Patent Description:
Long chain omega-<NUM> (LC-ω3) fatty acids such as docosahexaenoic acid (DHA) provide many health benefits, and can be included in human diets, e.g., by consuming algae-derived products or algae-eating fish. Alternative sources for LC-ω3 fatty acids are needed to satisfy increased human consumption needs. A genetically-modified canola line, DHA canola NS-B50027-<NUM>, accumulates a significant concentration of DHA in canola seed. There remains a need for efficient identification of NS-B50027-<NUM>, e.g., for plant stewardship purposes. <CIT> provides compositions and methods to identify DHA canola NS-B50027-<NUM> and progeny thereof.

The present embodiments provide methods and primers to identify DHA canola NS-B50027-<NUM> event.

The present invention provides a qualitative gel based method of identifying the presence of a DHA canola NS-B50027-<NUM> even or progeny thereof as defined in claim <NUM>. The dependent claims set out features of certain embodiments of the present invention.

One embodiment provides an agarose gel electrophoresis-based method to qualitatively detect the presence (or absence) of the Nuseed DHA Canola NS-B50027-<NUM> event. More specifically, two event-specific PCR assays targeting the T-DNA insertion junction sites in the canola genome can be used for adventitious presence testing, trait purity testing, and trait introgression, and to support DHA Canola NS-B50027-<NUM> plant stewardship.

It should be understood that this invention is not limited to the particular methodology, protocols, and reagents, etc., described herein and as such may vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined solely by the claims.

As used herein and in the claims, the singular forms "a," "an," and "the" include the plural reference unless the context clearly indicates otherwise. Throughout this specification, unless otherwise indicated, "comprise," "comprises" and "comprising" are used inclusively rather than exclusively, so that a stated integer or group of integers may include one or more other non-stated integers or groups of integers. The term "or" is inclusive unless modified, for example, by "either. " Thus, unless 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 fatty acid composition due to environmental conditions. Values are typically expressed as percent by weight of total fatty acid, or percent weight of the total seed. Accordingly, other than in the operating examples, or where otherwise indicated, all numbers expressing quantities or reaction conditions used herein should be understood as modified in all instances by the term "about.

NS-B50027-<NUM> DHA canola was generated, inter alia, via Agrobacterium tumefaciens-mediated transformation of canola cultivar AV Jade with a binary vector designated pJP3416_GA7-ModB. Vector pJP3416_GA7-ModB was designed specifically to convert oleic acid to DHA in seed, and contains expression cassettes for seven microalgae and yeast transgenes (abbreviated Micpu-Δ6D, Pyrco-Δ5E, Pavsa-Δ5D, Picpa-ω3D, Pavsa-Δ4D, Lackl-Δ12D and Pyrco-Δ6E) involved in the DHA biosynthesis pathway (and also includes a PAT gene for herbicide tolerance selection). Each transgene has its own expression cassette comprising seed-specific promoter, enhancer, and terminator. See <CIT> and <CIT>.

NS-B50027-<NUM> DHA canola was characterized with vector-targeted sequencing, whole-genome sequencing and Polymerase Chain Reaction (PCR)-amplicon sequencing. Sequencing data indicated that the DHA canola contained no vector backbone, no binary vector bacterial selectable marker gene Neomycin Phosphotransferase II (NPT II) or any A. tumefaciens genome sequence. Sequencing information also indicated that DHA canola contained two T-DNA inserts, one on chromosome A02 and the other on chromosome AOS. The full genomic DNA sequences of the two T-DNA inserts were verified and the sequence of each T-DNA insert perfectly matched the reference of vector pJP3416_GA7-ModB. Both T-DNA inserts were required to accumulate the desired amount of DHA in seed oil.

More specifically, molecular characterization of the NS-B50027-<NUM> canola indicated that it carried two transgenic inserts, one on chromosome A02 and the other on chromosome A05, and that both transgenic inserts were required to accumulate about <NUM>% DHA in canola seed. See <NPL>).

The A02 T-DNA insertion is a partial insert, containing complete gene expression cassettes for genes Micpu-Δ6D, Pyrco-Δ5E, Pavsa-Δ5D and Picpa-ω3D but not for genes Pavsa-Δ4D, Lackl-Δ12D, Pyrco-Δ6E, nor PAT. The sequence of the A02 T-DNA insert otherwise matches the reference of vector pJP3416_GA7-ModB. The A02 T-DNA insert is located within the <NUM>' UTR of a hypothetical protein gene on chromosome A02.

The A05 T-DNA insertion contains two T-DNA transgene sets from the binary vector that formed a palindromic structure in the right border-transgenes-left border: left border-transgenes-right border orientation. The sequence of the A05 T-DNA insert also matches the reference sequence in vector pJP3416_GA7-ModB. The A05 T-DNA insert is located within the second exon of a Pto-Interacting (PTI) gene on chromosome A05.

The present embodiments provide methods and primers for the relatively easy identification of the Nuseed DHA canola NS-B50027-<NUM> event. More specifically, the present embodiments provides a qualitative detection method for determining the presence of DHA Canola (Event NS-B50027-<NUM>) in oilseeds DNA sample. The assay can be used for adventitious presence testing, trait purity testing, and trait introgression, and to support DHA Canola NS-B50027-<NUM> regulatory submission and commercialization. See also <CIT>.

Event-specific gel-based assay, targeting an insert in DHA canola NS-B50027-<NUM> on chromosomes A05 and A02, have been successfully developed and validated. The HMG reference gene PCR profile was designed to confirm the PCR ability of DNA sample.

The Limit of Detection (LOD) of the event-specific assay is determined at least <NUM>% NS-B50027-<NUM> DNA to total DNA, or less than <NUM> genome copies. The specificity of the assay was validated by testing <NUM> CRM materials available from AOCS and <NUM> different Nuseed non- GM conventional oilseeds varieties.

DNA were extracted from seeds using CTAB DNA extraction method briefly described below:.

Store the DNA samples at <NUM> for temporary storage up to a week or at -<NUM> for long term storage.

Prepare <NUM> ng/µl DNA solutions from DHA canola NS-B50027-<NUM> DNA and negative control AV Jade before making up the following spike samples, as provided in Table <NUM>. Six DHA canola NS-B50027-<NUM> spike levels were made as described below, the genome copies number of reference gene HMG and DHA canola is listed in Table <NUM>.

Assemble all reaction (<NUM>µL/reaction) components (Cat Log #: M0480L) on ice as illustrated in Table <NUM>.

PCR was used for amplification with the following profile parameters:.

Internal reference gene: Canola HMG is used as the internal reference gene for this qualitative detection method. Primer sequences are provided in Table <NUM>, below. The amplicon size for HMG is <NUM> bp.

GeneRuler low range DNA ladder: Thermo Scientific™ GeneRuler™ Low Range DNA Ladder, ready-to-use, contains a mix often chromatography-purified individual DNA fragments (in base pairs): <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, and <NUM> (<FIG>). Agarose gel electrophoresis.

PCR products are resolved through agarose gel electrophoresis and analyzed with the Life Technology Image System. For each PCR reaction, <NUM>µL PCR products is mixed with <NUM>µL H2O and <NUM>µL loading dye (6x; Thermo Fisher Scientific Cat. Log# R1161) and run on a <NUM>% agarose gel (gel size <NUM> long and <NUM> wide) at <NUM> Volts for <NUM> minutes. DNA ladder (<NUM>µl per lane) is run on proper number of lanes to enable PCR amplicon sizes to be identified. Gel image is captured with the Life Technology Image system.

Chemicals and equipment that are needed for DNA extraction: CTAB, NaCl, Tris-HCl, EDTA, DNase-free RNase, Ethanol, Grinder, Centrifuge, Tubes, etc..

The qualitative event-specific assay (A05-<NUM>) was designed to detect the junction between T-DNA insert and genomic DNA on Chromosome A05. The qualitative event-specific assay (A02-<NUM>) was designed to detect the junction between T-DNA insert and genomic DNA on Chromosome A02, see <CIT>. The primer sequences, locations, and product sizes are shown in Table <NUM> and <FIG> and <FIG>. The Brassica HMG gene was used as the reference gene for the internal control recommended by Chinese National Standard (MARA <NUM>-<NUM>-<NUM>). Primer sequences and the expected amplicon sizes are listed in the following table. The primer pair A02-258F and A02-258R is used to detect the insert or junction on chromosome A02, and the amplicon size is <NUM> bp. The primer pair A05-216F and A05-216R is used to detect the insert or junction on chromosome A05, and the amplicon size is <NUM> bp. The primer pair Hmg206F and Hmg206R is used to detect the endogenous HMG gene (internal reference gene HMG as control), and the amplicon size is <NUM> bp.

A hundred nanogram (ng) genomic DNA template of the following samples were subject to event-specific qualitative PCR (Assay A05-<NUM> for junction in chromosome A05 and Assay HMG for Reference Gene):.

The results from the qualitative event-specific assay with <NUM> different DHA canola NS-B50027-<NUM> spike levels.

The qualitative event-specific assay (A05-<NUM>) was tested with AV Jade, NTC, six different DHA canola NS-B50027-<NUM> spike levels, and eight conventional Nuseed canola lines (<FIG>). The results from <NUM> replicates showed the assay can consistently detect the expected amplicons at least <NUM>% spike level (<FIG>). Gel lanes for <FIG> and <FIG> from the assays are provided in Table <NUM> and Table <NUM>.

The qualitative event-specific Assay (A05-<NUM>) and reference gene assay HMG206 were tested with six different commercial canola GMO events ordered from AOCS. The results showed assay A05-<NUM> (<FIG>) did not have any amplicons from six commercial canola events. The reference gene assay HMG206 amplified the expected amplicons from the six commercial canola events as expected. The results demonstrated the assay A05-<NUM> developed is specific for NS-<NUM>-<NUM> event in canola.

The qualitative event-specific Assay A05-<NUM> were tested with twenty-seven CRM materials ordered from AOCS including twenty-five different commercial GMO events. The results showed assay A05-<NUM> did not have any amplicons from all these materials, including in eight canola CRM (seven GMO events and one non-GMO canola (<FIG>), seven soybean events, seven maize events, four cotton events and one non-GMO cotton (<FIG>). The DHA canola positive controls amplified the expected amplicons in the same experiment. The results demonstrated the assay A05-<NUM> provided herein is event specific for only canola NS-B50027-<NUM> event.

No amplicons of the qualitative event-specific Assay A05-<NUM> were present in non-GM canola materials. More specifically, the qualitative event-specific Assay A05-<NUM> was also validated with eight non-GM conventional canola varieties. Seven were selected from Nuseed germplasm pool with various genetic backgrounds (<FIG>) and one was ordered from AOCS. The results showed A05-<NUM> assay did not amplify any amplicons from these <NUM> non-GM conventional canola varieties.

The event specific assay A05-<NUM> was tested with NS-B50027-<NUM> event line DNA at six different spike levels and sensitivity (%) was calculated based on <NUM> replicates at <NUM>% spike level. All samples containing event NS-B50027-<NUM> DNA from <NUM>% to <NUM>% (<NUM>% equals to <NUM> genome copies for NS-B50027-<NUM>, see Table <NUM>) showed the expected amplicons consistently from A05-<NUM> assay. So, the Limit of Detection (LOD) is at least <NUM>% NS-B50027-<NUM> DNA to total DNA or less than <NUM> genome copies.

The event specific assay A05-<NUM> was also tested with twenty-five commercially available GM events from AOCS and eight different non-GM conventional oilseeds (Brassica napus) varieties. The results showed the assay can amplify the expected amplicons only from NS-B50027-<NUM>. The event specific assay A05-<NUM> can specifically detect the DNA of DHA canola.

The HMG reference gene PCR profile was designed as internal control, all oilseed samples tested showed the expected <NUM>-bp amplicon in the same lab setting and PCR condition.

Event-specific gel-based assay A05-<NUM>, targeting the insert in DHA canola NS-B50027-<NUM> on chromosome A05, has been successfully developed and validated. The assay can be used for adventitious presence testing, trait purity testing, and trait introgression, and to support DHA Canola NS-B50027-<NUM> regulatory submission and commercialization.

This example provides a qualitative detection method developed to determine the presence of DHA Canola (Event NS-B50027-<NUM>) in oilseeds DNA sample. As shown herein, the assay can be used for adventitious presence testing, trait purity testing, and trait introgression, and to support DHA Canola NS-B50027-<NUM> regulatory submission and commercialization.

Event-specific gel-based assay A05-<NUM>, targeting the insert in DHA canola NS-B50027-<NUM> on chromosome A05 has been successfully developed. The HMG reference gene PCR profile was designed as reference gene to confirm the PCR ability of DNA sample.

The Limit of Detection (LOD) of the event-specific A02 assay is determined at least <NUM>% NS-B50027-<NUM> DNA to total DNA, or less than <NUM> genome copies.

DNAs were extracted from the seeds using CTAB DNA extraction method, as described in Example <NUM>.

Samples with different DHA canola DNA spikes were prepared according to the protocol, as described in Example <NUM>.

Six DHA canola NS-B50027-<NUM> spike levels were made as described below, the genome copies number of reference gene HMG and DHA canola is listed in Table <NUM>.

PCR was conducted as described in Example <NUM>. For PCR assembly: Assemble all reaction (<NUM>µL/reaction) components (New England BioLabs (NEB) Inc. , Catalog Log # M0480L) on ice, as illustrated in Table <NUM>.

For PCR cycling profile: PCR was used for amplification with the following profile parameters, as described in Example <NUM>, Table <NUM>.

The canola HMG gene is used as the internal reference gene for this qualitative detection method. The Thermo Scientific™ GeneRuler™ Low Range DNA Ladder (ready-to-use) containing a mix often chromatography-purified individual DNA fragments (in base pairs: <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>, <NUM>) is used as a reference guide.

Agarose gel electrophoresis was conducted according to this protocol: For each sample, <NUM>µL PCR products, <NUM>µL H<NUM>O and <NUM>µL loading dye (6x; Thermo Fisher Scientific #R1161) were mixed before loading, and <NUM>µL DNA Ladder was used. A <NUM>% agarose gel was run at <NUM> Volts for <NUM>. An image of the gel was captured using the Life Technology Image system.

Two primer pairs were employed for this qualitative detection method. The amplicon size for HMG is <NUM> bp. Assay A05-<NUM> was designed to detect the junction between T-DNA insert and genomic DNA on chromosome A05. The primer sequences, locations, and product sizes are shown in Table <NUM> and <FIG>. The Brassica HMG gene was used as the reference gene for the internal control recommended by Chinese National Standard (MARA <NUM>-<NUM>-<NUM>).

Samples of <NUM> ng of genomic DNA template, as provided herein, were subjected to event-specific qualitative PCR (Assay A05-<NUM> for junction in chromosome A05 and Assay HMG for Reference Gene):.

Assay A05-<NUM> was tested along with AV Jade, NTC (no template control) and six different DHA canola NS-B50027-<NUM> spike levels (see <FIG>). In addition, the results from fifteen replicates showed both assays can consistently detect the expected amplicons at least <NUM>% spike level (see <FIG>). There was no amplicon from all reactions from AV Jade (negative control) for event specific assay A05-<NUM>.

The sample layout of the gel images for <FIG> are described in Tables <NUM> and <NUM>, respectively.

The event specific assay A05-<NUM> was tested with NS-B50027-<NUM> event line DNA at six different spike levels and sensitivity (%) was calculated based on fifteen replicates. All samples containing event NS-B50027-<NUM> DNA from <NUM>% to <NUM>% (<NUM>% equals <NUM> genome copies for NS-B50027-<NUM>, see Table <NUM>) showed the expected amplicons consistently from A05-<NUM> assay. The Limit of Detection (LOD) is at least <NUM>% NS-B50027-<NUM> DNA to total DNA or less than <NUM> genome copies. No amplicon was detected from AV Jade (a negative control) for event-specific assay A05-<NUM>.

Event-specific gel-based assay A05-<NUM>, targeting the insert in DHA canola NS-B50027-<NUM> on chromosome A05 has been successfully developed. The assay may be used for adventitious presence testing, trait purity testing, and trait introgression, and to support Nuseed DHA Canola NS-B50027-<NUM> regulatory submission and commercialization.

The specificity of the event-specific A05-<NUM> assay, as discussed above, was further validated by testing twenty-five commercially available GM events (including seven canola events, seven soybean events, seven maize events and four cotton events) from AOCS and seven different non-GM conventional oilseeds varieties. The validation data showed that the assays can detect positive results only from DHA samples. All CRM materials and conventional oilseeds varieties showed negative results for Event NS-B50027-<NUM>.

To carry out the validation assays, DNA were extracted from seeds using CTAB DNA extraction, as described in Example <NUM>.

Samples of <NUM> ng genomic DNA template, as described herein, were subjected to event-specific qualitative PCR (assay A05-<NUM> for junction in chromosome A05 and assay HMG for Reference Gene):.

The parameters for PCR assembly, PCR cycling profile and primers, as used herein, were identical with those described in Tables <NUM>, <NUM> and <NUM>, respectively.

For each sample, <NUM>µL PCR products, <NUM>µL H<NUM>O, and <NUM>µL loading dye (6x; Thermo Fisher Scientific #R1161) were mixed before loading, and <NUM>µL DNA Ladder was used. A <NUM>% agarose gel was run at <NUM> Volts for <NUM>. The gel was then photographed using a Life Technology Image system.

Assays A05-<NUM> and HMG were validated with six different commercial canola GMO events ordered from AOCS. The results showed assay A05-<NUM> (see <FIG>) did not have any amplicons from six commercial canola events. The control gene assay HMG amplified the expected amplicons from these six commercial canola events as expected. The results demonstrated that assay A05-<NUM> developed at Nuseed was event-specific for canola. The sample layout for <FIG> is described in Table <NUM>.

Assays A05-<NUM> and HMG were further validated with all twenty-five different commercial GMO events ordered from AOCS. The results showed assay A05-<NUM> did not have any amplicons from all these events, including in seven canola events, seven soybean events, seven maize events, and four cotton events. The DHA canola-positive controls amplified the expected amplicons in the same experiment (see <FIG> and <FIG> for assay A05-<NUM>). The results demonstrated that assay A05-<NUM> developed at Nuseed was event-specific for only canola NS-B50027-<NUM> event. The sample layout for <FIG> and <FIG> is described below in Tables 15A and 15B, respectively.

Assay A05-<NUM> was further validated with seven non-GM conventional canola varieties selected from Nuseed germplasm pool with various genetic backgrounds. The results showed that assay A05-<NUM> did not amplify any amplicon from these seven non-GM conventional canola varieties (see <FIG>). Sample layout of <FIG> is described in Table <NUM>.

The specificity of the A05-<NUM> assay was validated from testing twenty-five commercially available GM events (including seven canola events, seven soybean events, seven maize events and four cotton events) from AOCS and eight different non-GM conventional oilseeds varieties (one from AOCS and seven from Nuseed germplasm pool). Accordingly, the event-specific gel-based assay, A05-<NUM>, targeting the insert in DHA canola NS-B50027-<NUM> on chromosome A05 was successfully developed and validated. The assay can be used for adventitious presence testing, trait purity testing, and trait introgression, and to support regulatory submission, stewardship, and commercialization.

The twenty-seven Certified Reference Materials (CRM) from American Oil Chemists' Society (AOCS) employed and tested in the above study are listed in Table A:.

Claim 1:
A qualitative gel-based method of identifying the presence of a DHA canola NS-B50027-<NUM> event or progeny thereof in a biological sample, said method comprising detecting a two event-specific detection method that comprises
(a) extracting a DNA sample from said biological sample;
(b) providing a first DNA primer pair combination, wherein said first DNA primer pair combination is from nucleotides as set forth in SEQ ID NO: <NUM> (A05-216F) and SEQ ID NO:<NUM> (A05-216R);
(c) providing a second DNA primer pair combination, wherein said second DNA primer pair combination is from nucleotides as set forth in SEQ ID NO:<NUM> (A02-258F) and SEQ ID NO:<NUM> (A02-258R);
(d) providing DNA amplification reaction conditions; and
(e) performing two separate DNA amplification reactions with said first (as in step (b)) and second (as in step (c)) DNA primer pair combinations, thereby producing an amplicon pair consisting of a <NUM>-bp amplicon and a <NUM>-bp amplicon, respectively;
wherein the presence of said amplicon pair identifies the presence of the DHA canola NS- B50027-<NUM> event or progeny thereof in the biological sample.