y NO:73 GAAGGTCGGAGTCAACGGATTGTCCTTAC ATGGTGACCTCCAC PrimerCommon NO :74 GCAGGAGGTACTTTAAGCTTATA NBN41 Connection area downstream insert A05 Primer_Allele X NO:75 GAAGGTGACCAAGTTCATGCTTTTTTTATT CAACCGTTGGCTAAGGTA Primer Allele Y NO:76 GAAGGTCGGAGTC AACGGA H l 111A AND C AACGTTGGCTAAGGTC PrimerCommon NO:77 GATTGTCGTTTCCCGCCTTCAGTTTT Primer_Common2 NO:78 TTCTTGGGTGGGTCTGTCCTTACAT NBN43 Connection area downstream insert A05 Primer_Allele X NO:79 GAAGGTGACCAAGTTCATGCTTTTTTTATT CAACCGTTGGCTAAGGTA Primer Allele Y NO:80 GAAGGTCGGAGTCAACGGATTTTTTATTC AACCGTTGGCTAAGGTC PrimerCommon NO:81 GGAGATTCCACTAGCAGATTGTCGTT Primer_Common2 NO:82 TTCTTGGGTGGGTCTGTCCTTACAT NBN11 Connection area downstream insert A05 Primer_Allele X NO:87 GAAGGTGACCAAGTTCATGCTACTTTTTT TTCAACTGTTGGCTAAGGTA PrimerAllele Y NO:88 GAAGGTCGGAGTCAACGGATTACTTTTT TTCAACTGTTGGCTAAGGTC PrimerCommon NO:89 GTGTGTTCTTGGGTGGGTCTG Primer_Common2 NO:90 GTCGTTTCCCGCCTTCAGTTTT

The KASP genotyping system requires two components: Assay mix and Master mix. The assay mix is a mixture of the required primers and the master mix contains all other required components including PCR buffer, universal fluorescent imaging system and Taq polymerase.

The KASP reaction was run in a 4.0 µl volume consisting of 2.0 µl (10.0 ng) genomic DNA, 2.0 µl 2x KASP master mix, and 0.06 µl Assay mix (primers). The assay mix (primers) is a combination of 12 µM allele-specific Primer_Allele X and 12 µM Primer_Common for dual primer assays, a combination of 12 µM allele-specific Primer_Allele X, 12 µM allele-specific Primer_Allele Y, and 30 µM Primer_Common for triple primer assays, and a combination of 12 μM allele-specific Primer_Allele X, 12 μM allele-specific PrimerAllele Y, 12 μM PrimerCommon and 12 μM Primer_Common2 for four primer assays.

Reactions were run in a 384-well plate in an LGC Hydrocycler 16 using the following cycle settings: 1 cycle at 94°C for 15 min followed by eight cycles at 94°C for 30 s and at 64°C-57°C (drop 1.0°C per cycle) for 60 s, followed by thirty cycles at 94°C for 30 s and at 57°C for 60 s. If no clear genotyping clusters were obtained, the plate was further temperature cycled using three additional cycles at 94°C for 30 seconds and at 57°C for 60 seconds.

After completion of the KASP reactions, the transgenic allele was labeled with FAM with Primer_Allele X, and the non-transgenic wild-type allele was labeled with HEX with PrimerAllele Y. Fluorescent signals were read on a PheraStar microplate reader at 485 nm excitation wavelength and 520 nm emission wavelength for FAM and at 535/556 nm for HEX. Data was analyzed using the LGC Kraken database.

Eight gene-specific dominant assays (NBN01-NBN08; one assay/gene) were designed to detect eight genes in the construct cassette. A total of twenty insert-specific codominant KASP assays targeting the upstream (NBN57, NBN68, NBN58, NBN85, and NBN14) and downstream (NBN16, NBN62, and NBN64) insertion junction regions at A02, and upstream (NBN52, NBN51, NBN09 , NBN50, NBN48, and NBN10) and downstream (NBN83, NBN82, NBN84, NBN66, NBN41, and NBN43) insertion junction regions on A05, was developed and validated using 1200 progeny of introgression-derived NS-B50027-4 populations (Table 14) . Over 10,000 samples have been genotyped with these markers.

Thirty competitive allele-specific PCR (KASP) assays were designed and validated targeting eight genes and four junction regions of two DHA-enriched canola NS-B50027-4 event inserts. These assays provided a simple, cost effective, high throughput and flexible approach to detect and monitor NS-B50027-4 in a breeding program.

Example 4 Detailed comparison of NS-B50027-4 and non-transgenic canola.

Data relating to canola seed production at the experimental field plots for 2014-2016 are summarized in the table. The DHA range and total EPA+DPA+DHA levels are based on several observations in the field. The content of essential fatty acids in both NS-B50027-4 and non-transgenic Control canola can vary by several percentage points depending on growing conditions. In the next table. 15 0.0 may refer to a trace amount, which is defined as an amount less than needed to accurately determine the amount of a component.

Table 15

Detailed comparison of fatty acid content in NS-B50027-4 with control

Fatty acid NS-B50027-4 (%) Control Canola (%) Myristic C14:0 0.1 0.1 palmitic С16:0 4.3 3.9 Palmitoleic C16.1 0.2 0.2 Stearic С18:0 2.2 1.6 Oleic S18:1p9s 38.7 63.6 Cis-vaccinal S18:1p7s 4.2 3.5 Linoleic From 18:2p6s 7.8 13.1 GLA S18:3p6 0.6 0.0 ALA S18:ZpZ 21.7 10.3 Arachidonic S20:0 0.6 0.6 SDA S18:4pZ 2.2 0.0 Gondoeva S20:1p9s 1.3 1.5 Geneicosanoic С21:0 0.0 0.0 DGLA S20:Zp6 0.0 0.0 ETE S20:ZpZ 0.7 0.0 Begenovaya C22:0 0.3 0.3 ETA S20:4p3 0.0 0.0 Erucovaya S22:1p9s 0.0 0.0 ERA S20:5pZ 0.4 0.0 Lignoceric С24:0 0.2 0.1 DPA6 S22:5p6 0.0 0.0 neuron S24:1p9s 0.1 0.2 DPA3 S22:5pZ 0.9 0.0 DHA S22:6pZ 9.8 (8-10) 0.0 Other 3.8 1.3 Sum: EPA+DPA+DHA 11.1 (10-12) 0.0 Total Omega 3 35.7 10.4 Total Omega 6 8.4 11.3 ω3/ω6 4.3 0.9 Total saturated 7.7 6.7 Total 44.5 68.9 monounsaturated Total polyunsaturated 44.1 23.5

The seeds collected from the experimental cultivation of NS-B50027-4 were crushed, and the oil was obtained by cold pressing. Seeds harvested from the isogenic parent line, AV Jade, were treated in a similar manner and the composition of each oil was compared as shown in Table 1. 16.

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Table 16

NS-B50027-4 Oil composition

Component (units) | NS-B50027-4 | AV Jade Saturated TAG (%) С4:0 Oily <0.1 <0.1 С6:0 Nylon <0.1 <0.1 С8:0 Caprylic <0.1 <0.1 From 10:0 Capric <0.1 <0.1 From 12:0 Lauric <0.1 <0.1 From 14:0 Myristic <0.1 <0.1 С15:0 Pentadecanoic <0.1 <0.1 From 16:0 Palmitic 4.3 3.9 From 17:0 Margarine <0.1 <0.1 С18:0 Stearic 2.9 2.5 С20:0 Arachidonic 0.8 0.5 C22:0 Begen 0.4 0.2 C24:0 Lignoceric 0.1 0.1 All saturated 8.7 7.3 Monounsaturated TAG (%) C 14:1 Myristoleic <0.1 <0.1 C 16:1 Palmitoleic 0.2 0.1 С17:1 Heptade price <0.1 <0.1 С18:1 Oleic 44.9 58.8 S20L Eicosene 1.3 1.0 C22:1 Docosene <0.1 <0.1 C24:1 Nervonic <0.1 <0.1 PUFA TAG (%) C18:2w6 Linolenic 7.6 18.9 C18:3sh6 gamma linolenic 0.5 <0.1 C18: Zsh3 alpha-linolenic 20.9 10.5 С20:2w6 Eicosadiene <0.1 <0.1 С20:Зш6 Eikosatry new <0.1 <0.1 C20: Zsh3 Eicosatriene 0.6 <0.1 S20:4sh6 Arachidon <0.1 <0.1 C20:5w3 Eicosapentaenoic 0.4 <0.1 C22:2w6 Docosadiene <0.1 <0.1 C22:4w6 Docosatetraenoic <0.1 <0.1 C22:5w3 Docosapentaenoic 1.0 <0.1 S22:6shZ Docosahexaenoic 9.4 0.2 Total PUFA (%) 40.6 29.9 Total Mono Trans Fatty Acids Total Poly Trans Fat acids P : M : S ratio PUFA (%) Omega 3 fatty acids Omega 6 fatty acids ω3:ω6 vitamins beta-carotene (mcg/100g) alpha-tocopherol (mg/100g) beta-tocopherol (mg/100g) delta-tocopherol (mg/100g) 0.1 0.8 4.7:5.4:1 32.3 8.2 3.94 BY 19 <0.1 0.6 0.2 0.2 4.1:8.2:1 10.9 19.0 0.57 82 15 <0.1 0.8 gamma-tocopherol (mg/100g) 43 42 Astaxanthin (mg/kg) <0.05 <0.05 Vitamin K1 (µg/100g) 17 15 Phytosterols (mg/100g) Cholesterol <5.0 <5.0 Brassicasterin 29 67 campesterol 250 170 Campestanol <5.0 <5.0 Stigmasterin <5.0 <5.0 beta-sitosterol 370 320 beta-sitostanol 34 27 Total Phytosterols 690 600

Under the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure (1977), Applicants have deposited at least 2,500 Canola seeds NS-B50027-4 with the American Type Culture Collection (ATCC®), Manassas, VA., 20110-2209 , USA, June 9, 2016. Accession number PTA-123186. During the course of this application, access to the invention may be made available to the U.S. Patent and Trademark Office upon request; all restrictions on access to the public are irrevocably lifted upon the issuance of patents

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Claims (15)

that; deposit of line NS-B50027-4 will be held at ATCC® Depository, which is a public depository, for 30 or 5 years after the most recent seed request or for the duration of the patent, whichever is longer; and seeds will be replaced with viable seeds if the deposited seeds become non-viable during this period. Seed viability was tested at the time of application. Appendix A contains the deposit date, access number and proof of viability. Applicants complied with all requirements of 37 C.F.R. §§ 1.8011.809. Applicants place no restrictions on the availability of deposited material from ATCC®; however, the Applicant is not entitled to waive any restrictions imposed by law on the transfer of biological material or its transport in trade. Applicant does not waive any infringement of its rights under the patent granted under this application or under the Plant Variety Protection Act (7 U.S.C. § 2321 et seq.). While the foregoing embodiments have been described in detail by way of illustration and examples for clarity and understanding, it will be apparent to those skilled in the art that certain changes and modifications, such as single gene modifications and mutations, somoclonal variants, variants of individuals selected from large populations of plants of a given inbred line and the like, may be practiced within the scope of the invention, which is limited solely by the appended claims. CLAIM 1. An in vitro method for detecting the presence of a transgenic locus in a sample containing plant DNA, said method comprising (a) contacting said sample with at least one first primer pair that binds to a first transgene flanking junction region of said transgenic locus, wherein said at least one first primer is selected from the primer group consisting of SEQ ID NOs: 17-31 and SEQ ID NOs: 83-86, the sequences of which target the upstream junction region of the transgenic locus of chromosome A02; or at least one second primer pair that binds to a second flanking transgene junction region of said transgenic locus, said at least one second primer being selected from the group of primers consisting of SEQ ID NOs: 32-41 whose sequences target the located below the junction region of the transgenic locus of chromosome A02; or at least ten adjacent nucleotides of any of these primers or their complements; (b) subjecting said sample to a polymerase chain reaction; and (c) evaluating the amplicons generated between said primers, said generated amplicons identifying a transgenic locus of chromosome A02 of a canola plant derived from NS-B50027-4, or a portion thereof, wherein a representative seed of said inbred canola line NS-B50027-4 is deposited under access number АТСС РТА-123186. 2. The in vitro method of claim 1, said method being a genotyping assay based on competitive allele-specific PCR. 3. The in vitro method of claim 1, wherein said transgenic locus of chromosome A02 contains one copy of each of the genes from M. pusilla A6 desaturase, P. cordata A5 elongase, P. salina A5 desaturase, and P. pastoris A15/sh3 -desaturase, and the specified transgenic locus is located on the A02 chromosome. 4. The in vitro method of claim 1, further comprising the step of contacting said sample with at least one third primer that binds to the transgenic sequence to generate an amplicon that identifies the transgenic A02 chromosome locus, said at least one third primer selected from the group of primers consisting of those shown in SEQ ID NOs: 1-8, the sequences of which are targeted to the genes of M. pusilla A6-desaturase, P. cordata A5-elongase, P. salina A5-desaturase and P. pastoris A15 /m3-desaturase, or at least ten consecutive nucleotides, or their complements. 5. A method for detecting the presence of DNA corresponding to the A02 segregant of the object Brassica NS-B50027-4 in a sample containing plant DNA, comprising the following steps: (a) contacting said sample with a primer pair which, when used in a nucleic acid amplification reaction with genomic DNA from Brassica event A02 segregant NS-B50027-4, produces an amplicon that is diagnostic for Brassica event A02 segregant, said primer pair being selected from the group consisting of SEQ ID NOs: 17-41 and SEQ ID NOs: 83-86, the sequences of which are targeted at the upstream junction region of the transgenic locus of chromosome A02; (b) performing a nucleic acid amplification reaction, thereby producing an amplicon; And - 58 041446 (c) detection of the amplicon, wherein the detected amplicon identifies the junction region of the segregant A02 flanking the transgenic locus of chromosome A02, which is diagnostic for the Brassica NS-B50027-4 object, the typical seed of which was deposited with the ATCC under accession number PTA-123186. 6. The method of claim 5, said method being a genotyping analysis based on competitive allele-specific PCR. 7. The method of claim 5, further comprising in step (a) contacting said sample with a primer pair selected from the group consisting of SEQ ID NOs: 1-8 to generate an amplicon that identifies segregant A02. 8. Segregant obtained from transgenic canola variety NS-B50027-4 deposited under accession number ATCC PTA-123186, containing junction regions flanking the transgenic locus of chromosome A02 NS-B50027, identified in the in vitro method according to any one of claims 1-7. 9. A method for increasing the production of long chain polyunsaturated fatty acid (LC PUFA) in Brassica that produces LC-PUFA, comprising the steps of (a) segregating the NS-B50027-4 genome to obtain a segregant containing the specified NS-B50027-4 A02 locus; and (b) introgressing the segregant with the recipient Brassica that contains the LC-PUFA biosynthetic genes to produce introgressed Brassica progeny containing the recipient LC-PUFA biosynthetic genes and the NS-B50027-4 A02 locus, said introgressed Brassica progeny producing increased amounts of LC -PUFA, and moreover, the specified introgressed progeny of Brassica is identified by the method according to claim 1. 10. The method of claim 9, wherein the LC-PUFA is at least one of eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA), or docosahexaenoic acid (DHA). 11. A Brassica plant or Brassica seed containing the A02 transgenic locus of object NS B50027-4 deposited under ATCC Accession PTA-123186, its progeny having an A02 transgene flanking junction region containing SEQ ID NO: 17-41 or SEQ ID NO: 83-86. 12. A Brassica plant or Brassica seed containing the A02 transgenic locus of object NS B50027-4 deposited under ATCC accession number PTA-123186, and its progeny identified using primers containing SEQ ID NO: 17-41 or SEQ ID NO: 83- 86 whose sequences target the upstream junction region of the transgenic locus of chromosome A02, or at least ten consecutive nucleotides thereof, or their complements. 13. Canola inbred segregant NS-B50027-4, a representative seed of said canola inbred line is deposited under ATCC accession number PTA-123186, said segregant containing in its genome a transgenic locus containing one copy of each of the M. pusilla A6 desaturase gene, P. cordata A5 elongases, P. salina A5 desaturases and P. pastoris A15/m3 desaturases, wherein said segregant is characterized by a nucleic acid comprising SEQ ID NOs: 1-8 and SEQ ID NOs: 17-41, and SEQ ID NO: 83-86, the sequences of which are targeted to the upstream junction region of the transgenic locus of chromosome A02. 14. A method for producing an inbred canola line, comprising (a) obtaining a hybrid plant by crossing (i) a first segregant plant grown from the seed of an inbred canola line NS-B50027-4, wherein a representative seed of said inbred canola line is deposited under accession number ATCC PTA- 123186, moreover, the specified segregant contains in its genome a transgenic locus containing one copy of each of the M. pusilla A6-desaturase, P. cordata A5-elongase, P. salina A5-desaturase, and P. pastoris A15/m3-desaturase genes, c ( ii) a second line canola plant, said hybrid plant containing a transgenic segregant locus; (b) crossing said hybrid with a second line canola plant; and (c) repeating step (b) at least once to obtain said canola inbred line, wherein said canola inbred line contains at least one of said transgenic loci, wherein the resulting canola inbred line is characterized by SEQ ID NOs: 1-8, SEQ ID NO: 17-41 and SEQ ID NO: 83-86. 15. An inbred canola line obtained by the method of claim 14, wherein said inbred canola line contains at least one trait selected from herbicide resistance, insect resistance, bacterial disease resistance, fungal disease resistance, viral disease resistance, or sterility. -